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[ "Characterization of a New Member of Alphacoronavirus with Unique Genomic Features in Rhinolophus Bats\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6521148/\n\nSHA: ee14de143337eec0e9708f8139bfac2b7b8fdd27\n\nAuthors: Wang, Ning; Luo, Chuming; Liu, Haizhou; Yang, Xinglou; Hu, Ben; Zhang, Wei; Li, Bei; Zhu, Yan; Zhu, Guangjian; Shen, Xurui; Peng, Cheng; Shi, Zhengli\nDate: 2019-04-24\nDOI: 10.3390/v11040379\nLicense: cc-by", "Abstract: Bats have been identified as a natural reservoir of a variety of coronaviruses (CoVs). Several of them have caused diseases in humans and domestic animals by interspecies transmission. Considering the diversity of bat coronaviruses, bat species and populations, we expect to discover more bat CoVs through virus surveillance. In this study, we described a new member of alphaCoV (BtCoV/Rh/YN2012) in bats with unique genome features. Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene. All the putative genes were further confirmed by reverse-transcription analyses. One unique gene at the 3’ end of the BtCoV/Rh/YN2012 genome, ORF9, exhibits ~30% amino acid identity to ORF7a of the SARS-related coronavirus. Functional analysis showed ORF4a protein can activate IFN-β production, whereas ORF3a can regulate NF-κB production. We also screened the spike-mediated virus entry using", "the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells. Our results expand the knowledge on the genetic diversity of bat coronaviruses. Continuous screening of bat viruses will help us further understand the important role played by bats in coronavirus evolution and transmission.", "Text: Members of the Coronaviridae family are enveloped, non-segmented, positive-strand RNA viruses with genome sizes ranging from 26-32 kb [1] . These viruses are classified into two subfamilies: Letovirinae, which contains the only genus: Alphaletovirus; and Orthocoronavirinae (CoV), which consists of alpha, beta, gamma, and deltacoronaviruses (CoVs) [2, 3] . Alpha and betacoronaviruses mainly infect mammals and cause human and animal diseases. Gamma-and delta-CoVs mainly infect birds, but some can also infect mammals [4, 5] . Six human CoVs (HCoVs) are known to cause human diseases. HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus (SARS-CoV) and", "All sampling procedures were performed by veterinarians, with approval from Animal Ethics Committee of the Wuhan Institute of Virology (WIVH5210201). The study was conducted in accordance with the Guide for the Care and Use of Wild Mammals in Research of the People's Republic of China.\n\nBat fecal swab and pellet samples were collected from November 2004 to November 2014 in different seasons in Southern China, as described previously [16] .\n\nViral RNA was extracted from 200 µL of fecal swab or pellet samples using the High Pure Viral RNA Kit (Roche Diagnostics GmbH, Mannheim, Germany) as per the manufacturer's instructions. RNA was eluted in 50 µL of elution buffer, aliquoted, and stored at -80 • C. One-step hemi-nested reverse-transcription (RT-) PCR (Invitrogen, San Diego, CA, USA) was employed to detect coronavirus, as previously described [17, 18] .", "To confirm the bat species of an individual sample, we PCR amplified the cytochrome b (Cytob) and/or NADH dehydrogenase subunit 1 (ND1) gene using DNA extracted from the feces or swabs [19, 20] . The gene sequences were assembled excluding the primer sequences. BLASTN was used to identify host species based on the most closely related sequences with the highest query coverage and a minimum identity of 95%.", "Full genomic sequences were determined by one-step PCR (Invitrogen, San Diego, CA, USA) amplification with degenerate primers (Table S1 ) designed on the basis of multiple alignments of available alpha-CoV sequences deposited in GenBank or amplified with SuperScript IV Reverse Transcriptase (Invitrogen) and Expand Long Template PCR System (Roche Diagnostics GmbH, Mannheim, Germany) with specific primers (primer sequences are available upon request). Sequences of the 5' and 3' genomic ends were obtained by 5' and 3' rapid amplification of cDNA ends (SMARTer Viruses 2019, 11, 379 3 of 19 RACE 5'/3' Kit; Clontech, Mountain View, CA, USA), respectively. PCR products were gel-purified and subjected directly to sequencing. PCR products over 5kb were subjected to deep sequencing using Hiseq2500 system. For some fragments, the PCR products were cloned into the pGEM-T Easy Vector (Promega, Madison, WI, USA) for sequencing. At least five independent clones were sequenced to obtain a consensus", "sequence.", "The Next Generation Sequencing (NGS) data were filtered and mapped to the reference sequence of BatCoV HKU10 (GenBank accession number NC_018871) using Geneious 7.1.8 [21] . Genomes were preliminarily assembled using DNAStar lasergene V7 (DNAStar, Madison, WI, USA). Putative open reading frames (ORFs) were predicted using NCBI's ORF finder (https://www.ncbi.nlm.nih.gov/ orffinder/) with a minimal ORF length of 150 nt, followed by manual inspection. The sequences of the 5' untranslated region (5'-UTR) and 3'-UTR were defined, and the leader sequence, the leader and body transcriptional regulatory sequence (TRS) were identified as previously described [22] . The cleavage of the 16 nonstructural proteins coded by ORF1ab was determined by alignment of aa sequences of other CoVs and the recognition pattern of the 3C-like proteinase and papain-like proteinase. Phylogenetic trees based on nt or aa sequences were constructed using the maximum likelihood algorithm with bootstrap values", "determined by 1000 replicates in the MEGA 6 software package [23] . Full-length genome sequences obtained in this study were aligned with those of previously reported alpha-CoVs using MUSCLE [24] . The aligned sequences were scanned for recombination events by using Recombination Detection Program [25] . Potential recombination events as suggested by strong p-values (<10 -20 ) were confirmed using similarity plot and bootscan analyses implemented in Simplot 3.5.1 [26] . The number of synonymous substitutions per synonymous site, Ks, and the number of nonsynonymous substitutions per nonsynonymous site, Ka, for each coding region were calculated using the Ka/Ks calculation tool of the Norwegian Bioinformatics Platform (http://services.cbu.uib.no/tools/kaks) with default parameters [27] . The protein homology detection was analyzed using HHpred (https://toolkit.tuebingen.mpg.de/#/tools/hhpred) with default parameters [28] .", "A set of nested RT-PCRs was employed to determine the presence of viral subgenomic mRNAs in the CoV-positive samples [29] . Forward primers were designed targeting the leader sequence at the 5'-end of the complete genome, while reverse primers were designed within the ORFs. Specific and suspected amplicons of expected sizes were purified and then cloned into the pGEM-T Easy vector for sequencing.", "Bat primary or immortalized cells (Rhinolophus sinicus kidney immortalized cells, RsKT; Rhinolophus sinicus Lung primary cells, RsLu4323; Rhinolophus sinicus brain immortalized cells, RsBrT; Rhinolophus affinis kidney primary cells, RaK4324; Rousettus leschenaultii Kidney immortalized cells, RlKT; Hipposideros pratti lung immortalized cells, HpLuT) generated in our laboratory were all cultured in DMEM/F12 with 15% FBS. Pteropus alecto kidney cells (Paki) was maintained in DMEM/F12 supplemented with 10% FBS. Other cells were maintained according to the recommendations of American Type Culture Collection (ATCC, www.atcc.org).", "The putative accessory genes of the newly detected virus were generated by RT-PCR from viral RNA extracted from fecal samples, as described previously [30] . The influenza virus NS1 plasmid was generated in our lab [31] . The human bocavirus (HBoV) VP2 plasmid was kindly provided by prof. Hanzhong Wang of the Wuhan Institute of Virology, Chinese Academy of Sciences. SARS-CoV ORF7a was synthesized by Sangon Biotech. The transfections were performed with Lipofectamine 3000 Reagent (Life Technologies). Expression of these accessory genes were analyzed by Western blotting using an mAb (Roche Diagnostics GmbH, Mannheim, Germany) against the HA tag.", "The virus isolation was performed as previously described [12] . Briefly, fecal supernatant was acquired via gradient centrifugation and then added to Vero E6 cells, 1:10 diluted in DMEM. After incubation at 37°C for 1 h the inoculum was replaced by fresh DMEM containing 2% FBS and the antibiotic-antimycotic (Gibco, Grand Island, NY, USA). Three blind passages were carried out. Cells were checked daily for cytopathic effect. Both culture supernatant and cell pellet were examined for CoV by RT-PCR [17] .", "Apoptosis was analyzed as previously described [18] . Briefly, 293T cells in 12-well plates were transfected with 3 µg of expression plasmid or empty vector, and the cells were collected 24 h post transfection. Apoptosis was detected by flow cytometry using by the Annexin V-FITC/PI Apoptosis Detection Kit (YEASEN, Shanghai, China) following the manufacturer's instructions. Annexin-V-positive and PI-negative cells were considered to be in the early apoptotic phase and those stained for both Annexin V and PI were deemed to undergo late apoptosis or necrosis. All experiments were repeated three times. Student's t-test was used to evaluate the data, with p < 0.05 considered significant.", "HEK 293T cells were seeded in 24-well plates and then co-transfected with reporter plasmids (pRL-TK and pIFN-βIFN-or pNF-κB-Luc) [30] , as well as plasmids expressing accessory genes, empty vector plasmid pcAGGS, influenza virus NS1 [32] , SARS-CoV ORF7a [33] , or HBoV VP2 [34] . At 24 h post transfection, cells were treated with Sendai virus (SeV) (100 hemagglutinin units [HAU]/mL) or human tumor necrosis factor alpha (TNF-α; R&D system) for 6 h to activate IFNβ or NF-κB, respectively. Cell lysates were prepared, and luciferase activity was measured using the dual-luciferase assay kit (Promega, Madison, WI, USA) according to the manufacturer's instructions.", "Retroviruses pseudotyped with BtCoV/Rh/YN2012 RsYN1, RsYN3, RaGD, or MERS-CoV spike, or no spike (mock) were used to infect human, bat or other mammalian cells in 96-well plates. The pseudovirus particles were confirmed with Western blotting and negative-staining electromicroscopy. The production process, measurements of infection and luciferase activity were conducted, as described previously [35, 36] .\n\nThe complete genome nucleotide sequences of BtCoV/Rh/YN2012 strains RsYN1, RsYN2, RsYN3, and RaGD obtained in this study have been submitted to the GenBank under MG916901 to MG916904.", "The surveillance was performed between November 2004 to November 2014 in 19 provinces of China. In total, 2061 fecal samples were collected from at least 12 Rhinolophus bat species ( Figure 1A ). CoVs were detected in 209 of these samples ( Figure 1B and Table 1 ). Partial RdRp sequences suggested the presence of at least 8 different CoVs. Five of these viruses are related to known species: Mi-BatCoV 1 (>94% nt identity), Mi-BatCoV HKU8 [37] (>93% nt identity), BtRf-AlphaCoV/HuB2013 [11] (>99% nt identity), SARSr-CoV [38] (>89% nt identity), and HKU2-related CoV [39] (>85% nt identity). While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species. Virus isolation was performed as previously described [12] , but was not successful. identity). While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species. Virus isolation", "was performed as previously described [12] , but was not successful.", "We next characterized a novel alpha-CoV, BtCoV/Rh/YN2012. It was detected in 3 R.affinis and 6 R.sinicus, respectively. Based on the sequences, we defined three genotypes, which represented by RsYN1, RsYN3, and RaGD, respectively. Strain RsYN2 was classified into the RsYN3 genotype. Four full-length genomes were obtained. Three of them were from R.sinicus (Strain RsYN1, RsYN2, and RsYN3), while the other one was from R.affinis (Strain RaGD). The sizes of these 4 genomes are between 28,715 to 29,102, with G+C contents between 39.0% to 41.3%. The genomes exhibit similar structures and transcription regulatory sequences (TRS) that are identical to those of other alpha-CoVs ( Figure 2 and Table 2 ). Exceptions including three additional ORFs (ORF3b, ORF4a and ORF4b) were observed. All the 4 strains have ORF4a & ORF4b, while only strain RsYN1 has ORF3b.", "The replicase gene, ORF1ab, occupies~20.4 kb of the genome. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. It encodes polyproteins 1a and 1ab, which could be cleaved into 16 non-structural proteins (Nsp1-Nsp16). The 3'-end of the cleavage sites recognized by 3C-like proteinase (Nsp4-Nsp10, Nsp12-Nsp16) and papain-like proteinase (Nsp1-Nsp3) were confirmed. The proteins including Nsp3 (papain-like 2 proteas, PL2pro), Nsp5 (chymotrypsin-like protease, 3CLpro), Nsp12 (RdRp), Nsp13 (helicase), and other proteins of unknown function ( Table 3 ). The 7 concatenated domains of polyprotein 1 shared <90% aa sequence identity with those of other known alpha-CoVs ( Table 2 ), suggesting that these viruses represent a novel CoV species within the alpha-CoV. The closest assigned CoV species to BtCoV/Rh/YN2012 are BtCoV-HKU10 and BtRf-AlphaCoV/Hub2013. The three strains from Yunnan Province were clustered into two genotypes (83% genome identity) correlated to their sampling location.", "The third genotype represented by strain RaGD was isolated to strains found in Yunnan (<75.4% genome identity). We then examined the individual genes ( Table 2) . All of the genes showed low aa sequence identity to known CoVs. The four strains of BtCoV/Rh/YN2012 showed genetic diversity among all different genes except ORF1ab (>83.7% aa identity). Notably, the spike proteins are highly divergent among these strains. Other structure proteins (E, M, and N) are more conserved than the spike and other accessory proteins. Comparing the accessory genes among these four strains revealed that the strains of the same genotype shared a 100% identical ORF3a. However, the proteins encoded by ORF3as were highly divergent among different genotypes (<65% aa identity). The putative accessory genes were also BLASTed against GenBank records. Most accessory genes have no homologues in GenBank-database, except for ORF3a (52.0-55.5% aa identity with BatCoV HKU10 ORF3) and ORF9 (28.1-32.0% aa identity with", "SARSr-CoV ORF7a). We analyzed the protein homology with HHpred software. The results showed that ORF9s and SARS-CoV OR7a are homologues (possibility: 100%, E value <10 −48 ). We further screened the genomes for potential recombination evidence. No significant recombination breakpoint was detected by bootscan analysis.", "To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing ( Figure 3 and Table 2 ). The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. \n\nTo confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing ( Figure 3 and Table 2 ). The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b.", "Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs (Figure 4) . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history.", "Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs (Figure 4) . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history.", "Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs (Figure 4) . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history.", "The Ka/Ks ratios (Ks is the number of synonymous substitutions per synonymous sites and Ka is the number of nonsynonymous substitutions per nonsynonymous site) were calculated for all genes. The Ka/Ks ratios for most of the genes were generally low, which indicates these genes were under purified selection. However, the Ka/Ks ratios of ORF4a, ORF4b, and ORF9 (0.727, 0.623, and 0.843, respectively) were significantly higher than those of other ORFs (Table 4 ). For further selection pressure evaluation of the ORF4a and ORF4b gene, we sequenced another four ORF4a and ORF4b genes (strain Rs4223, Rs4236, Rs4240, and Ra13576 was shown in Figure 1B", "As SARS-CoV ORF7a was reported to induce apoptosis, we conducted apoptosis analysis on BtCoV/Rh/YN2012 ORF9, a~30% aa identity homologue of SARSr-CoV ORF7a. We transiently transfected ORF9 of BtCoV/Rh/YN2012 into HEK293T cells to examine whether this ORF9 triggers apoptosis. Western blot was performed to confirm the expression of ORF9s and SARS-CoV ORF7a ( Figure S1 ). ORF9 couldn't induce apoptosis as the ORF7a of SARS-CoV Tor2 ( Figure S2 ). The results indicated that BtCoV/Rh/YN2012 ORF9 was not involved in apoptosis induction.", "To determine whether these accessory proteins modulate IFN induction, we transfected reporter plasmids (pIFNβ-Luc and pRL-TK) and expression plasmids to 293T cells. All the cells over-expressing the accessory genes, as well as influenza virus NS1 (strain PR8), HBoV VP2, or empty vector were tested for luciferase activity after SeV infection. Luciferase activity stimulated by SeV was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 ( Figure 5A ). Other accessory proteins showed no effect on IFN production ( Figure S3 ). Expression of these accessory genes were confirmed by Western blot ( Figure S1 ). was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those", "controls, the ORF4a proteins exhibit an active effect as HBoV VP2 ( Figure 5A ). Other accessory proteins showed no effect on IFN production ( Figure S3 ). Expression of these accessory genes were confirmed by Western blot (Figure S1 ). Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. (B) ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector (500 ng), an NS1-expressing plasmid (500 ng), a SARS-CoV ORF7a-expressing plasmid (500 ng), or ORF3a-expressing plasmids (500 ng). After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each", "determination performed in triplicate (mean ± SD of fold change). Asterisks indicate significant differences between groups (compared with Empty vector-NC, p < 0.05, as determined by student t test).", "NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. (B) ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector (500 ng), an NS1-expressing plasmid (500 ng), a SARS-CoV ORF7a-expressing plasmid (500 ng), or ORF3a-expressing plasmids (500 ng). After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative", "of at least three independent experiments, with each determination performed in triplicate (mean ± SD of fold change). Asterisks indicate significant differences between groups (compared with Empty vector-NC, p < 0.05, as determined by student t test).", "NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter plasmids (pNF-κB-Luc and pRL-TK), as well as accessory protein-expressing plasmids, or controls (empty vector, NS1, SARS-CoV Tor2-ORF7a). The cells were mock treated or treated with TNF-α for 6 h at 24 h post-transfection. The luciferase activity was determined. RsYN1-ORF3a and RaGD-ORF3a activated NF-κB as SARS-CoV ORF7a, whereas RsYN2-ORF3a inhibited NF-κB as NS1 ( Figure 5B ). Expressions of ORF3as were confirmed with Western blot ( Figure S1 ). Other accessory proteins did not modulate NF-κB production ( Figure S4 ).", "To understand the infectivity of these newly detected BtCoV/Rh/YN2012, we selected the RsYN1, RsYN3 and RaGD spike proteins for spike-mediated pseudovirus entry studies. Both Western blot analysis and negative-staining electron microscopy observation confirmed the preparation of BtCoV/Rh/YN2012 successfully ( Figure S5 ). A total of 11 human cell lines, 8 bat cells, and 9 other mammal cell lines were tested, and no strong positive was found (Table S2) .", "In this study, a novel alpha-CoV species, BtCoV/Rh/YN2012, was identified in two Rhinolophus species. The 4 strains with full-length genome were sequences. The 7 conserved replicase domains of these viruses possessed <90% aa sequence identity to those of other known alpha-CoVs, which defines a new species in accordance with the ICTV taxonomy standard [42] . These novel alpha-CoVs showed high genetic diversity in their structural and non-structural genes. Strain RaGD from R. affinis, collected in Guangdong province, formed a divergent independent branch from the other 3 strains from R. sinicus, sampled in Yunnan Province, indicating an independent evolution process associated with geographic isolation and host restrain. Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations. These two genotypes had low genome sequence identity, especially in the S gene and accessory genes. Considering the remote geographic location of the", "host bat habitat, the host tropism, and the virus diversity, we suppose BtCoV/Rh/YN2012 may have spread in these two provinces with a long history of circulation in their natural reservoir, Rhinolophus bats. With the sequence evidence, we suppose that these viruses are still rapidly evolving.", "Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs. First, novel accessory genes, which had no homologues, were identified in the genomes. Second, multiple TRSs were found between S and E genes while other alphacoronavirus only had one TRS there. These TRSs precede ORF3a, ORF3b (only in RsYN1), and ORF4a/b respectively. Third, accessory gene ORF9 showed homology with those of other known CoV species in another coronavirus genus, especially with accessory genes from SARSr-CoV.", "Accessory genes are usually involved in virus-host interactions during CoV infection [43] . In most CoVs, accessory genes are dispensable for virus replication. However, an intact 3c gene of feline CoV was required for viral replication in the gut [44] [45] [46] . Deletion of the genus-specific genes in mouse hepatitis virus led to a reduction in virulence [47] . SARS-CoV ORF7a, which was identified to be involved in the suppression of RNA silencing [48] , inhibition of cellular protein synthesis [49] , cell-cycle blockage [50] , and apoptosis induction [51, 52] . In this study, we found that BtCoV/Rh/YN2012 ORF9 shares~30% aa sequence identity with SARS-CoV ORF7a. Interestingly, BtCoV/Rh/YN2012 and SARSr-CoV were both detected in R. sinicus from the same cave. We suppose that SARS-CoV and BtCoV/Rh/YN2012 may have acquired ORF7a or ORF9 from a common ancestor through genome recombination or horizontal gene transfer. Whereas, ORF9 of BtCoV/Rh/YN2012 failed to induce apoptosis or", "activate NF-κB production, these differences may be induced by the divergent evolution of these proteins in different pressure.", "Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β. ORF3a from RsYN1 and RaGD upregulated NF-κB, but the homologue from RsYN2 downregulated NF-κB expression. These differences may be caused by amino acid sequence variations and may contribute to a viruses' pathogenicity with a different pathway.", "Though lacking of intestinal cell lines from the natural host of BtCoV/Rh/YN2012, we screened the cell tropism of their spike protein through pseudotyped retrovirus entry with human, bat and other mammalian cell lines. Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals. Multiple reasons may lead to failed infection of coronavirus spike-pseudotyped retrovirus system, including receptor absence in target cells, failed recognition to the receptor homologue from non-host species, maladaptation in non-host cells during the spike maturation or virus entry, or the limitation of retrovirus system in stimulating coronavirus entry. The weak infectivity of RsYN1 pseudotyped retrovirus in Huh-7 cells could be explained by the binding of spike protein to polysaccharide secreted to the surface. The assumption needs to be further confirmed by experiments.", "Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs. Coincidently, the two highly pathogenic agents, SARS-CoV and Rh-BatCoV HKU2 both originated from Rhinolophus bats. Considering the diversity of CoVs carried by this bat genus and their wide geographical distribution, there may be a low risk of spillover of these viruses to other animals and humans. Long-term surveillances and pathogenesis studies will help to prevent future human and animal diseases caused by these bat CoVs.", "Supplementary Materials: The following are available online at http://www.mdpi.com/1999-4915/11/4/379/s1, Figure S1 : western blot analysis of the expression of accessory proteins. Figure S2 : Apoptosis analysis of ORF9 proteins of BtCoV/Rh/YN2012. Figure S3 : Functional analysis of ORF3a, ORF3b, ORF4b, ORF8 and ORF9 proteins on the production of Type I interferon. Figure S4 : Functional analysis of ORF3b, ORF4a, ORF4b, ORF8 and ORF9 proteins on the production of NF-κB. Figure S5 : Characteristic of BtCoV/Rh/YN2012 spike mediated pseudovirus. Table S1 : General primers for AlphaCoVs genome sequencing. Table S2 : Primers for the detection of viral sugbenomic mRNAs. Table S3" ]

[ 3 ]

[ "NF-κB" ]

[ "Characterization of a New Member of Alphacoronavirus with Unique Genomic Features in Rhinolophus Bats\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6521148/\n\nSHA: ee14de143337eec0e9708f8139bfac2b7b8fdd27\n\nAuthors: Wang, Ning; Luo, Chuming; Liu, Haizhou; Yang, Xinglou; Hu, Ben; Zhang, Wei; Li, Bei; Zhu, Yan; Zhu, Guangjian; Shen, Xurui; Peng, Cheng; Shi, Zhengli\nDate: 2019-04-24\nDOI: 10.3390/v11040379\nLicense: cc-by", "Abstract: Bats have been identified as a natural reservoir of a variety of coronaviruses (CoVs). Several of them have caused diseases in humans and domestic animals by interspecies transmission. Considering the diversity of bat coronaviruses, bat species and populations, we expect to discover more bat CoVs through virus surveillance. In this study, we described a new member of alphaCoV (BtCoV/Rh/YN2012) in bats with unique genome features. Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene. All the putative genes were further confirmed by reverse-transcription analyses. One unique gene at the 3’ end of the BtCoV/Rh/YN2012 genome, ORF9, exhibits ~30% amino acid identity to ORF7a of the SARS-related coronavirus. Functional analysis showed ORF4a protein can activate IFN-β production, whereas ORF3a can regulate NF-κB production. We also screened the spike-mediated virus entry using", "the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells. Our results expand the knowledge on the genetic diversity of bat coronaviruses. Continuous screening of bat viruses will help us further understand the important role played by bats in coronavirus evolution and transmission.", "Text: Members of the Coronaviridae family are enveloped, non-segmented, positive-strand RNA viruses with genome sizes ranging from 26-32 kb [1] . These viruses are classified into two subfamilies: Letovirinae, which contains the only genus: Alphaletovirus; and Orthocoronavirinae (CoV), which consists of alpha, beta, gamma, and deltacoronaviruses (CoVs) [2, 3] . Alpha and betacoronaviruses mainly infect mammals and cause human and animal diseases. Gamma-and delta-CoVs mainly infect birds, but some can also infect mammals [4, 5] . Six human CoVs (HCoVs) are known to cause human diseases. HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus (SARS-CoV) and", "All sampling procedures were performed by veterinarians, with approval from Animal Ethics Committee of the Wuhan Institute of Virology (WIVH5210201). The study was conducted in accordance with the Guide for the Care and Use of Wild Mammals in Research of the People's Republic of China.\n\nBat fecal swab and pellet samples were collected from November 2004 to November 2014 in different seasons in Southern China, as described previously [16] .\n\nViral RNA was extracted from 200 µL of fecal swab or pellet samples using the High Pure Viral RNA Kit (Roche Diagnostics GmbH, Mannheim, Germany) as per the manufacturer's instructions. RNA was eluted in 50 µL of elution buffer, aliquoted, and stored at -80 • C. One-step hemi-nested reverse-transcription (RT-) PCR (Invitrogen, San Diego, CA, USA) was employed to detect coronavirus, as previously described [17, 18] .", "To confirm the bat species of an individual sample, we PCR amplified the cytochrome b (Cytob) and/or NADH dehydrogenase subunit 1 (ND1) gene using DNA extracted from the feces or swabs [19, 20] . The gene sequences were assembled excluding the primer sequences. BLASTN was used to identify host species based on the most closely related sequences with the highest query coverage and a minimum identity of 95%.", "Full genomic sequences were determined by one-step PCR (Invitrogen, San Diego, CA, USA) amplification with degenerate primers (Table S1 ) designed on the basis of multiple alignments of available alpha-CoV sequences deposited in GenBank or amplified with SuperScript IV Reverse Transcriptase (Invitrogen) and Expand Long Template PCR System (Roche Diagnostics GmbH, Mannheim, Germany) with specific primers (primer sequences are available upon request). Sequences of the 5' and 3' genomic ends were obtained by 5' and 3' rapid amplification of cDNA ends (SMARTer Viruses 2019, 11, 379 3 of 19 RACE 5'/3' Kit; Clontech, Mountain View, CA, USA), respectively. PCR products were gel-purified and subjected directly to sequencing. PCR products over 5kb were subjected to deep sequencing using Hiseq2500 system. For some fragments, the PCR products were cloned into the pGEM-T Easy Vector (Promega, Madison, WI, USA) for sequencing. At least five independent clones were sequenced to obtain a consensus", "sequence.", "The Next Generation Sequencing (NGS) data were filtered and mapped to the reference sequence of BatCoV HKU10 (GenBank accession number NC_018871) using Geneious 7.1.8 [21] . Genomes were preliminarily assembled using DNAStar lasergene V7 (DNAStar, Madison, WI, USA). Putative open reading frames (ORFs) were predicted using NCBI's ORF finder (https://www.ncbi.nlm.nih.gov/ orffinder/) with a minimal ORF length of 150 nt, followed by manual inspection. The sequences of the 5' untranslated region (5'-UTR) and 3'-UTR were defined, and the leader sequence, the leader and body transcriptional regulatory sequence (TRS) were identified as previously described [22] . The cleavage of the 16 nonstructural proteins coded by ORF1ab was determined by alignment of aa sequences of other CoVs and the recognition pattern of the 3C-like proteinase and papain-like proteinase. Phylogenetic trees based on nt or aa sequences were constructed using the maximum likelihood algorithm with bootstrap values", "determined by 1000 replicates in the MEGA 6 software package [23] . Full-length genome sequences obtained in this study were aligned with those of previously reported alpha-CoVs using MUSCLE [24] . The aligned sequences were scanned for recombination events by using Recombination Detection Program [25] . Potential recombination events as suggested by strong p-values (<10 -20 ) were confirmed using similarity plot and bootscan analyses implemented in Simplot 3.5.1 [26] . The number of synonymous substitutions per synonymous site, Ks, and the number of nonsynonymous substitutions per nonsynonymous site, Ka, for each coding region were calculated using the Ka/Ks calculation tool of the Norwegian Bioinformatics Platform (http://services.cbu.uib.no/tools/kaks) with default parameters [27] . The protein homology detection was analyzed using HHpred (https://toolkit.tuebingen.mpg.de/#/tools/hhpred) with default parameters [28] .", "A set of nested RT-PCRs was employed to determine the presence of viral subgenomic mRNAs in the CoV-positive samples [29] . Forward primers were designed targeting the leader sequence at the 5'-end of the complete genome, while reverse primers were designed within the ORFs. Specific and suspected amplicons of expected sizes were purified and then cloned into the pGEM-T Easy vector for sequencing.", "Bat primary or immortalized cells (Rhinolophus sinicus kidney immortalized cells, RsKT; Rhinolophus sinicus Lung primary cells, RsLu4323; Rhinolophus sinicus brain immortalized cells, RsBrT; Rhinolophus affinis kidney primary cells, RaK4324; Rousettus leschenaultii Kidney immortalized cells, RlKT; Hipposideros pratti lung immortalized cells, HpLuT) generated in our laboratory were all cultured in DMEM/F12 with 15% FBS. Pteropus alecto kidney cells (Paki) was maintained in DMEM/F12 supplemented with 10% FBS. Other cells were maintained according to the recommendations of American Type Culture Collection (ATCC, www.atcc.org).", "The putative accessory genes of the newly detected virus were generated by RT-PCR from viral RNA extracted from fecal samples, as described previously [30] . The influenza virus NS1 plasmid was generated in our lab [31] . The human bocavirus (HBoV) VP2 plasmid was kindly provided by prof. Hanzhong Wang of the Wuhan Institute of Virology, Chinese Academy of Sciences. SARS-CoV ORF7a was synthesized by Sangon Biotech. The transfections were performed with Lipofectamine 3000 Reagent (Life Technologies). Expression of these accessory genes were analyzed by Western blotting using an mAb (Roche Diagnostics GmbH, Mannheim, Germany) against the HA tag.", "The virus isolation was performed as previously described [12] . Briefly, fecal supernatant was acquired via gradient centrifugation and then added to Vero E6 cells, 1:10 diluted in DMEM. After incubation at 37°C for 1 h the inoculum was replaced by fresh DMEM containing 2% FBS and the antibiotic-antimycotic (Gibco, Grand Island, NY, USA). Three blind passages were carried out. Cells were checked daily for cytopathic effect. Both culture supernatant and cell pellet were examined for CoV by RT-PCR [17] .", "Apoptosis was analyzed as previously described [18] . Briefly, 293T cells in 12-well plates were transfected with 3 µg of expression plasmid or empty vector, and the cells were collected 24 h post transfection. Apoptosis was detected by flow cytometry using by the Annexin V-FITC/PI Apoptosis Detection Kit (YEASEN, Shanghai, China) following the manufacturer's instructions. Annexin-V-positive and PI-negative cells were considered to be in the early apoptotic phase and those stained for both Annexin V and PI were deemed to undergo late apoptosis or necrosis. All experiments were repeated three times. Student's t-test was used to evaluate the data, with p < 0.05 considered significant.", "HEK 293T cells were seeded in 24-well plates and then co-transfected with reporter plasmids (pRL-TK and pIFN-βIFN-or pNF-κB-Luc) [30] , as well as plasmids expressing accessory genes, empty vector plasmid pcAGGS, influenza virus NS1 [32] , SARS-CoV ORF7a [33] , or HBoV VP2 [34] . At 24 h post transfection, cells were treated with Sendai virus (SeV) (100 hemagglutinin units [HAU]/mL) or human tumor necrosis factor alpha (TNF-α; R&D system) for 6 h to activate IFNβ or NF-κB, respectively. Cell lysates were prepared, and luciferase activity was measured using the dual-luciferase assay kit (Promega, Madison, WI, USA) according to the manufacturer's instructions.", "Retroviruses pseudotyped with BtCoV/Rh/YN2012 RsYN1, RsYN3, RaGD, or MERS-CoV spike, or no spike (mock) were used to infect human, bat or other mammalian cells in 96-well plates. The pseudovirus particles were confirmed with Western blotting and negative-staining electromicroscopy. The production process, measurements of infection and luciferase activity were conducted, as described previously [35, 36] .\n\nThe complete genome nucleotide sequences of BtCoV/Rh/YN2012 strains RsYN1, RsYN2, RsYN3, and RaGD obtained in this study have been submitted to the GenBank under MG916901 to MG916904.", "The surveillance was performed between November 2004 to November 2014 in 19 provinces of China. In total, 2061 fecal samples were collected from at least 12 Rhinolophus bat species ( Figure 1A ). CoVs were detected in 209 of these samples ( Figure 1B and Table 1 ). Partial RdRp sequences suggested the presence of at least 8 different CoVs. Five of these viruses are related to known species: Mi-BatCoV 1 (>94% nt identity), Mi-BatCoV HKU8 [37] (>93% nt identity), BtRf-AlphaCoV/HuB2013 [11] (>99% nt identity), SARSr-CoV [38] (>89% nt identity), and HKU2-related CoV [39] (>85% nt identity). While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species. Virus isolation was performed as previously described [12] , but was not successful. identity). While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species. Virus isolation", "was performed as previously described [12] , but was not successful.", "We next characterized a novel alpha-CoV, BtCoV/Rh/YN2012. It was detected in 3 R.affinis and 6 R.sinicus, respectively. Based on the sequences, we defined three genotypes, which represented by RsYN1, RsYN3, and RaGD, respectively. Strain RsYN2 was classified into the RsYN3 genotype. Four full-length genomes were obtained. Three of them were from R.sinicus (Strain RsYN1, RsYN2, and RsYN3), while the other one was from R.affinis (Strain RaGD). The sizes of these 4 genomes are between 28,715 to 29,102, with G+C contents between 39.0% to 41.3%. The genomes exhibit similar structures and transcription regulatory sequences (TRS) that are identical to those of other alpha-CoVs ( Figure 2 and Table 2 ). Exceptions including three additional ORFs (ORF3b, ORF4a and ORF4b) were observed. All the 4 strains have ORF4a & ORF4b, while only strain RsYN1 has ORF3b.", "The replicase gene, ORF1ab, occupies~20.4 kb of the genome. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. It encodes polyproteins 1a and 1ab, which could be cleaved into 16 non-structural proteins (Nsp1-Nsp16). The 3'-end of the cleavage sites recognized by 3C-like proteinase (Nsp4-Nsp10, Nsp12-Nsp16) and papain-like proteinase (Nsp1-Nsp3) were confirmed. The proteins including Nsp3 (papain-like 2 proteas, PL2pro), Nsp5 (chymotrypsin-like protease, 3CLpro), Nsp12 (RdRp), Nsp13 (helicase), and other proteins of unknown function ( Table 3 ). The 7 concatenated domains of polyprotein 1 shared <90% aa sequence identity with those of other known alpha-CoVs ( Table 2 ), suggesting that these viruses represent a novel CoV species within the alpha-CoV. The closest assigned CoV species to BtCoV/Rh/YN2012 are BtCoV-HKU10 and BtRf-AlphaCoV/Hub2013. The three strains from Yunnan Province were clustered into two genotypes (83% genome identity) correlated to their sampling location.", "The third genotype represented by strain RaGD was isolated to strains found in Yunnan (<75.4% genome identity). We then examined the individual genes ( Table 2) . All of the genes showed low aa sequence identity to known CoVs. The four strains of BtCoV/Rh/YN2012 showed genetic diversity among all different genes except ORF1ab (>83.7% aa identity). Notably, the spike proteins are highly divergent among these strains. Other structure proteins (E, M, and N) are more conserved than the spike and other accessory proteins. Comparing the accessory genes among these four strains revealed that the strains of the same genotype shared a 100% identical ORF3a. However, the proteins encoded by ORF3as were highly divergent among different genotypes (<65% aa identity). The putative accessory genes were also BLASTed against GenBank records. Most accessory genes have no homologues in GenBank-database, except for ORF3a (52.0-55.5% aa identity with BatCoV HKU10 ORF3) and ORF9 (28.1-32.0% aa identity with", "SARSr-CoV ORF7a). We analyzed the protein homology with HHpred software. The results showed that ORF9s and SARS-CoV OR7a are homologues (possibility: 100%, E value <10 −48 ). We further screened the genomes for potential recombination evidence. No significant recombination breakpoint was detected by bootscan analysis.", "To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing ( Figure 3 and Table 2 ). The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. \n\nTo confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing ( Figure 3 and Table 2 ). The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b.", "Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs (Figure 4) . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history.", "Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs (Figure 4) . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history.", "Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs (Figure 4) . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history.", "The Ka/Ks ratios (Ks is the number of synonymous substitutions per synonymous sites and Ka is the number of nonsynonymous substitutions per nonsynonymous site) were calculated for all genes. The Ka/Ks ratios for most of the genes were generally low, which indicates these genes were under purified selection. However, the Ka/Ks ratios of ORF4a, ORF4b, and ORF9 (0.727, 0.623, and 0.843, respectively) were significantly higher than those of other ORFs (Table 4 ). For further selection pressure evaluation of the ORF4a and ORF4b gene, we sequenced another four ORF4a and ORF4b genes (strain Rs4223, Rs4236, Rs4240, and Ra13576 was shown in Figure 1B", "As SARS-CoV ORF7a was reported to induce apoptosis, we conducted apoptosis analysis on BtCoV/Rh/YN2012 ORF9, a~30% aa identity homologue of SARSr-CoV ORF7a. We transiently transfected ORF9 of BtCoV/Rh/YN2012 into HEK293T cells to examine whether this ORF9 triggers apoptosis. Western blot was performed to confirm the expression of ORF9s and SARS-CoV ORF7a ( Figure S1 ). ORF9 couldn't induce apoptosis as the ORF7a of SARS-CoV Tor2 ( Figure S2 ). The results indicated that BtCoV/Rh/YN2012 ORF9 was not involved in apoptosis induction.", "To determine whether these accessory proteins modulate IFN induction, we transfected reporter plasmids (pIFNβ-Luc and pRL-TK) and expression plasmids to 293T cells. All the cells over-expressing the accessory genes, as well as influenza virus NS1 (strain PR8), HBoV VP2, or empty vector were tested for luciferase activity after SeV infection. Luciferase activity stimulated by SeV was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 ( Figure 5A ). Other accessory proteins showed no effect on IFN production ( Figure S3 ). Expression of these accessory genes were confirmed by Western blot ( Figure S1 ). was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those", "controls, the ORF4a proteins exhibit an active effect as HBoV VP2 ( Figure 5A ). Other accessory proteins showed no effect on IFN production ( Figure S3 ). Expression of these accessory genes were confirmed by Western blot (Figure S1 ). Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. (B) ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector (500 ng), an NS1-expressing plasmid (500 ng), a SARS-CoV ORF7a-expressing plasmid (500 ng), or ORF3a-expressing plasmids (500 ng). After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each", "determination performed in triplicate (mean ± SD of fold change). Asterisks indicate significant differences between groups (compared with Empty vector-NC, p < 0.05, as determined by student t test).", "NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. (B) ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector (500 ng), an NS1-expressing plasmid (500 ng), a SARS-CoV ORF7a-expressing plasmid (500 ng), or ORF3a-expressing plasmids (500 ng). After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative", "of at least three independent experiments, with each determination performed in triplicate (mean ± SD of fold change). Asterisks indicate significant differences between groups (compared with Empty vector-NC, p < 0.05, as determined by student t test).", "NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter plasmids (pNF-κB-Luc and pRL-TK), as well as accessory protein-expressing plasmids, or controls (empty vector, NS1, SARS-CoV Tor2-ORF7a). The cells were mock treated or treated with TNF-α for 6 h at 24 h post-transfection. The luciferase activity was determined. RsYN1-ORF3a and RaGD-ORF3a activated NF-κB as SARS-CoV ORF7a, whereas RsYN2-ORF3a inhibited NF-κB as NS1 ( Figure 5B ). Expressions of ORF3as were confirmed with Western blot ( Figure S1 ). Other accessory proteins did not modulate NF-κB production ( Figure S4 ).", "To understand the infectivity of these newly detected BtCoV/Rh/YN2012, we selected the RsYN1, RsYN3 and RaGD spike proteins for spike-mediated pseudovirus entry studies. Both Western blot analysis and negative-staining electron microscopy observation confirmed the preparation of BtCoV/Rh/YN2012 successfully ( Figure S5 ). A total of 11 human cell lines, 8 bat cells, and 9 other mammal cell lines were tested, and no strong positive was found (Table S2) .", "In this study, a novel alpha-CoV species, BtCoV/Rh/YN2012, was identified in two Rhinolophus species. The 4 strains with full-length genome were sequences. The 7 conserved replicase domains of these viruses possessed <90% aa sequence identity to those of other known alpha-CoVs, which defines a new species in accordance with the ICTV taxonomy standard [42] . These novel alpha-CoVs showed high genetic diversity in their structural and non-structural genes. Strain RaGD from R. affinis, collected in Guangdong province, formed a divergent independent branch from the other 3 strains from R. sinicus, sampled in Yunnan Province, indicating an independent evolution process associated with geographic isolation and host restrain. Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations. These two genotypes had low genome sequence identity, especially in the S gene and accessory genes. Considering the remote geographic location of the", "host bat habitat, the host tropism, and the virus diversity, we suppose BtCoV/Rh/YN2012 may have spread in these two provinces with a long history of circulation in their natural reservoir, Rhinolophus bats. With the sequence evidence, we suppose that these viruses are still rapidly evolving.", "Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs. First, novel accessory genes, which had no homologues, were identified in the genomes. Second, multiple TRSs were found between S and E genes while other alphacoronavirus only had one TRS there. These TRSs precede ORF3a, ORF3b (only in RsYN1), and ORF4a/b respectively. Third, accessory gene ORF9 showed homology with those of other known CoV species in another coronavirus genus, especially with accessory genes from SARSr-CoV.", "Accessory genes are usually involved in virus-host interactions during CoV infection [43] . In most CoVs, accessory genes are dispensable for virus replication. However, an intact 3c gene of feline CoV was required for viral replication in the gut [44] [45] [46] . Deletion of the genus-specific genes in mouse hepatitis virus led to a reduction in virulence [47] . SARS-CoV ORF7a, which was identified to be involved in the suppression of RNA silencing [48] , inhibition of cellular protein synthesis [49] , cell-cycle blockage [50] , and apoptosis induction [51, 52] . In this study, we found that BtCoV/Rh/YN2012 ORF9 shares~30% aa sequence identity with SARS-CoV ORF7a. Interestingly, BtCoV/Rh/YN2012 and SARSr-CoV were both detected in R. sinicus from the same cave. We suppose that SARS-CoV and BtCoV/Rh/YN2012 may have acquired ORF7a or ORF9 from a common ancestor through genome recombination or horizontal gene transfer. Whereas, ORF9 of BtCoV/Rh/YN2012 failed to induce apoptosis or", "activate NF-κB production, these differences may be induced by the divergent evolution of these proteins in different pressure.", "Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β. ORF3a from RsYN1 and RaGD upregulated NF-κB, but the homologue from RsYN2 downregulated NF-κB expression. These differences may be caused by amino acid sequence variations and may contribute to a viruses' pathogenicity with a different pathway.", "Though lacking of intestinal cell lines from the natural host of BtCoV/Rh/YN2012, we screened the cell tropism of their spike protein through pseudotyped retrovirus entry with human, bat and other mammalian cell lines. Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals. Multiple reasons may lead to failed infection of coronavirus spike-pseudotyped retrovirus system, including receptor absence in target cells, failed recognition to the receptor homologue from non-host species, maladaptation in non-host cells during the spike maturation or virus entry, or the limitation of retrovirus system in stimulating coronavirus entry. The weak infectivity of RsYN1 pseudotyped retrovirus in Huh-7 cells could be explained by the binding of spike protein to polysaccharide secreted to the surface. The assumption needs to be further confirmed by experiments.", "Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs. Coincidently, the two highly pathogenic agents, SARS-CoV and Rh-BatCoV HKU2 both originated from Rhinolophus bats. Considering the diversity of CoVs carried by this bat genus and their wide geographical distribution, there may be a low risk of spillover of these viruses to other animals and humans. Long-term surveillances and pathogenesis studies will help to prevent future human and animal diseases caused by these bat CoVs.", "Supplementary Materials: The following are available online at http://www.mdpi.com/1999-4915/11/4/379/s1, Figure S1 : western blot analysis of the expression of accessory proteins. Figure S2 : Apoptosis analysis of ORF9 proteins of BtCoV/Rh/YN2012. Figure S3 : Functional analysis of ORF3a, ORF3b, ORF4b, ORF8 and ORF9 proteins on the production of Type I interferon. Figure S4 : Functional analysis of ORF3b, ORF4a, ORF4b, ORF8 and ORF9 proteins on the production of NF-κB. Figure S5 : Characteristic of BtCoV/Rh/YN2012 spike mediated pseudovirus. Table S1 : General primers for AlphaCoVs genome sequencing. Table S2 : Primers for the detection of viral sugbenomic mRNAs. Table S3" ]

[ 1 ]

[ "Rhinolophus bats seem to harbor a wide diversity of CoVs" ]

[ "Characterization of a New Member of Alphacoronavirus with Unique Genomic Features in Rhinolophus Bats\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6521148/\n\nSHA: ee14de143337eec0e9708f8139bfac2b7b8fdd27\n\nAuthors: Wang, Ning; Luo, Chuming; Liu, Haizhou; Yang, Xinglou; Hu, Ben; Zhang, Wei; Li, Bei; Zhu, Yan; Zhu, Guangjian; Shen, Xurui; Peng, Cheng; Shi, Zhengli\nDate: 2019-04-24\nDOI: 10.3390/v11040379\nLicense: cc-by", "Abstract: Bats have been identified as a natural reservoir of a variety of coronaviruses (CoVs). Several of them have caused diseases in humans and domestic animals by interspecies transmission. Considering the diversity of bat coronaviruses, bat species and populations, we expect to discover more bat CoVs through virus surveillance. In this study, we described a new member of alphaCoV (BtCoV/Rh/YN2012) in bats with unique genome features. Unique accessory genes, ORF4a and ORF4b were found between the spike gene and the envelope gene, while ORF8 gene was found downstream of the nucleocapsid gene. All the putative genes were further confirmed by reverse-transcription analyses. One unique gene at the 3’ end of the BtCoV/Rh/YN2012 genome, ORF9, exhibits ~30% amino acid identity to ORF7a of the SARS-related coronavirus. Functional analysis showed ORF4a protein can activate IFN-β production, whereas ORF3a can regulate NF-κB production. We also screened the spike-mediated virus entry using", "the spike-pseudotyped retroviruses system, although failed to find any fully permissive cells. Our results expand the knowledge on the genetic diversity of bat coronaviruses. Continuous screening of bat viruses will help us further understand the important role played by bats in coronavirus evolution and transmission.", "Text: Members of the Coronaviridae family are enveloped, non-segmented, positive-strand RNA viruses with genome sizes ranging from 26-32 kb [1] . These viruses are classified into two subfamilies: Letovirinae, which contains the only genus: Alphaletovirus; and Orthocoronavirinae (CoV), which consists of alpha, beta, gamma, and deltacoronaviruses (CoVs) [2, 3] . Alpha and betacoronaviruses mainly infect mammals and cause human and animal diseases. Gamma-and delta-CoVs mainly infect birds, but some can also infect mammals [4, 5] . Six human CoVs (HCoVs) are known to cause human diseases. HCoV-HKU1, HCoV-OC43, HCoV-229E, and HCoV-NL63 commonly cause mild respiratory illness or asymptomatic infection; however, severe acute respiratory syndrome coronavirus (SARS-CoV) and", "All sampling procedures were performed by veterinarians, with approval from Animal Ethics Committee of the Wuhan Institute of Virology (WIVH5210201). The study was conducted in accordance with the Guide for the Care and Use of Wild Mammals in Research of the People's Republic of China.\n\nBat fecal swab and pellet samples were collected from November 2004 to November 2014 in different seasons in Southern China, as described previously [16] .\n\nViral RNA was extracted from 200 µL of fecal swab or pellet samples using the High Pure Viral RNA Kit (Roche Diagnostics GmbH, Mannheim, Germany) as per the manufacturer's instructions. RNA was eluted in 50 µL of elution buffer, aliquoted, and stored at -80 • C. One-step hemi-nested reverse-transcription (RT-) PCR (Invitrogen, San Diego, CA, USA) was employed to detect coronavirus, as previously described [17, 18] .", "To confirm the bat species of an individual sample, we PCR amplified the cytochrome b (Cytob) and/or NADH dehydrogenase subunit 1 (ND1) gene using DNA extracted from the feces or swabs [19, 20] . The gene sequences were assembled excluding the primer sequences. BLASTN was used to identify host species based on the most closely related sequences with the highest query coverage and a minimum identity of 95%.", "Full genomic sequences were determined by one-step PCR (Invitrogen, San Diego, CA, USA) amplification with degenerate primers (Table S1 ) designed on the basis of multiple alignments of available alpha-CoV sequences deposited in GenBank or amplified with SuperScript IV Reverse Transcriptase (Invitrogen) and Expand Long Template PCR System (Roche Diagnostics GmbH, Mannheim, Germany) with specific primers (primer sequences are available upon request). Sequences of the 5' and 3' genomic ends were obtained by 5' and 3' rapid amplification of cDNA ends (SMARTer Viruses 2019, 11, 379 3 of 19 RACE 5'/3' Kit; Clontech, Mountain View, CA, USA), respectively. PCR products were gel-purified and subjected directly to sequencing. PCR products over 5kb were subjected to deep sequencing using Hiseq2500 system. For some fragments, the PCR products were cloned into the pGEM-T Easy Vector (Promega, Madison, WI, USA) for sequencing. At least five independent clones were sequenced to obtain a consensus", "sequence.", "The Next Generation Sequencing (NGS) data were filtered and mapped to the reference sequence of BatCoV HKU10 (GenBank accession number NC_018871) using Geneious 7.1.8 [21] . Genomes were preliminarily assembled using DNAStar lasergene V7 (DNAStar, Madison, WI, USA). Putative open reading frames (ORFs) were predicted using NCBI's ORF finder (https://www.ncbi.nlm.nih.gov/ orffinder/) with a minimal ORF length of 150 nt, followed by manual inspection. The sequences of the 5' untranslated region (5'-UTR) and 3'-UTR were defined, and the leader sequence, the leader and body transcriptional regulatory sequence (TRS) were identified as previously described [22] . The cleavage of the 16 nonstructural proteins coded by ORF1ab was determined by alignment of aa sequences of other CoVs and the recognition pattern of the 3C-like proteinase and papain-like proteinase. Phylogenetic trees based on nt or aa sequences were constructed using the maximum likelihood algorithm with bootstrap values", "determined by 1000 replicates in the MEGA 6 software package [23] . Full-length genome sequences obtained in this study were aligned with those of previously reported alpha-CoVs using MUSCLE [24] . The aligned sequences were scanned for recombination events by using Recombination Detection Program [25] . Potential recombination events as suggested by strong p-values (<10 -20 ) were confirmed using similarity plot and bootscan analyses implemented in Simplot 3.5.1 [26] . The number of synonymous substitutions per synonymous site, Ks, and the number of nonsynonymous substitutions per nonsynonymous site, Ka, for each coding region were calculated using the Ka/Ks calculation tool of the Norwegian Bioinformatics Platform (http://services.cbu.uib.no/tools/kaks) with default parameters [27] . The protein homology detection was analyzed using HHpred (https://toolkit.tuebingen.mpg.de/#/tools/hhpred) with default parameters [28] .", "A set of nested RT-PCRs was employed to determine the presence of viral subgenomic mRNAs in the CoV-positive samples [29] . Forward primers were designed targeting the leader sequence at the 5'-end of the complete genome, while reverse primers were designed within the ORFs. Specific and suspected amplicons of expected sizes were purified and then cloned into the pGEM-T Easy vector for sequencing.", "Bat primary or immortalized cells (Rhinolophus sinicus kidney immortalized cells, RsKT; Rhinolophus sinicus Lung primary cells, RsLu4323; Rhinolophus sinicus brain immortalized cells, RsBrT; Rhinolophus affinis kidney primary cells, RaK4324; Rousettus leschenaultii Kidney immortalized cells, RlKT; Hipposideros pratti lung immortalized cells, HpLuT) generated in our laboratory were all cultured in DMEM/F12 with 15% FBS. Pteropus alecto kidney cells (Paki) was maintained in DMEM/F12 supplemented with 10% FBS. Other cells were maintained according to the recommendations of American Type Culture Collection (ATCC, www.atcc.org).", "The putative accessory genes of the newly detected virus were generated by RT-PCR from viral RNA extracted from fecal samples, as described previously [30] . The influenza virus NS1 plasmid was generated in our lab [31] . The human bocavirus (HBoV) VP2 plasmid was kindly provided by prof. Hanzhong Wang of the Wuhan Institute of Virology, Chinese Academy of Sciences. SARS-CoV ORF7a was synthesized by Sangon Biotech. The transfections were performed with Lipofectamine 3000 Reagent (Life Technologies). Expression of these accessory genes were analyzed by Western blotting using an mAb (Roche Diagnostics GmbH, Mannheim, Germany) against the HA tag.", "The virus isolation was performed as previously described [12] . Briefly, fecal supernatant was acquired via gradient centrifugation and then added to Vero E6 cells, 1:10 diluted in DMEM. After incubation at 37°C for 1 h the inoculum was replaced by fresh DMEM containing 2% FBS and the antibiotic-antimycotic (Gibco, Grand Island, NY, USA). Three blind passages were carried out. Cells were checked daily for cytopathic effect. Both culture supernatant and cell pellet were examined for CoV by RT-PCR [17] .", "Apoptosis was analyzed as previously described [18] . Briefly, 293T cells in 12-well plates were transfected with 3 µg of expression plasmid or empty vector, and the cells were collected 24 h post transfection. Apoptosis was detected by flow cytometry using by the Annexin V-FITC/PI Apoptosis Detection Kit (YEASEN, Shanghai, China) following the manufacturer's instructions. Annexin-V-positive and PI-negative cells were considered to be in the early apoptotic phase and those stained for both Annexin V and PI were deemed to undergo late apoptosis or necrosis. All experiments were repeated three times. Student's t-test was used to evaluate the data, with p < 0.05 considered significant.", "HEK 293T cells were seeded in 24-well plates and then co-transfected with reporter plasmids (pRL-TK and pIFN-βIFN-or pNF-κB-Luc) [30] , as well as plasmids expressing accessory genes, empty vector plasmid pcAGGS, influenza virus NS1 [32] , SARS-CoV ORF7a [33] , or HBoV VP2 [34] . At 24 h post transfection, cells were treated with Sendai virus (SeV) (100 hemagglutinin units [HAU]/mL) or human tumor necrosis factor alpha (TNF-α; R&D system) for 6 h to activate IFNβ or NF-κB, respectively. Cell lysates were prepared, and luciferase activity was measured using the dual-luciferase assay kit (Promega, Madison, WI, USA) according to the manufacturer's instructions.", "Retroviruses pseudotyped with BtCoV/Rh/YN2012 RsYN1, RsYN3, RaGD, or MERS-CoV spike, or no spike (mock) were used to infect human, bat or other mammalian cells in 96-well plates. The pseudovirus particles were confirmed with Western blotting and negative-staining electromicroscopy. The production process, measurements of infection and luciferase activity were conducted, as described previously [35, 36] .\n\nThe complete genome nucleotide sequences of BtCoV/Rh/YN2012 strains RsYN1, RsYN2, RsYN3, and RaGD obtained in this study have been submitted to the GenBank under MG916901 to MG916904.", "The surveillance was performed between November 2004 to November 2014 in 19 provinces of China. In total, 2061 fecal samples were collected from at least 12 Rhinolophus bat species ( Figure 1A ). CoVs were detected in 209 of these samples ( Figure 1B and Table 1 ). Partial RdRp sequences suggested the presence of at least 8 different CoVs. Five of these viruses are related to known species: Mi-BatCoV 1 (>94% nt identity), Mi-BatCoV HKU8 [37] (>93% nt identity), BtRf-AlphaCoV/HuB2013 [11] (>99% nt identity), SARSr-CoV [38] (>89% nt identity), and HKU2-related CoV [39] (>85% nt identity). While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species. Virus isolation was performed as previously described [12] , but was not successful. identity). While the other three CoV sequences showed less than 83% nt identity to known CoV species. These three viruses should represent novel CoV species. Virus isolation", "was performed as previously described [12] , but was not successful.", "We next characterized a novel alpha-CoV, BtCoV/Rh/YN2012. It was detected in 3 R.affinis and 6 R.sinicus, respectively. Based on the sequences, we defined three genotypes, which represented by RsYN1, RsYN3, and RaGD, respectively. Strain RsYN2 was classified into the RsYN3 genotype. Four full-length genomes were obtained. Three of them were from R.sinicus (Strain RsYN1, RsYN2, and RsYN3), while the other one was from R.affinis (Strain RaGD). The sizes of these 4 genomes are between 28,715 to 29,102, with G+C contents between 39.0% to 41.3%. The genomes exhibit similar structures and transcription regulatory sequences (TRS) that are identical to those of other alpha-CoVs ( Figure 2 and Table 2 ). Exceptions including three additional ORFs (ORF3b, ORF4a and ORF4b) were observed. All the 4 strains have ORF4a & ORF4b, while only strain RsYN1 has ORF3b.", "The replicase gene, ORF1ab, occupies~20.4 kb of the genome. The replicase gene, ORF1ab, occupies~20.4 kb of the genome. It encodes polyproteins 1a and 1ab, which could be cleaved into 16 non-structural proteins (Nsp1-Nsp16). The 3'-end of the cleavage sites recognized by 3C-like proteinase (Nsp4-Nsp10, Nsp12-Nsp16) and papain-like proteinase (Nsp1-Nsp3) were confirmed. The proteins including Nsp3 (papain-like 2 proteas, PL2pro), Nsp5 (chymotrypsin-like protease, 3CLpro), Nsp12 (RdRp), Nsp13 (helicase), and other proteins of unknown function ( Table 3 ). The 7 concatenated domains of polyprotein 1 shared <90% aa sequence identity with those of other known alpha-CoVs ( Table 2 ), suggesting that these viruses represent a novel CoV species within the alpha-CoV. The closest assigned CoV species to BtCoV/Rh/YN2012 are BtCoV-HKU10 and BtRf-AlphaCoV/Hub2013. The three strains from Yunnan Province were clustered into two genotypes (83% genome identity) correlated to their sampling location.", "The third genotype represented by strain RaGD was isolated to strains found in Yunnan (<75.4% genome identity). We then examined the individual genes ( Table 2) . All of the genes showed low aa sequence identity to known CoVs. The four strains of BtCoV/Rh/YN2012 showed genetic diversity among all different genes except ORF1ab (>83.7% aa identity). Notably, the spike proteins are highly divergent among these strains. Other structure proteins (E, M, and N) are more conserved than the spike and other accessory proteins. Comparing the accessory genes among these four strains revealed that the strains of the same genotype shared a 100% identical ORF3a. However, the proteins encoded by ORF3as were highly divergent among different genotypes (<65% aa identity). The putative accessory genes were also BLASTed against GenBank records. Most accessory genes have no homologues in GenBank-database, except for ORF3a (52.0-55.5% aa identity with BatCoV HKU10 ORF3) and ORF9 (28.1-32.0% aa identity with", "SARSr-CoV ORF7a). We analyzed the protein homology with HHpred software. The results showed that ORF9s and SARS-CoV OR7a are homologues (possibility: 100%, E value <10 −48 ). We further screened the genomes for potential recombination evidence. No significant recombination breakpoint was detected by bootscan analysis.", "To confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing ( Figure 3 and Table 2 ). The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b. \n\nTo confirm the presence of subgenomic RNA, we designed a set of primers targeting all the predicted ORFs as described. The amplicons were firstly confirmed via agarose-gel electrophoresis and then sequencing ( Figure 3 and Table 2 ). The sequences showed that all the ORFs, except ORF4b, had preceding TRS. Hence, the ORF4b may be translated from bicistronic mRNAs. In RsYN1, an additional subgenomic RNA starting inside the ORF3a was found through sequencing, which led to a unique ORF3b.", "Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs (Figure 4) . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history.", "Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs (Figure 4) . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history.", "Phylogenetic trees were constructed using the aa sequences of RdRp and S of BtCoV/Rh/YN2012 and other representative CoVs (Figure 4) . In both trees, all BtCoV/Rh/YN2012 were clustered together and formed a distinct lineage to other known coronavirus species. Two distinct sublineages were observed within BtCoV/Rh/YN2012. One was from Ra sampled in Guangdong, while the other was from Rs sampled in Yunnan Among the strains from Yunnan, RsYN2 and RsYN3 were clustered together, while RsYN1 was isolated. The topology of these four strains was correlated to the sampling location. The relatively long branches reflect a high diversity among these strains, indicating a long independent evolution history.", "The Ka/Ks ratios (Ks is the number of synonymous substitutions per synonymous sites and Ka is the number of nonsynonymous substitutions per nonsynonymous site) were calculated for all genes. The Ka/Ks ratios for most of the genes were generally low, which indicates these genes were under purified selection. However, the Ka/Ks ratios of ORF4a, ORF4b, and ORF9 (0.727, 0.623, and 0.843, respectively) were significantly higher than those of other ORFs (Table 4 ). For further selection pressure evaluation of the ORF4a and ORF4b gene, we sequenced another four ORF4a and ORF4b genes (strain Rs4223, Rs4236, Rs4240, and Ra13576 was shown in Figure 1B", "As SARS-CoV ORF7a was reported to induce apoptosis, we conducted apoptosis analysis on BtCoV/Rh/YN2012 ORF9, a~30% aa identity homologue of SARSr-CoV ORF7a. We transiently transfected ORF9 of BtCoV/Rh/YN2012 into HEK293T cells to examine whether this ORF9 triggers apoptosis. Western blot was performed to confirm the expression of ORF9s and SARS-CoV ORF7a ( Figure S1 ). ORF9 couldn't induce apoptosis as the ORF7a of SARS-CoV Tor2 ( Figure S2 ). The results indicated that BtCoV/Rh/YN2012 ORF9 was not involved in apoptosis induction.", "To determine whether these accessory proteins modulate IFN induction, we transfected reporter plasmids (pIFNβ-Luc and pRL-TK) and expression plasmids to 293T cells. All the cells over-expressing the accessory genes, as well as influenza virus NS1 (strain PR8), HBoV VP2, or empty vector were tested for luciferase activity after SeV infection. Luciferase activity stimulated by SeV was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those controls, the ORF4a proteins exhibit an active effect as HBoV VP2 ( Figure 5A ). Other accessory proteins showed no effect on IFN production ( Figure S3 ). Expression of these accessory genes were confirmed by Western blot ( Figure S1 ). was remarkably higher than that without SeV treatment as expected. Influenza virus NS1 inhibits the expression from IFN promoter, while HBoV VP2 activate the expression. Compared to those", "controls, the ORF4a proteins exhibit an active effect as HBoV VP2 ( Figure 5A ). Other accessory proteins showed no effect on IFN production ( Figure S3 ). Expression of these accessory genes were confirmed by Western blot (Figure S1 ). Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. (B) ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector (500 ng), an NS1-expressing plasmid (500 ng), a SARS-CoV ORF7a-expressing plasmid (500 ng), or ORF3a-expressing plasmids (500 ng). After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative of at least three independent experiments, with each", "determination performed in triplicate (mean ± SD of fold change). Asterisks indicate significant differences between groups (compared with Empty vector-NC, p < 0.05, as determined by student t test).", "NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter Samples were collected at 6 h postinfection, followed by dual-luciferase assay. The results were expressed as the firefly luciferase value normalized to that of Renilla luciferase. (B) ORF3a protein activate NF-κB. 293T cells were transfected with 100 ng pNF-κB-Luc, 10 ng pRL-TK, empty vector (500 ng), an NS1-expressing plasmid (500 ng), a SARS-CoV ORF7a-expressing plasmid (500 ng), or ORF3a-expressing plasmids (500 ng). After 24 h, the cells were treated with TNF-α. Dual-luciferase activity was determined after 6 h. The results were expressed as the firefly luciferase activity normalized to that of Renilla luciferase. The experiments were performed three times independently. Data are representative", "of at least three independent experiments, with each determination performed in triplicate (mean ± SD of fold change). Asterisks indicate significant differences between groups (compared with Empty vector-NC, p < 0.05, as determined by student t test).", "NF-κB plays an important role in regulating the immune response to viral infection and is also a key factor frequently targeted by viruses for taking over the host cell. In this study, we tested if these accessory proteins could modulate NF-κB. 293T cells were co-transfected with reporter plasmids (pNF-κB-Luc and pRL-TK), as well as accessory protein-expressing plasmids, or controls (empty vector, NS1, SARS-CoV Tor2-ORF7a). The cells were mock treated or treated with TNF-α for 6 h at 24 h post-transfection. The luciferase activity was determined. RsYN1-ORF3a and RaGD-ORF3a activated NF-κB as SARS-CoV ORF7a, whereas RsYN2-ORF3a inhibited NF-κB as NS1 ( Figure 5B ). Expressions of ORF3as were confirmed with Western blot ( Figure S1 ). Other accessory proteins did not modulate NF-κB production ( Figure S4 ).", "To understand the infectivity of these newly detected BtCoV/Rh/YN2012, we selected the RsYN1, RsYN3 and RaGD spike proteins for spike-mediated pseudovirus entry studies. Both Western blot analysis and negative-staining electron microscopy observation confirmed the preparation of BtCoV/Rh/YN2012 successfully ( Figure S5 ). A total of 11 human cell lines, 8 bat cells, and 9 other mammal cell lines were tested, and no strong positive was found (Table S2) .", "In this study, a novel alpha-CoV species, BtCoV/Rh/YN2012, was identified in two Rhinolophus species. The 4 strains with full-length genome were sequences. The 7 conserved replicase domains of these viruses possessed <90% aa sequence identity to those of other known alpha-CoVs, which defines a new species in accordance with the ICTV taxonomy standard [42] . These novel alpha-CoVs showed high genetic diversity in their structural and non-structural genes. Strain RaGD from R. affinis, collected in Guangdong province, formed a divergent independent branch from the other 3 strains from R. sinicus, sampled in Yunnan Province, indicating an independent evolution process associated with geographic isolation and host restrain. Though collected from same province, these three virus strains formed two genotypes correlated to sampling locations. These two genotypes had low genome sequence identity, especially in the S gene and accessory genes. Considering the remote geographic location of the", "host bat habitat, the host tropism, and the virus diversity, we suppose BtCoV/Rh/YN2012 may have spread in these two provinces with a long history of circulation in their natural reservoir, Rhinolophus bats. With the sequence evidence, we suppose that these viruses are still rapidly evolving.", "Our study revealed that BtCoV/Rh/YN2012 has a unique genome structure compared to other alpha-CoVs. First, novel accessory genes, which had no homologues, were identified in the genomes. Second, multiple TRSs were found between S and E genes while other alphacoronavirus only had one TRS there. These TRSs precede ORF3a, ORF3b (only in RsYN1), and ORF4a/b respectively. Third, accessory gene ORF9 showed homology with those of other known CoV species in another coronavirus genus, especially with accessory genes from SARSr-CoV.", "Accessory genes are usually involved in virus-host interactions during CoV infection [43] . In most CoVs, accessory genes are dispensable for virus replication. However, an intact 3c gene of feline CoV was required for viral replication in the gut [44] [45] [46] . Deletion of the genus-specific genes in mouse hepatitis virus led to a reduction in virulence [47] . SARS-CoV ORF7a, which was identified to be involved in the suppression of RNA silencing [48] , inhibition of cellular protein synthesis [49] , cell-cycle blockage [50] , and apoptosis induction [51, 52] . In this study, we found that BtCoV/Rh/YN2012 ORF9 shares~30% aa sequence identity with SARS-CoV ORF7a. Interestingly, BtCoV/Rh/YN2012 and SARSr-CoV were both detected in R. sinicus from the same cave. We suppose that SARS-CoV and BtCoV/Rh/YN2012 may have acquired ORF7a or ORF9 from a common ancestor through genome recombination or horizontal gene transfer. Whereas, ORF9 of BtCoV/Rh/YN2012 failed to induce apoptosis or", "activate NF-κB production, these differences may be induced by the divergent evolution of these proteins in different pressure.", "Though different BtCoV/Rh/YN2012 ORF4a share <64.4% amino acid identity, all of them could activate IFN-β. ORF3a from RsYN1 and RaGD upregulated NF-κB, but the homologue from RsYN2 downregulated NF-κB expression. These differences may be caused by amino acid sequence variations and may contribute to a viruses' pathogenicity with a different pathway.", "Though lacking of intestinal cell lines from the natural host of BtCoV/Rh/YN2012, we screened the cell tropism of their spike protein through pseudotyped retrovirus entry with human, bat and other mammalian cell lines. Most of cell lines screened were unsusceptible to BtCoV/Rh/YN2012, indicating a low risk of interspecies transmission to human and other animals. Multiple reasons may lead to failed infection of coronavirus spike-pseudotyped retrovirus system, including receptor absence in target cells, failed recognition to the receptor homologue from non-host species, maladaptation in non-host cells during the spike maturation or virus entry, or the limitation of retrovirus system in stimulating coronavirus entry. The weak infectivity of RsYN1 pseudotyped retrovirus in Huh-7 cells could be explained by the binding of spike protein to polysaccharide secreted to the surface. The assumption needs to be further confirmed by experiments.", "Our long-term surveillances suggest that Rhinolophus bats seem to harbor a wide diversity of CoVs. Coincidently, the two highly pathogenic agents, SARS-CoV and Rh-BatCoV HKU2 both originated from Rhinolophus bats. Considering the diversity of CoVs carried by this bat genus and their wide geographical distribution, there may be a low risk of spillover of these viruses to other animals and humans. Long-term surveillances and pathogenesis studies will help to prevent future human and animal diseases caused by these bat CoVs.", "Supplementary Materials: The following are available online at http://www.mdpi.com/1999-4915/11/4/379/s1, Figure S1 : western blot analysis of the expression of accessory proteins. Figure S2 : Apoptosis analysis of ORF9 proteins of BtCoV/Rh/YN2012. Figure S3 : Functional analysis of ORF3a, ORF3b, ORF4b, ORF8 and ORF9 proteins on the production of Type I interferon. Figure S4 : Functional analysis of ORF3b, ORF4a, ORF4b, ORF8 and ORF9 proteins on the production of NF-κB. Figure S5 : Characteristic of BtCoV/Rh/YN2012 spike mediated pseudovirus. Table S1 : General primers for AlphaCoVs genome sequencing. Table S2 : Primers for the detection of viral sugbenomic mRNAs. Table S3" ]

[ 43 ]

[ "FIP virus (FIPV)" ]

[ "In Vitro Antiviral Activity of Circular Triple Helix Forming Oligonucleotide RNA towards Feline Infectious Peritonitis Virus Replication\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3950953/\n\nSHA: f5ad2323eb387f6e271e2842bb2cc4a33504fde3\n\nAuthors: Choong, Oi Kuan; Mehrbod, Parvaneh; Tejo, Bimo Ario; Omar, Abdul Rahman\nDate: 2014-02-20\nDOI: 10.1155/2014/654712\nLicense: cc-by", "Abstract: Feline Infectious Peritonitis (FIP) is a severe fatal immune-augmented disease in cat population. It is caused by FIP virus (FIPV), a virulent mutant strain of Feline Enteric Coronavirus (FECV). Current treatments and prophylactics are not effective. The in vitro antiviral properties of five circular Triple-Helix Forming Oligonucleotide (TFO) RNAs (TFO1 to TFO5), which target the different regions of virulent feline coronavirus (FCoV) strain FIPV WSU 79-1146 genome, were tested in FIPV-infected Crandell-Rees Feline Kidney (CRFK) cells. RT-qPCR results showed that the circular TFO RNAs, except TFO2, inhibit FIPV replication, where the viral genome copy numbers decreased significantly by 5-fold log(10) from 10(14) in the virus-inoculated cells to 10(9) in the circular TFO RNAs-transfected cells. Furthermore, the binding of the circular TFO RNA with the targeted viral genome segment was also confirmed using electrophoretic mobility shift assay. The strength of binding kinetics", "between the TFO RNAs and their target regions was demonstrated by NanoITC assay. In conclusion, the circular TFOs have the potential to be further developed as antiviral agents against FIPV infection.", "Text: Feline Infectious Peritonitis Virus (FIPV) is an enveloped virus with a nonsegmented, positive sense, single-stranded RNA genome. FIPV is grouped as feline coronavirus (FCoV), under the family Coronaviridae. FCoV is divided into two biotypes, namely, Feline Enteric Coronavirus (FECV), a ubiquitous enteric biotype of FCoV, and FIPV, a virulent biotype of FCoV [1] . The relationship between these two biotypes still remains unclear. Two hypotheses have been proposed, (i) internal mutation theory and (ii) circulating high virulent-low virulent theory. Internal mutation theory stated that the development of FIP is due to the exposure of cat to variants of FCoV which have been mutated by gaining the ability to replicate within the macrophages [2] , while the circulating high virulent-low virulent theory explains the existence of both distinctive pathogenic and benign lineages of viruses within the cat population [3] .", "Study has shown that about 40-80% of cats are detected with FECV shedding in their faeces [4] . About 12% of these FECV-positive cats have developed immune-mediated fatal FIP disease [4] . The prevalence of FIP among felines is due to continual cycles of infection and reinfection of FECV and indiscernible clinical symptoms of infected cats with FECV at an early stage before the progressive development of FIPV.", "Vaccination against FIPV with an attenuated, temperature-sensitive strain of type II FIPV induces low antibody titre in kittens that have not been exposed to FCoV. However, there is considerable controversy on the safety and efficacy of this vaccine, since the vaccine contains type 2 strain, whereas type 1 viruses are more prevalent in the field [4] . In addition, antibodies against FIPV do not protect infected cats but enhance the infection of monocytes and macrophages via a mechanism known as Antibody-Dependent Enhancement [1] . Besides vaccines, several antiviral drugs such as ribavirin, 2 BioMed Research International interferons, and immunosuppressive drugs have been used as treatments for FIPV-infected cats, mainly to suppress the inflammatory and detrimental immune response [5] [6] [7] [8] . However, those treatments were ineffective. Hence, there is still significant unmet medical need to develop effective treatments and prophylactics for FIPV infection.", "Triple Helix Forming Oligonucleotide (TFO) is defined as homopyrimidine oligonucleotides, which can form a sequence-specific triple helix by Hoogsteen bonds to the major groove of a complementary homopyrimidinehomopurine stretch in duplex DNA [9] . Furthermore, double helical RNA or DNA-RNA hybrids can be targeted as a template for triple helix formation, once the strand composition on the stabilities of triple helical complexes is determined [10] . Hence, TFO has been used to impede gene expressions by transcription inhibition of viral genes or oncogenes [11] [12] [13] [14] [15] [16] . The main purpose of this study is to develop and evaluate the in vitro antiviral properties of circular TFO RNAs against FIPV replication.", "serotype II strain WSU 79-1146 (ATCC no. VR-1777) was grown in CRFK cells. A serial 10-fold dilution of FIPV was prepared from the working stock. Confluent 96-well plate was inoculated with 100 L of each virus dilution/well. The plate was incubated in a humidified incubator at 37 ∘ C, 5% CO 2 . Cytopathic effects (CPE) development was observed. The results were recorded after 72 hours and the virus tissue culture infective dose 50 (TCID 50 ) was calculated using Reed and Muench's method [17] .", "Oligonucleotide RNA. The Triple Helix Forming Oligonucleotides (TFOs) were designed based on the genome sequence of FIPV serotype II strain WSU 79-1146 (Accession no: AY994055) [18] . TFOs, which specifically target the different regions of the FIPV genome, and one unrelated TFO were constructed ( Table 1 ). The specificity of the TFOs was identified using BLAST search in the NCBI database. The designed linear TFOs were synthesized by Dharmacon Research (USA), whereby the 5 and 3 ends of the linear TFOs were modified with phosphate (PO 4 ) group and hydroxide (OH) group, respectively. These modifications were necessary for the circularization of linear TFO. The process of circularization, using the T4 RNA ligase 1 (ssRNA ligase) (New England Biolabs Inc., England), was carried out according to the manufacturer's protocol. After ligation, the circular TFO RNAs were recovered by ethanol precipitation and the purity of the circular TFO RNAs was measured using spectrophotometer.", "Denaturing of urea polyacrylamide gel electrophoresis was performed as described before [19] with modification. Briefly, 20% of denatured urea polyacrylamide gel was prepared and polymerized for 30 minutes. Then, the gel was prerun at 20 to 40 V for 45 minutes. Five L of TFO RNA mixed with 5 L of urea loading buffer was heated at 92 ∘ C for 2 minutes and immediately chilled on ice. It was run on the gel at 200 V for 45 minutes. Finally, the gel was stained with ethidium bromide (Sigma, USA) and viewed with a Bio-Rad Gel Doc XR system (CA, USA). (EMSA) . The target regions of the FIPV genome were synthesized by Dharmacon Research (USA) ( Table 1) . Each TFO RNA was mixed with the target region in 1X binding buffer containing 25 mM Tris-HCl, 6 mM MgCl 2 , and 10 mMNaCl in a final volume of 10 L and subsequently incubated at 37 ∘ C for 2 hours. The sample was run on 15% native polyacrylamide gel at 80 V, in cool condition. The stained gel was viewed by a Bio-Rad Gel Doc XR system.", "Regions. The binding strength was measured using a nano Isothermal Titration Calorimeter (ITC) (TA instruments, Newcastle, UK). The RNA sample mixtures, consisting of circular TFOs (0.0002 mM), were incubated with their respective synthetic target regions (0.015 mM) using 1X binding buffer as the diluent. The experiment was run at 37 ∘ C with 2 L/injection, for a total of 25 injections. Data was collected every 250 seconds and analyzed using the NanoAnalyze software v2.3.6 provided by the manufacturer.", "This experiment was conducted in CRFK cells, where 3 × 10 4 cell/well was seeded in 96-well plate to reach 80% confluency 24 hours prior to transfection. One hundred nM of TFO RNAs was separately transfected into the CRFK cells using a HiPerFect Transfection Reagent (Qiagen, Germany), as per the manufacturer's protocol. The plate was incubated at 37 ∘ C with 5% CO 2 for 6 hours. Then, the cultures were infected with 100TCID 50 of FIPV serotype II strain WSU 79-1146 for 1 hour at 37 ∘ C (100 L/well). Finally, the viral inoculum was replaced by fresh maintenance media (MEM containing 1% FBS and 1% pen/strep). Virus-infected and uninfected cells were maintained as positive and negative controls, respectively. The morphology of the cultures was recorded 72 hours after infection and samples were harvested at this time point and stored at −80 ∘ C prior to RNA extraction.", "Inhibition. Different concentrations of circular TFO1 RNA (25 nM, 50 nM, 100 nM, and 500 nM) were transfected into CRFK cells. The plate was incubated for 6 hours followed by virus inoculation for 1 hour at 37 ∘ C with 5% CO2. The cells were processed as described above.\n\nMadin-Darby Canine Kidney (MDCK) cell (ATCC no. CCL-34), at a concentration of 4 × 10 4 cell/well, was seeded in 96-well plate to reach 80% confluency 24 hours prior to transfection. Transfection was performed the same as before. One hundred nM of circular TFO RNA was transfected into MDCK cells. Following 6 hours \n\nORF1a/1b and 530-541\n\nORF1a/1b and 7399-7411\n\nORF1a/1b and 14048-14061", "- * Highlighted in bold indicated the binding region. * * Unrelated circular TFO. [20, 21] , respectively. The reverse transcriptase quantitative real-time PCR (RT-qPCR) was performed using a Bio-Rad CFX96 real-time system (BioRad, USA). The reaction was amplified in a final volume of 25 L using a SensiMix SYBR No-ROX One-Step Kit (Bioline, UK), which consisted of 12.5 L 2X SensiMix SYBR No-Rox One-\n\nStep reaction buffer, 10 M forward and reverse primers, 10 units RiboSafe RNase inhibitor, and 5 L template RNA. Absolute quantification approach was used to quantify qPCR results where a standard curve of a serial dilution of virus was plotted before the quantification. Amount of the virus in the samples was quantified based on this standard curve.", "Analysis. Data statistical analysis was performed using SPSS 18.0. Data were represented as mean ± SE of three independent tests. One-way ANOVA, Tukey post hoc test was used to analyze the significant level among the data. ≤ 0.05 was considered significant. genome, which play important roles in viral replication, were selected as the target binding sites for the triplex formation. The target regions were 5 untranslated region (5 UTR), Open Reading Frames (ORFs) 1a and 1b, and 3 untranslated region (3 UTR) ( Table 1 ). The TFOs were designed in duplex, as they can bind with the single stranded target region and reshape into triplex. Both ends of the duplex TFOs were ligated with a linker sequence or clamps (C-C) to construct circular TFO RNA.\n\nDenaturing PAGE assay was carried out after the ligation process to determine the formation of the circular TFO. As shown in Figure 1 , the circular TFO RNAs migrated faster than the linear TFO RNAs, when subjected to 20% denaturing PAGE.", "Target Region. The binding ability was determined using Electrophoretic Mobility Shift Assay (EMSA) [23] . The appearance of the slow mobility band indicates the successful hybridization of circular TFO RNA with its target region. The binding ability of different TFO RNAs (TFO1 to TFO5) against their target regions was determined by EMSA (Figure 2) . TFO1, TFO3, TFO4, and TFO5 showed slow mobility band, while TFO2 showed the lack of an upward shifted band. This indicates the possession of triplex binding ability for all circular TFO RNAs, except TFO2.\n\nTFO RNA. Study on the interaction and hybridization of TFO towards its target region is crucial, since the stronger the binding is, the more stable the triplex structure forms. As shown in supplementary Figure 1 (Table 3) .", "The antiviral effect of circular TFO RNAs was investigated by RT-qPCR assay at 72 hours after transfection. The results showed viral RNA genome copy numbers of 3.65 × 10 9 , 3.22 × 10 14 , 5.04 × 10 9 , 5.01 × 10 9 , 4.41 × 10 9 , and 3.96 × 10 14 in cells treated with TFO1, TFO2, TFO3, TFO4, TFO5, and TFO7, respectively. The data analyzed by one-way ANOVA, Tukey post hoc test showed significant high viral RNA genome copy number of 4.03 × 10 14 for virus inoculated cells as compared to circular TFO1, TFO3, TFO4, and TFO5 treatments ( ≤ 0.05). The viral RNA copies of circular TFO2, linear TFO3 and TFO4, and unrelated circular TFO7 RNAs transfected cells also showed high viral RNA copy numbers which did not show significant differences to the infected cells ( ≥ 0.05) ( Figure 3 ). The morphological changes of the cells were also captured 72 hours after transfection. The cells transfected with circular TFO1, TFO3, TFO4, and TFO5 appeared to be in good condition following virus", "inoculation, while the cells transfected with circular TFO2 and linear TFO3 and TFO4 showed visible cytopathic effect (CPE), the same as virus inoculated cells (supplementary Figure 2) . Furthermore, cells transfected with TFO only remain viable indicating that TFO treatment is generally not toxic to the cells. Hence, these results illustrated the capacity of circular TFO RNAs (except TFO2) to inhibit FIPV replication.", "Concentrations on FIPV Replication. Circular TFO1 was used to examine the dose-response relationship as a representative to other TFOs. The experimental conditions were identical to that of the previous experiment, except for TFO1 concentrations of 25 nM, 50 nM, 100 nM, and 500 nM. There was no significant reduction in viral RNA genome copies using the concentration of 25 nM TFO1. The other concentrations caused significant reductions in copy numbers as compared to the virus-infected cells. However, no significant difference was detected in copy numbers from all of these concentrations ( Figure 4 ).", "The specificity of the TFO towards FIPV was tested, using TFO1 and TFO5, as the proper representatives of TFOs, on influenza A virus H1N1 New Jersey 8/76. The analyzed data using one-way ANOVA, Tukey post hoc test did not show significant reductions in the copies of viral RNA for both TFOs compared to the influenza virus inoculated cells ( ≥ 0.05) (supplementary Figure 3 ). Complex structure G4/Cir4 Figure 2 : EMSA analysis. EMSA analysis illustrated the binding of circular TFO 1, 3, 4, and 5 to the target regions as evidenced by upward band shift. Binding of each circular TFO except circular TFO2 to its respective target forms a complex that migrates slower than unbound TFO. G1 to G5 represent the target region for circular TFO1 to TFO5 and Cir1 to Cir5 represent the circular TFO1 to TFO5, respectively. in the replication process [24] . Meanwhile, the ORF1a/1b of FIPV are translated into polyproteins that are cleaved into nonstructural proteins which assemble into", "replicationtranscription complexes together with other viral proteins [24] . Hence, the development of molecular therapy targeting these critical regions may provide the possibility to inhibit FIPV replication.", "Development of antiviral therapies against FIPV using siRNA [25] and viral protease inhibitors [26] Figure 4 : TFO1 dose-response study for inhibiting FIPV replication. The concentrations of 50 nM and higher showed significant antiviral effects. 50 nM of circular TFO1 RNA was able to reduce viral copy number by 5-fold log 10 from 10 14 to 10 9 , while 100 and 500 nM showed 4-fold reduction. Data are averages of 3 independent tests (mean ± SE). * Significantly different from FIPV-infected group.", "as potential new treatments against FIPV infection. In this study, circular Triple Helix Forming Oligonucleotide (TFO) RNAs, specifically targeting the short regions of viral genome for triplex formation, were designed and evaluated. TFO1 and TFO2 targeted the 5 and 3 UTRs of the viral genome, respectively. TFO3 to TFO5 targeted different regions of the ORF1a/1b on FIPV genome. Prior to in vitro antiviral study, the ligated circular TFOs were evaluated using PAGE analysis. All of the circularised TFO showed faster migration pattern compared to the linear TFO; however, only slight variation was detected for some of the TFO (Figure 1 ). The reason for this is not clear but probably due to the differences in length and the tertiary structures of the TFOs leading to differences in the migration rate. EMSA was used to show the binding capability of each circular TFO towards the target region in the FIPV genome except for TFO2 which showed lack of formation of complex structure upon", "hybridization ( Figure 2) . The EMSA result also concurred with the antiviral study, where all circular TFOs (except TFO2) were able to demonstrate a significant reduction in the viral RNA genome copy numbers by 5-fold log 10 from 10 14 in virus inoculated cells to 10 9 in TFO-transfected cells (Figure 3 ). However, no antiviral properties were detected from the linear TFOs and unrelated circular TFO7 RNA, confirming that the antiviral activity is associated with specific binding of circular TFOs towards targeted regions.", "Furthermore, the binding of the circular TFO to the target region was confirmed by nanoITC analysis; where the low value and high stability allowed TFOs to compete effectively with the target regions for inhibiting transcription in cell-free systems. Since, TFO1 shows the lowest value (Table 3) , the antiviral properties of this TFO were evaluated in doseresponse study. As shown in Figure 4 , 50 and 100 nM of TFO1 showed similar antiviral effects indicating the potential therapeutic application of TFO1 on FIPV replication. However, increasing the concentration of TFO1 to 500 nm failed to reduce the viral load further probably due to inefficiency of the transfection reagent to transfect the TFO into the cells. In addition, the virus has fast replication rate upon in vitro infection, where previous study on the growth of FIPV in CRFK cells showed that by 2 hours approximately 67% of FIPV 79-1146 were internalized by CRFK cells by endocytosis increasing to more than 70% at 3 hours [27,", "28] . The above finding probably also explained the reason why no antiviral effect was detected when the transfection of the TFO was performed on virus-infected cells (data not shown).", "The antiviral properties, as demonstrated by the circular TFOs, were probably associated with the binding of the TFO to the target region, based on both the Watson-Crick and Hoogsteen hydrogen bonds, which enhance the stability in terms of enthalpy, which is brought about by joining together two out of three strands of the triple helix in the proper orientation [29] . Therefore, the triplex formation is tightly bonded and not easy to detach. Furthermore, the circular TFOs were designed in such way that the presence of hydrogen bonding donors and acceptors in the purines is able to form two hydrogen bonds, while the pyrimidine bases can only form one additional hydrogen bond with incoming third bases [30] . However, there are various factors that may limit the activity of TFOs in cells like intracellular degradation of the TFO and limited accessibility of the TFO to the target sites which can prevent triplex formation [31] . These findings may also explain the inability of the designed", "TFO1 to inhibit further virus replication in dose-response study (Figure 4) .", "Various molecular-based therapies against infectious diseases and cancer have been developed and tested. However, only the siRNA-based therapy has been studied extensively as a novel antiviral and anticancer therapy [32, 33] . Recently, McDonagh et al. [25] developed siRNA with antiviral activity against the FIPV 79-1146, where the designed siRNA was able to reduce the copy number of viral genome compared with virus-infected cells. The potential therapeutic application of TFOs, such as linear TFO conjugated with psoralen to inhibit the transcription of human immunodeficiency provirus [13] and TFO to inhibit the transcription of 1(I) collagen in rat fibroblasts [14] , has also been reported. In addition, short TFO conjugated with daunomycin targeting the promoter region of oncogene has been designed and evaluated on human cancer cells [31] . These studies indicated the flexibility of using TFO-based oligonucleotides as a potential molecular-based therapy. In this study, we demonstrated", "short circular TFO RNAs between 28 and 34 mers (Table 1) , which are able to inhibit FIPV replication by binding to specific target regions of the FIPV genome. All designed circular TFOs (except TFO2) showed significant inhibitory effects against FIPV replication. The TFOs that formed triplex structures showed antiviral effects towards FIPV replication. The reason why TFO2 failed to show any interaction with the target region or antiviral activity is probably due to the length of TFO2 (i.e., 24 mers), which might be insufficient to a triplex formation upon hybridization (Figure 2 ), be effective enough to suppress viral RNA transcription, and eventually inhibit virus replication. Nevertheless, the inability of TFO2 to show antiviral effect due to failure in the formation of functional tertiary structure of the triplex formation cannot be ruled out. In vitro antiviral study which showed no antiviral property for unrelated TFO (TFO7) and also inability of circular TFO1 and TFO5 to", "inhibit influenza A virus H1N1 infected cells confirms the specificity of the TFOs' activity.", "In conclusion, the circular TFO RNA has the potential to be developed as a therapy against FIPV in cats. However, further studies on TFO specificity, actual mechanism of circular TFO RNA in the transcription alteration consequence of inhibiting the viral transcription process, and in vivo animal studies are important for this approach to work as a therapy in the future." ]

[ 1 ]

[ "enveloped virus with a nonsegmented, positive sense, single-stranded RNA genome" ]

[ "In Vitro Antiviral Activity of Circular Triple Helix Forming Oligonucleotide RNA towards Feline Infectious Peritonitis Virus Replication\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3950953/\n\nSHA: f5ad2323eb387f6e271e2842bb2cc4a33504fde3\n\nAuthors: Choong, Oi Kuan; Mehrbod, Parvaneh; Tejo, Bimo Ario; Omar, Abdul Rahman\nDate: 2014-02-20\nDOI: 10.1155/2014/654712\nLicense: cc-by", "Abstract: Feline Infectious Peritonitis (FIP) is a severe fatal immune-augmented disease in cat population. It is caused by FIP virus (FIPV), a virulent mutant strain of Feline Enteric Coronavirus (FECV). Current treatments and prophylactics are not effective. The in vitro antiviral properties of five circular Triple-Helix Forming Oligonucleotide (TFO) RNAs (TFO1 to TFO5), which target the different regions of virulent feline coronavirus (FCoV) strain FIPV WSU 79-1146 genome, were tested in FIPV-infected Crandell-Rees Feline Kidney (CRFK) cells. RT-qPCR results showed that the circular TFO RNAs, except TFO2, inhibit FIPV replication, where the viral genome copy numbers decreased significantly by 5-fold log(10) from 10(14) in the virus-inoculated cells to 10(9) in the circular TFO RNAs-transfected cells. Furthermore, the binding of the circular TFO RNA with the targeted viral genome segment was also confirmed using electrophoretic mobility shift assay. The strength of binding kinetics", "between the TFO RNAs and their target regions was demonstrated by NanoITC assay. In conclusion, the circular TFOs have the potential to be further developed as antiviral agents against FIPV infection.", "Text: Feline Infectious Peritonitis Virus (FIPV) is an enveloped virus with a nonsegmented, positive sense, single-stranded RNA genome. FIPV is grouped as feline coronavirus (FCoV), under the family Coronaviridae. FCoV is divided into two biotypes, namely, Feline Enteric Coronavirus (FECV), a ubiquitous enteric biotype of FCoV, and FIPV, a virulent biotype of FCoV [1] . The relationship between these two biotypes still remains unclear. Two hypotheses have been proposed, (i) internal mutation theory and (ii) circulating high virulent-low virulent theory. Internal mutation theory stated that the development of FIP is due to the exposure of cat to variants of FCoV which have been mutated by gaining the ability to replicate within the macrophages [2] , while the circulating high virulent-low virulent theory explains the existence of both distinctive pathogenic and benign lineages of viruses within the cat population [3] .", "Study has shown that about 40-80% of cats are detected with FECV shedding in their faeces [4] . About 12% of these FECV-positive cats have developed immune-mediated fatal FIP disease [4] . The prevalence of FIP among felines is due to continual cycles of infection and reinfection of FECV and indiscernible clinical symptoms of infected cats with FECV at an early stage before the progressive development of FIPV.", "Vaccination against FIPV with an attenuated, temperature-sensitive strain of type II FIPV induces low antibody titre in kittens that have not been exposed to FCoV. However, there is considerable controversy on the safety and efficacy of this vaccine, since the vaccine contains type 2 strain, whereas type 1 viruses are more prevalent in the field [4] . In addition, antibodies against FIPV do not protect infected cats but enhance the infection of monocytes and macrophages via a mechanism known as Antibody-Dependent Enhancement [1] . Besides vaccines, several antiviral drugs such as ribavirin, 2 BioMed Research International interferons, and immunosuppressive drugs have been used as treatments for FIPV-infected cats, mainly to suppress the inflammatory and detrimental immune response [5] [6] [7] [8] . However, those treatments were ineffective. Hence, there is still significant unmet medical need to develop effective treatments and prophylactics for FIPV infection.", "Triple Helix Forming Oligonucleotide (TFO) is defined as homopyrimidine oligonucleotides, which can form a sequence-specific triple helix by Hoogsteen bonds to the major groove of a complementary homopyrimidinehomopurine stretch in duplex DNA [9] . Furthermore, double helical RNA or DNA-RNA hybrids can be targeted as a template for triple helix formation, once the strand composition on the stabilities of triple helical complexes is determined [10] . Hence, TFO has been used to impede gene expressions by transcription inhibition of viral genes or oncogenes [11] [12] [13] [14] [15] [16] . The main purpose of this study is to develop and evaluate the in vitro antiviral properties of circular TFO RNAs against FIPV replication.", "serotype II strain WSU 79-1146 (ATCC no. VR-1777) was grown in CRFK cells. A serial 10-fold dilution of FIPV was prepared from the working stock. Confluent 96-well plate was inoculated with 100 L of each virus dilution/well. The plate was incubated in a humidified incubator at 37 ∘ C, 5% CO 2 . Cytopathic effects (CPE) development was observed. The results were recorded after 72 hours and the virus tissue culture infective dose 50 (TCID 50 ) was calculated using Reed and Muench's method [17] .", "Oligonucleotide RNA. The Triple Helix Forming Oligonucleotides (TFOs) were designed based on the genome sequence of FIPV serotype II strain WSU 79-1146 (Accession no: AY994055) [18] . TFOs, which specifically target the different regions of the FIPV genome, and one unrelated TFO were constructed ( Table 1 ). The specificity of the TFOs was identified using BLAST search in the NCBI database. The designed linear TFOs were synthesized by Dharmacon Research (USA), whereby the 5 and 3 ends of the linear TFOs were modified with phosphate (PO 4 ) group and hydroxide (OH) group, respectively. These modifications were necessary for the circularization of linear TFO. The process of circularization, using the T4 RNA ligase 1 (ssRNA ligase) (New England Biolabs Inc., England), was carried out according to the manufacturer's protocol. After ligation, the circular TFO RNAs were recovered by ethanol precipitation and the purity of the circular TFO RNAs was measured using spectrophotometer.", "Denaturing of urea polyacrylamide gel electrophoresis was performed as described before [19] with modification. Briefly, 20% of denatured urea polyacrylamide gel was prepared and polymerized for 30 minutes. Then, the gel was prerun at 20 to 40 V for 45 minutes. Five L of TFO RNA mixed with 5 L of urea loading buffer was heated at 92 ∘ C for 2 minutes and immediately chilled on ice. It was run on the gel at 200 V for 45 minutes. Finally, the gel was stained with ethidium bromide (Sigma, USA) and viewed with a Bio-Rad Gel Doc XR system (CA, USA). (EMSA) . The target regions of the FIPV genome were synthesized by Dharmacon Research (USA) ( Table 1) . Each TFO RNA was mixed with the target region in 1X binding buffer containing 25 mM Tris-HCl, 6 mM MgCl 2 , and 10 mMNaCl in a final volume of 10 L and subsequently incubated at 37 ∘ C for 2 hours. The sample was run on 15% native polyacrylamide gel at 80 V, in cool condition. The stained gel was viewed by a Bio-Rad Gel Doc XR system.", "Regions. The binding strength was measured using a nano Isothermal Titration Calorimeter (ITC) (TA instruments, Newcastle, UK). The RNA sample mixtures, consisting of circular TFOs (0.0002 mM), were incubated with their respective synthetic target regions (0.015 mM) using 1X binding buffer as the diluent. The experiment was run at 37 ∘ C with 2 L/injection, for a total of 25 injections. Data was collected every 250 seconds and analyzed using the NanoAnalyze software v2.3.6 provided by the manufacturer.", "This experiment was conducted in CRFK cells, where 3 × 10 4 cell/well was seeded in 96-well plate to reach 80% confluency 24 hours prior to transfection. One hundred nM of TFO RNAs was separately transfected into the CRFK cells using a HiPerFect Transfection Reagent (Qiagen, Germany), as per the manufacturer's protocol. The plate was incubated at 37 ∘ C with 5% CO 2 for 6 hours. Then, the cultures were infected with 100TCID 50 of FIPV serotype II strain WSU 79-1146 for 1 hour at 37 ∘ C (100 L/well). Finally, the viral inoculum was replaced by fresh maintenance media (MEM containing 1% FBS and 1% pen/strep). Virus-infected and uninfected cells were maintained as positive and negative controls, respectively. The morphology of the cultures was recorded 72 hours after infection and samples were harvested at this time point and stored at −80 ∘ C prior to RNA extraction.", "Inhibition. Different concentrations of circular TFO1 RNA (25 nM, 50 nM, 100 nM, and 500 nM) were transfected into CRFK cells. The plate was incubated for 6 hours followed by virus inoculation for 1 hour at 37 ∘ C with 5% CO2. The cells were processed as described above.\n\nMadin-Darby Canine Kidney (MDCK) cell (ATCC no. CCL-34), at a concentration of 4 × 10 4 cell/well, was seeded in 96-well plate to reach 80% confluency 24 hours prior to transfection. Transfection was performed the same as before. One hundred nM of circular TFO RNA was transfected into MDCK cells. Following 6 hours \n\nORF1a/1b and 530-541\n\nORF1a/1b and 7399-7411\n\nORF1a/1b and 14048-14061", "- * Highlighted in bold indicated the binding region. * * Unrelated circular TFO. [20, 21] , respectively. The reverse transcriptase quantitative real-time PCR (RT-qPCR) was performed using a Bio-Rad CFX96 real-time system (BioRad, USA). The reaction was amplified in a final volume of 25 L using a SensiMix SYBR No-ROX One-Step Kit (Bioline, UK), which consisted of 12.5 L 2X SensiMix SYBR No-Rox One-\n\nStep reaction buffer, 10 M forward and reverse primers, 10 units RiboSafe RNase inhibitor, and 5 L template RNA. Absolute quantification approach was used to quantify qPCR results where a standard curve of a serial dilution of virus was plotted before the quantification. Amount of the virus in the samples was quantified based on this standard curve.", "Analysis. Data statistical analysis was performed using SPSS 18.0. Data were represented as mean ± SE of three independent tests. One-way ANOVA, Tukey post hoc test was used to analyze the significant level among the data. ≤ 0.05 was considered significant. genome, which play important roles in viral replication, were selected as the target binding sites for the triplex formation. The target regions were 5 untranslated region (5 UTR), Open Reading Frames (ORFs) 1a and 1b, and 3 untranslated region (3 UTR) ( Table 1 ). The TFOs were designed in duplex, as they can bind with the single stranded target region and reshape into triplex. Both ends of the duplex TFOs were ligated with a linker sequence or clamps (C-C) to construct circular TFO RNA.\n\nDenaturing PAGE assay was carried out after the ligation process to determine the formation of the circular TFO. As shown in Figure 1 , the circular TFO RNAs migrated faster than the linear TFO RNAs, when subjected to 20% denaturing PAGE.", "Target Region. The binding ability was determined using Electrophoretic Mobility Shift Assay (EMSA) [23] . The appearance of the slow mobility band indicates the successful hybridization of circular TFO RNA with its target region. The binding ability of different TFO RNAs (TFO1 to TFO5) against their target regions was determined by EMSA (Figure 2) . TFO1, TFO3, TFO4, and TFO5 showed slow mobility band, while TFO2 showed the lack of an upward shifted band. This indicates the possession of triplex binding ability for all circular TFO RNAs, except TFO2.\n\nTFO RNA. Study on the interaction and hybridization of TFO towards its target region is crucial, since the stronger the binding is, the more stable the triplex structure forms. As shown in supplementary Figure 1 (Table 3) .", "The antiviral effect of circular TFO RNAs was investigated by RT-qPCR assay at 72 hours after transfection. The results showed viral RNA genome copy numbers of 3.65 × 10 9 , 3.22 × 10 14 , 5.04 × 10 9 , 5.01 × 10 9 , 4.41 × 10 9 , and 3.96 × 10 14 in cells treated with TFO1, TFO2, TFO3, TFO4, TFO5, and TFO7, respectively. The data analyzed by one-way ANOVA, Tukey post hoc test showed significant high viral RNA genome copy number of 4.03 × 10 14 for virus inoculated cells as compared to circular TFO1, TFO3, TFO4, and TFO5 treatments ( ≤ 0.05). The viral RNA copies of circular TFO2, linear TFO3 and TFO4, and unrelated circular TFO7 RNAs transfected cells also showed high viral RNA copy numbers which did not show significant differences to the infected cells ( ≥ 0.05) ( Figure 3 ). The morphological changes of the cells were also captured 72 hours after transfection. The cells transfected with circular TFO1, TFO3, TFO4, and TFO5 appeared to be in good condition following virus", "inoculation, while the cells transfected with circular TFO2 and linear TFO3 and TFO4 showed visible cytopathic effect (CPE), the same as virus inoculated cells (supplementary Figure 2) . Furthermore, cells transfected with TFO only remain viable indicating that TFO treatment is generally not toxic to the cells. Hence, these results illustrated the capacity of circular TFO RNAs (except TFO2) to inhibit FIPV replication.", "Concentrations on FIPV Replication. Circular TFO1 was used to examine the dose-response relationship as a representative to other TFOs. The experimental conditions were identical to that of the previous experiment, except for TFO1 concentrations of 25 nM, 50 nM, 100 nM, and 500 nM. There was no significant reduction in viral RNA genome copies using the concentration of 25 nM TFO1. The other concentrations caused significant reductions in copy numbers as compared to the virus-infected cells. However, no significant difference was detected in copy numbers from all of these concentrations ( Figure 4 ).", "The specificity of the TFO towards FIPV was tested, using TFO1 and TFO5, as the proper representatives of TFOs, on influenza A virus H1N1 New Jersey 8/76. The analyzed data using one-way ANOVA, Tukey post hoc test did not show significant reductions in the copies of viral RNA for both TFOs compared to the influenza virus inoculated cells ( ≥ 0.05) (supplementary Figure 3 ). Complex structure G4/Cir4 Figure 2 : EMSA analysis. EMSA analysis illustrated the binding of circular TFO 1, 3, 4, and 5 to the target regions as evidenced by upward band shift. Binding of each circular TFO except circular TFO2 to its respective target forms a complex that migrates slower than unbound TFO. G1 to G5 represent the target region for circular TFO1 to TFO5 and Cir1 to Cir5 represent the circular TFO1 to TFO5, respectively. in the replication process [24] . Meanwhile, the ORF1a/1b of FIPV are translated into polyproteins that are cleaved into nonstructural proteins which assemble into", "replicationtranscription complexes together with other viral proteins [24] . Hence, the development of molecular therapy targeting these critical regions may provide the possibility to inhibit FIPV replication.", "Development of antiviral therapies against FIPV using siRNA [25] and viral protease inhibitors [26] Figure 4 : TFO1 dose-response study for inhibiting FIPV replication. The concentrations of 50 nM and higher showed significant antiviral effects. 50 nM of circular TFO1 RNA was able to reduce viral copy number by 5-fold log 10 from 10 14 to 10 9 , while 100 and 500 nM showed 4-fold reduction. Data are averages of 3 independent tests (mean ± SE). * Significantly different from FIPV-infected group.", "as potential new treatments against FIPV infection. In this study, circular Triple Helix Forming Oligonucleotide (TFO) RNAs, specifically targeting the short regions of viral genome for triplex formation, were designed and evaluated. TFO1 and TFO2 targeted the 5 and 3 UTRs of the viral genome, respectively. TFO3 to TFO5 targeted different regions of the ORF1a/1b on FIPV genome. Prior to in vitro antiviral study, the ligated circular TFOs were evaluated using PAGE analysis. All of the circularised TFO showed faster migration pattern compared to the linear TFO; however, only slight variation was detected for some of the TFO (Figure 1 ). The reason for this is not clear but probably due to the differences in length and the tertiary structures of the TFOs leading to differences in the migration rate. EMSA was used to show the binding capability of each circular TFO towards the target region in the FIPV genome except for TFO2 which showed lack of formation of complex structure upon", "hybridization ( Figure 2) . The EMSA result also concurred with the antiviral study, where all circular TFOs (except TFO2) were able to demonstrate a significant reduction in the viral RNA genome copy numbers by 5-fold log 10 from 10 14 in virus inoculated cells to 10 9 in TFO-transfected cells (Figure 3 ). However, no antiviral properties were detected from the linear TFOs and unrelated circular TFO7 RNA, confirming that the antiviral activity is associated with specific binding of circular TFOs towards targeted regions.", "Furthermore, the binding of the circular TFO to the target region was confirmed by nanoITC analysis; where the low value and high stability allowed TFOs to compete effectively with the target regions for inhibiting transcription in cell-free systems. Since, TFO1 shows the lowest value (Table 3) , the antiviral properties of this TFO were evaluated in doseresponse study. As shown in Figure 4 , 50 and 100 nM of TFO1 showed similar antiviral effects indicating the potential therapeutic application of TFO1 on FIPV replication. However, increasing the concentration of TFO1 to 500 nm failed to reduce the viral load further probably due to inefficiency of the transfection reagent to transfect the TFO into the cells. In addition, the virus has fast replication rate upon in vitro infection, where previous study on the growth of FIPV in CRFK cells showed that by 2 hours approximately 67% of FIPV 79-1146 were internalized by CRFK cells by endocytosis increasing to more than 70% at 3 hours [27,", "28] . The above finding probably also explained the reason why no antiviral effect was detected when the transfection of the TFO was performed on virus-infected cells (data not shown).", "The antiviral properties, as demonstrated by the circular TFOs, were probably associated with the binding of the TFO to the target region, based on both the Watson-Crick and Hoogsteen hydrogen bonds, which enhance the stability in terms of enthalpy, which is brought about by joining together two out of three strands of the triple helix in the proper orientation [29] . Therefore, the triplex formation is tightly bonded and not easy to detach. Furthermore, the circular TFOs were designed in such way that the presence of hydrogen bonding donors and acceptors in the purines is able to form two hydrogen bonds, while the pyrimidine bases can only form one additional hydrogen bond with incoming third bases [30] . However, there are various factors that may limit the activity of TFOs in cells like intracellular degradation of the TFO and limited accessibility of the TFO to the target sites which can prevent triplex formation [31] . These findings may also explain the inability of the designed", "TFO1 to inhibit further virus replication in dose-response study (Figure 4) .", "Various molecular-based therapies against infectious diseases and cancer have been developed and tested. However, only the siRNA-based therapy has been studied extensively as a novel antiviral and anticancer therapy [32, 33] . Recently, McDonagh et al. [25] developed siRNA with antiviral activity against the FIPV 79-1146, where the designed siRNA was able to reduce the copy number of viral genome compared with virus-infected cells. The potential therapeutic application of TFOs, such as linear TFO conjugated with psoralen to inhibit the transcription of human immunodeficiency provirus [13] and TFO to inhibit the transcription of 1(I) collagen in rat fibroblasts [14] , has also been reported. In addition, short TFO conjugated with daunomycin targeting the promoter region of oncogene has been designed and evaluated on human cancer cells [31] . These studies indicated the flexibility of using TFO-based oligonucleotides as a potential molecular-based therapy. In this study, we demonstrated", "short circular TFO RNAs between 28 and 34 mers (Table 1) , which are able to inhibit FIPV replication by binding to specific target regions of the FIPV genome. All designed circular TFOs (except TFO2) showed significant inhibitory effects against FIPV replication. The TFOs that formed triplex structures showed antiviral effects towards FIPV replication. The reason why TFO2 failed to show any interaction with the target region or antiviral activity is probably due to the length of TFO2 (i.e., 24 mers), which might be insufficient to a triplex formation upon hybridization (Figure 2 ), be effective enough to suppress viral RNA transcription, and eventually inhibit virus replication. Nevertheless, the inability of TFO2 to show antiviral effect due to failure in the formation of functional tertiary structure of the triplex formation cannot be ruled out. In vitro antiviral study which showed no antiviral property for unrelated TFO (TFO7) and also inability of circular TFO1 and TFO5 to", "inhibit influenza A virus H1N1 infected cells confirms the specificity of the TFOs' activity.", "In conclusion, the circular TFO RNA has the potential to be developed as a therapy against FIPV in cats. However, further studies on TFO specificity, actual mechanism of circular TFO RNA in the transcription alteration consequence of inhibiting the viral transcription process, and in vivo animal studies are important for this approach to work as a therapy in the future." ]

[ 3 ]

[ "shedding in their faeces" ]

[ "In Vitro Antiviral Activity of Circular Triple Helix Forming Oligonucleotide RNA towards Feline Infectious Peritonitis Virus Replication\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3950953/\n\nSHA: f5ad2323eb387f6e271e2842bb2cc4a33504fde3\n\nAuthors: Choong, Oi Kuan; Mehrbod, Parvaneh; Tejo, Bimo Ario; Omar, Abdul Rahman\nDate: 2014-02-20\nDOI: 10.1155/2014/654712\nLicense: cc-by", "Abstract: Feline Infectious Peritonitis (FIP) is a severe fatal immune-augmented disease in cat population. It is caused by FIP virus (FIPV), a virulent mutant strain of Feline Enteric Coronavirus (FECV). Current treatments and prophylactics are not effective. The in vitro antiviral properties of five circular Triple-Helix Forming Oligonucleotide (TFO) RNAs (TFO1 to TFO5), which target the different regions of virulent feline coronavirus (FCoV) strain FIPV WSU 79-1146 genome, were tested in FIPV-infected Crandell-Rees Feline Kidney (CRFK) cells. RT-qPCR results showed that the circular TFO RNAs, except TFO2, inhibit FIPV replication, where the viral genome copy numbers decreased significantly by 5-fold log(10) from 10(14) in the virus-inoculated cells to 10(9) in the circular TFO RNAs-transfected cells. Furthermore, the binding of the circular TFO RNA with the targeted viral genome segment was also confirmed using electrophoretic mobility shift assay. The strength of binding kinetics", "between the TFO RNAs and their target regions was demonstrated by NanoITC assay. In conclusion, the circular TFOs have the potential to be further developed as antiviral agents against FIPV infection.", "Text: Feline Infectious Peritonitis Virus (FIPV) is an enveloped virus with a nonsegmented, positive sense, single-stranded RNA genome. FIPV is grouped as feline coronavirus (FCoV), under the family Coronaviridae. FCoV is divided into two biotypes, namely, Feline Enteric Coronavirus (FECV), a ubiquitous enteric biotype of FCoV, and FIPV, a virulent biotype of FCoV [1] . The relationship between these two biotypes still remains unclear. Two hypotheses have been proposed, (i) internal mutation theory and (ii) circulating high virulent-low virulent theory. Internal mutation theory stated that the development of FIP is due to the exposure of cat to variants of FCoV which have been mutated by gaining the ability to replicate within the macrophages [2] , while the circulating high virulent-low virulent theory explains the existence of both distinctive pathogenic and benign lineages of viruses within the cat population [3] .", "Study has shown that about 40-80% of cats are detected with FECV shedding in their faeces [4] . About 12% of these FECV-positive cats have developed immune-mediated fatal FIP disease [4] . The prevalence of FIP among felines is due to continual cycles of infection and reinfection of FECV and indiscernible clinical symptoms of infected cats with FECV at an early stage before the progressive development of FIPV.", "Vaccination against FIPV with an attenuated, temperature-sensitive strain of type II FIPV induces low antibody titre in kittens that have not been exposed to FCoV. However, there is considerable controversy on the safety and efficacy of this vaccine, since the vaccine contains type 2 strain, whereas type 1 viruses are more prevalent in the field [4] . In addition, antibodies against FIPV do not protect infected cats but enhance the infection of monocytes and macrophages via a mechanism known as Antibody-Dependent Enhancement [1] . Besides vaccines, several antiviral drugs such as ribavirin, 2 BioMed Research International interferons, and immunosuppressive drugs have been used as treatments for FIPV-infected cats, mainly to suppress the inflammatory and detrimental immune response [5] [6] [7] [8] . However, those treatments were ineffective. Hence, there is still significant unmet medical need to develop effective treatments and prophylactics for FIPV infection.", "Triple Helix Forming Oligonucleotide (TFO) is defined as homopyrimidine oligonucleotides, which can form a sequence-specific triple helix by Hoogsteen bonds to the major groove of a complementary homopyrimidinehomopurine stretch in duplex DNA [9] . Furthermore, double helical RNA or DNA-RNA hybrids can be targeted as a template for triple helix formation, once the strand composition on the stabilities of triple helical complexes is determined [10] . Hence, TFO has been used to impede gene expressions by transcription inhibition of viral genes or oncogenes [11] [12] [13] [14] [15] [16] . The main purpose of this study is to develop and evaluate the in vitro antiviral properties of circular TFO RNAs against FIPV replication.", "serotype II strain WSU 79-1146 (ATCC no. VR-1777) was grown in CRFK cells. A serial 10-fold dilution of FIPV was prepared from the working stock. Confluent 96-well plate was inoculated with 100 L of each virus dilution/well. The plate was incubated in a humidified incubator at 37 ∘ C, 5% CO 2 . Cytopathic effects (CPE) development was observed. The results were recorded after 72 hours and the virus tissue culture infective dose 50 (TCID 50 ) was calculated using Reed and Muench's method [17] .", "Oligonucleotide RNA. The Triple Helix Forming Oligonucleotides (TFOs) were designed based on the genome sequence of FIPV serotype II strain WSU 79-1146 (Accession no: AY994055) [18] . TFOs, which specifically target the different regions of the FIPV genome, and one unrelated TFO were constructed ( Table 1 ). The specificity of the TFOs was identified using BLAST search in the NCBI database. The designed linear TFOs were synthesized by Dharmacon Research (USA), whereby the 5 and 3 ends of the linear TFOs were modified with phosphate (PO 4 ) group and hydroxide (OH) group, respectively. These modifications were necessary for the circularization of linear TFO. The process of circularization, using the T4 RNA ligase 1 (ssRNA ligase) (New England Biolabs Inc., England), was carried out according to the manufacturer's protocol. After ligation, the circular TFO RNAs were recovered by ethanol precipitation and the purity of the circular TFO RNAs was measured using spectrophotometer.", "Denaturing of urea polyacrylamide gel electrophoresis was performed as described before [19] with modification. Briefly, 20% of denatured urea polyacrylamide gel was prepared and polymerized for 30 minutes. Then, the gel was prerun at 20 to 40 V for 45 minutes. Five L of TFO RNA mixed with 5 L of urea loading buffer was heated at 92 ∘ C for 2 minutes and immediately chilled on ice. It was run on the gel at 200 V for 45 minutes. Finally, the gel was stained with ethidium bromide (Sigma, USA) and viewed with a Bio-Rad Gel Doc XR system (CA, USA). (EMSA) . The target regions of the FIPV genome were synthesized by Dharmacon Research (USA) ( Table 1) . Each TFO RNA was mixed with the target region in 1X binding buffer containing 25 mM Tris-HCl, 6 mM MgCl 2 , and 10 mMNaCl in a final volume of 10 L and subsequently incubated at 37 ∘ C for 2 hours. The sample was run on 15% native polyacrylamide gel at 80 V, in cool condition. The stained gel was viewed by a Bio-Rad Gel Doc XR system.", "Regions. The binding strength was measured using a nano Isothermal Titration Calorimeter (ITC) (TA instruments, Newcastle, UK). The RNA sample mixtures, consisting of circular TFOs (0.0002 mM), were incubated with their respective synthetic target regions (0.015 mM) using 1X binding buffer as the diluent. The experiment was run at 37 ∘ C with 2 L/injection, for a total of 25 injections. Data was collected every 250 seconds and analyzed using the NanoAnalyze software v2.3.6 provided by the manufacturer.", "This experiment was conducted in CRFK cells, where 3 × 10 4 cell/well was seeded in 96-well plate to reach 80% confluency 24 hours prior to transfection. One hundred nM of TFO RNAs was separately transfected into the CRFK cells using a HiPerFect Transfection Reagent (Qiagen, Germany), as per the manufacturer's protocol. The plate was incubated at 37 ∘ C with 5% CO 2 for 6 hours. Then, the cultures were infected with 100TCID 50 of FIPV serotype II strain WSU 79-1146 for 1 hour at 37 ∘ C (100 L/well). Finally, the viral inoculum was replaced by fresh maintenance media (MEM containing 1% FBS and 1% pen/strep). Virus-infected and uninfected cells were maintained as positive and negative controls, respectively. The morphology of the cultures was recorded 72 hours after infection and samples were harvested at this time point and stored at −80 ∘ C prior to RNA extraction.", "Inhibition. Different concentrations of circular TFO1 RNA (25 nM, 50 nM, 100 nM, and 500 nM) were transfected into CRFK cells. The plate was incubated for 6 hours followed by virus inoculation for 1 hour at 37 ∘ C with 5% CO2. The cells were processed as described above.\n\nMadin-Darby Canine Kidney (MDCK) cell (ATCC no. CCL-34), at a concentration of 4 × 10 4 cell/well, was seeded in 96-well plate to reach 80% confluency 24 hours prior to transfection. Transfection was performed the same as before. One hundred nM of circular TFO RNA was transfected into MDCK cells. Following 6 hours \n\nORF1a/1b and 530-541\n\nORF1a/1b and 7399-7411\n\nORF1a/1b and 14048-14061", "- * Highlighted in bold indicated the binding region. * * Unrelated circular TFO. [20, 21] , respectively. The reverse transcriptase quantitative real-time PCR (RT-qPCR) was performed using a Bio-Rad CFX96 real-time system (BioRad, USA). The reaction was amplified in a final volume of 25 L using a SensiMix SYBR No-ROX One-Step Kit (Bioline, UK), which consisted of 12.5 L 2X SensiMix SYBR No-Rox One-\n\nStep reaction buffer, 10 M forward and reverse primers, 10 units RiboSafe RNase inhibitor, and 5 L template RNA. Absolute quantification approach was used to quantify qPCR results where a standard curve of a serial dilution of virus was plotted before the quantification. Amount of the virus in the samples was quantified based on this standard curve.", "Analysis. Data statistical analysis was performed using SPSS 18.0. Data were represented as mean ± SE of three independent tests. One-way ANOVA, Tukey post hoc test was used to analyze the significant level among the data. ≤ 0.05 was considered significant. genome, which play important roles in viral replication, were selected as the target binding sites for the triplex formation. The target regions were 5 untranslated region (5 UTR), Open Reading Frames (ORFs) 1a and 1b, and 3 untranslated region (3 UTR) ( Table 1 ). The TFOs were designed in duplex, as they can bind with the single stranded target region and reshape into triplex. Both ends of the duplex TFOs were ligated with a linker sequence or clamps (C-C) to construct circular TFO RNA.\n\nDenaturing PAGE assay was carried out after the ligation process to determine the formation of the circular TFO. As shown in Figure 1 , the circular TFO RNAs migrated faster than the linear TFO RNAs, when subjected to 20% denaturing PAGE.", "Target Region. The binding ability was determined using Electrophoretic Mobility Shift Assay (EMSA) [23] . The appearance of the slow mobility band indicates the successful hybridization of circular TFO RNA with its target region. The binding ability of different TFO RNAs (TFO1 to TFO5) against their target regions was determined by EMSA (Figure 2) . TFO1, TFO3, TFO4, and TFO5 showed slow mobility band, while TFO2 showed the lack of an upward shifted band. This indicates the possession of triplex binding ability for all circular TFO RNAs, except TFO2.\n\nTFO RNA. Study on the interaction and hybridization of TFO towards its target region is crucial, since the stronger the binding is, the more stable the triplex structure forms. As shown in supplementary Figure 1 (Table 3) .", "The antiviral effect of circular TFO RNAs was investigated by RT-qPCR assay at 72 hours after transfection. The results showed viral RNA genome copy numbers of 3.65 × 10 9 , 3.22 × 10 14 , 5.04 × 10 9 , 5.01 × 10 9 , 4.41 × 10 9 , and 3.96 × 10 14 in cells treated with TFO1, TFO2, TFO3, TFO4, TFO5, and TFO7, respectively. The data analyzed by one-way ANOVA, Tukey post hoc test showed significant high viral RNA genome copy number of 4.03 × 10 14 for virus inoculated cells as compared to circular TFO1, TFO3, TFO4, and TFO5 treatments ( ≤ 0.05). The viral RNA copies of circular TFO2, linear TFO3 and TFO4, and unrelated circular TFO7 RNAs transfected cells also showed high viral RNA copy numbers which did not show significant differences to the infected cells ( ≥ 0.05) ( Figure 3 ). The morphological changes of the cells were also captured 72 hours after transfection. The cells transfected with circular TFO1, TFO3, TFO4, and TFO5 appeared to be in good condition following virus", "inoculation, while the cells transfected with circular TFO2 and linear TFO3 and TFO4 showed visible cytopathic effect (CPE), the same as virus inoculated cells (supplementary Figure 2) . Furthermore, cells transfected with TFO only remain viable indicating that TFO treatment is generally not toxic to the cells. Hence, these results illustrated the capacity of circular TFO RNAs (except TFO2) to inhibit FIPV replication.", "Concentrations on FIPV Replication. Circular TFO1 was used to examine the dose-response relationship as a representative to other TFOs. The experimental conditions were identical to that of the previous experiment, except for TFO1 concentrations of 25 nM, 50 nM, 100 nM, and 500 nM. There was no significant reduction in viral RNA genome copies using the concentration of 25 nM TFO1. The other concentrations caused significant reductions in copy numbers as compared to the virus-infected cells. However, no significant difference was detected in copy numbers from all of these concentrations ( Figure 4 ).", "The specificity of the TFO towards FIPV was tested, using TFO1 and TFO5, as the proper representatives of TFOs, on influenza A virus H1N1 New Jersey 8/76. The analyzed data using one-way ANOVA, Tukey post hoc test did not show significant reductions in the copies of viral RNA for both TFOs compared to the influenza virus inoculated cells ( ≥ 0.05) (supplementary Figure 3 ). Complex structure G4/Cir4 Figure 2 : EMSA analysis. EMSA analysis illustrated the binding of circular TFO 1, 3, 4, and 5 to the target regions as evidenced by upward band shift. Binding of each circular TFO except circular TFO2 to its respective target forms a complex that migrates slower than unbound TFO. G1 to G5 represent the target region for circular TFO1 to TFO5 and Cir1 to Cir5 represent the circular TFO1 to TFO5, respectively. in the replication process [24] . Meanwhile, the ORF1a/1b of FIPV are translated into polyproteins that are cleaved into nonstructural proteins which assemble into", "replicationtranscription complexes together with other viral proteins [24] . Hence, the development of molecular therapy targeting these critical regions may provide the possibility to inhibit FIPV replication.", "Development of antiviral therapies against FIPV using siRNA [25] and viral protease inhibitors [26] Figure 4 : TFO1 dose-response study for inhibiting FIPV replication. The concentrations of 50 nM and higher showed significant antiviral effects. 50 nM of circular TFO1 RNA was able to reduce viral copy number by 5-fold log 10 from 10 14 to 10 9 , while 100 and 500 nM showed 4-fold reduction. Data are averages of 3 independent tests (mean ± SE). * Significantly different from FIPV-infected group.", "as potential new treatments against FIPV infection. In this study, circular Triple Helix Forming Oligonucleotide (TFO) RNAs, specifically targeting the short regions of viral genome for triplex formation, were designed and evaluated. TFO1 and TFO2 targeted the 5 and 3 UTRs of the viral genome, respectively. TFO3 to TFO5 targeted different regions of the ORF1a/1b on FIPV genome. Prior to in vitro antiviral study, the ligated circular TFOs were evaluated using PAGE analysis. All of the circularised TFO showed faster migration pattern compared to the linear TFO; however, only slight variation was detected for some of the TFO (Figure 1 ). The reason for this is not clear but probably due to the differences in length and the tertiary structures of the TFOs leading to differences in the migration rate. EMSA was used to show the binding capability of each circular TFO towards the target region in the FIPV genome except for TFO2 which showed lack of formation of complex structure upon", "hybridization ( Figure 2) . The EMSA result also concurred with the antiviral study, where all circular TFOs (except TFO2) were able to demonstrate a significant reduction in the viral RNA genome copy numbers by 5-fold log 10 from 10 14 in virus inoculated cells to 10 9 in TFO-transfected cells (Figure 3 ). However, no antiviral properties were detected from the linear TFOs and unrelated circular TFO7 RNA, confirming that the antiviral activity is associated with specific binding of circular TFOs towards targeted regions.", "Furthermore, the binding of the circular TFO to the target region was confirmed by nanoITC analysis; where the low value and high stability allowed TFOs to compete effectively with the target regions for inhibiting transcription in cell-free systems. Since, TFO1 shows the lowest value (Table 3) , the antiviral properties of this TFO were evaluated in doseresponse study. As shown in Figure 4 , 50 and 100 nM of TFO1 showed similar antiviral effects indicating the potential therapeutic application of TFO1 on FIPV replication. However, increasing the concentration of TFO1 to 500 nm failed to reduce the viral load further probably due to inefficiency of the transfection reagent to transfect the TFO into the cells. In addition, the virus has fast replication rate upon in vitro infection, where previous study on the growth of FIPV in CRFK cells showed that by 2 hours approximately 67% of FIPV 79-1146 were internalized by CRFK cells by endocytosis increasing to more than 70% at 3 hours [27,", "28] . The above finding probably also explained the reason why no antiviral effect was detected when the transfection of the TFO was performed on virus-infected cells (data not shown).", "The antiviral properties, as demonstrated by the circular TFOs, were probably associated with the binding of the TFO to the target region, based on both the Watson-Crick and Hoogsteen hydrogen bonds, which enhance the stability in terms of enthalpy, which is brought about by joining together two out of three strands of the triple helix in the proper orientation [29] . Therefore, the triplex formation is tightly bonded and not easy to detach. Furthermore, the circular TFOs were designed in such way that the presence of hydrogen bonding donors and acceptors in the purines is able to form two hydrogen bonds, while the pyrimidine bases can only form one additional hydrogen bond with incoming third bases [30] . However, there are various factors that may limit the activity of TFOs in cells like intracellular degradation of the TFO and limited accessibility of the TFO to the target sites which can prevent triplex formation [31] . These findings may also explain the inability of the designed", "TFO1 to inhibit further virus replication in dose-response study (Figure 4) .", "Various molecular-based therapies against infectious diseases and cancer have been developed and tested. However, only the siRNA-based therapy has been studied extensively as a novel antiviral and anticancer therapy [32, 33] . Recently, McDonagh et al. [25] developed siRNA with antiviral activity against the FIPV 79-1146, where the designed siRNA was able to reduce the copy number of viral genome compared with virus-infected cells. The potential therapeutic application of TFOs, such as linear TFO conjugated with psoralen to inhibit the transcription of human immunodeficiency provirus [13] and TFO to inhibit the transcription of 1(I) collagen in rat fibroblasts [14] , has also been reported. In addition, short TFO conjugated with daunomycin targeting the promoter region of oncogene has been designed and evaluated on human cancer cells [31] . These studies indicated the flexibility of using TFO-based oligonucleotides as a potential molecular-based therapy. In this study, we demonstrated", "short circular TFO RNAs between 28 and 34 mers (Table 1) , which are able to inhibit FIPV replication by binding to specific target regions of the FIPV genome. All designed circular TFOs (except TFO2) showed significant inhibitory effects against FIPV replication. The TFOs that formed triplex structures showed antiviral effects towards FIPV replication. The reason why TFO2 failed to show any interaction with the target region or antiviral activity is probably due to the length of TFO2 (i.e., 24 mers), which might be insufficient to a triplex formation upon hybridization (Figure 2 ), be effective enough to suppress viral RNA transcription, and eventually inhibit virus replication. Nevertheless, the inability of TFO2 to show antiviral effect due to failure in the formation of functional tertiary structure of the triplex formation cannot be ruled out. In vitro antiviral study which showed no antiviral property for unrelated TFO (TFO7) and also inability of circular TFO1 and TFO5 to", "inhibit influenza A virus H1N1 infected cells confirms the specificity of the TFOs' activity.", "In conclusion, the circular TFO RNA has the potential to be developed as a therapy against FIPV in cats. However, further studies on TFO specificity, actual mechanism of circular TFO RNA in the transcription alteration consequence of inhibiting the viral transcription process, and in vivo animal studies are important for this approach to work as a therapy in the future." ]

[ 4 ]

[ "an attenuated, temperature-sensitive strain of type II FIPV" ]

[ "In Vitro Antiviral Activity of Circular Triple Helix Forming Oligonucleotide RNA towards Feline Infectious Peritonitis Virus Replication\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3950953/\n\nSHA: f5ad2323eb387f6e271e2842bb2cc4a33504fde3\n\nAuthors: Choong, Oi Kuan; Mehrbod, Parvaneh; Tejo, Bimo Ario; Omar, Abdul Rahman\nDate: 2014-02-20\nDOI: 10.1155/2014/654712\nLicense: cc-by", "Abstract: Feline Infectious Peritonitis (FIP) is a severe fatal immune-augmented disease in cat population. It is caused by FIP virus (FIPV), a virulent mutant strain of Feline Enteric Coronavirus (FECV). Current treatments and prophylactics are not effective. The in vitro antiviral properties of five circular Triple-Helix Forming Oligonucleotide (TFO) RNAs (TFO1 to TFO5), which target the different regions of virulent feline coronavirus (FCoV) strain FIPV WSU 79-1146 genome, were tested in FIPV-infected Crandell-Rees Feline Kidney (CRFK) cells. RT-qPCR results showed that the circular TFO RNAs, except TFO2, inhibit FIPV replication, where the viral genome copy numbers decreased significantly by 5-fold log(10) from 10(14) in the virus-inoculated cells to 10(9) in the circular TFO RNAs-transfected cells. Furthermore, the binding of the circular TFO RNA with the targeted viral genome segment was also confirmed using electrophoretic mobility shift assay. The strength of binding kinetics", "between the TFO RNAs and their target regions was demonstrated by NanoITC assay. In conclusion, the circular TFOs have the potential to be further developed as antiviral agents against FIPV infection.", "Text: Feline Infectious Peritonitis Virus (FIPV) is an enveloped virus with a nonsegmented, positive sense, single-stranded RNA genome. FIPV is grouped as feline coronavirus (FCoV), under the family Coronaviridae. FCoV is divided into two biotypes, namely, Feline Enteric Coronavirus (FECV), a ubiquitous enteric biotype of FCoV, and FIPV, a virulent biotype of FCoV [1] . The relationship between these two biotypes still remains unclear. Two hypotheses have been proposed, (i) internal mutation theory and (ii) circulating high virulent-low virulent theory. Internal mutation theory stated that the development of FIP is due to the exposure of cat to variants of FCoV which have been mutated by gaining the ability to replicate within the macrophages [2] , while the circulating high virulent-low virulent theory explains the existence of both distinctive pathogenic and benign lineages of viruses within the cat population [3] .", "Study has shown that about 40-80% of cats are detected with FECV shedding in their faeces [4] . About 12% of these FECV-positive cats have developed immune-mediated fatal FIP disease [4] . The prevalence of FIP among felines is due to continual cycles of infection and reinfection of FECV and indiscernible clinical symptoms of infected cats with FECV at an early stage before the progressive development of FIPV.", "Vaccination against FIPV with an attenuated, temperature-sensitive strain of type II FIPV induces low antibody titre in kittens that have not been exposed to FCoV. However, there is considerable controversy on the safety and efficacy of this vaccine, since the vaccine contains type 2 strain, whereas type 1 viruses are more prevalent in the field [4] . In addition, antibodies against FIPV do not protect infected cats but enhance the infection of monocytes and macrophages via a mechanism known as Antibody-Dependent Enhancement [1] . Besides vaccines, several antiviral drugs such as ribavirin, 2 BioMed Research International interferons, and immunosuppressive drugs have been used as treatments for FIPV-infected cats, mainly to suppress the inflammatory and detrimental immune response [5] [6] [7] [8] . However, those treatments were ineffective. Hence, there is still significant unmet medical need to develop effective treatments and prophylactics for FIPV infection.", "Triple Helix Forming Oligonucleotide (TFO) is defined as homopyrimidine oligonucleotides, which can form a sequence-specific triple helix by Hoogsteen bonds to the major groove of a complementary homopyrimidinehomopurine stretch in duplex DNA [9] . Furthermore, double helical RNA or DNA-RNA hybrids can be targeted as a template for triple helix formation, once the strand composition on the stabilities of triple helical complexes is determined [10] . Hence, TFO has been used to impede gene expressions by transcription inhibition of viral genes or oncogenes [11] [12] [13] [14] [15] [16] . The main purpose of this study is to develop and evaluate the in vitro antiviral properties of circular TFO RNAs against FIPV replication.", "serotype II strain WSU 79-1146 (ATCC no. VR-1777) was grown in CRFK cells. A serial 10-fold dilution of FIPV was prepared from the working stock. Confluent 96-well plate was inoculated with 100 L of each virus dilution/well. The plate was incubated in a humidified incubator at 37 ∘ C, 5% CO 2 . Cytopathic effects (CPE) development was observed. The results were recorded after 72 hours and the virus tissue culture infective dose 50 (TCID 50 ) was calculated using Reed and Muench's method [17] .", "Oligonucleotide RNA. The Triple Helix Forming Oligonucleotides (TFOs) were designed based on the genome sequence of FIPV serotype II strain WSU 79-1146 (Accession no: AY994055) [18] . TFOs, which specifically target the different regions of the FIPV genome, and one unrelated TFO were constructed ( Table 1 ). The specificity of the TFOs was identified using BLAST search in the NCBI database. The designed linear TFOs were synthesized by Dharmacon Research (USA), whereby the 5 and 3 ends of the linear TFOs were modified with phosphate (PO 4 ) group and hydroxide (OH) group, respectively. These modifications were necessary for the circularization of linear TFO. The process of circularization, using the T4 RNA ligase 1 (ssRNA ligase) (New England Biolabs Inc., England), was carried out according to the manufacturer's protocol. After ligation, the circular TFO RNAs were recovered by ethanol precipitation and the purity of the circular TFO RNAs was measured using spectrophotometer.", "Denaturing of urea polyacrylamide gel electrophoresis was performed as described before [19] with modification. Briefly, 20% of denatured urea polyacrylamide gel was prepared and polymerized for 30 minutes. Then, the gel was prerun at 20 to 40 V for 45 minutes. Five L of TFO RNA mixed with 5 L of urea loading buffer was heated at 92 ∘ C for 2 minutes and immediately chilled on ice. It was run on the gel at 200 V for 45 minutes. Finally, the gel was stained with ethidium bromide (Sigma, USA) and viewed with a Bio-Rad Gel Doc XR system (CA, USA). (EMSA) . The target regions of the FIPV genome were synthesized by Dharmacon Research (USA) ( Table 1) . Each TFO RNA was mixed with the target region in 1X binding buffer containing 25 mM Tris-HCl, 6 mM MgCl 2 , and 10 mMNaCl in a final volume of 10 L and subsequently incubated at 37 ∘ C for 2 hours. The sample was run on 15% native polyacrylamide gel at 80 V, in cool condition. The stained gel was viewed by a Bio-Rad Gel Doc XR system.", "Regions. The binding strength was measured using a nano Isothermal Titration Calorimeter (ITC) (TA instruments, Newcastle, UK). The RNA sample mixtures, consisting of circular TFOs (0.0002 mM), were incubated with their respective synthetic target regions (0.015 mM) using 1X binding buffer as the diluent. The experiment was run at 37 ∘ C with 2 L/injection, for a total of 25 injections. Data was collected every 250 seconds and analyzed using the NanoAnalyze software v2.3.6 provided by the manufacturer.", "This experiment was conducted in CRFK cells, where 3 × 10 4 cell/well was seeded in 96-well plate to reach 80% confluency 24 hours prior to transfection. One hundred nM of TFO RNAs was separately transfected into the CRFK cells using a HiPerFect Transfection Reagent (Qiagen, Germany), as per the manufacturer's protocol. The plate was incubated at 37 ∘ C with 5% CO 2 for 6 hours. Then, the cultures were infected with 100TCID 50 of FIPV serotype II strain WSU 79-1146 for 1 hour at 37 ∘ C (100 L/well). Finally, the viral inoculum was replaced by fresh maintenance media (MEM containing 1% FBS and 1% pen/strep). Virus-infected and uninfected cells were maintained as positive and negative controls, respectively. The morphology of the cultures was recorded 72 hours after infection and samples were harvested at this time point and stored at −80 ∘ C prior to RNA extraction.", "Inhibition. Different concentrations of circular TFO1 RNA (25 nM, 50 nM, 100 nM, and 500 nM) were transfected into CRFK cells. The plate was incubated for 6 hours followed by virus inoculation for 1 hour at 37 ∘ C with 5% CO2. The cells were processed as described above.\n\nMadin-Darby Canine Kidney (MDCK) cell (ATCC no. CCL-34), at a concentration of 4 × 10 4 cell/well, was seeded in 96-well plate to reach 80% confluency 24 hours prior to transfection. Transfection was performed the same as before. One hundred nM of circular TFO RNA was transfected into MDCK cells. Following 6 hours \n\nORF1a/1b and 530-541\n\nORF1a/1b and 7399-7411\n\nORF1a/1b and 14048-14061", "- * Highlighted in bold indicated the binding region. * * Unrelated circular TFO. [20, 21] , respectively. The reverse transcriptase quantitative real-time PCR (RT-qPCR) was performed using a Bio-Rad CFX96 real-time system (BioRad, USA). The reaction was amplified in a final volume of 25 L using a SensiMix SYBR No-ROX One-Step Kit (Bioline, UK), which consisted of 12.5 L 2X SensiMix SYBR No-Rox One-\n\nStep reaction buffer, 10 M forward and reverse primers, 10 units RiboSafe RNase inhibitor, and 5 L template RNA. Absolute quantification approach was used to quantify qPCR results where a standard curve of a serial dilution of virus was plotted before the quantification. Amount of the virus in the samples was quantified based on this standard curve.", "Analysis. Data statistical analysis was performed using SPSS 18.0. Data were represented as mean ± SE of three independent tests. One-way ANOVA, Tukey post hoc test was used to analyze the significant level among the data. ≤ 0.05 was considered significant. genome, which play important roles in viral replication, were selected as the target binding sites for the triplex formation. The target regions were 5 untranslated region (5 UTR), Open Reading Frames (ORFs) 1a and 1b, and 3 untranslated region (3 UTR) ( Table 1 ). The TFOs were designed in duplex, as they can bind with the single stranded target region and reshape into triplex. Both ends of the duplex TFOs were ligated with a linker sequence or clamps (C-C) to construct circular TFO RNA.\n\nDenaturing PAGE assay was carried out after the ligation process to determine the formation of the circular TFO. As shown in Figure 1 , the circular TFO RNAs migrated faster than the linear TFO RNAs, when subjected to 20% denaturing PAGE.", "Target Region. The binding ability was determined using Electrophoretic Mobility Shift Assay (EMSA) [23] . The appearance of the slow mobility band indicates the successful hybridization of circular TFO RNA with its target region. The binding ability of different TFO RNAs (TFO1 to TFO5) against their target regions was determined by EMSA (Figure 2) . TFO1, TFO3, TFO4, and TFO5 showed slow mobility band, while TFO2 showed the lack of an upward shifted band. This indicates the possession of triplex binding ability for all circular TFO RNAs, except TFO2.\n\nTFO RNA. Study on the interaction and hybridization of TFO towards its target region is crucial, since the stronger the binding is, the more stable the triplex structure forms. As shown in supplementary Figure 1 (Table 3) .", "The antiviral effect of circular TFO RNAs was investigated by RT-qPCR assay at 72 hours after transfection. The results showed viral RNA genome copy numbers of 3.65 × 10 9 , 3.22 × 10 14 , 5.04 × 10 9 , 5.01 × 10 9 , 4.41 × 10 9 , and 3.96 × 10 14 in cells treated with TFO1, TFO2, TFO3, TFO4, TFO5, and TFO7, respectively. The data analyzed by one-way ANOVA, Tukey post hoc test showed significant high viral RNA genome copy number of 4.03 × 10 14 for virus inoculated cells as compared to circular TFO1, TFO3, TFO4, and TFO5 treatments ( ≤ 0.05). The viral RNA copies of circular TFO2, linear TFO3 and TFO4, and unrelated circular TFO7 RNAs transfected cells also showed high viral RNA copy numbers which did not show significant differences to the infected cells ( ≥ 0.05) ( Figure 3 ). The morphological changes of the cells were also captured 72 hours after transfection. The cells transfected with circular TFO1, TFO3, TFO4, and TFO5 appeared to be in good condition following virus", "inoculation, while the cells transfected with circular TFO2 and linear TFO3 and TFO4 showed visible cytopathic effect (CPE), the same as virus inoculated cells (supplementary Figure 2) . Furthermore, cells transfected with TFO only remain viable indicating that TFO treatment is generally not toxic to the cells. Hence, these results illustrated the capacity of circular TFO RNAs (except TFO2) to inhibit FIPV replication.", "Concentrations on FIPV Replication. Circular TFO1 was used to examine the dose-response relationship as a representative to other TFOs. The experimental conditions were identical to that of the previous experiment, except for TFO1 concentrations of 25 nM, 50 nM, 100 nM, and 500 nM. There was no significant reduction in viral RNA genome copies using the concentration of 25 nM TFO1. The other concentrations caused significant reductions in copy numbers as compared to the virus-infected cells. However, no significant difference was detected in copy numbers from all of these concentrations ( Figure 4 ).", "The specificity of the TFO towards FIPV was tested, using TFO1 and TFO5, as the proper representatives of TFOs, on influenza A virus H1N1 New Jersey 8/76. The analyzed data using one-way ANOVA, Tukey post hoc test did not show significant reductions in the copies of viral RNA for both TFOs compared to the influenza virus inoculated cells ( ≥ 0.05) (supplementary Figure 3 ). Complex structure G4/Cir4 Figure 2 : EMSA analysis. EMSA analysis illustrated the binding of circular TFO 1, 3, 4, and 5 to the target regions as evidenced by upward band shift. Binding of each circular TFO except circular TFO2 to its respective target forms a complex that migrates slower than unbound TFO. G1 to G5 represent the target region for circular TFO1 to TFO5 and Cir1 to Cir5 represent the circular TFO1 to TFO5, respectively. in the replication process [24] . Meanwhile, the ORF1a/1b of FIPV are translated into polyproteins that are cleaved into nonstructural proteins which assemble into", "replicationtranscription complexes together with other viral proteins [24] . Hence, the development of molecular therapy targeting these critical regions may provide the possibility to inhibit FIPV replication.", "Development of antiviral therapies against FIPV using siRNA [25] and viral protease inhibitors [26] Figure 4 : TFO1 dose-response study for inhibiting FIPV replication. The concentrations of 50 nM and higher showed significant antiviral effects. 50 nM of circular TFO1 RNA was able to reduce viral copy number by 5-fold log 10 from 10 14 to 10 9 , while 100 and 500 nM showed 4-fold reduction. Data are averages of 3 independent tests (mean ± SE). * Significantly different from FIPV-infected group.", "as potential new treatments against FIPV infection. In this study, circular Triple Helix Forming Oligonucleotide (TFO) RNAs, specifically targeting the short regions of viral genome for triplex formation, were designed and evaluated. TFO1 and TFO2 targeted the 5 and 3 UTRs of the viral genome, respectively. TFO3 to TFO5 targeted different regions of the ORF1a/1b on FIPV genome. Prior to in vitro antiviral study, the ligated circular TFOs were evaluated using PAGE analysis. All of the circularised TFO showed faster migration pattern compared to the linear TFO; however, only slight variation was detected for some of the TFO (Figure 1 ). The reason for this is not clear but probably due to the differences in length and the tertiary structures of the TFOs leading to differences in the migration rate. EMSA was used to show the binding capability of each circular TFO towards the target region in the FIPV genome except for TFO2 which showed lack of formation of complex structure upon", "hybridization ( Figure 2) . The EMSA result also concurred with the antiviral study, where all circular TFOs (except TFO2) were able to demonstrate a significant reduction in the viral RNA genome copy numbers by 5-fold log 10 from 10 14 in virus inoculated cells to 10 9 in TFO-transfected cells (Figure 3 ). However, no antiviral properties were detected from the linear TFOs and unrelated circular TFO7 RNA, confirming that the antiviral activity is associated with specific binding of circular TFOs towards targeted regions.", "Furthermore, the binding of the circular TFO to the target region was confirmed by nanoITC analysis; where the low value and high stability allowed TFOs to compete effectively with the target regions for inhibiting transcription in cell-free systems. Since, TFO1 shows the lowest value (Table 3) , the antiviral properties of this TFO were evaluated in doseresponse study. As shown in Figure 4 , 50 and 100 nM of TFO1 showed similar antiviral effects indicating the potential therapeutic application of TFO1 on FIPV replication. However, increasing the concentration of TFO1 to 500 nm failed to reduce the viral load further probably due to inefficiency of the transfection reagent to transfect the TFO into the cells. In addition, the virus has fast replication rate upon in vitro infection, where previous study on the growth of FIPV in CRFK cells showed that by 2 hours approximately 67% of FIPV 79-1146 were internalized by CRFK cells by endocytosis increasing to more than 70% at 3 hours [27,", "28] . The above finding probably also explained the reason why no antiviral effect was detected when the transfection of the TFO was performed on virus-infected cells (data not shown).", "The antiviral properties, as demonstrated by the circular TFOs, were probably associated with the binding of the TFO to the target region, based on both the Watson-Crick and Hoogsteen hydrogen bonds, which enhance the stability in terms of enthalpy, which is brought about by joining together two out of three strands of the triple helix in the proper orientation [29] . Therefore, the triplex formation is tightly bonded and not easy to detach. Furthermore, the circular TFOs were designed in such way that the presence of hydrogen bonding donors and acceptors in the purines is able to form two hydrogen bonds, while the pyrimidine bases can only form one additional hydrogen bond with incoming third bases [30] . However, there are various factors that may limit the activity of TFOs in cells like intracellular degradation of the TFO and limited accessibility of the TFO to the target sites which can prevent triplex formation [31] . These findings may also explain the inability of the designed", "TFO1 to inhibit further virus replication in dose-response study (Figure 4) .", "Various molecular-based therapies against infectious diseases and cancer have been developed and tested. However, only the siRNA-based therapy has been studied extensively as a novel antiviral and anticancer therapy [32, 33] . Recently, McDonagh et al. [25] developed siRNA with antiviral activity against the FIPV 79-1146, where the designed siRNA was able to reduce the copy number of viral genome compared with virus-infected cells. The potential therapeutic application of TFOs, such as linear TFO conjugated with psoralen to inhibit the transcription of human immunodeficiency provirus [13] and TFO to inhibit the transcription of 1(I) collagen in rat fibroblasts [14] , has also been reported. In addition, short TFO conjugated with daunomycin targeting the promoter region of oncogene has been designed and evaluated on human cancer cells [31] . These studies indicated the flexibility of using TFO-based oligonucleotides as a potential molecular-based therapy. In this study, we demonstrated", "short circular TFO RNAs between 28 and 34 mers (Table 1) , which are able to inhibit FIPV replication by binding to specific target regions of the FIPV genome. All designed circular TFOs (except TFO2) showed significant inhibitory effects against FIPV replication. The TFOs that formed triplex structures showed antiviral effects towards FIPV replication. The reason why TFO2 failed to show any interaction with the target region or antiviral activity is probably due to the length of TFO2 (i.e., 24 mers), which might be insufficient to a triplex formation upon hybridization (Figure 2 ), be effective enough to suppress viral RNA transcription, and eventually inhibit virus replication. Nevertheless, the inability of TFO2 to show antiviral effect due to failure in the formation of functional tertiary structure of the triplex formation cannot be ruled out. In vitro antiviral study which showed no antiviral property for unrelated TFO (TFO7) and also inability of circular TFO1 and TFO5 to", "inhibit influenza A virus H1N1 infected cells confirms the specificity of the TFOs' activity.", "In conclusion, the circular TFO RNA has the potential to be developed as a therapy against FIPV in cats. However, further studies on TFO specificity, actual mechanism of circular TFO RNA in the transcription alteration consequence of inhibiting the viral transcription process, and in vivo animal studies are important for this approach to work as a therapy in the future." ]

[ 5 ]

[ "the vaccine contains type 2 strain, whereas type 1 viruses are more prevalent in the field" ]

[ "In Vitro Antiviral Activity of Circular Triple Helix Forming Oligonucleotide RNA towards Feline Infectious Peritonitis Virus Replication\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3950953/\n\nSHA: f5ad2323eb387f6e271e2842bb2cc4a33504fde3\n\nAuthors: Choong, Oi Kuan; Mehrbod, Parvaneh; Tejo, Bimo Ario; Omar, Abdul Rahman\nDate: 2014-02-20\nDOI: 10.1155/2014/654712\nLicense: cc-by", "Abstract: Feline Infectious Peritonitis (FIP) is a severe fatal immune-augmented disease in cat population. It is caused by FIP virus (FIPV), a virulent mutant strain of Feline Enteric Coronavirus (FECV). Current treatments and prophylactics are not effective. The in vitro antiviral properties of five circular Triple-Helix Forming Oligonucleotide (TFO) RNAs (TFO1 to TFO5), which target the different regions of virulent feline coronavirus (FCoV) strain FIPV WSU 79-1146 genome, were tested in FIPV-infected Crandell-Rees Feline Kidney (CRFK) cells. RT-qPCR results showed that the circular TFO RNAs, except TFO2, inhibit FIPV replication, where the viral genome copy numbers decreased significantly by 5-fold log(10) from 10(14) in the virus-inoculated cells to 10(9) in the circular TFO RNAs-transfected cells. Furthermore, the binding of the circular TFO RNA with the targeted viral genome segment was also confirmed using electrophoretic mobility shift assay. The strength of binding kinetics", "between the TFO RNAs and their target regions was demonstrated by NanoITC assay. In conclusion, the circular TFOs have the potential to be further developed as antiviral agents against FIPV infection.", "Text: Feline Infectious Peritonitis Virus (FIPV) is an enveloped virus with a nonsegmented, positive sense, single-stranded RNA genome. FIPV is grouped as feline coronavirus (FCoV), under the family Coronaviridae. FCoV is divided into two biotypes, namely, Feline Enteric Coronavirus (FECV), a ubiquitous enteric biotype of FCoV, and FIPV, a virulent biotype of FCoV [1] . The relationship between these two biotypes still remains unclear. Two hypotheses have been proposed, (i) internal mutation theory and (ii) circulating high virulent-low virulent theory. Internal mutation theory stated that the development of FIP is due to the exposure of cat to variants of FCoV which have been mutated by gaining the ability to replicate within the macrophages [2] , while the circulating high virulent-low virulent theory explains the existence of both distinctive pathogenic and benign lineages of viruses within the cat population [3] .", "Study has shown that about 40-80% of cats are detected with FECV shedding in their faeces [4] . About 12% of these FECV-positive cats have developed immune-mediated fatal FIP disease [4] . The prevalence of FIP among felines is due to continual cycles of infection and reinfection of FECV and indiscernible clinical symptoms of infected cats with FECV at an early stage before the progressive development of FIPV.", "Vaccination against FIPV with an attenuated, temperature-sensitive strain of type II FIPV induces low antibody titre in kittens that have not been exposed to FCoV. However, there is considerable controversy on the safety and efficacy of this vaccine, since the vaccine contains type 2 strain, whereas type 1 viruses are more prevalent in the field [4] . In addition, antibodies against FIPV do not protect infected cats but enhance the infection of monocytes and macrophages via a mechanism known as Antibody-Dependent Enhancement [1] . Besides vaccines, several antiviral drugs such as ribavirin, 2 BioMed Research International interferons, and immunosuppressive drugs have been used as treatments for FIPV-infected cats, mainly to suppress the inflammatory and detrimental immune response [5] [6] [7] [8] . However, those treatments were ineffective. Hence, there is still significant unmet medical need to develop effective treatments and prophylactics for FIPV infection.", "Triple Helix Forming Oligonucleotide (TFO) is defined as homopyrimidine oligonucleotides, which can form a sequence-specific triple helix by Hoogsteen bonds to the major groove of a complementary homopyrimidinehomopurine stretch in duplex DNA [9] . Furthermore, double helical RNA or DNA-RNA hybrids can be targeted as a template for triple helix formation, once the strand composition on the stabilities of triple helical complexes is determined [10] . Hence, TFO has been used to impede gene expressions by transcription inhibition of viral genes or oncogenes [11] [12] [13] [14] [15] [16] . The main purpose of this study is to develop and evaluate the in vitro antiviral properties of circular TFO RNAs against FIPV replication.", "serotype II strain WSU 79-1146 (ATCC no. VR-1777) was grown in CRFK cells. A serial 10-fold dilution of FIPV was prepared from the working stock. Confluent 96-well plate was inoculated with 100 L of each virus dilution/well. The plate was incubated in a humidified incubator at 37 ∘ C, 5% CO 2 . Cytopathic effects (CPE) development was observed. The results were recorded after 72 hours and the virus tissue culture infective dose 50 (TCID 50 ) was calculated using Reed and Muench's method [17] .", "Oligonucleotide RNA. The Triple Helix Forming Oligonucleotides (TFOs) were designed based on the genome sequence of FIPV serotype II strain WSU 79-1146 (Accession no: AY994055) [18] . TFOs, which specifically target the different regions of the FIPV genome, and one unrelated TFO were constructed ( Table 1 ). The specificity of the TFOs was identified using BLAST search in the NCBI database. The designed linear TFOs were synthesized by Dharmacon Research (USA), whereby the 5 and 3 ends of the linear TFOs were modified with phosphate (PO 4 ) group and hydroxide (OH) group, respectively. These modifications were necessary for the circularization of linear TFO. The process of circularization, using the T4 RNA ligase 1 (ssRNA ligase) (New England Biolabs Inc., England), was carried out according to the manufacturer's protocol. After ligation, the circular TFO RNAs were recovered by ethanol precipitation and the purity of the circular TFO RNAs was measured using spectrophotometer.", "Denaturing of urea polyacrylamide gel electrophoresis was performed as described before [19] with modification. Briefly, 20% of denatured urea polyacrylamide gel was prepared and polymerized for 30 minutes. Then, the gel was prerun at 20 to 40 V for 45 minutes. Five L of TFO RNA mixed with 5 L of urea loading buffer was heated at 92 ∘ C for 2 minutes and immediately chilled on ice. It was run on the gel at 200 V for 45 minutes. Finally, the gel was stained with ethidium bromide (Sigma, USA) and viewed with a Bio-Rad Gel Doc XR system (CA, USA). (EMSA) . The target regions of the FIPV genome were synthesized by Dharmacon Research (USA) ( Table 1) . Each TFO RNA was mixed with the target region in 1X binding buffer containing 25 mM Tris-HCl, 6 mM MgCl 2 , and 10 mMNaCl in a final volume of 10 L and subsequently incubated at 37 ∘ C for 2 hours. The sample was run on 15% native polyacrylamide gel at 80 V, in cool condition. The stained gel was viewed by a Bio-Rad Gel Doc XR system.", "Regions. The binding strength was measured using a nano Isothermal Titration Calorimeter (ITC) (TA instruments, Newcastle, UK). The RNA sample mixtures, consisting of circular TFOs (0.0002 mM), were incubated with their respective synthetic target regions (0.015 mM) using 1X binding buffer as the diluent. The experiment was run at 37 ∘ C with 2 L/injection, for a total of 25 injections. Data was collected every 250 seconds and analyzed using the NanoAnalyze software v2.3.6 provided by the manufacturer.", "This experiment was conducted in CRFK cells, where 3 × 10 4 cell/well was seeded in 96-well plate to reach 80% confluency 24 hours prior to transfection. One hundred nM of TFO RNAs was separately transfected into the CRFK cells using a HiPerFect Transfection Reagent (Qiagen, Germany), as per the manufacturer's protocol. The plate was incubated at 37 ∘ C with 5% CO 2 for 6 hours. Then, the cultures were infected with 100TCID 50 of FIPV serotype II strain WSU 79-1146 for 1 hour at 37 ∘ C (100 L/well). Finally, the viral inoculum was replaced by fresh maintenance media (MEM containing 1% FBS and 1% pen/strep). Virus-infected and uninfected cells were maintained as positive and negative controls, respectively. The morphology of the cultures was recorded 72 hours after infection and samples were harvested at this time point and stored at −80 ∘ C prior to RNA extraction.", "Inhibition. Different concentrations of circular TFO1 RNA (25 nM, 50 nM, 100 nM, and 500 nM) were transfected into CRFK cells. The plate was incubated for 6 hours followed by virus inoculation for 1 hour at 37 ∘ C with 5% CO2. The cells were processed as described above.\n\nMadin-Darby Canine Kidney (MDCK) cell (ATCC no. CCL-34), at a concentration of 4 × 10 4 cell/well, was seeded in 96-well plate to reach 80% confluency 24 hours prior to transfection. Transfection was performed the same as before. One hundred nM of circular TFO RNA was transfected into MDCK cells. Following 6 hours \n\nORF1a/1b and 530-541\n\nORF1a/1b and 7399-7411\n\nORF1a/1b and 14048-14061", "- * Highlighted in bold indicated the binding region. * * Unrelated circular TFO. [20, 21] , respectively. The reverse transcriptase quantitative real-time PCR (RT-qPCR) was performed using a Bio-Rad CFX96 real-time system (BioRad, USA). The reaction was amplified in a final volume of 25 L using a SensiMix SYBR No-ROX One-Step Kit (Bioline, UK), which consisted of 12.5 L 2X SensiMix SYBR No-Rox One-\n\nStep reaction buffer, 10 M forward and reverse primers, 10 units RiboSafe RNase inhibitor, and 5 L template RNA. Absolute quantification approach was used to quantify qPCR results where a standard curve of a serial dilution of virus was plotted before the quantification. Amount of the virus in the samples was quantified based on this standard curve.", "Analysis. Data statistical analysis was performed using SPSS 18.0. Data were represented as mean ± SE of three independent tests. One-way ANOVA, Tukey post hoc test was used to analyze the significant level among the data. ≤ 0.05 was considered significant. genome, which play important roles in viral replication, were selected as the target binding sites for the triplex formation. The target regions were 5 untranslated region (5 UTR), Open Reading Frames (ORFs) 1a and 1b, and 3 untranslated region (3 UTR) ( Table 1 ). The TFOs were designed in duplex, as they can bind with the single stranded target region and reshape into triplex. Both ends of the duplex TFOs were ligated with a linker sequence or clamps (C-C) to construct circular TFO RNA.\n\nDenaturing PAGE assay was carried out after the ligation process to determine the formation of the circular TFO. As shown in Figure 1 , the circular TFO RNAs migrated faster than the linear TFO RNAs, when subjected to 20% denaturing PAGE.", "Target Region. The binding ability was determined using Electrophoretic Mobility Shift Assay (EMSA) [23] . The appearance of the slow mobility band indicates the successful hybridization of circular TFO RNA with its target region. The binding ability of different TFO RNAs (TFO1 to TFO5) against their target regions was determined by EMSA (Figure 2) . TFO1, TFO3, TFO4, and TFO5 showed slow mobility band, while TFO2 showed the lack of an upward shifted band. This indicates the possession of triplex binding ability for all circular TFO RNAs, except TFO2.\n\nTFO RNA. Study on the interaction and hybridization of TFO towards its target region is crucial, since the stronger the binding is, the more stable the triplex structure forms. As shown in supplementary Figure 1 (Table 3) .", "The antiviral effect of circular TFO RNAs was investigated by RT-qPCR assay at 72 hours after transfection. The results showed viral RNA genome copy numbers of 3.65 × 10 9 , 3.22 × 10 14 , 5.04 × 10 9 , 5.01 × 10 9 , 4.41 × 10 9 , and 3.96 × 10 14 in cells treated with TFO1, TFO2, TFO3, TFO4, TFO5, and TFO7, respectively. The data analyzed by one-way ANOVA, Tukey post hoc test showed significant high viral RNA genome copy number of 4.03 × 10 14 for virus inoculated cells as compared to circular TFO1, TFO3, TFO4, and TFO5 treatments ( ≤ 0.05). The viral RNA copies of circular TFO2, linear TFO3 and TFO4, and unrelated circular TFO7 RNAs transfected cells also showed high viral RNA copy numbers which did not show significant differences to the infected cells ( ≥ 0.05) ( Figure 3 ). The morphological changes of the cells were also captured 72 hours after transfection. The cells transfected with circular TFO1, TFO3, TFO4, and TFO5 appeared to be in good condition following virus", "inoculation, while the cells transfected with circular TFO2 and linear TFO3 and TFO4 showed visible cytopathic effect (CPE), the same as virus inoculated cells (supplementary Figure 2) . Furthermore, cells transfected with TFO only remain viable indicating that TFO treatment is generally not toxic to the cells. Hence, these results illustrated the capacity of circular TFO RNAs (except TFO2) to inhibit FIPV replication.", "Concentrations on FIPV Replication. Circular TFO1 was used to examine the dose-response relationship as a representative to other TFOs. The experimental conditions were identical to that of the previous experiment, except for TFO1 concentrations of 25 nM, 50 nM, 100 nM, and 500 nM. There was no significant reduction in viral RNA genome copies using the concentration of 25 nM TFO1. The other concentrations caused significant reductions in copy numbers as compared to the virus-infected cells. However, no significant difference was detected in copy numbers from all of these concentrations ( Figure 4 ).", "The specificity of the TFO towards FIPV was tested, using TFO1 and TFO5, as the proper representatives of TFOs, on influenza A virus H1N1 New Jersey 8/76. The analyzed data using one-way ANOVA, Tukey post hoc test did not show significant reductions in the copies of viral RNA for both TFOs compared to the influenza virus inoculated cells ( ≥ 0.05) (supplementary Figure 3 ). Complex structure G4/Cir4 Figure 2 : EMSA analysis. EMSA analysis illustrated the binding of circular TFO 1, 3, 4, and 5 to the target regions as evidenced by upward band shift. Binding of each circular TFO except circular TFO2 to its respective target forms a complex that migrates slower than unbound TFO. G1 to G5 represent the target region for circular TFO1 to TFO5 and Cir1 to Cir5 represent the circular TFO1 to TFO5, respectively. in the replication process [24] . Meanwhile, the ORF1a/1b of FIPV are translated into polyproteins that are cleaved into nonstructural proteins which assemble into", "replicationtranscription complexes together with other viral proteins [24] . Hence, the development of molecular therapy targeting these critical regions may provide the possibility to inhibit FIPV replication.", "Development of antiviral therapies against FIPV using siRNA [25] and viral protease inhibitors [26] Figure 4 : TFO1 dose-response study for inhibiting FIPV replication. The concentrations of 50 nM and higher showed significant antiviral effects. 50 nM of circular TFO1 RNA was able to reduce viral copy number by 5-fold log 10 from 10 14 to 10 9 , while 100 and 500 nM showed 4-fold reduction. Data are averages of 3 independent tests (mean ± SE). * Significantly different from FIPV-infected group.", "as potential new treatments against FIPV infection. In this study, circular Triple Helix Forming Oligonucleotide (TFO) RNAs, specifically targeting the short regions of viral genome for triplex formation, were designed and evaluated. TFO1 and TFO2 targeted the 5 and 3 UTRs of the viral genome, respectively. TFO3 to TFO5 targeted different regions of the ORF1a/1b on FIPV genome. Prior to in vitro antiviral study, the ligated circular TFOs were evaluated using PAGE analysis. All of the circularised TFO showed faster migration pattern compared to the linear TFO; however, only slight variation was detected for some of the TFO (Figure 1 ). The reason for this is not clear but probably due to the differences in length and the tertiary structures of the TFOs leading to differences in the migration rate. EMSA was used to show the binding capability of each circular TFO towards the target region in the FIPV genome except for TFO2 which showed lack of formation of complex structure upon", "hybridization ( Figure 2) . The EMSA result also concurred with the antiviral study, where all circular TFOs (except TFO2) were able to demonstrate a significant reduction in the viral RNA genome copy numbers by 5-fold log 10 from 10 14 in virus inoculated cells to 10 9 in TFO-transfected cells (Figure 3 ). However, no antiviral properties were detected from the linear TFOs and unrelated circular TFO7 RNA, confirming that the antiviral activity is associated with specific binding of circular TFOs towards targeted regions.", "Furthermore, the binding of the circular TFO to the target region was confirmed by nanoITC analysis; where the low value and high stability allowed TFOs to compete effectively with the target regions for inhibiting transcription in cell-free systems. Since, TFO1 shows the lowest value (Table 3) , the antiviral properties of this TFO were evaluated in doseresponse study. As shown in Figure 4 , 50 and 100 nM of TFO1 showed similar antiviral effects indicating the potential therapeutic application of TFO1 on FIPV replication. However, increasing the concentration of TFO1 to 500 nm failed to reduce the viral load further probably due to inefficiency of the transfection reagent to transfect the TFO into the cells. In addition, the virus has fast replication rate upon in vitro infection, where previous study on the growth of FIPV in CRFK cells showed that by 2 hours approximately 67% of FIPV 79-1146 were internalized by CRFK cells by endocytosis increasing to more than 70% at 3 hours [27,", "28] . The above finding probably also explained the reason why no antiviral effect was detected when the transfection of the TFO was performed on virus-infected cells (data not shown).", "The antiviral properties, as demonstrated by the circular TFOs, were probably associated with the binding of the TFO to the target region, based on both the Watson-Crick and Hoogsteen hydrogen bonds, which enhance the stability in terms of enthalpy, which is brought about by joining together two out of three strands of the triple helix in the proper orientation [29] . Therefore, the triplex formation is tightly bonded and not easy to detach. Furthermore, the circular TFOs were designed in such way that the presence of hydrogen bonding donors and acceptors in the purines is able to form two hydrogen bonds, while the pyrimidine bases can only form one additional hydrogen bond with incoming third bases [30] . However, there are various factors that may limit the activity of TFOs in cells like intracellular degradation of the TFO and limited accessibility of the TFO to the target sites which can prevent triplex formation [31] . These findings may also explain the inability of the designed", "TFO1 to inhibit further virus replication in dose-response study (Figure 4) .", "Various molecular-based therapies against infectious diseases and cancer have been developed and tested. However, only the siRNA-based therapy has been studied extensively as a novel antiviral and anticancer therapy [32, 33] . Recently, McDonagh et al. [25] developed siRNA with antiviral activity against the FIPV 79-1146, where the designed siRNA was able to reduce the copy number of viral genome compared with virus-infected cells. The potential therapeutic application of TFOs, such as linear TFO conjugated with psoralen to inhibit the transcription of human immunodeficiency provirus [13] and TFO to inhibit the transcription of 1(I) collagen in rat fibroblasts [14] , has also been reported. In addition, short TFO conjugated with daunomycin targeting the promoter region of oncogene has been designed and evaluated on human cancer cells [31] . These studies indicated the flexibility of using TFO-based oligonucleotides as a potential molecular-based therapy. In this study, we demonstrated", "short circular TFO RNAs between 28 and 34 mers (Table 1) , which are able to inhibit FIPV replication by binding to specific target regions of the FIPV genome. All designed circular TFOs (except TFO2) showed significant inhibitory effects against FIPV replication. The TFOs that formed triplex structures showed antiviral effects towards FIPV replication. The reason why TFO2 failed to show any interaction with the target region or antiviral activity is probably due to the length of TFO2 (i.e., 24 mers), which might be insufficient to a triplex formation upon hybridization (Figure 2 ), be effective enough to suppress viral RNA transcription, and eventually inhibit virus replication. Nevertheless, the inability of TFO2 to show antiviral effect due to failure in the formation of functional tertiary structure of the triplex formation cannot be ruled out. In vitro antiviral study which showed no antiviral property for unrelated TFO (TFO7) and also inability of circular TFO1 and TFO5 to", "inhibit influenza A virus H1N1 infected cells confirms the specificity of the TFOs' activity.", "In conclusion, the circular TFO RNA has the potential to be developed as a therapy against FIPV in cats. However, further studies on TFO specificity, actual mechanism of circular TFO RNA in the transcription alteration consequence of inhibiting the viral transcription process, and in vivo animal studies are important for this approach to work as a therapy in the future." ]

[ 5 ]

[ "30 minutes" ]

[ "In Vitro Antiviral Activity of Circular Triple Helix Forming Oligonucleotide RNA towards Feline Infectious Peritonitis Virus Replication\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3950953/\n\nSHA: f5ad2323eb387f6e271e2842bb2cc4a33504fde3\n\nAuthors: Choong, Oi Kuan; Mehrbod, Parvaneh; Tejo, Bimo Ario; Omar, Abdul Rahman\nDate: 2014-02-20\nDOI: 10.1155/2014/654712\nLicense: cc-by", "Abstract: Feline Infectious Peritonitis (FIP) is a severe fatal immune-augmented disease in cat population. It is caused by FIP virus (FIPV), a virulent mutant strain of Feline Enteric Coronavirus (FECV). Current treatments and prophylactics are not effective. The in vitro antiviral properties of five circular Triple-Helix Forming Oligonucleotide (TFO) RNAs (TFO1 to TFO5), which target the different regions of virulent feline coronavirus (FCoV) strain FIPV WSU 79-1146 genome, were tested in FIPV-infected Crandell-Rees Feline Kidney (CRFK) cells. RT-qPCR results showed that the circular TFO RNAs, except TFO2, inhibit FIPV replication, where the viral genome copy numbers decreased significantly by 5-fold log(10) from 10(14) in the virus-inoculated cells to 10(9) in the circular TFO RNAs-transfected cells. Furthermore, the binding of the circular TFO RNA with the targeted viral genome segment was also confirmed using electrophoretic mobility shift assay. The strength of binding kinetics", "between the TFO RNAs and their target regions was demonstrated by NanoITC assay. In conclusion, the circular TFOs have the potential to be further developed as antiviral agents against FIPV infection.", "Text: Feline Infectious Peritonitis Virus (FIPV) is an enveloped virus with a nonsegmented, positive sense, single-stranded RNA genome. FIPV is grouped as feline coronavirus (FCoV), under the family Coronaviridae. FCoV is divided into two biotypes, namely, Feline Enteric Coronavirus (FECV), a ubiquitous enteric biotype of FCoV, and FIPV, a virulent biotype of FCoV [1] . The relationship between these two biotypes still remains unclear. Two hypotheses have been proposed, (i) internal mutation theory and (ii) circulating high virulent-low virulent theory. Internal mutation theory stated that the development of FIP is due to the exposure of cat to variants of FCoV which have been mutated by gaining the ability to replicate within the macrophages [2] , while the circulating high virulent-low virulent theory explains the existence of both distinctive pathogenic and benign lineages of viruses within the cat population [3] .", "Study has shown that about 40-80% of cats are detected with FECV shedding in their faeces [4] . About 12% of these FECV-positive cats have developed immune-mediated fatal FIP disease [4] . The prevalence of FIP among felines is due to continual cycles of infection and reinfection of FECV and indiscernible clinical symptoms of infected cats with FECV at an early stage before the progressive development of FIPV.", "Vaccination against FIPV with an attenuated, temperature-sensitive strain of type II FIPV induces low antibody titre in kittens that have not been exposed to FCoV. However, there is considerable controversy on the safety and efficacy of this vaccine, since the vaccine contains type 2 strain, whereas type 1 viruses are more prevalent in the field [4] . In addition, antibodies against FIPV do not protect infected cats but enhance the infection of monocytes and macrophages via a mechanism known as Antibody-Dependent Enhancement [1] . Besides vaccines, several antiviral drugs such as ribavirin, 2 BioMed Research International interferons, and immunosuppressive drugs have been used as treatments for FIPV-infected cats, mainly to suppress the inflammatory and detrimental immune response [5] [6] [7] [8] . However, those treatments were ineffective. Hence, there is still significant unmet medical need to develop effective treatments and prophylactics for FIPV infection.", "Triple Helix Forming Oligonucleotide (TFO) is defined as homopyrimidine oligonucleotides, which can form a sequence-specific triple helix by Hoogsteen bonds to the major groove of a complementary homopyrimidinehomopurine stretch in duplex DNA [9] . Furthermore, double helical RNA or DNA-RNA hybrids can be targeted as a template for triple helix formation, once the strand composition on the stabilities of triple helical complexes is determined [10] . Hence, TFO has been used to impede gene expressions by transcription inhibition of viral genes or oncogenes [11] [12] [13] [14] [15] [16] . The main purpose of this study is to develop and evaluate the in vitro antiviral properties of circular TFO RNAs against FIPV replication.", "serotype II strain WSU 79-1146 (ATCC no. VR-1777) was grown in CRFK cells. A serial 10-fold dilution of FIPV was prepared from the working stock. Confluent 96-well plate was inoculated with 100 L of each virus dilution/well. The plate was incubated in a humidified incubator at 37 ∘ C, 5% CO 2 . Cytopathic effects (CPE) development was observed. The results were recorded after 72 hours and the virus tissue culture infective dose 50 (TCID 50 ) was calculated using Reed and Muench's method [17] .", "Oligonucleotide RNA. The Triple Helix Forming Oligonucleotides (TFOs) were designed based on the genome sequence of FIPV serotype II strain WSU 79-1146 (Accession no: AY994055) [18] . TFOs, which specifically target the different regions of the FIPV genome, and one unrelated TFO were constructed ( Table 1 ). The specificity of the TFOs was identified using BLAST search in the NCBI database. The designed linear TFOs were synthesized by Dharmacon Research (USA), whereby the 5 and 3 ends of the linear TFOs were modified with phosphate (PO 4 ) group and hydroxide (OH) group, respectively. These modifications were necessary for the circularization of linear TFO. The process of circularization, using the T4 RNA ligase 1 (ssRNA ligase) (New England Biolabs Inc., England), was carried out according to the manufacturer's protocol. After ligation, the circular TFO RNAs were recovered by ethanol precipitation and the purity of the circular TFO RNAs was measured using spectrophotometer.", "Denaturing of urea polyacrylamide gel electrophoresis was performed as described before [19] with modification. Briefly, 20% of denatured urea polyacrylamide gel was prepared and polymerized for 30 minutes. Then, the gel was prerun at 20 to 40 V for 45 minutes. Five L of TFO RNA mixed with 5 L of urea loading buffer was heated at 92 ∘ C for 2 minutes and immediately chilled on ice. It was run on the gel at 200 V for 45 minutes. Finally, the gel was stained with ethidium bromide (Sigma, USA) and viewed with a Bio-Rad Gel Doc XR system (CA, USA). (EMSA) . The target regions of the FIPV genome were synthesized by Dharmacon Research (USA) ( Table 1) . Each TFO RNA was mixed with the target region in 1X binding buffer containing 25 mM Tris-HCl, 6 mM MgCl 2 , and 10 mMNaCl in a final volume of 10 L and subsequently incubated at 37 ∘ C for 2 hours. The sample was run on 15% native polyacrylamide gel at 80 V, in cool condition. The stained gel was viewed by a Bio-Rad Gel Doc XR system.", "Regions. The binding strength was measured using a nano Isothermal Titration Calorimeter (ITC) (TA instruments, Newcastle, UK). The RNA sample mixtures, consisting of circular TFOs (0.0002 mM), were incubated with their respective synthetic target regions (0.015 mM) using 1X binding buffer as the diluent. The experiment was run at 37 ∘ C with 2 L/injection, for a total of 25 injections. Data was collected every 250 seconds and analyzed using the NanoAnalyze software v2.3.6 provided by the manufacturer.", "This experiment was conducted in CRFK cells, where 3 × 10 4 cell/well was seeded in 96-well plate to reach 80% confluency 24 hours prior to transfection. One hundred nM of TFO RNAs was separately transfected into the CRFK cells using a HiPerFect Transfection Reagent (Qiagen, Germany), as per the manufacturer's protocol. The plate was incubated at 37 ∘ C with 5% CO 2 for 6 hours. Then, the cultures were infected with 100TCID 50 of FIPV serotype II strain WSU 79-1146 for 1 hour at 37 ∘ C (100 L/well). Finally, the viral inoculum was replaced by fresh maintenance media (MEM containing 1% FBS and 1% pen/strep). Virus-infected and uninfected cells were maintained as positive and negative controls, respectively. The morphology of the cultures was recorded 72 hours after infection and samples were harvested at this time point and stored at −80 ∘ C prior to RNA extraction.", "Inhibition. Different concentrations of circular TFO1 RNA (25 nM, 50 nM, 100 nM, and 500 nM) were transfected into CRFK cells. The plate was incubated for 6 hours followed by virus inoculation for 1 hour at 37 ∘ C with 5% CO2. The cells were processed as described above.\n\nMadin-Darby Canine Kidney (MDCK) cell (ATCC no. CCL-34), at a concentration of 4 × 10 4 cell/well, was seeded in 96-well plate to reach 80% confluency 24 hours prior to transfection. Transfection was performed the same as before. One hundred nM of circular TFO RNA was transfected into MDCK cells. Following 6 hours \n\nORF1a/1b and 530-541\n\nORF1a/1b and 7399-7411\n\nORF1a/1b and 14048-14061", "- * Highlighted in bold indicated the binding region. * * Unrelated circular TFO. [20, 21] , respectively. The reverse transcriptase quantitative real-time PCR (RT-qPCR) was performed using a Bio-Rad CFX96 real-time system (BioRad, USA). The reaction was amplified in a final volume of 25 L using a SensiMix SYBR No-ROX One-Step Kit (Bioline, UK), which consisted of 12.5 L 2X SensiMix SYBR No-Rox One-\n\nStep reaction buffer, 10 M forward and reverse primers, 10 units RiboSafe RNase inhibitor, and 5 L template RNA. Absolute quantification approach was used to quantify qPCR results where a standard curve of a serial dilution of virus was plotted before the quantification. Amount of the virus in the samples was quantified based on this standard curve.", "Analysis. Data statistical analysis was performed using SPSS 18.0. Data were represented as mean ± SE of three independent tests. One-way ANOVA, Tukey post hoc test was used to analyze the significant level among the data. ≤ 0.05 was considered significant. genome, which play important roles in viral replication, were selected as the target binding sites for the triplex formation. The target regions were 5 untranslated region (5 UTR), Open Reading Frames (ORFs) 1a and 1b, and 3 untranslated region (3 UTR) ( Table 1 ). The TFOs were designed in duplex, as they can bind with the single stranded target region and reshape into triplex. Both ends of the duplex TFOs were ligated with a linker sequence or clamps (C-C) to construct circular TFO RNA.\n\nDenaturing PAGE assay was carried out after the ligation process to determine the formation of the circular TFO. As shown in Figure 1 , the circular TFO RNAs migrated faster than the linear TFO RNAs, when subjected to 20% denaturing PAGE.", "Target Region. The binding ability was determined using Electrophoretic Mobility Shift Assay (EMSA) [23] . The appearance of the slow mobility band indicates the successful hybridization of circular TFO RNA with its target region. The binding ability of different TFO RNAs (TFO1 to TFO5) against their target regions was determined by EMSA (Figure 2) . TFO1, TFO3, TFO4, and TFO5 showed slow mobility band, while TFO2 showed the lack of an upward shifted band. This indicates the possession of triplex binding ability for all circular TFO RNAs, except TFO2.\n\nTFO RNA. Study on the interaction and hybridization of TFO towards its target region is crucial, since the stronger the binding is, the more stable the triplex structure forms. As shown in supplementary Figure 1 (Table 3) .", "The antiviral effect of circular TFO RNAs was investigated by RT-qPCR assay at 72 hours after transfection. The results showed viral RNA genome copy numbers of 3.65 × 10 9 , 3.22 × 10 14 , 5.04 × 10 9 , 5.01 × 10 9 , 4.41 × 10 9 , and 3.96 × 10 14 in cells treated with TFO1, TFO2, TFO3, TFO4, TFO5, and TFO7, respectively. The data analyzed by one-way ANOVA, Tukey post hoc test showed significant high viral RNA genome copy number of 4.03 × 10 14 for virus inoculated cells as compared to circular TFO1, TFO3, TFO4, and TFO5 treatments ( ≤ 0.05). The viral RNA copies of circular TFO2, linear TFO3 and TFO4, and unrelated circular TFO7 RNAs transfected cells also showed high viral RNA copy numbers which did not show significant differences to the infected cells ( ≥ 0.05) ( Figure 3 ). The morphological changes of the cells were also captured 72 hours after transfection. The cells transfected with circular TFO1, TFO3, TFO4, and TFO5 appeared to be in good condition following virus", "inoculation, while the cells transfected with circular TFO2 and linear TFO3 and TFO4 showed visible cytopathic effect (CPE), the same as virus inoculated cells (supplementary Figure 2) . Furthermore, cells transfected with TFO only remain viable indicating that TFO treatment is generally not toxic to the cells. Hence, these results illustrated the capacity of circular TFO RNAs (except TFO2) to inhibit FIPV replication.", "Concentrations on FIPV Replication. Circular TFO1 was used to examine the dose-response relationship as a representative to other TFOs. The experimental conditions were identical to that of the previous experiment, except for TFO1 concentrations of 25 nM, 50 nM, 100 nM, and 500 nM. There was no significant reduction in viral RNA genome copies using the concentration of 25 nM TFO1. The other concentrations caused significant reductions in copy numbers as compared to the virus-infected cells. However, no significant difference was detected in copy numbers from all of these concentrations ( Figure 4 ).", "The specificity of the TFO towards FIPV was tested, using TFO1 and TFO5, as the proper representatives of TFOs, on influenza A virus H1N1 New Jersey 8/76. The analyzed data using one-way ANOVA, Tukey post hoc test did not show significant reductions in the copies of viral RNA for both TFOs compared to the influenza virus inoculated cells ( ≥ 0.05) (supplementary Figure 3 ). Complex structure G4/Cir4 Figure 2 : EMSA analysis. EMSA analysis illustrated the binding of circular TFO 1, 3, 4, and 5 to the target regions as evidenced by upward band shift. Binding of each circular TFO except circular TFO2 to its respective target forms a complex that migrates slower than unbound TFO. G1 to G5 represent the target region for circular TFO1 to TFO5 and Cir1 to Cir5 represent the circular TFO1 to TFO5, respectively. in the replication process [24] . Meanwhile, the ORF1a/1b of FIPV are translated into polyproteins that are cleaved into nonstructural proteins which assemble into", "replicationtranscription complexes together with other viral proteins [24] . Hence, the development of molecular therapy targeting these critical regions may provide the possibility to inhibit FIPV replication.", "Development of antiviral therapies against FIPV using siRNA [25] and viral protease inhibitors [26] Figure 4 : TFO1 dose-response study for inhibiting FIPV replication. The concentrations of 50 nM and higher showed significant antiviral effects. 50 nM of circular TFO1 RNA was able to reduce viral copy number by 5-fold log 10 from 10 14 to 10 9 , while 100 and 500 nM showed 4-fold reduction. Data are averages of 3 independent tests (mean ± SE). * Significantly different from FIPV-infected group.", "as potential new treatments against FIPV infection. In this study, circular Triple Helix Forming Oligonucleotide (TFO) RNAs, specifically targeting the short regions of viral genome for triplex formation, were designed and evaluated. TFO1 and TFO2 targeted the 5 and 3 UTRs of the viral genome, respectively. TFO3 to TFO5 targeted different regions of the ORF1a/1b on FIPV genome. Prior to in vitro antiviral study, the ligated circular TFOs were evaluated using PAGE analysis. All of the circularised TFO showed faster migration pattern compared to the linear TFO; however, only slight variation was detected for some of the TFO (Figure 1 ). The reason for this is not clear but probably due to the differences in length and the tertiary structures of the TFOs leading to differences in the migration rate. EMSA was used to show the binding capability of each circular TFO towards the target region in the FIPV genome except for TFO2 which showed lack of formation of complex structure upon", "hybridization ( Figure 2) . The EMSA result also concurred with the antiviral study, where all circular TFOs (except TFO2) were able to demonstrate a significant reduction in the viral RNA genome copy numbers by 5-fold log 10 from 10 14 in virus inoculated cells to 10 9 in TFO-transfected cells (Figure 3 ). However, no antiviral properties were detected from the linear TFOs and unrelated circular TFO7 RNA, confirming that the antiviral activity is associated with specific binding of circular TFOs towards targeted regions.", "Furthermore, the binding of the circular TFO to the target region was confirmed by nanoITC analysis; where the low value and high stability allowed TFOs to compete effectively with the target regions for inhibiting transcription in cell-free systems. Since, TFO1 shows the lowest value (Table 3) , the antiviral properties of this TFO were evaluated in doseresponse study. As shown in Figure 4 , 50 and 100 nM of TFO1 showed similar antiviral effects indicating the potential therapeutic application of TFO1 on FIPV replication. However, increasing the concentration of TFO1 to 500 nm failed to reduce the viral load further probably due to inefficiency of the transfection reagent to transfect the TFO into the cells. In addition, the virus has fast replication rate upon in vitro infection, where previous study on the growth of FIPV in CRFK cells showed that by 2 hours approximately 67% of FIPV 79-1146 were internalized by CRFK cells by endocytosis increasing to more than 70% at 3 hours [27,", "28] . The above finding probably also explained the reason why no antiviral effect was detected when the transfection of the TFO was performed on virus-infected cells (data not shown).", "The antiviral properties, as demonstrated by the circular TFOs, were probably associated with the binding of the TFO to the target region, based on both the Watson-Crick and Hoogsteen hydrogen bonds, which enhance the stability in terms of enthalpy, which is brought about by joining together two out of three strands of the triple helix in the proper orientation [29] . Therefore, the triplex formation is tightly bonded and not easy to detach. Furthermore, the circular TFOs were designed in such way that the presence of hydrogen bonding donors and acceptors in the purines is able to form two hydrogen bonds, while the pyrimidine bases can only form one additional hydrogen bond with incoming third bases [30] . However, there are various factors that may limit the activity of TFOs in cells like intracellular degradation of the TFO and limited accessibility of the TFO to the target sites which can prevent triplex formation [31] . These findings may also explain the inability of the designed", "TFO1 to inhibit further virus replication in dose-response study (Figure 4) .", "Various molecular-based therapies against infectious diseases and cancer have been developed and tested. However, only the siRNA-based therapy has been studied extensively as a novel antiviral and anticancer therapy [32, 33] . Recently, McDonagh et al. [25] developed siRNA with antiviral activity against the FIPV 79-1146, where the designed siRNA was able to reduce the copy number of viral genome compared with virus-infected cells. The potential therapeutic application of TFOs, such as linear TFO conjugated with psoralen to inhibit the transcription of human immunodeficiency provirus [13] and TFO to inhibit the transcription of 1(I) collagen in rat fibroblasts [14] , has also been reported. In addition, short TFO conjugated with daunomycin targeting the promoter region of oncogene has been designed and evaluated on human cancer cells [31] . These studies indicated the flexibility of using TFO-based oligonucleotides as a potential molecular-based therapy. In this study, we demonstrated", "short circular TFO RNAs between 28 and 34 mers (Table 1) , which are able to inhibit FIPV replication by binding to specific target regions of the FIPV genome. All designed circular TFOs (except TFO2) showed significant inhibitory effects against FIPV replication. The TFOs that formed triplex structures showed antiviral effects towards FIPV replication. The reason why TFO2 failed to show any interaction with the target region or antiviral activity is probably due to the length of TFO2 (i.e., 24 mers), which might be insufficient to a triplex formation upon hybridization (Figure 2 ), be effective enough to suppress viral RNA transcription, and eventually inhibit virus replication. Nevertheless, the inability of TFO2 to show antiviral effect due to failure in the formation of functional tertiary structure of the triplex formation cannot be ruled out. In vitro antiviral study which showed no antiviral property for unrelated TFO (TFO7) and also inability of circular TFO1 and TFO5 to", "inhibit influenza A virus H1N1 infected cells confirms the specificity of the TFOs' activity.", "In conclusion, the circular TFO RNA has the potential to be developed as a therapy against FIPV in cats. However, further studies on TFO specificity, actual mechanism of circular TFO RNA in the transcription alteration consequence of inhibiting the viral transcription process, and in vivo animal studies are important for this approach to work as a therapy in the future." ]

[ 9 ]

[ "nano Isothermal Titration Calorimeter (ITC)" ]

[ "In Vitro Antiviral Activity of Circular Triple Helix Forming Oligonucleotide RNA towards Feline Infectious Peritonitis Virus Replication\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3950953/\n\nSHA: f5ad2323eb387f6e271e2842bb2cc4a33504fde3\n\nAuthors: Choong, Oi Kuan; Mehrbod, Parvaneh; Tejo, Bimo Ario; Omar, Abdul Rahman\nDate: 2014-02-20\nDOI: 10.1155/2014/654712\nLicense: cc-by", "Abstract: Feline Infectious Peritonitis (FIP) is a severe fatal immune-augmented disease in cat population. It is caused by FIP virus (FIPV), a virulent mutant strain of Feline Enteric Coronavirus (FECV). Current treatments and prophylactics are not effective. The in vitro antiviral properties of five circular Triple-Helix Forming Oligonucleotide (TFO) RNAs (TFO1 to TFO5), which target the different regions of virulent feline coronavirus (FCoV) strain FIPV WSU 79-1146 genome, were tested in FIPV-infected Crandell-Rees Feline Kidney (CRFK) cells. RT-qPCR results showed that the circular TFO RNAs, except TFO2, inhibit FIPV replication, where the viral genome copy numbers decreased significantly by 5-fold log(10) from 10(14) in the virus-inoculated cells to 10(9) in the circular TFO RNAs-transfected cells. Furthermore, the binding of the circular TFO RNA with the targeted viral genome segment was also confirmed using electrophoretic mobility shift assay. The strength of binding kinetics", "between the TFO RNAs and their target regions was demonstrated by NanoITC assay. In conclusion, the circular TFOs have the potential to be further developed as antiviral agents against FIPV infection.", "Text: Feline Infectious Peritonitis Virus (FIPV) is an enveloped virus with a nonsegmented, positive sense, single-stranded RNA genome. FIPV is grouped as feline coronavirus (FCoV), under the family Coronaviridae. FCoV is divided into two biotypes, namely, Feline Enteric Coronavirus (FECV), a ubiquitous enteric biotype of FCoV, and FIPV, a virulent biotype of FCoV [1] . The relationship between these two biotypes still remains unclear. Two hypotheses have been proposed, (i) internal mutation theory and (ii) circulating high virulent-low virulent theory. Internal mutation theory stated that the development of FIP is due to the exposure of cat to variants of FCoV which have been mutated by gaining the ability to replicate within the macrophages [2] , while the circulating high virulent-low virulent theory explains the existence of both distinctive pathogenic and benign lineages of viruses within the cat population [3] .", "Study has shown that about 40-80% of cats are detected with FECV shedding in their faeces [4] . About 12% of these FECV-positive cats have developed immune-mediated fatal FIP disease [4] . The prevalence of FIP among felines is due to continual cycles of infection and reinfection of FECV and indiscernible clinical symptoms of infected cats with FECV at an early stage before the progressive development of FIPV.", "Vaccination against FIPV with an attenuated, temperature-sensitive strain of type II FIPV induces low antibody titre in kittens that have not been exposed to FCoV. However, there is considerable controversy on the safety and efficacy of this vaccine, since the vaccine contains type 2 strain, whereas type 1 viruses are more prevalent in the field [4] . In addition, antibodies against FIPV do not protect infected cats but enhance the infection of monocytes and macrophages via a mechanism known as Antibody-Dependent Enhancement [1] . Besides vaccines, several antiviral drugs such as ribavirin, 2 BioMed Research International interferons, and immunosuppressive drugs have been used as treatments for FIPV-infected cats, mainly to suppress the inflammatory and detrimental immune response [5] [6] [7] [8] . However, those treatments were ineffective. Hence, there is still significant unmet medical need to develop effective treatments and prophylactics for FIPV infection.", "Triple Helix Forming Oligonucleotide (TFO) is defined as homopyrimidine oligonucleotides, which can form a sequence-specific triple helix by Hoogsteen bonds to the major groove of a complementary homopyrimidinehomopurine stretch in duplex DNA [9] . Furthermore, double helical RNA or DNA-RNA hybrids can be targeted as a template for triple helix formation, once the strand composition on the stabilities of triple helical complexes is determined [10] . Hence, TFO has been used to impede gene expressions by transcription inhibition of viral genes or oncogenes [11] [12] [13] [14] [15] [16] . The main purpose of this study is to develop and evaluate the in vitro antiviral properties of circular TFO RNAs against FIPV replication.", "serotype II strain WSU 79-1146 (ATCC no. VR-1777) was grown in CRFK cells. A serial 10-fold dilution of FIPV was prepared from the working stock. Confluent 96-well plate was inoculated with 100 L of each virus dilution/well. The plate was incubated in a humidified incubator at 37 ∘ C, 5% CO 2 . Cytopathic effects (CPE) development was observed. The results were recorded after 72 hours and the virus tissue culture infective dose 50 (TCID 50 ) was calculated using Reed and Muench's method [17] .", "Oligonucleotide RNA. The Triple Helix Forming Oligonucleotides (TFOs) were designed based on the genome sequence of FIPV serotype II strain WSU 79-1146 (Accession no: AY994055) [18] . TFOs, which specifically target the different regions of the FIPV genome, and one unrelated TFO were constructed ( Table 1 ). The specificity of the TFOs was identified using BLAST search in the NCBI database. The designed linear TFOs were synthesized by Dharmacon Research (USA), whereby the 5 and 3 ends of the linear TFOs were modified with phosphate (PO 4 ) group and hydroxide (OH) group, respectively. These modifications were necessary for the circularization of linear TFO. The process of circularization, using the T4 RNA ligase 1 (ssRNA ligase) (New England Biolabs Inc., England), was carried out according to the manufacturer's protocol. After ligation, the circular TFO RNAs were recovered by ethanol precipitation and the purity of the circular TFO RNAs was measured using spectrophotometer.", "Denaturing of urea polyacrylamide gel electrophoresis was performed as described before [19] with modification. Briefly, 20% of denatured urea polyacrylamide gel was prepared and polymerized for 30 minutes. Then, the gel was prerun at 20 to 40 V for 45 minutes. Five L of TFO RNA mixed with 5 L of urea loading buffer was heated at 92 ∘ C for 2 minutes and immediately chilled on ice. It was run on the gel at 200 V for 45 minutes. Finally, the gel was stained with ethidium bromide (Sigma, USA) and viewed with a Bio-Rad Gel Doc XR system (CA, USA). (EMSA) . The target regions of the FIPV genome were synthesized by Dharmacon Research (USA) ( Table 1) . Each TFO RNA was mixed with the target region in 1X binding buffer containing 25 mM Tris-HCl, 6 mM MgCl 2 , and 10 mMNaCl in a final volume of 10 L and subsequently incubated at 37 ∘ C for 2 hours. The sample was run on 15% native polyacrylamide gel at 80 V, in cool condition. The stained gel was viewed by a Bio-Rad Gel Doc XR system.", "Regions. The binding strength was measured using a nano Isothermal Titration Calorimeter (ITC) (TA instruments, Newcastle, UK). The RNA sample mixtures, consisting of circular TFOs (0.0002 mM), were incubated with their respective synthetic target regions (0.015 mM) using 1X binding buffer as the diluent. The experiment was run at 37 ∘ C with 2 L/injection, for a total of 25 injections. Data was collected every 250 seconds and analyzed using the NanoAnalyze software v2.3.6 provided by the manufacturer.", "This experiment was conducted in CRFK cells, where 3 × 10 4 cell/well was seeded in 96-well plate to reach 80% confluency 24 hours prior to transfection. One hundred nM of TFO RNAs was separately transfected into the CRFK cells using a HiPerFect Transfection Reagent (Qiagen, Germany), as per the manufacturer's protocol. The plate was incubated at 37 ∘ C with 5% CO 2 for 6 hours. Then, the cultures were infected with 100TCID 50 of FIPV serotype II strain WSU 79-1146 for 1 hour at 37 ∘ C (100 L/well). Finally, the viral inoculum was replaced by fresh maintenance media (MEM containing 1% FBS and 1% pen/strep). Virus-infected and uninfected cells were maintained as positive and negative controls, respectively. The morphology of the cultures was recorded 72 hours after infection and samples were harvested at this time point and stored at −80 ∘ C prior to RNA extraction.", "Inhibition. Different concentrations of circular TFO1 RNA (25 nM, 50 nM, 100 nM, and 500 nM) were transfected into CRFK cells. The plate was incubated for 6 hours followed by virus inoculation for 1 hour at 37 ∘ C with 5% CO2. The cells were processed as described above.\n\nMadin-Darby Canine Kidney (MDCK) cell (ATCC no. CCL-34), at a concentration of 4 × 10 4 cell/well, was seeded in 96-well plate to reach 80% confluency 24 hours prior to transfection. Transfection was performed the same as before. One hundred nM of circular TFO RNA was transfected into MDCK cells. Following 6 hours \n\nORF1a/1b and 530-541\n\nORF1a/1b and 7399-7411\n\nORF1a/1b and 14048-14061", "- * Highlighted in bold indicated the binding region. * * Unrelated circular TFO. [20, 21] , respectively. The reverse transcriptase quantitative real-time PCR (RT-qPCR) was performed using a Bio-Rad CFX96 real-time system (BioRad, USA). The reaction was amplified in a final volume of 25 L using a SensiMix SYBR No-ROX One-Step Kit (Bioline, UK), which consisted of 12.5 L 2X SensiMix SYBR No-Rox One-\n\nStep reaction buffer, 10 M forward and reverse primers, 10 units RiboSafe RNase inhibitor, and 5 L template RNA. Absolute quantification approach was used to quantify qPCR results where a standard curve of a serial dilution of virus was plotted before the quantification. Amount of the virus in the samples was quantified based on this standard curve.", "Analysis. Data statistical analysis was performed using SPSS 18.0. Data were represented as mean ± SE of three independent tests. One-way ANOVA, Tukey post hoc test was used to analyze the significant level among the data. ≤ 0.05 was considered significant. genome, which play important roles in viral replication, were selected as the target binding sites for the triplex formation. The target regions were 5 untranslated region (5 UTR), Open Reading Frames (ORFs) 1a and 1b, and 3 untranslated region (3 UTR) ( Table 1 ). The TFOs were designed in duplex, as they can bind with the single stranded target region and reshape into triplex. Both ends of the duplex TFOs were ligated with a linker sequence or clamps (C-C) to construct circular TFO RNA.\n\nDenaturing PAGE assay was carried out after the ligation process to determine the formation of the circular TFO. As shown in Figure 1 , the circular TFO RNAs migrated faster than the linear TFO RNAs, when subjected to 20% denaturing PAGE.", "Target Region. The binding ability was determined using Electrophoretic Mobility Shift Assay (EMSA) [23] . The appearance of the slow mobility band indicates the successful hybridization of circular TFO RNA with its target region. The binding ability of different TFO RNAs (TFO1 to TFO5) against their target regions was determined by EMSA (Figure 2) . TFO1, TFO3, TFO4, and TFO5 showed slow mobility band, while TFO2 showed the lack of an upward shifted band. This indicates the possession of triplex binding ability for all circular TFO RNAs, except TFO2.\n\nTFO RNA. Study on the interaction and hybridization of TFO towards its target region is crucial, since the stronger the binding is, the more stable the triplex structure forms. As shown in supplementary Figure 1 (Table 3) .", "The antiviral effect of circular TFO RNAs was investigated by RT-qPCR assay at 72 hours after transfection. The results showed viral RNA genome copy numbers of 3.65 × 10 9 , 3.22 × 10 14 , 5.04 × 10 9 , 5.01 × 10 9 , 4.41 × 10 9 , and 3.96 × 10 14 in cells treated with TFO1, TFO2, TFO3, TFO4, TFO5, and TFO7, respectively. The data analyzed by one-way ANOVA, Tukey post hoc test showed significant high viral RNA genome copy number of 4.03 × 10 14 for virus inoculated cells as compared to circular TFO1, TFO3, TFO4, and TFO5 treatments ( ≤ 0.05). The viral RNA copies of circular TFO2, linear TFO3 and TFO4, and unrelated circular TFO7 RNAs transfected cells also showed high viral RNA copy numbers which did not show significant differences to the infected cells ( ≥ 0.05) ( Figure 3 ). The morphological changes of the cells were also captured 72 hours after transfection. The cells transfected with circular TFO1, TFO3, TFO4, and TFO5 appeared to be in good condition following virus", "inoculation, while the cells transfected with circular TFO2 and linear TFO3 and TFO4 showed visible cytopathic effect (CPE), the same as virus inoculated cells (supplementary Figure 2) . Furthermore, cells transfected with TFO only remain viable indicating that TFO treatment is generally not toxic to the cells. Hence, these results illustrated the capacity of circular TFO RNAs (except TFO2) to inhibit FIPV replication.", "Concentrations on FIPV Replication. Circular TFO1 was used to examine the dose-response relationship as a representative to other TFOs. The experimental conditions were identical to that of the previous experiment, except for TFO1 concentrations of 25 nM, 50 nM, 100 nM, and 500 nM. There was no significant reduction in viral RNA genome copies using the concentration of 25 nM TFO1. The other concentrations caused significant reductions in copy numbers as compared to the virus-infected cells. However, no significant difference was detected in copy numbers from all of these concentrations ( Figure 4 ).", "The specificity of the TFO towards FIPV was tested, using TFO1 and TFO5, as the proper representatives of TFOs, on influenza A virus H1N1 New Jersey 8/76. The analyzed data using one-way ANOVA, Tukey post hoc test did not show significant reductions in the copies of viral RNA for both TFOs compared to the influenza virus inoculated cells ( ≥ 0.05) (supplementary Figure 3 ). Complex structure G4/Cir4 Figure 2 : EMSA analysis. EMSA analysis illustrated the binding of circular TFO 1, 3, 4, and 5 to the target regions as evidenced by upward band shift. Binding of each circular TFO except circular TFO2 to its respective target forms a complex that migrates slower than unbound TFO. G1 to G5 represent the target region for circular TFO1 to TFO5 and Cir1 to Cir5 represent the circular TFO1 to TFO5, respectively. in the replication process [24] . Meanwhile, the ORF1a/1b of FIPV are translated into polyproteins that are cleaved into nonstructural proteins which assemble into", "replicationtranscription complexes together with other viral proteins [24] . Hence, the development of molecular therapy targeting these critical regions may provide the possibility to inhibit FIPV replication.", "Development of antiviral therapies against FIPV using siRNA [25] and viral protease inhibitors [26] Figure 4 : TFO1 dose-response study for inhibiting FIPV replication. The concentrations of 50 nM and higher showed significant antiviral effects. 50 nM of circular TFO1 RNA was able to reduce viral copy number by 5-fold log 10 from 10 14 to 10 9 , while 100 and 500 nM showed 4-fold reduction. Data are averages of 3 independent tests (mean ± SE). * Significantly different from FIPV-infected group.", "as potential new treatments against FIPV infection. In this study, circular Triple Helix Forming Oligonucleotide (TFO) RNAs, specifically targeting the short regions of viral genome for triplex formation, were designed and evaluated. TFO1 and TFO2 targeted the 5 and 3 UTRs of the viral genome, respectively. TFO3 to TFO5 targeted different regions of the ORF1a/1b on FIPV genome. Prior to in vitro antiviral study, the ligated circular TFOs were evaluated using PAGE analysis. All of the circularised TFO showed faster migration pattern compared to the linear TFO; however, only slight variation was detected for some of the TFO (Figure 1 ). The reason for this is not clear but probably due to the differences in length and the tertiary structures of the TFOs leading to differences in the migration rate. EMSA was used to show the binding capability of each circular TFO towards the target region in the FIPV genome except for TFO2 which showed lack of formation of complex structure upon", "hybridization ( Figure 2) . The EMSA result also concurred with the antiviral study, where all circular TFOs (except TFO2) were able to demonstrate a significant reduction in the viral RNA genome copy numbers by 5-fold log 10 from 10 14 in virus inoculated cells to 10 9 in TFO-transfected cells (Figure 3 ). However, no antiviral properties were detected from the linear TFOs and unrelated circular TFO7 RNA, confirming that the antiviral activity is associated with specific binding of circular TFOs towards targeted regions.", "Furthermore, the binding of the circular TFO to the target region was confirmed by nanoITC analysis; where the low value and high stability allowed TFOs to compete effectively with the target regions for inhibiting transcription in cell-free systems. Since, TFO1 shows the lowest value (Table 3) , the antiviral properties of this TFO were evaluated in doseresponse study. As shown in Figure 4 , 50 and 100 nM of TFO1 showed similar antiviral effects indicating the potential therapeutic application of TFO1 on FIPV replication. However, increasing the concentration of TFO1 to 500 nm failed to reduce the viral load further probably due to inefficiency of the transfection reagent to transfect the TFO into the cells. In addition, the virus has fast replication rate upon in vitro infection, where previous study on the growth of FIPV in CRFK cells showed that by 2 hours approximately 67% of FIPV 79-1146 were internalized by CRFK cells by endocytosis increasing to more than 70% at 3 hours [27,", "28] . The above finding probably also explained the reason why no antiviral effect was detected when the transfection of the TFO was performed on virus-infected cells (data not shown).", "The antiviral properties, as demonstrated by the circular TFOs, were probably associated with the binding of the TFO to the target region, based on both the Watson-Crick and Hoogsteen hydrogen bonds, which enhance the stability in terms of enthalpy, which is brought about by joining together two out of three strands of the triple helix in the proper orientation [29] . Therefore, the triplex formation is tightly bonded and not easy to detach. Furthermore, the circular TFOs were designed in such way that the presence of hydrogen bonding donors and acceptors in the purines is able to form two hydrogen bonds, while the pyrimidine bases can only form one additional hydrogen bond with incoming third bases [30] . However, there are various factors that may limit the activity of TFOs in cells like intracellular degradation of the TFO and limited accessibility of the TFO to the target sites which can prevent triplex formation [31] . These findings may also explain the inability of the designed", "TFO1 to inhibit further virus replication in dose-response study (Figure 4) .", "Various molecular-based therapies against infectious diseases and cancer have been developed and tested. However, only the siRNA-based therapy has been studied extensively as a novel antiviral and anticancer therapy [32, 33] . Recently, McDonagh et al. [25] developed siRNA with antiviral activity against the FIPV 79-1146, where the designed siRNA was able to reduce the copy number of viral genome compared with virus-infected cells. The potential therapeutic application of TFOs, such as linear TFO conjugated with psoralen to inhibit the transcription of human immunodeficiency provirus [13] and TFO to inhibit the transcription of 1(I) collagen in rat fibroblasts [14] , has also been reported. In addition, short TFO conjugated with daunomycin targeting the promoter region of oncogene has been designed and evaluated on human cancer cells [31] . These studies indicated the flexibility of using TFO-based oligonucleotides as a potential molecular-based therapy. In this study, we demonstrated", "short circular TFO RNAs between 28 and 34 mers (Table 1) , which are able to inhibit FIPV replication by binding to specific target regions of the FIPV genome. All designed circular TFOs (except TFO2) showed significant inhibitory effects against FIPV replication. The TFOs that formed triplex structures showed antiviral effects towards FIPV replication. The reason why TFO2 failed to show any interaction with the target region or antiviral activity is probably due to the length of TFO2 (i.e., 24 mers), which might be insufficient to a triplex formation upon hybridization (Figure 2 ), be effective enough to suppress viral RNA transcription, and eventually inhibit virus replication. Nevertheless, the inability of TFO2 to show antiviral effect due to failure in the formation of functional tertiary structure of the triplex formation cannot be ruled out. In vitro antiviral study which showed no antiviral property for unrelated TFO (TFO7) and also inability of circular TFO1 and TFO5 to", "inhibit influenza A virus H1N1 infected cells confirms the specificity of the TFOs' activity.", "In conclusion, the circular TFO RNA has the potential to be developed as a therapy against FIPV in cats. However, further studies on TFO specificity, actual mechanism of circular TFO RNA in the transcription alteration consequence of inhibiting the viral transcription process, and in vivo animal studies are important for this approach to work as a therapy in the future." ]

[ 10 ]

[ "the anti-influenza A (H2N2) virus activity of patchouli alcohol" ]

[ "Inhibitory Effect and Possible Mechanism of Action of Patchouli Alcohol against Influenza A (H2N2) Virus\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6264369/\n\nSHA: f2d842780b9928cc70f38a4458553f2431877603\n\nAuthors: Wu, Huaxing; Li, Beili; Wang, Xue; Jin, Mingyuan; Wang, Guonian\nDate: 2011-08-03\nDOI: 10.3390/molecules16086489\nLicense: cc-by", "Abstract: In the present study, the anti-influenza A (H2N2) virus activity of patchouli alcohol was studied in vitro, in vivo and in silico. The CC(50) of patchouli alcohol was above 20 µM. Patchouli alcohol could inhibit influenza virus with an IC(50) of 4.03 ± 0.23 µM. MTT assay showed that the inhibition by patchouli alcohol appears strongly after penetration of the virus into the cell. In the influenza mouse model, patchouli alcohol showed obvious protection against the viral infection at a dose of 5 mg/kg/day. Flexible docking and molecular dynamic simulations indicated that patchouli alcohol was bound to the neuraminidase protein of influenza virus, with an interaction energy of –40.38 kcal mol(–1). The invariant key active-site residues Asp151, Arg152, Glu119, Glu276 and Tyr406 played important roles during the binding process. Based on spatial and energetic criteria, patchouli alcohol interfered with the NA functions. Results presented here suggest that patchouli alcohol", "possesses anti-influenza A (H2N2) virus properties, and therefore is a potential source of anti-influenza agents for the pharmaceutical industry.", "Text: The influenza virus, which is one of the main causes of acute respiratory infections in humans, can lead to annual epidemics and infrequent pandemics. The two influenza pandemics of the 20 th century, \"Asian Influenza (1957/H2N2)\" and \"Hong Kong Influenza (1968/H3N2)\" resulted in the deaths of an estimated 2-3 million people globally [1, 2] . Today, their descendants continue to cause the majority of influenza infections in humans [3] . So far as it is learned that the most effective antiviral drug is the neuraminidase (NA) inhibitor, which target the NA glycoproteins of influenza A and B virus [4, 5] .", "The release of new virions from the infected cell is a key step in the influenza life cycle and need neuraminidase (NA) to cleave the α-ketosidic linkage between terminal sialic acid and an adjacent sugar residue [6] . The NA inhibitors were designed to prevent the key step by blocking the active site of enzyme and thus allow sufficient time for the host immune systems to remove infected viruses [7] . Consistent efforts have been devoted to the development of NA inhibitors, using the crystal structure of the N2 sub-type NA protein [8] [9] [10] [11] [12] [13] [14] [15] . Indeed, oseltamivir (Tamiflu) is the representative NA inhibitor that has proven to be uniquely applicable oral drug in clinical practice for the treatment of influenza infection [4, 8, 9] . However, with an increase in medical use, the oseltamivir-resistant strains have been found and probably lead to a large scale outbreak of novel pandemic flu [16, 17] .", "Patchouli alcohol ( Figure 1 ) has been well known for over a century. It is a major constituent of the pungent oil from the East Indian shrub Pogostemon cablin (Blanco) Benth, and widely used in fragrances. Patchouli oil is an important essential oil in the perfume industry, used to give a base and lasting character to a fragrance [16, 17] . The essential oil is very appreciated for its characteristic pleasant and long lasting woody, earthy, and camphoraceous odor, as well as for its fixative properties, being suitable for use in soaps and cosmetic products [16, 17] . The aerial part of Pogostemon cablin has wildly been used for the treatment of the common cold and as an antifungal agent in China [16, 17] . Moreover, the plant is widely used in Traditional Chinese Medicine as it presents various types of pharmacological activity according to the composition of the oil [16, 17] . Patchouli alcohol, as the major volatile constituent of patchouli oil, has been found to strongly inhibit", "H1N1 replication and weakly inhibit B/Ibaraki/2/85 replication [18] . To the best of our knowledge, the anti-influenza virus (H2N2) activities of patchouli alcohol have not been evaluated yet. Therefore, the aim of the present study was to evaluate the anti-influenza A virus (H2N2) activity of patchouli alcohol by MTT assay and mouse influenza model. On such basis, explicitly solvated docking and molecular dynamic (MD) methods were applied to investigative the binding mode involving patchouli alcohol with influenza virus NA protein. We anticipate that the insight into the understanding of inhibiting mechanism will be of value in the rational design of novel anti-influenza drugs.", "First the efficacy of patchouli alcohol on influenza A (H2N2) virus replication and cell viability were examined. CC 50 was used to express the cytotoxicity of patchouli alcohol on MDCK. The CC 50 of patchouli alcohol was above 20 mM, which indicated that patchouli alcohol did not affect the growth of MDCK (Table 1) . Thus, it seems that the antiviral effects of patchouli alcohol were not due to the cytotoxicity. Moreover, patchouli alcohol was found to inhibit influenza A (H2N2) virus with an IC 50 of 4.03 ± 0.23 µM. Based on the IC 50 and CC 50 values, the selectivity index (SI) was calculated as >4.96. It is reported that a SI of 4 or more is appropriate for an antiviral agent [18] , suggesting that patchouli alcohol can be judged to have anti-influenza A (H2N2) virus activity.", "Until now, it has been found that patchouli alcohol showed dose-dependent anti-influenza virus (A/PR/8/34, H1N1) activity, with an IC 50 value of 2.635 µM. Furthermore, it showed weak activity against B/Ibaraki/2/85 (IC 50 = 40.82 µM) [19] . With the addition of the above H2N2 inhibitory activity, we have a comprehensively view of the anti-influenza activity of patchouli alcohol.\n\nCells were pretreated with patchouli alcohol prior to virus infection (pretreatment cells), viruses were pretreated prior to infection (pretreatment virus), and patchouli alcohol was added during the adsorption period (adsorption) or after penetration of the viruses into cells (replication). Experiments were repeated independently three times and data presented are the average of three experiments. The symbols * indicated very significant difference p < 0.01 with respect to other mode (pretreatment virus, adsorption and pretreatment cell).", "As shown in Figure 2 , patchouli alcohol showed anti-influenza A (H2N2) virus activity in a timedependent manner. It showed best antiviral activity when added at a concentration of 8 µM during the replication period with inhibition of the viral replication of 97.68% ± 2.09% for influenza A (H2N2) at 72 h. However, no significant effect was detected when patchouli alcohol was used for pretreatment of cells or viruses or when patchouli alcohol was only added during the adsorption phase. These results suggested that the inhibition of influenza A (H2N2) virus by patchouli alcohol appears to occur much more strongly after penetration of the virus into the cell. Besides, biochemical studies have indicated that the bioactivity of NA protein is essential determinant after the replication of influenza A (H2N2) virus [20] [21] [22] . Hence, we conclude that the function of NA protein may be suppressed by patchouli alcohol.", "To evaluate the toxicity of patchouli alcohol, the mean value of body weight of mice in each group was statistically analyzed. The mean weights of mice administered at the 2 mg/kg/dose oseltamivir, 2 mg/kg/dose patchouli alcohol and 10 mg/kg/dose of patchouli alcohol one time daily for 7 days were not significantly different compared with the normal control mice, showing no toxicity of patchouli alcohol and oseltamivir within the testing concentration (P > 0.05). Physiological status was observed in virus infection mice. Three days after viral infection, some mice, especially mice in the H2N2 infected control group showed changes in behavior, such as a tendency to huddle, diminished vitality, and ruffled fur, etc. In the mouse influenza model, viral infection leads to loss of body weight and high mortality. Therefore, the efficacy of patchouli alcohol and oseltamivir were evaluated on the basis of survival rate measured for 15 days post-infection, for treated infected animals relative", "to untreated infected (control) animals. A comparison of efficacy of patchouli alcohol and oseltamivir in vivo mouse influenza model (oral treatment) showed that at a dose of 5 mg/kg/day, patchouli alcohol showed obvious protection against the influenza virus, as the mean day to death was detected as 11.8 ± 1.1 (Table 2) . When the dose was lowered to 1 mg/kg/day, patchouli alcohol showed weaker protection (measured by Survivors/total) than that of 5 mg/kg/day, the mean day to death was 7.5 ± 1.8. Whereas oseltamivir at this dose level (1 mg/kg/day) showed 50% protection (measured by survivors/total) against the influenza virus. In the H2N2 infected control group, there were no survivors. In view of both in vitro and in vivo data, we conclude that patchouli alcohol could be used in the treatment of human influenza virus infections.", "Based on the above experiment data, patchouli alcohol is determined to be bound within NA protein. As the total energies and backbone root-mean-square-deviations (RMSD) in Figure 3 indicate, the energy-minimized patchouli alcohol-NA complex has been in equilibrium since about 0.5 ns, and then retains quite stable in the last 19.5 ns. It is consistent with the previous MD results of other NA inhibitors [23] [24] [25] [26] [27] [28] . Accordingly, the geometric and energetic analyses were made on the average structures of 0.5~20.0 ns MD trajectories, where the system has been already at equilibrium. The interaction energy (E inter ) of patchouli alcohol with NA was calculated at −40.38 kcal mol −1 , where the vdW rather than electrostatic interactions were found to play a dominant role, contribute to about 72% (−29.18 kcal mol −1 ). As shown in Figure 4 , the patchouli alcohol was bound at the active site which also bound to oseltamivir and zanamivir [28] . As Figure 5 shows, the oxygen", "atom of patchouli alcohol was oriented towards the sidechains of residues Glu119 and Tyr406, with one H-bond formed with each residue. The values of distances in Figure 6 further reveal that the docked complex remains rather stable throughout the simulation, with the average distances of Glu119:OE2patchouli alcohol:O and Tyr406:OH -patchouli alcohol:O less than 2.8 Å. The sum contributions (E sum ) of residues Glu119 and Tyr406 amounted to −8.46 and −7.37 kcal mol −1 , respectively (Table 3) . Besides, patchouli alcohol was stabilized by residues Arg118, Asp151, Arg152, Trp178, Ala246, Glu276, Arg292, Asn294 and Gln347, especially residues Asp151, Arg152 and Glu276 ( Figure 5 and Table 3 ). As a matter of fact, residues Asp151, Arg152, Glu119, Glu276 and Tyr406 of the NA protein have already received enough attention from rational drug designs [14, 30, 31] . The catalytic residues Asp151, Arg152 and Glu276 are crucial to the NA functions and the residues Glu119 and Tyr406 are", "important to stabilize the NA active sites [32, 33] . It suggests that the NA functions will be affected by the presence of patchouli alcohol, consistent with the above experiments. Patchouli alcohol matches with the NA active site and has an acceptable interaction energy. Considering the obvious structure discrepancies against current NA inhibitors, it represents an ideal lead compound for the designs of novel anti-influenza agents.", "Patchouli alcohol and oseltamivir were obtained from Sigma Chemical Co. (St. Louis, MO, USA, purity > 99%) and was stored in glass vials with Teflon sealed caps at −20 ± 0.5 °C in the absence of light.\n\nMDCK (Madin-Darby canine kidney) was purchased from Harbin Veterinary Research Institute (Harbin, Heilongjiang, China). The cells were grown in monolayer culture with Eagle's minimum essential medium (EMEM) supplemented with 10% fetal calf serum (FCS), 100 U/mL penicillin and 100 μg/mL streptomycin. The monolayers were removed from their plastic surfaces and serially passaged whenever they became confluent. Cells were plated out onto 96-well culture plates for cytotoxicity and anti-influenza assays, and propagated at 37 °C in an atmosphere of 5% CO 2 .", "The influenza strain A/Leningrad/134/17/1957 H2N2) was purchased from National Control Institute of Veterinary Bioproducts and Pharmaceuticals (Beijing, China). Virus was routinely grown on MDCK cells. The stock cultures were prepared from supernatants of infected cells and stored at −80 °C.", "The cellular toxicity of patchouli alcohol on MDCK cells was assessed by the MTT method. Briefly, cells were seeded on a microtiter plate in the absence or presence of various concentrations (20 µM -0.0098 µM) of patchouli alcohol (eight replicates) and incubated at 37 °C in a humidified atmosphere of 5% CO 2 for 72 h. The supernatants were discarded, washed with PBS twice and MTT reagent (5 mg/mL in PBS) was added to each well. After incubation at 37 °C for 4 h, the supernatants were removed, then 200 μL DMSO was added and incubated at 37 °C for another 30 min. After that the plates were read on an ELISA reader (Thermo Molecular Devices Co., Union City, USA) at 570/630 nm. The mean OD of the cell control wells was assigned a value of 100%. The maximal non-toxic concentration (TD 0 ) and 50% cytotoxic concentration (CC 50 ) were calculated by linear regression analysis of the dose-response curves generated from the data.", "Inhibition of virus replication was measured by the MTT method. Serial dilution of the treated virus was adsorbed to the cells for 1 h at 37 °C. The residual inoculum was discared and infected cells were added with EMEM containing 2% FCS. Each assay was performed in eight replicates. After incubation for 72 h at 37 °C, the cultures were measured by MTT method as described above. The concentration of patchouli alcohol and oseltamivir which inhibited virus numbers by 50% (IC 50 ) was determined from dose-response curves.", "Cells and viruses were incubated with patchouli alcohol at different stages during the viral infection cycle in order to determine the mode of antiviral action. Cells were pretreated with patchouli alcohol before viral infection, viruses were incubated with patchouli alcohol before infection and cells and viruses were incubated together with patchouli alcohol during adsorption or after penetration of the virus into the host cells. Patchouli alcohol was always used at the nontoxic concentration. Cell monolayers were pretreated with patchouli alcohol prior to inoculation with virus by adding patchouli alcohol to the culture medium and incubation for 1 h at 37 °C. The compound was aspirated and cells were washed immediately before the influenza A (H2N2) inoculum was added. For pretreatment virus, Influenza A (H2N2) was incubated in medium containing patchouli alcohol for 1h at room temperature prior to infection of MDCK cells. For analyzing the anti-influenza A (H2N2) inhibition during", "the adsorption period, the same amount of influenza A (H2N2) was mixed with the drug and added to the cells immediately. After 1 h of adsorption at 37 °C, the inoculum was removed and DMEM supplemented with 2 % FCS were added to the cells. The effect of patchouli alcohol against influenza A (H2N2) was also tested during the replication period by adding it after adsorption, as typical performed in anti-influenza A (H2N2) susceptibility studies. Each assay was run in eight replicates.", "Kunming mice, weighing 18-22 g (6 weeks of age) were purchased from Harbin Veterinary Research Institute Animal Co., Ltd. (Harbin, Heilongjiang, China) . First, the toxicity of patchouli alcohol and oseltamivir was assessed in the healthy mice by the loss of body weight compared with the control group (2% DMSO in physiological saline). The mice were orally administered with 10 mg/kg/dose patchouli alcohol, 2 mg/kg/dose patchouli alcohol or 2 mg/kg/dose oseltamivir (dissolved in 2% DMSO in physiological saline) one time daily for 7 days. The weight of mice was determined daily. We conducted procedures according to Principle of Laboratory Animal Care (NIH Publication No. 85 -23, revised 1985) and the guidelines of the Peking University Animal Research Committee.", "Kunming mice were anesthetized with isoflurane and exposed to virus (A/Leningrad/134/17/1957) by intranasal instillation. Drugs were prepared in 2% DMSO in physiological saline and administered 4 h prior to virus exposure and continued daily for 5 days. All mice were observed daily for changes in weight and for any deaths. Parameters for evaluation of antiviral activity included weight loss, reduction in mortality and/or increase in mean day to death (MDD) determined through 15 days.", "The N2 sub-type neuraminidase crystal structure (PDB code 1IVD) was obtained from the RCSB Protein Data Bank [34] . For convenience, the structure is named as NA hereafter. Geometry and partial atomic charges of the patchouli alcohol ( Figure 1) were calculated with the Discover 3.0 module (Insight II 2005) [35] by applying the BFGS algorithm [36] and the consistent-valence force-field (CVFF), with a convergence criterion of 0.01 kcal mol −1 Å −1 . The docking and molecular dynamics (MD) simulations were performed by the general protocols in the Insight II 2005 software packages, consistent with the previous literatures [24, 26, 28, 35, [37] [38] [39] . During the MD simulations, the canonical ensemble (NVT) was employed at normal temperature (300 K). The MD temperature was controlled by the velocity scaling thermostat [40] . Integrations of the classical equations of motion were achieved using the Verlet algorithm. The systems were solvated in a large sphere of TIP3P water molecules", "[40] with the radius of 35.0 Å, which is enough to hold the ensembles [40] . The MD trajectories were generated using a 1.0-fs time step for a total of 20.0 ns, saved at 5.0-ps intervals. The interaction energies of patchouli alcohol with NA and the respective residues at the NA active site were calculated by the Docking module [35], over the 0.5~20.0 ns MD trajectories.", "All results are expressed as mean values ± standard deviations (SDs) (n = 3). The significance of difference was calculated by one-way analysis of variance, and values p < 0.001 were considered to be significant.\n\nIn conclusion, patchouli alcohol possesses anti-influenza A (H2N2) virus activity via interference with the NA function that cleaves the α-glycosidic bond between sialic acid and glycoconjugate. Our results provide the promising information for the potential use of patchouli alcohol in the treatment of influenza A (H2N2) virus infectious disease. Further mechanistic studies on the anti-influenza A virus activity are needed to support this point of view." ]

[ 1 ]

[ "NA glycoproteins of influenza A and B virus" ]

[ "Inhibitory Effect and Possible Mechanism of Action of Patchouli Alcohol against Influenza A (H2N2) Virus\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6264369/\n\nSHA: f2d842780b9928cc70f38a4458553f2431877603\n\nAuthors: Wu, Huaxing; Li, Beili; Wang, Xue; Jin, Mingyuan; Wang, Guonian\nDate: 2011-08-03\nDOI: 10.3390/molecules16086489\nLicense: cc-by", "Abstract: In the present study, the anti-influenza A (H2N2) virus activity of patchouli alcohol was studied in vitro, in vivo and in silico. The CC(50) of patchouli alcohol was above 20 µM. Patchouli alcohol could inhibit influenza virus with an IC(50) of 4.03 ± 0.23 µM. MTT assay showed that the inhibition by patchouli alcohol appears strongly after penetration of the virus into the cell. In the influenza mouse model, patchouli alcohol showed obvious protection against the viral infection at a dose of 5 mg/kg/day. Flexible docking and molecular dynamic simulations indicated that patchouli alcohol was bound to the neuraminidase protein of influenza virus, with an interaction energy of –40.38 kcal mol(–1). The invariant key active-site residues Asp151, Arg152, Glu119, Glu276 and Tyr406 played important roles during the binding process. Based on spatial and energetic criteria, patchouli alcohol interfered with the NA functions. Results presented here suggest that patchouli alcohol", "possesses anti-influenza A (H2N2) virus properties, and therefore is a potential source of anti-influenza agents for the pharmaceutical industry.", "Text: The influenza virus, which is one of the main causes of acute respiratory infections in humans, can lead to annual epidemics and infrequent pandemics. The two influenza pandemics of the 20 th century, \"Asian Influenza (1957/H2N2)\" and \"Hong Kong Influenza (1968/H3N2)\" resulted in the deaths of an estimated 2-3 million people globally [1, 2] . Today, their descendants continue to cause the majority of influenza infections in humans [3] . So far as it is learned that the most effective antiviral drug is the neuraminidase (NA) inhibitor, which target the NA glycoproteins of influenza A and B virus [4, 5] .", "The release of new virions from the infected cell is a key step in the influenza life cycle and need neuraminidase (NA) to cleave the α-ketosidic linkage between terminal sialic acid and an adjacent sugar residue [6] . The NA inhibitors were designed to prevent the key step by blocking the active site of enzyme and thus allow sufficient time for the host immune systems to remove infected viruses [7] . Consistent efforts have been devoted to the development of NA inhibitors, using the crystal structure of the N2 sub-type NA protein [8] [9] [10] [11] [12] [13] [14] [15] . Indeed, oseltamivir (Tamiflu) is the representative NA inhibitor that has proven to be uniquely applicable oral drug in clinical practice for the treatment of influenza infection [4, 8, 9] . However, with an increase in medical use, the oseltamivir-resistant strains have been found and probably lead to a large scale outbreak of novel pandemic flu [16, 17] .", "Patchouli alcohol ( Figure 1 ) has been well known for over a century. It is a major constituent of the pungent oil from the East Indian shrub Pogostemon cablin (Blanco) Benth, and widely used in fragrances. Patchouli oil is an important essential oil in the perfume industry, used to give a base and lasting character to a fragrance [16, 17] . The essential oil is very appreciated for its characteristic pleasant and long lasting woody, earthy, and camphoraceous odor, as well as for its fixative properties, being suitable for use in soaps and cosmetic products [16, 17] . The aerial part of Pogostemon cablin has wildly been used for the treatment of the common cold and as an antifungal agent in China [16, 17] . Moreover, the plant is widely used in Traditional Chinese Medicine as it presents various types of pharmacological activity according to the composition of the oil [16, 17] . Patchouli alcohol, as the major volatile constituent of patchouli oil, has been found to strongly inhibit", "H1N1 replication and weakly inhibit B/Ibaraki/2/85 replication [18] . To the best of our knowledge, the anti-influenza virus (H2N2) activities of patchouli alcohol have not been evaluated yet. Therefore, the aim of the present study was to evaluate the anti-influenza A virus (H2N2) activity of patchouli alcohol by MTT assay and mouse influenza model. On such basis, explicitly solvated docking and molecular dynamic (MD) methods were applied to investigative the binding mode involving patchouli alcohol with influenza virus NA protein. We anticipate that the insight into the understanding of inhibiting mechanism will be of value in the rational design of novel anti-influenza drugs.", "First the efficacy of patchouli alcohol on influenza A (H2N2) virus replication and cell viability were examined. CC 50 was used to express the cytotoxicity of patchouli alcohol on MDCK. The CC 50 of patchouli alcohol was above 20 mM, which indicated that patchouli alcohol did not affect the growth of MDCK (Table 1) . Thus, it seems that the antiviral effects of patchouli alcohol were not due to the cytotoxicity. Moreover, patchouli alcohol was found to inhibit influenza A (H2N2) virus with an IC 50 of 4.03 ± 0.23 µM. Based on the IC 50 and CC 50 values, the selectivity index (SI) was calculated as >4.96. It is reported that a SI of 4 or more is appropriate for an antiviral agent [18] , suggesting that patchouli alcohol can be judged to have anti-influenza A (H2N2) virus activity.", "Until now, it has been found that patchouli alcohol showed dose-dependent anti-influenza virus (A/PR/8/34, H1N1) activity, with an IC 50 value of 2.635 µM. Furthermore, it showed weak activity against B/Ibaraki/2/85 (IC 50 = 40.82 µM) [19] . With the addition of the above H2N2 inhibitory activity, we have a comprehensively view of the anti-influenza activity of patchouli alcohol.\n\nCells were pretreated with patchouli alcohol prior to virus infection (pretreatment cells), viruses were pretreated prior to infection (pretreatment virus), and patchouli alcohol was added during the adsorption period (adsorption) or after penetration of the viruses into cells (replication). Experiments were repeated independently three times and data presented are the average of three experiments. The symbols * indicated very significant difference p < 0.01 with respect to other mode (pretreatment virus, adsorption and pretreatment cell).", "As shown in Figure 2 , patchouli alcohol showed anti-influenza A (H2N2) virus activity in a timedependent manner. It showed best antiviral activity when added at a concentration of 8 µM during the replication period with inhibition of the viral replication of 97.68% ± 2.09% for influenza A (H2N2) at 72 h. However, no significant effect was detected when patchouli alcohol was used for pretreatment of cells or viruses or when patchouli alcohol was only added during the adsorption phase. These results suggested that the inhibition of influenza A (H2N2) virus by patchouli alcohol appears to occur much more strongly after penetration of the virus into the cell. Besides, biochemical studies have indicated that the bioactivity of NA protein is essential determinant after the replication of influenza A (H2N2) virus [20] [21] [22] . Hence, we conclude that the function of NA protein may be suppressed by patchouli alcohol.", "To evaluate the toxicity of patchouli alcohol, the mean value of body weight of mice in each group was statistically analyzed. The mean weights of mice administered at the 2 mg/kg/dose oseltamivir, 2 mg/kg/dose patchouli alcohol and 10 mg/kg/dose of patchouli alcohol one time daily for 7 days were not significantly different compared with the normal control mice, showing no toxicity of patchouli alcohol and oseltamivir within the testing concentration (P > 0.05). Physiological status was observed in virus infection mice. Three days after viral infection, some mice, especially mice in the H2N2 infected control group showed changes in behavior, such as a tendency to huddle, diminished vitality, and ruffled fur, etc. In the mouse influenza model, viral infection leads to loss of body weight and high mortality. Therefore, the efficacy of patchouli alcohol and oseltamivir were evaluated on the basis of survival rate measured for 15 days post-infection, for treated infected animals relative", "to untreated infected (control) animals. A comparison of efficacy of patchouli alcohol and oseltamivir in vivo mouse influenza model (oral treatment) showed that at a dose of 5 mg/kg/day, patchouli alcohol showed obvious protection against the influenza virus, as the mean day to death was detected as 11.8 ± 1.1 (Table 2) . When the dose was lowered to 1 mg/kg/day, patchouli alcohol showed weaker protection (measured by Survivors/total) than that of 5 mg/kg/day, the mean day to death was 7.5 ± 1.8. Whereas oseltamivir at this dose level (1 mg/kg/day) showed 50% protection (measured by survivors/total) against the influenza virus. In the H2N2 infected control group, there were no survivors. In view of both in vitro and in vivo data, we conclude that patchouli alcohol could be used in the treatment of human influenza virus infections.", "Based on the above experiment data, patchouli alcohol is determined to be bound within NA protein. As the total energies and backbone root-mean-square-deviations (RMSD) in Figure 3 indicate, the energy-minimized patchouli alcohol-NA complex has been in equilibrium since about 0.5 ns, and then retains quite stable in the last 19.5 ns. It is consistent with the previous MD results of other NA inhibitors [23] [24] [25] [26] [27] [28] . Accordingly, the geometric and energetic analyses were made on the average structures of 0.5~20.0 ns MD trajectories, where the system has been already at equilibrium. The interaction energy (E inter ) of patchouli alcohol with NA was calculated at −40.38 kcal mol −1 , where the vdW rather than electrostatic interactions were found to play a dominant role, contribute to about 72% (−29.18 kcal mol −1 ). As shown in Figure 4 , the patchouli alcohol was bound at the active site which also bound to oseltamivir and zanamivir [28] . As Figure 5 shows, the oxygen", "atom of patchouli alcohol was oriented towards the sidechains of residues Glu119 and Tyr406, with one H-bond formed with each residue. The values of distances in Figure 6 further reveal that the docked complex remains rather stable throughout the simulation, with the average distances of Glu119:OE2patchouli alcohol:O and Tyr406:OH -patchouli alcohol:O less than 2.8 Å. The sum contributions (E sum ) of residues Glu119 and Tyr406 amounted to −8.46 and −7.37 kcal mol −1 , respectively (Table 3) . Besides, patchouli alcohol was stabilized by residues Arg118, Asp151, Arg152, Trp178, Ala246, Glu276, Arg292, Asn294 and Gln347, especially residues Asp151, Arg152 and Glu276 ( Figure 5 and Table 3 ). As a matter of fact, residues Asp151, Arg152, Glu119, Glu276 and Tyr406 of the NA protein have already received enough attention from rational drug designs [14, 30, 31] . The catalytic residues Asp151, Arg152 and Glu276 are crucial to the NA functions and the residues Glu119 and Tyr406 are", "important to stabilize the NA active sites [32, 33] . It suggests that the NA functions will be affected by the presence of patchouli alcohol, consistent with the above experiments. Patchouli alcohol matches with the NA active site and has an acceptable interaction energy. Considering the obvious structure discrepancies against current NA inhibitors, it represents an ideal lead compound for the designs of novel anti-influenza agents.", "Patchouli alcohol and oseltamivir were obtained from Sigma Chemical Co. (St. Louis, MO, USA, purity > 99%) and was stored in glass vials with Teflon sealed caps at −20 ± 0.5 °C in the absence of light.\n\nMDCK (Madin-Darby canine kidney) was purchased from Harbin Veterinary Research Institute (Harbin, Heilongjiang, China). The cells were grown in monolayer culture with Eagle's minimum essential medium (EMEM) supplemented with 10% fetal calf serum (FCS), 100 U/mL penicillin and 100 μg/mL streptomycin. The monolayers were removed from their plastic surfaces and serially passaged whenever they became confluent. Cells were plated out onto 96-well culture plates for cytotoxicity and anti-influenza assays, and propagated at 37 °C in an atmosphere of 5% CO 2 .", "The influenza strain A/Leningrad/134/17/1957 H2N2) was purchased from National Control Institute of Veterinary Bioproducts and Pharmaceuticals (Beijing, China). Virus was routinely grown on MDCK cells. The stock cultures were prepared from supernatants of infected cells and stored at −80 °C.", "The cellular toxicity of patchouli alcohol on MDCK cells was assessed by the MTT method. Briefly, cells were seeded on a microtiter plate in the absence or presence of various concentrations (20 µM -0.0098 µM) of patchouli alcohol (eight replicates) and incubated at 37 °C in a humidified atmosphere of 5% CO 2 for 72 h. The supernatants were discarded, washed with PBS twice and MTT reagent (5 mg/mL in PBS) was added to each well. After incubation at 37 °C for 4 h, the supernatants were removed, then 200 μL DMSO was added and incubated at 37 °C for another 30 min. After that the plates were read on an ELISA reader (Thermo Molecular Devices Co., Union City, USA) at 570/630 nm. The mean OD of the cell control wells was assigned a value of 100%. The maximal non-toxic concentration (TD 0 ) and 50% cytotoxic concentration (CC 50 ) were calculated by linear regression analysis of the dose-response curves generated from the data.", "Inhibition of virus replication was measured by the MTT method. Serial dilution of the treated virus was adsorbed to the cells for 1 h at 37 °C. The residual inoculum was discared and infected cells were added with EMEM containing 2% FCS. Each assay was performed in eight replicates. After incubation for 72 h at 37 °C, the cultures were measured by MTT method as described above. The concentration of patchouli alcohol and oseltamivir which inhibited virus numbers by 50% (IC 50 ) was determined from dose-response curves.", "Cells and viruses were incubated with patchouli alcohol at different stages during the viral infection cycle in order to determine the mode of antiviral action. Cells were pretreated with patchouli alcohol before viral infection, viruses were incubated with patchouli alcohol before infection and cells and viruses were incubated together with patchouli alcohol during adsorption or after penetration of the virus into the host cells. Patchouli alcohol was always used at the nontoxic concentration. Cell monolayers were pretreated with patchouli alcohol prior to inoculation with virus by adding patchouli alcohol to the culture medium and incubation for 1 h at 37 °C. The compound was aspirated and cells were washed immediately before the influenza A (H2N2) inoculum was added. For pretreatment virus, Influenza A (H2N2) was incubated in medium containing patchouli alcohol for 1h at room temperature prior to infection of MDCK cells. For analyzing the anti-influenza A (H2N2) inhibition during", "the adsorption period, the same amount of influenza A (H2N2) was mixed with the drug and added to the cells immediately. After 1 h of adsorption at 37 °C, the inoculum was removed and DMEM supplemented with 2 % FCS were added to the cells. The effect of patchouli alcohol against influenza A (H2N2) was also tested during the replication period by adding it after adsorption, as typical performed in anti-influenza A (H2N2) susceptibility studies. Each assay was run in eight replicates.", "Kunming mice, weighing 18-22 g (6 weeks of age) were purchased from Harbin Veterinary Research Institute Animal Co., Ltd. (Harbin, Heilongjiang, China) . First, the toxicity of patchouli alcohol and oseltamivir was assessed in the healthy mice by the loss of body weight compared with the control group (2% DMSO in physiological saline). The mice were orally administered with 10 mg/kg/dose patchouli alcohol, 2 mg/kg/dose patchouli alcohol or 2 mg/kg/dose oseltamivir (dissolved in 2% DMSO in physiological saline) one time daily for 7 days. The weight of mice was determined daily. We conducted procedures according to Principle of Laboratory Animal Care (NIH Publication No. 85 -23, revised 1985) and the guidelines of the Peking University Animal Research Committee.", "Kunming mice were anesthetized with isoflurane and exposed to virus (A/Leningrad/134/17/1957) by intranasal instillation. Drugs were prepared in 2% DMSO in physiological saline and administered 4 h prior to virus exposure and continued daily for 5 days. All mice were observed daily for changes in weight and for any deaths. Parameters for evaluation of antiviral activity included weight loss, reduction in mortality and/or increase in mean day to death (MDD) determined through 15 days.", "The N2 sub-type neuraminidase crystal structure (PDB code 1IVD) was obtained from the RCSB Protein Data Bank [34] . For convenience, the structure is named as NA hereafter. Geometry and partial atomic charges of the patchouli alcohol ( Figure 1) were calculated with the Discover 3.0 module (Insight II 2005) [35] by applying the BFGS algorithm [36] and the consistent-valence force-field (CVFF), with a convergence criterion of 0.01 kcal mol −1 Å −1 . The docking and molecular dynamics (MD) simulations were performed by the general protocols in the Insight II 2005 software packages, consistent with the previous literatures [24, 26, 28, 35, [37] [38] [39] . During the MD simulations, the canonical ensemble (NVT) was employed at normal temperature (300 K). The MD temperature was controlled by the velocity scaling thermostat [40] . Integrations of the classical equations of motion were achieved using the Verlet algorithm. The systems were solvated in a large sphere of TIP3P water molecules", "[40] with the radius of 35.0 Å, which is enough to hold the ensembles [40] . The MD trajectories were generated using a 1.0-fs time step for a total of 20.0 ns, saved at 5.0-ps intervals. The interaction energies of patchouli alcohol with NA and the respective residues at the NA active site were calculated by the Docking module [35], over the 0.5~20.0 ns MD trajectories.", "All results are expressed as mean values ± standard deviations (SDs) (n = 3). The significance of difference was calculated by one-way analysis of variance, and values p < 0.001 were considered to be significant.\n\nIn conclusion, patchouli alcohol possesses anti-influenza A (H2N2) virus activity via interference with the NA function that cleaves the α-glycosidic bond between sialic acid and glycoconjugate. Our results provide the promising information for the potential use of patchouli alcohol in the treatment of influenza A (H2N2) virus infectious disease. Further mechanistic studies on the anti-influenza A virus activity are needed to support this point of view." ]

[ 3 ]

[ "cleave the α-ketosidic linkage between terminal sialic acid and an adjacent sugar residue" ]

[ "Inhibitory Effect and Possible Mechanism of Action of Patchouli Alcohol against Influenza A (H2N2) Virus\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6264369/\n\nSHA: f2d842780b9928cc70f38a4458553f2431877603\n\nAuthors: Wu, Huaxing; Li, Beili; Wang, Xue; Jin, Mingyuan; Wang, Guonian\nDate: 2011-08-03\nDOI: 10.3390/molecules16086489\nLicense: cc-by", "Abstract: In the present study, the anti-influenza A (H2N2) virus activity of patchouli alcohol was studied in vitro, in vivo and in silico. The CC(50) of patchouli alcohol was above 20 µM. Patchouli alcohol could inhibit influenza virus with an IC(50) of 4.03 ± 0.23 µM. MTT assay showed that the inhibition by patchouli alcohol appears strongly after penetration of the virus into the cell. In the influenza mouse model, patchouli alcohol showed obvious protection against the viral infection at a dose of 5 mg/kg/day. Flexible docking and molecular dynamic simulations indicated that patchouli alcohol was bound to the neuraminidase protein of influenza virus, with an interaction energy of –40.38 kcal mol(–1). The invariant key active-site residues Asp151, Arg152, Glu119, Glu276 and Tyr406 played important roles during the binding process. Based on spatial and energetic criteria, patchouli alcohol interfered with the NA functions. Results presented here suggest that patchouli alcohol", "possesses anti-influenza A (H2N2) virus properties, and therefore is a potential source of anti-influenza agents for the pharmaceutical industry.", "Text: The influenza virus, which is one of the main causes of acute respiratory infections in humans, can lead to annual epidemics and infrequent pandemics. The two influenza pandemics of the 20 th century, \"Asian Influenza (1957/H2N2)\" and \"Hong Kong Influenza (1968/H3N2)\" resulted in the deaths of an estimated 2-3 million people globally [1, 2] . Today, their descendants continue to cause the majority of influenza infections in humans [3] . So far as it is learned that the most effective antiviral drug is the neuraminidase (NA) inhibitor, which target the NA glycoproteins of influenza A and B virus [4, 5] .", "The release of new virions from the infected cell is a key step in the influenza life cycle and need neuraminidase (NA) to cleave the α-ketosidic linkage between terminal sialic acid and an adjacent sugar residue [6] . The NA inhibitors were designed to prevent the key step by blocking the active site of enzyme and thus allow sufficient time for the host immune systems to remove infected viruses [7] . Consistent efforts have been devoted to the development of NA inhibitors, using the crystal structure of the N2 sub-type NA protein [8] [9] [10] [11] [12] [13] [14] [15] . Indeed, oseltamivir (Tamiflu) is the representative NA inhibitor that has proven to be uniquely applicable oral drug in clinical practice for the treatment of influenza infection [4, 8, 9] . However, with an increase in medical use, the oseltamivir-resistant strains have been found and probably lead to a large scale outbreak of novel pandemic flu [16, 17] .", "Patchouli alcohol ( Figure 1 ) has been well known for over a century. It is a major constituent of the pungent oil from the East Indian shrub Pogostemon cablin (Blanco) Benth, and widely used in fragrances. Patchouli oil is an important essential oil in the perfume industry, used to give a base and lasting character to a fragrance [16, 17] . The essential oil is very appreciated for its characteristic pleasant and long lasting woody, earthy, and camphoraceous odor, as well as for its fixative properties, being suitable for use in soaps and cosmetic products [16, 17] . The aerial part of Pogostemon cablin has wildly been used for the treatment of the common cold and as an antifungal agent in China [16, 17] . Moreover, the plant is widely used in Traditional Chinese Medicine as it presents various types of pharmacological activity according to the composition of the oil [16, 17] . Patchouli alcohol, as the major volatile constituent of patchouli oil, has been found to strongly inhibit", "H1N1 replication and weakly inhibit B/Ibaraki/2/85 replication [18] . To the best of our knowledge, the anti-influenza virus (H2N2) activities of patchouli alcohol have not been evaluated yet. Therefore, the aim of the present study was to evaluate the anti-influenza A virus (H2N2) activity of patchouli alcohol by MTT assay and mouse influenza model. On such basis, explicitly solvated docking and molecular dynamic (MD) methods were applied to investigative the binding mode involving patchouli alcohol with influenza virus NA protein. We anticipate that the insight into the understanding of inhibiting mechanism will be of value in the rational design of novel anti-influenza drugs.", "First the efficacy of patchouli alcohol on influenza A (H2N2) virus replication and cell viability were examined. CC 50 was used to express the cytotoxicity of patchouli alcohol on MDCK. The CC 50 of patchouli alcohol was above 20 mM, which indicated that patchouli alcohol did not affect the growth of MDCK (Table 1) . Thus, it seems that the antiviral effects of patchouli alcohol were not due to the cytotoxicity. Moreover, patchouli alcohol was found to inhibit influenza A (H2N2) virus with an IC 50 of 4.03 ± 0.23 µM. Based on the IC 50 and CC 50 values, the selectivity index (SI) was calculated as >4.96. It is reported that a SI of 4 or more is appropriate for an antiviral agent [18] , suggesting that patchouli alcohol can be judged to have anti-influenza A (H2N2) virus activity.", "Until now, it has been found that patchouli alcohol showed dose-dependent anti-influenza virus (A/PR/8/34, H1N1) activity, with an IC 50 value of 2.635 µM. Furthermore, it showed weak activity against B/Ibaraki/2/85 (IC 50 = 40.82 µM) [19] . With the addition of the above H2N2 inhibitory activity, we have a comprehensively view of the anti-influenza activity of patchouli alcohol.\n\nCells were pretreated with patchouli alcohol prior to virus infection (pretreatment cells), viruses were pretreated prior to infection (pretreatment virus), and patchouli alcohol was added during the adsorption period (adsorption) or after penetration of the viruses into cells (replication). Experiments were repeated independently three times and data presented are the average of three experiments. The symbols * indicated very significant difference p < 0.01 with respect to other mode (pretreatment virus, adsorption and pretreatment cell).", "As shown in Figure 2 , patchouli alcohol showed anti-influenza A (H2N2) virus activity in a timedependent manner. It showed best antiviral activity when added at a concentration of 8 µM during the replication period with inhibition of the viral replication of 97.68% ± 2.09% for influenza A (H2N2) at 72 h. However, no significant effect was detected when patchouli alcohol was used for pretreatment of cells or viruses or when patchouli alcohol was only added during the adsorption phase. These results suggested that the inhibition of influenza A (H2N2) virus by patchouli alcohol appears to occur much more strongly after penetration of the virus into the cell. Besides, biochemical studies have indicated that the bioactivity of NA protein is essential determinant after the replication of influenza A (H2N2) virus [20] [21] [22] . Hence, we conclude that the function of NA protein may be suppressed by patchouli alcohol.", "To evaluate the toxicity of patchouli alcohol, the mean value of body weight of mice in each group was statistically analyzed. The mean weights of mice administered at the 2 mg/kg/dose oseltamivir, 2 mg/kg/dose patchouli alcohol and 10 mg/kg/dose of patchouli alcohol one time daily for 7 days were not significantly different compared with the normal control mice, showing no toxicity of patchouli alcohol and oseltamivir within the testing concentration (P > 0.05). Physiological status was observed in virus infection mice. Three days after viral infection, some mice, especially mice in the H2N2 infected control group showed changes in behavior, such as a tendency to huddle, diminished vitality, and ruffled fur, etc. In the mouse influenza model, viral infection leads to loss of body weight and high mortality. Therefore, the efficacy of patchouli alcohol and oseltamivir were evaluated on the basis of survival rate measured for 15 days post-infection, for treated infected animals relative", "to untreated infected (control) animals. A comparison of efficacy of patchouli alcohol and oseltamivir in vivo mouse influenza model (oral treatment) showed that at a dose of 5 mg/kg/day, patchouli alcohol showed obvious protection against the influenza virus, as the mean day to death was detected as 11.8 ± 1.1 (Table 2) . When the dose was lowered to 1 mg/kg/day, patchouli alcohol showed weaker protection (measured by Survivors/total) than that of 5 mg/kg/day, the mean day to death was 7.5 ± 1.8. Whereas oseltamivir at this dose level (1 mg/kg/day) showed 50% protection (measured by survivors/total) against the influenza virus. In the H2N2 infected control group, there were no survivors. In view of both in vitro and in vivo data, we conclude that patchouli alcohol could be used in the treatment of human influenza virus infections.", "Based on the above experiment data, patchouli alcohol is determined to be bound within NA protein. As the total energies and backbone root-mean-square-deviations (RMSD) in Figure 3 indicate, the energy-minimized patchouli alcohol-NA complex has been in equilibrium since about 0.5 ns, and then retains quite stable in the last 19.5 ns. It is consistent with the previous MD results of other NA inhibitors [23] [24] [25] [26] [27] [28] . Accordingly, the geometric and energetic analyses were made on the average structures of 0.5~20.0 ns MD trajectories, where the system has been already at equilibrium. The interaction energy (E inter ) of patchouli alcohol with NA was calculated at −40.38 kcal mol −1 , where the vdW rather than electrostatic interactions were found to play a dominant role, contribute to about 72% (−29.18 kcal mol −1 ). As shown in Figure 4 , the patchouli alcohol was bound at the active site which also bound to oseltamivir and zanamivir [28] . As Figure 5 shows, the oxygen", "atom of patchouli alcohol was oriented towards the sidechains of residues Glu119 and Tyr406, with one H-bond formed with each residue. The values of distances in Figure 6 further reveal that the docked complex remains rather stable throughout the simulation, with the average distances of Glu119:OE2patchouli alcohol:O and Tyr406:OH -patchouli alcohol:O less than 2.8 Å. The sum contributions (E sum ) of residues Glu119 and Tyr406 amounted to −8.46 and −7.37 kcal mol −1 , respectively (Table 3) . Besides, patchouli alcohol was stabilized by residues Arg118, Asp151, Arg152, Trp178, Ala246, Glu276, Arg292, Asn294 and Gln347, especially residues Asp151, Arg152 and Glu276 ( Figure 5 and Table 3 ). As a matter of fact, residues Asp151, Arg152, Glu119, Glu276 and Tyr406 of the NA protein have already received enough attention from rational drug designs [14, 30, 31] . The catalytic residues Asp151, Arg152 and Glu276 are crucial to the NA functions and the residues Glu119 and Tyr406 are", "important to stabilize the NA active sites [32, 33] . It suggests that the NA functions will be affected by the presence of patchouli alcohol, consistent with the above experiments. Patchouli alcohol matches with the NA active site and has an acceptable interaction energy. Considering the obvious structure discrepancies against current NA inhibitors, it represents an ideal lead compound for the designs of novel anti-influenza agents.", "Patchouli alcohol and oseltamivir were obtained from Sigma Chemical Co. (St. Louis, MO, USA, purity > 99%) and was stored in glass vials with Teflon sealed caps at −20 ± 0.5 °C in the absence of light.\n\nMDCK (Madin-Darby canine kidney) was purchased from Harbin Veterinary Research Institute (Harbin, Heilongjiang, China). The cells were grown in monolayer culture with Eagle's minimum essential medium (EMEM) supplemented with 10% fetal calf serum (FCS), 100 U/mL penicillin and 100 μg/mL streptomycin. The monolayers were removed from their plastic surfaces and serially passaged whenever they became confluent. Cells were plated out onto 96-well culture plates for cytotoxicity and anti-influenza assays, and propagated at 37 °C in an atmosphere of 5% CO 2 .", "The influenza strain A/Leningrad/134/17/1957 H2N2) was purchased from National Control Institute of Veterinary Bioproducts and Pharmaceuticals (Beijing, China). Virus was routinely grown on MDCK cells. The stock cultures were prepared from supernatants of infected cells and stored at −80 °C.", "The cellular toxicity of patchouli alcohol on MDCK cells was assessed by the MTT method. Briefly, cells were seeded on a microtiter plate in the absence or presence of various concentrations (20 µM -0.0098 µM) of patchouli alcohol (eight replicates) and incubated at 37 °C in a humidified atmosphere of 5% CO 2 for 72 h. The supernatants were discarded, washed with PBS twice and MTT reagent (5 mg/mL in PBS) was added to each well. After incubation at 37 °C for 4 h, the supernatants were removed, then 200 μL DMSO was added and incubated at 37 °C for another 30 min. After that the plates were read on an ELISA reader (Thermo Molecular Devices Co., Union City, USA) at 570/630 nm. The mean OD of the cell control wells was assigned a value of 100%. The maximal non-toxic concentration (TD 0 ) and 50% cytotoxic concentration (CC 50 ) were calculated by linear regression analysis of the dose-response curves generated from the data.", "Inhibition of virus replication was measured by the MTT method. Serial dilution of the treated virus was adsorbed to the cells for 1 h at 37 °C. The residual inoculum was discared and infected cells were added with EMEM containing 2% FCS. Each assay was performed in eight replicates. After incubation for 72 h at 37 °C, the cultures were measured by MTT method as described above. The concentration of patchouli alcohol and oseltamivir which inhibited virus numbers by 50% (IC 50 ) was determined from dose-response curves.", "Cells and viruses were incubated with patchouli alcohol at different stages during the viral infection cycle in order to determine the mode of antiviral action. Cells were pretreated with patchouli alcohol before viral infection, viruses were incubated with patchouli alcohol before infection and cells and viruses were incubated together with patchouli alcohol during adsorption or after penetration of the virus into the host cells. Patchouli alcohol was always used at the nontoxic concentration. Cell monolayers were pretreated with patchouli alcohol prior to inoculation with virus by adding patchouli alcohol to the culture medium and incubation for 1 h at 37 °C. The compound was aspirated and cells were washed immediately before the influenza A (H2N2) inoculum was added. For pretreatment virus, Influenza A (H2N2) was incubated in medium containing patchouli alcohol for 1h at room temperature prior to infection of MDCK cells. For analyzing the anti-influenza A (H2N2) inhibition during", "the adsorption period, the same amount of influenza A (H2N2) was mixed with the drug and added to the cells immediately. After 1 h of adsorption at 37 °C, the inoculum was removed and DMEM supplemented with 2 % FCS were added to the cells. The effect of patchouli alcohol against influenza A (H2N2) was also tested during the replication period by adding it after adsorption, as typical performed in anti-influenza A (H2N2) susceptibility studies. Each assay was run in eight replicates.", "Kunming mice, weighing 18-22 g (6 weeks of age) were purchased from Harbin Veterinary Research Institute Animal Co., Ltd. (Harbin, Heilongjiang, China) . First, the toxicity of patchouli alcohol and oseltamivir was assessed in the healthy mice by the loss of body weight compared with the control group (2% DMSO in physiological saline). The mice were orally administered with 10 mg/kg/dose patchouli alcohol, 2 mg/kg/dose patchouli alcohol or 2 mg/kg/dose oseltamivir (dissolved in 2% DMSO in physiological saline) one time daily for 7 days. The weight of mice was determined daily. We conducted procedures according to Principle of Laboratory Animal Care (NIH Publication No. 85 -23, revised 1985) and the guidelines of the Peking University Animal Research Committee.", "Kunming mice were anesthetized with isoflurane and exposed to virus (A/Leningrad/134/17/1957) by intranasal instillation. Drugs were prepared in 2% DMSO in physiological saline and administered 4 h prior to virus exposure and continued daily for 5 days. All mice were observed daily for changes in weight and for any deaths. Parameters for evaluation of antiviral activity included weight loss, reduction in mortality and/or increase in mean day to death (MDD) determined through 15 days.", "The N2 sub-type neuraminidase crystal structure (PDB code 1IVD) was obtained from the RCSB Protein Data Bank [34] . For convenience, the structure is named as NA hereafter. Geometry and partial atomic charges of the patchouli alcohol ( Figure 1) were calculated with the Discover 3.0 module (Insight II 2005) [35] by applying the BFGS algorithm [36] and the consistent-valence force-field (CVFF), with a convergence criterion of 0.01 kcal mol −1 Å −1 . The docking and molecular dynamics (MD) simulations were performed by the general protocols in the Insight II 2005 software packages, consistent with the previous literatures [24, 26, 28, 35, [37] [38] [39] . During the MD simulations, the canonical ensemble (NVT) was employed at normal temperature (300 K). The MD temperature was controlled by the velocity scaling thermostat [40] . Integrations of the classical equations of motion were achieved using the Verlet algorithm. The systems were solvated in a large sphere of TIP3P water molecules", "[40] with the radius of 35.0 Å, which is enough to hold the ensembles [40] . The MD trajectories were generated using a 1.0-fs time step for a total of 20.0 ns, saved at 5.0-ps intervals. The interaction energies of patchouli alcohol with NA and the respective residues at the NA active site were calculated by the Docking module [35], over the 0.5~20.0 ns MD trajectories.", "All results are expressed as mean values ± standard deviations (SDs) (n = 3). The significance of difference was calculated by one-way analysis of variance, and values p < 0.001 were considered to be significant.\n\nIn conclusion, patchouli alcohol possesses anti-influenza A (H2N2) virus activity via interference with the NA function that cleaves the α-glycosidic bond between sialic acid and glycoconjugate. Our results provide the promising information for the potential use of patchouli alcohol in the treatment of influenza A (H2N2) virus infectious disease. Further mechanistic studies on the anti-influenza A virus activity are needed to support this point of view." ]

[ 4 ]

[ "NA inhibitor" ]

[ "Inhibitory Effect and Possible Mechanism of Action of Patchouli Alcohol against Influenza A (H2N2) Virus\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6264369/\n\nSHA: f2d842780b9928cc70f38a4458553f2431877603\n\nAuthors: Wu, Huaxing; Li, Beili; Wang, Xue; Jin, Mingyuan; Wang, Guonian\nDate: 2011-08-03\nDOI: 10.3390/molecules16086489\nLicense: cc-by", "Abstract: In the present study, the anti-influenza A (H2N2) virus activity of patchouli alcohol was studied in vitro, in vivo and in silico. The CC(50) of patchouli alcohol was above 20 µM. Patchouli alcohol could inhibit influenza virus with an IC(50) of 4.03 ± 0.23 µM. MTT assay showed that the inhibition by patchouli alcohol appears strongly after penetration of the virus into the cell. In the influenza mouse model, patchouli alcohol showed obvious protection against the viral infection at a dose of 5 mg/kg/day. Flexible docking and molecular dynamic simulations indicated that patchouli alcohol was bound to the neuraminidase protein of influenza virus, with an interaction energy of –40.38 kcal mol(–1). The invariant key active-site residues Asp151, Arg152, Glu119, Glu276 and Tyr406 played important roles during the binding process. Based on spatial and energetic criteria, patchouli alcohol interfered with the NA functions. Results presented here suggest that patchouli alcohol", "possesses anti-influenza A (H2N2) virus properties, and therefore is a potential source of anti-influenza agents for the pharmaceutical industry.", "Text: The influenza virus, which is one of the main causes of acute respiratory infections in humans, can lead to annual epidemics and infrequent pandemics. The two influenza pandemics of the 20 th century, \"Asian Influenza (1957/H2N2)\" and \"Hong Kong Influenza (1968/H3N2)\" resulted in the deaths of an estimated 2-3 million people globally [1, 2] . Today, their descendants continue to cause the majority of influenza infections in humans [3] . So far as it is learned that the most effective antiviral drug is the neuraminidase (NA) inhibitor, which target the NA glycoproteins of influenza A and B virus [4, 5] .", "The release of new virions from the infected cell is a key step in the influenza life cycle and need neuraminidase (NA) to cleave the α-ketosidic linkage between terminal sialic acid and an adjacent sugar residue [6] . The NA inhibitors were designed to prevent the key step by blocking the active site of enzyme and thus allow sufficient time for the host immune systems to remove infected viruses [7] . Consistent efforts have been devoted to the development of NA inhibitors, using the crystal structure of the N2 sub-type NA protein [8] [9] [10] [11] [12] [13] [14] [15] . Indeed, oseltamivir (Tamiflu) is the representative NA inhibitor that has proven to be uniquely applicable oral drug in clinical practice for the treatment of influenza infection [4, 8, 9] . However, with an increase in medical use, the oseltamivir-resistant strains have been found and probably lead to a large scale outbreak of novel pandemic flu [16, 17] .", "Patchouli alcohol ( Figure 1 ) has been well known for over a century. It is a major constituent of the pungent oil from the East Indian shrub Pogostemon cablin (Blanco) Benth, and widely used in fragrances. Patchouli oil is an important essential oil in the perfume industry, used to give a base and lasting character to a fragrance [16, 17] . The essential oil is very appreciated for its characteristic pleasant and long lasting woody, earthy, and camphoraceous odor, as well as for its fixative properties, being suitable for use in soaps and cosmetic products [16, 17] . The aerial part of Pogostemon cablin has wildly been used for the treatment of the common cold and as an antifungal agent in China [16, 17] . Moreover, the plant is widely used in Traditional Chinese Medicine as it presents various types of pharmacological activity according to the composition of the oil [16, 17] . Patchouli alcohol, as the major volatile constituent of patchouli oil, has been found to strongly inhibit", "H1N1 replication and weakly inhibit B/Ibaraki/2/85 replication [18] . To the best of our knowledge, the anti-influenza virus (H2N2) activities of patchouli alcohol have not been evaluated yet. Therefore, the aim of the present study was to evaluate the anti-influenza A virus (H2N2) activity of patchouli alcohol by MTT assay and mouse influenza model. On such basis, explicitly solvated docking and molecular dynamic (MD) methods were applied to investigative the binding mode involving patchouli alcohol with influenza virus NA protein. We anticipate that the insight into the understanding of inhibiting mechanism will be of value in the rational design of novel anti-influenza drugs.", "First the efficacy of patchouli alcohol on influenza A (H2N2) virus replication and cell viability were examined. CC 50 was used to express the cytotoxicity of patchouli alcohol on MDCK. The CC 50 of patchouli alcohol was above 20 mM, which indicated that patchouli alcohol did not affect the growth of MDCK (Table 1) . Thus, it seems that the antiviral effects of patchouli alcohol were not due to the cytotoxicity. Moreover, patchouli alcohol was found to inhibit influenza A (H2N2) virus with an IC 50 of 4.03 ± 0.23 µM. Based on the IC 50 and CC 50 values, the selectivity index (SI) was calculated as >4.96. It is reported that a SI of 4 or more is appropriate for an antiviral agent [18] , suggesting that patchouli alcohol can be judged to have anti-influenza A (H2N2) virus activity.", "Until now, it has been found that patchouli alcohol showed dose-dependent anti-influenza virus (A/PR/8/34, H1N1) activity, with an IC 50 value of 2.635 µM. Furthermore, it showed weak activity against B/Ibaraki/2/85 (IC 50 = 40.82 µM) [19] . With the addition of the above H2N2 inhibitory activity, we have a comprehensively view of the anti-influenza activity of patchouli alcohol.\n\nCells were pretreated with patchouli alcohol prior to virus infection (pretreatment cells), viruses were pretreated prior to infection (pretreatment virus), and patchouli alcohol was added during the adsorption period (adsorption) or after penetration of the viruses into cells (replication). Experiments were repeated independently three times and data presented are the average of three experiments. The symbols * indicated very significant difference p < 0.01 with respect to other mode (pretreatment virus, adsorption and pretreatment cell).", "As shown in Figure 2 , patchouli alcohol showed anti-influenza A (H2N2) virus activity in a timedependent manner. It showed best antiviral activity when added at a concentration of 8 µM during the replication period with inhibition of the viral replication of 97.68% ± 2.09% for influenza A (H2N2) at 72 h. However, no significant effect was detected when patchouli alcohol was used for pretreatment of cells or viruses or when patchouli alcohol was only added during the adsorption phase. These results suggested that the inhibition of influenza A (H2N2) virus by patchouli alcohol appears to occur much more strongly after penetration of the virus into the cell. Besides, biochemical studies have indicated that the bioactivity of NA protein is essential determinant after the replication of influenza A (H2N2) virus [20] [21] [22] . Hence, we conclude that the function of NA protein may be suppressed by patchouli alcohol.", "To evaluate the toxicity of patchouli alcohol, the mean value of body weight of mice in each group was statistically analyzed. The mean weights of mice administered at the 2 mg/kg/dose oseltamivir, 2 mg/kg/dose patchouli alcohol and 10 mg/kg/dose of patchouli alcohol one time daily for 7 days were not significantly different compared with the normal control mice, showing no toxicity of patchouli alcohol and oseltamivir within the testing concentration (P > 0.05). Physiological status was observed in virus infection mice. Three days after viral infection, some mice, especially mice in the H2N2 infected control group showed changes in behavior, such as a tendency to huddle, diminished vitality, and ruffled fur, etc. In the mouse influenza model, viral infection leads to loss of body weight and high mortality. Therefore, the efficacy of patchouli alcohol and oseltamivir were evaluated on the basis of survival rate measured for 15 days post-infection, for treated infected animals relative", "to untreated infected (control) animals. A comparison of efficacy of patchouli alcohol and oseltamivir in vivo mouse influenza model (oral treatment) showed that at a dose of 5 mg/kg/day, patchouli alcohol showed obvious protection against the influenza virus, as the mean day to death was detected as 11.8 ± 1.1 (Table 2) . When the dose was lowered to 1 mg/kg/day, patchouli alcohol showed weaker protection (measured by Survivors/total) than that of 5 mg/kg/day, the mean day to death was 7.5 ± 1.8. Whereas oseltamivir at this dose level (1 mg/kg/day) showed 50% protection (measured by survivors/total) against the influenza virus. In the H2N2 infected control group, there were no survivors. In view of both in vitro and in vivo data, we conclude that patchouli alcohol could be used in the treatment of human influenza virus infections.", "Based on the above experiment data, patchouli alcohol is determined to be bound within NA protein. As the total energies and backbone root-mean-square-deviations (RMSD) in Figure 3 indicate, the energy-minimized patchouli alcohol-NA complex has been in equilibrium since about 0.5 ns, and then retains quite stable in the last 19.5 ns. It is consistent with the previous MD results of other NA inhibitors [23] [24] [25] [26] [27] [28] . Accordingly, the geometric and energetic analyses were made on the average structures of 0.5~20.0 ns MD trajectories, where the system has been already at equilibrium. The interaction energy (E inter ) of patchouli alcohol with NA was calculated at −40.38 kcal mol −1 , where the vdW rather than electrostatic interactions were found to play a dominant role, contribute to about 72% (−29.18 kcal mol −1 ). As shown in Figure 4 , the patchouli alcohol was bound at the active site which also bound to oseltamivir and zanamivir [28] . As Figure 5 shows, the oxygen", "atom of patchouli alcohol was oriented towards the sidechains of residues Glu119 and Tyr406, with one H-bond formed with each residue. The values of distances in Figure 6 further reveal that the docked complex remains rather stable throughout the simulation, with the average distances of Glu119:OE2patchouli alcohol:O and Tyr406:OH -patchouli alcohol:O less than 2.8 Å. The sum contributions (E sum ) of residues Glu119 and Tyr406 amounted to −8.46 and −7.37 kcal mol −1 , respectively (Table 3) . Besides, patchouli alcohol was stabilized by residues Arg118, Asp151, Arg152, Trp178, Ala246, Glu276, Arg292, Asn294 and Gln347, especially residues Asp151, Arg152 and Glu276 ( Figure 5 and Table 3 ). As a matter of fact, residues Asp151, Arg152, Glu119, Glu276 and Tyr406 of the NA protein have already received enough attention from rational drug designs [14, 30, 31] . The catalytic residues Asp151, Arg152 and Glu276 are crucial to the NA functions and the residues Glu119 and Tyr406 are", "important to stabilize the NA active sites [32, 33] . It suggests that the NA functions will be affected by the presence of patchouli alcohol, consistent with the above experiments. Patchouli alcohol matches with the NA active site and has an acceptable interaction energy. Considering the obvious structure discrepancies against current NA inhibitors, it represents an ideal lead compound for the designs of novel anti-influenza agents.", "Patchouli alcohol and oseltamivir were obtained from Sigma Chemical Co. (St. Louis, MO, USA, purity > 99%) and was stored in glass vials with Teflon sealed caps at −20 ± 0.5 °C in the absence of light.\n\nMDCK (Madin-Darby canine kidney) was purchased from Harbin Veterinary Research Institute (Harbin, Heilongjiang, China). The cells were grown in monolayer culture with Eagle's minimum essential medium (EMEM) supplemented with 10% fetal calf serum (FCS), 100 U/mL penicillin and 100 μg/mL streptomycin. The monolayers were removed from their plastic surfaces and serially passaged whenever they became confluent. Cells were plated out onto 96-well culture plates for cytotoxicity and anti-influenza assays, and propagated at 37 °C in an atmosphere of 5% CO 2 .", "The influenza strain A/Leningrad/134/17/1957 H2N2) was purchased from National Control Institute of Veterinary Bioproducts and Pharmaceuticals (Beijing, China). Virus was routinely grown on MDCK cells. The stock cultures were prepared from supernatants of infected cells and stored at −80 °C.", "The cellular toxicity of patchouli alcohol on MDCK cells was assessed by the MTT method. Briefly, cells were seeded on a microtiter plate in the absence or presence of various concentrations (20 µM -0.0098 µM) of patchouli alcohol (eight replicates) and incubated at 37 °C in a humidified atmosphere of 5% CO 2 for 72 h. The supernatants were discarded, washed with PBS twice and MTT reagent (5 mg/mL in PBS) was added to each well. After incubation at 37 °C for 4 h, the supernatants were removed, then 200 μL DMSO was added and incubated at 37 °C for another 30 min. After that the plates were read on an ELISA reader (Thermo Molecular Devices Co., Union City, USA) at 570/630 nm. The mean OD of the cell control wells was assigned a value of 100%. The maximal non-toxic concentration (TD 0 ) and 50% cytotoxic concentration (CC 50 ) were calculated by linear regression analysis of the dose-response curves generated from the data.", "Inhibition of virus replication was measured by the MTT method. Serial dilution of the treated virus was adsorbed to the cells for 1 h at 37 °C. The residual inoculum was discared and infected cells were added with EMEM containing 2% FCS. Each assay was performed in eight replicates. After incubation for 72 h at 37 °C, the cultures were measured by MTT method as described above. The concentration of patchouli alcohol and oseltamivir which inhibited virus numbers by 50% (IC 50 ) was determined from dose-response curves.", "Cells and viruses were incubated with patchouli alcohol at different stages during the viral infection cycle in order to determine the mode of antiviral action. Cells were pretreated with patchouli alcohol before viral infection, viruses were incubated with patchouli alcohol before infection and cells and viruses were incubated together with patchouli alcohol during adsorption or after penetration of the virus into the host cells. Patchouli alcohol was always used at the nontoxic concentration. Cell monolayers were pretreated with patchouli alcohol prior to inoculation with virus by adding patchouli alcohol to the culture medium and incubation for 1 h at 37 °C. The compound was aspirated and cells were washed immediately before the influenza A (H2N2) inoculum was added. For pretreatment virus, Influenza A (H2N2) was incubated in medium containing patchouli alcohol for 1h at room temperature prior to infection of MDCK cells. For analyzing the anti-influenza A (H2N2) inhibition during", "the adsorption period, the same amount of influenza A (H2N2) was mixed with the drug and added to the cells immediately. After 1 h of adsorption at 37 °C, the inoculum was removed and DMEM supplemented with 2 % FCS were added to the cells. The effect of patchouli alcohol against influenza A (H2N2) was also tested during the replication period by adding it after adsorption, as typical performed in anti-influenza A (H2N2) susceptibility studies. Each assay was run in eight replicates.", "Kunming mice, weighing 18-22 g (6 weeks of age) were purchased from Harbin Veterinary Research Institute Animal Co., Ltd. (Harbin, Heilongjiang, China) . First, the toxicity of patchouli alcohol and oseltamivir was assessed in the healthy mice by the loss of body weight compared with the control group (2% DMSO in physiological saline). The mice were orally administered with 10 mg/kg/dose patchouli alcohol, 2 mg/kg/dose patchouli alcohol or 2 mg/kg/dose oseltamivir (dissolved in 2% DMSO in physiological saline) one time daily for 7 days. The weight of mice was determined daily. We conducted procedures according to Principle of Laboratory Animal Care (NIH Publication No. 85 -23, revised 1985) and the guidelines of the Peking University Animal Research Committee.", "Kunming mice were anesthetized with isoflurane and exposed to virus (A/Leningrad/134/17/1957) by intranasal instillation. Drugs were prepared in 2% DMSO in physiological saline and administered 4 h prior to virus exposure and continued daily for 5 days. All mice were observed daily for changes in weight and for any deaths. Parameters for evaluation of antiviral activity included weight loss, reduction in mortality and/or increase in mean day to death (MDD) determined through 15 days.", "The N2 sub-type neuraminidase crystal structure (PDB code 1IVD) was obtained from the RCSB Protein Data Bank [34] . For convenience, the structure is named as NA hereafter. Geometry and partial atomic charges of the patchouli alcohol ( Figure 1) were calculated with the Discover 3.0 module (Insight II 2005) [35] by applying the BFGS algorithm [36] and the consistent-valence force-field (CVFF), with a convergence criterion of 0.01 kcal mol −1 Å −1 . The docking and molecular dynamics (MD) simulations were performed by the general protocols in the Insight II 2005 software packages, consistent with the previous literatures [24, 26, 28, 35, [37] [38] [39] . During the MD simulations, the canonical ensemble (NVT) was employed at normal temperature (300 K). The MD temperature was controlled by the velocity scaling thermostat [40] . Integrations of the classical equations of motion were achieved using the Verlet algorithm. The systems were solvated in a large sphere of TIP3P water molecules", "[40] with the radius of 35.0 Å, which is enough to hold the ensembles [40] . The MD trajectories were generated using a 1.0-fs time step for a total of 20.0 ns, saved at 5.0-ps intervals. The interaction energies of patchouli alcohol with NA and the respective residues at the NA active site were calculated by the Docking module [35], over the 0.5~20.0 ns MD trajectories.", "All results are expressed as mean values ± standard deviations (SDs) (n = 3). The significance of difference was calculated by one-way analysis of variance, and values p < 0.001 were considered to be significant.\n\nIn conclusion, patchouli alcohol possesses anti-influenza A (H2N2) virus activity via interference with the NA function that cleaves the α-glycosidic bond between sialic acid and glycoconjugate. Our results provide the promising information for the potential use of patchouli alcohol in the treatment of influenza A (H2N2) virus infectious disease. Further mechanistic studies on the anti-influenza A virus activity are needed to support this point of view." ]

[ 4 ]

[ "CC 50" ]

[ "Inhibitory Effect and Possible Mechanism of Action of Patchouli Alcohol against Influenza A (H2N2) Virus\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6264369/\n\nSHA: f2d842780b9928cc70f38a4458553f2431877603\n\nAuthors: Wu, Huaxing; Li, Beili; Wang, Xue; Jin, Mingyuan; Wang, Guonian\nDate: 2011-08-03\nDOI: 10.3390/molecules16086489\nLicense: cc-by", "Abstract: In the present study, the anti-influenza A (H2N2) virus activity of patchouli alcohol was studied in vitro, in vivo and in silico. The CC(50) of patchouli alcohol was above 20 µM. Patchouli alcohol could inhibit influenza virus with an IC(50) of 4.03 ± 0.23 µM. MTT assay showed that the inhibition by patchouli alcohol appears strongly after penetration of the virus into the cell. In the influenza mouse model, patchouli alcohol showed obvious protection against the viral infection at a dose of 5 mg/kg/day. Flexible docking and molecular dynamic simulations indicated that patchouli alcohol was bound to the neuraminidase protein of influenza virus, with an interaction energy of –40.38 kcal mol(–1). The invariant key active-site residues Asp151, Arg152, Glu119, Glu276 and Tyr406 played important roles during the binding process. Based on spatial and energetic criteria, patchouli alcohol interfered with the NA functions. Results presented here suggest that patchouli alcohol", "possesses anti-influenza A (H2N2) virus properties, and therefore is a potential source of anti-influenza agents for the pharmaceutical industry.", "Text: The influenza virus, which is one of the main causes of acute respiratory infections in humans, can lead to annual epidemics and infrequent pandemics. The two influenza pandemics of the 20 th century, \"Asian Influenza (1957/H2N2)\" and \"Hong Kong Influenza (1968/H3N2)\" resulted in the deaths of an estimated 2-3 million people globally [1, 2] . Today, their descendants continue to cause the majority of influenza infections in humans [3] . So far as it is learned that the most effective antiviral drug is the neuraminidase (NA) inhibitor, which target the NA glycoproteins of influenza A and B virus [4, 5] .", "The release of new virions from the infected cell is a key step in the influenza life cycle and need neuraminidase (NA) to cleave the α-ketosidic linkage between terminal sialic acid and an adjacent sugar residue [6] . The NA inhibitors were designed to prevent the key step by blocking the active site of enzyme and thus allow sufficient time for the host immune systems to remove infected viruses [7] . Consistent efforts have been devoted to the development of NA inhibitors, using the crystal structure of the N2 sub-type NA protein [8] [9] [10] [11] [12] [13] [14] [15] . Indeed, oseltamivir (Tamiflu) is the representative NA inhibitor that has proven to be uniquely applicable oral drug in clinical practice for the treatment of influenza infection [4, 8, 9] . However, with an increase in medical use, the oseltamivir-resistant strains have been found and probably lead to a large scale outbreak of novel pandemic flu [16, 17] .", "Patchouli alcohol ( Figure 1 ) has been well known for over a century. It is a major constituent of the pungent oil from the East Indian shrub Pogostemon cablin (Blanco) Benth, and widely used in fragrances. Patchouli oil is an important essential oil in the perfume industry, used to give a base and lasting character to a fragrance [16, 17] . The essential oil is very appreciated for its characteristic pleasant and long lasting woody, earthy, and camphoraceous odor, as well as for its fixative properties, being suitable for use in soaps and cosmetic products [16, 17] . The aerial part of Pogostemon cablin has wildly been used for the treatment of the common cold and as an antifungal agent in China [16, 17] . Moreover, the plant is widely used in Traditional Chinese Medicine as it presents various types of pharmacological activity according to the composition of the oil [16, 17] . Patchouli alcohol, as the major volatile constituent of patchouli oil, has been found to strongly inhibit", "H1N1 replication and weakly inhibit B/Ibaraki/2/85 replication [18] . To the best of our knowledge, the anti-influenza virus (H2N2) activities of patchouli alcohol have not been evaluated yet. Therefore, the aim of the present study was to evaluate the anti-influenza A virus (H2N2) activity of patchouli alcohol by MTT assay and mouse influenza model. On such basis, explicitly solvated docking and molecular dynamic (MD) methods were applied to investigative the binding mode involving patchouli alcohol with influenza virus NA protein. We anticipate that the insight into the understanding of inhibiting mechanism will be of value in the rational design of novel anti-influenza drugs.", "First the efficacy of patchouli alcohol on influenza A (H2N2) virus replication and cell viability were examined. CC 50 was used to express the cytotoxicity of patchouli alcohol on MDCK. The CC 50 of patchouli alcohol was above 20 mM, which indicated that patchouli alcohol did not affect the growth of MDCK (Table 1) . Thus, it seems that the antiviral effects of patchouli alcohol were not due to the cytotoxicity. Moreover, patchouli alcohol was found to inhibit influenza A (H2N2) virus with an IC 50 of 4.03 ± 0.23 µM. Based on the IC 50 and CC 50 values, the selectivity index (SI) was calculated as >4.96. It is reported that a SI of 4 or more is appropriate for an antiviral agent [18] , suggesting that patchouli alcohol can be judged to have anti-influenza A (H2N2) virus activity.", "Until now, it has been found that patchouli alcohol showed dose-dependent anti-influenza virus (A/PR/8/34, H1N1) activity, with an IC 50 value of 2.635 µM. Furthermore, it showed weak activity against B/Ibaraki/2/85 (IC 50 = 40.82 µM) [19] . With the addition of the above H2N2 inhibitory activity, we have a comprehensively view of the anti-influenza activity of patchouli alcohol.\n\nCells were pretreated with patchouli alcohol prior to virus infection (pretreatment cells), viruses were pretreated prior to infection (pretreatment virus), and patchouli alcohol was added during the adsorption period (adsorption) or after penetration of the viruses into cells (replication). Experiments were repeated independently three times and data presented are the average of three experiments. The symbols * indicated very significant difference p < 0.01 with respect to other mode (pretreatment virus, adsorption and pretreatment cell).", "As shown in Figure 2 , patchouli alcohol showed anti-influenza A (H2N2) virus activity in a timedependent manner. It showed best antiviral activity when added at a concentration of 8 µM during the replication period with inhibition of the viral replication of 97.68% ± 2.09% for influenza A (H2N2) at 72 h. However, no significant effect was detected when patchouli alcohol was used for pretreatment of cells or viruses or when patchouli alcohol was only added during the adsorption phase. These results suggested that the inhibition of influenza A (H2N2) virus by patchouli alcohol appears to occur much more strongly after penetration of the virus into the cell. Besides, biochemical studies have indicated that the bioactivity of NA protein is essential determinant after the replication of influenza A (H2N2) virus [20] [21] [22] . Hence, we conclude that the function of NA protein may be suppressed by patchouli alcohol.", "To evaluate the toxicity of patchouli alcohol, the mean value of body weight of mice in each group was statistically analyzed. The mean weights of mice administered at the 2 mg/kg/dose oseltamivir, 2 mg/kg/dose patchouli alcohol and 10 mg/kg/dose of patchouli alcohol one time daily for 7 days were not significantly different compared with the normal control mice, showing no toxicity of patchouli alcohol and oseltamivir within the testing concentration (P > 0.05). Physiological status was observed in virus infection mice. Three days after viral infection, some mice, especially mice in the H2N2 infected control group showed changes in behavior, such as a tendency to huddle, diminished vitality, and ruffled fur, etc. In the mouse influenza model, viral infection leads to loss of body weight and high mortality. Therefore, the efficacy of patchouli alcohol and oseltamivir were evaluated on the basis of survival rate measured for 15 days post-infection, for treated infected animals relative", "to untreated infected (control) animals. A comparison of efficacy of patchouli alcohol and oseltamivir in vivo mouse influenza model (oral treatment) showed that at a dose of 5 mg/kg/day, patchouli alcohol showed obvious protection against the influenza virus, as the mean day to death was detected as 11.8 ± 1.1 (Table 2) . When the dose was lowered to 1 mg/kg/day, patchouli alcohol showed weaker protection (measured by Survivors/total) than that of 5 mg/kg/day, the mean day to death was 7.5 ± 1.8. Whereas oseltamivir at this dose level (1 mg/kg/day) showed 50% protection (measured by survivors/total) against the influenza virus. In the H2N2 infected control group, there were no survivors. In view of both in vitro and in vivo data, we conclude that patchouli alcohol could be used in the treatment of human influenza virus infections.", "Based on the above experiment data, patchouli alcohol is determined to be bound within NA protein. As the total energies and backbone root-mean-square-deviations (RMSD) in Figure 3 indicate, the energy-minimized patchouli alcohol-NA complex has been in equilibrium since about 0.5 ns, and then retains quite stable in the last 19.5 ns. It is consistent with the previous MD results of other NA inhibitors [23] [24] [25] [26] [27] [28] . Accordingly, the geometric and energetic analyses were made on the average structures of 0.5~20.0 ns MD trajectories, where the system has been already at equilibrium. The interaction energy (E inter ) of patchouli alcohol with NA was calculated at −40.38 kcal mol −1 , where the vdW rather than electrostatic interactions were found to play a dominant role, contribute to about 72% (−29.18 kcal mol −1 ). As shown in Figure 4 , the patchouli alcohol was bound at the active site which also bound to oseltamivir and zanamivir [28] . As Figure 5 shows, the oxygen", "atom of patchouli alcohol was oriented towards the sidechains of residues Glu119 and Tyr406, with one H-bond formed with each residue. The values of distances in Figure 6 further reveal that the docked complex remains rather stable throughout the simulation, with the average distances of Glu119:OE2patchouli alcohol:O and Tyr406:OH -patchouli alcohol:O less than 2.8 Å. The sum contributions (E sum ) of residues Glu119 and Tyr406 amounted to −8.46 and −7.37 kcal mol −1 , respectively (Table 3) . Besides, patchouli alcohol was stabilized by residues Arg118, Asp151, Arg152, Trp178, Ala246, Glu276, Arg292, Asn294 and Gln347, especially residues Asp151, Arg152 and Glu276 ( Figure 5 and Table 3 ). As a matter of fact, residues Asp151, Arg152, Glu119, Glu276 and Tyr406 of the NA protein have already received enough attention from rational drug designs [14, 30, 31] . The catalytic residues Asp151, Arg152 and Glu276 are crucial to the NA functions and the residues Glu119 and Tyr406 are", "important to stabilize the NA active sites [32, 33] . It suggests that the NA functions will be affected by the presence of patchouli alcohol, consistent with the above experiments. Patchouli alcohol matches with the NA active site and has an acceptable interaction energy. Considering the obvious structure discrepancies against current NA inhibitors, it represents an ideal lead compound for the designs of novel anti-influenza agents.", "Patchouli alcohol and oseltamivir were obtained from Sigma Chemical Co. (St. Louis, MO, USA, purity > 99%) and was stored in glass vials with Teflon sealed caps at −20 ± 0.5 °C in the absence of light.\n\nMDCK (Madin-Darby canine kidney) was purchased from Harbin Veterinary Research Institute (Harbin, Heilongjiang, China). The cells were grown in monolayer culture with Eagle's minimum essential medium (EMEM) supplemented with 10% fetal calf serum (FCS), 100 U/mL penicillin and 100 μg/mL streptomycin. The monolayers were removed from their plastic surfaces and serially passaged whenever they became confluent. Cells were plated out onto 96-well culture plates for cytotoxicity and anti-influenza assays, and propagated at 37 °C in an atmosphere of 5% CO 2 .", "The influenza strain A/Leningrad/134/17/1957 H2N2) was purchased from National Control Institute of Veterinary Bioproducts and Pharmaceuticals (Beijing, China). Virus was routinely grown on MDCK cells. The stock cultures were prepared from supernatants of infected cells and stored at −80 °C.", "The cellular toxicity of patchouli alcohol on MDCK cells was assessed by the MTT method. Briefly, cells were seeded on a microtiter plate in the absence or presence of various concentrations (20 µM -0.0098 µM) of patchouli alcohol (eight replicates) and incubated at 37 °C in a humidified atmosphere of 5% CO 2 for 72 h. The supernatants were discarded, washed with PBS twice and MTT reagent (5 mg/mL in PBS) was added to each well. After incubation at 37 °C for 4 h, the supernatants were removed, then 200 μL DMSO was added and incubated at 37 °C for another 30 min. After that the plates were read on an ELISA reader (Thermo Molecular Devices Co., Union City, USA) at 570/630 nm. The mean OD of the cell control wells was assigned a value of 100%. The maximal non-toxic concentration (TD 0 ) and 50% cytotoxic concentration (CC 50 ) were calculated by linear regression analysis of the dose-response curves generated from the data.", "Inhibition of virus replication was measured by the MTT method. Serial dilution of the treated virus was adsorbed to the cells for 1 h at 37 °C. The residual inoculum was discared and infected cells were added with EMEM containing 2% FCS. Each assay was performed in eight replicates. After incubation for 72 h at 37 °C, the cultures were measured by MTT method as described above. The concentration of patchouli alcohol and oseltamivir which inhibited virus numbers by 50% (IC 50 ) was determined from dose-response curves.", "Cells and viruses were incubated with patchouli alcohol at different stages during the viral infection cycle in order to determine the mode of antiviral action. Cells were pretreated with patchouli alcohol before viral infection, viruses were incubated with patchouli alcohol before infection and cells and viruses were incubated together with patchouli alcohol during adsorption or after penetration of the virus into the host cells. Patchouli alcohol was always used at the nontoxic concentration. Cell monolayers were pretreated with patchouli alcohol prior to inoculation with virus by adding patchouli alcohol to the culture medium and incubation for 1 h at 37 °C. The compound was aspirated and cells were washed immediately before the influenza A (H2N2) inoculum was added. For pretreatment virus, Influenza A (H2N2) was incubated in medium containing patchouli alcohol for 1h at room temperature prior to infection of MDCK cells. For analyzing the anti-influenza A (H2N2) inhibition during", "the adsorption period, the same amount of influenza A (H2N2) was mixed with the drug and added to the cells immediately. After 1 h of adsorption at 37 °C, the inoculum was removed and DMEM supplemented with 2 % FCS were added to the cells. The effect of patchouli alcohol against influenza A (H2N2) was also tested during the replication period by adding it after adsorption, as typical performed in anti-influenza A (H2N2) susceptibility studies. Each assay was run in eight replicates.", "Kunming mice, weighing 18-22 g (6 weeks of age) were purchased from Harbin Veterinary Research Institute Animal Co., Ltd. (Harbin, Heilongjiang, China) . First, the toxicity of patchouli alcohol and oseltamivir was assessed in the healthy mice by the loss of body weight compared with the control group (2% DMSO in physiological saline). The mice were orally administered with 10 mg/kg/dose patchouli alcohol, 2 mg/kg/dose patchouli alcohol or 2 mg/kg/dose oseltamivir (dissolved in 2% DMSO in physiological saline) one time daily for 7 days. The weight of mice was determined daily. We conducted procedures according to Principle of Laboratory Animal Care (NIH Publication No. 85 -23, revised 1985) and the guidelines of the Peking University Animal Research Committee.", "Kunming mice were anesthetized with isoflurane and exposed to virus (A/Leningrad/134/17/1957) by intranasal instillation. Drugs were prepared in 2% DMSO in physiological saline and administered 4 h prior to virus exposure and continued daily for 5 days. All mice were observed daily for changes in weight and for any deaths. Parameters for evaluation of antiviral activity included weight loss, reduction in mortality and/or increase in mean day to death (MDD) determined through 15 days.", "The N2 sub-type neuraminidase crystal structure (PDB code 1IVD) was obtained from the RCSB Protein Data Bank [34] . For convenience, the structure is named as NA hereafter. Geometry and partial atomic charges of the patchouli alcohol ( Figure 1) were calculated with the Discover 3.0 module (Insight II 2005) [35] by applying the BFGS algorithm [36] and the consistent-valence force-field (CVFF), with a convergence criterion of 0.01 kcal mol −1 Å −1 . The docking and molecular dynamics (MD) simulations were performed by the general protocols in the Insight II 2005 software packages, consistent with the previous literatures [24, 26, 28, 35, [37] [38] [39] . During the MD simulations, the canonical ensemble (NVT) was employed at normal temperature (300 K). The MD temperature was controlled by the velocity scaling thermostat [40] . Integrations of the classical equations of motion were achieved using the Verlet algorithm. The systems were solvated in a large sphere of TIP3P water molecules", "[40] with the radius of 35.0 Å, which is enough to hold the ensembles [40] . The MD trajectories were generated using a 1.0-fs time step for a total of 20.0 ns, saved at 5.0-ps intervals. The interaction energies of patchouli alcohol with NA and the respective residues at the NA active site were calculated by the Docking module [35], over the 0.5~20.0 ns MD trajectories.", "All results are expressed as mean values ± standard deviations (SDs) (n = 3). The significance of difference was calculated by one-way analysis of variance, and values p < 0.001 were considered to be significant.\n\nIn conclusion, patchouli alcohol possesses anti-influenza A (H2N2) virus activity via interference with the NA function that cleaves the α-glycosidic bond between sialic acid and glycoconjugate. Our results provide the promising information for the potential use of patchouli alcohol in the treatment of influenza A (H2N2) virus infectious disease. Further mechanistic studies on the anti-influenza A virus activity are needed to support this point of view." ]

[ 7 ]

[ "MTT method" ]

[ "Inhibitory Effect and Possible Mechanism of Action of Patchouli Alcohol against Influenza A (H2N2) Virus\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6264369/\n\nSHA: f2d842780b9928cc70f38a4458553f2431877603\n\nAuthors: Wu, Huaxing; Li, Beili; Wang, Xue; Jin, Mingyuan; Wang, Guonian\nDate: 2011-08-03\nDOI: 10.3390/molecules16086489\nLicense: cc-by", "Abstract: In the present study, the anti-influenza A (H2N2) virus activity of patchouli alcohol was studied in vitro, in vivo and in silico. The CC(50) of patchouli alcohol was above 20 µM. Patchouli alcohol could inhibit influenza virus with an IC(50) of 4.03 ± 0.23 µM. MTT assay showed that the inhibition by patchouli alcohol appears strongly after penetration of the virus into the cell. In the influenza mouse model, patchouli alcohol showed obvious protection against the viral infection at a dose of 5 mg/kg/day. Flexible docking and molecular dynamic simulations indicated that patchouli alcohol was bound to the neuraminidase protein of influenza virus, with an interaction energy of –40.38 kcal mol(–1). The invariant key active-site residues Asp151, Arg152, Glu119, Glu276 and Tyr406 played important roles during the binding process. Based on spatial and energetic criteria, patchouli alcohol interfered with the NA functions. Results presented here suggest that patchouli alcohol", "possesses anti-influenza A (H2N2) virus properties, and therefore is a potential source of anti-influenza agents for the pharmaceutical industry.", "Text: The influenza virus, which is one of the main causes of acute respiratory infections in humans, can lead to annual epidemics and infrequent pandemics. The two influenza pandemics of the 20 th century, \"Asian Influenza (1957/H2N2)\" and \"Hong Kong Influenza (1968/H3N2)\" resulted in the deaths of an estimated 2-3 million people globally [1, 2] . Today, their descendants continue to cause the majority of influenza infections in humans [3] . So far as it is learned that the most effective antiviral drug is the neuraminidase (NA) inhibitor, which target the NA glycoproteins of influenza A and B virus [4, 5] .", "The release of new virions from the infected cell is a key step in the influenza life cycle and need neuraminidase (NA) to cleave the α-ketosidic linkage between terminal sialic acid and an adjacent sugar residue [6] . The NA inhibitors were designed to prevent the key step by blocking the active site of enzyme and thus allow sufficient time for the host immune systems to remove infected viruses [7] . Consistent efforts have been devoted to the development of NA inhibitors, using the crystal structure of the N2 sub-type NA protein [8] [9] [10] [11] [12] [13] [14] [15] . Indeed, oseltamivir (Tamiflu) is the representative NA inhibitor that has proven to be uniquely applicable oral drug in clinical practice for the treatment of influenza infection [4, 8, 9] . However, with an increase in medical use, the oseltamivir-resistant strains have been found and probably lead to a large scale outbreak of novel pandemic flu [16, 17] .", "Patchouli alcohol ( Figure 1 ) has been well known for over a century. It is a major constituent of the pungent oil from the East Indian shrub Pogostemon cablin (Blanco) Benth, and widely used in fragrances. Patchouli oil is an important essential oil in the perfume industry, used to give a base and lasting character to a fragrance [16, 17] . The essential oil is very appreciated for its characteristic pleasant and long lasting woody, earthy, and camphoraceous odor, as well as for its fixative properties, being suitable for use in soaps and cosmetic products [16, 17] . The aerial part of Pogostemon cablin has wildly been used for the treatment of the common cold and as an antifungal agent in China [16, 17] . Moreover, the plant is widely used in Traditional Chinese Medicine as it presents various types of pharmacological activity according to the composition of the oil [16, 17] . Patchouli alcohol, as the major volatile constituent of patchouli oil, has been found to strongly inhibit", "H1N1 replication and weakly inhibit B/Ibaraki/2/85 replication [18] . To the best of our knowledge, the anti-influenza virus (H2N2) activities of patchouli alcohol have not been evaluated yet. Therefore, the aim of the present study was to evaluate the anti-influenza A virus (H2N2) activity of patchouli alcohol by MTT assay and mouse influenza model. On such basis, explicitly solvated docking and molecular dynamic (MD) methods were applied to investigative the binding mode involving patchouli alcohol with influenza virus NA protein. We anticipate that the insight into the understanding of inhibiting mechanism will be of value in the rational design of novel anti-influenza drugs.", "First the efficacy of patchouli alcohol on influenza A (H2N2) virus replication and cell viability were examined. CC 50 was used to express the cytotoxicity of patchouli alcohol on MDCK. The CC 50 of patchouli alcohol was above 20 mM, which indicated that patchouli alcohol did not affect the growth of MDCK (Table 1) . Thus, it seems that the antiviral effects of patchouli alcohol were not due to the cytotoxicity. Moreover, patchouli alcohol was found to inhibit influenza A (H2N2) virus with an IC 50 of 4.03 ± 0.23 µM. Based on the IC 50 and CC 50 values, the selectivity index (SI) was calculated as >4.96. It is reported that a SI of 4 or more is appropriate for an antiviral agent [18] , suggesting that patchouli alcohol can be judged to have anti-influenza A (H2N2) virus activity.", "Until now, it has been found that patchouli alcohol showed dose-dependent anti-influenza virus (A/PR/8/34, H1N1) activity, with an IC 50 value of 2.635 µM. Furthermore, it showed weak activity against B/Ibaraki/2/85 (IC 50 = 40.82 µM) [19] . With the addition of the above H2N2 inhibitory activity, we have a comprehensively view of the anti-influenza activity of patchouli alcohol.\n\nCells were pretreated with patchouli alcohol prior to virus infection (pretreatment cells), viruses were pretreated prior to infection (pretreatment virus), and patchouli alcohol was added during the adsorption period (adsorption) or after penetration of the viruses into cells (replication). Experiments were repeated independently three times and data presented are the average of three experiments. The symbols * indicated very significant difference p < 0.01 with respect to other mode (pretreatment virus, adsorption and pretreatment cell).", "As shown in Figure 2 , patchouli alcohol showed anti-influenza A (H2N2) virus activity in a timedependent manner. It showed best antiviral activity when added at a concentration of 8 µM during the replication period with inhibition of the viral replication of 97.68% ± 2.09% for influenza A (H2N2) at 72 h. However, no significant effect was detected when patchouli alcohol was used for pretreatment of cells or viruses or when patchouli alcohol was only added during the adsorption phase. These results suggested that the inhibition of influenza A (H2N2) virus by patchouli alcohol appears to occur much more strongly after penetration of the virus into the cell. Besides, biochemical studies have indicated that the bioactivity of NA protein is essential determinant after the replication of influenza A (H2N2) virus [20] [21] [22] . Hence, we conclude that the function of NA protein may be suppressed by patchouli alcohol.", "To evaluate the toxicity of patchouli alcohol, the mean value of body weight of mice in each group was statistically analyzed. The mean weights of mice administered at the 2 mg/kg/dose oseltamivir, 2 mg/kg/dose patchouli alcohol and 10 mg/kg/dose of patchouli alcohol one time daily for 7 days were not significantly different compared with the normal control mice, showing no toxicity of patchouli alcohol and oseltamivir within the testing concentration (P > 0.05). Physiological status was observed in virus infection mice. Three days after viral infection, some mice, especially mice in the H2N2 infected control group showed changes in behavior, such as a tendency to huddle, diminished vitality, and ruffled fur, etc. In the mouse influenza model, viral infection leads to loss of body weight and high mortality. Therefore, the efficacy of patchouli alcohol and oseltamivir were evaluated on the basis of survival rate measured for 15 days post-infection, for treated infected animals relative", "to untreated infected (control) animals. A comparison of efficacy of patchouli alcohol and oseltamivir in vivo mouse influenza model (oral treatment) showed that at a dose of 5 mg/kg/day, patchouli alcohol showed obvious protection against the influenza virus, as the mean day to death was detected as 11.8 ± 1.1 (Table 2) . When the dose was lowered to 1 mg/kg/day, patchouli alcohol showed weaker protection (measured by Survivors/total) than that of 5 mg/kg/day, the mean day to death was 7.5 ± 1.8. Whereas oseltamivir at this dose level (1 mg/kg/day) showed 50% protection (measured by survivors/total) against the influenza virus. In the H2N2 infected control group, there were no survivors. In view of both in vitro and in vivo data, we conclude that patchouli alcohol could be used in the treatment of human influenza virus infections.", "Based on the above experiment data, patchouli alcohol is determined to be bound within NA protein. As the total energies and backbone root-mean-square-deviations (RMSD) in Figure 3 indicate, the energy-minimized patchouli alcohol-NA complex has been in equilibrium since about 0.5 ns, and then retains quite stable in the last 19.5 ns. It is consistent with the previous MD results of other NA inhibitors [23] [24] [25] [26] [27] [28] . Accordingly, the geometric and energetic analyses were made on the average structures of 0.5~20.0 ns MD trajectories, where the system has been already at equilibrium. The interaction energy (E inter ) of patchouli alcohol with NA was calculated at −40.38 kcal mol −1 , where the vdW rather than electrostatic interactions were found to play a dominant role, contribute to about 72% (−29.18 kcal mol −1 ). As shown in Figure 4 , the patchouli alcohol was bound at the active site which also bound to oseltamivir and zanamivir [28] . As Figure 5 shows, the oxygen", "atom of patchouli alcohol was oriented towards the sidechains of residues Glu119 and Tyr406, with one H-bond formed with each residue. The values of distances in Figure 6 further reveal that the docked complex remains rather stable throughout the simulation, with the average distances of Glu119:OE2patchouli alcohol:O and Tyr406:OH -patchouli alcohol:O less than 2.8 Å. The sum contributions (E sum ) of residues Glu119 and Tyr406 amounted to −8.46 and −7.37 kcal mol −1 , respectively (Table 3) . Besides, patchouli alcohol was stabilized by residues Arg118, Asp151, Arg152, Trp178, Ala246, Glu276, Arg292, Asn294 and Gln347, especially residues Asp151, Arg152 and Glu276 ( Figure 5 and Table 3 ). As a matter of fact, residues Asp151, Arg152, Glu119, Glu276 and Tyr406 of the NA protein have already received enough attention from rational drug designs [14, 30, 31] . The catalytic residues Asp151, Arg152 and Glu276 are crucial to the NA functions and the residues Glu119 and Tyr406 are", "important to stabilize the NA active sites [32, 33] . It suggests that the NA functions will be affected by the presence of patchouli alcohol, consistent with the above experiments. Patchouli alcohol matches with the NA active site and has an acceptable interaction energy. Considering the obvious structure discrepancies against current NA inhibitors, it represents an ideal lead compound for the designs of novel anti-influenza agents.", "Patchouli alcohol and oseltamivir were obtained from Sigma Chemical Co. (St. Louis, MO, USA, purity > 99%) and was stored in glass vials with Teflon sealed caps at −20 ± 0.5 °C in the absence of light.\n\nMDCK (Madin-Darby canine kidney) was purchased from Harbin Veterinary Research Institute (Harbin, Heilongjiang, China). The cells were grown in monolayer culture with Eagle's minimum essential medium (EMEM) supplemented with 10% fetal calf serum (FCS), 100 U/mL penicillin and 100 μg/mL streptomycin. The monolayers were removed from their plastic surfaces and serially passaged whenever they became confluent. Cells were plated out onto 96-well culture plates for cytotoxicity and anti-influenza assays, and propagated at 37 °C in an atmosphere of 5% CO 2 .", "The influenza strain A/Leningrad/134/17/1957 H2N2) was purchased from National Control Institute of Veterinary Bioproducts and Pharmaceuticals (Beijing, China). Virus was routinely grown on MDCK cells. The stock cultures were prepared from supernatants of infected cells and stored at −80 °C.", "The cellular toxicity of patchouli alcohol on MDCK cells was assessed by the MTT method. Briefly, cells were seeded on a microtiter plate in the absence or presence of various concentrations (20 µM -0.0098 µM) of patchouli alcohol (eight replicates) and incubated at 37 °C in a humidified atmosphere of 5% CO 2 for 72 h. The supernatants were discarded, washed with PBS twice and MTT reagent (5 mg/mL in PBS) was added to each well. After incubation at 37 °C for 4 h, the supernatants were removed, then 200 μL DMSO was added and incubated at 37 °C for another 30 min. After that the plates were read on an ELISA reader (Thermo Molecular Devices Co., Union City, USA) at 570/630 nm. The mean OD of the cell control wells was assigned a value of 100%. The maximal non-toxic concentration (TD 0 ) and 50% cytotoxic concentration (CC 50 ) were calculated by linear regression analysis of the dose-response curves generated from the data.", "Inhibition of virus replication was measured by the MTT method. Serial dilution of the treated virus was adsorbed to the cells for 1 h at 37 °C. The residual inoculum was discared and infected cells were added with EMEM containing 2% FCS. Each assay was performed in eight replicates. After incubation for 72 h at 37 °C, the cultures were measured by MTT method as described above. The concentration of patchouli alcohol and oseltamivir which inhibited virus numbers by 50% (IC 50 ) was determined from dose-response curves.", "Cells and viruses were incubated with patchouli alcohol at different stages during the viral infection cycle in order to determine the mode of antiviral action. Cells were pretreated with patchouli alcohol before viral infection, viruses were incubated with patchouli alcohol before infection and cells and viruses were incubated together with patchouli alcohol during adsorption or after penetration of the virus into the host cells. Patchouli alcohol was always used at the nontoxic concentration. Cell monolayers were pretreated with patchouli alcohol prior to inoculation with virus by adding patchouli alcohol to the culture medium and incubation for 1 h at 37 °C. The compound was aspirated and cells were washed immediately before the influenza A (H2N2) inoculum was added. For pretreatment virus, Influenza A (H2N2) was incubated in medium containing patchouli alcohol for 1h at room temperature prior to infection of MDCK cells. For analyzing the anti-influenza A (H2N2) inhibition during", "the adsorption period, the same amount of influenza A (H2N2) was mixed with the drug and added to the cells immediately. After 1 h of adsorption at 37 °C, the inoculum was removed and DMEM supplemented with 2 % FCS were added to the cells. The effect of patchouli alcohol against influenza A (H2N2) was also tested during the replication period by adding it after adsorption, as typical performed in anti-influenza A (H2N2) susceptibility studies. Each assay was run in eight replicates.", "Kunming mice, weighing 18-22 g (6 weeks of age) were purchased from Harbin Veterinary Research Institute Animal Co., Ltd. (Harbin, Heilongjiang, China) . First, the toxicity of patchouli alcohol and oseltamivir was assessed in the healthy mice by the loss of body weight compared with the control group (2% DMSO in physiological saline). The mice were orally administered with 10 mg/kg/dose patchouli alcohol, 2 mg/kg/dose patchouli alcohol or 2 mg/kg/dose oseltamivir (dissolved in 2% DMSO in physiological saline) one time daily for 7 days. The weight of mice was determined daily. We conducted procedures according to Principle of Laboratory Animal Care (NIH Publication No. 85 -23, revised 1985) and the guidelines of the Peking University Animal Research Committee.", "Kunming mice were anesthetized with isoflurane and exposed to virus (A/Leningrad/134/17/1957) by intranasal instillation. Drugs were prepared in 2% DMSO in physiological saline and administered 4 h prior to virus exposure and continued daily for 5 days. All mice were observed daily for changes in weight and for any deaths. Parameters for evaluation of antiviral activity included weight loss, reduction in mortality and/or increase in mean day to death (MDD) determined through 15 days.", "The N2 sub-type neuraminidase crystal structure (PDB code 1IVD) was obtained from the RCSB Protein Data Bank [34] . For convenience, the structure is named as NA hereafter. Geometry and partial atomic charges of the patchouli alcohol ( Figure 1) were calculated with the Discover 3.0 module (Insight II 2005) [35] by applying the BFGS algorithm [36] and the consistent-valence force-field (CVFF), with a convergence criterion of 0.01 kcal mol −1 Å −1 . The docking and molecular dynamics (MD) simulations were performed by the general protocols in the Insight II 2005 software packages, consistent with the previous literatures [24, 26, 28, 35, [37] [38] [39] . During the MD simulations, the canonical ensemble (NVT) was employed at normal temperature (300 K). The MD temperature was controlled by the velocity scaling thermostat [40] . Integrations of the classical equations of motion were achieved using the Verlet algorithm. The systems were solvated in a large sphere of TIP3P water molecules", "[40] with the radius of 35.0 Å, which is enough to hold the ensembles [40] . The MD trajectories were generated using a 1.0-fs time step for a total of 20.0 ns, saved at 5.0-ps intervals. The interaction energies of patchouli alcohol with NA and the respective residues at the NA active site were calculated by the Docking module [35], over the 0.5~20.0 ns MD trajectories.", "All results are expressed as mean values ± standard deviations (SDs) (n = 3). The significance of difference was calculated by one-way analysis of variance, and values p < 0.001 were considered to be significant.\n\nIn conclusion, patchouli alcohol possesses anti-influenza A (H2N2) virus activity via interference with the NA function that cleaves the α-glycosidic bond between sialic acid and glycoconjugate. Our results provide the promising information for the potential use of patchouli alcohol in the treatment of influenza A (H2N2) virus infectious disease. Further mechanistic studies on the anti-influenza A virus activity are needed to support this point of view." ]

[ 17 ]

[ "patchouli alcohol possesses anti-influenza A (H2N2) virus activity" ]

[ "Inhibitory Effect and Possible Mechanism of Action of Patchouli Alcohol against Influenza A (H2N2) Virus\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6264369/\n\nSHA: f2d842780b9928cc70f38a4458553f2431877603\n\nAuthors: Wu, Huaxing; Li, Beili; Wang, Xue; Jin, Mingyuan; Wang, Guonian\nDate: 2011-08-03\nDOI: 10.3390/molecules16086489\nLicense: cc-by", "Abstract: In the present study, the anti-influenza A (H2N2) virus activity of patchouli alcohol was studied in vitro, in vivo and in silico. The CC(50) of patchouli alcohol was above 20 µM. Patchouli alcohol could inhibit influenza virus with an IC(50) of 4.03 ± 0.23 µM. MTT assay showed that the inhibition by patchouli alcohol appears strongly after penetration of the virus into the cell. In the influenza mouse model, patchouli alcohol showed obvious protection against the viral infection at a dose of 5 mg/kg/day. Flexible docking and molecular dynamic simulations indicated that patchouli alcohol was bound to the neuraminidase protein of influenza virus, with an interaction energy of –40.38 kcal mol(–1). The invariant key active-site residues Asp151, Arg152, Glu119, Glu276 and Tyr406 played important roles during the binding process. Based on spatial and energetic criteria, patchouli alcohol interfered with the NA functions. Results presented here suggest that patchouli alcohol", "possesses anti-influenza A (H2N2) virus properties, and therefore is a potential source of anti-influenza agents for the pharmaceutical industry.", "Text: The influenza virus, which is one of the main causes of acute respiratory infections in humans, can lead to annual epidemics and infrequent pandemics. The two influenza pandemics of the 20 th century, \"Asian Influenza (1957/H2N2)\" and \"Hong Kong Influenza (1968/H3N2)\" resulted in the deaths of an estimated 2-3 million people globally [1, 2] . Today, their descendants continue to cause the majority of influenza infections in humans [3] . So far as it is learned that the most effective antiviral drug is the neuraminidase (NA) inhibitor, which target the NA glycoproteins of influenza A and B virus [4, 5] .", "The release of new virions from the infected cell is a key step in the influenza life cycle and need neuraminidase (NA) to cleave the α-ketosidic linkage between terminal sialic acid and an adjacent sugar residue [6] . The NA inhibitors were designed to prevent the key step by blocking the active site of enzyme and thus allow sufficient time for the host immune systems to remove infected viruses [7] . Consistent efforts have been devoted to the development of NA inhibitors, using the crystal structure of the N2 sub-type NA protein [8] [9] [10] [11] [12] [13] [14] [15] . Indeed, oseltamivir (Tamiflu) is the representative NA inhibitor that has proven to be uniquely applicable oral drug in clinical practice for the treatment of influenza infection [4, 8, 9] . However, with an increase in medical use, the oseltamivir-resistant strains have been found and probably lead to a large scale outbreak of novel pandemic flu [16, 17] .", "Patchouli alcohol ( Figure 1 ) has been well known for over a century. It is a major constituent of the pungent oil from the East Indian shrub Pogostemon cablin (Blanco) Benth, and widely used in fragrances. Patchouli oil is an important essential oil in the perfume industry, used to give a base and lasting character to a fragrance [16, 17] . The essential oil is very appreciated for its characteristic pleasant and long lasting woody, earthy, and camphoraceous odor, as well as for its fixative properties, being suitable for use in soaps and cosmetic products [16, 17] . The aerial part of Pogostemon cablin has wildly been used for the treatment of the common cold and as an antifungal agent in China [16, 17] . Moreover, the plant is widely used in Traditional Chinese Medicine as it presents various types of pharmacological activity according to the composition of the oil [16, 17] . Patchouli alcohol, as the major volatile constituent of patchouli oil, has been found to strongly inhibit", "H1N1 replication and weakly inhibit B/Ibaraki/2/85 replication [18] . To the best of our knowledge, the anti-influenza virus (H2N2) activities of patchouli alcohol have not been evaluated yet. Therefore, the aim of the present study was to evaluate the anti-influenza A virus (H2N2) activity of patchouli alcohol by MTT assay and mouse influenza model. On such basis, explicitly solvated docking and molecular dynamic (MD) methods were applied to investigative the binding mode involving patchouli alcohol with influenza virus NA protein. We anticipate that the insight into the understanding of inhibiting mechanism will be of value in the rational design of novel anti-influenza drugs.", "First the efficacy of patchouli alcohol on influenza A (H2N2) virus replication and cell viability were examined. CC 50 was used to express the cytotoxicity of patchouli alcohol on MDCK. The CC 50 of patchouli alcohol was above 20 mM, which indicated that patchouli alcohol did not affect the growth of MDCK (Table 1) . Thus, it seems that the antiviral effects of patchouli alcohol were not due to the cytotoxicity. Moreover, patchouli alcohol was found to inhibit influenza A (H2N2) virus with an IC 50 of 4.03 ± 0.23 µM. Based on the IC 50 and CC 50 values, the selectivity index (SI) was calculated as >4.96. It is reported that a SI of 4 or more is appropriate for an antiviral agent [18] , suggesting that patchouli alcohol can be judged to have anti-influenza A (H2N2) virus activity.", "Until now, it has been found that patchouli alcohol showed dose-dependent anti-influenza virus (A/PR/8/34, H1N1) activity, with an IC 50 value of 2.635 µM. Furthermore, it showed weak activity against B/Ibaraki/2/85 (IC 50 = 40.82 µM) [19] . With the addition of the above H2N2 inhibitory activity, we have a comprehensively view of the anti-influenza activity of patchouli alcohol.\n\nCells were pretreated with patchouli alcohol prior to virus infection (pretreatment cells), viruses were pretreated prior to infection (pretreatment virus), and patchouli alcohol was added during the adsorption period (adsorption) or after penetration of the viruses into cells (replication). Experiments were repeated independently three times and data presented are the average of three experiments. The symbols * indicated very significant difference p < 0.01 with respect to other mode (pretreatment virus, adsorption and pretreatment cell).", "As shown in Figure 2 , patchouli alcohol showed anti-influenza A (H2N2) virus activity in a timedependent manner. It showed best antiviral activity when added at a concentration of 8 µM during the replication period with inhibition of the viral replication of 97.68% ± 2.09% for influenza A (H2N2) at 72 h. However, no significant effect was detected when patchouli alcohol was used for pretreatment of cells or viruses or when patchouli alcohol was only added during the adsorption phase. These results suggested that the inhibition of influenza A (H2N2) virus by patchouli alcohol appears to occur much more strongly after penetration of the virus into the cell. Besides, biochemical studies have indicated that the bioactivity of NA protein is essential determinant after the replication of influenza A (H2N2) virus [20] [21] [22] . Hence, we conclude that the function of NA protein may be suppressed by patchouli alcohol.", "To evaluate the toxicity of patchouli alcohol, the mean value of body weight of mice in each group was statistically analyzed. The mean weights of mice administered at the 2 mg/kg/dose oseltamivir, 2 mg/kg/dose patchouli alcohol and 10 mg/kg/dose of patchouli alcohol one time daily for 7 days were not significantly different compared with the normal control mice, showing no toxicity of patchouli alcohol and oseltamivir within the testing concentration (P > 0.05). Physiological status was observed in virus infection mice. Three days after viral infection, some mice, especially mice in the H2N2 infected control group showed changes in behavior, such as a tendency to huddle, diminished vitality, and ruffled fur, etc. In the mouse influenza model, viral infection leads to loss of body weight and high mortality. Therefore, the efficacy of patchouli alcohol and oseltamivir were evaluated on the basis of survival rate measured for 15 days post-infection, for treated infected animals relative", "to untreated infected (control) animals. A comparison of efficacy of patchouli alcohol and oseltamivir in vivo mouse influenza model (oral treatment) showed that at a dose of 5 mg/kg/day, patchouli alcohol showed obvious protection against the influenza virus, as the mean day to death was detected as 11.8 ± 1.1 (Table 2) . When the dose was lowered to 1 mg/kg/day, patchouli alcohol showed weaker protection (measured by Survivors/total) than that of 5 mg/kg/day, the mean day to death was 7.5 ± 1.8. Whereas oseltamivir at this dose level (1 mg/kg/day) showed 50% protection (measured by survivors/total) against the influenza virus. In the H2N2 infected control group, there were no survivors. In view of both in vitro and in vivo data, we conclude that patchouli alcohol could be used in the treatment of human influenza virus infections.", "Based on the above experiment data, patchouli alcohol is determined to be bound within NA protein. As the total energies and backbone root-mean-square-deviations (RMSD) in Figure 3 indicate, the energy-minimized patchouli alcohol-NA complex has been in equilibrium since about 0.5 ns, and then retains quite stable in the last 19.5 ns. It is consistent with the previous MD results of other NA inhibitors [23] [24] [25] [26] [27] [28] . Accordingly, the geometric and energetic analyses were made on the average structures of 0.5~20.0 ns MD trajectories, where the system has been already at equilibrium. The interaction energy (E inter ) of patchouli alcohol with NA was calculated at −40.38 kcal mol −1 , where the vdW rather than electrostatic interactions were found to play a dominant role, contribute to about 72% (−29.18 kcal mol −1 ). As shown in Figure 4 , the patchouli alcohol was bound at the active site which also bound to oseltamivir and zanamivir [28] . As Figure 5 shows, the oxygen", "atom of patchouli alcohol was oriented towards the sidechains of residues Glu119 and Tyr406, with one H-bond formed with each residue. The values of distances in Figure 6 further reveal that the docked complex remains rather stable throughout the simulation, with the average distances of Glu119:OE2patchouli alcohol:O and Tyr406:OH -patchouli alcohol:O less than 2.8 Å. The sum contributions (E sum ) of residues Glu119 and Tyr406 amounted to −8.46 and −7.37 kcal mol −1 , respectively (Table 3) . Besides, patchouli alcohol was stabilized by residues Arg118, Asp151, Arg152, Trp178, Ala246, Glu276, Arg292, Asn294 and Gln347, especially residues Asp151, Arg152 and Glu276 ( Figure 5 and Table 3 ). As a matter of fact, residues Asp151, Arg152, Glu119, Glu276 and Tyr406 of the NA protein have already received enough attention from rational drug designs [14, 30, 31] . The catalytic residues Asp151, Arg152 and Glu276 are crucial to the NA functions and the residues Glu119 and Tyr406 are", "important to stabilize the NA active sites [32, 33] . It suggests that the NA functions will be affected by the presence of patchouli alcohol, consistent with the above experiments. Patchouli alcohol matches with the NA active site and has an acceptable interaction energy. Considering the obvious structure discrepancies against current NA inhibitors, it represents an ideal lead compound for the designs of novel anti-influenza agents.", "Patchouli alcohol and oseltamivir were obtained from Sigma Chemical Co. (St. Louis, MO, USA, purity > 99%) and was stored in glass vials with Teflon sealed caps at −20 ± 0.5 °C in the absence of light.\n\nMDCK (Madin-Darby canine kidney) was purchased from Harbin Veterinary Research Institute (Harbin, Heilongjiang, China). The cells were grown in monolayer culture with Eagle's minimum essential medium (EMEM) supplemented with 10% fetal calf serum (FCS), 100 U/mL penicillin and 100 μg/mL streptomycin. The monolayers were removed from their plastic surfaces and serially passaged whenever they became confluent. Cells were plated out onto 96-well culture plates for cytotoxicity and anti-influenza assays, and propagated at 37 °C in an atmosphere of 5% CO 2 .", "The influenza strain A/Leningrad/134/17/1957 H2N2) was purchased from National Control Institute of Veterinary Bioproducts and Pharmaceuticals (Beijing, China). Virus was routinely grown on MDCK cells. The stock cultures were prepared from supernatants of infected cells and stored at −80 °C.", "The cellular toxicity of patchouli alcohol on MDCK cells was assessed by the MTT method. Briefly, cells were seeded on a microtiter plate in the absence or presence of various concentrations (20 µM -0.0098 µM) of patchouli alcohol (eight replicates) and incubated at 37 °C in a humidified atmosphere of 5% CO 2 for 72 h. The supernatants were discarded, washed with PBS twice and MTT reagent (5 mg/mL in PBS) was added to each well. After incubation at 37 °C for 4 h, the supernatants were removed, then 200 μL DMSO was added and incubated at 37 °C for another 30 min. After that the plates were read on an ELISA reader (Thermo Molecular Devices Co., Union City, USA) at 570/630 nm. The mean OD of the cell control wells was assigned a value of 100%. The maximal non-toxic concentration (TD 0 ) and 50% cytotoxic concentration (CC 50 ) were calculated by linear regression analysis of the dose-response curves generated from the data.", "Inhibition of virus replication was measured by the MTT method. Serial dilution of the treated virus was adsorbed to the cells for 1 h at 37 °C. The residual inoculum was discared and infected cells were added with EMEM containing 2% FCS. Each assay was performed in eight replicates. After incubation for 72 h at 37 °C, the cultures were measured by MTT method as described above. The concentration of patchouli alcohol and oseltamivir which inhibited virus numbers by 50% (IC 50 ) was determined from dose-response curves.", "Cells and viruses were incubated with patchouli alcohol at different stages during the viral infection cycle in order to determine the mode of antiviral action. Cells were pretreated with patchouli alcohol before viral infection, viruses were incubated with patchouli alcohol before infection and cells and viruses were incubated together with patchouli alcohol during adsorption or after penetration of the virus into the host cells. Patchouli alcohol was always used at the nontoxic concentration. Cell monolayers were pretreated with patchouli alcohol prior to inoculation with virus by adding patchouli alcohol to the culture medium and incubation for 1 h at 37 °C. The compound was aspirated and cells were washed immediately before the influenza A (H2N2) inoculum was added. For pretreatment virus, Influenza A (H2N2) was incubated in medium containing patchouli alcohol for 1h at room temperature prior to infection of MDCK cells. For analyzing the anti-influenza A (H2N2) inhibition during", "the adsorption period, the same amount of influenza A (H2N2) was mixed with the drug and added to the cells immediately. After 1 h of adsorption at 37 °C, the inoculum was removed and DMEM supplemented with 2 % FCS were added to the cells. The effect of patchouli alcohol against influenza A (H2N2) was also tested during the replication period by adding it after adsorption, as typical performed in anti-influenza A (H2N2) susceptibility studies. Each assay was run in eight replicates.", "Kunming mice, weighing 18-22 g (6 weeks of age) were purchased from Harbin Veterinary Research Institute Animal Co., Ltd. (Harbin, Heilongjiang, China) . First, the toxicity of patchouli alcohol and oseltamivir was assessed in the healthy mice by the loss of body weight compared with the control group (2% DMSO in physiological saline). The mice were orally administered with 10 mg/kg/dose patchouli alcohol, 2 mg/kg/dose patchouli alcohol or 2 mg/kg/dose oseltamivir (dissolved in 2% DMSO in physiological saline) one time daily for 7 days. The weight of mice was determined daily. We conducted procedures according to Principle of Laboratory Animal Care (NIH Publication No. 85 -23, revised 1985) and the guidelines of the Peking University Animal Research Committee.", "Kunming mice were anesthetized with isoflurane and exposed to virus (A/Leningrad/134/17/1957) by intranasal instillation. Drugs were prepared in 2% DMSO in physiological saline and administered 4 h prior to virus exposure and continued daily for 5 days. All mice were observed daily for changes in weight and for any deaths. Parameters for evaluation of antiviral activity included weight loss, reduction in mortality and/or increase in mean day to death (MDD) determined through 15 days.", "The N2 sub-type neuraminidase crystal structure (PDB code 1IVD) was obtained from the RCSB Protein Data Bank [34] . For convenience, the structure is named as NA hereafter. Geometry and partial atomic charges of the patchouli alcohol ( Figure 1) were calculated with the Discover 3.0 module (Insight II 2005) [35] by applying the BFGS algorithm [36] and the consistent-valence force-field (CVFF), with a convergence criterion of 0.01 kcal mol −1 Å −1 . The docking and molecular dynamics (MD) simulations were performed by the general protocols in the Insight II 2005 software packages, consistent with the previous literatures [24, 26, 28, 35, [37] [38] [39] . During the MD simulations, the canonical ensemble (NVT) was employed at normal temperature (300 K). The MD temperature was controlled by the velocity scaling thermostat [40] . Integrations of the classical equations of motion were achieved using the Verlet algorithm. The systems were solvated in a large sphere of TIP3P water molecules", "[40] with the radius of 35.0 Å, which is enough to hold the ensembles [40] . The MD trajectories were generated using a 1.0-fs time step for a total of 20.0 ns, saved at 5.0-ps intervals. The interaction energies of patchouli alcohol with NA and the respective residues at the NA active site were calculated by the Docking module [35], over the 0.5~20.0 ns MD trajectories.", "All results are expressed as mean values ± standard deviations (SDs) (n = 3). The significance of difference was calculated by one-way analysis of variance, and values p < 0.001 were considered to be significant.\n\nIn conclusion, patchouli alcohol possesses anti-influenza A (H2N2) virus activity via interference with the NA function that cleaves the α-glycosidic bond between sialic acid and glycoconjugate. Our results provide the promising information for the potential use of patchouli alcohol in the treatment of influenza A (H2N2) virus infectious disease. Further mechanistic studies on the anti-influenza A virus activity are needed to support this point of view." ]

[ 25 ]

[ "time, equipment, and technical expertise requirements" ]

[ "Isothermal Amplification Using a Chemical Heating Device for Point-of-Care Detection of HIV-1\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3285652/\n\nSHA: ef7110a9022bac2e50c995b0f6b826ff071e48f8\n\nAuthors: Curtis, Kelly A.; Rudolph, Donna L.; Nejad, Irene; Singleton, Jered; Beddoe, Andy; Weigl, Bernhard; LaBarre, Paul; Owen, S. Michele\nDate: 2012-02-23\nDOI: 10.1371/journal.pone.0031432\nLicense: cc0", "Abstract: BACKGROUND: To date, the use of traditional nucleic acid amplification tests (NAAT) for detection of HIV-1 DNA or RNA has been restricted to laboratory settings due to time, equipment, and technical expertise requirements. The availability of a rapid NAAT with applicability for resource-limited or point-of-care (POC) settings would fill a great need in HIV diagnostics, allowing for timely diagnosis or confirmation of infection status, as well as facilitating the diagnosis of acute infection, screening and evaluation of infants born to HIV-infected mothers. Isothermal amplification methods, such as reverse-transcription, loop-mediated isothermal amplification (RT-LAMP), exhibit characteristics that are ideal for POC settings, since they are typically quicker, easier to perform, and allow for integration into low-tech, portable heating devices. METHODOLOGY/SIGNIFICANT FINDINGS: In this study, we evaluated the HIV-1 RT-LAMP assay using portable, non-instrumented nucleic acid", "amplification (NINA) heating devices that generate heat from the exothermic reaction of calcium oxide and water. The NINA heating devices exhibited stable temperatures throughout the amplification reaction and consistent amplification results between three separate devices and a thermalcycler. The performance of the NINA heaters was validated using whole blood specimens from HIV-1 infected patients. CONCLUSION: The RT-LAMP isothermal amplification method used in conjunction with a chemical heating device provides a portable, rapid and robust NAAT platform that has the potential to facilitate HIV-1 testing in resource-limited settings and POC.", "Text: HIV-1 diagnostic tests are held to a high standard of performance, as diagnosis has a direct impact on patient care and reduction of transmission. Despite technological advances in the field of HIV diagnostics and the high sensitivity and specificity associated with most HIV diagnostic tests that are currently available, it is estimated that approximately 20% of HIV-infected individuals living in the United States remain undiagnosed [1] . Furthermore, testing sites have reported as many as 35 to 50% of individuals with an initial positive test result will not return for a confirmatory diagnosis if follow-up laboratory testing is required [2] . Rapid HIV antibodybased tests, which can be performed with minimal training and typically provide results in under 30 minutes [3] , have facilitated HIV testing at the point-of-care and subsequently increased the numbers of individuals aware of their serostatus [4] . Rapid tests are currently a key component of HIV screening at the", "point-of-care (POC), significantly expanding the diagnostic capabilities of testing sites in developed countries, as well as resource-limited settings.", "Despite the advances made by the widespread availability of rapid tests, all antibody-based tests for the detection of HIV exhibit some limitations. HIV-specific antibody typically begins to appear around three weeks post-infection, allowing for detection by most antibody-based assays within 3-6 weeks [3, 5] . The window of time prior to or during early seroconversion may lead to false-negative test results in recently infected individuals. Additionally, accurate diagnosis of infants born to HIV-infected mothers can be challenging if based solely on antibody positivity, since vertically transferred maternal antibodies may persist for 12-18 months after birth [6, 7] . For confirmatory diagnosis of early HIV infection or infant diagnosis, nucleic acid amplification tests (NAAT) are preferred, as HIV-1 RNA can be detected as early as 10-12 days post infection and HIV-1 DNA and/or RNA are definitive indicators of active infection [5] . In their current form, however, NAAT's are not", "feasible for POC testing, because they are timeconsuming, expensive, and technically complicated. To date, the Aptima HIV-1 RNA assay (Gen-Probe, Inc., http://www.fda.gov/ BiologicsBloodVaccines/BloodBloodProducts/ApprovedProducts/ LicensedProductsBLAs/BloodDonorScreening/InfectiousDisease/ UCM080466) is the only FDA-approved NAAT for the diagnosis or confirmation of HIV-1 infection and it is only suitable for laboratory testing.", "To meet the needs of HIV-1 diagnosis at the POC, a rapid NAAT that can be performed with minimal training, limited equipment, and a relatively short turnaround time (,1 hour)is desirable [8] . The development of a rapid NAAT has proven to be especially challenging since the technology involved in simplifying the test procedure often equates to increased equipment and material costs [8] . Additionally, the reduction in technical complexity should not compromise test sensitivity and specificity. For increased applicability at the POC, an increasing number of novel isothermal amplification techniques have been developed [9] . Isothermal amplification is an attractive alternative to traditional PCR or RT-PCR since thermalcycling is not required, allowing for greater versatility in terms of heating or amplification devices. One such amplification method, termed Loop-Mediated Isothermal Amplification (LAMP) [10] , has been optimized for the detection of DNA and/or RNA (RT-LAMP) from a wide", "range of bacterial and viral pathogens [11, 12, 13, 14, 15, 16, 17, 18, 19] , including HIV [20, 21] .", "LAMP or RT-LAMP exhibits several characteristics that are ideal for integration into a rapid nucleic-acid based diagnostic test. The amplification reaction requires six primers specific for eight separate regions within the target sequence, contributing to the high specificity of the amplification method. Amplified material can typically be detected within 15-60 minutes when incubated at a constant reaction temperature of 60-65uC [22] . LAMP has also proven to be less sensitive to biological inhibitors than PCR [23, 24] , which enables direct amplification from clinical specimens, thereby eliminating the need for an additional nucleic acid extraction step. Direct amplification from plasma, whole blood, and oral fluid has previously been demonstrated for HIV-1 [20, 21, 25] . Lastly, immediate visual detection of amplified products is facilitated by the large amount of DNA that is generated by each reaction. Several groups have incorporated fluorescent detection methods into the LAMP", "assay for real-time or immediate naked-eye detection [15, 17, 21, 22, 26] .", "The simplicity and isothermal nature of the LAMP procedure opens the door for the evaluation of low-tech integrated devices or novel heating elements, which are appropriate for low-resource settings, where costly equipment and electricity cannot be obtained. In this study, the HIV-1 RT-LAMP assay was evaluated using portable, non-instrumented nucleic acid amplification (NINA) devices that generate heat from the exothermic reaction of calcium oxide and water [27, 28] . We demonstrated the temperature stability of the NINA heating devices and feasibility for POC testing of whole blood specimens from HIV-1 infected individuals.", "Prototype NINA heaters were designed and provided by Program for Appropriate Technology in Health (PATH, Seattle, WA), as described [27, 28] . Briefly, an amplification temperature of approximately 60uC was provided by the exothermic reaction of calcium oxide (CaO; Sigma-Aldrich, St. Louis, MO) and water. The heating devices, containing the chemical reaction, were designed using thermally insulated, stainless-steel canisters with plastic screw-top lids (Fig. 1) . The lids were modified to contain three sample wells that fit standard 200 ml PCR tubes and were filled with a proprietary phase-change material (PCM) that was used to buffer the heat derived from the exothermic reaction, thereby providing a constant temperature. Lastly, plastic caps containing foam insulation were designed to fit on the top of the canister lids. The thermal profiles of the sample wells were measured and recorded using a digital thermometer (DaqPRO 5300 Data recorder; OMEGA Engineering, Inc., Stamford, CT).", "DNA and RNA linearity panels were prepared to determine the sensitivity of the HIV-specific RT-LAMP assay. A DNA panel was generated from DNA extracted from the human monocytic cell line OM-10.1 [29] , using a QIAamp DNA blood mini kit (QIAGEN, Valencia, CA). Cell count was used to quantify the input DNA copy number, as a single integrated provirus is contained in each cell [29] . The extracted DNA was diluted tenfold in RNase-free water to create a linearity panel, ranging from 10 5 copies/ml to 10 3 copies/ml. An RNA linearity panel was obtained commercially (PRD801; SeraCare Life Sciences, Mil- ford, MA) and ranged from 2.9610 6 copies/ml to 8 copies/ml, as determined by Roche AMPLICOR HIV MONITOR TM v 1.5, Bayer VERSANT HIV-1 RNA bDNA 3.0 Assay, bioMerieux NucliSensH HIV-1 QT, and Abbott Real Time HIV-1 m2000 TM . RNA was extracted from the panel members using a Viral RNA mini kit (QIAGEN). Negative controls included DNA extracted from PBMC infected with HIV-2 SLRHC [30] and RNA", "extracted from HIV-2 NIH-Z purified virus (Advanced Biotechnologies Inc., Columbia, MD).", "Whole blood from HIV-1 infected individuals was collected as part of a separate, IRB-approved study [31] , or obtained commercially (SeraCare Life Sciences). All HIV-positive samples were confirmed using the following tests: Genetic Systems HIV-1/ HIV-2 plus O EIA (Bio-Rad Laboratories, Redmond, WA), GS HIV-1 Western blot (Bio-Rad Laboratories), Aptima HIV-1 RNA assay (Gen-Probe, Inc., San Diego, CA), and Amplicor HIV-1 DNA assay (Roche Diagnostics, Branchburg, NJ ). Viral and proviral loads are unknown, since the samples were tested with qualitative, nucleic acid-based assays. All clinical specimens evaluated in this study were obtained from individuals infected with subtype B HIV-1 virus. As a negative control, HIV-1 seronegative blood samples (SeraCare Life Sciences) were included in every experiment involving whole blood. A positive control included HIV-1 seronegative blood spiked with 5610 6 virus particles/ml of HIV-1 BaL (Advanced Biotechnologies Inc.).", "HIV-1-specific RT-LAMP primers were designed to recognize a conserved sequence within the reverse transcriptase (RT) gene. The six primers required for the RT-LAMP reaction, forward outer (F3), backward outer (B3), forward inner (FIP), backward inner (BIP), and the loop primers (LoopF and LoopB), were designed using the PrimerExplorer V4 software (Eiken Chemical Co. Ltd.; http:// primerexplorer.jp/e/). The LAMP primers and amplification cycle have been described in detail by Nagamine et al. [32] . Additional modifications included a linker sequence of four thymidines inserted between the F2 and F1c sequences of the FIP primer, as described [20] , and the addition of the fluorescent molecule HEX to the 59 end of the LoopF primer. The labeled primer, along with a quencher probe, allowed for immediate visual detection of amplified products [21] . The quencher probe consisted of the complementary sequence of the LoopF primer with Black Hole Quencher-1 (BHQ-1) added to the 39 end. The", "HIV-1 HXB2 sequence (GenBank accession number AF033819) was used as the reference for generating the RT-LAMP primers. The sequences of the HIV-1 RT-specific primers and quencher are listed in Table 1 .", "The RT-LAMP reaction was performed using the following reaction mix: 0.2 mM (final concentration) of each F3 and B3 primers, 1.6 mM of each FIP and BIP primers, 0.8 mM of each LoopF and HEX-LoopB primers, 0.8 M betaine (Sigma-Aldrich), 10 mM MgSO 4 , 1.4 mM dNTPs, 16 ThermoPol reaction buffer (New England Biolabs, Ipswich, MA), 16 U Bst DNA polymerase (New England Biolabs) and 2 U AMV reverse transcriptase (Invitrogen, Carlsbad, CA). The reaction was carried out in a total volume of 25 ml for amplification of extracted nucleic acid, 10 ml of which constituted the sample. For amplification of whole blood specimens, a 100 ml reaction volume was used to facilitate visual detection of amplified products. Whole blood was added directly into the reaction at a total volume of 40 ml, following a 1:4 dilution with red blood cell lysis buffer (2.5 mM KHCO 3 , 37.5 mM NH 4 Cl, and 0.025 mM EDTA), as previously described [21] . The reaction mixture was incubated at 60uC for 60 minutes, using a", "GeneAmpH PCR System (Applied Biosystems, Foster City, CA) or the NINA heaters. For reactions amplified in the thermalcylcer, an additional two minute heating step of 80uC was added at the end of the amplification cycle to terminate the reaction.", "The reaction tubes were evaluated for the presence of amplification, following addition of the quencher probe at a 2:1 ratio of quencher to labeled-primer, as previously described [21] . Amplification was determined visually by observing fluorescence in the reaction tubes, using the UV lamp from a ChemiDoc XRS system (Bio-Rad Laboratories, Hercules, CA). Amplification was confirmed by electrophoresis using a 1.2% agarose gel containing SYBRH Safe gel stain (Invitrogen), which was subsequently visualized using the ChemiDoc XRS system.", "To compare temperature and amplification consistency, three NINA heaters were tested in parallel. The heating reaction was initiated by adding 18 g of CaO to each NINA canister, followed by 6 ml of water. The lid of each canister was then sealed to contain the exothermic reaction. After adding 200 ml of water to each of the sample wells, temperature recording was initiated. Reaction tubes were added to the sample wells once each reaction chamber reached a temperature of 58.5uC. For all samples incubated in the NINA heater, 15 ml of mineral oil was added to the reaction tube during the reaction mix preparation. The samples were incubated in the heaters for a total of 60 minutes. All reactions were carried out in a temperature-controlled laboratory with an ambient temperature of 28uC, unless otherwise stated. Following the amplification reaction, the samples were incubated for two minutes in a heat block set to 80uC. After each amplification cycle, the temperature profile of each device", "was analyzed by calculating the temperature mean, standard deviation, median, minimum, and maximum from the data provided by the DaqPRO 5300.", "The stability of the NINA heaters at extreme low and high temperatures was evaluated by placing the canisters in a refrigerator set to 4uC or a 37uC incubator during the length of the amplification reaction. The temperature profiles were recorded and compared to those of reactions that occurred at the laboratory room temperature of 28uC.", "To determine the sensitivity of RT-LAMP reaction using RTspecific primers, DNA and RNA linearity panels were tested in a thermalcycler. The limit of detection for HIV-1 DNA was 10 copies/reaction. For the RNA linearity panel, the sample containing 1700 copies/reaction was detected in all of the three replicates, while the sample containing 140 copies/reaction was detected in three out of five replicates (60%). For both DNA and RNA linearity panels, the two samples nearest the limit of detection were chosen to further evaluate the performance consistency between the thermalcycler and NINA heaters. In terms of positivity, the amplification results were consistent between all three heaters and the thermalcycler ( Table 2) . Since the RT-LAMP assay requires a constant temperature of 60uC for the length of the amplification reaction, the temperature profiles of the sample wells were compared over the course of the incubation and between all three NINA heaters. A representative temperature", "profile is displayed in Figure 2 , showing a steady reaction temperature at or close to 60uC for length of amplification reaction. During the 60 minute incubation, the average temperature for each device was 60.2, 59.8, and 59.7 (Table 3 ). The minimum temperature achieved during the reaction reflects the fact that the temperature of the sample port dropped temporarily after the sample tubes are added to the device, as shown in Figure 2 . The maximum temperature of the devices deviated from the desired reaction temperature of 60uC by less than one degree.", "The ability of the NINA heaters to maintain a steady reaction temperature in a wide range of ambient temperatures is essential for POC testing, whether referring to an air-conditioned laboratory or high-temperature field site. To evaluate the performance of the NINA heaters at extreme low or high temperatures, the canisters were placed in a 4uC refrigerator or a 37uC incubator for the length of the amplification reaction. The limit of detection for the DNA and RNA linearity panels was similar to the results obtained in our temperature-controlled laboratory (28uC; Table 2 ). The greatest degree of temperature variation of the sample wells was observed at the ambient temperature of 4uC ( Table 3 ). The average temperature was approximately two degrees lower than the desired reaction temperature of 60uC. Additionally, the temperature of the devices tended to decline from their steady state during the last 20 minutes of the reaction (data not shown). The temperature profiles at the", "ambient temperature of 37uC, however, were similar to those at 28uC.", "Whole blood samples from HIV-1 infected individuals were added directly into the RT-LAMP reaction and tested in the NINA heaters. Positivity of the clinical specimens was consistent between the thermalcycler and devices (Table 4 ). Amplification consistency was most evident with two of the patient samples (patient #4 and #5) that were only positive in one of the three replicates, regardless of the heating device that was used. All HIVnegative blood samples, included in each reaction, were negative (data not shown). A representative experiment using the NINA heaters is displayed in Figure 3 , showing detection by agarose gel and visual identification of fluorescence in the reaction tubes.", "In this study, we demonstrate the performance of portable, inexpensive, non-instrumented nucleic acid (NINA) heaters for amplification of HIV-1 using RT-LAMP. The isothermal amplification reaction coupled with a device that generates heat from an exothermic chemical reaction, as opposed to grid electricity or battery power, comprises a point-of-care NAAT that is practical for use in resource-limited settings. The heating devices require minimal training and technical expertise to operate and take approximately 10-15 minutes to reach a reaction temperature of 60uC once the chemical reaction has been initiated [27, 28] . Furthermore, the temperature of the sample wells remain relatively stable at the desired reaction temperature of 60uC throughout the amplification reaction, as demonstrated by the heating profiles and the consistency in amplification between the devices and thermalcycler.", "Since point-of-care testing may refer to an air-conditioned laboratory or a field site with high temperatures and humidity, the stability of the temperature generated by the heating devices must be reliable. Though the temperature profiles at a representative cold temperature of 4uC indicated a loss in reaction temperature towards the end of the 60 minute incubation, the temperature fluctuations were not significant enough to affect the amplification reaction. Regardless, this thermal effect could be mitigated with small modifications to the device to reduce heat loss at lower temperatures. It should be possible to extend the temperature range of the NINA heaters to 4uC and below by either adding a larger quantity of heating mixture, better insulation, or both. Of greater concern is the performance of the NINA heaters in hightemperature field sites, where temperature control is not an option.", "We demonstrate no difference in the temperature stability of the NINA heaters and amplification consistency at an ambient temperature of 37uC as compared to our temperature-controlled laboratory.", "For increased applicability for use at the POC, several modifications can be made to the NINA heaters. The prototype devices evaluated in this study contained only three sample wells; however, up to 16 sample wells can be added to the lid of the insulated canisters for a larger testing volume. In this study, samples were removed from the NINA heaters after the amplification reaction and heated for an additional two minutes in an 80uC heat block to terminate the reaction. While the additional heating step is not necessary to observe the amplified products from extracted nucleic acid, the short, high-temperature incubation facilitates the visual observation of the fluorescent label in the whole blood samples. Modifications may be made to the whole blood sample preparation method to eliminate the need for the heating step. Alternatively, a second temperature-moderating compartment can be added to the alternate end of the NINA canisters, so the samples can be removed from the", "amplification compartment and reinserted into the 80uC compartment. Lastly, the DaqPRO data recorder was used in this study for validation purposes only and would not be necessary for the final POC product.", "The feasibility of using LAMP as a diagnostic method in resource-limited settings has been demonstrated for tuberculosis [33] . To reduce hands-on time and preparation error, the authors describe the use of reaction tubes pre-prepared with lyophilized reaction mix. For POC use, limited sample manipulation and reagent preparation is desired and, therefore, it is anticipated that the test procedure of the end product will include reconstituting the amplification reagents in water and adding the sample directly into the reaction tube. We demonstrate the use of the NINA heaters for amplification directly from whole blood specimens, eliminating the need for a time-consuming, nucleic acid extraction procedure and reducing the volume of sample needed for the amplification reaction. A total volume of 10 ml of whole blood was added to each reaction tube, which can easily be obtained by finger-stick in settings where venipuncture is not feasible. Additionally, our fluorescent detection method", "enables immediate visualization of amplified products in the absence of specialized equipment. To avoid cross-contamination of amplified material, it is preferred that the reaction tubes remain closed post-amplification. Future modifications will include optimizing the labeledprimer/quencher sequences so that all components can be added into the reaction mix prior to amplification. Due to availability, the Bio-Rad ChemiDoc system was used as the UV source in this study; however, an inexpensive keychain light would be more suitable for naked-eye detection at the POC. For sensitive and specific detection of diverse HIV-1 isolates, including non-B subtypes, identification of the optimal primer set/sets is a key step in the development of the RT-LAMP assay. Although all experiments performed in this study involved subtype B standards and specimens, ongoing research involves the continued development and optimization of RT-LAMP primers based on regions of the HIV-1 genome that are", "conserved among diverse subtypes. Future studies will include large-scale evaluation of clinical specimens with the optimized RT-LAMP assay and NINA device. In summary, the RT-LAMP isothermal amplification method used in conjunction with a simplified, chemical heating device exhibits characteristics that are ideal for a rapid NAAT for POC testing. The simplified, portable assay has the potential to fill an important gap in HIV-1 diagnostics, providing immediate knowledge or confirmation of HIV-1 infection status at the POC." ]

[ 1 ]

[ "HIV-1 RT-LAMP assay" ]

[ "Isothermal Amplification Using a Chemical Heating Device for Point-of-Care Detection of HIV-1\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3285652/\n\nSHA: ef7110a9022bac2e50c995b0f6b826ff071e48f8\n\nAuthors: Curtis, Kelly A.; Rudolph, Donna L.; Nejad, Irene; Singleton, Jered; Beddoe, Andy; Weigl, Bernhard; LaBarre, Paul; Owen, S. Michele\nDate: 2012-02-23\nDOI: 10.1371/journal.pone.0031432\nLicense: cc0", "Abstract: BACKGROUND: To date, the use of traditional nucleic acid amplification tests (NAAT) for detection of HIV-1 DNA or RNA has been restricted to laboratory settings due to time, equipment, and technical expertise requirements. The availability of a rapid NAAT with applicability for resource-limited or point-of-care (POC) settings would fill a great need in HIV diagnostics, allowing for timely diagnosis or confirmation of infection status, as well as facilitating the diagnosis of acute infection, screening and evaluation of infants born to HIV-infected mothers. Isothermal amplification methods, such as reverse-transcription, loop-mediated isothermal amplification (RT-LAMP), exhibit characteristics that are ideal for POC settings, since they are typically quicker, easier to perform, and allow for integration into low-tech, portable heating devices. METHODOLOGY/SIGNIFICANT FINDINGS: In this study, we evaluated the HIV-1 RT-LAMP assay using portable, non-instrumented nucleic acid", "amplification (NINA) heating devices that generate heat from the exothermic reaction of calcium oxide and water. The NINA heating devices exhibited stable temperatures throughout the amplification reaction and consistent amplification results between three separate devices and a thermalcycler. The performance of the NINA heaters was validated using whole blood specimens from HIV-1 infected patients. CONCLUSION: The RT-LAMP isothermal amplification method used in conjunction with a chemical heating device provides a portable, rapid and robust NAAT platform that has the potential to facilitate HIV-1 testing in resource-limited settings and POC.", "Text: HIV-1 diagnostic tests are held to a high standard of performance, as diagnosis has a direct impact on patient care and reduction of transmission. Despite technological advances in the field of HIV diagnostics and the high sensitivity and specificity associated with most HIV diagnostic tests that are currently available, it is estimated that approximately 20% of HIV-infected individuals living in the United States remain undiagnosed [1] . Furthermore, testing sites have reported as many as 35 to 50% of individuals with an initial positive test result will not return for a confirmatory diagnosis if follow-up laboratory testing is required [2] . Rapid HIV antibodybased tests, which can be performed with minimal training and typically provide results in under 30 minutes [3] , have facilitated HIV testing at the point-of-care and subsequently increased the numbers of individuals aware of their serostatus [4] . Rapid tests are currently a key component of HIV screening at the", "point-of-care (POC), significantly expanding the diagnostic capabilities of testing sites in developed countries, as well as resource-limited settings.", "Despite the advances made by the widespread availability of rapid tests, all antibody-based tests for the detection of HIV exhibit some limitations. HIV-specific antibody typically begins to appear around three weeks post-infection, allowing for detection by most antibody-based assays within 3-6 weeks [3, 5] . The window of time prior to or during early seroconversion may lead to false-negative test results in recently infected individuals. Additionally, accurate diagnosis of infants born to HIV-infected mothers can be challenging if based solely on antibody positivity, since vertically transferred maternal antibodies may persist for 12-18 months after birth [6, 7] . For confirmatory diagnosis of early HIV infection or infant diagnosis, nucleic acid amplification tests (NAAT) are preferred, as HIV-1 RNA can be detected as early as 10-12 days post infection and HIV-1 DNA and/or RNA are definitive indicators of active infection [5] . In their current form, however, NAAT's are not", "feasible for POC testing, because they are timeconsuming, expensive, and technically complicated. To date, the Aptima HIV-1 RNA assay (Gen-Probe, Inc., http://www.fda.gov/ BiologicsBloodVaccines/BloodBloodProducts/ApprovedProducts/ LicensedProductsBLAs/BloodDonorScreening/InfectiousDisease/ UCM080466) is the only FDA-approved NAAT for the diagnosis or confirmation of HIV-1 infection and it is only suitable for laboratory testing.", "To meet the needs of HIV-1 diagnosis at the POC, a rapid NAAT that can be performed with minimal training, limited equipment, and a relatively short turnaround time (,1 hour)is desirable [8] . The development of a rapid NAAT has proven to be especially challenging since the technology involved in simplifying the test procedure often equates to increased equipment and material costs [8] . Additionally, the reduction in technical complexity should not compromise test sensitivity and specificity. For increased applicability at the POC, an increasing number of novel isothermal amplification techniques have been developed [9] . Isothermal amplification is an attractive alternative to traditional PCR or RT-PCR since thermalcycling is not required, allowing for greater versatility in terms of heating or amplification devices. One such amplification method, termed Loop-Mediated Isothermal Amplification (LAMP) [10] , has been optimized for the detection of DNA and/or RNA (RT-LAMP) from a wide", "range of bacterial and viral pathogens [11, 12, 13, 14, 15, 16, 17, 18, 19] , including HIV [20, 21] .", "LAMP or RT-LAMP exhibits several characteristics that are ideal for integration into a rapid nucleic-acid based diagnostic test. The amplification reaction requires six primers specific for eight separate regions within the target sequence, contributing to the high specificity of the amplification method. Amplified material can typically be detected within 15-60 minutes when incubated at a constant reaction temperature of 60-65uC [22] . LAMP has also proven to be less sensitive to biological inhibitors than PCR [23, 24] , which enables direct amplification from clinical specimens, thereby eliminating the need for an additional nucleic acid extraction step. Direct amplification from plasma, whole blood, and oral fluid has previously been demonstrated for HIV-1 [20, 21, 25] . Lastly, immediate visual detection of amplified products is facilitated by the large amount of DNA that is generated by each reaction. Several groups have incorporated fluorescent detection methods into the LAMP", "assay for real-time or immediate naked-eye detection [15, 17, 21, 22, 26] .", "The simplicity and isothermal nature of the LAMP procedure opens the door for the evaluation of low-tech integrated devices or novel heating elements, which are appropriate for low-resource settings, where costly equipment and electricity cannot be obtained. In this study, the HIV-1 RT-LAMP assay was evaluated using portable, non-instrumented nucleic acid amplification (NINA) devices that generate heat from the exothermic reaction of calcium oxide and water [27, 28] . We demonstrated the temperature stability of the NINA heating devices and feasibility for POC testing of whole blood specimens from HIV-1 infected individuals.", "Prototype NINA heaters were designed and provided by Program for Appropriate Technology in Health (PATH, Seattle, WA), as described [27, 28] . Briefly, an amplification temperature of approximately 60uC was provided by the exothermic reaction of calcium oxide (CaO; Sigma-Aldrich, St. Louis, MO) and water. The heating devices, containing the chemical reaction, were designed using thermally insulated, stainless-steel canisters with plastic screw-top lids (Fig. 1) . The lids were modified to contain three sample wells that fit standard 200 ml PCR tubes and were filled with a proprietary phase-change material (PCM) that was used to buffer the heat derived from the exothermic reaction, thereby providing a constant temperature. Lastly, plastic caps containing foam insulation were designed to fit on the top of the canister lids. The thermal profiles of the sample wells were measured and recorded using a digital thermometer (DaqPRO 5300 Data recorder; OMEGA Engineering, Inc., Stamford, CT).", "DNA and RNA linearity panels were prepared to determine the sensitivity of the HIV-specific RT-LAMP assay. A DNA panel was generated from DNA extracted from the human monocytic cell line OM-10.1 [29] , using a QIAamp DNA blood mini kit (QIAGEN, Valencia, CA). Cell count was used to quantify the input DNA copy number, as a single integrated provirus is contained in each cell [29] . The extracted DNA was diluted tenfold in RNase-free water to create a linearity panel, ranging from 10 5 copies/ml to 10 3 copies/ml. An RNA linearity panel was obtained commercially (PRD801; SeraCare Life Sciences, Mil- ford, MA) and ranged from 2.9610 6 copies/ml to 8 copies/ml, as determined by Roche AMPLICOR HIV MONITOR TM v 1.5, Bayer VERSANT HIV-1 RNA bDNA 3.0 Assay, bioMerieux NucliSensH HIV-1 QT, and Abbott Real Time HIV-1 m2000 TM . RNA was extracted from the panel members using a Viral RNA mini kit (QIAGEN). Negative controls included DNA extracted from PBMC infected with HIV-2 SLRHC [30] and RNA", "extracted from HIV-2 NIH-Z purified virus (Advanced Biotechnologies Inc., Columbia, MD).", "Whole blood from HIV-1 infected individuals was collected as part of a separate, IRB-approved study [31] , or obtained commercially (SeraCare Life Sciences). All HIV-positive samples were confirmed using the following tests: Genetic Systems HIV-1/ HIV-2 plus O EIA (Bio-Rad Laboratories, Redmond, WA), GS HIV-1 Western blot (Bio-Rad Laboratories), Aptima HIV-1 RNA assay (Gen-Probe, Inc., San Diego, CA), and Amplicor HIV-1 DNA assay (Roche Diagnostics, Branchburg, NJ ). Viral and proviral loads are unknown, since the samples were tested with qualitative, nucleic acid-based assays. All clinical specimens evaluated in this study were obtained from individuals infected with subtype B HIV-1 virus. As a negative control, HIV-1 seronegative blood samples (SeraCare Life Sciences) were included in every experiment involving whole blood. A positive control included HIV-1 seronegative blood spiked with 5610 6 virus particles/ml of HIV-1 BaL (Advanced Biotechnologies Inc.).", "HIV-1-specific RT-LAMP primers were designed to recognize a conserved sequence within the reverse transcriptase (RT) gene. The six primers required for the RT-LAMP reaction, forward outer (F3), backward outer (B3), forward inner (FIP), backward inner (BIP), and the loop primers (LoopF and LoopB), were designed using the PrimerExplorer V4 software (Eiken Chemical Co. Ltd.; http:// primerexplorer.jp/e/). The LAMP primers and amplification cycle have been described in detail by Nagamine et al. [32] . Additional modifications included a linker sequence of four thymidines inserted between the F2 and F1c sequences of the FIP primer, as described [20] , and the addition of the fluorescent molecule HEX to the 59 end of the LoopF primer. The labeled primer, along with a quencher probe, allowed for immediate visual detection of amplified products [21] . The quencher probe consisted of the complementary sequence of the LoopF primer with Black Hole Quencher-1 (BHQ-1) added to the 39 end. The", "HIV-1 HXB2 sequence (GenBank accession number AF033819) was used as the reference for generating the RT-LAMP primers. The sequences of the HIV-1 RT-specific primers and quencher are listed in Table 1 .", "The RT-LAMP reaction was performed using the following reaction mix: 0.2 mM (final concentration) of each F3 and B3 primers, 1.6 mM of each FIP and BIP primers, 0.8 mM of each LoopF and HEX-LoopB primers, 0.8 M betaine (Sigma-Aldrich), 10 mM MgSO 4 , 1.4 mM dNTPs, 16 ThermoPol reaction buffer (New England Biolabs, Ipswich, MA), 16 U Bst DNA polymerase (New England Biolabs) and 2 U AMV reverse transcriptase (Invitrogen, Carlsbad, CA). The reaction was carried out in a total volume of 25 ml for amplification of extracted nucleic acid, 10 ml of which constituted the sample. For amplification of whole blood specimens, a 100 ml reaction volume was used to facilitate visual detection of amplified products. Whole blood was added directly into the reaction at a total volume of 40 ml, following a 1:4 dilution with red blood cell lysis buffer (2.5 mM KHCO 3 , 37.5 mM NH 4 Cl, and 0.025 mM EDTA), as previously described [21] . The reaction mixture was incubated at 60uC for 60 minutes, using a", "GeneAmpH PCR System (Applied Biosystems, Foster City, CA) or the NINA heaters. For reactions amplified in the thermalcylcer, an additional two minute heating step of 80uC was added at the end of the amplification cycle to terminate the reaction.", "The reaction tubes were evaluated for the presence of amplification, following addition of the quencher probe at a 2:1 ratio of quencher to labeled-primer, as previously described [21] . Amplification was determined visually by observing fluorescence in the reaction tubes, using the UV lamp from a ChemiDoc XRS system (Bio-Rad Laboratories, Hercules, CA). Amplification was confirmed by electrophoresis using a 1.2% agarose gel containing SYBRH Safe gel stain (Invitrogen), which was subsequently visualized using the ChemiDoc XRS system.", "To compare temperature and amplification consistency, three NINA heaters were tested in parallel. The heating reaction was initiated by adding 18 g of CaO to each NINA canister, followed by 6 ml of water. The lid of each canister was then sealed to contain the exothermic reaction. After adding 200 ml of water to each of the sample wells, temperature recording was initiated. Reaction tubes were added to the sample wells once each reaction chamber reached a temperature of 58.5uC. For all samples incubated in the NINA heater, 15 ml of mineral oil was added to the reaction tube during the reaction mix preparation. The samples were incubated in the heaters for a total of 60 minutes. All reactions were carried out in a temperature-controlled laboratory with an ambient temperature of 28uC, unless otherwise stated. Following the amplification reaction, the samples were incubated for two minutes in a heat block set to 80uC. After each amplification cycle, the temperature profile of each device", "was analyzed by calculating the temperature mean, standard deviation, median, minimum, and maximum from the data provided by the DaqPRO 5300.", "The stability of the NINA heaters at extreme low and high temperatures was evaluated by placing the canisters in a refrigerator set to 4uC or a 37uC incubator during the length of the amplification reaction. The temperature profiles were recorded and compared to those of reactions that occurred at the laboratory room temperature of 28uC.", "To determine the sensitivity of RT-LAMP reaction using RTspecific primers, DNA and RNA linearity panels were tested in a thermalcycler. The limit of detection for HIV-1 DNA was 10 copies/reaction. For the RNA linearity panel, the sample containing 1700 copies/reaction was detected in all of the three replicates, while the sample containing 140 copies/reaction was detected in three out of five replicates (60%). For both DNA and RNA linearity panels, the two samples nearest the limit of detection were chosen to further evaluate the performance consistency between the thermalcycler and NINA heaters. In terms of positivity, the amplification results were consistent between all three heaters and the thermalcycler ( Table 2) . Since the RT-LAMP assay requires a constant temperature of 60uC for the length of the amplification reaction, the temperature profiles of the sample wells were compared over the course of the incubation and between all three NINA heaters. A representative temperature", "profile is displayed in Figure 2 , showing a steady reaction temperature at or close to 60uC for length of amplification reaction. During the 60 minute incubation, the average temperature for each device was 60.2, 59.8, and 59.7 (Table 3 ). The minimum temperature achieved during the reaction reflects the fact that the temperature of the sample port dropped temporarily after the sample tubes are added to the device, as shown in Figure 2 . The maximum temperature of the devices deviated from the desired reaction temperature of 60uC by less than one degree.", "The ability of the NINA heaters to maintain a steady reaction temperature in a wide range of ambient temperatures is essential for POC testing, whether referring to an air-conditioned laboratory or high-temperature field site. To evaluate the performance of the NINA heaters at extreme low or high temperatures, the canisters were placed in a 4uC refrigerator or a 37uC incubator for the length of the amplification reaction. The limit of detection for the DNA and RNA linearity panels was similar to the results obtained in our temperature-controlled laboratory (28uC; Table 2 ). The greatest degree of temperature variation of the sample wells was observed at the ambient temperature of 4uC ( Table 3 ). The average temperature was approximately two degrees lower than the desired reaction temperature of 60uC. Additionally, the temperature of the devices tended to decline from their steady state during the last 20 minutes of the reaction (data not shown). The temperature profiles at the", "ambient temperature of 37uC, however, were similar to those at 28uC.", "Whole blood samples from HIV-1 infected individuals were added directly into the RT-LAMP reaction and tested in the NINA heaters. Positivity of the clinical specimens was consistent between the thermalcycler and devices (Table 4 ). Amplification consistency was most evident with two of the patient samples (patient #4 and #5) that were only positive in one of the three replicates, regardless of the heating device that was used. All HIVnegative blood samples, included in each reaction, were negative (data not shown). A representative experiment using the NINA heaters is displayed in Figure 3 , showing detection by agarose gel and visual identification of fluorescence in the reaction tubes.", "In this study, we demonstrate the performance of portable, inexpensive, non-instrumented nucleic acid (NINA) heaters for amplification of HIV-1 using RT-LAMP. The isothermal amplification reaction coupled with a device that generates heat from an exothermic chemical reaction, as opposed to grid electricity or battery power, comprises a point-of-care NAAT that is practical for use in resource-limited settings. The heating devices require minimal training and technical expertise to operate and take approximately 10-15 minutes to reach a reaction temperature of 60uC once the chemical reaction has been initiated [27, 28] . Furthermore, the temperature of the sample wells remain relatively stable at the desired reaction temperature of 60uC throughout the amplification reaction, as demonstrated by the heating profiles and the consistency in amplification between the devices and thermalcycler.", "Since point-of-care testing may refer to an air-conditioned laboratory or a field site with high temperatures and humidity, the stability of the temperature generated by the heating devices must be reliable. Though the temperature profiles at a representative cold temperature of 4uC indicated a loss in reaction temperature towards the end of the 60 minute incubation, the temperature fluctuations were not significant enough to affect the amplification reaction. Regardless, this thermal effect could be mitigated with small modifications to the device to reduce heat loss at lower temperatures. It should be possible to extend the temperature range of the NINA heaters to 4uC and below by either adding a larger quantity of heating mixture, better insulation, or both. Of greater concern is the performance of the NINA heaters in hightemperature field sites, where temperature control is not an option.", "We demonstrate no difference in the temperature stability of the NINA heaters and amplification consistency at an ambient temperature of 37uC as compared to our temperature-controlled laboratory.", "For increased applicability for use at the POC, several modifications can be made to the NINA heaters. The prototype devices evaluated in this study contained only three sample wells; however, up to 16 sample wells can be added to the lid of the insulated canisters for a larger testing volume. In this study, samples were removed from the NINA heaters after the amplification reaction and heated for an additional two minutes in an 80uC heat block to terminate the reaction. While the additional heating step is not necessary to observe the amplified products from extracted nucleic acid, the short, high-temperature incubation facilitates the visual observation of the fluorescent label in the whole blood samples. Modifications may be made to the whole blood sample preparation method to eliminate the need for the heating step. Alternatively, a second temperature-moderating compartment can be added to the alternate end of the NINA canisters, so the samples can be removed from the", "amplification compartment and reinserted into the 80uC compartment. Lastly, the DaqPRO data recorder was used in this study for validation purposes only and would not be necessary for the final POC product.", "The feasibility of using LAMP as a diagnostic method in resource-limited settings has been demonstrated for tuberculosis [33] . To reduce hands-on time and preparation error, the authors describe the use of reaction tubes pre-prepared with lyophilized reaction mix. For POC use, limited sample manipulation and reagent preparation is desired and, therefore, it is anticipated that the test procedure of the end product will include reconstituting the amplification reagents in water and adding the sample directly into the reaction tube. We demonstrate the use of the NINA heaters for amplification directly from whole blood specimens, eliminating the need for a time-consuming, nucleic acid extraction procedure and reducing the volume of sample needed for the amplification reaction. A total volume of 10 ml of whole blood was added to each reaction tube, which can easily be obtained by finger-stick in settings where venipuncture is not feasible. Additionally, our fluorescent detection method", "enables immediate visualization of amplified products in the absence of specialized equipment. To avoid cross-contamination of amplified material, it is preferred that the reaction tubes remain closed post-amplification. Future modifications will include optimizing the labeledprimer/quencher sequences so that all components can be added into the reaction mix prior to amplification. Due to availability, the Bio-Rad ChemiDoc system was used as the UV source in this study; however, an inexpensive keychain light would be more suitable for naked-eye detection at the POC. For sensitive and specific detection of diverse HIV-1 isolates, including non-B subtypes, identification of the optimal primer set/sets is a key step in the development of the RT-LAMP assay. Although all experiments performed in this study involved subtype B standards and specimens, ongoing research involves the continued development and optimization of RT-LAMP primers based on regions of the HIV-1 genome that are", "conserved among diverse subtypes. Future studies will include large-scale evaluation of clinical specimens with the optimized RT-LAMP assay and NINA device. In summary, the RT-LAMP isothermal amplification method used in conjunction with a simplified, chemical heating device exhibits characteristics that are ideal for a rapid NAAT for POC testing. The simplified, portable assay has the potential to fill an important gap in HIV-1 diagnostics, providing immediate knowledge or confirmation of HIV-1 infection status at the POC." ]

[ 1 ]

[ "whole blood specimens" ]

[ "Isothermal Amplification Using a Chemical Heating Device for Point-of-Care Detection of HIV-1\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3285652/\n\nSHA: ef7110a9022bac2e50c995b0f6b826ff071e48f8\n\nAuthors: Curtis, Kelly A.; Rudolph, Donna L.; Nejad, Irene; Singleton, Jered; Beddoe, Andy; Weigl, Bernhard; LaBarre, Paul; Owen, S. Michele\nDate: 2012-02-23\nDOI: 10.1371/journal.pone.0031432\nLicense: cc0", "Abstract: BACKGROUND: To date, the use of traditional nucleic acid amplification tests (NAAT) for detection of HIV-1 DNA or RNA has been restricted to laboratory settings due to time, equipment, and technical expertise requirements. The availability of a rapid NAAT with applicability for resource-limited or point-of-care (POC) settings would fill a great need in HIV diagnostics, allowing for timely diagnosis or confirmation of infection status, as well as facilitating the diagnosis of acute infection, screening and evaluation of infants born to HIV-infected mothers. Isothermal amplification methods, such as reverse-transcription, loop-mediated isothermal amplification (RT-LAMP), exhibit characteristics that are ideal for POC settings, since they are typically quicker, easier to perform, and allow for integration into low-tech, portable heating devices. METHODOLOGY/SIGNIFICANT FINDINGS: In this study, we evaluated the HIV-1 RT-LAMP assay using portable, non-instrumented nucleic acid", "amplification (NINA) heating devices that generate heat from the exothermic reaction of calcium oxide and water. The NINA heating devices exhibited stable temperatures throughout the amplification reaction and consistent amplification results between three separate devices and a thermalcycler. The performance of the NINA heaters was validated using whole blood specimens from HIV-1 infected patients. CONCLUSION: The RT-LAMP isothermal amplification method used in conjunction with a chemical heating device provides a portable, rapid and robust NAAT platform that has the potential to facilitate HIV-1 testing in resource-limited settings and POC.", "Text: HIV-1 diagnostic tests are held to a high standard of performance, as diagnosis has a direct impact on patient care and reduction of transmission. Despite technological advances in the field of HIV diagnostics and the high sensitivity and specificity associated with most HIV diagnostic tests that are currently available, it is estimated that approximately 20% of HIV-infected individuals living in the United States remain undiagnosed [1] . Furthermore, testing sites have reported as many as 35 to 50% of individuals with an initial positive test result will not return for a confirmatory diagnosis if follow-up laboratory testing is required [2] . Rapid HIV antibodybased tests, which can be performed with minimal training and typically provide results in under 30 minutes [3] , have facilitated HIV testing at the point-of-care and subsequently increased the numbers of individuals aware of their serostatus [4] . Rapid tests are currently a key component of HIV screening at the", "point-of-care (POC), significantly expanding the diagnostic capabilities of testing sites in developed countries, as well as resource-limited settings.", "Despite the advances made by the widespread availability of rapid tests, all antibody-based tests for the detection of HIV exhibit some limitations. HIV-specific antibody typically begins to appear around three weeks post-infection, allowing for detection by most antibody-based assays within 3-6 weeks [3, 5] . The window of time prior to or during early seroconversion may lead to false-negative test results in recently infected individuals. Additionally, accurate diagnosis of infants born to HIV-infected mothers can be challenging if based solely on antibody positivity, since vertically transferred maternal antibodies may persist for 12-18 months after birth [6, 7] . For confirmatory diagnosis of early HIV infection or infant diagnosis, nucleic acid amplification tests (NAAT) are preferred, as HIV-1 RNA can be detected as early as 10-12 days post infection and HIV-1 DNA and/or RNA are definitive indicators of active infection [5] . In their current form, however, NAAT's are not", "feasible for POC testing, because they are timeconsuming, expensive, and technically complicated. To date, the Aptima HIV-1 RNA assay (Gen-Probe, Inc., http://www.fda.gov/ BiologicsBloodVaccines/BloodBloodProducts/ApprovedProducts/ LicensedProductsBLAs/BloodDonorScreening/InfectiousDisease/ UCM080466) is the only FDA-approved NAAT for the diagnosis or confirmation of HIV-1 infection and it is only suitable for laboratory testing.", "To meet the needs of HIV-1 diagnosis at the POC, a rapid NAAT that can be performed with minimal training, limited equipment, and a relatively short turnaround time (,1 hour)is desirable [8] . The development of a rapid NAAT has proven to be especially challenging since the technology involved in simplifying the test procedure often equates to increased equipment and material costs [8] . Additionally, the reduction in technical complexity should not compromise test sensitivity and specificity. For increased applicability at the POC, an increasing number of novel isothermal amplification techniques have been developed [9] . Isothermal amplification is an attractive alternative to traditional PCR or RT-PCR since thermalcycling is not required, allowing for greater versatility in terms of heating or amplification devices. One such amplification method, termed Loop-Mediated Isothermal Amplification (LAMP) [10] , has been optimized for the detection of DNA and/or RNA (RT-LAMP) from a wide", "range of bacterial and viral pathogens [11, 12, 13, 14, 15, 16, 17, 18, 19] , including HIV [20, 21] .", "LAMP or RT-LAMP exhibits several characteristics that are ideal for integration into a rapid nucleic-acid based diagnostic test. The amplification reaction requires six primers specific for eight separate regions within the target sequence, contributing to the high specificity of the amplification method. Amplified material can typically be detected within 15-60 minutes when incubated at a constant reaction temperature of 60-65uC [22] . LAMP has also proven to be less sensitive to biological inhibitors than PCR [23, 24] , which enables direct amplification from clinical specimens, thereby eliminating the need for an additional nucleic acid extraction step. Direct amplification from plasma, whole blood, and oral fluid has previously been demonstrated for HIV-1 [20, 21, 25] . Lastly, immediate visual detection of amplified products is facilitated by the large amount of DNA that is generated by each reaction. Several groups have incorporated fluorescent detection methods into the LAMP", "assay for real-time or immediate naked-eye detection [15, 17, 21, 22, 26] .", "The simplicity and isothermal nature of the LAMP procedure opens the door for the evaluation of low-tech integrated devices or novel heating elements, which are appropriate for low-resource settings, where costly equipment and electricity cannot be obtained. In this study, the HIV-1 RT-LAMP assay was evaluated using portable, non-instrumented nucleic acid amplification (NINA) devices that generate heat from the exothermic reaction of calcium oxide and water [27, 28] . We demonstrated the temperature stability of the NINA heating devices and feasibility for POC testing of whole blood specimens from HIV-1 infected individuals.", "Prototype NINA heaters were designed and provided by Program for Appropriate Technology in Health (PATH, Seattle, WA), as described [27, 28] . Briefly, an amplification temperature of approximately 60uC was provided by the exothermic reaction of calcium oxide (CaO; Sigma-Aldrich, St. Louis, MO) and water. The heating devices, containing the chemical reaction, were designed using thermally insulated, stainless-steel canisters with plastic screw-top lids (Fig. 1) . The lids were modified to contain three sample wells that fit standard 200 ml PCR tubes and were filled with a proprietary phase-change material (PCM) that was used to buffer the heat derived from the exothermic reaction, thereby providing a constant temperature. Lastly, plastic caps containing foam insulation were designed to fit on the top of the canister lids. The thermal profiles of the sample wells were measured and recorded using a digital thermometer (DaqPRO 5300 Data recorder; OMEGA Engineering, Inc., Stamford, CT).", "DNA and RNA linearity panels were prepared to determine the sensitivity of the HIV-specific RT-LAMP assay. A DNA panel was generated from DNA extracted from the human monocytic cell line OM-10.1 [29] , using a QIAamp DNA blood mini kit (QIAGEN, Valencia, CA). Cell count was used to quantify the input DNA copy number, as a single integrated provirus is contained in each cell [29] . The extracted DNA was diluted tenfold in RNase-free water to create a linearity panel, ranging from 10 5 copies/ml to 10 3 copies/ml. An RNA linearity panel was obtained commercially (PRD801; SeraCare Life Sciences, Mil- ford, MA) and ranged from 2.9610 6 copies/ml to 8 copies/ml, as determined by Roche AMPLICOR HIV MONITOR TM v 1.5, Bayer VERSANT HIV-1 RNA bDNA 3.0 Assay, bioMerieux NucliSensH HIV-1 QT, and Abbott Real Time HIV-1 m2000 TM . RNA was extracted from the panel members using a Viral RNA mini kit (QIAGEN). Negative controls included DNA extracted from PBMC infected with HIV-2 SLRHC [30] and RNA", "extracted from HIV-2 NIH-Z purified virus (Advanced Biotechnologies Inc., Columbia, MD).", "Whole blood from HIV-1 infected individuals was collected as part of a separate, IRB-approved study [31] , or obtained commercially (SeraCare Life Sciences). All HIV-positive samples were confirmed using the following tests: Genetic Systems HIV-1/ HIV-2 plus O EIA (Bio-Rad Laboratories, Redmond, WA), GS HIV-1 Western blot (Bio-Rad Laboratories), Aptima HIV-1 RNA assay (Gen-Probe, Inc., San Diego, CA), and Amplicor HIV-1 DNA assay (Roche Diagnostics, Branchburg, NJ ). Viral and proviral loads are unknown, since the samples were tested with qualitative, nucleic acid-based assays. All clinical specimens evaluated in this study were obtained from individuals infected with subtype B HIV-1 virus. As a negative control, HIV-1 seronegative blood samples (SeraCare Life Sciences) were included in every experiment involving whole blood. A positive control included HIV-1 seronegative blood spiked with 5610 6 virus particles/ml of HIV-1 BaL (Advanced Biotechnologies Inc.).", "HIV-1-specific RT-LAMP primers were designed to recognize a conserved sequence within the reverse transcriptase (RT) gene. The six primers required for the RT-LAMP reaction, forward outer (F3), backward outer (B3), forward inner (FIP), backward inner (BIP), and the loop primers (LoopF and LoopB), were designed using the PrimerExplorer V4 software (Eiken Chemical Co. Ltd.; http:// primerexplorer.jp/e/). The LAMP primers and amplification cycle have been described in detail by Nagamine et al. [32] . Additional modifications included a linker sequence of four thymidines inserted between the F2 and F1c sequences of the FIP primer, as described [20] , and the addition of the fluorescent molecule HEX to the 59 end of the LoopF primer. The labeled primer, along with a quencher probe, allowed for immediate visual detection of amplified products [21] . The quencher probe consisted of the complementary sequence of the LoopF primer with Black Hole Quencher-1 (BHQ-1) added to the 39 end. The", "HIV-1 HXB2 sequence (GenBank accession number AF033819) was used as the reference for generating the RT-LAMP primers. The sequences of the HIV-1 RT-specific primers and quencher are listed in Table 1 .", "The RT-LAMP reaction was performed using the following reaction mix: 0.2 mM (final concentration) of each F3 and B3 primers, 1.6 mM of each FIP and BIP primers, 0.8 mM of each LoopF and HEX-LoopB primers, 0.8 M betaine (Sigma-Aldrich), 10 mM MgSO 4 , 1.4 mM dNTPs, 16 ThermoPol reaction buffer (New England Biolabs, Ipswich, MA), 16 U Bst DNA polymerase (New England Biolabs) and 2 U AMV reverse transcriptase (Invitrogen, Carlsbad, CA). The reaction was carried out in a total volume of 25 ml for amplification of extracted nucleic acid, 10 ml of which constituted the sample. For amplification of whole blood specimens, a 100 ml reaction volume was used to facilitate visual detection of amplified products. Whole blood was added directly into the reaction at a total volume of 40 ml, following a 1:4 dilution with red blood cell lysis buffer (2.5 mM KHCO 3 , 37.5 mM NH 4 Cl, and 0.025 mM EDTA), as previously described [21] . The reaction mixture was incubated at 60uC for 60 minutes, using a", "GeneAmpH PCR System (Applied Biosystems, Foster City, CA) or the NINA heaters. For reactions amplified in the thermalcylcer, an additional two minute heating step of 80uC was added at the end of the amplification cycle to terminate the reaction.", "The reaction tubes were evaluated for the presence of amplification, following addition of the quencher probe at a 2:1 ratio of quencher to labeled-primer, as previously described [21] . Amplification was determined visually by observing fluorescence in the reaction tubes, using the UV lamp from a ChemiDoc XRS system (Bio-Rad Laboratories, Hercules, CA). Amplification was confirmed by electrophoresis using a 1.2% agarose gel containing SYBRH Safe gel stain (Invitrogen), which was subsequently visualized using the ChemiDoc XRS system.", "To compare temperature and amplification consistency, three NINA heaters were tested in parallel. The heating reaction was initiated by adding 18 g of CaO to each NINA canister, followed by 6 ml of water. The lid of each canister was then sealed to contain the exothermic reaction. After adding 200 ml of water to each of the sample wells, temperature recording was initiated. Reaction tubes were added to the sample wells once each reaction chamber reached a temperature of 58.5uC. For all samples incubated in the NINA heater, 15 ml of mineral oil was added to the reaction tube during the reaction mix preparation. The samples were incubated in the heaters for a total of 60 minutes. All reactions were carried out in a temperature-controlled laboratory with an ambient temperature of 28uC, unless otherwise stated. Following the amplification reaction, the samples were incubated for two minutes in a heat block set to 80uC. After each amplification cycle, the temperature profile of each device", "was analyzed by calculating the temperature mean, standard deviation, median, minimum, and maximum from the data provided by the DaqPRO 5300.", "The stability of the NINA heaters at extreme low and high temperatures was evaluated by placing the canisters in a refrigerator set to 4uC or a 37uC incubator during the length of the amplification reaction. The temperature profiles were recorded and compared to those of reactions that occurred at the laboratory room temperature of 28uC.", "To determine the sensitivity of RT-LAMP reaction using RTspecific primers, DNA and RNA linearity panels were tested in a thermalcycler. The limit of detection for HIV-1 DNA was 10 copies/reaction. For the RNA linearity panel, the sample containing 1700 copies/reaction was detected in all of the three replicates, while the sample containing 140 copies/reaction was detected in three out of five replicates (60%). For both DNA and RNA linearity panels, the two samples nearest the limit of detection were chosen to further evaluate the performance consistency between the thermalcycler and NINA heaters. In terms of positivity, the amplification results were consistent between all three heaters and the thermalcycler ( Table 2) . Since the RT-LAMP assay requires a constant temperature of 60uC for the length of the amplification reaction, the temperature profiles of the sample wells were compared over the course of the incubation and between all three NINA heaters. A representative temperature", "profile is displayed in Figure 2 , showing a steady reaction temperature at or close to 60uC for length of amplification reaction. During the 60 minute incubation, the average temperature for each device was 60.2, 59.8, and 59.7 (Table 3 ). The minimum temperature achieved during the reaction reflects the fact that the temperature of the sample port dropped temporarily after the sample tubes are added to the device, as shown in Figure 2 . The maximum temperature of the devices deviated from the desired reaction temperature of 60uC by less than one degree.", "The ability of the NINA heaters to maintain a steady reaction temperature in a wide range of ambient temperatures is essential for POC testing, whether referring to an air-conditioned laboratory or high-temperature field site. To evaluate the performance of the NINA heaters at extreme low or high temperatures, the canisters were placed in a 4uC refrigerator or a 37uC incubator for the length of the amplification reaction. The limit of detection for the DNA and RNA linearity panels was similar to the results obtained in our temperature-controlled laboratory (28uC; Table 2 ). The greatest degree of temperature variation of the sample wells was observed at the ambient temperature of 4uC ( Table 3 ). The average temperature was approximately two degrees lower than the desired reaction temperature of 60uC. Additionally, the temperature of the devices tended to decline from their steady state during the last 20 minutes of the reaction (data not shown). The temperature profiles at the", "ambient temperature of 37uC, however, were similar to those at 28uC.", "Whole blood samples from HIV-1 infected individuals were added directly into the RT-LAMP reaction and tested in the NINA heaters. Positivity of the clinical specimens was consistent between the thermalcycler and devices (Table 4 ). Amplification consistency was most evident with two of the patient samples (patient #4 and #5) that were only positive in one of the three replicates, regardless of the heating device that was used. All HIVnegative blood samples, included in each reaction, were negative (data not shown). A representative experiment using the NINA heaters is displayed in Figure 3 , showing detection by agarose gel and visual identification of fluorescence in the reaction tubes.", "In this study, we demonstrate the performance of portable, inexpensive, non-instrumented nucleic acid (NINA) heaters for amplification of HIV-1 using RT-LAMP. The isothermal amplification reaction coupled with a device that generates heat from an exothermic chemical reaction, as opposed to grid electricity or battery power, comprises a point-of-care NAAT that is practical for use in resource-limited settings. The heating devices require minimal training and technical expertise to operate and take approximately 10-15 minutes to reach a reaction temperature of 60uC once the chemical reaction has been initiated [27, 28] . Furthermore, the temperature of the sample wells remain relatively stable at the desired reaction temperature of 60uC throughout the amplification reaction, as demonstrated by the heating profiles and the consistency in amplification between the devices and thermalcycler.", "Since point-of-care testing may refer to an air-conditioned laboratory or a field site with high temperatures and humidity, the stability of the temperature generated by the heating devices must be reliable. Though the temperature profiles at a representative cold temperature of 4uC indicated a loss in reaction temperature towards the end of the 60 minute incubation, the temperature fluctuations were not significant enough to affect the amplification reaction. Regardless, this thermal effect could be mitigated with small modifications to the device to reduce heat loss at lower temperatures. It should be possible to extend the temperature range of the NINA heaters to 4uC and below by either adding a larger quantity of heating mixture, better insulation, or both. Of greater concern is the performance of the NINA heaters in hightemperature field sites, where temperature control is not an option.", "We demonstrate no difference in the temperature stability of the NINA heaters and amplification consistency at an ambient temperature of 37uC as compared to our temperature-controlled laboratory.", "For increased applicability for use at the POC, several modifications can be made to the NINA heaters. The prototype devices evaluated in this study contained only three sample wells; however, up to 16 sample wells can be added to the lid of the insulated canisters for a larger testing volume. In this study, samples were removed from the NINA heaters after the amplification reaction and heated for an additional two minutes in an 80uC heat block to terminate the reaction. While the additional heating step is not necessary to observe the amplified products from extracted nucleic acid, the short, high-temperature incubation facilitates the visual observation of the fluorescent label in the whole blood samples. Modifications may be made to the whole blood sample preparation method to eliminate the need for the heating step. Alternatively, a second temperature-moderating compartment can be added to the alternate end of the NINA canisters, so the samples can be removed from the", "amplification compartment and reinserted into the 80uC compartment. Lastly, the DaqPRO data recorder was used in this study for validation purposes only and would not be necessary for the final POC product.", "The feasibility of using LAMP as a diagnostic method in resource-limited settings has been demonstrated for tuberculosis [33] . To reduce hands-on time and preparation error, the authors describe the use of reaction tubes pre-prepared with lyophilized reaction mix. For POC use, limited sample manipulation and reagent preparation is desired and, therefore, it is anticipated that the test procedure of the end product will include reconstituting the amplification reagents in water and adding the sample directly into the reaction tube. We demonstrate the use of the NINA heaters for amplification directly from whole blood specimens, eliminating the need for a time-consuming, nucleic acid extraction procedure and reducing the volume of sample needed for the amplification reaction. A total volume of 10 ml of whole blood was added to each reaction tube, which can easily be obtained by finger-stick in settings where venipuncture is not feasible. Additionally, our fluorescent detection method", "enables immediate visualization of amplified products in the absence of specialized equipment. To avoid cross-contamination of amplified material, it is preferred that the reaction tubes remain closed post-amplification. Future modifications will include optimizing the labeledprimer/quencher sequences so that all components can be added into the reaction mix prior to amplification. Due to availability, the Bio-Rad ChemiDoc system was used as the UV source in this study; however, an inexpensive keychain light would be more suitable for naked-eye detection at the POC. For sensitive and specific detection of diverse HIV-1 isolates, including non-B subtypes, identification of the optimal primer set/sets is a key step in the development of the RT-LAMP assay. Although all experiments performed in this study involved subtype B standards and specimens, ongoing research involves the continued development and optimization of RT-LAMP primers based on regions of the HIV-1 genome that are", "conserved among diverse subtypes. Future studies will include large-scale evaluation of clinical specimens with the optimized RT-LAMP assay and NINA device. In summary, the RT-LAMP isothermal amplification method used in conjunction with a simplified, chemical heating device exhibits characteristics that are ideal for a rapid NAAT for POC testing. The simplified, portable assay has the potential to fill an important gap in HIV-1 diagnostics, providing immediate knowledge or confirmation of HIV-1 infection status at the POC." ]

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[ "Isothermal Amplification Using a Chemical Heating Device for Point-of-Care Detection of HIV-1\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3285652/\n\nSHA: ef7110a9022bac2e50c995b0f6b826ff071e48f8\n\nAuthors: Curtis, Kelly A.; Rudolph, Donna L.; Nejad, Irene; Singleton, Jered; Beddoe, Andy; Weigl, Bernhard; LaBarre, Paul; Owen, S. Michele\nDate: 2012-02-23\nDOI: 10.1371/journal.pone.0031432\nLicense: cc0", "Abstract: BACKGROUND: To date, the use of traditional nucleic acid amplification tests (NAAT) for detection of HIV-1 DNA or RNA has been restricted to laboratory settings due to time, equipment, and technical expertise requirements. The availability of a rapid NAAT with applicability for resource-limited or point-of-care (POC) settings would fill a great need in HIV diagnostics, allowing for timely diagnosis or confirmation of infection status, as well as facilitating the diagnosis of acute infection, screening and evaluation of infants born to HIV-infected mothers. Isothermal amplification methods, such as reverse-transcription, loop-mediated isothermal amplification (RT-LAMP), exhibit characteristics that are ideal for POC settings, since they are typically quicker, easier to perform, and allow for integration into low-tech, portable heating devices. METHODOLOGY/SIGNIFICANT FINDINGS: In this study, we evaluated the HIV-1 RT-LAMP assay using portable, non-instrumented nucleic acid", "amplification (NINA) heating devices that generate heat from the exothermic reaction of calcium oxide and water. The NINA heating devices exhibited stable temperatures throughout the amplification reaction and consistent amplification results between three separate devices and a thermalcycler. The performance of the NINA heaters was validated using whole blood specimens from HIV-1 infected patients. CONCLUSION: The RT-LAMP isothermal amplification method used in conjunction with a chemical heating device provides a portable, rapid and robust NAAT platform that has the potential to facilitate HIV-1 testing in resource-limited settings and POC.", "Text: HIV-1 diagnostic tests are held to a high standard of performance, as diagnosis has a direct impact on patient care and reduction of transmission. Despite technological advances in the field of HIV diagnostics and the high sensitivity and specificity associated with most HIV diagnostic tests that are currently available, it is estimated that approximately 20% of HIV-infected individuals living in the United States remain undiagnosed [1] . Furthermore, testing sites have reported as many as 35 to 50% of individuals with an initial positive test result will not return for a confirmatory diagnosis if follow-up laboratory testing is required [2] . Rapid HIV antibodybased tests, which can be performed with minimal training and typically provide results in under 30 minutes [3] , have facilitated HIV testing at the point-of-care and subsequently increased the numbers of individuals aware of their serostatus [4] . Rapid tests are currently a key component of HIV screening at the", "point-of-care (POC), significantly expanding the diagnostic capabilities of testing sites in developed countries, as well as resource-limited settings.", "Despite the advances made by the widespread availability of rapid tests, all antibody-based tests for the detection of HIV exhibit some limitations. HIV-specific antibody typically begins to appear around three weeks post-infection, allowing for detection by most antibody-based assays within 3-6 weeks [3, 5] . The window of time prior to or during early seroconversion may lead to false-negative test results in recently infected individuals. Additionally, accurate diagnosis of infants born to HIV-infected mothers can be challenging if based solely on antibody positivity, since vertically transferred maternal antibodies may persist for 12-18 months after birth [6, 7] . For confirmatory diagnosis of early HIV infection or infant diagnosis, nucleic acid amplification tests (NAAT) are preferred, as HIV-1 RNA can be detected as early as 10-12 days post infection and HIV-1 DNA and/or RNA are definitive indicators of active infection [5] . In their current form, however, NAAT's are not", "feasible for POC testing, because they are timeconsuming, expensive, and technically complicated. To date, the Aptima HIV-1 RNA assay (Gen-Probe, Inc., http://www.fda.gov/ BiologicsBloodVaccines/BloodBloodProducts/ApprovedProducts/ LicensedProductsBLAs/BloodDonorScreening/InfectiousDisease/ UCM080466) is the only FDA-approved NAAT for the diagnosis or confirmation of HIV-1 infection and it is only suitable for laboratory testing.", "To meet the needs of HIV-1 diagnosis at the POC, a rapid NAAT that can be performed with minimal training, limited equipment, and a relatively short turnaround time (,1 hour)is desirable [8] . The development of a rapid NAAT has proven to be especially challenging since the technology involved in simplifying the test procedure often equates to increased equipment and material costs [8] . Additionally, the reduction in technical complexity should not compromise test sensitivity and specificity. For increased applicability at the POC, an increasing number of novel isothermal amplification techniques have been developed [9] . Isothermal amplification is an attractive alternative to traditional PCR or RT-PCR since thermalcycling is not required, allowing for greater versatility in terms of heating or amplification devices. One such amplification method, termed Loop-Mediated Isothermal Amplification (LAMP) [10] , has been optimized for the detection of DNA and/or RNA (RT-LAMP) from a wide", "range of bacterial and viral pathogens [11, 12, 13, 14, 15, 16, 17, 18, 19] , including HIV [20, 21] .", "LAMP or RT-LAMP exhibits several characteristics that are ideal for integration into a rapid nucleic-acid based diagnostic test. The amplification reaction requires six primers specific for eight separate regions within the target sequence, contributing to the high specificity of the amplification method. Amplified material can typically be detected within 15-60 minutes when incubated at a constant reaction temperature of 60-65uC [22] . LAMP has also proven to be less sensitive to biological inhibitors than PCR [23, 24] , which enables direct amplification from clinical specimens, thereby eliminating the need for an additional nucleic acid extraction step. Direct amplification from plasma, whole blood, and oral fluid has previously been demonstrated for HIV-1 [20, 21, 25] . Lastly, immediate visual detection of amplified products is facilitated by the large amount of DNA that is generated by each reaction. Several groups have incorporated fluorescent detection methods into the LAMP", "assay for real-time or immediate naked-eye detection [15, 17, 21, 22, 26] .", "The simplicity and isothermal nature of the LAMP procedure opens the door for the evaluation of low-tech integrated devices or novel heating elements, which are appropriate for low-resource settings, where costly equipment and electricity cannot be obtained. In this study, the HIV-1 RT-LAMP assay was evaluated using portable, non-instrumented nucleic acid amplification (NINA) devices that generate heat from the exothermic reaction of calcium oxide and water [27, 28] . We demonstrated the temperature stability of the NINA heating devices and feasibility for POC testing of whole blood specimens from HIV-1 infected individuals.", "Prototype NINA heaters were designed and provided by Program for Appropriate Technology in Health (PATH, Seattle, WA), as described [27, 28] . Briefly, an amplification temperature of approximately 60uC was provided by the exothermic reaction of calcium oxide (CaO; Sigma-Aldrich, St. Louis, MO) and water. The heating devices, containing the chemical reaction, were designed using thermally insulated, stainless-steel canisters with plastic screw-top lids (Fig. 1) . The lids were modified to contain three sample wells that fit standard 200 ml PCR tubes and were filled with a proprietary phase-change material (PCM) that was used to buffer the heat derived from the exothermic reaction, thereby providing a constant temperature. Lastly, plastic caps containing foam insulation were designed to fit on the top of the canister lids. The thermal profiles of the sample wells were measured and recorded using a digital thermometer (DaqPRO 5300 Data recorder; OMEGA Engineering, Inc., Stamford, CT).", "DNA and RNA linearity panels were prepared to determine the sensitivity of the HIV-specific RT-LAMP assay. A DNA panel was generated from DNA extracted from the human monocytic cell line OM-10.1 [29] , using a QIAamp DNA blood mini kit (QIAGEN, Valencia, CA). Cell count was used to quantify the input DNA copy number, as a single integrated provirus is contained in each cell [29] . The extracted DNA was diluted tenfold in RNase-free water to create a linearity panel, ranging from 10 5 copies/ml to 10 3 copies/ml. An RNA linearity panel was obtained commercially (PRD801; SeraCare Life Sciences, Mil- ford, MA) and ranged from 2.9610 6 copies/ml to 8 copies/ml, as determined by Roche AMPLICOR HIV MONITOR TM v 1.5, Bayer VERSANT HIV-1 RNA bDNA 3.0 Assay, bioMerieux NucliSensH HIV-1 QT, and Abbott Real Time HIV-1 m2000 TM . RNA was extracted from the panel members using a Viral RNA mini kit (QIAGEN). Negative controls included DNA extracted from PBMC infected with HIV-2 SLRHC [30] and RNA", "extracted from HIV-2 NIH-Z purified virus (Advanced Biotechnologies Inc., Columbia, MD).", "Whole blood from HIV-1 infected individuals was collected as part of a separate, IRB-approved study [31] , or obtained commercially (SeraCare Life Sciences). All HIV-positive samples were confirmed using the following tests: Genetic Systems HIV-1/ HIV-2 plus O EIA (Bio-Rad Laboratories, Redmond, WA), GS HIV-1 Western blot (Bio-Rad Laboratories), Aptima HIV-1 RNA assay (Gen-Probe, Inc., San Diego, CA), and Amplicor HIV-1 DNA assay (Roche Diagnostics, Branchburg, NJ ). Viral and proviral loads are unknown, since the samples were tested with qualitative, nucleic acid-based assays. All clinical specimens evaluated in this study were obtained from individuals infected with subtype B HIV-1 virus. As a negative control, HIV-1 seronegative blood samples (SeraCare Life Sciences) were included in every experiment involving whole blood. A positive control included HIV-1 seronegative blood spiked with 5610 6 virus particles/ml of HIV-1 BaL (Advanced Biotechnologies Inc.).", "HIV-1-specific RT-LAMP primers were designed to recognize a conserved sequence within the reverse transcriptase (RT) gene. The six primers required for the RT-LAMP reaction, forward outer (F3), backward outer (B3), forward inner (FIP), backward inner (BIP), and the loop primers (LoopF and LoopB), were designed using the PrimerExplorer V4 software (Eiken Chemical Co. Ltd.; http:// primerexplorer.jp/e/). The LAMP primers and amplification cycle have been described in detail by Nagamine et al. [32] . Additional modifications included a linker sequence of four thymidines inserted between the F2 and F1c sequences of the FIP primer, as described [20] , and the addition of the fluorescent molecule HEX to the 59 end of the LoopF primer. The labeled primer, along with a quencher probe, allowed for immediate visual detection of amplified products [21] . The quencher probe consisted of the complementary sequence of the LoopF primer with Black Hole Quencher-1 (BHQ-1) added to the 39 end. The", "HIV-1 HXB2 sequence (GenBank accession number AF033819) was used as the reference for generating the RT-LAMP primers. The sequences of the HIV-1 RT-specific primers and quencher are listed in Table 1 .", "The RT-LAMP reaction was performed using the following reaction mix: 0.2 mM (final concentration) of each F3 and B3 primers, 1.6 mM of each FIP and BIP primers, 0.8 mM of each LoopF and HEX-LoopB primers, 0.8 M betaine (Sigma-Aldrich), 10 mM MgSO 4 , 1.4 mM dNTPs, 16 ThermoPol reaction buffer (New England Biolabs, Ipswich, MA), 16 U Bst DNA polymerase (New England Biolabs) and 2 U AMV reverse transcriptase (Invitrogen, Carlsbad, CA). The reaction was carried out in a total volume of 25 ml for amplification of extracted nucleic acid, 10 ml of which constituted the sample. For amplification of whole blood specimens, a 100 ml reaction volume was used to facilitate visual detection of amplified products. Whole blood was added directly into the reaction at a total volume of 40 ml, following a 1:4 dilution with red blood cell lysis buffer (2.5 mM KHCO 3 , 37.5 mM NH 4 Cl, and 0.025 mM EDTA), as previously described [21] . The reaction mixture was incubated at 60uC for 60 minutes, using a", "GeneAmpH PCR System (Applied Biosystems, Foster City, CA) or the NINA heaters. For reactions amplified in the thermalcylcer, an additional two minute heating step of 80uC was added at the end of the amplification cycle to terminate the reaction.", "The reaction tubes were evaluated for the presence of amplification, following addition of the quencher probe at a 2:1 ratio of quencher to labeled-primer, as previously described [21] . Amplification was determined visually by observing fluorescence in the reaction tubes, using the UV lamp from a ChemiDoc XRS system (Bio-Rad Laboratories, Hercules, CA). Amplification was confirmed by electrophoresis using a 1.2% agarose gel containing SYBRH Safe gel stain (Invitrogen), which was subsequently visualized using the ChemiDoc XRS system.", "To compare temperature and amplification consistency, three NINA heaters were tested in parallel. The heating reaction was initiated by adding 18 g of CaO to each NINA canister, followed by 6 ml of water. The lid of each canister was then sealed to contain the exothermic reaction. After adding 200 ml of water to each of the sample wells, temperature recording was initiated. Reaction tubes were added to the sample wells once each reaction chamber reached a temperature of 58.5uC. For all samples incubated in the NINA heater, 15 ml of mineral oil was added to the reaction tube during the reaction mix preparation. The samples were incubated in the heaters for a total of 60 minutes. All reactions were carried out in a temperature-controlled laboratory with an ambient temperature of 28uC, unless otherwise stated. Following the amplification reaction, the samples were incubated for two minutes in a heat block set to 80uC. After each amplification cycle, the temperature profile of each device", "was analyzed by calculating the temperature mean, standard deviation, median, minimum, and maximum from the data provided by the DaqPRO 5300.", "The stability of the NINA heaters at extreme low and high temperatures was evaluated by placing the canisters in a refrigerator set to 4uC or a 37uC incubator during the length of the amplification reaction. The temperature profiles were recorded and compared to those of reactions that occurred at the laboratory room temperature of 28uC.", "To determine the sensitivity of RT-LAMP reaction using RTspecific primers, DNA and RNA linearity panels were tested in a thermalcycler. The limit of detection for HIV-1 DNA was 10 copies/reaction. For the RNA linearity panel, the sample containing 1700 copies/reaction was detected in all of the three replicates, while the sample containing 140 copies/reaction was detected in three out of five replicates (60%). For both DNA and RNA linearity panels, the two samples nearest the limit of detection were chosen to further evaluate the performance consistency between the thermalcycler and NINA heaters. In terms of positivity, the amplification results were consistent between all three heaters and the thermalcycler ( Table 2) . Since the RT-LAMP assay requires a constant temperature of 60uC for the length of the amplification reaction, the temperature profiles of the sample wells were compared over the course of the incubation and between all three NINA heaters. A representative temperature", "profile is displayed in Figure 2 , showing a steady reaction temperature at or close to 60uC for length of amplification reaction. During the 60 minute incubation, the average temperature for each device was 60.2, 59.8, and 59.7 (Table 3 ). The minimum temperature achieved during the reaction reflects the fact that the temperature of the sample port dropped temporarily after the sample tubes are added to the device, as shown in Figure 2 . The maximum temperature of the devices deviated from the desired reaction temperature of 60uC by less than one degree.", "The ability of the NINA heaters to maintain a steady reaction temperature in a wide range of ambient temperatures is essential for POC testing, whether referring to an air-conditioned laboratory or high-temperature field site. To evaluate the performance of the NINA heaters at extreme low or high temperatures, the canisters were placed in a 4uC refrigerator or a 37uC incubator for the length of the amplification reaction. The limit of detection for the DNA and RNA linearity panels was similar to the results obtained in our temperature-controlled laboratory (28uC; Table 2 ). The greatest degree of temperature variation of the sample wells was observed at the ambient temperature of 4uC ( Table 3 ). The average temperature was approximately two degrees lower than the desired reaction temperature of 60uC. Additionally, the temperature of the devices tended to decline from their steady state during the last 20 minutes of the reaction (data not shown). The temperature profiles at the", "ambient temperature of 37uC, however, were similar to those at 28uC.", "Whole blood samples from HIV-1 infected individuals were added directly into the RT-LAMP reaction and tested in the NINA heaters. Positivity of the clinical specimens was consistent between the thermalcycler and devices (Table 4 ). Amplification consistency was most evident with two of the patient samples (patient #4 and #5) that were only positive in one of the three replicates, regardless of the heating device that was used. All HIVnegative blood samples, included in each reaction, were negative (data not shown). A representative experiment using the NINA heaters is displayed in Figure 3 , showing detection by agarose gel and visual identification of fluorescence in the reaction tubes.", "In this study, we demonstrate the performance of portable, inexpensive, non-instrumented nucleic acid (NINA) heaters for amplification of HIV-1 using RT-LAMP. The isothermal amplification reaction coupled with a device that generates heat from an exothermic chemical reaction, as opposed to grid electricity or battery power, comprises a point-of-care NAAT that is practical for use in resource-limited settings. The heating devices require minimal training and technical expertise to operate and take approximately 10-15 minutes to reach a reaction temperature of 60uC once the chemical reaction has been initiated [27, 28] . Furthermore, the temperature of the sample wells remain relatively stable at the desired reaction temperature of 60uC throughout the amplification reaction, as demonstrated by the heating profiles and the consistency in amplification between the devices and thermalcycler.", "Since point-of-care testing may refer to an air-conditioned laboratory or a field site with high temperatures and humidity, the stability of the temperature generated by the heating devices must be reliable. Though the temperature profiles at a representative cold temperature of 4uC indicated a loss in reaction temperature towards the end of the 60 minute incubation, the temperature fluctuations were not significant enough to affect the amplification reaction. Regardless, this thermal effect could be mitigated with small modifications to the device to reduce heat loss at lower temperatures. It should be possible to extend the temperature range of the NINA heaters to 4uC and below by either adding a larger quantity of heating mixture, better insulation, or both. Of greater concern is the performance of the NINA heaters in hightemperature field sites, where temperature control is not an option.", "We demonstrate no difference in the temperature stability of the NINA heaters and amplification consistency at an ambient temperature of 37uC as compared to our temperature-controlled laboratory.", "For increased applicability for use at the POC, several modifications can be made to the NINA heaters. The prototype devices evaluated in this study contained only three sample wells; however, up to 16 sample wells can be added to the lid of the insulated canisters for a larger testing volume. In this study, samples were removed from the NINA heaters after the amplification reaction and heated for an additional two minutes in an 80uC heat block to terminate the reaction. While the additional heating step is not necessary to observe the amplified products from extracted nucleic acid, the short, high-temperature incubation facilitates the visual observation of the fluorescent label in the whole blood samples. Modifications may be made to the whole blood sample preparation method to eliminate the need for the heating step. Alternatively, a second temperature-moderating compartment can be added to the alternate end of the NINA canisters, so the samples can be removed from the", "amplification compartment and reinserted into the 80uC compartment. Lastly, the DaqPRO data recorder was used in this study for validation purposes only and would not be necessary for the final POC product.", "The feasibility of using LAMP as a diagnostic method in resource-limited settings has been demonstrated for tuberculosis [33] . To reduce hands-on time and preparation error, the authors describe the use of reaction tubes pre-prepared with lyophilized reaction mix. For POC use, limited sample manipulation and reagent preparation is desired and, therefore, it is anticipated that the test procedure of the end product will include reconstituting the amplification reagents in water and adding the sample directly into the reaction tube. We demonstrate the use of the NINA heaters for amplification directly from whole blood specimens, eliminating the need for a time-consuming, nucleic acid extraction procedure and reducing the volume of sample needed for the amplification reaction. A total volume of 10 ml of whole blood was added to each reaction tube, which can easily be obtained by finger-stick in settings where venipuncture is not feasible. Additionally, our fluorescent detection method", "enables immediate visualization of amplified products in the absence of specialized equipment. To avoid cross-contamination of amplified material, it is preferred that the reaction tubes remain closed post-amplification. Future modifications will include optimizing the labeledprimer/quencher sequences so that all components can be added into the reaction mix prior to amplification. Due to availability, the Bio-Rad ChemiDoc system was used as the UV source in this study; however, an inexpensive keychain light would be more suitable for naked-eye detection at the POC. For sensitive and specific detection of diverse HIV-1 isolates, including non-B subtypes, identification of the optimal primer set/sets is a key step in the development of the RT-LAMP assay. Although all experiments performed in this study involved subtype B standards and specimens, ongoing research involves the continued development and optimization of RT-LAMP primers based on regions of the HIV-1 genome that are", "conserved among diverse subtypes. Future studies will include large-scale evaluation of clinical specimens with the optimized RT-LAMP assay and NINA device. In summary, the RT-LAMP isothermal amplification method used in conjunction with a simplified, chemical heating device exhibits characteristics that are ideal for a rapid NAAT for POC testing. The simplified, portable assay has the potential to fill an important gap in HIV-1 diagnostics, providing immediate knowledge or confirmation of HIV-1 infection status at the POC." ]

[ 3 ]

[ "35 to 50%" ]

[ "Isothermal Amplification Using a Chemical Heating Device for Point-of-Care Detection of HIV-1\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3285652/\n\nSHA: ef7110a9022bac2e50c995b0f6b826ff071e48f8\n\nAuthors: Curtis, Kelly A.; Rudolph, Donna L.; Nejad, Irene; Singleton, Jered; Beddoe, Andy; Weigl, Bernhard; LaBarre, Paul; Owen, S. Michele\nDate: 2012-02-23\nDOI: 10.1371/journal.pone.0031432\nLicense: cc0", "Abstract: BACKGROUND: To date, the use of traditional nucleic acid amplification tests (NAAT) for detection of HIV-1 DNA or RNA has been restricted to laboratory settings due to time, equipment, and technical expertise requirements. The availability of a rapid NAAT with applicability for resource-limited or point-of-care (POC) settings would fill a great need in HIV diagnostics, allowing for timely diagnosis or confirmation of infection status, as well as facilitating the diagnosis of acute infection, screening and evaluation of infants born to HIV-infected mothers. Isothermal amplification methods, such as reverse-transcription, loop-mediated isothermal amplification (RT-LAMP), exhibit characteristics that are ideal for POC settings, since they are typically quicker, easier to perform, and allow for integration into low-tech, portable heating devices. METHODOLOGY/SIGNIFICANT FINDINGS: In this study, we evaluated the HIV-1 RT-LAMP assay using portable, non-instrumented nucleic acid", "amplification (NINA) heating devices that generate heat from the exothermic reaction of calcium oxide and water. The NINA heating devices exhibited stable temperatures throughout the amplification reaction and consistent amplification results between three separate devices and a thermalcycler. The performance of the NINA heaters was validated using whole blood specimens from HIV-1 infected patients. CONCLUSION: The RT-LAMP isothermal amplification method used in conjunction with a chemical heating device provides a portable, rapid and robust NAAT platform that has the potential to facilitate HIV-1 testing in resource-limited settings and POC.", "Text: HIV-1 diagnostic tests are held to a high standard of performance, as diagnosis has a direct impact on patient care and reduction of transmission. Despite technological advances in the field of HIV diagnostics and the high sensitivity and specificity associated with most HIV diagnostic tests that are currently available, it is estimated that approximately 20% of HIV-infected individuals living in the United States remain undiagnosed [1] . Furthermore, testing sites have reported as many as 35 to 50% of individuals with an initial positive test result will not return for a confirmatory diagnosis if follow-up laboratory testing is required [2] . Rapid HIV antibodybased tests, which can be performed with minimal training and typically provide results in under 30 minutes [3] , have facilitated HIV testing at the point-of-care and subsequently increased the numbers of individuals aware of their serostatus [4] . Rapid tests are currently a key component of HIV screening at the", "point-of-care (POC), significantly expanding the diagnostic capabilities of testing sites in developed countries, as well as resource-limited settings.", "Despite the advances made by the widespread availability of rapid tests, all antibody-based tests for the detection of HIV exhibit some limitations. HIV-specific antibody typically begins to appear around three weeks post-infection, allowing for detection by most antibody-based assays within 3-6 weeks [3, 5] . The window of time prior to or during early seroconversion may lead to false-negative test results in recently infected individuals. Additionally, accurate diagnosis of infants born to HIV-infected mothers can be challenging if based solely on antibody positivity, since vertically transferred maternal antibodies may persist for 12-18 months after birth [6, 7] . For confirmatory diagnosis of early HIV infection or infant diagnosis, nucleic acid amplification tests (NAAT) are preferred, as HIV-1 RNA can be detected as early as 10-12 days post infection and HIV-1 DNA and/or RNA are definitive indicators of active infection [5] . In their current form, however, NAAT's are not", "feasible for POC testing, because they are timeconsuming, expensive, and technically complicated. To date, the Aptima HIV-1 RNA assay (Gen-Probe, Inc., http://www.fda.gov/ BiologicsBloodVaccines/BloodBloodProducts/ApprovedProducts/ LicensedProductsBLAs/BloodDonorScreening/InfectiousDisease/ UCM080466) is the only FDA-approved NAAT for the diagnosis or confirmation of HIV-1 infection and it is only suitable for laboratory testing.", "To meet the needs of HIV-1 diagnosis at the POC, a rapid NAAT that can be performed with minimal training, limited equipment, and a relatively short turnaround time (,1 hour)is desirable [8] . The development of a rapid NAAT has proven to be especially challenging since the technology involved in simplifying the test procedure often equates to increased equipment and material costs [8] . Additionally, the reduction in technical complexity should not compromise test sensitivity and specificity. For increased applicability at the POC, an increasing number of novel isothermal amplification techniques have been developed [9] . Isothermal amplification is an attractive alternative to traditional PCR or RT-PCR since thermalcycling is not required, allowing for greater versatility in terms of heating or amplification devices. One such amplification method, termed Loop-Mediated Isothermal Amplification (LAMP) [10] , has been optimized for the detection of DNA and/or RNA (RT-LAMP) from a wide", "range of bacterial and viral pathogens [11, 12, 13, 14, 15, 16, 17, 18, 19] , including HIV [20, 21] .", "LAMP or RT-LAMP exhibits several characteristics that are ideal for integration into a rapid nucleic-acid based diagnostic test. The amplification reaction requires six primers specific for eight separate regions within the target sequence, contributing to the high specificity of the amplification method. Amplified material can typically be detected within 15-60 minutes when incubated at a constant reaction temperature of 60-65uC [22] . LAMP has also proven to be less sensitive to biological inhibitors than PCR [23, 24] , which enables direct amplification from clinical specimens, thereby eliminating the need for an additional nucleic acid extraction step. Direct amplification from plasma, whole blood, and oral fluid has previously been demonstrated for HIV-1 [20, 21, 25] . Lastly, immediate visual detection of amplified products is facilitated by the large amount of DNA that is generated by each reaction. Several groups have incorporated fluorescent detection methods into the LAMP", "assay for real-time or immediate naked-eye detection [15, 17, 21, 22, 26] .", "The simplicity and isothermal nature of the LAMP procedure opens the door for the evaluation of low-tech integrated devices or novel heating elements, which are appropriate for low-resource settings, where costly equipment and electricity cannot be obtained. In this study, the HIV-1 RT-LAMP assay was evaluated using portable, non-instrumented nucleic acid amplification (NINA) devices that generate heat from the exothermic reaction of calcium oxide and water [27, 28] . We demonstrated the temperature stability of the NINA heating devices and feasibility for POC testing of whole blood specimens from HIV-1 infected individuals.", "Prototype NINA heaters were designed and provided by Program for Appropriate Technology in Health (PATH, Seattle, WA), as described [27, 28] . Briefly, an amplification temperature of approximately 60uC was provided by the exothermic reaction of calcium oxide (CaO; Sigma-Aldrich, St. Louis, MO) and water. The heating devices, containing the chemical reaction, were designed using thermally insulated, stainless-steel canisters with plastic screw-top lids (Fig. 1) . The lids were modified to contain three sample wells that fit standard 200 ml PCR tubes and were filled with a proprietary phase-change material (PCM) that was used to buffer the heat derived from the exothermic reaction, thereby providing a constant temperature. Lastly, plastic caps containing foam insulation were designed to fit on the top of the canister lids. The thermal profiles of the sample wells were measured and recorded using a digital thermometer (DaqPRO 5300 Data recorder; OMEGA Engineering, Inc., Stamford, CT).", "DNA and RNA linearity panels were prepared to determine the sensitivity of the HIV-specific RT-LAMP assay. A DNA panel was generated from DNA extracted from the human monocytic cell line OM-10.1 [29] , using a QIAamp DNA blood mini kit (QIAGEN, Valencia, CA). Cell count was used to quantify the input DNA copy number, as a single integrated provirus is contained in each cell [29] . The extracted DNA was diluted tenfold in RNase-free water to create a linearity panel, ranging from 10 5 copies/ml to 10 3 copies/ml. An RNA linearity panel was obtained commercially (PRD801; SeraCare Life Sciences, Mil- ford, MA) and ranged from 2.9610 6 copies/ml to 8 copies/ml, as determined by Roche AMPLICOR HIV MONITOR TM v 1.5, Bayer VERSANT HIV-1 RNA bDNA 3.0 Assay, bioMerieux NucliSensH HIV-1 QT, and Abbott Real Time HIV-1 m2000 TM . RNA was extracted from the panel members using a Viral RNA mini kit (QIAGEN). Negative controls included DNA extracted from PBMC infected with HIV-2 SLRHC [30] and RNA", "extracted from HIV-2 NIH-Z purified virus (Advanced Biotechnologies Inc., Columbia, MD).", "Whole blood from HIV-1 infected individuals was collected as part of a separate, IRB-approved study [31] , or obtained commercially (SeraCare Life Sciences). All HIV-positive samples were confirmed using the following tests: Genetic Systems HIV-1/ HIV-2 plus O EIA (Bio-Rad Laboratories, Redmond, WA), GS HIV-1 Western blot (Bio-Rad Laboratories), Aptima HIV-1 RNA assay (Gen-Probe, Inc., San Diego, CA), and Amplicor HIV-1 DNA assay (Roche Diagnostics, Branchburg, NJ ). Viral and proviral loads are unknown, since the samples were tested with qualitative, nucleic acid-based assays. All clinical specimens evaluated in this study were obtained from individuals infected with subtype B HIV-1 virus. As a negative control, HIV-1 seronegative blood samples (SeraCare Life Sciences) were included in every experiment involving whole blood. A positive control included HIV-1 seronegative blood spiked with 5610 6 virus particles/ml of HIV-1 BaL (Advanced Biotechnologies Inc.).", "HIV-1-specific RT-LAMP primers were designed to recognize a conserved sequence within the reverse transcriptase (RT) gene. The six primers required for the RT-LAMP reaction, forward outer (F3), backward outer (B3), forward inner (FIP), backward inner (BIP), and the loop primers (LoopF and LoopB), were designed using the PrimerExplorer V4 software (Eiken Chemical Co. Ltd.; http:// primerexplorer.jp/e/). The LAMP primers and amplification cycle have been described in detail by Nagamine et al. [32] . Additional modifications included a linker sequence of four thymidines inserted between the F2 and F1c sequences of the FIP primer, as described [20] , and the addition of the fluorescent molecule HEX to the 59 end of the LoopF primer. The labeled primer, along with a quencher probe, allowed for immediate visual detection of amplified products [21] . The quencher probe consisted of the complementary sequence of the LoopF primer with Black Hole Quencher-1 (BHQ-1) added to the 39 end. The", "HIV-1 HXB2 sequence (GenBank accession number AF033819) was used as the reference for generating the RT-LAMP primers. The sequences of the HIV-1 RT-specific primers and quencher are listed in Table 1 .", "The RT-LAMP reaction was performed using the following reaction mix: 0.2 mM (final concentration) of each F3 and B3 primers, 1.6 mM of each FIP and BIP primers, 0.8 mM of each LoopF and HEX-LoopB primers, 0.8 M betaine (Sigma-Aldrich), 10 mM MgSO 4 , 1.4 mM dNTPs, 16 ThermoPol reaction buffer (New England Biolabs, Ipswich, MA), 16 U Bst DNA polymerase (New England Biolabs) and 2 U AMV reverse transcriptase (Invitrogen, Carlsbad, CA). The reaction was carried out in a total volume of 25 ml for amplification of extracted nucleic acid, 10 ml of which constituted the sample. For amplification of whole blood specimens, a 100 ml reaction volume was used to facilitate visual detection of amplified products. Whole blood was added directly into the reaction at a total volume of 40 ml, following a 1:4 dilution with red blood cell lysis buffer (2.5 mM KHCO 3 , 37.5 mM NH 4 Cl, and 0.025 mM EDTA), as previously described [21] . The reaction mixture was incubated at 60uC for 60 minutes, using a", "GeneAmpH PCR System (Applied Biosystems, Foster City, CA) or the NINA heaters. For reactions amplified in the thermalcylcer, an additional two minute heating step of 80uC was added at the end of the amplification cycle to terminate the reaction.", "The reaction tubes were evaluated for the presence of amplification, following addition of the quencher probe at a 2:1 ratio of quencher to labeled-primer, as previously described [21] . Amplification was determined visually by observing fluorescence in the reaction tubes, using the UV lamp from a ChemiDoc XRS system (Bio-Rad Laboratories, Hercules, CA). Amplification was confirmed by electrophoresis using a 1.2% agarose gel containing SYBRH Safe gel stain (Invitrogen), which was subsequently visualized using the ChemiDoc XRS system.", "To compare temperature and amplification consistency, three NINA heaters were tested in parallel. The heating reaction was initiated by adding 18 g of CaO to each NINA canister, followed by 6 ml of water. The lid of each canister was then sealed to contain the exothermic reaction. After adding 200 ml of water to each of the sample wells, temperature recording was initiated. Reaction tubes were added to the sample wells once each reaction chamber reached a temperature of 58.5uC. For all samples incubated in the NINA heater, 15 ml of mineral oil was added to the reaction tube during the reaction mix preparation. The samples were incubated in the heaters for a total of 60 minutes. All reactions were carried out in a temperature-controlled laboratory with an ambient temperature of 28uC, unless otherwise stated. Following the amplification reaction, the samples were incubated for two minutes in a heat block set to 80uC. After each amplification cycle, the temperature profile of each device", "was analyzed by calculating the temperature mean, standard deviation, median, minimum, and maximum from the data provided by the DaqPRO 5300.", "The stability of the NINA heaters at extreme low and high temperatures was evaluated by placing the canisters in a refrigerator set to 4uC or a 37uC incubator during the length of the amplification reaction. The temperature profiles were recorded and compared to those of reactions that occurred at the laboratory room temperature of 28uC.", "To determine the sensitivity of RT-LAMP reaction using RTspecific primers, DNA and RNA linearity panels were tested in a thermalcycler. The limit of detection for HIV-1 DNA was 10 copies/reaction. For the RNA linearity panel, the sample containing 1700 copies/reaction was detected in all of the three replicates, while the sample containing 140 copies/reaction was detected in three out of five replicates (60%). For both DNA and RNA linearity panels, the two samples nearest the limit of detection were chosen to further evaluate the performance consistency between the thermalcycler and NINA heaters. In terms of positivity, the amplification results were consistent between all three heaters and the thermalcycler ( Table 2) . Since the RT-LAMP assay requires a constant temperature of 60uC for the length of the amplification reaction, the temperature profiles of the sample wells were compared over the course of the incubation and between all three NINA heaters. A representative temperature", "profile is displayed in Figure 2 , showing a steady reaction temperature at or close to 60uC for length of amplification reaction. During the 60 minute incubation, the average temperature for each device was 60.2, 59.8, and 59.7 (Table 3 ). The minimum temperature achieved during the reaction reflects the fact that the temperature of the sample port dropped temporarily after the sample tubes are added to the device, as shown in Figure 2 . The maximum temperature of the devices deviated from the desired reaction temperature of 60uC by less than one degree.", "The ability of the NINA heaters to maintain a steady reaction temperature in a wide range of ambient temperatures is essential for POC testing, whether referring to an air-conditioned laboratory or high-temperature field site. To evaluate the performance of the NINA heaters at extreme low or high temperatures, the canisters were placed in a 4uC refrigerator or a 37uC incubator for the length of the amplification reaction. The limit of detection for the DNA and RNA linearity panels was similar to the results obtained in our temperature-controlled laboratory (28uC; Table 2 ). The greatest degree of temperature variation of the sample wells was observed at the ambient temperature of 4uC ( Table 3 ). The average temperature was approximately two degrees lower than the desired reaction temperature of 60uC. Additionally, the temperature of the devices tended to decline from their steady state during the last 20 minutes of the reaction (data not shown). The temperature profiles at the", "ambient temperature of 37uC, however, were similar to those at 28uC.", "Whole blood samples from HIV-1 infected individuals were added directly into the RT-LAMP reaction and tested in the NINA heaters. Positivity of the clinical specimens was consistent between the thermalcycler and devices (Table 4 ). Amplification consistency was most evident with two of the patient samples (patient #4 and #5) that were only positive in one of the three replicates, regardless of the heating device that was used. All HIVnegative blood samples, included in each reaction, were negative (data not shown). A representative experiment using the NINA heaters is displayed in Figure 3 , showing detection by agarose gel and visual identification of fluorescence in the reaction tubes.", "In this study, we demonstrate the performance of portable, inexpensive, non-instrumented nucleic acid (NINA) heaters for amplification of HIV-1 using RT-LAMP. The isothermal amplification reaction coupled with a device that generates heat from an exothermic chemical reaction, as opposed to grid electricity or battery power, comprises a point-of-care NAAT that is practical for use in resource-limited settings. The heating devices require minimal training and technical expertise to operate and take approximately 10-15 minutes to reach a reaction temperature of 60uC once the chemical reaction has been initiated [27, 28] . Furthermore, the temperature of the sample wells remain relatively stable at the desired reaction temperature of 60uC throughout the amplification reaction, as demonstrated by the heating profiles and the consistency in amplification between the devices and thermalcycler.", "Since point-of-care testing may refer to an air-conditioned laboratory or a field site with high temperatures and humidity, the stability of the temperature generated by the heating devices must be reliable. Though the temperature profiles at a representative cold temperature of 4uC indicated a loss in reaction temperature towards the end of the 60 minute incubation, the temperature fluctuations were not significant enough to affect the amplification reaction. Regardless, this thermal effect could be mitigated with small modifications to the device to reduce heat loss at lower temperatures. It should be possible to extend the temperature range of the NINA heaters to 4uC and below by either adding a larger quantity of heating mixture, better insulation, or both. Of greater concern is the performance of the NINA heaters in hightemperature field sites, where temperature control is not an option.", "We demonstrate no difference in the temperature stability of the NINA heaters and amplification consistency at an ambient temperature of 37uC as compared to our temperature-controlled laboratory.", "For increased applicability for use at the POC, several modifications can be made to the NINA heaters. The prototype devices evaluated in this study contained only three sample wells; however, up to 16 sample wells can be added to the lid of the insulated canisters for a larger testing volume. In this study, samples were removed from the NINA heaters after the amplification reaction and heated for an additional two minutes in an 80uC heat block to terminate the reaction. While the additional heating step is not necessary to observe the amplified products from extracted nucleic acid, the short, high-temperature incubation facilitates the visual observation of the fluorescent label in the whole blood samples. Modifications may be made to the whole blood sample preparation method to eliminate the need for the heating step. Alternatively, a second temperature-moderating compartment can be added to the alternate end of the NINA canisters, so the samples can be removed from the", "amplification compartment and reinserted into the 80uC compartment. Lastly, the DaqPRO data recorder was used in this study for validation purposes only and would not be necessary for the final POC product.", "The feasibility of using LAMP as a diagnostic method in resource-limited settings has been demonstrated for tuberculosis [33] . To reduce hands-on time and preparation error, the authors describe the use of reaction tubes pre-prepared with lyophilized reaction mix. For POC use, limited sample manipulation and reagent preparation is desired and, therefore, it is anticipated that the test procedure of the end product will include reconstituting the amplification reagents in water and adding the sample directly into the reaction tube. We demonstrate the use of the NINA heaters for amplification directly from whole blood specimens, eliminating the need for a time-consuming, nucleic acid extraction procedure and reducing the volume of sample needed for the amplification reaction. A total volume of 10 ml of whole blood was added to each reaction tube, which can easily be obtained by finger-stick in settings where venipuncture is not feasible. Additionally, our fluorescent detection method", "enables immediate visualization of amplified products in the absence of specialized equipment. To avoid cross-contamination of amplified material, it is preferred that the reaction tubes remain closed post-amplification. Future modifications will include optimizing the labeledprimer/quencher sequences so that all components can be added into the reaction mix prior to amplification. Due to availability, the Bio-Rad ChemiDoc system was used as the UV source in this study; however, an inexpensive keychain light would be more suitable for naked-eye detection at the POC. For sensitive and specific detection of diverse HIV-1 isolates, including non-B subtypes, identification of the optimal primer set/sets is a key step in the development of the RT-LAMP assay. Although all experiments performed in this study involved subtype B standards and specimens, ongoing research involves the continued development and optimization of RT-LAMP primers based on regions of the HIV-1 genome that are", "conserved among diverse subtypes. Future studies will include large-scale evaluation of clinical specimens with the optimized RT-LAMP assay and NINA device. In summary, the RT-LAMP isothermal amplification method used in conjunction with a simplified, chemical heating device exhibits characteristics that are ideal for a rapid NAAT for POC testing. The simplified, portable assay has the potential to fill an important gap in HIV-1 diagnostics, providing immediate knowledge or confirmation of HIV-1 infection status at the POC." ]

[ 3 ]

[ "Chi-square test and Fisher's exact test" ]

[ "Viral and bacterial co-infection in severe pneumonia triggers innate immune responses and specifically enhances IP-10: a translational study\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5138590/\n\nSHA: ef3d6cabc804e5eb587b34249b539c1b5efa4cc4\n\nAuthors: Hoffmann, Jonathan; Machado, Daniela; Terrier, Olivier; Pouzol, Stephane; Messaoudi, Mélina; Basualdo, Wilma; Espínola, Emilio E; Guillen, Rosa M.; Rosa-Calatrava, Manuel; Picot, Valentina; Bénet, Thomas; Endtz, Hubert; Russomando, Graciela; Paranhos-Baccalà, Gláucia\nDate: 2016-12-06\nDOI: 10.1038/srep38532\nLicense: cc-by", "Abstract: Mixed viral and bacterial infections are widely described in community-acquired pneumonia; however, the clinical implications of co-infection on the associated immunopathology remain poorly studied. In this study, microRNA, mRNA and cytokine/chemokine secretion profiling were investigated for human monocyte-derived macrophages infected in-vitro with Influenza virus A/H1N1 and/or Streptococcus pneumoniae. We observed that the in-vitro co-infection synergistically increased interferon-γ-induced protein-10 (CXCL10, IP-10) expression compared to the singly-infected cells conditions. We demonstrated that endogenous miRNA-200a-3p, whose expression was synergistically induced following co-infection, indirectly regulates CXCL10 expression by targeting suppressor of cytokine signaling-6 (SOCS-6), a well-known regulator of the JAK-STAT signaling pathway. Additionally, in a subsequent clinical pilot study, immunomodulators levels were evaluated in samples from 74 children (≤5", "years-old) hospitalized with viral and/or bacterial community-acquired pneumonia. Clinically, among the 74 cases of pneumonia, patients with identified mixed-detection had significantly higher (3.6-fold) serum IP-10 levels than those with a single detection (P = 0.03), and were significantly associated with severe pneumonia (P < 0.01). This study demonstrates that viral and bacterial co-infection modulates the JAK-STAT signaling pathway and leads to exacerbated IP-10 expression, which could play a major role in the pathogenesis of pneumonia.", "Text: Scientific RepoRts | 6:38532 | DOI: 10 .1038/srep38532 pathogenesis of several diseases and has been suggested as a potential biomarker of viral infection 10, 11 , late-onset bacterial infection in premature infants 12 , and a promising biomarker of sepsis and septic shock 13, 14 . Combined analysis of IP-10 and IFN-γ has also been reported as a useful biomarker for diagnosis and monitoring therapeutic efficacy in patients with active tuberculosis [15] [16] [17] , and both remain detectable in the urine of patients with pulmonary diseases in the absence of renal dysfunction 18 .", "With airway epithelial cells 19 , resident alveolar macrophages (AMs) and blood monocytes-derived macrophages (recruited into tissues under inflammatory conditions 20, 21 ) represent a major line of defense against both pneumococcal (through their high phagocytic capacity [22] [23] [24] ) and influenza infection 25, 26 . So far, no studies have yet focused on the intracellular mechanisms that regulate IP-10 in human blood leukocytes during mixed IAV and SP infection. Several studies indicated that host non-coding small RNAs (including microRNAs) may function as immunomodulators by regulating several pivotal intracellular processes, such as the innate immune response 27 and antiviral activity 28, 29 ; both of these processes are closely related to toll-like receptor (TLR) signaling pathways.", "In this study, we firstly investigated the in vitro intracellular mechanisms that mediate the innate immune response in IAV and/or SP infected human monocyte-derived macrophages (MDMs). Using this approach, we observed that mixed-infection of MDMs induces a synergistic production of IP-10 which can be related to a miRNA-200a/JAK-STAT/SOCS-6 regulatory pathway. Subsequently, in a retrospective analysis of clinical samples collected from children ≤ 5 years-old hospitalized with pneumonia, we confirmed that serum IP-10 level could be related to both viral and/or bacterial etiologies and disease severity.", "Characteristics of MDMs infected by IAV and/or SP. Initially, we investigated in vitro the impact of single and mixed IAV and SP infection on MDMs. Firstly, active replication of IAV was assessed by qRT-PCR and quantification of new infectious viral particles in the cell supernatants ( Fig. 1a,b ). IAV titer increased over time after single infection with IAV and correlated with increased production of negative-strand IAV RNA. Maximum viral replication was observed at 18-24 hours post-infection, after which time both RNA replication and the quantity of infectious particles decreased. In this in vitro model, subsequent challenge of IAV-infected MDMs with SP had no significant impact on the production of new infectious viral particles (Fig. 1b) . Together, these results indicate permissive and productive infection of MDMs by IAV. Secondly, we evaluated whether MDMs are permissive for both IAV and SP infection. The presence of pneumococci within IAV-and SP-infected primary MDMs was", "confirmed at 8 h post-infection (Fig. 1c) , suggesting that MDMs are permissive for viral and bacterial co-infection in the early steps of infection. Importantly, confocal co-detection of mixed IAV and SP was only effective following 8 h post-infection due to the bactericidal impact of SP internalization within human macrophages (after 24 h, data not shown). Thirdly, we evaluated the impact of single and mixed infection with IAV and SP on MDM viability. Mixed infection significantly decreased cell viability (65.2 ± 4.5% total cell death at 48 hours post-infection; P < 0.0001) compared to single SP and IAV infection (39.6 ± 1.7% and 17.4 ± 1.1% total cell death, respectively; Fig. 1d ). Taken together, these results confirmed human MDMs are permissive to mixed viral and bacterial infection. mRNA, microRNA and protein expression profiling reveal an overall induction of the host innate immune response following IAV and/or SP infection of MDMs. To investigate the innate immune response", "orchestrated by IAV-and SP-infected human MDMs, we firstly evaluated the expression of 84 genes involved in the innate and adaptive immune responses (Table S1) ; the major differentially-expressed genes are summarized in Fig. 2a . Expression profiling indicated an overall induction of genes related to the JAK-STAT, NF-Κ β and TLR signaling pathways. Indeed, all interferon-stimulated genes (ISGs) screened, including CXCL10 (fold-change [FC] = 240.9), CCL-2 (FC = 34.2) and MX-1 (FC = 151.4) were upregulated following mixed infection compared to uninfected cells, most of which are closely related to STAT-1 (FC = 52.3), IRF-7 (FC = 6.8) and IFNB1 (FC = 5.2) also found upregulated in mixed infected cells. Secondly, we investigated the endogenous microRNA expression profiles of IAV-and SP-infected MDMs. A selection of microRNAs that were found to be differentially-expressed under different infection conditions are shown in Fig. 2b and Table S2 . MiRNA-200a-3p was overexpressed after both", "single IAV (FC = 6.9), single SP (FC = 3.7) and mixed IAV/SP infection (FC = 7.3), indicating this miRNA may play a role in the innate immune response to viral and bacterial co-infection. Similar miRNA-200a-3p dysregulation profiles were obtained following IAV and/or SP infections of human macrophages-like (THP-1 monocytes-derived macrophages) or primary MDMs (data not shown). Thirdly, the secreted levels of various antiviral, pro-inflammatory and immunomodulatory cytokines/chemokines were assayed in IAV-and SP-infected-THP-1 and primary MDM cell supernatants. We observed a remarkable correlation between the mRNA and protein expression profiles of single or mixed infected MDMs especially regarding CXCL-10 and IP-10 expression. Indeed, the level of IP-10 was synergistically increased in the supernatant of IAV-infected THP-1 MDMs exposed to SP (mean: 30,589 ± 16,484 pg ml −1 ) compared to single IAV infection (1,439 ± 566.5 pg ml −1 ) and single SP infection (4,472 ± 2,001 pg ml −1 ; P≤", "0.05; Fig. 2c ) at 24 hours after infection. In those cells, IP-10 expression reduced over time (48 to 72 hours), coinciding with a significant higher proportion of necrotic and apoptotic cells (Fig. 1d) . The synergistic expression of IP-10 was similarly observed at 24 hours post-infection using primary MDMs (Fig. 2d) . Significantly increased secretion of the other tested cytokines and chemokines was not observed post-infection, even in mixed infected MDMs (Fig. S1 ). Interestingly, a significant production of IP-10 was also observed in supernatants of primary human airway epithelial cells (HAEC) mixed-infected by IAV and SP compared to the single infections (Fig. 2e) . Taken together, the mRNA and protein profiling results suggested that mixed viral and bacterial infection of MDMs induces a synergistic pro-inflammatory response related to the type-1 interferon and JAK-STAT signaling pathways, with IP-10 as signature of IAV/SP co-infection. Among all microRNAs screened, miR-200a-3p", "was the most Scientific RepoRts | 6:38532 | DOI: 10.1038/srep38532 overexpressed in IAV/SP co-infection of human MDMs. In the remainder of this study, we decided to investigate the interconnection between miR-200a-3p expression and the innate immune response.", "Endogenous miRNA-200a-3p expression correlates with CXCL10 (IP-10) induction following mixed IAV and SP infection of human MDMs. Using a specific Taqman probe assay targeting miR-200a-3p, we confirmed a significant upregulation of miR-200a-3p following mixed IAV and SP infection of human MDMs (Fig. 3a) . In this experiment, a more marked up-regulation of miR-200a-3p was observed following IAV+ SP compared to results obtained previously (Fig. 2b) . This discrepancy has been attributed to the use of two different approaches to quantify miR-200a-3p expression. The use of a target-specific stem-loop reverse transcription primer in Fig. 3a allows a better sensitivity of miR-200a-3p detection compared to the non-specific fluorescent dye used in Fig. 2b . As the general trend was suggestive of a synergistic induction of miR-200a-3p in response to mixed infection (Fig. 3a) , we hypothesized microRNA-200a-3p may play a role in the regulation of CXCL10 (IP-10), which was also synergistically", "upregulated in mixed-infected MDMs ( Fig. 2c and d) and primary HAEC ( Statistical analyses were performed using two-way ANOVA with Tukey's post-hoc test; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.", "Scientific RepoRts | 6:38532 | DOI: 10.1038/srep38532 CXCL10 (Fig. 3d) . These results suggested miR-200a-3p indirectly regulates CXCL10 and led us to hypothesize that miR-200a-3p controls a potential repressor of the JAK-STAT signaling pathway. . At 18 h after transfection, the MDMs were singly or mixed infected as described previously. At 8 h post-IAV and/or SP infection, total mRNA was extracted and amplified by PCR using specific primers for the indicated genes. Values represent median ± IQR (a, c) or mean ± SEM (d, e) of three biological replicates. Statistical analyses were performed using a Kruskal-Wallis test (non-parametric, one-way ANOVA with Dunn's post-hoc test) for data presented in (a, c). An ordinary two-way ANOVA (with Tukey's post-hoc multiple comparison test) was used for data presented in (d, e). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. MiRNA-200a-3p indirectly regulates IP-10 expression by targeting SOCS6. As shown in Fig. 2a , several JAK-STAT", "signaling pathway genes were deregulated in mixed IAV-and SP-infected human MDMs; therefore, we hypothesized that miR-200a-3p directly regulates a regulator of the JAK-STAT signaling pathway. Predictive target analysis indicated that the 3' UTR of suppressor of cytokine signaling-6 (SOCS6) may be targeted by miR-200a-3p (Fig. 3b) . SOCS proteins constitute a class of negative regulators of JAK-STAT signaling pathways that are induced by both cytokines and TLR signaling. MiRNA-200a-3p was not predicted to target any of the other six members of the SOCS gene family. Transfection of human MDMs with MIM-200a downregulated SOCS6 (FC = 0.57) while inhibition of miR-200a-3p (INH-200a) upregulated SOCS6 (FC = 1.55), confirming that miR-200a-3p effectively regulates the expression of SOCS6 (Fig. 3e) . Moreover, SOCS6 was synergistically downregulated in IAV-or IAV/SP-infected MDMs overexpressing miRNA-200a (Fig. 3e) , suggesting that both infection and miR-200a-3p negatively regulate the", "expression of SOCS6. Finally, western blotting confirmed that expression of SOCS-6 sharply reduced following infection, especially after mixed IAV and SP infection (Fig. 3f) .", "These results indicate miR-200a-3p is strongly induced in response to mixed viral and bacterial co-infection, which in turn leads to downregulation of the JAK-STAT regulator SOCS-6 at both the mRNA and protein levels and subsequent upregulation of IP-10.", "analyses demonstrated mixed IAV and SP infection of human MDMs and HAEC induced significant production of IP-10. As blood leukocytes and respiratory tract epithelial cells actively contribute to inflammation during pneumonia, we hypothesized the level of IP-10 in serum of patient with pneumonia may be both indicative of mixed respiratory infection and disease severity. As part of a prospective, hospital-based, multicenter case-control study on the etiology of pneumonia among children under 5-years-old, a total of 74 patients (44 male, 30 female) were included in this pilot evaluation. According to WHO guidelines, retrospective analysis indicated 44 (59.5%) children had clinical signs of non-severe pneumonia and 30 (40.5%) children had signs of severe pneumonia. The main patient characteristics at inclusion are shown in Table 1 . Patients with severe pneumonia had significant more recorded episodes of dyspnea (P < 0.001), cyanosis (P = 0.03), lower chest indrawing (P < 0.001), dullness", "to percussion (P < 0.001) and lethargy (P < 0.001) during chest examination than patient with non-severe pneumonia. Moreover, pleural effusions were significantly more observed among critically ill patients and the duration of hospitalization was significantly longer for the children with severe pneumonia than for those with non-severe pneumonia (P = 0.0015). Two deaths occurred within the group of children retrospectively defined with severe pneumonia. Evaluation of the systemic inflammatory response of the 74 cases is shown in Table 2 . Serum level of CRP, IP-10, PCT, G-CSF, IL-6, IL-8 and MIP-1β were significantly more elevated in serum samples from critically ill patients. Patients with severe pneumonia had significantly higher (4.2-fold) serum IP-10 levels than those with a non-severe pneumonia (P < 0.001) suggesting IP-10 as a promising prognostic marker in pneumonia. Diagnostic accuracy measures for predicting pneumonia severity using blood-based biomarkers are summarized in", "Table S3 . Briefly, in this study, the optimal IP-10 cut-off value for identifying patient with severe pneumonia was 4,240 pg ml −1 , with an area under the receiver operating characteristic curve of 0.69 (95% CI, 0.57 to 0.82, P < 0.001). Defining as positive a serum IP-10 level above this cut-off resulted in a sensitivity of 63.3%, specificity of 63.6% and a positive likelihood ratio of 1.74. Prognostic values of IP-10 were closed to procalcitonin (PCT; AUC = 0.70; 95% IC, 0.58 to 0.82, P < 0.001) and IL-6 (AUC = 0.70; 95% IC, 0.58-0.83, P < 0.001).", "Multiplex PCR-based screening of respiratory and blood samples reveal a high variety of pathogen associations (Table 3) . Respiratory viruses were detected in the nasal aspirates (NAs) of 63/74 patients (85.1%). Etiological bacteria of pneumonia (S. pneumoniae, n = 19; S. aureus, n = 1; or H. influenzae type B, n = 7) were identified via real-time PCR in the blood samples of 27/74 (36.5%) of the patients. Multiplex PCR assays allowed the identification of respiratory bacteria in the blood of 19 patients with negative blood culture results. Among the 74 cases PCR-positive for respiratory pathogens, a single virus or bacteria were detected in the NAs of 7 (9.4%) and 3 (4.0%) patients, respectively; these 10/74 (13.5%) cases were defined as the single infection group. The mixed infection group included the 62/74 (83.8%) cases in which (1) multiple viruses and/or bacteria were identified in NAs (38/74; 51.3%) without any bacteria identified in blood samples or (2) one or more viruses", "and/or bacteria were identified in NAs and associated with a blood bacteremia (24/74; 32.4%). We evaluated whether IP-10 serum level could correlate with the viral and bacterial etiologies of pneumonia. Patients with mixed infection had significant higher (3.6-fold) IP-10 serum level than patient with single detection (P = 0.03; Table 4 ). A stratified analysis reveals that the highest IP-10 serum level was observed among patients with both several respiratory pathogens identified (mixed-detection group) and severe pneumonia (14,427 pg ml −1 , IQR (3,981-82,994). In detail, a remarkable IP-10 serum level (142,531 pg ml −1 ), representing 33-fold higher above cut-off value predicting pneumonia severity was observed in patient with hRV in NA co-detected with S. pneumoniae (serotype 14) in pleural effusion and blood. In concordance with our in-vitro model of co-infection, a significant IP-10 level (90,338 pg ml −1 ) was quantified in blood sample of patient with severe bacteremic", "pneumococcal (serotype 14) pneumonia with a positive co-detection of Influenza B virus in NA. Taken together, these results suggest that high serum IP-10 levels are significantly associated with mixed viral and bacterial detection and also related to pneumonia pathogenesis.", "This study provides additional in vitro and clinical data to improve our understanding of the immunopathology of mixed viral and bacterial pneumonia (Fig. 4) .", "The in vitro model of influenza and pneumococcal superinfection of human MDMs demonstrated that mixed infection synergistically induced release of the pro-inflammatory chemokine IP-10, strongly suggesting human Scientific RepoRts | 6:38532 | DOI: 10.1038/srep38532 blood leukocytes contribute to the immunopathology of pneumonia. Additionally, transcriptomics and omics analyses provided new data on the inflammatory pathways that are activated during mixed infection and related to synergistic induction of the pro-inflammatory chemokine IP-10 in mixed infected cells. Our observations are consistent with a recent study describing IP-10 induction as host-proteome signature of both viral and bacterial infections 30 . Of the differentially-expressed genes observed in mixed infected MDMs, the transcription factors STAT-1 and IRF-7 appear to play crucial roles in the regulation of interferon-stimulated genes including CXCL10 (IP-10). By focusing on the intracellular mechanisms that regulate", "inflammatory pathways, we demonstrated a novel role for miRNA-200a-3p in the regulation of CXCL10 (IP-10). These observations are consistent with previous reports showing that RNA virus infection upregulates miR-155 in macrophages and dendritic cells and also regulates suppressor of cytokine signaling 1 (SOCS1), suggesting the existence of a miRNA/JAK-STAT/SOCS regulatory pathway during viral infection 29 . Our study suggests co-infection leads to overexpression of miR-200a-3p, which in turn targets and downregulates the JAK-STAT regulator SOCS-6 and consequently increases CXCL10 (IP-10) expression. Interestingly, a complementary in-silico approach reveals that several microRNAs that were found dysregulated in our experiments of IAV and SP co-infection of MDMs or HAEC, might target several genes of SOCS family and play similar role than miR-200a-3p. Indeed, miRNA-142-3p might target SOCS4, 5, 6 mRNA while miRNA-194-5p might target SOCS2, 3, 4, 5 and 7 mRNA. These observations", "underline that intra-cellular regulation of IP-10 is not limited to the contribution of a sole microRNA. A complex inter-relationship between numerous host microRNAs and inhibitors of the JAK-STAT signaling pathway occur to control host innate inflammatory response against viral and/or bacterial infections. Clinically, the majority of pediatric CAP cases in this study were associated with both positive viral and/or bacterial detection. Respiratory microorganisms were detected in 97% of cases; 51.3% of which were viral-viral, viral-bacterial or bacterial-bacterial co-detected only in nasal aspirates, 32.4% of which co-detected in both nasal aspirates and blood samples. These data are consistent with previous etiological studies of pediatric CAP 3,31-33 . S. pneumoniae was the major bacteria identified in blood (19/74; 25.7%) and mainly co-detected with respiratory viruses in NAs (16/19; 84.2%). We observed a very high diversity of viral and bacterial associations in biological samples", "from children with pneumonia. In comparison with IAV and SP14 combination evaluated in-vitro, no pneumonia cases were singly influenza and pneumococcus infected, and no similar co-detection with those two pathogens has been clinically observed. Nevertheless, Influenza B (IVB) virus was identified in 5 patients and two of them had a positive SP co-detection in blood (one non-typable strain and one serotype 14 using our molecular typing test). IVB and SP14 combination seems to be the nearest pathogen co-detection to that in-vitro investigated. Clinically, this co-detection was associated with both a very high IP-10 expression and a very severe pneumonia case definition. Interestingly, our translational pilot evaluation reveals IP-10 expression can be induced by several different viral and/or bacterial combinations. As immune response to each pathogen is different, further in-vitro investigations using different pathogens associations are needed to better characterize the mechanisms", "involved in the immunopathology of pneumonia.", "In this cohort, highest serum IP-10 levels were identified among patients with both several pathogen detected and severe pneumonia, suggesting a significant role of IP-10 on pneumonia pathogenesis. Indeed, high plasma levels of IP-10 have previously been reported in patients with sepsis 12 , and were associated with high mortality rate, especially among patients with CAP 34 . Additionally, the IP-10-CXCR3 axis has been related to acute immune lung injury and lymphocyte apoptosis during the development of severe acute respiratory syndrome (SARS) 35, 36 . Moreover, an in vivo study that modeled influenza and pneumococcal superinfection in mice indicated that pro-inflammatory chemokines, including IP-10, play a crucial role in influenza-induced susceptibility to lung neutrophilia, severe immunopathology and mortality 37 . In this study, markedly elevated IP-10 (92,809 pg ml −1 ) combined with the highest PCT level (74.4 pg ml −1 ) were quantified in the serum sample of a child who died,", "in whom S. pneumoniae (serotype 9 V) was identified in the blood (PCR and blood culture) and co-detected with Haemophilus influenzae type B in nasal aspirate. These observations suggest an interrelationship between co-detection, elevated serum IP-10 and the pathogenesis of pneumonia.", "Several limitations of this pilot translational study need to be acknowledged before concluding mixed infection is related to elevated IP-10 and disease severity. Indeed, although viral shedding (e.g., of HRV and HBoV) is common in asymptomatic children, we were unable to evaluate the levels of immunomodulators in the serum samples of a control group. Moreover, although the samples were collected within the first 24 hours after admission, only a single blood sample was processed for each patient. Therefore, a larger, longitudinal study on the etiology and severity of pneumonia will be necessary to confirm these results. In conclusion, the present findings suggest that mixed respiratory infections and IP-10 may play major, interconnected roles in the pathogenesis of pneumonia. Clinically, assessment and monitoring of induced IP-10 serum level may assist clinicians to improve diagnosis and patient management of severe community-acquired pneumonia.", "Viral and bacterial strains. The 10 ng ml −1 M-CSF (Miltenyi Biotec). THP− 1 MDMs were obtained by culturing cells with 10 ng ml -1 phorbol myristate acetate (PMA; Invivogen, Toulouse, France) for 72 hours. Human airway epithelial cells (HAEC, bronchial cell type) originated from a 54-years old woman with no pathology reported (batch number MD056501) were provided by Mucilair (Epithelix, Geneva, Switzerland). Sterility, tissue integrity (TEER), mucus production and cilia beating frequency have been certified by the company. Gene expression profiling. Total cellular mRNA was purified using the RNeasy kit (Qiagen, Hilden, Germany). Reverse-transcription of total mRNA was performed using the RT 2 First Strand Kit (SABiosciences, Hilden, Germany). The expression of 84 genes involved in the human innate and adaptive immune responses was evaluated using the RT 2 profiler ™ PCR Array (SABiosciences) according to the manufacturer's recommendations. The Δ Δ Ct method was applied to calculate", "the fold changes in gene expression for each gene relative to uninfected control cells using the web-based RT 2 profiler PCR Array Data Analysis software (SABiosciences).", "MicroRNA profiling array. Total cellular microRNAs were purified using the miRNeasy Mini kit (Qiagen) and reverse-transcribed using the miScript Reverse Transcription kit (Qiagen). The profiling of 84 miRNAs was performed using the Human Immunopathology miScript miRNA PCR Array kit (Qiagen) according to the manufacturer's instructions. Data were analyzed using the miScript miRNA PCR array data analysis web portal.\n\nIn silico miRNA target prediction. MiRNA target genes were retrieved and compiled using TargetScan 38 and microRNA.org resource 39 . The interactions between miRNAs and intracellular pathways were predicted using DIANA-miRPath v2.0 40 .", "THP-1 MDMs were seeded in 24-well plates (0.5 × 10 6 per well) in triplicate, exposed to Influenza A H1N1 (A/Solomon islands/3/2006) virus (IAV) under serum-free conditions for 1 hour and then cultured for 3 hours in fresh RPMI-1640 containing 2% FBS. Streptococcus pneumoniae (SP) serotype 14 was added at 4 hours after IAV infection. Gentamicin (10 μ g ml −1 ) was added 2 hours after SP infection (i.e. 6 hours post-influenza infection) and maintained in the culture media throughout the experiment to kill extracellular bacteria and limit bacterial growth. Cell viability was determined by flow-cytometry using the FITC/Annexin V apoptosis detection kit (BD Biosciences), according to the manufacturer's instructions. #4427975) . In this assay, fold changes have been defined by the Δ Δ Ct method using control RNU-44 and -48 as reference microRNAs. Total mRNA was purified from transfected and infected MDMs using the RNeasy kit (Qiagen) and specific primers were used to amplify transforming", "growth factor beta-2 (TGFB2; F: 5′ -CCATCCCGCCCACTTTCTAC-3′ , R: 5′ -AGCTCAATCCGTTGTTCAGGC-3′ ), SOCS6 (F: 5′ -AAGAATTCATCCCTTGGATTAGGTAAC-3′ , R: 5′ -CAGACTGGAGGTCGTGGAA-3′ ) 41 43 , and 3) absence of wheezing at auscultation, and, 4) first symptoms appearing within the last 14 days, and 5) radiological confirmation of pneumonia as per WHO guidelines 44 . Based on these primary criteria defining pneumonia cases, all 74 cases were retrospectively re-evaluated according to the WHO \"Pocket book of hospital care for children\" 45 criteria to evaluate pneumonia severity. Cases that died during the study, or who had at least one additional clinical signs including central cyanosis, dullness to percussion during chest examination, prostration/lethargy, pleural effusion observed on chest radiography were retrospectively included in the severe pneumonia group. Patients without any of these additional clinical signs were included in the non-severe pneumonia group. Table 4 . a IP-10 values are", "expressed in pg ml -1 . IP-10 concentration differences between groups were compared using unpaired Mann-Whitney tests; significant changes (P < 0.05) are in bold. Clinical and molecular analysis. Nasopharyngeal aspirates (NAs) and whole blood samples were collected from children within 24 hours of admission. Whole blood samples were used for complete blood counts, blood culture and multiplex real-time PCR to identify Staphylococcus aureus, Streptococcus pneumoniae and Haemophilus influenzae type B 46 . S. pneumoniae serotypes were defined using a 11 multiplex real-time PCR assay targeting the 40 most frequently represented serotypes or serogroups according to protocol developed by Messaoudi et al. 47 . Serum C-reactive protein (CRP; AssayPro, St. Charles, Missouri, United States) and Procalcitonin (PCT; VIDAS B.R.A.H.M.S; bioMérieux) were quantified from whole-blood samples. Multiplex real-time non quantitative PCR (Fast-Track Diagnostic, Sliema, Malta) was used to detect 19 viruses", "and five bacteria in the respiratory specimens (NAs and pleural effusions). Mixed detection was defined as 1) PCR-positive for multiple viruses in NAs, 2) positive blood culture or PCR-positive for multiple bacteria in blood or 3) PCR-positive for one or multiple viruses in NAs and one or multiple bacteria in blood (identified by PCR and blood culture).", "Ethical approval. The study protocol, informed consent statement, clinical research form, any amendments and all other study documents were submitted to and approved by the Ethical Committee of the Instituto de Investigaciones en Ciencias de la Salud, the Universidad Nacional de Asunción (IICS-UNA) and the Hospital Pediátrico Niños de Acosta Ñu. Informed consent was obtained from all subjects involved in this study. The clinical investigation was conducted according to the principles expressed in the Declaration of Helsinki.", "Statistical analysis. The Chi-square test and Fisher's exact test were used to compare categorical variables; continuous variables and non-normally distributed data were compared using the Mann-Whitney U-test; normally distributed data were compared using unpaired t-tests. Comparative analyses between experimental conditions (i.e., MOCK, IAV, SP or IAV + SP) were performed using one-way ANOVA with Tukey's post-hoc test or Kruskal-Wallis analysis with Dunn's post-hoc tests. Receiver operating curve (ROC) analysis was used to determine the optimal cut-off value for IP-10 to differentiate between non-severe and severe pneumonia cases. P < 0.05 was considered statistically significant. All statistical analyses were performed using GraphPad Prism (La Jolla, California, United States)." ]

[ 41 ]

[ "unable to evaluate the levels of immunomodulators in the serum samples of a control group" ]

[ "Viral and bacterial co-infection in severe pneumonia triggers innate immune responses and specifically enhances IP-10: a translational study\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5138590/\n\nSHA: ef3d6cabc804e5eb587b34249b539c1b5efa4cc4\n\nAuthors: Hoffmann, Jonathan; Machado, Daniela; Terrier, Olivier; Pouzol, Stephane; Messaoudi, Mélina; Basualdo, Wilma; Espínola, Emilio E; Guillen, Rosa M.; Rosa-Calatrava, Manuel; Picot, Valentina; Bénet, Thomas; Endtz, Hubert; Russomando, Graciela; Paranhos-Baccalà, Gláucia\nDate: 2016-12-06\nDOI: 10.1038/srep38532\nLicense: cc-by", "Abstract: Mixed viral and bacterial infections are widely described in community-acquired pneumonia; however, the clinical implications of co-infection on the associated immunopathology remain poorly studied. In this study, microRNA, mRNA and cytokine/chemokine secretion profiling were investigated for human monocyte-derived macrophages infected in-vitro with Influenza virus A/H1N1 and/or Streptococcus pneumoniae. We observed that the in-vitro co-infection synergistically increased interferon-γ-induced protein-10 (CXCL10, IP-10) expression compared to the singly-infected cells conditions. We demonstrated that endogenous miRNA-200a-3p, whose expression was synergistically induced following co-infection, indirectly regulates CXCL10 expression by targeting suppressor of cytokine signaling-6 (SOCS-6), a well-known regulator of the JAK-STAT signaling pathway. Additionally, in a subsequent clinical pilot study, immunomodulators levels were evaluated in samples from 74 children (≤5", "years-old) hospitalized with viral and/or bacterial community-acquired pneumonia. Clinically, among the 74 cases of pneumonia, patients with identified mixed-detection had significantly higher (3.6-fold) serum IP-10 levels than those with a single detection (P = 0.03), and were significantly associated with severe pneumonia (P < 0.01). This study demonstrates that viral and bacterial co-infection modulates the JAK-STAT signaling pathway and leads to exacerbated IP-10 expression, which could play a major role in the pathogenesis of pneumonia.", "Text: Scientific RepoRts | 6:38532 | DOI: 10 .1038/srep38532 pathogenesis of several diseases and has been suggested as a potential biomarker of viral infection 10, 11 , late-onset bacterial infection in premature infants 12 , and a promising biomarker of sepsis and septic shock 13, 14 . Combined analysis of IP-10 and IFN-γ has also been reported as a useful biomarker for diagnosis and monitoring therapeutic efficacy in patients with active tuberculosis [15] [16] [17] , and both remain detectable in the urine of patients with pulmonary diseases in the absence of renal dysfunction 18 .", "With airway epithelial cells 19 , resident alveolar macrophages (AMs) and blood monocytes-derived macrophages (recruited into tissues under inflammatory conditions 20, 21 ) represent a major line of defense against both pneumococcal (through their high phagocytic capacity [22] [23] [24] ) and influenza infection 25, 26 . So far, no studies have yet focused on the intracellular mechanisms that regulate IP-10 in human blood leukocytes during mixed IAV and SP infection. Several studies indicated that host non-coding small RNAs (including microRNAs) may function as immunomodulators by regulating several pivotal intracellular processes, such as the innate immune response 27 and antiviral activity 28, 29 ; both of these processes are closely related to toll-like receptor (TLR) signaling pathways.", "In this study, we firstly investigated the in vitro intracellular mechanisms that mediate the innate immune response in IAV and/or SP infected human monocyte-derived macrophages (MDMs). Using this approach, we observed that mixed-infection of MDMs induces a synergistic production of IP-10 which can be related to a miRNA-200a/JAK-STAT/SOCS-6 regulatory pathway. Subsequently, in a retrospective analysis of clinical samples collected from children ≤ 5 years-old hospitalized with pneumonia, we confirmed that serum IP-10 level could be related to both viral and/or bacterial etiologies and disease severity.", "Characteristics of MDMs infected by IAV and/or SP. Initially, we investigated in vitro the impact of single and mixed IAV and SP infection on MDMs. Firstly, active replication of IAV was assessed by qRT-PCR and quantification of new infectious viral particles in the cell supernatants ( Fig. 1a,b ). IAV titer increased over time after single infection with IAV and correlated with increased production of negative-strand IAV RNA. Maximum viral replication was observed at 18-24 hours post-infection, after which time both RNA replication and the quantity of infectious particles decreased. In this in vitro model, subsequent challenge of IAV-infected MDMs with SP had no significant impact on the production of new infectious viral particles (Fig. 1b) . Together, these results indicate permissive and productive infection of MDMs by IAV. Secondly, we evaluated whether MDMs are permissive for both IAV and SP infection. The presence of pneumococci within IAV-and SP-infected primary MDMs was", "confirmed at 8 h post-infection (Fig. 1c) , suggesting that MDMs are permissive for viral and bacterial co-infection in the early steps of infection. Importantly, confocal co-detection of mixed IAV and SP was only effective following 8 h post-infection due to the bactericidal impact of SP internalization within human macrophages (after 24 h, data not shown). Thirdly, we evaluated the impact of single and mixed infection with IAV and SP on MDM viability. Mixed infection significantly decreased cell viability (65.2 ± 4.5% total cell death at 48 hours post-infection; P < 0.0001) compared to single SP and IAV infection (39.6 ± 1.7% and 17.4 ± 1.1% total cell death, respectively; Fig. 1d ). Taken together, these results confirmed human MDMs are permissive to mixed viral and bacterial infection. mRNA, microRNA and protein expression profiling reveal an overall induction of the host innate immune response following IAV and/or SP infection of MDMs. To investigate the innate immune response", "orchestrated by IAV-and SP-infected human MDMs, we firstly evaluated the expression of 84 genes involved in the innate and adaptive immune responses (Table S1) ; the major differentially-expressed genes are summarized in Fig. 2a . Expression profiling indicated an overall induction of genes related to the JAK-STAT, NF-Κ β and TLR signaling pathways. Indeed, all interferon-stimulated genes (ISGs) screened, including CXCL10 (fold-change [FC] = 240.9), CCL-2 (FC = 34.2) and MX-1 (FC = 151.4) were upregulated following mixed infection compared to uninfected cells, most of which are closely related to STAT-1 (FC = 52.3), IRF-7 (FC = 6.8) and IFNB1 (FC = 5.2) also found upregulated in mixed infected cells. Secondly, we investigated the endogenous microRNA expression profiles of IAV-and SP-infected MDMs. A selection of microRNAs that were found to be differentially-expressed under different infection conditions are shown in Fig. 2b and Table S2 . MiRNA-200a-3p was overexpressed after both", "single IAV (FC = 6.9), single SP (FC = 3.7) and mixed IAV/SP infection (FC = 7.3), indicating this miRNA may play a role in the innate immune response to viral and bacterial co-infection. Similar miRNA-200a-3p dysregulation profiles were obtained following IAV and/or SP infections of human macrophages-like (THP-1 monocytes-derived macrophages) or primary MDMs (data not shown). Thirdly, the secreted levels of various antiviral, pro-inflammatory and immunomodulatory cytokines/chemokines were assayed in IAV-and SP-infected-THP-1 and primary MDM cell supernatants. We observed a remarkable correlation between the mRNA and protein expression profiles of single or mixed infected MDMs especially regarding CXCL-10 and IP-10 expression. Indeed, the level of IP-10 was synergistically increased in the supernatant of IAV-infected THP-1 MDMs exposed to SP (mean: 30,589 ± 16,484 pg ml −1 ) compared to single IAV infection (1,439 ± 566.5 pg ml −1 ) and single SP infection (4,472 ± 2,001 pg ml −1 ; P≤", "0.05; Fig. 2c ) at 24 hours after infection. In those cells, IP-10 expression reduced over time (48 to 72 hours), coinciding with a significant higher proportion of necrotic and apoptotic cells (Fig. 1d) . The synergistic expression of IP-10 was similarly observed at 24 hours post-infection using primary MDMs (Fig. 2d) . Significantly increased secretion of the other tested cytokines and chemokines was not observed post-infection, even in mixed infected MDMs (Fig. S1 ). Interestingly, a significant production of IP-10 was also observed in supernatants of primary human airway epithelial cells (HAEC) mixed-infected by IAV and SP compared to the single infections (Fig. 2e) . Taken together, the mRNA and protein profiling results suggested that mixed viral and bacterial infection of MDMs induces a synergistic pro-inflammatory response related to the type-1 interferon and JAK-STAT signaling pathways, with IP-10 as signature of IAV/SP co-infection. Among all microRNAs screened, miR-200a-3p", "was the most Scientific RepoRts | 6:38532 | DOI: 10.1038/srep38532 overexpressed in IAV/SP co-infection of human MDMs. In the remainder of this study, we decided to investigate the interconnection between miR-200a-3p expression and the innate immune response.", "Endogenous miRNA-200a-3p expression correlates with CXCL10 (IP-10) induction following mixed IAV and SP infection of human MDMs. Using a specific Taqman probe assay targeting miR-200a-3p, we confirmed a significant upregulation of miR-200a-3p following mixed IAV and SP infection of human MDMs (Fig. 3a) . In this experiment, a more marked up-regulation of miR-200a-3p was observed following IAV+ SP compared to results obtained previously (Fig. 2b) . This discrepancy has been attributed to the use of two different approaches to quantify miR-200a-3p expression. The use of a target-specific stem-loop reverse transcription primer in Fig. 3a allows a better sensitivity of miR-200a-3p detection compared to the non-specific fluorescent dye used in Fig. 2b . As the general trend was suggestive of a synergistic induction of miR-200a-3p in response to mixed infection (Fig. 3a) , we hypothesized microRNA-200a-3p may play a role in the regulation of CXCL10 (IP-10), which was also synergistically", "upregulated in mixed-infected MDMs ( Fig. 2c and d) and primary HAEC ( Statistical analyses were performed using two-way ANOVA with Tukey's post-hoc test; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.", "Scientific RepoRts | 6:38532 | DOI: 10.1038/srep38532 CXCL10 (Fig. 3d) . These results suggested miR-200a-3p indirectly regulates CXCL10 and led us to hypothesize that miR-200a-3p controls a potential repressor of the JAK-STAT signaling pathway. . At 18 h after transfection, the MDMs were singly or mixed infected as described previously. At 8 h post-IAV and/or SP infection, total mRNA was extracted and amplified by PCR using specific primers for the indicated genes. Values represent median ± IQR (a, c) or mean ± SEM (d, e) of three biological replicates. Statistical analyses were performed using a Kruskal-Wallis test (non-parametric, one-way ANOVA with Dunn's post-hoc test) for data presented in (a, c). An ordinary two-way ANOVA (with Tukey's post-hoc multiple comparison test) was used for data presented in (d, e). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. MiRNA-200a-3p indirectly regulates IP-10 expression by targeting SOCS6. As shown in Fig. 2a , several JAK-STAT", "signaling pathway genes were deregulated in mixed IAV-and SP-infected human MDMs; therefore, we hypothesized that miR-200a-3p directly regulates a regulator of the JAK-STAT signaling pathway. Predictive target analysis indicated that the 3' UTR of suppressor of cytokine signaling-6 (SOCS6) may be targeted by miR-200a-3p (Fig. 3b) . SOCS proteins constitute a class of negative regulators of JAK-STAT signaling pathways that are induced by both cytokines and TLR signaling. MiRNA-200a-3p was not predicted to target any of the other six members of the SOCS gene family. Transfection of human MDMs with MIM-200a downregulated SOCS6 (FC = 0.57) while inhibition of miR-200a-3p (INH-200a) upregulated SOCS6 (FC = 1.55), confirming that miR-200a-3p effectively regulates the expression of SOCS6 (Fig. 3e) . Moreover, SOCS6 was synergistically downregulated in IAV-or IAV/SP-infected MDMs overexpressing miRNA-200a (Fig. 3e) , suggesting that both infection and miR-200a-3p negatively regulate the", "expression of SOCS6. Finally, western blotting confirmed that expression of SOCS-6 sharply reduced following infection, especially after mixed IAV and SP infection (Fig. 3f) .", "These results indicate miR-200a-3p is strongly induced in response to mixed viral and bacterial co-infection, which in turn leads to downregulation of the JAK-STAT regulator SOCS-6 at both the mRNA and protein levels and subsequent upregulation of IP-10.", "analyses demonstrated mixed IAV and SP infection of human MDMs and HAEC induced significant production of IP-10. As blood leukocytes and respiratory tract epithelial cells actively contribute to inflammation during pneumonia, we hypothesized the level of IP-10 in serum of patient with pneumonia may be both indicative of mixed respiratory infection and disease severity. As part of a prospective, hospital-based, multicenter case-control study on the etiology of pneumonia among children under 5-years-old, a total of 74 patients (44 male, 30 female) were included in this pilot evaluation. According to WHO guidelines, retrospective analysis indicated 44 (59.5%) children had clinical signs of non-severe pneumonia and 30 (40.5%) children had signs of severe pneumonia. The main patient characteristics at inclusion are shown in Table 1 . Patients with severe pneumonia had significant more recorded episodes of dyspnea (P < 0.001), cyanosis (P = 0.03), lower chest indrawing (P < 0.001), dullness", "to percussion (P < 0.001) and lethargy (P < 0.001) during chest examination than patient with non-severe pneumonia. Moreover, pleural effusions were significantly more observed among critically ill patients and the duration of hospitalization was significantly longer for the children with severe pneumonia than for those with non-severe pneumonia (P = 0.0015). Two deaths occurred within the group of children retrospectively defined with severe pneumonia. Evaluation of the systemic inflammatory response of the 74 cases is shown in Table 2 . Serum level of CRP, IP-10, PCT, G-CSF, IL-6, IL-8 and MIP-1β were significantly more elevated in serum samples from critically ill patients. Patients with severe pneumonia had significantly higher (4.2-fold) serum IP-10 levels than those with a non-severe pneumonia (P < 0.001) suggesting IP-10 as a promising prognostic marker in pneumonia. Diagnostic accuracy measures for predicting pneumonia severity using blood-based biomarkers are summarized in", "Table S3 . Briefly, in this study, the optimal IP-10 cut-off value for identifying patient with severe pneumonia was 4,240 pg ml −1 , with an area under the receiver operating characteristic curve of 0.69 (95% CI, 0.57 to 0.82, P < 0.001). Defining as positive a serum IP-10 level above this cut-off resulted in a sensitivity of 63.3%, specificity of 63.6% and a positive likelihood ratio of 1.74. Prognostic values of IP-10 were closed to procalcitonin (PCT; AUC = 0.70; 95% IC, 0.58 to 0.82, P < 0.001) and IL-6 (AUC = 0.70; 95% IC, 0.58-0.83, P < 0.001).", "Multiplex PCR-based screening of respiratory and blood samples reveal a high variety of pathogen associations (Table 3) . Respiratory viruses were detected in the nasal aspirates (NAs) of 63/74 patients (85.1%). Etiological bacteria of pneumonia (S. pneumoniae, n = 19; S. aureus, n = 1; or H. influenzae type B, n = 7) were identified via real-time PCR in the blood samples of 27/74 (36.5%) of the patients. Multiplex PCR assays allowed the identification of respiratory bacteria in the blood of 19 patients with negative blood culture results. Among the 74 cases PCR-positive for respiratory pathogens, a single virus or bacteria were detected in the NAs of 7 (9.4%) and 3 (4.0%) patients, respectively; these 10/74 (13.5%) cases were defined as the single infection group. The mixed infection group included the 62/74 (83.8%) cases in which (1) multiple viruses and/or bacteria were identified in NAs (38/74; 51.3%) without any bacteria identified in blood samples or (2) one or more viruses", "and/or bacteria were identified in NAs and associated with a blood bacteremia (24/74; 32.4%). We evaluated whether IP-10 serum level could correlate with the viral and bacterial etiologies of pneumonia. Patients with mixed infection had significant higher (3.6-fold) IP-10 serum level than patient with single detection (P = 0.03; Table 4 ). A stratified analysis reveals that the highest IP-10 serum level was observed among patients with both several respiratory pathogens identified (mixed-detection group) and severe pneumonia (14,427 pg ml −1 , IQR (3,981-82,994). In detail, a remarkable IP-10 serum level (142,531 pg ml −1 ), representing 33-fold higher above cut-off value predicting pneumonia severity was observed in patient with hRV in NA co-detected with S. pneumoniae (serotype 14) in pleural effusion and blood. In concordance with our in-vitro model of co-infection, a significant IP-10 level (90,338 pg ml −1 ) was quantified in blood sample of patient with severe bacteremic", "pneumococcal (serotype 14) pneumonia with a positive co-detection of Influenza B virus in NA. Taken together, these results suggest that high serum IP-10 levels are significantly associated with mixed viral and bacterial detection and also related to pneumonia pathogenesis.", "This study provides additional in vitro and clinical data to improve our understanding of the immunopathology of mixed viral and bacterial pneumonia (Fig. 4) .", "The in vitro model of influenza and pneumococcal superinfection of human MDMs demonstrated that mixed infection synergistically induced release of the pro-inflammatory chemokine IP-10, strongly suggesting human Scientific RepoRts | 6:38532 | DOI: 10.1038/srep38532 blood leukocytes contribute to the immunopathology of pneumonia. Additionally, transcriptomics and omics analyses provided new data on the inflammatory pathways that are activated during mixed infection and related to synergistic induction of the pro-inflammatory chemokine IP-10 in mixed infected cells. Our observations are consistent with a recent study describing IP-10 induction as host-proteome signature of both viral and bacterial infections 30 . Of the differentially-expressed genes observed in mixed infected MDMs, the transcription factors STAT-1 and IRF-7 appear to play crucial roles in the regulation of interferon-stimulated genes including CXCL10 (IP-10). By focusing on the intracellular mechanisms that regulate", "inflammatory pathways, we demonstrated a novel role for miRNA-200a-3p in the regulation of CXCL10 (IP-10). These observations are consistent with previous reports showing that RNA virus infection upregulates miR-155 in macrophages and dendritic cells and also regulates suppressor of cytokine signaling 1 (SOCS1), suggesting the existence of a miRNA/JAK-STAT/SOCS regulatory pathway during viral infection 29 . Our study suggests co-infection leads to overexpression of miR-200a-3p, which in turn targets and downregulates the JAK-STAT regulator SOCS-6 and consequently increases CXCL10 (IP-10) expression. Interestingly, a complementary in-silico approach reveals that several microRNAs that were found dysregulated in our experiments of IAV and SP co-infection of MDMs or HAEC, might target several genes of SOCS family and play similar role than miR-200a-3p. Indeed, miRNA-142-3p might target SOCS4, 5, 6 mRNA while miRNA-194-5p might target SOCS2, 3, 4, 5 and 7 mRNA. These observations", "underline that intra-cellular regulation of IP-10 is not limited to the contribution of a sole microRNA. A complex inter-relationship between numerous host microRNAs and inhibitors of the JAK-STAT signaling pathway occur to control host innate inflammatory response against viral and/or bacterial infections. Clinically, the majority of pediatric CAP cases in this study were associated with both positive viral and/or bacterial detection. Respiratory microorganisms were detected in 97% of cases; 51.3% of which were viral-viral, viral-bacterial or bacterial-bacterial co-detected only in nasal aspirates, 32.4% of which co-detected in both nasal aspirates and blood samples. These data are consistent with previous etiological studies of pediatric CAP 3,31-33 . S. pneumoniae was the major bacteria identified in blood (19/74; 25.7%) and mainly co-detected with respiratory viruses in NAs (16/19; 84.2%). We observed a very high diversity of viral and bacterial associations in biological samples", "from children with pneumonia. In comparison with IAV and SP14 combination evaluated in-vitro, no pneumonia cases were singly influenza and pneumococcus infected, and no similar co-detection with those two pathogens has been clinically observed. Nevertheless, Influenza B (IVB) virus was identified in 5 patients and two of them had a positive SP co-detection in blood (one non-typable strain and one serotype 14 using our molecular typing test). IVB and SP14 combination seems to be the nearest pathogen co-detection to that in-vitro investigated. Clinically, this co-detection was associated with both a very high IP-10 expression and a very severe pneumonia case definition. Interestingly, our translational pilot evaluation reveals IP-10 expression can be induced by several different viral and/or bacterial combinations. As immune response to each pathogen is different, further in-vitro investigations using different pathogens associations are needed to better characterize the mechanisms", "involved in the immunopathology of pneumonia.", "In this cohort, highest serum IP-10 levels were identified among patients with both several pathogen detected and severe pneumonia, suggesting a significant role of IP-10 on pneumonia pathogenesis. Indeed, high plasma levels of IP-10 have previously been reported in patients with sepsis 12 , and were associated with high mortality rate, especially among patients with CAP 34 . Additionally, the IP-10-CXCR3 axis has been related to acute immune lung injury and lymphocyte apoptosis during the development of severe acute respiratory syndrome (SARS) 35, 36 . Moreover, an in vivo study that modeled influenza and pneumococcal superinfection in mice indicated that pro-inflammatory chemokines, including IP-10, play a crucial role in influenza-induced susceptibility to lung neutrophilia, severe immunopathology and mortality 37 . In this study, markedly elevated IP-10 (92,809 pg ml −1 ) combined with the highest PCT level (74.4 pg ml −1 ) were quantified in the serum sample of a child who died,", "in whom S. pneumoniae (serotype 9 V) was identified in the blood (PCR and blood culture) and co-detected with Haemophilus influenzae type B in nasal aspirate. These observations suggest an interrelationship between co-detection, elevated serum IP-10 and the pathogenesis of pneumonia.", "Several limitations of this pilot translational study need to be acknowledged before concluding mixed infection is related to elevated IP-10 and disease severity. Indeed, although viral shedding (e.g., of HRV and HBoV) is common in asymptomatic children, we were unable to evaluate the levels of immunomodulators in the serum samples of a control group. Moreover, although the samples were collected within the first 24 hours after admission, only a single blood sample was processed for each patient. Therefore, a larger, longitudinal study on the etiology and severity of pneumonia will be necessary to confirm these results. In conclusion, the present findings suggest that mixed respiratory infections and IP-10 may play major, interconnected roles in the pathogenesis of pneumonia. Clinically, assessment and monitoring of induced IP-10 serum level may assist clinicians to improve diagnosis and patient management of severe community-acquired pneumonia.", "Viral and bacterial strains. The 10 ng ml −1 M-CSF (Miltenyi Biotec). THP− 1 MDMs were obtained by culturing cells with 10 ng ml -1 phorbol myristate acetate (PMA; Invivogen, Toulouse, France) for 72 hours. Human airway epithelial cells (HAEC, bronchial cell type) originated from a 54-years old woman with no pathology reported (batch number MD056501) were provided by Mucilair (Epithelix, Geneva, Switzerland). Sterility, tissue integrity (TEER), mucus production and cilia beating frequency have been certified by the company. Gene expression profiling. Total cellular mRNA was purified using the RNeasy kit (Qiagen, Hilden, Germany). Reverse-transcription of total mRNA was performed using the RT 2 First Strand Kit (SABiosciences, Hilden, Germany). The expression of 84 genes involved in the human innate and adaptive immune responses was evaluated using the RT 2 profiler ™ PCR Array (SABiosciences) according to the manufacturer's recommendations. The Δ Δ Ct method was applied to calculate", "the fold changes in gene expression for each gene relative to uninfected control cells using the web-based RT 2 profiler PCR Array Data Analysis software (SABiosciences).", "MicroRNA profiling array. Total cellular microRNAs were purified using the miRNeasy Mini kit (Qiagen) and reverse-transcribed using the miScript Reverse Transcription kit (Qiagen). The profiling of 84 miRNAs was performed using the Human Immunopathology miScript miRNA PCR Array kit (Qiagen) according to the manufacturer's instructions. Data were analyzed using the miScript miRNA PCR array data analysis web portal.\n\nIn silico miRNA target prediction. MiRNA target genes were retrieved and compiled using TargetScan 38 and microRNA.org resource 39 . The interactions between miRNAs and intracellular pathways were predicted using DIANA-miRPath v2.0 40 .", "THP-1 MDMs were seeded in 24-well plates (0.5 × 10 6 per well) in triplicate, exposed to Influenza A H1N1 (A/Solomon islands/3/2006) virus (IAV) under serum-free conditions for 1 hour and then cultured for 3 hours in fresh RPMI-1640 containing 2% FBS. Streptococcus pneumoniae (SP) serotype 14 was added at 4 hours after IAV infection. Gentamicin (10 μ g ml −1 ) was added 2 hours after SP infection (i.e. 6 hours post-influenza infection) and maintained in the culture media throughout the experiment to kill extracellular bacteria and limit bacterial growth. Cell viability was determined by flow-cytometry using the FITC/Annexin V apoptosis detection kit (BD Biosciences), according to the manufacturer's instructions. #4427975) . In this assay, fold changes have been defined by the Δ Δ Ct method using control RNU-44 and -48 as reference microRNAs. Total mRNA was purified from transfected and infected MDMs using the RNeasy kit (Qiagen) and specific primers were used to amplify transforming", "growth factor beta-2 (TGFB2; F: 5′ -CCATCCCGCCCACTTTCTAC-3′ , R: 5′ -AGCTCAATCCGTTGTTCAGGC-3′ ), SOCS6 (F: 5′ -AAGAATTCATCCCTTGGATTAGGTAAC-3′ , R: 5′ -CAGACTGGAGGTCGTGGAA-3′ ) 41 43 , and 3) absence of wheezing at auscultation, and, 4) first symptoms appearing within the last 14 days, and 5) radiological confirmation of pneumonia as per WHO guidelines 44 . Based on these primary criteria defining pneumonia cases, all 74 cases were retrospectively re-evaluated according to the WHO \"Pocket book of hospital care for children\" 45 criteria to evaluate pneumonia severity. Cases that died during the study, or who had at least one additional clinical signs including central cyanosis, dullness to percussion during chest examination, prostration/lethargy, pleural effusion observed on chest radiography were retrospectively included in the severe pneumonia group. Patients without any of these additional clinical signs were included in the non-severe pneumonia group. Table 4 . a IP-10 values are", "expressed in pg ml -1 . IP-10 concentration differences between groups were compared using unpaired Mann-Whitney tests; significant changes (P < 0.05) are in bold. Clinical and molecular analysis. Nasopharyngeal aspirates (NAs) and whole blood samples were collected from children within 24 hours of admission. Whole blood samples were used for complete blood counts, blood culture and multiplex real-time PCR to identify Staphylococcus aureus, Streptococcus pneumoniae and Haemophilus influenzae type B 46 . S. pneumoniae serotypes were defined using a 11 multiplex real-time PCR assay targeting the 40 most frequently represented serotypes or serogroups according to protocol developed by Messaoudi et al. 47 . Serum C-reactive protein (CRP; AssayPro, St. Charles, Missouri, United States) and Procalcitonin (PCT; VIDAS B.R.A.H.M.S; bioMérieux) were quantified from whole-blood samples. Multiplex real-time non quantitative PCR (Fast-Track Diagnostic, Sliema, Malta) was used to detect 19 viruses", "and five bacteria in the respiratory specimens (NAs and pleural effusions). Mixed detection was defined as 1) PCR-positive for multiple viruses in NAs, 2) positive blood culture or PCR-positive for multiple bacteria in blood or 3) PCR-positive for one or multiple viruses in NAs and one or multiple bacteria in blood (identified by PCR and blood culture).", "Ethical approval. The study protocol, informed consent statement, clinical research form, any amendments and all other study documents were submitted to and approved by the Ethical Committee of the Instituto de Investigaciones en Ciencias de la Salud, the Universidad Nacional de Asunción (IICS-UNA) and the Hospital Pediátrico Niños de Acosta Ñu. Informed consent was obtained from all subjects involved in this study. The clinical investigation was conducted according to the principles expressed in the Declaration of Helsinki.", "Statistical analysis. The Chi-square test and Fisher's exact test were used to compare categorical variables; continuous variables and non-normally distributed data were compared using the Mann-Whitney U-test; normally distributed data were compared using unpaired t-tests. Comparative analyses between experimental conditions (i.e., MOCK, IAV, SP or IAV + SP) were performed using one-way ANOVA with Tukey's post-hoc test or Kruskal-Wallis analysis with Dunn's post-hoc tests. Receiver operating curve (ROC) analysis was used to determine the optimal cut-off value for IP-10 to differentiate between non-severe and severe pneumonia cases. P < 0.05 was considered statistically significant. All statistical analyses were performed using GraphPad Prism (La Jolla, California, United States)." ]

[ 32 ]

[ "a larger, longitudinal study on the etiology and severity of pneumonia" ]

[ "Viral and bacterial co-infection in severe pneumonia triggers innate immune responses and specifically enhances IP-10: a translational study\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5138590/\n\nSHA: ef3d6cabc804e5eb587b34249b539c1b5efa4cc4\n\nAuthors: Hoffmann, Jonathan; Machado, Daniela; Terrier, Olivier; Pouzol, Stephane; Messaoudi, Mélina; Basualdo, Wilma; Espínola, Emilio E; Guillen, Rosa M.; Rosa-Calatrava, Manuel; Picot, Valentina; Bénet, Thomas; Endtz, Hubert; Russomando, Graciela; Paranhos-Baccalà, Gláucia\nDate: 2016-12-06\nDOI: 10.1038/srep38532\nLicense: cc-by", "Abstract: Mixed viral and bacterial infections are widely described in community-acquired pneumonia; however, the clinical implications of co-infection on the associated immunopathology remain poorly studied. In this study, microRNA, mRNA and cytokine/chemokine secretion profiling were investigated for human monocyte-derived macrophages infected in-vitro with Influenza virus A/H1N1 and/or Streptococcus pneumoniae. We observed that the in-vitro co-infection synergistically increased interferon-γ-induced protein-10 (CXCL10, IP-10) expression compared to the singly-infected cells conditions. We demonstrated that endogenous miRNA-200a-3p, whose expression was synergistically induced following co-infection, indirectly regulates CXCL10 expression by targeting suppressor of cytokine signaling-6 (SOCS-6), a well-known regulator of the JAK-STAT signaling pathway. Additionally, in a subsequent clinical pilot study, immunomodulators levels were evaluated in samples from 74 children (≤5", "years-old) hospitalized with viral and/or bacterial community-acquired pneumonia. Clinically, among the 74 cases of pneumonia, patients with identified mixed-detection had significantly higher (3.6-fold) serum IP-10 levels than those with a single detection (P = 0.03), and were significantly associated with severe pneumonia (P < 0.01). This study demonstrates that viral and bacterial co-infection modulates the JAK-STAT signaling pathway and leads to exacerbated IP-10 expression, which could play a major role in the pathogenesis of pneumonia.", "Text: Scientific RepoRts | 6:38532 | DOI: 10 .1038/srep38532 pathogenesis of several diseases and has been suggested as a potential biomarker of viral infection 10, 11 , late-onset bacterial infection in premature infants 12 , and a promising biomarker of sepsis and septic shock 13, 14 . Combined analysis of IP-10 and IFN-γ has also been reported as a useful biomarker for diagnosis and monitoring therapeutic efficacy in patients with active tuberculosis [15] [16] [17] , and both remain detectable in the urine of patients with pulmonary diseases in the absence of renal dysfunction 18 .", "With airway epithelial cells 19 , resident alveolar macrophages (AMs) and blood monocytes-derived macrophages (recruited into tissues under inflammatory conditions 20, 21 ) represent a major line of defense against both pneumococcal (through their high phagocytic capacity [22] [23] [24] ) and influenza infection 25, 26 . So far, no studies have yet focused on the intracellular mechanisms that regulate IP-10 in human blood leukocytes during mixed IAV and SP infection. Several studies indicated that host non-coding small RNAs (including microRNAs) may function as immunomodulators by regulating several pivotal intracellular processes, such as the innate immune response 27 and antiviral activity 28, 29 ; both of these processes are closely related to toll-like receptor (TLR) signaling pathways.", "In this study, we firstly investigated the in vitro intracellular mechanisms that mediate the innate immune response in IAV and/or SP infected human monocyte-derived macrophages (MDMs). Using this approach, we observed that mixed-infection of MDMs induces a synergistic production of IP-10 which can be related to a miRNA-200a/JAK-STAT/SOCS-6 regulatory pathway. Subsequently, in a retrospective analysis of clinical samples collected from children ≤ 5 years-old hospitalized with pneumonia, we confirmed that serum IP-10 level could be related to both viral and/or bacterial etiologies and disease severity.", "Characteristics of MDMs infected by IAV and/or SP. Initially, we investigated in vitro the impact of single and mixed IAV and SP infection on MDMs. Firstly, active replication of IAV was assessed by qRT-PCR and quantification of new infectious viral particles in the cell supernatants ( Fig. 1a,b ). IAV titer increased over time after single infection with IAV and correlated with increased production of negative-strand IAV RNA. Maximum viral replication was observed at 18-24 hours post-infection, after which time both RNA replication and the quantity of infectious particles decreased. In this in vitro model, subsequent challenge of IAV-infected MDMs with SP had no significant impact on the production of new infectious viral particles (Fig. 1b) . Together, these results indicate permissive and productive infection of MDMs by IAV. Secondly, we evaluated whether MDMs are permissive for both IAV and SP infection. The presence of pneumococci within IAV-and SP-infected primary MDMs was", "confirmed at 8 h post-infection (Fig. 1c) , suggesting that MDMs are permissive for viral and bacterial co-infection in the early steps of infection. Importantly, confocal co-detection of mixed IAV and SP was only effective following 8 h post-infection due to the bactericidal impact of SP internalization within human macrophages (after 24 h, data not shown). Thirdly, we evaluated the impact of single and mixed infection with IAV and SP on MDM viability. Mixed infection significantly decreased cell viability (65.2 ± 4.5% total cell death at 48 hours post-infection; P < 0.0001) compared to single SP and IAV infection (39.6 ± 1.7% and 17.4 ± 1.1% total cell death, respectively; Fig. 1d ). Taken together, these results confirmed human MDMs are permissive to mixed viral and bacterial infection. mRNA, microRNA and protein expression profiling reveal an overall induction of the host innate immune response following IAV and/or SP infection of MDMs. To investigate the innate immune response", "orchestrated by IAV-and SP-infected human MDMs, we firstly evaluated the expression of 84 genes involved in the innate and adaptive immune responses (Table S1) ; the major differentially-expressed genes are summarized in Fig. 2a . Expression profiling indicated an overall induction of genes related to the JAK-STAT, NF-Κ β and TLR signaling pathways. Indeed, all interferon-stimulated genes (ISGs) screened, including CXCL10 (fold-change [FC] = 240.9), CCL-2 (FC = 34.2) and MX-1 (FC = 151.4) were upregulated following mixed infection compared to uninfected cells, most of which are closely related to STAT-1 (FC = 52.3), IRF-7 (FC = 6.8) and IFNB1 (FC = 5.2) also found upregulated in mixed infected cells. Secondly, we investigated the endogenous microRNA expression profiles of IAV-and SP-infected MDMs. A selection of microRNAs that were found to be differentially-expressed under different infection conditions are shown in Fig. 2b and Table S2 . MiRNA-200a-3p was overexpressed after both", "single IAV (FC = 6.9), single SP (FC = 3.7) and mixed IAV/SP infection (FC = 7.3), indicating this miRNA may play a role in the innate immune response to viral and bacterial co-infection. Similar miRNA-200a-3p dysregulation profiles were obtained following IAV and/or SP infections of human macrophages-like (THP-1 monocytes-derived macrophages) or primary MDMs (data not shown). Thirdly, the secreted levels of various antiviral, pro-inflammatory and immunomodulatory cytokines/chemokines were assayed in IAV-and SP-infected-THP-1 and primary MDM cell supernatants. We observed a remarkable correlation between the mRNA and protein expression profiles of single or mixed infected MDMs especially regarding CXCL-10 and IP-10 expression. Indeed, the level of IP-10 was synergistically increased in the supernatant of IAV-infected THP-1 MDMs exposed to SP (mean: 30,589 ± 16,484 pg ml −1 ) compared to single IAV infection (1,439 ± 566.5 pg ml −1 ) and single SP infection (4,472 ± 2,001 pg ml −1 ; P≤", "0.05; Fig. 2c ) at 24 hours after infection. In those cells, IP-10 expression reduced over time (48 to 72 hours), coinciding with a significant higher proportion of necrotic and apoptotic cells (Fig. 1d) . The synergistic expression of IP-10 was similarly observed at 24 hours post-infection using primary MDMs (Fig. 2d) . Significantly increased secretion of the other tested cytokines and chemokines was not observed post-infection, even in mixed infected MDMs (Fig. S1 ). Interestingly, a significant production of IP-10 was also observed in supernatants of primary human airway epithelial cells (HAEC) mixed-infected by IAV and SP compared to the single infections (Fig. 2e) . Taken together, the mRNA and protein profiling results suggested that mixed viral and bacterial infection of MDMs induces a synergistic pro-inflammatory response related to the type-1 interferon and JAK-STAT signaling pathways, with IP-10 as signature of IAV/SP co-infection. Among all microRNAs screened, miR-200a-3p", "was the most Scientific RepoRts | 6:38532 | DOI: 10.1038/srep38532 overexpressed in IAV/SP co-infection of human MDMs. In the remainder of this study, we decided to investigate the interconnection between miR-200a-3p expression and the innate immune response.", "Endogenous miRNA-200a-3p expression correlates with CXCL10 (IP-10) induction following mixed IAV and SP infection of human MDMs. Using a specific Taqman probe assay targeting miR-200a-3p, we confirmed a significant upregulation of miR-200a-3p following mixed IAV and SP infection of human MDMs (Fig. 3a) . In this experiment, a more marked up-regulation of miR-200a-3p was observed following IAV+ SP compared to results obtained previously (Fig. 2b) . This discrepancy has been attributed to the use of two different approaches to quantify miR-200a-3p expression. The use of a target-specific stem-loop reverse transcription primer in Fig. 3a allows a better sensitivity of miR-200a-3p detection compared to the non-specific fluorescent dye used in Fig. 2b . As the general trend was suggestive of a synergistic induction of miR-200a-3p in response to mixed infection (Fig. 3a) , we hypothesized microRNA-200a-3p may play a role in the regulation of CXCL10 (IP-10), which was also synergistically", "upregulated in mixed-infected MDMs ( Fig. 2c and d) and primary HAEC ( Statistical analyses were performed using two-way ANOVA with Tukey's post-hoc test; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.", "Scientific RepoRts | 6:38532 | DOI: 10.1038/srep38532 CXCL10 (Fig. 3d) . These results suggested miR-200a-3p indirectly regulates CXCL10 and led us to hypothesize that miR-200a-3p controls a potential repressor of the JAK-STAT signaling pathway. . At 18 h after transfection, the MDMs were singly or mixed infected as described previously. At 8 h post-IAV and/or SP infection, total mRNA was extracted and amplified by PCR using specific primers for the indicated genes. Values represent median ± IQR (a, c) or mean ± SEM (d, e) of three biological replicates. Statistical analyses were performed using a Kruskal-Wallis test (non-parametric, one-way ANOVA with Dunn's post-hoc test) for data presented in (a, c). An ordinary two-way ANOVA (with Tukey's post-hoc multiple comparison test) was used for data presented in (d, e). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. MiRNA-200a-3p indirectly regulates IP-10 expression by targeting SOCS6. As shown in Fig. 2a , several JAK-STAT", "signaling pathway genes were deregulated in mixed IAV-and SP-infected human MDMs; therefore, we hypothesized that miR-200a-3p directly regulates a regulator of the JAK-STAT signaling pathway. Predictive target analysis indicated that the 3' UTR of suppressor of cytokine signaling-6 (SOCS6) may be targeted by miR-200a-3p (Fig. 3b) . SOCS proteins constitute a class of negative regulators of JAK-STAT signaling pathways that are induced by both cytokines and TLR signaling. MiRNA-200a-3p was not predicted to target any of the other six members of the SOCS gene family. Transfection of human MDMs with MIM-200a downregulated SOCS6 (FC = 0.57) while inhibition of miR-200a-3p (INH-200a) upregulated SOCS6 (FC = 1.55), confirming that miR-200a-3p effectively regulates the expression of SOCS6 (Fig. 3e) . Moreover, SOCS6 was synergistically downregulated in IAV-or IAV/SP-infected MDMs overexpressing miRNA-200a (Fig. 3e) , suggesting that both infection and miR-200a-3p negatively regulate the", "expression of SOCS6. Finally, western blotting confirmed that expression of SOCS-6 sharply reduced following infection, especially after mixed IAV and SP infection (Fig. 3f) .", "These results indicate miR-200a-3p is strongly induced in response to mixed viral and bacterial co-infection, which in turn leads to downregulation of the JAK-STAT regulator SOCS-6 at both the mRNA and protein levels and subsequent upregulation of IP-10.", "analyses demonstrated mixed IAV and SP infection of human MDMs and HAEC induced significant production of IP-10. As blood leukocytes and respiratory tract epithelial cells actively contribute to inflammation during pneumonia, we hypothesized the level of IP-10 in serum of patient with pneumonia may be both indicative of mixed respiratory infection and disease severity. As part of a prospective, hospital-based, multicenter case-control study on the etiology of pneumonia among children under 5-years-old, a total of 74 patients (44 male, 30 female) were included in this pilot evaluation. According to WHO guidelines, retrospective analysis indicated 44 (59.5%) children had clinical signs of non-severe pneumonia and 30 (40.5%) children had signs of severe pneumonia. The main patient characteristics at inclusion are shown in Table 1 . Patients with severe pneumonia had significant more recorded episodes of dyspnea (P < 0.001), cyanosis (P = 0.03), lower chest indrawing (P < 0.001), dullness", "to percussion (P < 0.001) and lethargy (P < 0.001) during chest examination than patient with non-severe pneumonia. Moreover, pleural effusions were significantly more observed among critically ill patients and the duration of hospitalization was significantly longer for the children with severe pneumonia than for those with non-severe pneumonia (P = 0.0015). Two deaths occurred within the group of children retrospectively defined with severe pneumonia. Evaluation of the systemic inflammatory response of the 74 cases is shown in Table 2 . Serum level of CRP, IP-10, PCT, G-CSF, IL-6, IL-8 and MIP-1β were significantly more elevated in serum samples from critically ill patients. Patients with severe pneumonia had significantly higher (4.2-fold) serum IP-10 levels than those with a non-severe pneumonia (P < 0.001) suggesting IP-10 as a promising prognostic marker in pneumonia. Diagnostic accuracy measures for predicting pneumonia severity using blood-based biomarkers are summarized in", "Table S3 . Briefly, in this study, the optimal IP-10 cut-off value for identifying patient with severe pneumonia was 4,240 pg ml −1 , with an area under the receiver operating characteristic curve of 0.69 (95% CI, 0.57 to 0.82, P < 0.001). Defining as positive a serum IP-10 level above this cut-off resulted in a sensitivity of 63.3%, specificity of 63.6% and a positive likelihood ratio of 1.74. Prognostic values of IP-10 were closed to procalcitonin (PCT; AUC = 0.70; 95% IC, 0.58 to 0.82, P < 0.001) and IL-6 (AUC = 0.70; 95% IC, 0.58-0.83, P < 0.001).", "Multiplex PCR-based screening of respiratory and blood samples reveal a high variety of pathogen associations (Table 3) . Respiratory viruses were detected in the nasal aspirates (NAs) of 63/74 patients (85.1%). Etiological bacteria of pneumonia (S. pneumoniae, n = 19; S. aureus, n = 1; or H. influenzae type B, n = 7) were identified via real-time PCR in the blood samples of 27/74 (36.5%) of the patients. Multiplex PCR assays allowed the identification of respiratory bacteria in the blood of 19 patients with negative blood culture results. Among the 74 cases PCR-positive for respiratory pathogens, a single virus or bacteria were detected in the NAs of 7 (9.4%) and 3 (4.0%) patients, respectively; these 10/74 (13.5%) cases were defined as the single infection group. The mixed infection group included the 62/74 (83.8%) cases in which (1) multiple viruses and/or bacteria were identified in NAs (38/74; 51.3%) without any bacteria identified in blood samples or (2) one or more viruses", "and/or bacteria were identified in NAs and associated with a blood bacteremia (24/74; 32.4%). We evaluated whether IP-10 serum level could correlate with the viral and bacterial etiologies of pneumonia. Patients with mixed infection had significant higher (3.6-fold) IP-10 serum level than patient with single detection (P = 0.03; Table 4 ). A stratified analysis reveals that the highest IP-10 serum level was observed among patients with both several respiratory pathogens identified (mixed-detection group) and severe pneumonia (14,427 pg ml −1 , IQR (3,981-82,994). In detail, a remarkable IP-10 serum level (142,531 pg ml −1 ), representing 33-fold higher above cut-off value predicting pneumonia severity was observed in patient with hRV in NA co-detected with S. pneumoniae (serotype 14) in pleural effusion and blood. In concordance with our in-vitro model of co-infection, a significant IP-10 level (90,338 pg ml −1 ) was quantified in blood sample of patient with severe bacteremic", "pneumococcal (serotype 14) pneumonia with a positive co-detection of Influenza B virus in NA. Taken together, these results suggest that high serum IP-10 levels are significantly associated with mixed viral and bacterial detection and also related to pneumonia pathogenesis.", "This study provides additional in vitro and clinical data to improve our understanding of the immunopathology of mixed viral and bacterial pneumonia (Fig. 4) .", "The in vitro model of influenza and pneumococcal superinfection of human MDMs demonstrated that mixed infection synergistically induced release of the pro-inflammatory chemokine IP-10, strongly suggesting human Scientific RepoRts | 6:38532 | DOI: 10.1038/srep38532 blood leukocytes contribute to the immunopathology of pneumonia. Additionally, transcriptomics and omics analyses provided new data on the inflammatory pathways that are activated during mixed infection and related to synergistic induction of the pro-inflammatory chemokine IP-10 in mixed infected cells. Our observations are consistent with a recent study describing IP-10 induction as host-proteome signature of both viral and bacterial infections 30 . Of the differentially-expressed genes observed in mixed infected MDMs, the transcription factors STAT-1 and IRF-7 appear to play crucial roles in the regulation of interferon-stimulated genes including CXCL10 (IP-10). By focusing on the intracellular mechanisms that regulate", "inflammatory pathways, we demonstrated a novel role for miRNA-200a-3p in the regulation of CXCL10 (IP-10). These observations are consistent with previous reports showing that RNA virus infection upregulates miR-155 in macrophages and dendritic cells and also regulates suppressor of cytokine signaling 1 (SOCS1), suggesting the existence of a miRNA/JAK-STAT/SOCS regulatory pathway during viral infection 29 . Our study suggests co-infection leads to overexpression of miR-200a-3p, which in turn targets and downregulates the JAK-STAT regulator SOCS-6 and consequently increases CXCL10 (IP-10) expression. Interestingly, a complementary in-silico approach reveals that several microRNAs that were found dysregulated in our experiments of IAV and SP co-infection of MDMs or HAEC, might target several genes of SOCS family and play similar role than miR-200a-3p. Indeed, miRNA-142-3p might target SOCS4, 5, 6 mRNA while miRNA-194-5p might target SOCS2, 3, 4, 5 and 7 mRNA. These observations", "underline that intra-cellular regulation of IP-10 is not limited to the contribution of a sole microRNA. A complex inter-relationship between numerous host microRNAs and inhibitors of the JAK-STAT signaling pathway occur to control host innate inflammatory response against viral and/or bacterial infections. Clinically, the majority of pediatric CAP cases in this study were associated with both positive viral and/or bacterial detection. Respiratory microorganisms were detected in 97% of cases; 51.3% of which were viral-viral, viral-bacterial or bacterial-bacterial co-detected only in nasal aspirates, 32.4% of which co-detected in both nasal aspirates and blood samples. These data are consistent with previous etiological studies of pediatric CAP 3,31-33 . S. pneumoniae was the major bacteria identified in blood (19/74; 25.7%) and mainly co-detected with respiratory viruses in NAs (16/19; 84.2%). We observed a very high diversity of viral and bacterial associations in biological samples", "from children with pneumonia. In comparison with IAV and SP14 combination evaluated in-vitro, no pneumonia cases were singly influenza and pneumococcus infected, and no similar co-detection with those two pathogens has been clinically observed. Nevertheless, Influenza B (IVB) virus was identified in 5 patients and two of them had a positive SP co-detection in blood (one non-typable strain and one serotype 14 using our molecular typing test). IVB and SP14 combination seems to be the nearest pathogen co-detection to that in-vitro investigated. Clinically, this co-detection was associated with both a very high IP-10 expression and a very severe pneumonia case definition. Interestingly, our translational pilot evaluation reveals IP-10 expression can be induced by several different viral and/or bacterial combinations. As immune response to each pathogen is different, further in-vitro investigations using different pathogens associations are needed to better characterize the mechanisms", "involved in the immunopathology of pneumonia.", "In this cohort, highest serum IP-10 levels were identified among patients with both several pathogen detected and severe pneumonia, suggesting a significant role of IP-10 on pneumonia pathogenesis. Indeed, high plasma levels of IP-10 have previously been reported in patients with sepsis 12 , and were associated with high mortality rate, especially among patients with CAP 34 . Additionally, the IP-10-CXCR3 axis has been related to acute immune lung injury and lymphocyte apoptosis during the development of severe acute respiratory syndrome (SARS) 35, 36 . Moreover, an in vivo study that modeled influenza and pneumococcal superinfection in mice indicated that pro-inflammatory chemokines, including IP-10, play a crucial role in influenza-induced susceptibility to lung neutrophilia, severe immunopathology and mortality 37 . In this study, markedly elevated IP-10 (92,809 pg ml −1 ) combined with the highest PCT level (74.4 pg ml −1 ) were quantified in the serum sample of a child who died,", "in whom S. pneumoniae (serotype 9 V) was identified in the blood (PCR and blood culture) and co-detected with Haemophilus influenzae type B in nasal aspirate. These observations suggest an interrelationship between co-detection, elevated serum IP-10 and the pathogenesis of pneumonia.", "Several limitations of this pilot translational study need to be acknowledged before concluding mixed infection is related to elevated IP-10 and disease severity. Indeed, although viral shedding (e.g., of HRV and HBoV) is common in asymptomatic children, we were unable to evaluate the levels of immunomodulators in the serum samples of a control group. Moreover, although the samples were collected within the first 24 hours after admission, only a single blood sample was processed for each patient. Therefore, a larger, longitudinal study on the etiology and severity of pneumonia will be necessary to confirm these results. In conclusion, the present findings suggest that mixed respiratory infections and IP-10 may play major, interconnected roles in the pathogenesis of pneumonia. Clinically, assessment and monitoring of induced IP-10 serum level may assist clinicians to improve diagnosis and patient management of severe community-acquired pneumonia.", "Viral and bacterial strains. The 10 ng ml −1 M-CSF (Miltenyi Biotec). THP− 1 MDMs were obtained by culturing cells with 10 ng ml -1 phorbol myristate acetate (PMA; Invivogen, Toulouse, France) for 72 hours. Human airway epithelial cells (HAEC, bronchial cell type) originated from a 54-years old woman with no pathology reported (batch number MD056501) were provided by Mucilair (Epithelix, Geneva, Switzerland). Sterility, tissue integrity (TEER), mucus production and cilia beating frequency have been certified by the company. Gene expression profiling. Total cellular mRNA was purified using the RNeasy kit (Qiagen, Hilden, Germany). Reverse-transcription of total mRNA was performed using the RT 2 First Strand Kit (SABiosciences, Hilden, Germany). The expression of 84 genes involved in the human innate and adaptive immune responses was evaluated using the RT 2 profiler ™ PCR Array (SABiosciences) according to the manufacturer's recommendations. The Δ Δ Ct method was applied to calculate", "the fold changes in gene expression for each gene relative to uninfected control cells using the web-based RT 2 profiler PCR Array Data Analysis software (SABiosciences).", "MicroRNA profiling array. Total cellular microRNAs were purified using the miRNeasy Mini kit (Qiagen) and reverse-transcribed using the miScript Reverse Transcription kit (Qiagen). The profiling of 84 miRNAs was performed using the Human Immunopathology miScript miRNA PCR Array kit (Qiagen) according to the manufacturer's instructions. Data were analyzed using the miScript miRNA PCR array data analysis web portal.\n\nIn silico miRNA target prediction. MiRNA target genes were retrieved and compiled using TargetScan 38 and microRNA.org resource 39 . The interactions between miRNAs and intracellular pathways were predicted using DIANA-miRPath v2.0 40 .", "THP-1 MDMs were seeded in 24-well plates (0.5 × 10 6 per well) in triplicate, exposed to Influenza A H1N1 (A/Solomon islands/3/2006) virus (IAV) under serum-free conditions for 1 hour and then cultured for 3 hours in fresh RPMI-1640 containing 2% FBS. Streptococcus pneumoniae (SP) serotype 14 was added at 4 hours after IAV infection. Gentamicin (10 μ g ml −1 ) was added 2 hours after SP infection (i.e. 6 hours post-influenza infection) and maintained in the culture media throughout the experiment to kill extracellular bacteria and limit bacterial growth. Cell viability was determined by flow-cytometry using the FITC/Annexin V apoptosis detection kit (BD Biosciences), according to the manufacturer's instructions. #4427975) . In this assay, fold changes have been defined by the Δ Δ Ct method using control RNU-44 and -48 as reference microRNAs. Total mRNA was purified from transfected and infected MDMs using the RNeasy kit (Qiagen) and specific primers were used to amplify transforming", "growth factor beta-2 (TGFB2; F: 5′ -CCATCCCGCCCACTTTCTAC-3′ , R: 5′ -AGCTCAATCCGTTGTTCAGGC-3′ ), SOCS6 (F: 5′ -AAGAATTCATCCCTTGGATTAGGTAAC-3′ , R: 5′ -CAGACTGGAGGTCGTGGAA-3′ ) 41 43 , and 3) absence of wheezing at auscultation, and, 4) first symptoms appearing within the last 14 days, and 5) radiological confirmation of pneumonia as per WHO guidelines 44 . Based on these primary criteria defining pneumonia cases, all 74 cases were retrospectively re-evaluated according to the WHO \"Pocket book of hospital care for children\" 45 criteria to evaluate pneumonia severity. Cases that died during the study, or who had at least one additional clinical signs including central cyanosis, dullness to percussion during chest examination, prostration/lethargy, pleural effusion observed on chest radiography were retrospectively included in the severe pneumonia group. Patients without any of these additional clinical signs were included in the non-severe pneumonia group. Table 4 . a IP-10 values are", "expressed in pg ml -1 . IP-10 concentration differences between groups were compared using unpaired Mann-Whitney tests; significant changes (P < 0.05) are in bold. Clinical and molecular analysis. Nasopharyngeal aspirates (NAs) and whole blood samples were collected from children within 24 hours of admission. Whole blood samples were used for complete blood counts, blood culture and multiplex real-time PCR to identify Staphylococcus aureus, Streptococcus pneumoniae and Haemophilus influenzae type B 46 . S. pneumoniae serotypes were defined using a 11 multiplex real-time PCR assay targeting the 40 most frequently represented serotypes or serogroups according to protocol developed by Messaoudi et al. 47 . Serum C-reactive protein (CRP; AssayPro, St. Charles, Missouri, United States) and Procalcitonin (PCT; VIDAS B.R.A.H.M.S; bioMérieux) were quantified from whole-blood samples. Multiplex real-time non quantitative PCR (Fast-Track Diagnostic, Sliema, Malta) was used to detect 19 viruses", "and five bacteria in the respiratory specimens (NAs and pleural effusions). Mixed detection was defined as 1) PCR-positive for multiple viruses in NAs, 2) positive blood culture or PCR-positive for multiple bacteria in blood or 3) PCR-positive for one or multiple viruses in NAs and one or multiple bacteria in blood (identified by PCR and blood culture).", "Ethical approval. The study protocol, informed consent statement, clinical research form, any amendments and all other study documents were submitted to and approved by the Ethical Committee of the Instituto de Investigaciones en Ciencias de la Salud, the Universidad Nacional de Asunción (IICS-UNA) and the Hospital Pediátrico Niños de Acosta Ñu. Informed consent was obtained from all subjects involved in this study. The clinical investigation was conducted according to the principles expressed in the Declaration of Helsinki.", "Statistical analysis. The Chi-square test and Fisher's exact test were used to compare categorical variables; continuous variables and non-normally distributed data were compared using the Mann-Whitney U-test; normally distributed data were compared using unpaired t-tests. Comparative analyses between experimental conditions (i.e., MOCK, IAV, SP or IAV + SP) were performed using one-way ANOVA with Tukey's post-hoc test or Kruskal-Wallis analysis with Dunn's post-hoc tests. Receiver operating curve (ROC) analysis was used to determine the optimal cut-off value for IP-10 to differentiate between non-severe and severe pneumonia cases. P < 0.05 was considered statistically significant. All statistical analyses were performed using GraphPad Prism (La Jolla, California, United States)." ]

[ 32 ]

[ "mixed respiratory infections and IP-10 may play major, interconnected roles in the pathogenesis of pneumonia" ]

[ "Viral and bacterial co-infection in severe pneumonia triggers innate immune responses and specifically enhances IP-10: a translational study\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5138590/\n\nSHA: ef3d6cabc804e5eb587b34249b539c1b5efa4cc4\n\nAuthors: Hoffmann, Jonathan; Machado, Daniela; Terrier, Olivier; Pouzol, Stephane; Messaoudi, Mélina; Basualdo, Wilma; Espínola, Emilio E; Guillen, Rosa M.; Rosa-Calatrava, Manuel; Picot, Valentina; Bénet, Thomas; Endtz, Hubert; Russomando, Graciela; Paranhos-Baccalà, Gláucia\nDate: 2016-12-06\nDOI: 10.1038/srep38532\nLicense: cc-by", "Abstract: Mixed viral and bacterial infections are widely described in community-acquired pneumonia; however, the clinical implications of co-infection on the associated immunopathology remain poorly studied. In this study, microRNA, mRNA and cytokine/chemokine secretion profiling were investigated for human monocyte-derived macrophages infected in-vitro with Influenza virus A/H1N1 and/or Streptococcus pneumoniae. We observed that the in-vitro co-infection synergistically increased interferon-γ-induced protein-10 (CXCL10, IP-10) expression compared to the singly-infected cells conditions. We demonstrated that endogenous miRNA-200a-3p, whose expression was synergistically induced following co-infection, indirectly regulates CXCL10 expression by targeting suppressor of cytokine signaling-6 (SOCS-6), a well-known regulator of the JAK-STAT signaling pathway. Additionally, in a subsequent clinical pilot study, immunomodulators levels were evaluated in samples from 74 children (≤5", "years-old) hospitalized with viral and/or bacterial community-acquired pneumonia. Clinically, among the 74 cases of pneumonia, patients with identified mixed-detection had significantly higher (3.6-fold) serum IP-10 levels than those with a single detection (P = 0.03), and were significantly associated with severe pneumonia (P < 0.01). This study demonstrates that viral and bacterial co-infection modulates the JAK-STAT signaling pathway and leads to exacerbated IP-10 expression, which could play a major role in the pathogenesis of pneumonia.", "Text: Scientific RepoRts | 6:38532 | DOI: 10 .1038/srep38532 pathogenesis of several diseases and has been suggested as a potential biomarker of viral infection 10, 11 , late-onset bacterial infection in premature infants 12 , and a promising biomarker of sepsis and septic shock 13, 14 . Combined analysis of IP-10 and IFN-γ has also been reported as a useful biomarker for diagnosis and monitoring therapeutic efficacy in patients with active tuberculosis [15] [16] [17] , and both remain detectable in the urine of patients with pulmonary diseases in the absence of renal dysfunction 18 .", "With airway epithelial cells 19 , resident alveolar macrophages (AMs) and blood monocytes-derived macrophages (recruited into tissues under inflammatory conditions 20, 21 ) represent a major line of defense against both pneumococcal (through their high phagocytic capacity [22] [23] [24] ) and influenza infection 25, 26 . So far, no studies have yet focused on the intracellular mechanisms that regulate IP-10 in human blood leukocytes during mixed IAV and SP infection. Several studies indicated that host non-coding small RNAs (including microRNAs) may function as immunomodulators by regulating several pivotal intracellular processes, such as the innate immune response 27 and antiviral activity 28, 29 ; both of these processes are closely related to toll-like receptor (TLR) signaling pathways.", "In this study, we firstly investigated the in vitro intracellular mechanisms that mediate the innate immune response in IAV and/or SP infected human monocyte-derived macrophages (MDMs). Using this approach, we observed that mixed-infection of MDMs induces a synergistic production of IP-10 which can be related to a miRNA-200a/JAK-STAT/SOCS-6 regulatory pathway. Subsequently, in a retrospective analysis of clinical samples collected from children ≤ 5 years-old hospitalized with pneumonia, we confirmed that serum IP-10 level could be related to both viral and/or bacterial etiologies and disease severity.", "Characteristics of MDMs infected by IAV and/or SP. Initially, we investigated in vitro the impact of single and mixed IAV and SP infection on MDMs. Firstly, active replication of IAV was assessed by qRT-PCR and quantification of new infectious viral particles in the cell supernatants ( Fig. 1a,b ). IAV titer increased over time after single infection with IAV and correlated with increased production of negative-strand IAV RNA. Maximum viral replication was observed at 18-24 hours post-infection, after which time both RNA replication and the quantity of infectious particles decreased. In this in vitro model, subsequent challenge of IAV-infected MDMs with SP had no significant impact on the production of new infectious viral particles (Fig. 1b) . Together, these results indicate permissive and productive infection of MDMs by IAV. Secondly, we evaluated whether MDMs are permissive for both IAV and SP infection. The presence of pneumococci within IAV-and SP-infected primary MDMs was", "confirmed at 8 h post-infection (Fig. 1c) , suggesting that MDMs are permissive for viral and bacterial co-infection in the early steps of infection. Importantly, confocal co-detection of mixed IAV and SP was only effective following 8 h post-infection due to the bactericidal impact of SP internalization within human macrophages (after 24 h, data not shown). Thirdly, we evaluated the impact of single and mixed infection with IAV and SP on MDM viability. Mixed infection significantly decreased cell viability (65.2 ± 4.5% total cell death at 48 hours post-infection; P < 0.0001) compared to single SP and IAV infection (39.6 ± 1.7% and 17.4 ± 1.1% total cell death, respectively; Fig. 1d ). Taken together, these results confirmed human MDMs are permissive to mixed viral and bacterial infection. mRNA, microRNA and protein expression profiling reveal an overall induction of the host innate immune response following IAV and/or SP infection of MDMs. To investigate the innate immune response", "orchestrated by IAV-and SP-infected human MDMs, we firstly evaluated the expression of 84 genes involved in the innate and adaptive immune responses (Table S1) ; the major differentially-expressed genes are summarized in Fig. 2a . Expression profiling indicated an overall induction of genes related to the JAK-STAT, NF-Κ β and TLR signaling pathways. Indeed, all interferon-stimulated genes (ISGs) screened, including CXCL10 (fold-change [FC] = 240.9), CCL-2 (FC = 34.2) and MX-1 (FC = 151.4) were upregulated following mixed infection compared to uninfected cells, most of which are closely related to STAT-1 (FC = 52.3), IRF-7 (FC = 6.8) and IFNB1 (FC = 5.2) also found upregulated in mixed infected cells. Secondly, we investigated the endogenous microRNA expression profiles of IAV-and SP-infected MDMs. A selection of microRNAs that were found to be differentially-expressed under different infection conditions are shown in Fig. 2b and Table S2 . MiRNA-200a-3p was overexpressed after both", "single IAV (FC = 6.9), single SP (FC = 3.7) and mixed IAV/SP infection (FC = 7.3), indicating this miRNA may play a role in the innate immune response to viral and bacterial co-infection. Similar miRNA-200a-3p dysregulation profiles were obtained following IAV and/or SP infections of human macrophages-like (THP-1 monocytes-derived macrophages) or primary MDMs (data not shown). Thirdly, the secreted levels of various antiviral, pro-inflammatory and immunomodulatory cytokines/chemokines were assayed in IAV-and SP-infected-THP-1 and primary MDM cell supernatants. We observed a remarkable correlation between the mRNA and protein expression profiles of single or mixed infected MDMs especially regarding CXCL-10 and IP-10 expression. Indeed, the level of IP-10 was synergistically increased in the supernatant of IAV-infected THP-1 MDMs exposed to SP (mean: 30,589 ± 16,484 pg ml −1 ) compared to single IAV infection (1,439 ± 566.5 pg ml −1 ) and single SP infection (4,472 ± 2,001 pg ml −1 ; P≤", "0.05; Fig. 2c ) at 24 hours after infection. In those cells, IP-10 expression reduced over time (48 to 72 hours), coinciding with a significant higher proportion of necrotic and apoptotic cells (Fig. 1d) . The synergistic expression of IP-10 was similarly observed at 24 hours post-infection using primary MDMs (Fig. 2d) . Significantly increased secretion of the other tested cytokines and chemokines was not observed post-infection, even in mixed infected MDMs (Fig. S1 ). Interestingly, a significant production of IP-10 was also observed in supernatants of primary human airway epithelial cells (HAEC) mixed-infected by IAV and SP compared to the single infections (Fig. 2e) . Taken together, the mRNA and protein profiling results suggested that mixed viral and bacterial infection of MDMs induces a synergistic pro-inflammatory response related to the type-1 interferon and JAK-STAT signaling pathways, with IP-10 as signature of IAV/SP co-infection. Among all microRNAs screened, miR-200a-3p", "was the most Scientific RepoRts | 6:38532 | DOI: 10.1038/srep38532 overexpressed in IAV/SP co-infection of human MDMs. In the remainder of this study, we decided to investigate the interconnection between miR-200a-3p expression and the innate immune response.", "Endogenous miRNA-200a-3p expression correlates with CXCL10 (IP-10) induction following mixed IAV and SP infection of human MDMs. Using a specific Taqman probe assay targeting miR-200a-3p, we confirmed a significant upregulation of miR-200a-3p following mixed IAV and SP infection of human MDMs (Fig. 3a) . In this experiment, a more marked up-regulation of miR-200a-3p was observed following IAV+ SP compared to results obtained previously (Fig. 2b) . This discrepancy has been attributed to the use of two different approaches to quantify miR-200a-3p expression. The use of a target-specific stem-loop reverse transcription primer in Fig. 3a allows a better sensitivity of miR-200a-3p detection compared to the non-specific fluorescent dye used in Fig. 2b . As the general trend was suggestive of a synergistic induction of miR-200a-3p in response to mixed infection (Fig. 3a) , we hypothesized microRNA-200a-3p may play a role in the regulation of CXCL10 (IP-10), which was also synergistically", "upregulated in mixed-infected MDMs ( Fig. 2c and d) and primary HAEC ( Statistical analyses were performed using two-way ANOVA with Tukey's post-hoc test; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.", "Scientific RepoRts | 6:38532 | DOI: 10.1038/srep38532 CXCL10 (Fig. 3d) . These results suggested miR-200a-3p indirectly regulates CXCL10 and led us to hypothesize that miR-200a-3p controls a potential repressor of the JAK-STAT signaling pathway. . At 18 h after transfection, the MDMs were singly or mixed infected as described previously. At 8 h post-IAV and/or SP infection, total mRNA was extracted and amplified by PCR using specific primers for the indicated genes. Values represent median ± IQR (a, c) or mean ± SEM (d, e) of three biological replicates. Statistical analyses were performed using a Kruskal-Wallis test (non-parametric, one-way ANOVA with Dunn's post-hoc test) for data presented in (a, c). An ordinary two-way ANOVA (with Tukey's post-hoc multiple comparison test) was used for data presented in (d, e). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. MiRNA-200a-3p indirectly regulates IP-10 expression by targeting SOCS6. As shown in Fig. 2a , several JAK-STAT", "signaling pathway genes were deregulated in mixed IAV-and SP-infected human MDMs; therefore, we hypothesized that miR-200a-3p directly regulates a regulator of the JAK-STAT signaling pathway. Predictive target analysis indicated that the 3' UTR of suppressor of cytokine signaling-6 (SOCS6) may be targeted by miR-200a-3p (Fig. 3b) . SOCS proteins constitute a class of negative regulators of JAK-STAT signaling pathways that are induced by both cytokines and TLR signaling. MiRNA-200a-3p was not predicted to target any of the other six members of the SOCS gene family. Transfection of human MDMs with MIM-200a downregulated SOCS6 (FC = 0.57) while inhibition of miR-200a-3p (INH-200a) upregulated SOCS6 (FC = 1.55), confirming that miR-200a-3p effectively regulates the expression of SOCS6 (Fig. 3e) . Moreover, SOCS6 was synergistically downregulated in IAV-or IAV/SP-infected MDMs overexpressing miRNA-200a (Fig. 3e) , suggesting that both infection and miR-200a-3p negatively regulate the", "expression of SOCS6. Finally, western blotting confirmed that expression of SOCS-6 sharply reduced following infection, especially after mixed IAV and SP infection (Fig. 3f) .", "These results indicate miR-200a-3p is strongly induced in response to mixed viral and bacterial co-infection, which in turn leads to downregulation of the JAK-STAT regulator SOCS-6 at both the mRNA and protein levels and subsequent upregulation of IP-10.", "analyses demonstrated mixed IAV and SP infection of human MDMs and HAEC induced significant production of IP-10. As blood leukocytes and respiratory tract epithelial cells actively contribute to inflammation during pneumonia, we hypothesized the level of IP-10 in serum of patient with pneumonia may be both indicative of mixed respiratory infection and disease severity. As part of a prospective, hospital-based, multicenter case-control study on the etiology of pneumonia among children under 5-years-old, a total of 74 patients (44 male, 30 female) were included in this pilot evaluation. According to WHO guidelines, retrospective analysis indicated 44 (59.5%) children had clinical signs of non-severe pneumonia and 30 (40.5%) children had signs of severe pneumonia. The main patient characteristics at inclusion are shown in Table 1 . Patients with severe pneumonia had significant more recorded episodes of dyspnea (P < 0.001), cyanosis (P = 0.03), lower chest indrawing (P < 0.001), dullness", "to percussion (P < 0.001) and lethargy (P < 0.001) during chest examination than patient with non-severe pneumonia. Moreover, pleural effusions were significantly more observed among critically ill patients and the duration of hospitalization was significantly longer for the children with severe pneumonia than for those with non-severe pneumonia (P = 0.0015). Two deaths occurred within the group of children retrospectively defined with severe pneumonia. Evaluation of the systemic inflammatory response of the 74 cases is shown in Table 2 . Serum level of CRP, IP-10, PCT, G-CSF, IL-6, IL-8 and MIP-1β were significantly more elevated in serum samples from critically ill patients. Patients with severe pneumonia had significantly higher (4.2-fold) serum IP-10 levels than those with a non-severe pneumonia (P < 0.001) suggesting IP-10 as a promising prognostic marker in pneumonia. Diagnostic accuracy measures for predicting pneumonia severity using blood-based biomarkers are summarized in", "Table S3 . Briefly, in this study, the optimal IP-10 cut-off value for identifying patient with severe pneumonia was 4,240 pg ml −1 , with an area under the receiver operating characteristic curve of 0.69 (95% CI, 0.57 to 0.82, P < 0.001). Defining as positive a serum IP-10 level above this cut-off resulted in a sensitivity of 63.3%, specificity of 63.6% and a positive likelihood ratio of 1.74. Prognostic values of IP-10 were closed to procalcitonin (PCT; AUC = 0.70; 95% IC, 0.58 to 0.82, P < 0.001) and IL-6 (AUC = 0.70; 95% IC, 0.58-0.83, P < 0.001).", "Multiplex PCR-based screening of respiratory and blood samples reveal a high variety of pathogen associations (Table 3) . Respiratory viruses were detected in the nasal aspirates (NAs) of 63/74 patients (85.1%). Etiological bacteria of pneumonia (S. pneumoniae, n = 19; S. aureus, n = 1; or H. influenzae type B, n = 7) were identified via real-time PCR in the blood samples of 27/74 (36.5%) of the patients. Multiplex PCR assays allowed the identification of respiratory bacteria in the blood of 19 patients with negative blood culture results. Among the 74 cases PCR-positive for respiratory pathogens, a single virus or bacteria were detected in the NAs of 7 (9.4%) and 3 (4.0%) patients, respectively; these 10/74 (13.5%) cases were defined as the single infection group. The mixed infection group included the 62/74 (83.8%) cases in which (1) multiple viruses and/or bacteria were identified in NAs (38/74; 51.3%) without any bacteria identified in blood samples or (2) one or more viruses", "and/or bacteria were identified in NAs and associated with a blood bacteremia (24/74; 32.4%). We evaluated whether IP-10 serum level could correlate with the viral and bacterial etiologies of pneumonia. Patients with mixed infection had significant higher (3.6-fold) IP-10 serum level than patient with single detection (P = 0.03; Table 4 ). A stratified analysis reveals that the highest IP-10 serum level was observed among patients with both several respiratory pathogens identified (mixed-detection group) and severe pneumonia (14,427 pg ml −1 , IQR (3,981-82,994). In detail, a remarkable IP-10 serum level (142,531 pg ml −1 ), representing 33-fold higher above cut-off value predicting pneumonia severity was observed in patient with hRV in NA co-detected with S. pneumoniae (serotype 14) in pleural effusion and blood. In concordance with our in-vitro model of co-infection, a significant IP-10 level (90,338 pg ml −1 ) was quantified in blood sample of patient with severe bacteremic", "pneumococcal (serotype 14) pneumonia with a positive co-detection of Influenza B virus in NA. Taken together, these results suggest that high serum IP-10 levels are significantly associated with mixed viral and bacterial detection and also related to pneumonia pathogenesis.", "This study provides additional in vitro and clinical data to improve our understanding of the immunopathology of mixed viral and bacterial pneumonia (Fig. 4) .", "The in vitro model of influenza and pneumococcal superinfection of human MDMs demonstrated that mixed infection synergistically induced release of the pro-inflammatory chemokine IP-10, strongly suggesting human Scientific RepoRts | 6:38532 | DOI: 10.1038/srep38532 blood leukocytes contribute to the immunopathology of pneumonia. Additionally, transcriptomics and omics analyses provided new data on the inflammatory pathways that are activated during mixed infection and related to synergistic induction of the pro-inflammatory chemokine IP-10 in mixed infected cells. Our observations are consistent with a recent study describing IP-10 induction as host-proteome signature of both viral and bacterial infections 30 . Of the differentially-expressed genes observed in mixed infected MDMs, the transcription factors STAT-1 and IRF-7 appear to play crucial roles in the regulation of interferon-stimulated genes including CXCL10 (IP-10). By focusing on the intracellular mechanisms that regulate", "inflammatory pathways, we demonstrated a novel role for miRNA-200a-3p in the regulation of CXCL10 (IP-10). These observations are consistent with previous reports showing that RNA virus infection upregulates miR-155 in macrophages and dendritic cells and also regulates suppressor of cytokine signaling 1 (SOCS1), suggesting the existence of a miRNA/JAK-STAT/SOCS regulatory pathway during viral infection 29 . Our study suggests co-infection leads to overexpression of miR-200a-3p, which in turn targets and downregulates the JAK-STAT regulator SOCS-6 and consequently increases CXCL10 (IP-10) expression. Interestingly, a complementary in-silico approach reveals that several microRNAs that were found dysregulated in our experiments of IAV and SP co-infection of MDMs or HAEC, might target several genes of SOCS family and play similar role than miR-200a-3p. Indeed, miRNA-142-3p might target SOCS4, 5, 6 mRNA while miRNA-194-5p might target SOCS2, 3, 4, 5 and 7 mRNA. These observations", "underline that intra-cellular regulation of IP-10 is not limited to the contribution of a sole microRNA. A complex inter-relationship between numerous host microRNAs and inhibitors of the JAK-STAT signaling pathway occur to control host innate inflammatory response against viral and/or bacterial infections. Clinically, the majority of pediatric CAP cases in this study were associated with both positive viral and/or bacterial detection. Respiratory microorganisms were detected in 97% of cases; 51.3% of which were viral-viral, viral-bacterial or bacterial-bacterial co-detected only in nasal aspirates, 32.4% of which co-detected in both nasal aspirates and blood samples. These data are consistent with previous etiological studies of pediatric CAP 3,31-33 . S. pneumoniae was the major bacteria identified in blood (19/74; 25.7%) and mainly co-detected with respiratory viruses in NAs (16/19; 84.2%). We observed a very high diversity of viral and bacterial associations in biological samples", "from children with pneumonia. In comparison with IAV and SP14 combination evaluated in-vitro, no pneumonia cases were singly influenza and pneumococcus infected, and no similar co-detection with those two pathogens has been clinically observed. Nevertheless, Influenza B (IVB) virus was identified in 5 patients and two of them had a positive SP co-detection in blood (one non-typable strain and one serotype 14 using our molecular typing test). IVB and SP14 combination seems to be the nearest pathogen co-detection to that in-vitro investigated. Clinically, this co-detection was associated with both a very high IP-10 expression and a very severe pneumonia case definition. Interestingly, our translational pilot evaluation reveals IP-10 expression can be induced by several different viral and/or bacterial combinations. As immune response to each pathogen is different, further in-vitro investigations using different pathogens associations are needed to better characterize the mechanisms", "involved in the immunopathology of pneumonia.", "In this cohort, highest serum IP-10 levels were identified among patients with both several pathogen detected and severe pneumonia, suggesting a significant role of IP-10 on pneumonia pathogenesis. Indeed, high plasma levels of IP-10 have previously been reported in patients with sepsis 12 , and were associated with high mortality rate, especially among patients with CAP 34 . Additionally, the IP-10-CXCR3 axis has been related to acute immune lung injury and lymphocyte apoptosis during the development of severe acute respiratory syndrome (SARS) 35, 36 . Moreover, an in vivo study that modeled influenza and pneumococcal superinfection in mice indicated that pro-inflammatory chemokines, including IP-10, play a crucial role in influenza-induced susceptibility to lung neutrophilia, severe immunopathology and mortality 37 . In this study, markedly elevated IP-10 (92,809 pg ml −1 ) combined with the highest PCT level (74.4 pg ml −1 ) were quantified in the serum sample of a child who died,", "in whom S. pneumoniae (serotype 9 V) was identified in the blood (PCR and blood culture) and co-detected with Haemophilus influenzae type B in nasal aspirate. These observations suggest an interrelationship between co-detection, elevated serum IP-10 and the pathogenesis of pneumonia.", "Several limitations of this pilot translational study need to be acknowledged before concluding mixed infection is related to elevated IP-10 and disease severity. Indeed, although viral shedding (e.g., of HRV and HBoV) is common in asymptomatic children, we were unable to evaluate the levels of immunomodulators in the serum samples of a control group. Moreover, although the samples were collected within the first 24 hours after admission, only a single blood sample was processed for each patient. Therefore, a larger, longitudinal study on the etiology and severity of pneumonia will be necessary to confirm these results. In conclusion, the present findings suggest that mixed respiratory infections and IP-10 may play major, interconnected roles in the pathogenesis of pneumonia. Clinically, assessment and monitoring of induced IP-10 serum level may assist clinicians to improve diagnosis and patient management of severe community-acquired pneumonia.", "Viral and bacterial strains. The 10 ng ml −1 M-CSF (Miltenyi Biotec). THP− 1 MDMs were obtained by culturing cells with 10 ng ml -1 phorbol myristate acetate (PMA; Invivogen, Toulouse, France) for 72 hours. Human airway epithelial cells (HAEC, bronchial cell type) originated from a 54-years old woman with no pathology reported (batch number MD056501) were provided by Mucilair (Epithelix, Geneva, Switzerland). Sterility, tissue integrity (TEER), mucus production and cilia beating frequency have been certified by the company. Gene expression profiling. Total cellular mRNA was purified using the RNeasy kit (Qiagen, Hilden, Germany). Reverse-transcription of total mRNA was performed using the RT 2 First Strand Kit (SABiosciences, Hilden, Germany). The expression of 84 genes involved in the human innate and adaptive immune responses was evaluated using the RT 2 profiler ™ PCR Array (SABiosciences) according to the manufacturer's recommendations. The Δ Δ Ct method was applied to calculate", "the fold changes in gene expression for each gene relative to uninfected control cells using the web-based RT 2 profiler PCR Array Data Analysis software (SABiosciences).", "MicroRNA profiling array. Total cellular microRNAs were purified using the miRNeasy Mini kit (Qiagen) and reverse-transcribed using the miScript Reverse Transcription kit (Qiagen). The profiling of 84 miRNAs was performed using the Human Immunopathology miScript miRNA PCR Array kit (Qiagen) according to the manufacturer's instructions. Data were analyzed using the miScript miRNA PCR array data analysis web portal.\n\nIn silico miRNA target prediction. MiRNA target genes were retrieved and compiled using TargetScan 38 and microRNA.org resource 39 . The interactions between miRNAs and intracellular pathways were predicted using DIANA-miRPath v2.0 40 .", "THP-1 MDMs were seeded in 24-well plates (0.5 × 10 6 per well) in triplicate, exposed to Influenza A H1N1 (A/Solomon islands/3/2006) virus (IAV) under serum-free conditions for 1 hour and then cultured for 3 hours in fresh RPMI-1640 containing 2% FBS. Streptococcus pneumoniae (SP) serotype 14 was added at 4 hours after IAV infection. Gentamicin (10 μ g ml −1 ) was added 2 hours after SP infection (i.e. 6 hours post-influenza infection) and maintained in the culture media throughout the experiment to kill extracellular bacteria and limit bacterial growth. Cell viability was determined by flow-cytometry using the FITC/Annexin V apoptosis detection kit (BD Biosciences), according to the manufacturer's instructions. #4427975) . In this assay, fold changes have been defined by the Δ Δ Ct method using control RNU-44 and -48 as reference microRNAs. Total mRNA was purified from transfected and infected MDMs using the RNeasy kit (Qiagen) and specific primers were used to amplify transforming", "growth factor beta-2 (TGFB2; F: 5′ -CCATCCCGCCCACTTTCTAC-3′ , R: 5′ -AGCTCAATCCGTTGTTCAGGC-3′ ), SOCS6 (F: 5′ -AAGAATTCATCCCTTGGATTAGGTAAC-3′ , R: 5′ -CAGACTGGAGGTCGTGGAA-3′ ) 41 43 , and 3) absence of wheezing at auscultation, and, 4) first symptoms appearing within the last 14 days, and 5) radiological confirmation of pneumonia as per WHO guidelines 44 . Based on these primary criteria defining pneumonia cases, all 74 cases were retrospectively re-evaluated according to the WHO \"Pocket book of hospital care for children\" 45 criteria to evaluate pneumonia severity. Cases that died during the study, or who had at least one additional clinical signs including central cyanosis, dullness to percussion during chest examination, prostration/lethargy, pleural effusion observed on chest radiography were retrospectively included in the severe pneumonia group. Patients without any of these additional clinical signs were included in the non-severe pneumonia group. Table 4 . a IP-10 values are", "expressed in pg ml -1 . IP-10 concentration differences between groups were compared using unpaired Mann-Whitney tests; significant changes (P < 0.05) are in bold. Clinical and molecular analysis. Nasopharyngeal aspirates (NAs) and whole blood samples were collected from children within 24 hours of admission. Whole blood samples were used for complete blood counts, blood culture and multiplex real-time PCR to identify Staphylococcus aureus, Streptococcus pneumoniae and Haemophilus influenzae type B 46 . S. pneumoniae serotypes were defined using a 11 multiplex real-time PCR assay targeting the 40 most frequently represented serotypes or serogroups according to protocol developed by Messaoudi et al. 47 . Serum C-reactive protein (CRP; AssayPro, St. Charles, Missouri, United States) and Procalcitonin (PCT; VIDAS B.R.A.H.M.S; bioMérieux) were quantified from whole-blood samples. Multiplex real-time non quantitative PCR (Fast-Track Diagnostic, Sliema, Malta) was used to detect 19 viruses", "and five bacteria in the respiratory specimens (NAs and pleural effusions). Mixed detection was defined as 1) PCR-positive for multiple viruses in NAs, 2) positive blood culture or PCR-positive for multiple bacteria in blood or 3) PCR-positive for one or multiple viruses in NAs and one or multiple bacteria in blood (identified by PCR and blood culture).", "Ethical approval. The study protocol, informed consent statement, clinical research form, any amendments and all other study documents were submitted to and approved by the Ethical Committee of the Instituto de Investigaciones en Ciencias de la Salud, the Universidad Nacional de Asunción (IICS-UNA) and the Hospital Pediátrico Niños de Acosta Ñu. Informed consent was obtained from all subjects involved in this study. The clinical investigation was conducted according to the principles expressed in the Declaration of Helsinki.", "Statistical analysis. The Chi-square test and Fisher's exact test were used to compare categorical variables; continuous variables and non-normally distributed data were compared using the Mann-Whitney U-test; normally distributed data were compared using unpaired t-tests. Comparative analyses between experimental conditions (i.e., MOCK, IAV, SP or IAV + SP) were performed using one-way ANOVA with Tukey's post-hoc test or Kruskal-Wallis analysis with Dunn's post-hoc tests. Receiver operating curve (ROC) analysis was used to determine the optimal cut-off value for IP-10 to differentiate between non-severe and severe pneumonia cases. P < 0.05 was considered statistically significant. All statistical analyses were performed using GraphPad Prism (La Jolla, California, United States)." ]

[ 32 ]

[ "highest serum IP-10 levels" ]

[ "Viral and bacterial co-infection in severe pneumonia triggers innate immune responses and specifically enhances IP-10: a translational study\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5138590/\n\nSHA: ef3d6cabc804e5eb587b34249b539c1b5efa4cc4\n\nAuthors: Hoffmann, Jonathan; Machado, Daniela; Terrier, Olivier; Pouzol, Stephane; Messaoudi, Mélina; Basualdo, Wilma; Espínola, Emilio E; Guillen, Rosa M.; Rosa-Calatrava, Manuel; Picot, Valentina; Bénet, Thomas; Endtz, Hubert; Russomando, Graciela; Paranhos-Baccalà, Gláucia\nDate: 2016-12-06\nDOI: 10.1038/srep38532\nLicense: cc-by", "Abstract: Mixed viral and bacterial infections are widely described in community-acquired pneumonia; however, the clinical implications of co-infection on the associated immunopathology remain poorly studied. In this study, microRNA, mRNA and cytokine/chemokine secretion profiling were investigated for human monocyte-derived macrophages infected in-vitro with Influenza virus A/H1N1 and/or Streptococcus pneumoniae. We observed that the in-vitro co-infection synergistically increased interferon-γ-induced protein-10 (CXCL10, IP-10) expression compared to the singly-infected cells conditions. We demonstrated that endogenous miRNA-200a-3p, whose expression was synergistically induced following co-infection, indirectly regulates CXCL10 expression by targeting suppressor of cytokine signaling-6 (SOCS-6), a well-known regulator of the JAK-STAT signaling pathway. Additionally, in a subsequent clinical pilot study, immunomodulators levels were evaluated in samples from 74 children (≤5", "years-old) hospitalized with viral and/or bacterial community-acquired pneumonia. Clinically, among the 74 cases of pneumonia, patients with identified mixed-detection had significantly higher (3.6-fold) serum IP-10 levels than those with a single detection (P = 0.03), and were significantly associated with severe pneumonia (P < 0.01). This study demonstrates that viral and bacterial co-infection modulates the JAK-STAT signaling pathway and leads to exacerbated IP-10 expression, which could play a major role in the pathogenesis of pneumonia.", "Text: Scientific RepoRts | 6:38532 | DOI: 10 .1038/srep38532 pathogenesis of several diseases and has been suggested as a potential biomarker of viral infection 10, 11 , late-onset bacterial infection in premature infants 12 , and a promising biomarker of sepsis and septic shock 13, 14 . Combined analysis of IP-10 and IFN-γ has also been reported as a useful biomarker for diagnosis and monitoring therapeutic efficacy in patients with active tuberculosis [15] [16] [17] , and both remain detectable in the urine of patients with pulmonary diseases in the absence of renal dysfunction 18 .", "With airway epithelial cells 19 , resident alveolar macrophages (AMs) and blood monocytes-derived macrophages (recruited into tissues under inflammatory conditions 20, 21 ) represent a major line of defense against both pneumococcal (through their high phagocytic capacity [22] [23] [24] ) and influenza infection 25, 26 . So far, no studies have yet focused on the intracellular mechanisms that regulate IP-10 in human blood leukocytes during mixed IAV and SP infection. Several studies indicated that host non-coding small RNAs (including microRNAs) may function as immunomodulators by regulating several pivotal intracellular processes, such as the innate immune response 27 and antiviral activity 28, 29 ; both of these processes are closely related to toll-like receptor (TLR) signaling pathways.", "In this study, we firstly investigated the in vitro intracellular mechanisms that mediate the innate immune response in IAV and/or SP infected human monocyte-derived macrophages (MDMs). Using this approach, we observed that mixed-infection of MDMs induces a synergistic production of IP-10 which can be related to a miRNA-200a/JAK-STAT/SOCS-6 regulatory pathway. Subsequently, in a retrospective analysis of clinical samples collected from children ≤ 5 years-old hospitalized with pneumonia, we confirmed that serum IP-10 level could be related to both viral and/or bacterial etiologies and disease severity.", "Characteristics of MDMs infected by IAV and/or SP. Initially, we investigated in vitro the impact of single and mixed IAV and SP infection on MDMs. Firstly, active replication of IAV was assessed by qRT-PCR and quantification of new infectious viral particles in the cell supernatants ( Fig. 1a,b ). IAV titer increased over time after single infection with IAV and correlated with increased production of negative-strand IAV RNA. Maximum viral replication was observed at 18-24 hours post-infection, after which time both RNA replication and the quantity of infectious particles decreased. In this in vitro model, subsequent challenge of IAV-infected MDMs with SP had no significant impact on the production of new infectious viral particles (Fig. 1b) . Together, these results indicate permissive and productive infection of MDMs by IAV. Secondly, we evaluated whether MDMs are permissive for both IAV and SP infection. The presence of pneumococci within IAV-and SP-infected primary MDMs was", "confirmed at 8 h post-infection (Fig. 1c) , suggesting that MDMs are permissive for viral and bacterial co-infection in the early steps of infection. Importantly, confocal co-detection of mixed IAV and SP was only effective following 8 h post-infection due to the bactericidal impact of SP internalization within human macrophages (after 24 h, data not shown). Thirdly, we evaluated the impact of single and mixed infection with IAV and SP on MDM viability. Mixed infection significantly decreased cell viability (65.2 ± 4.5% total cell death at 48 hours post-infection; P < 0.0001) compared to single SP and IAV infection (39.6 ± 1.7% and 17.4 ± 1.1% total cell death, respectively; Fig. 1d ). Taken together, these results confirmed human MDMs are permissive to mixed viral and bacterial infection. mRNA, microRNA and protein expression profiling reveal an overall induction of the host innate immune response following IAV and/or SP infection of MDMs. To investigate the innate immune response", "orchestrated by IAV-and SP-infected human MDMs, we firstly evaluated the expression of 84 genes involved in the innate and adaptive immune responses (Table S1) ; the major differentially-expressed genes are summarized in Fig. 2a . Expression profiling indicated an overall induction of genes related to the JAK-STAT, NF-Κ β and TLR signaling pathways. Indeed, all interferon-stimulated genes (ISGs) screened, including CXCL10 (fold-change [FC] = 240.9), CCL-2 (FC = 34.2) and MX-1 (FC = 151.4) were upregulated following mixed infection compared to uninfected cells, most of which are closely related to STAT-1 (FC = 52.3), IRF-7 (FC = 6.8) and IFNB1 (FC = 5.2) also found upregulated in mixed infected cells. Secondly, we investigated the endogenous microRNA expression profiles of IAV-and SP-infected MDMs. A selection of microRNAs that were found to be differentially-expressed under different infection conditions are shown in Fig. 2b and Table S2 . MiRNA-200a-3p was overexpressed after both", "single IAV (FC = 6.9), single SP (FC = 3.7) and mixed IAV/SP infection (FC = 7.3), indicating this miRNA may play a role in the innate immune response to viral and bacterial co-infection. Similar miRNA-200a-3p dysregulation profiles were obtained following IAV and/or SP infections of human macrophages-like (THP-1 monocytes-derived macrophages) or primary MDMs (data not shown). Thirdly, the secreted levels of various antiviral, pro-inflammatory and immunomodulatory cytokines/chemokines were assayed in IAV-and SP-infected-THP-1 and primary MDM cell supernatants. We observed a remarkable correlation between the mRNA and protein expression profiles of single or mixed infected MDMs especially regarding CXCL-10 and IP-10 expression. Indeed, the level of IP-10 was synergistically increased in the supernatant of IAV-infected THP-1 MDMs exposed to SP (mean: 30,589 ± 16,484 pg ml −1 ) compared to single IAV infection (1,439 ± 566.5 pg ml −1 ) and single SP infection (4,472 ± 2,001 pg ml −1 ; P≤", "0.05; Fig. 2c ) at 24 hours after infection. In those cells, IP-10 expression reduced over time (48 to 72 hours), coinciding with a significant higher proportion of necrotic and apoptotic cells (Fig. 1d) . The synergistic expression of IP-10 was similarly observed at 24 hours post-infection using primary MDMs (Fig. 2d) . Significantly increased secretion of the other tested cytokines and chemokines was not observed post-infection, even in mixed infected MDMs (Fig. S1 ). Interestingly, a significant production of IP-10 was also observed in supernatants of primary human airway epithelial cells (HAEC) mixed-infected by IAV and SP compared to the single infections (Fig. 2e) . Taken together, the mRNA and protein profiling results suggested that mixed viral and bacterial infection of MDMs induces a synergistic pro-inflammatory response related to the type-1 interferon and JAK-STAT signaling pathways, with IP-10 as signature of IAV/SP co-infection. Among all microRNAs screened, miR-200a-3p", "was the most Scientific RepoRts | 6:38532 | DOI: 10.1038/srep38532 overexpressed in IAV/SP co-infection of human MDMs. In the remainder of this study, we decided to investigate the interconnection between miR-200a-3p expression and the innate immune response.", "Endogenous miRNA-200a-3p expression correlates with CXCL10 (IP-10) induction following mixed IAV and SP infection of human MDMs. Using a specific Taqman probe assay targeting miR-200a-3p, we confirmed a significant upregulation of miR-200a-3p following mixed IAV and SP infection of human MDMs (Fig. 3a) . In this experiment, a more marked up-regulation of miR-200a-3p was observed following IAV+ SP compared to results obtained previously (Fig. 2b) . This discrepancy has been attributed to the use of two different approaches to quantify miR-200a-3p expression. The use of a target-specific stem-loop reverse transcription primer in Fig. 3a allows a better sensitivity of miR-200a-3p detection compared to the non-specific fluorescent dye used in Fig. 2b . As the general trend was suggestive of a synergistic induction of miR-200a-3p in response to mixed infection (Fig. 3a) , we hypothesized microRNA-200a-3p may play a role in the regulation of CXCL10 (IP-10), which was also synergistically", "upregulated in mixed-infected MDMs ( Fig. 2c and d) and primary HAEC ( Statistical analyses were performed using two-way ANOVA with Tukey's post-hoc test; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.", "Scientific RepoRts | 6:38532 | DOI: 10.1038/srep38532 CXCL10 (Fig. 3d) . These results suggested miR-200a-3p indirectly regulates CXCL10 and led us to hypothesize that miR-200a-3p controls a potential repressor of the JAK-STAT signaling pathway. . At 18 h after transfection, the MDMs were singly or mixed infected as described previously. At 8 h post-IAV and/or SP infection, total mRNA was extracted and amplified by PCR using specific primers for the indicated genes. Values represent median ± IQR (a, c) or mean ± SEM (d, e) of three biological replicates. Statistical analyses were performed using a Kruskal-Wallis test (non-parametric, one-way ANOVA with Dunn's post-hoc test) for data presented in (a, c). An ordinary two-way ANOVA (with Tukey's post-hoc multiple comparison test) was used for data presented in (d, e). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. MiRNA-200a-3p indirectly regulates IP-10 expression by targeting SOCS6. As shown in Fig. 2a , several JAK-STAT", "signaling pathway genes were deregulated in mixed IAV-and SP-infected human MDMs; therefore, we hypothesized that miR-200a-3p directly regulates a regulator of the JAK-STAT signaling pathway. Predictive target analysis indicated that the 3' UTR of suppressor of cytokine signaling-6 (SOCS6) may be targeted by miR-200a-3p (Fig. 3b) . SOCS proteins constitute a class of negative regulators of JAK-STAT signaling pathways that are induced by both cytokines and TLR signaling. MiRNA-200a-3p was not predicted to target any of the other six members of the SOCS gene family. Transfection of human MDMs with MIM-200a downregulated SOCS6 (FC = 0.57) while inhibition of miR-200a-3p (INH-200a) upregulated SOCS6 (FC = 1.55), confirming that miR-200a-3p effectively regulates the expression of SOCS6 (Fig. 3e) . Moreover, SOCS6 was synergistically downregulated in IAV-or IAV/SP-infected MDMs overexpressing miRNA-200a (Fig. 3e) , suggesting that both infection and miR-200a-3p negatively regulate the", "expression of SOCS6. Finally, western blotting confirmed that expression of SOCS-6 sharply reduced following infection, especially after mixed IAV and SP infection (Fig. 3f) .", "These results indicate miR-200a-3p is strongly induced in response to mixed viral and bacterial co-infection, which in turn leads to downregulation of the JAK-STAT regulator SOCS-6 at both the mRNA and protein levels and subsequent upregulation of IP-10.", "analyses demonstrated mixed IAV and SP infection of human MDMs and HAEC induced significant production of IP-10. As blood leukocytes and respiratory tract epithelial cells actively contribute to inflammation during pneumonia, we hypothesized the level of IP-10 in serum of patient with pneumonia may be both indicative of mixed respiratory infection and disease severity. As part of a prospective, hospital-based, multicenter case-control study on the etiology of pneumonia among children under 5-years-old, a total of 74 patients (44 male, 30 female) were included in this pilot evaluation. According to WHO guidelines, retrospective analysis indicated 44 (59.5%) children had clinical signs of non-severe pneumonia and 30 (40.5%) children had signs of severe pneumonia. The main patient characteristics at inclusion are shown in Table 1 . Patients with severe pneumonia had significant more recorded episodes of dyspnea (P < 0.001), cyanosis (P = 0.03), lower chest indrawing (P < 0.001), dullness", "to percussion (P < 0.001) and lethargy (P < 0.001) during chest examination than patient with non-severe pneumonia. Moreover, pleural effusions were significantly more observed among critically ill patients and the duration of hospitalization was significantly longer for the children with severe pneumonia than for those with non-severe pneumonia (P = 0.0015). Two deaths occurred within the group of children retrospectively defined with severe pneumonia. Evaluation of the systemic inflammatory response of the 74 cases is shown in Table 2 . Serum level of CRP, IP-10, PCT, G-CSF, IL-6, IL-8 and MIP-1β were significantly more elevated in serum samples from critically ill patients. Patients with severe pneumonia had significantly higher (4.2-fold) serum IP-10 levels than those with a non-severe pneumonia (P < 0.001) suggesting IP-10 as a promising prognostic marker in pneumonia. Diagnostic accuracy measures for predicting pneumonia severity using blood-based biomarkers are summarized in", "Table S3 . Briefly, in this study, the optimal IP-10 cut-off value for identifying patient with severe pneumonia was 4,240 pg ml −1 , with an area under the receiver operating characteristic curve of 0.69 (95% CI, 0.57 to 0.82, P < 0.001). Defining as positive a serum IP-10 level above this cut-off resulted in a sensitivity of 63.3%, specificity of 63.6% and a positive likelihood ratio of 1.74. Prognostic values of IP-10 were closed to procalcitonin (PCT; AUC = 0.70; 95% IC, 0.58 to 0.82, P < 0.001) and IL-6 (AUC = 0.70; 95% IC, 0.58-0.83, P < 0.001).", "Multiplex PCR-based screening of respiratory and blood samples reveal a high variety of pathogen associations (Table 3) . Respiratory viruses were detected in the nasal aspirates (NAs) of 63/74 patients (85.1%). Etiological bacteria of pneumonia (S. pneumoniae, n = 19; S. aureus, n = 1; or H. influenzae type B, n = 7) were identified via real-time PCR in the blood samples of 27/74 (36.5%) of the patients. Multiplex PCR assays allowed the identification of respiratory bacteria in the blood of 19 patients with negative blood culture results. Among the 74 cases PCR-positive for respiratory pathogens, a single virus or bacteria were detected in the NAs of 7 (9.4%) and 3 (4.0%) patients, respectively; these 10/74 (13.5%) cases were defined as the single infection group. The mixed infection group included the 62/74 (83.8%) cases in which (1) multiple viruses and/or bacteria were identified in NAs (38/74; 51.3%) without any bacteria identified in blood samples or (2) one or more viruses", "and/or bacteria were identified in NAs and associated with a blood bacteremia (24/74; 32.4%). We evaluated whether IP-10 serum level could correlate with the viral and bacterial etiologies of pneumonia. Patients with mixed infection had significant higher (3.6-fold) IP-10 serum level than patient with single detection (P = 0.03; Table 4 ). A stratified analysis reveals that the highest IP-10 serum level was observed among patients with both several respiratory pathogens identified (mixed-detection group) and severe pneumonia (14,427 pg ml −1 , IQR (3,981-82,994). In detail, a remarkable IP-10 serum level (142,531 pg ml −1 ), representing 33-fold higher above cut-off value predicting pneumonia severity was observed in patient with hRV in NA co-detected with S. pneumoniae (serotype 14) in pleural effusion and blood. In concordance with our in-vitro model of co-infection, a significant IP-10 level (90,338 pg ml −1 ) was quantified in blood sample of patient with severe bacteremic", "pneumococcal (serotype 14) pneumonia with a positive co-detection of Influenza B virus in NA. Taken together, these results suggest that high serum IP-10 levels are significantly associated with mixed viral and bacterial detection and also related to pneumonia pathogenesis.", "This study provides additional in vitro and clinical data to improve our understanding of the immunopathology of mixed viral and bacterial pneumonia (Fig. 4) .", "The in vitro model of influenza and pneumococcal superinfection of human MDMs demonstrated that mixed infection synergistically induced release of the pro-inflammatory chemokine IP-10, strongly suggesting human Scientific RepoRts | 6:38532 | DOI: 10.1038/srep38532 blood leukocytes contribute to the immunopathology of pneumonia. Additionally, transcriptomics and omics analyses provided new data on the inflammatory pathways that are activated during mixed infection and related to synergistic induction of the pro-inflammatory chemokine IP-10 in mixed infected cells. Our observations are consistent with a recent study describing IP-10 induction as host-proteome signature of both viral and bacterial infections 30 . Of the differentially-expressed genes observed in mixed infected MDMs, the transcription factors STAT-1 and IRF-7 appear to play crucial roles in the regulation of interferon-stimulated genes including CXCL10 (IP-10). By focusing on the intracellular mechanisms that regulate", "inflammatory pathways, we demonstrated a novel role for miRNA-200a-3p in the regulation of CXCL10 (IP-10). These observations are consistent with previous reports showing that RNA virus infection upregulates miR-155 in macrophages and dendritic cells and also regulates suppressor of cytokine signaling 1 (SOCS1), suggesting the existence of a miRNA/JAK-STAT/SOCS regulatory pathway during viral infection 29 . Our study suggests co-infection leads to overexpression of miR-200a-3p, which in turn targets and downregulates the JAK-STAT regulator SOCS-6 and consequently increases CXCL10 (IP-10) expression. Interestingly, a complementary in-silico approach reveals that several microRNAs that were found dysregulated in our experiments of IAV and SP co-infection of MDMs or HAEC, might target several genes of SOCS family and play similar role than miR-200a-3p. Indeed, miRNA-142-3p might target SOCS4, 5, 6 mRNA while miRNA-194-5p might target SOCS2, 3, 4, 5 and 7 mRNA. These observations", "underline that intra-cellular regulation of IP-10 is not limited to the contribution of a sole microRNA. A complex inter-relationship between numerous host microRNAs and inhibitors of the JAK-STAT signaling pathway occur to control host innate inflammatory response against viral and/or bacterial infections. Clinically, the majority of pediatric CAP cases in this study were associated with both positive viral and/or bacterial detection. Respiratory microorganisms were detected in 97% of cases; 51.3% of which were viral-viral, viral-bacterial or bacterial-bacterial co-detected only in nasal aspirates, 32.4% of which co-detected in both nasal aspirates and blood samples. These data are consistent with previous etiological studies of pediatric CAP 3,31-33 . S. pneumoniae was the major bacteria identified in blood (19/74; 25.7%) and mainly co-detected with respiratory viruses in NAs (16/19; 84.2%). We observed a very high diversity of viral and bacterial associations in biological samples", "from children with pneumonia. In comparison with IAV and SP14 combination evaluated in-vitro, no pneumonia cases were singly influenza and pneumococcus infected, and no similar co-detection with those two pathogens has been clinically observed. Nevertheless, Influenza B (IVB) virus was identified in 5 patients and two of them had a positive SP co-detection in blood (one non-typable strain and one serotype 14 using our molecular typing test). IVB and SP14 combination seems to be the nearest pathogen co-detection to that in-vitro investigated. Clinically, this co-detection was associated with both a very high IP-10 expression and a very severe pneumonia case definition. Interestingly, our translational pilot evaluation reveals IP-10 expression can be induced by several different viral and/or bacterial combinations. As immune response to each pathogen is different, further in-vitro investigations using different pathogens associations are needed to better characterize the mechanisms", "involved in the immunopathology of pneumonia.", "In this cohort, highest serum IP-10 levels were identified among patients with both several pathogen detected and severe pneumonia, suggesting a significant role of IP-10 on pneumonia pathogenesis. Indeed, high plasma levels of IP-10 have previously been reported in patients with sepsis 12 , and were associated with high mortality rate, especially among patients with CAP 34 . Additionally, the IP-10-CXCR3 axis has been related to acute immune lung injury and lymphocyte apoptosis during the development of severe acute respiratory syndrome (SARS) 35, 36 . Moreover, an in vivo study that modeled influenza and pneumococcal superinfection in mice indicated that pro-inflammatory chemokines, including IP-10, play a crucial role in influenza-induced susceptibility to lung neutrophilia, severe immunopathology and mortality 37 . In this study, markedly elevated IP-10 (92,809 pg ml −1 ) combined with the highest PCT level (74.4 pg ml −1 ) were quantified in the serum sample of a child who died,", "in whom S. pneumoniae (serotype 9 V) was identified in the blood (PCR and blood culture) and co-detected with Haemophilus influenzae type B in nasal aspirate. These observations suggest an interrelationship between co-detection, elevated serum IP-10 and the pathogenesis of pneumonia.", "Several limitations of this pilot translational study need to be acknowledged before concluding mixed infection is related to elevated IP-10 and disease severity. Indeed, although viral shedding (e.g., of HRV and HBoV) is common in asymptomatic children, we were unable to evaluate the levels of immunomodulators in the serum samples of a control group. Moreover, although the samples were collected within the first 24 hours after admission, only a single blood sample was processed for each patient. Therefore, a larger, longitudinal study on the etiology and severity of pneumonia will be necessary to confirm these results. In conclusion, the present findings suggest that mixed respiratory infections and IP-10 may play major, interconnected roles in the pathogenesis of pneumonia. Clinically, assessment and monitoring of induced IP-10 serum level may assist clinicians to improve diagnosis and patient management of severe community-acquired pneumonia.", "Viral and bacterial strains. The 10 ng ml −1 M-CSF (Miltenyi Biotec). THP− 1 MDMs were obtained by culturing cells with 10 ng ml -1 phorbol myristate acetate (PMA; Invivogen, Toulouse, France) for 72 hours. Human airway epithelial cells (HAEC, bronchial cell type) originated from a 54-years old woman with no pathology reported (batch number MD056501) were provided by Mucilair (Epithelix, Geneva, Switzerland). Sterility, tissue integrity (TEER), mucus production and cilia beating frequency have been certified by the company. Gene expression profiling. Total cellular mRNA was purified using the RNeasy kit (Qiagen, Hilden, Germany). Reverse-transcription of total mRNA was performed using the RT 2 First Strand Kit (SABiosciences, Hilden, Germany). The expression of 84 genes involved in the human innate and adaptive immune responses was evaluated using the RT 2 profiler ™ PCR Array (SABiosciences) according to the manufacturer's recommendations. The Δ Δ Ct method was applied to calculate", "the fold changes in gene expression for each gene relative to uninfected control cells using the web-based RT 2 profiler PCR Array Data Analysis software (SABiosciences).", "MicroRNA profiling array. Total cellular microRNAs were purified using the miRNeasy Mini kit (Qiagen) and reverse-transcribed using the miScript Reverse Transcription kit (Qiagen). The profiling of 84 miRNAs was performed using the Human Immunopathology miScript miRNA PCR Array kit (Qiagen) according to the manufacturer's instructions. Data were analyzed using the miScript miRNA PCR array data analysis web portal.\n\nIn silico miRNA target prediction. MiRNA target genes were retrieved and compiled using TargetScan 38 and microRNA.org resource 39 . The interactions between miRNAs and intracellular pathways were predicted using DIANA-miRPath v2.0 40 .", "THP-1 MDMs were seeded in 24-well plates (0.5 × 10 6 per well) in triplicate, exposed to Influenza A H1N1 (A/Solomon islands/3/2006) virus (IAV) under serum-free conditions for 1 hour and then cultured for 3 hours in fresh RPMI-1640 containing 2% FBS. Streptococcus pneumoniae (SP) serotype 14 was added at 4 hours after IAV infection. Gentamicin (10 μ g ml −1 ) was added 2 hours after SP infection (i.e. 6 hours post-influenza infection) and maintained in the culture media throughout the experiment to kill extracellular bacteria and limit bacterial growth. Cell viability was determined by flow-cytometry using the FITC/Annexin V apoptosis detection kit (BD Biosciences), according to the manufacturer's instructions. #4427975) . In this assay, fold changes have been defined by the Δ Δ Ct method using control RNU-44 and -48 as reference microRNAs. Total mRNA was purified from transfected and infected MDMs using the RNeasy kit (Qiagen) and specific primers were used to amplify transforming", "growth factor beta-2 (TGFB2; F: 5′ -CCATCCCGCCCACTTTCTAC-3′ , R: 5′ -AGCTCAATCCGTTGTTCAGGC-3′ ), SOCS6 (F: 5′ -AAGAATTCATCCCTTGGATTAGGTAAC-3′ , R: 5′ -CAGACTGGAGGTCGTGGAA-3′ ) 41 43 , and 3) absence of wheezing at auscultation, and, 4) first symptoms appearing within the last 14 days, and 5) radiological confirmation of pneumonia as per WHO guidelines 44 . Based on these primary criteria defining pneumonia cases, all 74 cases were retrospectively re-evaluated according to the WHO \"Pocket book of hospital care for children\" 45 criteria to evaluate pneumonia severity. Cases that died during the study, or who had at least one additional clinical signs including central cyanosis, dullness to percussion during chest examination, prostration/lethargy, pleural effusion observed on chest radiography were retrospectively included in the severe pneumonia group. Patients without any of these additional clinical signs were included in the non-severe pneumonia group. Table 4 . a IP-10 values are", "expressed in pg ml -1 . IP-10 concentration differences between groups were compared using unpaired Mann-Whitney tests; significant changes (P < 0.05) are in bold. Clinical and molecular analysis. Nasopharyngeal aspirates (NAs) and whole blood samples were collected from children within 24 hours of admission. Whole blood samples were used for complete blood counts, blood culture and multiplex real-time PCR to identify Staphylococcus aureus, Streptococcus pneumoniae and Haemophilus influenzae type B 46 . S. pneumoniae serotypes were defined using a 11 multiplex real-time PCR assay targeting the 40 most frequently represented serotypes or serogroups according to protocol developed by Messaoudi et al. 47 . Serum C-reactive protein (CRP; AssayPro, St. Charles, Missouri, United States) and Procalcitonin (PCT; VIDAS B.R.A.H.M.S; bioMérieux) were quantified from whole-blood samples. Multiplex real-time non quantitative PCR (Fast-Track Diagnostic, Sliema, Malta) was used to detect 19 viruses", "and five bacteria in the respiratory specimens (NAs and pleural effusions). Mixed detection was defined as 1) PCR-positive for multiple viruses in NAs, 2) positive blood culture or PCR-positive for multiple bacteria in blood or 3) PCR-positive for one or multiple viruses in NAs and one or multiple bacteria in blood (identified by PCR and blood culture).", "Ethical approval. The study protocol, informed consent statement, clinical research form, any amendments and all other study documents were submitted to and approved by the Ethical Committee of the Instituto de Investigaciones en Ciencias de la Salud, the Universidad Nacional de Asunción (IICS-UNA) and the Hospital Pediátrico Niños de Acosta Ñu. Informed consent was obtained from all subjects involved in this study. The clinical investigation was conducted according to the principles expressed in the Declaration of Helsinki.", "Statistical analysis. The Chi-square test and Fisher's exact test were used to compare categorical variables; continuous variables and non-normally distributed data were compared using the Mann-Whitney U-test; normally distributed data were compared using unpaired t-tests. Comparative analyses between experimental conditions (i.e., MOCK, IAV, SP or IAV + SP) were performed using one-way ANOVA with Tukey's post-hoc test or Kruskal-Wallis analysis with Dunn's post-hoc tests. Receiver operating curve (ROC) analysis was used to determine the optimal cut-off value for IP-10 to differentiate between non-severe and severe pneumonia cases. P < 0.05 was considered statistically significant. All statistical analyses were performed using GraphPad Prism (La Jolla, California, United States)." ]

[ 30 ]

[ "airway epithelial cells 19 , resident alveolar macrophages (AMs) and blood monocytes-derived macrophages" ]

[ "Viral and bacterial co-infection in severe pneumonia triggers innate immune responses and specifically enhances IP-10: a translational study\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5138590/\n\nSHA: ef3d6cabc804e5eb587b34249b539c1b5efa4cc4\n\nAuthors: Hoffmann, Jonathan; Machado, Daniela; Terrier, Olivier; Pouzol, Stephane; Messaoudi, Mélina; Basualdo, Wilma; Espínola, Emilio E; Guillen, Rosa M.; Rosa-Calatrava, Manuel; Picot, Valentina; Bénet, Thomas; Endtz, Hubert; Russomando, Graciela; Paranhos-Baccalà, Gláucia\nDate: 2016-12-06\nDOI: 10.1038/srep38532\nLicense: cc-by", "Abstract: Mixed viral and bacterial infections are widely described in community-acquired pneumonia; however, the clinical implications of co-infection on the associated immunopathology remain poorly studied. In this study, microRNA, mRNA and cytokine/chemokine secretion profiling were investigated for human monocyte-derived macrophages infected in-vitro with Influenza virus A/H1N1 and/or Streptococcus pneumoniae. We observed that the in-vitro co-infection synergistically increased interferon-γ-induced protein-10 (CXCL10, IP-10) expression compared to the singly-infected cells conditions. We demonstrated that endogenous miRNA-200a-3p, whose expression was synergistically induced following co-infection, indirectly regulates CXCL10 expression by targeting suppressor of cytokine signaling-6 (SOCS-6), a well-known regulator of the JAK-STAT signaling pathway. Additionally, in a subsequent clinical pilot study, immunomodulators levels were evaluated in samples from 74 children (≤5", "years-old) hospitalized with viral and/or bacterial community-acquired pneumonia. Clinically, among the 74 cases of pneumonia, patients with identified mixed-detection had significantly higher (3.6-fold) serum IP-10 levels than those with a single detection (P = 0.03), and were significantly associated with severe pneumonia (P < 0.01). This study demonstrates that viral and bacterial co-infection modulates the JAK-STAT signaling pathway and leads to exacerbated IP-10 expression, which could play a major role in the pathogenesis of pneumonia.", "Text: Scientific RepoRts | 6:38532 | DOI: 10 .1038/srep38532 pathogenesis of several diseases and has been suggested as a potential biomarker of viral infection 10, 11 , late-onset bacterial infection in premature infants 12 , and a promising biomarker of sepsis and septic shock 13, 14 . Combined analysis of IP-10 and IFN-γ has also been reported as a useful biomarker for diagnosis and monitoring therapeutic efficacy in patients with active tuberculosis [15] [16] [17] , and both remain detectable in the urine of patients with pulmonary diseases in the absence of renal dysfunction 18 .", "With airway epithelial cells 19 , resident alveolar macrophages (AMs) and blood monocytes-derived macrophages (recruited into tissues under inflammatory conditions 20, 21 ) represent a major line of defense against both pneumococcal (through their high phagocytic capacity [22] [23] [24] ) and influenza infection 25, 26 . So far, no studies have yet focused on the intracellular mechanisms that regulate IP-10 in human blood leukocytes during mixed IAV and SP infection. Several studies indicated that host non-coding small RNAs (including microRNAs) may function as immunomodulators by regulating several pivotal intracellular processes, such as the innate immune response 27 and antiviral activity 28, 29 ; both of these processes are closely related to toll-like receptor (TLR) signaling pathways.", "In this study, we firstly investigated the in vitro intracellular mechanisms that mediate the innate immune response in IAV and/or SP infected human monocyte-derived macrophages (MDMs). Using this approach, we observed that mixed-infection of MDMs induces a synergistic production of IP-10 which can be related to a miRNA-200a/JAK-STAT/SOCS-6 regulatory pathway. Subsequently, in a retrospective analysis of clinical samples collected from children ≤ 5 years-old hospitalized with pneumonia, we confirmed that serum IP-10 level could be related to both viral and/or bacterial etiologies and disease severity.", "Characteristics of MDMs infected by IAV and/or SP. Initially, we investigated in vitro the impact of single and mixed IAV and SP infection on MDMs. Firstly, active replication of IAV was assessed by qRT-PCR and quantification of new infectious viral particles in the cell supernatants ( Fig. 1a,b ). IAV titer increased over time after single infection with IAV and correlated with increased production of negative-strand IAV RNA. Maximum viral replication was observed at 18-24 hours post-infection, after which time both RNA replication and the quantity of infectious particles decreased. In this in vitro model, subsequent challenge of IAV-infected MDMs with SP had no significant impact on the production of new infectious viral particles (Fig. 1b) . Together, these results indicate permissive and productive infection of MDMs by IAV. Secondly, we evaluated whether MDMs are permissive for both IAV and SP infection. The presence of pneumococci within IAV-and SP-infected primary MDMs was", "confirmed at 8 h post-infection (Fig. 1c) , suggesting that MDMs are permissive for viral and bacterial co-infection in the early steps of infection. Importantly, confocal co-detection of mixed IAV and SP was only effective following 8 h post-infection due to the bactericidal impact of SP internalization within human macrophages (after 24 h, data not shown). Thirdly, we evaluated the impact of single and mixed infection with IAV and SP on MDM viability. Mixed infection significantly decreased cell viability (65.2 ± 4.5% total cell death at 48 hours post-infection; P < 0.0001) compared to single SP and IAV infection (39.6 ± 1.7% and 17.4 ± 1.1% total cell death, respectively; Fig. 1d ). Taken together, these results confirmed human MDMs are permissive to mixed viral and bacterial infection. mRNA, microRNA and protein expression profiling reveal an overall induction of the host innate immune response following IAV and/or SP infection of MDMs. To investigate the innate immune response", "orchestrated by IAV-and SP-infected human MDMs, we firstly evaluated the expression of 84 genes involved in the innate and adaptive immune responses (Table S1) ; the major differentially-expressed genes are summarized in Fig. 2a . Expression profiling indicated an overall induction of genes related to the JAK-STAT, NF-Κ β and TLR signaling pathways. Indeed, all interferon-stimulated genes (ISGs) screened, including CXCL10 (fold-change [FC] = 240.9), CCL-2 (FC = 34.2) and MX-1 (FC = 151.4) were upregulated following mixed infection compared to uninfected cells, most of which are closely related to STAT-1 (FC = 52.3), IRF-7 (FC = 6.8) and IFNB1 (FC = 5.2) also found upregulated in mixed infected cells. Secondly, we investigated the endogenous microRNA expression profiles of IAV-and SP-infected MDMs. A selection of microRNAs that were found to be differentially-expressed under different infection conditions are shown in Fig. 2b and Table S2 . MiRNA-200a-3p was overexpressed after both", "single IAV (FC = 6.9), single SP (FC = 3.7) and mixed IAV/SP infection (FC = 7.3), indicating this miRNA may play a role in the innate immune response to viral and bacterial co-infection. Similar miRNA-200a-3p dysregulation profiles were obtained following IAV and/or SP infections of human macrophages-like (THP-1 monocytes-derived macrophages) or primary MDMs (data not shown). Thirdly, the secreted levels of various antiviral, pro-inflammatory and immunomodulatory cytokines/chemokines were assayed in IAV-and SP-infected-THP-1 and primary MDM cell supernatants. We observed a remarkable correlation between the mRNA and protein expression profiles of single or mixed infected MDMs especially regarding CXCL-10 and IP-10 expression. Indeed, the level of IP-10 was synergistically increased in the supernatant of IAV-infected THP-1 MDMs exposed to SP (mean: 30,589 ± 16,484 pg ml −1 ) compared to single IAV infection (1,439 ± 566.5 pg ml −1 ) and single SP infection (4,472 ± 2,001 pg ml −1 ; P≤", "0.05; Fig. 2c ) at 24 hours after infection. In those cells, IP-10 expression reduced over time (48 to 72 hours), coinciding with a significant higher proportion of necrotic and apoptotic cells (Fig. 1d) . The synergistic expression of IP-10 was similarly observed at 24 hours post-infection using primary MDMs (Fig. 2d) . Significantly increased secretion of the other tested cytokines and chemokines was not observed post-infection, even in mixed infected MDMs (Fig. S1 ). Interestingly, a significant production of IP-10 was also observed in supernatants of primary human airway epithelial cells (HAEC) mixed-infected by IAV and SP compared to the single infections (Fig. 2e) . Taken together, the mRNA and protein profiling results suggested that mixed viral and bacterial infection of MDMs induces a synergistic pro-inflammatory response related to the type-1 interferon and JAK-STAT signaling pathways, with IP-10 as signature of IAV/SP co-infection. Among all microRNAs screened, miR-200a-3p", "was the most Scientific RepoRts | 6:38532 | DOI: 10.1038/srep38532 overexpressed in IAV/SP co-infection of human MDMs. In the remainder of this study, we decided to investigate the interconnection between miR-200a-3p expression and the innate immune response.", "Endogenous miRNA-200a-3p expression correlates with CXCL10 (IP-10) induction following mixed IAV and SP infection of human MDMs. Using a specific Taqman probe assay targeting miR-200a-3p, we confirmed a significant upregulation of miR-200a-3p following mixed IAV and SP infection of human MDMs (Fig. 3a) . In this experiment, a more marked up-regulation of miR-200a-3p was observed following IAV+ SP compared to results obtained previously (Fig. 2b) . This discrepancy has been attributed to the use of two different approaches to quantify miR-200a-3p expression. The use of a target-specific stem-loop reverse transcription primer in Fig. 3a allows a better sensitivity of miR-200a-3p detection compared to the non-specific fluorescent dye used in Fig. 2b . As the general trend was suggestive of a synergistic induction of miR-200a-3p in response to mixed infection (Fig. 3a) , we hypothesized microRNA-200a-3p may play a role in the regulation of CXCL10 (IP-10), which was also synergistically", "upregulated in mixed-infected MDMs ( Fig. 2c and d) and primary HAEC ( Statistical analyses were performed using two-way ANOVA with Tukey's post-hoc test; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.", "Scientific RepoRts | 6:38532 | DOI: 10.1038/srep38532 CXCL10 (Fig. 3d) . These results suggested miR-200a-3p indirectly regulates CXCL10 and led us to hypothesize that miR-200a-3p controls a potential repressor of the JAK-STAT signaling pathway. . At 18 h after transfection, the MDMs were singly or mixed infected as described previously. At 8 h post-IAV and/or SP infection, total mRNA was extracted and amplified by PCR using specific primers for the indicated genes. Values represent median ± IQR (a, c) or mean ± SEM (d, e) of three biological replicates. Statistical analyses were performed using a Kruskal-Wallis test (non-parametric, one-way ANOVA with Dunn's post-hoc test) for data presented in (a, c). An ordinary two-way ANOVA (with Tukey's post-hoc multiple comparison test) was used for data presented in (d, e). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. MiRNA-200a-3p indirectly regulates IP-10 expression by targeting SOCS6. As shown in Fig. 2a , several JAK-STAT", "signaling pathway genes were deregulated in mixed IAV-and SP-infected human MDMs; therefore, we hypothesized that miR-200a-3p directly regulates a regulator of the JAK-STAT signaling pathway. Predictive target analysis indicated that the 3' UTR of suppressor of cytokine signaling-6 (SOCS6) may be targeted by miR-200a-3p (Fig. 3b) . SOCS proteins constitute a class of negative regulators of JAK-STAT signaling pathways that are induced by both cytokines and TLR signaling. MiRNA-200a-3p was not predicted to target any of the other six members of the SOCS gene family. Transfection of human MDMs with MIM-200a downregulated SOCS6 (FC = 0.57) while inhibition of miR-200a-3p (INH-200a) upregulated SOCS6 (FC = 1.55), confirming that miR-200a-3p effectively regulates the expression of SOCS6 (Fig. 3e) . Moreover, SOCS6 was synergistically downregulated in IAV-or IAV/SP-infected MDMs overexpressing miRNA-200a (Fig. 3e) , suggesting that both infection and miR-200a-3p negatively regulate the", "expression of SOCS6. Finally, western blotting confirmed that expression of SOCS-6 sharply reduced following infection, especially after mixed IAV and SP infection (Fig. 3f) .", "These results indicate miR-200a-3p is strongly induced in response to mixed viral and bacterial co-infection, which in turn leads to downregulation of the JAK-STAT regulator SOCS-6 at both the mRNA and protein levels and subsequent upregulation of IP-10.", "analyses demonstrated mixed IAV and SP infection of human MDMs and HAEC induced significant production of IP-10. As blood leukocytes and respiratory tract epithelial cells actively contribute to inflammation during pneumonia, we hypothesized the level of IP-10 in serum of patient with pneumonia may be both indicative of mixed respiratory infection and disease severity. As part of a prospective, hospital-based, multicenter case-control study on the etiology of pneumonia among children under 5-years-old, a total of 74 patients (44 male, 30 female) were included in this pilot evaluation. According to WHO guidelines, retrospective analysis indicated 44 (59.5%) children had clinical signs of non-severe pneumonia and 30 (40.5%) children had signs of severe pneumonia. The main patient characteristics at inclusion are shown in Table 1 . Patients with severe pneumonia had significant more recorded episodes of dyspnea (P < 0.001), cyanosis (P = 0.03), lower chest indrawing (P < 0.001), dullness", "to percussion (P < 0.001) and lethargy (P < 0.001) during chest examination than patient with non-severe pneumonia. Moreover, pleural effusions were significantly more observed among critically ill patients and the duration of hospitalization was significantly longer for the children with severe pneumonia than for those with non-severe pneumonia (P = 0.0015). Two deaths occurred within the group of children retrospectively defined with severe pneumonia. Evaluation of the systemic inflammatory response of the 74 cases is shown in Table 2 . Serum level of CRP, IP-10, PCT, G-CSF, IL-6, IL-8 and MIP-1β were significantly more elevated in serum samples from critically ill patients. Patients with severe pneumonia had significantly higher (4.2-fold) serum IP-10 levels than those with a non-severe pneumonia (P < 0.001) suggesting IP-10 as a promising prognostic marker in pneumonia. Diagnostic accuracy measures for predicting pneumonia severity using blood-based biomarkers are summarized in", "Table S3 . Briefly, in this study, the optimal IP-10 cut-off value for identifying patient with severe pneumonia was 4,240 pg ml −1 , with an area under the receiver operating characteristic curve of 0.69 (95% CI, 0.57 to 0.82, P < 0.001). Defining as positive a serum IP-10 level above this cut-off resulted in a sensitivity of 63.3%, specificity of 63.6% and a positive likelihood ratio of 1.74. Prognostic values of IP-10 were closed to procalcitonin (PCT; AUC = 0.70; 95% IC, 0.58 to 0.82, P < 0.001) and IL-6 (AUC = 0.70; 95% IC, 0.58-0.83, P < 0.001).", "Multiplex PCR-based screening of respiratory and blood samples reveal a high variety of pathogen associations (Table 3) . Respiratory viruses were detected in the nasal aspirates (NAs) of 63/74 patients (85.1%). Etiological bacteria of pneumonia (S. pneumoniae, n = 19; S. aureus, n = 1; or H. influenzae type B, n = 7) were identified via real-time PCR in the blood samples of 27/74 (36.5%) of the patients. Multiplex PCR assays allowed the identification of respiratory bacteria in the blood of 19 patients with negative blood culture results. Among the 74 cases PCR-positive for respiratory pathogens, a single virus or bacteria were detected in the NAs of 7 (9.4%) and 3 (4.0%) patients, respectively; these 10/74 (13.5%) cases were defined as the single infection group. The mixed infection group included the 62/74 (83.8%) cases in which (1) multiple viruses and/or bacteria were identified in NAs (38/74; 51.3%) without any bacteria identified in blood samples or (2) one or more viruses", "and/or bacteria were identified in NAs and associated with a blood bacteremia (24/74; 32.4%). We evaluated whether IP-10 serum level could correlate with the viral and bacterial etiologies of pneumonia. Patients with mixed infection had significant higher (3.6-fold) IP-10 serum level than patient with single detection (P = 0.03; Table 4 ). A stratified analysis reveals that the highest IP-10 serum level was observed among patients with both several respiratory pathogens identified (mixed-detection group) and severe pneumonia (14,427 pg ml −1 , IQR (3,981-82,994). In detail, a remarkable IP-10 serum level (142,531 pg ml −1 ), representing 33-fold higher above cut-off value predicting pneumonia severity was observed in patient with hRV in NA co-detected with S. pneumoniae (serotype 14) in pleural effusion and blood. In concordance with our in-vitro model of co-infection, a significant IP-10 level (90,338 pg ml −1 ) was quantified in blood sample of patient with severe bacteremic", "pneumococcal (serotype 14) pneumonia with a positive co-detection of Influenza B virus in NA. Taken together, these results suggest that high serum IP-10 levels are significantly associated with mixed viral and bacterial detection and also related to pneumonia pathogenesis.", "This study provides additional in vitro and clinical data to improve our understanding of the immunopathology of mixed viral and bacterial pneumonia (Fig. 4) .", "The in vitro model of influenza and pneumococcal superinfection of human MDMs demonstrated that mixed infection synergistically induced release of the pro-inflammatory chemokine IP-10, strongly suggesting human Scientific RepoRts | 6:38532 | DOI: 10.1038/srep38532 blood leukocytes contribute to the immunopathology of pneumonia. Additionally, transcriptomics and omics analyses provided new data on the inflammatory pathways that are activated during mixed infection and related to synergistic induction of the pro-inflammatory chemokine IP-10 in mixed infected cells. Our observations are consistent with a recent study describing IP-10 induction as host-proteome signature of both viral and bacterial infections 30 . Of the differentially-expressed genes observed in mixed infected MDMs, the transcription factors STAT-1 and IRF-7 appear to play crucial roles in the regulation of interferon-stimulated genes including CXCL10 (IP-10). By focusing on the intracellular mechanisms that regulate", "inflammatory pathways, we demonstrated a novel role for miRNA-200a-3p in the regulation of CXCL10 (IP-10). These observations are consistent with previous reports showing that RNA virus infection upregulates miR-155 in macrophages and dendritic cells and also regulates suppressor of cytokine signaling 1 (SOCS1), suggesting the existence of a miRNA/JAK-STAT/SOCS regulatory pathway during viral infection 29 . Our study suggests co-infection leads to overexpression of miR-200a-3p, which in turn targets and downregulates the JAK-STAT regulator SOCS-6 and consequently increases CXCL10 (IP-10) expression. Interestingly, a complementary in-silico approach reveals that several microRNAs that were found dysregulated in our experiments of IAV and SP co-infection of MDMs or HAEC, might target several genes of SOCS family and play similar role than miR-200a-3p. Indeed, miRNA-142-3p might target SOCS4, 5, 6 mRNA while miRNA-194-5p might target SOCS2, 3, 4, 5 and 7 mRNA. These observations", "underline that intra-cellular regulation of IP-10 is not limited to the contribution of a sole microRNA. A complex inter-relationship between numerous host microRNAs and inhibitors of the JAK-STAT signaling pathway occur to control host innate inflammatory response against viral and/or bacterial infections. Clinically, the majority of pediatric CAP cases in this study were associated with both positive viral and/or bacterial detection. Respiratory microorganisms were detected in 97% of cases; 51.3% of which were viral-viral, viral-bacterial or bacterial-bacterial co-detected only in nasal aspirates, 32.4% of which co-detected in both nasal aspirates and blood samples. These data are consistent with previous etiological studies of pediatric CAP 3,31-33 . S. pneumoniae was the major bacteria identified in blood (19/74; 25.7%) and mainly co-detected with respiratory viruses in NAs (16/19; 84.2%). We observed a very high diversity of viral and bacterial associations in biological samples", "from children with pneumonia. In comparison with IAV and SP14 combination evaluated in-vitro, no pneumonia cases were singly influenza and pneumococcus infected, and no similar co-detection with those two pathogens has been clinically observed. Nevertheless, Influenza B (IVB) virus was identified in 5 patients and two of them had a positive SP co-detection in blood (one non-typable strain and one serotype 14 using our molecular typing test). IVB and SP14 combination seems to be the nearest pathogen co-detection to that in-vitro investigated. Clinically, this co-detection was associated with both a very high IP-10 expression and a very severe pneumonia case definition. Interestingly, our translational pilot evaluation reveals IP-10 expression can be induced by several different viral and/or bacterial combinations. As immune response to each pathogen is different, further in-vitro investigations using different pathogens associations are needed to better characterize the mechanisms", "involved in the immunopathology of pneumonia.", "In this cohort, highest serum IP-10 levels were identified among patients with both several pathogen detected and severe pneumonia, suggesting a significant role of IP-10 on pneumonia pathogenesis. Indeed, high plasma levels of IP-10 have previously been reported in patients with sepsis 12 , and were associated with high mortality rate, especially among patients with CAP 34 . Additionally, the IP-10-CXCR3 axis has been related to acute immune lung injury and lymphocyte apoptosis during the development of severe acute respiratory syndrome (SARS) 35, 36 . Moreover, an in vivo study that modeled influenza and pneumococcal superinfection in mice indicated that pro-inflammatory chemokines, including IP-10, play a crucial role in influenza-induced susceptibility to lung neutrophilia, severe immunopathology and mortality 37 . In this study, markedly elevated IP-10 (92,809 pg ml −1 ) combined with the highest PCT level (74.4 pg ml −1 ) were quantified in the serum sample of a child who died,", "in whom S. pneumoniae (serotype 9 V) was identified in the blood (PCR and blood culture) and co-detected with Haemophilus influenzae type B in nasal aspirate. These observations suggest an interrelationship between co-detection, elevated serum IP-10 and the pathogenesis of pneumonia.", "Several limitations of this pilot translational study need to be acknowledged before concluding mixed infection is related to elevated IP-10 and disease severity. Indeed, although viral shedding (e.g., of HRV and HBoV) is common in asymptomatic children, we were unable to evaluate the levels of immunomodulators in the serum samples of a control group. Moreover, although the samples were collected within the first 24 hours after admission, only a single blood sample was processed for each patient. Therefore, a larger, longitudinal study on the etiology and severity of pneumonia will be necessary to confirm these results. In conclusion, the present findings suggest that mixed respiratory infections and IP-10 may play major, interconnected roles in the pathogenesis of pneumonia. Clinically, assessment and monitoring of induced IP-10 serum level may assist clinicians to improve diagnosis and patient management of severe community-acquired pneumonia.", "Viral and bacterial strains. The 10 ng ml −1 M-CSF (Miltenyi Biotec). THP− 1 MDMs were obtained by culturing cells with 10 ng ml -1 phorbol myristate acetate (PMA; Invivogen, Toulouse, France) for 72 hours. Human airway epithelial cells (HAEC, bronchial cell type) originated from a 54-years old woman with no pathology reported (batch number MD056501) were provided by Mucilair (Epithelix, Geneva, Switzerland). Sterility, tissue integrity (TEER), mucus production and cilia beating frequency have been certified by the company. Gene expression profiling. Total cellular mRNA was purified using the RNeasy kit (Qiagen, Hilden, Germany). Reverse-transcription of total mRNA was performed using the RT 2 First Strand Kit (SABiosciences, Hilden, Germany). The expression of 84 genes involved in the human innate and adaptive immune responses was evaluated using the RT 2 profiler ™ PCR Array (SABiosciences) according to the manufacturer's recommendations. The Δ Δ Ct method was applied to calculate", "the fold changes in gene expression for each gene relative to uninfected control cells using the web-based RT 2 profiler PCR Array Data Analysis software (SABiosciences).", "MicroRNA profiling array. Total cellular microRNAs were purified using the miRNeasy Mini kit (Qiagen) and reverse-transcribed using the miScript Reverse Transcription kit (Qiagen). The profiling of 84 miRNAs was performed using the Human Immunopathology miScript miRNA PCR Array kit (Qiagen) according to the manufacturer's instructions. Data were analyzed using the miScript miRNA PCR array data analysis web portal.\n\nIn silico miRNA target prediction. MiRNA target genes were retrieved and compiled using TargetScan 38 and microRNA.org resource 39 . The interactions between miRNAs and intracellular pathways were predicted using DIANA-miRPath v2.0 40 .", "THP-1 MDMs were seeded in 24-well plates (0.5 × 10 6 per well) in triplicate, exposed to Influenza A H1N1 (A/Solomon islands/3/2006) virus (IAV) under serum-free conditions for 1 hour and then cultured for 3 hours in fresh RPMI-1640 containing 2% FBS. Streptococcus pneumoniae (SP) serotype 14 was added at 4 hours after IAV infection. Gentamicin (10 μ g ml −1 ) was added 2 hours after SP infection (i.e. 6 hours post-influenza infection) and maintained in the culture media throughout the experiment to kill extracellular bacteria and limit bacterial growth. Cell viability was determined by flow-cytometry using the FITC/Annexin V apoptosis detection kit (BD Biosciences), according to the manufacturer's instructions. #4427975) . In this assay, fold changes have been defined by the Δ Δ Ct method using control RNU-44 and -48 as reference microRNAs. Total mRNA was purified from transfected and infected MDMs using the RNeasy kit (Qiagen) and specific primers were used to amplify transforming", "growth factor beta-2 (TGFB2; F: 5′ -CCATCCCGCCCACTTTCTAC-3′ , R: 5′ -AGCTCAATCCGTTGTTCAGGC-3′ ), SOCS6 (F: 5′ -AAGAATTCATCCCTTGGATTAGGTAAC-3′ , R: 5′ -CAGACTGGAGGTCGTGGAA-3′ ) 41 43 , and 3) absence of wheezing at auscultation, and, 4) first symptoms appearing within the last 14 days, and 5) radiological confirmation of pneumonia as per WHO guidelines 44 . Based on these primary criteria defining pneumonia cases, all 74 cases were retrospectively re-evaluated according to the WHO \"Pocket book of hospital care for children\" 45 criteria to evaluate pneumonia severity. Cases that died during the study, or who had at least one additional clinical signs including central cyanosis, dullness to percussion during chest examination, prostration/lethargy, pleural effusion observed on chest radiography were retrospectively included in the severe pneumonia group. Patients without any of these additional clinical signs were included in the non-severe pneumonia group. Table 4 . a IP-10 values are", "expressed in pg ml -1 . IP-10 concentration differences between groups were compared using unpaired Mann-Whitney tests; significant changes (P < 0.05) are in bold. Clinical and molecular analysis. Nasopharyngeal aspirates (NAs) and whole blood samples were collected from children within 24 hours of admission. Whole blood samples were used for complete blood counts, blood culture and multiplex real-time PCR to identify Staphylococcus aureus, Streptococcus pneumoniae and Haemophilus influenzae type B 46 . S. pneumoniae serotypes were defined using a 11 multiplex real-time PCR assay targeting the 40 most frequently represented serotypes or serogroups according to protocol developed by Messaoudi et al. 47 . Serum C-reactive protein (CRP; AssayPro, St. Charles, Missouri, United States) and Procalcitonin (PCT; VIDAS B.R.A.H.M.S; bioMérieux) were quantified from whole-blood samples. Multiplex real-time non quantitative PCR (Fast-Track Diagnostic, Sliema, Malta) was used to detect 19 viruses", "and five bacteria in the respiratory specimens (NAs and pleural effusions). Mixed detection was defined as 1) PCR-positive for multiple viruses in NAs, 2) positive blood culture or PCR-positive for multiple bacteria in blood or 3) PCR-positive for one or multiple viruses in NAs and one or multiple bacteria in blood (identified by PCR and blood culture).", "Ethical approval. The study protocol, informed consent statement, clinical research form, any amendments and all other study documents were submitted to and approved by the Ethical Committee of the Instituto de Investigaciones en Ciencias de la Salud, the Universidad Nacional de Asunción (IICS-UNA) and the Hospital Pediátrico Niños de Acosta Ñu. Informed consent was obtained from all subjects involved in this study. The clinical investigation was conducted according to the principles expressed in the Declaration of Helsinki.", "Statistical analysis. The Chi-square test and Fisher's exact test were used to compare categorical variables; continuous variables and non-normally distributed data were compared using the Mann-Whitney U-test; normally distributed data were compared using unpaired t-tests. Comparative analyses between experimental conditions (i.e., MOCK, IAV, SP or IAV + SP) were performed using one-way ANOVA with Tukey's post-hoc test or Kruskal-Wallis analysis with Dunn's post-hoc tests. Receiver operating curve (ROC) analysis was used to determine the optimal cut-off value for IP-10 to differentiate between non-severe and severe pneumonia cases. P < 0.05 was considered statistically significant. All statistical analyses were performed using GraphPad Prism (La Jolla, California, United States)." ]

[ 4 ]

[ "Methicillin-resistant Staphylococcus aureus (MRSA)" ]

[ "In Vitro Bactericidal Activity of 4- and 5-Chloro-2-hydroxy-N-[1-oxo-1-(phenylamino)alkan-2-yl]benzamides against MRSA\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4321674/\n\nSHA: f0e6cef57dbae030aea2f324e21e00945ac659cf\n\nAuthors: Zadrazilova, Iveta; Pospisilova, Sarka; Pauk, Karel; Imramovsky, Ales; Vinsova, Jarmila; Cizek, Alois; Jampilek, Josef\nDate: 2015-01-15\nDOI: 10.1155/2015/349534\nLicense: cc-by", "Abstract: A series of nine substituted 2-hydroxy-N-[1-oxo-1-(phenylamino)alkan-2-yl]benzamides was assessed as prospective bactericidal agents against three clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and S. aureus ATCC 29213 as the reference and quality control strain. The minimum bactericidal concentration was determined by subculturing aliquots from MIC determination onto substance-free agar plates. The bactericidal kinetics of compounds 5-chloro-2-hydroxy-N-[(2S)-3-methyl-1-oxo-1-{[4-(trifluoromethyl)phenyl]amino}butan-2-yl]benzamide (1f), N-{(2S)-1-[(4-bromophenyl)amino]-3-methyl-1-oxobutan-2-yl}-4-chloro-2-hydroxybenzamide (1g), and 4-chloro-N-{(2S)-1-[(3,4-dichlorophenyl)amino]-3-methyl-1-oxobutan-2-yl}-2-hydroxybenzamide (1h) was established by time-kill assay with a final concentration of the compound equal to 1x, 2x, and 4x MIC; aliquots were removed at 0, 4, 6, 8, and 24 h time points. The most potent bactericidal agent was compound 1f exhibiting", "remarkable rapid concentration-dependent bactericidal effect even at 2x MIC at 4, 6, and 8 h (with a reduction in bacterial count ranging from 3.08 to 3.75 log(10) CFU/mL) and at 4x MIC at 4, 6, 8, and 24 h (5.30 log(10) CFU/mL reduction in bacterial count) after incubation against MRSA 63718. Reliable bactericidal effect against other strains was maintained at 4x MIC at 24 h.", "Text: The antibiotic resistance of invasive pathogens has become one of the most challenging and persistent health problems [1] . Methicillin-resistant Staphylococcus aureus (MRSA) has become the most common clinically relevant multiresistant pathogen [2] causing both healthcare-associated and community-acquired bloodstream infections with mortality rates up to 40% [3] .\n\nThe prevalence of MRSA is increasing worldwide and, according to the latest information of the European Centre for Disease Prevention and Control from 2012 [4] , can be considered alarming in some European countries, especially in Portugal and Romania, where ≥50% of all S. aureus isolates from invasive infections were identified as MRSA in 2012 (although, e.g., in Romania the prevalence of MRSA was 25-50% in 2010), followed by Italy, Greece, and Poland with 25-50% isolates being MRSA in 2012 (for comparison, in Poland MRSA isolates constituted 10-25% from all S. aureus isolates in 2010).", "The treatment failure of vancomycin, the therapeutic anti-MRSA agent of choice, due to the strains with elevated vancomycin minimum inhibitory concentration (MIC) values (i.e., the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism) within the susceptible range was described previously [5, 6] . Thus, the emergence of MRSA (and vancomycin-resistant S. aureus in the recent years as well [7] ) makes the discovery of new molecular scaffolds a priority, and the current situation even necessitates the reengineering and repositioning of some old drug families to achieve adequate control of these bacteria [8] . However, for the treatment of S. aureus bloodstream infections, bactericidal antimicrobial agents are considered to be superior to bacteriostatic drugs [9] . This fact should be considered during the development of effective and safe treatment options for MRSA infections.", "The history of clinical usage of salicylanilides (2-hydroxy-N-phenylbenzamides) dates back to the 1940s in therapy of tinea capitis, followed by the discovery of their anthelmintic properties in the mid 1950s [10] . Nowadays, salicylanilides (SALs) are a class of aromatic compounds possessing a wide range of interesting pharmacological activities, such as anthelmintic [11] , antibacterial [12, 13] , antimycobacterial [13] , antifungal [14] , and antiviral [15, 16] , among others. Despite being studied since the 1960s, the mechanism of action responsible for biological activities of these compounds has not been explained so far. SALs have been found to inhibit the two-component regulatory systems (TCS) of bacteria [17] . The latest studies specified them also as selective inhibitors of interleukin-12p40 production that plays a specific role in initiation, expansion, and control of cellular response to tuberculosis [18] . Furthermore, salicylanilides have been recognised as inhibitors", "of some bacterial enzymes, such as sortase A from S. aureus [19] , d-alanine-d-alanine ligase [20] , or transglycosylases from S. aureus (but not from M. tuberculosis) [12] . These enzymes participate in secretion of various proteins or in biosynthesis of bacterial cell wall. Recently, salicylanilides-like derivatives were described to inhibit two enzymes essential for mycobacteria: (i) methionine aminopeptidase, catalyzing a key step of the posttranslational modification of nascent proteins, and (ii) isocitrate lyase, which is essential for the metabolism of fatty acids [21] . Thus, SALs seem to be promising candidates for development of new antibacterial agents with a novel mechanism of action. Such new agents could be a solution to the resistance challenges.", "This study is a follow-up paper to a recently published article [13] . The synthesis of the series of novel derivatives of salicylamides, 4-and 5-chloro-2-hydroxy-N-[1-oxo-1-(phenylamino)alkan-2-yl]benzamides, called diamides due to their skeleton (for general structure see Table 1 ), was described previously [13, 22] , and their antimycobacterial and antibacterial activities against various bacterial species were reported [13] . As these compounds expressed very significant antibacterial activity with low MIC values against clinical isolates of MRSA as representatives of multidrugresistant bacteria, we decided to extend the knowledge about the antibacterial properties of these compounds against MRSA.", "The aim of the current study was to assess the overall in vitro bactericidal activity of nine newly synthesized diamides in dependence on time and concentration against clinical isolates of MRSA as representatives of multidrug-resistant bacteria. To the best of our knowledge, this is the first study dealing with the evaluation of novel microbiological characteristics of SAL analogues and revealing their bactericidal effect.", "The synthetic pathway of the series of novel diamides was described recently [13, 22] , and their structures (see Table 1 ) were confirmed by IR, NMR, and MS spectrometry, and the purity of the compounds was checked by CHN analysis [13, 22] . [27] ; and MRSA SA 3202 [27] (National Institute of Public Health, Prague, Czech Republic) both of human origin. Suspected colonies were confirmed by PCR; a 108 bp fragment specific for S. aureus was detected [28] . All isolates were tested for the presence of the mecA gene encoding methicillin resistance [29] . These three clinical isolates were classified as vancomycin-susceptible (but with higher MIC of vancomycin equal to 2 g/mL (VA2-MRSA) within the susceptible range for MRSA 63718) methicillinresistant S. aureus (VS-MRSA). For the MICs of vancomycin, see Table 1 . Vancomycin-susceptible methicillin-susceptible Staphylococcus aureus (VS-MSSA) ATCC 29213, obtained from the American Type Culture Collection, was used as the reference and", "quality control strain. The bacteria were stored at −80 ∘ C and were kept on blood agar plates (Columbia agar base with 5% ovine blood) between experiments. (MBCs) . The MBCs (i.e., the lowest concentrations of antibacterial agents required to kill a particular bacterium) were determined by subculturing aliquots (20 L) from wells with no visible bacterial growth and from control wells of MIC determination onto substance-free Mueller-Hinton agar (MHA) plates. The plates were incubated aerobically at 37 ∘ C for 24 h for colony count. The MBC was defined as the lowest concentration of substance, which produced ≥99.9% killing Table 1 : Chemical structures and in vitro MIC and MBC [ g/mL] values of tested 5-and 4-chloro-2-hydroxy-N-[1-oxo-1-(phenylamino)alkan-2-yl]benzamides (bactericidal effect of individual compounds against particular strains marked in bold). after 24 h of incubation as compared to the colony count of the starting inoculum [30] . To ensure reproducibility, each MBC", "assay was performed in at least triplicate on separate occasions.", "N H O H N O OH 1 2 R 1 R 3 R 2 Comp. R 1 R 2 R 3 MIC [ g/mL] MBC [ g/mL] 1 2 3 4 1 2 3 4 1a 5-Cl 4-CH 3 (S)-CH 3 >256 >256 >256 >256 >256 >256 >256 >256 1b 5-Cl 4-CH 3 (S)-CH(CH 3 ) 2 >256 >256 32 32 >256 >256 128 >256 1c 5-Cl 4-CH 3 (S)-benzyl >256 >256 >256 >256 >256 >256 >256 >256 1d 5-Cl 4-CH 3 (R)-CH 2 -indolyl >256 >256 >256 >256 >256 >256 >256 >256 1e 5-Cl 4-OCH 3 (S)-CH(CH 3 ) 2 >256 >256 >256 >256 >256 >256 >256 >256 1f 5-Cl 4-CF 3 (S)-CH(CH 3 ) 2 4 2 2 2 4 4 8 4 1g 4-Cl 4-Br (S)-CH(CH 3 ) 2 8 4 4 4 1 6 8 8 8 1h 4-Cl 3,4-Cl (S)-CH(CH 3 ) 2 2 1 1 1 4 1 4 2 1i 4-Cl 3,4-Cl (S)-benzyl 1 1 0.5 0.5 8 1 8 1 AMP - - - >16 >16 >16 0.25 >16 >16 >16 0.25 CPX - - - >16 >16 >16 0.5 >16 >16 >16 0.5 VAN - - - 2 1 1 1 2 1 1 1", "Time-kill assays were performed by the broth macrodilution method according to previously described methodology [30] with some modifications. Briefly, flasks containing sterile fresh Mueller-Hinton broth (MHB) with the appropriate antimicrobial agent were inoculated with the test organism in logarithmic growth phase to obtain the starting inoculum with the concentration of approximately 7.5 × 10 6 CFU/mL (actual inoculum concentrations ranged from 0.9 × 10 5 to 2.9 × 10 6 CFU/mL) and a final concentration of the antibiotic equal to 1x, 2x, and 4x MIC in 10 mL volume. For the determination of viable counts, aliquots were removed at 0, 4, 6, 8, and 24 h time points after inoculation, serially diluted in sterile phosphate buffered saline, and aliquots (20 L) were plated on MHA plates in duplicate. Colony counts were performed on plates yielding 6 to 60 colonies, and the mean was calculated. Antimicrobial carry-over was controlled by dilution and visual inspection of the distribution of", "colonies on the plates with observation of possible inhibition of growth at the site of the initial streaks. The plates were incubated at 37 ∘ C for 24 to 48 h, and the number of colonies was determined. To ensure reproducibility, each time-kill experiment was carried out in duplicate on separate occasions with results presented as the mean of all experiments. The growth control without the addition of antimicrobial agents and the control containing DMSO without any antimicrobial agent to exclude antibacterial activity of this solvent were included. Time-kill curves were constructed by plotting the log 10 CFU per millilitre versus time (over 24 h), and the change in bacterial concentration was determined. The results were analysed by evaluating the numbers of strains that yielded Δ(log 10 CFU/mL) values of −1 (corresponding to 90% killing), −2 (99% killing), and −3 (99.9% killing) at 4, 6, 8, and 24 h compared to counts at 0 h. Bactericidal activity was defined as a reduction of at", "least 99.9% (≥3 log 10 ) of the total count of CFU/mL in the original inoculum.", "Diamides seem to be promising candidates for antibacterial agents with very strong anti-MRSA activity, as it was published recently [13] . In the present study the series of nine newly synthesized diamides was evaluated as prospective bactericidal agents against representatives of multidrugresistant bacteria, three clinical isolates of MRSA, and Staphylococcus aureus ATCC 29213 (methicillin-susceptible) as the reference and quality control strain. Since SALs and their analogues are known as compounds with bacteriostatic effect [31] , this is the first study where SAL-like compounds were considered as prospective bactericidal agents and the dependence of bactericidal effect of these compounds on time and concentration was evaluated. Thus, absolutely novel microbiological characteristics of these compounds were revealed in the present study.", "Recently MIC values of diamides expressed as molar concentrations in mol/L were published [13] . To allow comparison with MBC values of the present study, MICs in g/mL were calculated and are recorded in Table 1 along with the activity of reference antibacterial drugs, ampicillin, ciprofloxacin, and vancomycin. Potential bactericidal activity of diamides was assessed using MBC assay [26] . MBC values of all tested compounds are recorded in Table 1 as well.\n\nBased on the obtained results, all compounds assessed as active according to MIC values in our previous study (1f-i) showed low or moderate MBC values against all four strains. The MBC values of these compounds did not exceed the highest tested drug concentration and ranged from 1 to 16 g/mL. In all cases, there were comparable MBC values for the clinical isolates of MRSA and the S. aureus reference strain.\n\nBactericidal activity is defined as a ratio of MBC to MIC of ≤4 [32] . Table 1 bactericidal activity is expressed in bold.", "As mentioned above, SALs are known to exhibit a bacteriostatic effect [31] , so it was very interesting to discover that diamides possess bactericidal activity. The amide bond (-CONH-) can cause interactions with a variety of enzymes [33] ; therefore the presence of two amide bonds could be responsible for the bactericidal effect of diamides against MRSA. The activity of SALs and their analogues results from multiple mechanisms, which are still under investigation; for example, it was found that SALs are capable of inhibiting transglycosylases in later stages of S. aureus (including MRSA) cell wall biosynthesis [12] . These enzymes catalyse the step prior to the transpeptidation in the peptidoglycan biosynthesis and are responsible for polymerization of lipid II, which occurs at the outer face of the membrane [12] . Since antibacterial agents targeting cell wall biosynthesis act as bactericidal agents [30, 34] , the failure in the cell wall biosynthesis due to the inhibition of", "transglycosylases could be responsible for bactericidal activity of diamides against MRSA.", "Based on these findings, antibacterial active diamides with bactericidal effect against all four tested strains as prospective bactericidal agents were chosen for subsequent timekill curve studies to determine the real dependence of bactericidal effect on concentration over time.", "1-oxobutan-2-yl}-2-hydroxybenzamide (1h) were tested in time-kill studies at 1x, 2x, and 4x MIC against all MRSA isolates and the S. aureus reference strain. The antibacterial effect of DMSO [35] used as the solvent of the tested compounds was excluded in this assay, as time-kill curves of this solvent were identical or very similar to those of the growth control. The extent of bacterial killing was estimated by the number of these strains showing a decrease ranging from 1 to 3 log 10 CFU/mL in viable cell count at different times after incubation. A summary of these data is presented in Table 2 . Based on these data it can be concluded that the bactericidal potency of tested diamides against all four strains decreased as follows: 1f > 1h > 1g. No bactericidal activity (i.e., ≥3 log 10 CFU/mL decrease) was observed at 1x MIC for any strain and time after incubation tested. At 4x MIC from the four strains, compounds 1f, 1 g, and 1h killed 2, 1, and 2 strains, respectively, at 8 h after", "incubation and 4, 2, and 2 strains, respectively, at 24 h after incubation.", "The findings of time-kill studies for each of the four staphylococci strains at exposure to compounds 1f, 1g, and 1h are summarized in Table 3 . Bactericidal activity (i.e., ≥3 log 10 CFU/mL decrease) is expressed in bold.", "For compound 1f rapid concentration-dependent antibacterial effect was recorded against clinical isolate of MRSA 63718. Time was not the predictive factor influencing the antibacterial activity because log 10 differences in CFU/mL from the starting inoculum were the same for 4x MIC (with the highest efficiency with a reduction in bacterial count of 5.30 log 10 CFU/mL) or very similar for 2x MIC (with a moderate regrowth after 24 h causing a loss of bactericidal activity) over 24 h. The bactericidal effect was maintained even at 2x MIC at 4 h after incubation for this strain (reduction of 3.08 log 10 CFU/mL). For the remaining strains, clinical isolates of MRSA SA 630, MRSA SA 3202, and S. aureus ATCC 29213, reliable bactericidal effect was recorded at 4x MIC at 24 h after incubation for all these strains with a reduction in bacterial count of 3.22, 3.30, and 3.65 log 10 CFU/mL, respectively.", "For compound 1g bactericidal effect against MRSA 63718 was noticed at 2x MIC at 6 and 8 h after incubation and at 4x MIC at 4, 6, and 8 h after incubation with a reduction in bacterial count ranging from 3.10 to 3.58 log 10 CFU/mL. The most effective killing was achieved at 6 h for both concentrations. As in the case of compound 1f, a regrowth was observed after 24 h after incubation. For the remaining isolates of MRSA, SA 630 and SA 3202, bactericidal effect occurred only at 4x MIC at 24 h after incubation with a reduction in bacterial count of 3.38 and 4.01 log 10 CFU/mL, respectively. The highest bactericidal effect was recorded for MRSA SA 3202 at 4x MIC at 24 h after incubation. A reduction consistent with bacteriostatic effect (0.03 to 2.37 log 10 CFU/mL) was observed at other concentrations over time for both isolates. No bactericidal effect was observed for the S. aureus reference strain; compound 1g demonstrated a pattern of bacteriostatic activity against this strain with a", "reduction in bacterial count ranging from 0.07 to 2.33 log 10 CFU/mL at 4x MIC over time. In other cases, a slight increase in bacterial counts (i.e., overgrowth) compared with the starting inoculum was observed with values ranging from 0.10 to 1.57 log 10 CFU/mL for this reference strain.", "For compound 1h bactericidal effect against MRSA 63718 was maintained at 4x MIC at 6 and 8 h after incubation with a reduction in bacterial count of 3.54 and 3.31 log 10 CFU/mL, respectively. The same as for 1g, the most potent bactericidal effect was maintained at 6 h after incubation. Regrowth at 24 h after incubation causing a loss of bactericidal activity was recorded similarly as with previous compounds. The reason for regrowth of the test organism at 24 h in the experiment is unknown. Most probably, selection of resistant mutants is responsible for this phenomenon [30] ; degradation of the drug in the growth medium is not assumed, as regrowth was", "Number of strains showing the following log 10 CFU/mL decrease a at the designated incubation time not observed for any other tested strain. For MRSA SA 630 concentration-dependent killing was recorded at 4x MIC at 6, 8, and 24 h after incubation with log 10 differences in CFU/mL from the starting inoculum being very similar over time (ranging from 3.18 to 3.39 log 10 CFU/mL). For MRSA SA 3202 reliable bactericidal effect was maintained only at 4x MIC at 24 h after incubation with a reduction in bacterial count of 3.02 log 10 CFU/mL. As for compound 1g, bacteriostatic activity against S. aureus reference strain was observed with a reduction in bacterial count ranging from 0.34 to 2.62 log 10 CFU/mL at 2x and 4x MIC. Overgrowth (values ranging from 0.04 to 1.43 log 10 CFU/mL) was recorded at 1x MIC for this strain. It is of note that in all staphylococci strains with similar MICs and MBCs for compounds 1g and 1h the responsiveness to antibacterial activity of these compounds varied", "with clinical strains of MRSA being effectively killed and the reference strain remaining unaffected at 4x MIC.", "There is a discrepancy between bactericidal results of MBC assay compared with time-kill kinetics. This difference could be caused by comparing microtiter (MBC assay) to macrobroth (time-kill assay) dilutions [36] . Moreover, although time-kill assays are more labour intensive and time consuming than MBC assays, they are recognised to provide a greater degree of characterisation of the cell eradication potential of antibacterial agents [37] .", "Concerning antibacterial effect, it is not generally important if the antibacterial agent is also bactericidal at higher concentrations, because the inhibition of bacterial proliferation usually achieves a therapeutic effect; the patient's immune system is capable of coping with the infection then [34] . However, bactericidal therapy could produce a better treatment result by rapid reduction of the bacterial load [38] . Moreover, in the case of an immune system disorder (e.g., immunosuppressive therapy, AIDS patients, etc.) bactericidal agents are unequivocally indicated. Considering steadily escalating numbers of immunocompromised patients with endocarditis, meningitis, or osteomyelitis in recent years, it is necessary to achieve bacterial killing and broaden the spectrum of antimicrobial agents with bactericidal active compounds [30] .", "The clinical outcome of MRSA bacteraemia is significantly influenced by vancomycin MIC. Treatment failure exceeding 60% for S. aureus with vancomycin MIC of 4 g/mL resulted in the change of susceptibility breakpoint from 4 g/mL to 2 g/mL by the Clinical and Laboratory Standards Institute (CLSI) in 2006 [23] as well as by the US Food and Drug Administration (FDA) in 2008 [39] . It has been recommended that for infections caused by MRSA strains with elevated vancomycin MICs (2 g/mL), alternative therapy should be considered [40] . It is of note that based on time-kill assays in the present study, all tested diamides (particularly compound 1f exhibiting rapid bactericidal concentration-dependent effect even at 2x MIC) were most effective against isolate MRSA 63718, which is the strain with elevated vancomycin MIC of 2 g/mL. The activity against the remaining isolates with vancomycin MIC of 1 g/mL was lower.", "Considering the emergence of decreasing vancomycin susceptibility of MRSA isolates and thus the therapeutic efficacy of vancomycin therapy, our aim was to determine the potential bactericidal role of novel antibacterial compounds against MRSA in vitro. Based on the obtained results, diamides can be suitable candidates for such novel bactericidal active compounds presenting a promising starting point for further investigations to ascertain real in vivo activity and the exact mechanism of action. \n\nThe present study is the first evidence of bactericidal effect of SAL analogues. Against other strains, reliable bactericidal effect was maintained at 4x MIC at 24 h after incubation. Considering the necessity to broaden the spectrum of bactericidal agents, diamides from the current study with a novel mechanism of action could present a very promising and interesting solution to this challenge for the future." ]

[ 3 ]

[ "vancomycin" ]

[ "In Vitro Bactericidal Activity of 4- and 5-Chloro-2-hydroxy-N-[1-oxo-1-(phenylamino)alkan-2-yl]benzamides against MRSA\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4321674/\n\nSHA: f0e6cef57dbae030aea2f324e21e00945ac659cf\n\nAuthors: Zadrazilova, Iveta; Pospisilova, Sarka; Pauk, Karel; Imramovsky, Ales; Vinsova, Jarmila; Cizek, Alois; Jampilek, Josef\nDate: 2015-01-15\nDOI: 10.1155/2015/349534\nLicense: cc-by", "Abstract: A series of nine substituted 2-hydroxy-N-[1-oxo-1-(phenylamino)alkan-2-yl]benzamides was assessed as prospective bactericidal agents against three clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and S. aureus ATCC 29213 as the reference and quality control strain. The minimum bactericidal concentration was determined by subculturing aliquots from MIC determination onto substance-free agar plates. The bactericidal kinetics of compounds 5-chloro-2-hydroxy-N-[(2S)-3-methyl-1-oxo-1-{[4-(trifluoromethyl)phenyl]amino}butan-2-yl]benzamide (1f), N-{(2S)-1-[(4-bromophenyl)amino]-3-methyl-1-oxobutan-2-yl}-4-chloro-2-hydroxybenzamide (1g), and 4-chloro-N-{(2S)-1-[(3,4-dichlorophenyl)amino]-3-methyl-1-oxobutan-2-yl}-2-hydroxybenzamide (1h) was established by time-kill assay with a final concentration of the compound equal to 1x, 2x, and 4x MIC; aliquots were removed at 0, 4, 6, 8, and 24 h time points. The most potent bactericidal agent was compound 1f exhibiting", "remarkable rapid concentration-dependent bactericidal effect even at 2x MIC at 4, 6, and 8 h (with a reduction in bacterial count ranging from 3.08 to 3.75 log(10) CFU/mL) and at 4x MIC at 4, 6, 8, and 24 h (5.30 log(10) CFU/mL reduction in bacterial count) after incubation against MRSA 63718. Reliable bactericidal effect against other strains was maintained at 4x MIC at 24 h.", "Text: The antibiotic resistance of invasive pathogens has become one of the most challenging and persistent health problems [1] . Methicillin-resistant Staphylococcus aureus (MRSA) has become the most common clinically relevant multiresistant pathogen [2] causing both healthcare-associated and community-acquired bloodstream infections with mortality rates up to 40% [3] .\n\nThe prevalence of MRSA is increasing worldwide and, according to the latest information of the European Centre for Disease Prevention and Control from 2012 [4] , can be considered alarming in some European countries, especially in Portugal and Romania, where ≥50% of all S. aureus isolates from invasive infections were identified as MRSA in 2012 (although, e.g., in Romania the prevalence of MRSA was 25-50% in 2010), followed by Italy, Greece, and Poland with 25-50% isolates being MRSA in 2012 (for comparison, in Poland MRSA isolates constituted 10-25% from all S. aureus isolates in 2010).", "The treatment failure of vancomycin, the therapeutic anti-MRSA agent of choice, due to the strains with elevated vancomycin minimum inhibitory concentration (MIC) values (i.e., the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism) within the susceptible range was described previously [5, 6] . Thus, the emergence of MRSA (and vancomycin-resistant S. aureus in the recent years as well [7] ) makes the discovery of new molecular scaffolds a priority, and the current situation even necessitates the reengineering and repositioning of some old drug families to achieve adequate control of these bacteria [8] . However, for the treatment of S. aureus bloodstream infections, bactericidal antimicrobial agents are considered to be superior to bacteriostatic drugs [9] . This fact should be considered during the development of effective and safe treatment options for MRSA infections.", "The history of clinical usage of salicylanilides (2-hydroxy-N-phenylbenzamides) dates back to the 1940s in therapy of tinea capitis, followed by the discovery of their anthelmintic properties in the mid 1950s [10] . Nowadays, salicylanilides (SALs) are a class of aromatic compounds possessing a wide range of interesting pharmacological activities, such as anthelmintic [11] , antibacterial [12, 13] , antimycobacterial [13] , antifungal [14] , and antiviral [15, 16] , among others. Despite being studied since the 1960s, the mechanism of action responsible for biological activities of these compounds has not been explained so far. SALs have been found to inhibit the two-component regulatory systems (TCS) of bacteria [17] . The latest studies specified them also as selective inhibitors of interleukin-12p40 production that plays a specific role in initiation, expansion, and control of cellular response to tuberculosis [18] . Furthermore, salicylanilides have been recognised as inhibitors", "of some bacterial enzymes, such as sortase A from S. aureus [19] , d-alanine-d-alanine ligase [20] , or transglycosylases from S. aureus (but not from M. tuberculosis) [12] . These enzymes participate in secretion of various proteins or in biosynthesis of bacterial cell wall. Recently, salicylanilides-like derivatives were described to inhibit two enzymes essential for mycobacteria: (i) methionine aminopeptidase, catalyzing a key step of the posttranslational modification of nascent proteins, and (ii) isocitrate lyase, which is essential for the metabolism of fatty acids [21] . Thus, SALs seem to be promising candidates for development of new antibacterial agents with a novel mechanism of action. Such new agents could be a solution to the resistance challenges.", "This study is a follow-up paper to a recently published article [13] . The synthesis of the series of novel derivatives of salicylamides, 4-and 5-chloro-2-hydroxy-N-[1-oxo-1-(phenylamino)alkan-2-yl]benzamides, called diamides due to their skeleton (for general structure see Table 1 ), was described previously [13, 22] , and their antimycobacterial and antibacterial activities against various bacterial species were reported [13] . As these compounds expressed very significant antibacterial activity with low MIC values against clinical isolates of MRSA as representatives of multidrugresistant bacteria, we decided to extend the knowledge about the antibacterial properties of these compounds against MRSA.", "The aim of the current study was to assess the overall in vitro bactericidal activity of nine newly synthesized diamides in dependence on time and concentration against clinical isolates of MRSA as representatives of multidrug-resistant bacteria. To the best of our knowledge, this is the first study dealing with the evaluation of novel microbiological characteristics of SAL analogues and revealing their bactericidal effect.", "The synthetic pathway of the series of novel diamides was described recently [13, 22] , and their structures (see Table 1 ) were confirmed by IR, NMR, and MS spectrometry, and the purity of the compounds was checked by CHN analysis [13, 22] . [27] ; and MRSA SA 3202 [27] (National Institute of Public Health, Prague, Czech Republic) both of human origin. Suspected colonies were confirmed by PCR; a 108 bp fragment specific for S. aureus was detected [28] . All isolates were tested for the presence of the mecA gene encoding methicillin resistance [29] . These three clinical isolates were classified as vancomycin-susceptible (but with higher MIC of vancomycin equal to 2 g/mL (VA2-MRSA) within the susceptible range for MRSA 63718) methicillinresistant S. aureus (VS-MRSA). For the MICs of vancomycin, see Table 1 . Vancomycin-susceptible methicillin-susceptible Staphylococcus aureus (VS-MSSA) ATCC 29213, obtained from the American Type Culture Collection, was used as the reference and", "quality control strain. The bacteria were stored at −80 ∘ C and were kept on blood agar plates (Columbia agar base with 5% ovine blood) between experiments. (MBCs) . The MBCs (i.e., the lowest concentrations of antibacterial agents required to kill a particular bacterium) were determined by subculturing aliquots (20 L) from wells with no visible bacterial growth and from control wells of MIC determination onto substance-free Mueller-Hinton agar (MHA) plates. The plates were incubated aerobically at 37 ∘ C for 24 h for colony count. The MBC was defined as the lowest concentration of substance, which produced ≥99.9% killing Table 1 : Chemical structures and in vitro MIC and MBC [ g/mL] values of tested 5-and 4-chloro-2-hydroxy-N-[1-oxo-1-(phenylamino)alkan-2-yl]benzamides (bactericidal effect of individual compounds against particular strains marked in bold). after 24 h of incubation as compared to the colony count of the starting inoculum [30] . To ensure reproducibility, each MBC", "assay was performed in at least triplicate on separate occasions.", "N H O H N O OH 1 2 R 1 R 3 R 2 Comp. R 1 R 2 R 3 MIC [ g/mL] MBC [ g/mL] 1 2 3 4 1 2 3 4 1a 5-Cl 4-CH 3 (S)-CH 3 >256 >256 >256 >256 >256 >256 >256 >256 1b 5-Cl 4-CH 3 (S)-CH(CH 3 ) 2 >256 >256 32 32 >256 >256 128 >256 1c 5-Cl 4-CH 3 (S)-benzyl >256 >256 >256 >256 >256 >256 >256 >256 1d 5-Cl 4-CH 3 (R)-CH 2 -indolyl >256 >256 >256 >256 >256 >256 >256 >256 1e 5-Cl 4-OCH 3 (S)-CH(CH 3 ) 2 >256 >256 >256 >256 >256 >256 >256 >256 1f 5-Cl 4-CF 3 (S)-CH(CH 3 ) 2 4 2 2 2 4 4 8 4 1g 4-Cl 4-Br (S)-CH(CH 3 ) 2 8 4 4 4 1 6 8 8 8 1h 4-Cl 3,4-Cl (S)-CH(CH 3 ) 2 2 1 1 1 4 1 4 2 1i 4-Cl 3,4-Cl (S)-benzyl 1 1 0.5 0.5 8 1 8 1 AMP - - - >16 >16 >16 0.25 >16 >16 >16 0.25 CPX - - - >16 >16 >16 0.5 >16 >16 >16 0.5 VAN - - - 2 1 1 1 2 1 1 1", "Time-kill assays were performed by the broth macrodilution method according to previously described methodology [30] with some modifications. Briefly, flasks containing sterile fresh Mueller-Hinton broth (MHB) with the appropriate antimicrobial agent were inoculated with the test organism in logarithmic growth phase to obtain the starting inoculum with the concentration of approximately 7.5 × 10 6 CFU/mL (actual inoculum concentrations ranged from 0.9 × 10 5 to 2.9 × 10 6 CFU/mL) and a final concentration of the antibiotic equal to 1x, 2x, and 4x MIC in 10 mL volume. For the determination of viable counts, aliquots were removed at 0, 4, 6, 8, and 24 h time points after inoculation, serially diluted in sterile phosphate buffered saline, and aliquots (20 L) were plated on MHA plates in duplicate. Colony counts were performed on plates yielding 6 to 60 colonies, and the mean was calculated. Antimicrobial carry-over was controlled by dilution and visual inspection of the distribution of", "colonies on the plates with observation of possible inhibition of growth at the site of the initial streaks. The plates were incubated at 37 ∘ C for 24 to 48 h, and the number of colonies was determined. To ensure reproducibility, each time-kill experiment was carried out in duplicate on separate occasions with results presented as the mean of all experiments. The growth control without the addition of antimicrobial agents and the control containing DMSO without any antimicrobial agent to exclude antibacterial activity of this solvent were included. Time-kill curves were constructed by plotting the log 10 CFU per millilitre versus time (over 24 h), and the change in bacterial concentration was determined. The results were analysed by evaluating the numbers of strains that yielded Δ(log 10 CFU/mL) values of −1 (corresponding to 90% killing), −2 (99% killing), and −3 (99.9% killing) at 4, 6, 8, and 24 h compared to counts at 0 h. Bactericidal activity was defined as a reduction of at", "least 99.9% (≥3 log 10 ) of the total count of CFU/mL in the original inoculum.", "Diamides seem to be promising candidates for antibacterial agents with very strong anti-MRSA activity, as it was published recently [13] . In the present study the series of nine newly synthesized diamides was evaluated as prospective bactericidal agents against representatives of multidrugresistant bacteria, three clinical isolates of MRSA, and Staphylococcus aureus ATCC 29213 (methicillin-susceptible) as the reference and quality control strain. Since SALs and their analogues are known as compounds with bacteriostatic effect [31] , this is the first study where SAL-like compounds were considered as prospective bactericidal agents and the dependence of bactericidal effect of these compounds on time and concentration was evaluated. Thus, absolutely novel microbiological characteristics of these compounds were revealed in the present study.", "Recently MIC values of diamides expressed as molar concentrations in mol/L were published [13] . To allow comparison with MBC values of the present study, MICs in g/mL were calculated and are recorded in Table 1 along with the activity of reference antibacterial drugs, ampicillin, ciprofloxacin, and vancomycin. Potential bactericidal activity of diamides was assessed using MBC assay [26] . MBC values of all tested compounds are recorded in Table 1 as well.\n\nBased on the obtained results, all compounds assessed as active according to MIC values in our previous study (1f-i) showed low or moderate MBC values against all four strains. The MBC values of these compounds did not exceed the highest tested drug concentration and ranged from 1 to 16 g/mL. In all cases, there were comparable MBC values for the clinical isolates of MRSA and the S. aureus reference strain.\n\nBactericidal activity is defined as a ratio of MBC to MIC of ≤4 [32] . Table 1 bactericidal activity is expressed in bold.", "As mentioned above, SALs are known to exhibit a bacteriostatic effect [31] , so it was very interesting to discover that diamides possess bactericidal activity. The amide bond (-CONH-) can cause interactions with a variety of enzymes [33] ; therefore the presence of two amide bonds could be responsible for the bactericidal effect of diamides against MRSA. The activity of SALs and their analogues results from multiple mechanisms, which are still under investigation; for example, it was found that SALs are capable of inhibiting transglycosylases in later stages of S. aureus (including MRSA) cell wall biosynthesis [12] . These enzymes catalyse the step prior to the transpeptidation in the peptidoglycan biosynthesis and are responsible for polymerization of lipid II, which occurs at the outer face of the membrane [12] . Since antibacterial agents targeting cell wall biosynthesis act as bactericidal agents [30, 34] , the failure in the cell wall biosynthesis due to the inhibition of", "transglycosylases could be responsible for bactericidal activity of diamides against MRSA.", "Based on these findings, antibacterial active diamides with bactericidal effect against all four tested strains as prospective bactericidal agents were chosen for subsequent timekill curve studies to determine the real dependence of bactericidal effect on concentration over time.", "1-oxobutan-2-yl}-2-hydroxybenzamide (1h) were tested in time-kill studies at 1x, 2x, and 4x MIC against all MRSA isolates and the S. aureus reference strain. The antibacterial effect of DMSO [35] used as the solvent of the tested compounds was excluded in this assay, as time-kill curves of this solvent were identical or very similar to those of the growth control. The extent of bacterial killing was estimated by the number of these strains showing a decrease ranging from 1 to 3 log 10 CFU/mL in viable cell count at different times after incubation. A summary of these data is presented in Table 2 . Based on these data it can be concluded that the bactericidal potency of tested diamides against all four strains decreased as follows: 1f > 1h > 1g. No bactericidal activity (i.e., ≥3 log 10 CFU/mL decrease) was observed at 1x MIC for any strain and time after incubation tested. At 4x MIC from the four strains, compounds 1f, 1 g, and 1h killed 2, 1, and 2 strains, respectively, at 8 h after", "incubation and 4, 2, and 2 strains, respectively, at 24 h after incubation.", "The findings of time-kill studies for each of the four staphylococci strains at exposure to compounds 1f, 1g, and 1h are summarized in Table 3 . Bactericidal activity (i.e., ≥3 log 10 CFU/mL decrease) is expressed in bold.", "For compound 1f rapid concentration-dependent antibacterial effect was recorded against clinical isolate of MRSA 63718. Time was not the predictive factor influencing the antibacterial activity because log 10 differences in CFU/mL from the starting inoculum were the same for 4x MIC (with the highest efficiency with a reduction in bacterial count of 5.30 log 10 CFU/mL) or very similar for 2x MIC (with a moderate regrowth after 24 h causing a loss of bactericidal activity) over 24 h. The bactericidal effect was maintained even at 2x MIC at 4 h after incubation for this strain (reduction of 3.08 log 10 CFU/mL). For the remaining strains, clinical isolates of MRSA SA 630, MRSA SA 3202, and S. aureus ATCC 29213, reliable bactericidal effect was recorded at 4x MIC at 24 h after incubation for all these strains with a reduction in bacterial count of 3.22, 3.30, and 3.65 log 10 CFU/mL, respectively.", "For compound 1g bactericidal effect against MRSA 63718 was noticed at 2x MIC at 6 and 8 h after incubation and at 4x MIC at 4, 6, and 8 h after incubation with a reduction in bacterial count ranging from 3.10 to 3.58 log 10 CFU/mL. The most effective killing was achieved at 6 h for both concentrations. As in the case of compound 1f, a regrowth was observed after 24 h after incubation. For the remaining isolates of MRSA, SA 630 and SA 3202, bactericidal effect occurred only at 4x MIC at 24 h after incubation with a reduction in bacterial count of 3.38 and 4.01 log 10 CFU/mL, respectively. The highest bactericidal effect was recorded for MRSA SA 3202 at 4x MIC at 24 h after incubation. A reduction consistent with bacteriostatic effect (0.03 to 2.37 log 10 CFU/mL) was observed at other concentrations over time for both isolates. No bactericidal effect was observed for the S. aureus reference strain; compound 1g demonstrated a pattern of bacteriostatic activity against this strain with a", "reduction in bacterial count ranging from 0.07 to 2.33 log 10 CFU/mL at 4x MIC over time. In other cases, a slight increase in bacterial counts (i.e., overgrowth) compared with the starting inoculum was observed with values ranging from 0.10 to 1.57 log 10 CFU/mL for this reference strain.", "For compound 1h bactericidal effect against MRSA 63718 was maintained at 4x MIC at 6 and 8 h after incubation with a reduction in bacterial count of 3.54 and 3.31 log 10 CFU/mL, respectively. The same as for 1g, the most potent bactericidal effect was maintained at 6 h after incubation. Regrowth at 24 h after incubation causing a loss of bactericidal activity was recorded similarly as with previous compounds. The reason for regrowth of the test organism at 24 h in the experiment is unknown. Most probably, selection of resistant mutants is responsible for this phenomenon [30] ; degradation of the drug in the growth medium is not assumed, as regrowth was", "Number of strains showing the following log 10 CFU/mL decrease a at the designated incubation time not observed for any other tested strain. For MRSA SA 630 concentration-dependent killing was recorded at 4x MIC at 6, 8, and 24 h after incubation with log 10 differences in CFU/mL from the starting inoculum being very similar over time (ranging from 3.18 to 3.39 log 10 CFU/mL). For MRSA SA 3202 reliable bactericidal effect was maintained only at 4x MIC at 24 h after incubation with a reduction in bacterial count of 3.02 log 10 CFU/mL. As for compound 1g, bacteriostatic activity against S. aureus reference strain was observed with a reduction in bacterial count ranging from 0.34 to 2.62 log 10 CFU/mL at 2x and 4x MIC. Overgrowth (values ranging from 0.04 to 1.43 log 10 CFU/mL) was recorded at 1x MIC for this strain. It is of note that in all staphylococci strains with similar MICs and MBCs for compounds 1g and 1h the responsiveness to antibacterial activity of these compounds varied", "with clinical strains of MRSA being effectively killed and the reference strain remaining unaffected at 4x MIC.", "There is a discrepancy between bactericidal results of MBC assay compared with time-kill kinetics. This difference could be caused by comparing microtiter (MBC assay) to macrobroth (time-kill assay) dilutions [36] . Moreover, although time-kill assays are more labour intensive and time consuming than MBC assays, they are recognised to provide a greater degree of characterisation of the cell eradication potential of antibacterial agents [37] .", "Concerning antibacterial effect, it is not generally important if the antibacterial agent is also bactericidal at higher concentrations, because the inhibition of bacterial proliferation usually achieves a therapeutic effect; the patient's immune system is capable of coping with the infection then [34] . However, bactericidal therapy could produce a better treatment result by rapid reduction of the bacterial load [38] . Moreover, in the case of an immune system disorder (e.g., immunosuppressive therapy, AIDS patients, etc.) bactericidal agents are unequivocally indicated. Considering steadily escalating numbers of immunocompromised patients with endocarditis, meningitis, or osteomyelitis in recent years, it is necessary to achieve bacterial killing and broaden the spectrum of antimicrobial agents with bactericidal active compounds [30] .", "The clinical outcome of MRSA bacteraemia is significantly influenced by vancomycin MIC. Treatment failure exceeding 60% for S. aureus with vancomycin MIC of 4 g/mL resulted in the change of susceptibility breakpoint from 4 g/mL to 2 g/mL by the Clinical and Laboratory Standards Institute (CLSI) in 2006 [23] as well as by the US Food and Drug Administration (FDA) in 2008 [39] . It has been recommended that for infections caused by MRSA strains with elevated vancomycin MICs (2 g/mL), alternative therapy should be considered [40] . It is of note that based on time-kill assays in the present study, all tested diamides (particularly compound 1f exhibiting rapid bactericidal concentration-dependent effect even at 2x MIC) were most effective against isolate MRSA 63718, which is the strain with elevated vancomycin MIC of 2 g/mL. The activity against the remaining isolates with vancomycin MIC of 1 g/mL was lower.", "Considering the emergence of decreasing vancomycin susceptibility of MRSA isolates and thus the therapeutic efficacy of vancomycin therapy, our aim was to determine the potential bactericidal role of novel antibacterial compounds against MRSA in vitro. Based on the obtained results, diamides can be suitable candidates for such novel bactericidal active compounds presenting a promising starting point for further investigations to ascertain real in vivo activity and the exact mechanism of action. \n\nThe present study is the first evidence of bactericidal effect of SAL analogues. Against other strains, reliable bactericidal effect was maintained at 4x MIC at 24 h after incubation. Considering the necessity to broaden the spectrum of bactericidal agents, diamides from the current study with a novel mechanism of action could present a very promising and interesting solution to this challenge for the future." ]

[ 4 ]

[ "isocitrate lyase" ]

[ "In Vitro Bactericidal Activity of 4- and 5-Chloro-2-hydroxy-N-[1-oxo-1-(phenylamino)alkan-2-yl]benzamides against MRSA\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4321674/\n\nSHA: f0e6cef57dbae030aea2f324e21e00945ac659cf\n\nAuthors: Zadrazilova, Iveta; Pospisilova, Sarka; Pauk, Karel; Imramovsky, Ales; Vinsova, Jarmila; Cizek, Alois; Jampilek, Josef\nDate: 2015-01-15\nDOI: 10.1155/2015/349534\nLicense: cc-by", "Abstract: A series of nine substituted 2-hydroxy-N-[1-oxo-1-(phenylamino)alkan-2-yl]benzamides was assessed as prospective bactericidal agents against three clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and S. aureus ATCC 29213 as the reference and quality control strain. The minimum bactericidal concentration was determined by subculturing aliquots from MIC determination onto substance-free agar plates. The bactericidal kinetics of compounds 5-chloro-2-hydroxy-N-[(2S)-3-methyl-1-oxo-1-{[4-(trifluoromethyl)phenyl]amino}butan-2-yl]benzamide (1f), N-{(2S)-1-[(4-bromophenyl)amino]-3-methyl-1-oxobutan-2-yl}-4-chloro-2-hydroxybenzamide (1g), and 4-chloro-N-{(2S)-1-[(3,4-dichlorophenyl)amino]-3-methyl-1-oxobutan-2-yl}-2-hydroxybenzamide (1h) was established by time-kill assay with a final concentration of the compound equal to 1x, 2x, and 4x MIC; aliquots were removed at 0, 4, 6, 8, and 24 h time points. The most potent bactericidal agent was compound 1f exhibiting", "remarkable rapid concentration-dependent bactericidal effect even at 2x MIC at 4, 6, and 8 h (with a reduction in bacterial count ranging from 3.08 to 3.75 log(10) CFU/mL) and at 4x MIC at 4, 6, 8, and 24 h (5.30 log(10) CFU/mL reduction in bacterial count) after incubation against MRSA 63718. Reliable bactericidal effect against other strains was maintained at 4x MIC at 24 h.", "Text: The antibiotic resistance of invasive pathogens has become one of the most challenging and persistent health problems [1] . Methicillin-resistant Staphylococcus aureus (MRSA) has become the most common clinically relevant multiresistant pathogen [2] causing both healthcare-associated and community-acquired bloodstream infections with mortality rates up to 40% [3] .\n\nThe prevalence of MRSA is increasing worldwide and, according to the latest information of the European Centre for Disease Prevention and Control from 2012 [4] , can be considered alarming in some European countries, especially in Portugal and Romania, where ≥50% of all S. aureus isolates from invasive infections were identified as MRSA in 2012 (although, e.g., in Romania the prevalence of MRSA was 25-50% in 2010), followed by Italy, Greece, and Poland with 25-50% isolates being MRSA in 2012 (for comparison, in Poland MRSA isolates constituted 10-25% from all S. aureus isolates in 2010).", "The treatment failure of vancomycin, the therapeutic anti-MRSA agent of choice, due to the strains with elevated vancomycin minimum inhibitory concentration (MIC) values (i.e., the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism) within the susceptible range was described previously [5, 6] . Thus, the emergence of MRSA (and vancomycin-resistant S. aureus in the recent years as well [7] ) makes the discovery of new molecular scaffolds a priority, and the current situation even necessitates the reengineering and repositioning of some old drug families to achieve adequate control of these bacteria [8] . However, for the treatment of S. aureus bloodstream infections, bactericidal antimicrobial agents are considered to be superior to bacteriostatic drugs [9] . This fact should be considered during the development of effective and safe treatment options for MRSA infections.", "The history of clinical usage of salicylanilides (2-hydroxy-N-phenylbenzamides) dates back to the 1940s in therapy of tinea capitis, followed by the discovery of their anthelmintic properties in the mid 1950s [10] . Nowadays, salicylanilides (SALs) are a class of aromatic compounds possessing a wide range of interesting pharmacological activities, such as anthelmintic [11] , antibacterial [12, 13] , antimycobacterial [13] , antifungal [14] , and antiviral [15, 16] , among others. Despite being studied since the 1960s, the mechanism of action responsible for biological activities of these compounds has not been explained so far. SALs have been found to inhibit the two-component regulatory systems (TCS) of bacteria [17] . The latest studies specified them also as selective inhibitors of interleukin-12p40 production that plays a specific role in initiation, expansion, and control of cellular response to tuberculosis [18] . Furthermore, salicylanilides have been recognised as inhibitors", "of some bacterial enzymes, such as sortase A from S. aureus [19] , d-alanine-d-alanine ligase [20] , or transglycosylases from S. aureus (but not from M. tuberculosis) [12] . These enzymes participate in secretion of various proteins or in biosynthesis of bacterial cell wall. Recently, salicylanilides-like derivatives were described to inhibit two enzymes essential for mycobacteria: (i) methionine aminopeptidase, catalyzing a key step of the posttranslational modification of nascent proteins, and (ii) isocitrate lyase, which is essential for the metabolism of fatty acids [21] . Thus, SALs seem to be promising candidates for development of new antibacterial agents with a novel mechanism of action. Such new agents could be a solution to the resistance challenges.", "This study is a follow-up paper to a recently published article [13] . The synthesis of the series of novel derivatives of salicylamides, 4-and 5-chloro-2-hydroxy-N-[1-oxo-1-(phenylamino)alkan-2-yl]benzamides, called diamides due to their skeleton (for general structure see Table 1 ), was described previously [13, 22] , and their antimycobacterial and antibacterial activities against various bacterial species were reported [13] . As these compounds expressed very significant antibacterial activity with low MIC values against clinical isolates of MRSA as representatives of multidrugresistant bacteria, we decided to extend the knowledge about the antibacterial properties of these compounds against MRSA.", "The aim of the current study was to assess the overall in vitro bactericidal activity of nine newly synthesized diamides in dependence on time and concentration against clinical isolates of MRSA as representatives of multidrug-resistant bacteria. To the best of our knowledge, this is the first study dealing with the evaluation of novel microbiological characteristics of SAL analogues and revealing their bactericidal effect.", "The synthetic pathway of the series of novel diamides was described recently [13, 22] , and their structures (see Table 1 ) were confirmed by IR, NMR, and MS spectrometry, and the purity of the compounds was checked by CHN analysis [13, 22] . [27] ; and MRSA SA 3202 [27] (National Institute of Public Health, Prague, Czech Republic) both of human origin. Suspected colonies were confirmed by PCR; a 108 bp fragment specific for S. aureus was detected [28] . All isolates were tested for the presence of the mecA gene encoding methicillin resistance [29] . These three clinical isolates were classified as vancomycin-susceptible (but with higher MIC of vancomycin equal to 2 g/mL (VA2-MRSA) within the susceptible range for MRSA 63718) methicillinresistant S. aureus (VS-MRSA). For the MICs of vancomycin, see Table 1 . Vancomycin-susceptible methicillin-susceptible Staphylococcus aureus (VS-MSSA) ATCC 29213, obtained from the American Type Culture Collection, was used as the reference and", "quality control strain. The bacteria were stored at −80 ∘ C and were kept on blood agar plates (Columbia agar base with 5% ovine blood) between experiments. (MBCs) . The MBCs (i.e., the lowest concentrations of antibacterial agents required to kill a particular bacterium) were determined by subculturing aliquots (20 L) from wells with no visible bacterial growth and from control wells of MIC determination onto substance-free Mueller-Hinton agar (MHA) plates. The plates were incubated aerobically at 37 ∘ C for 24 h for colony count. The MBC was defined as the lowest concentration of substance, which produced ≥99.9% killing Table 1 : Chemical structures and in vitro MIC and MBC [ g/mL] values of tested 5-and 4-chloro-2-hydroxy-N-[1-oxo-1-(phenylamino)alkan-2-yl]benzamides (bactericidal effect of individual compounds against particular strains marked in bold). after 24 h of incubation as compared to the colony count of the starting inoculum [30] . To ensure reproducibility, each MBC", "assay was performed in at least triplicate on separate occasions.", "N H O H N O OH 1 2 R 1 R 3 R 2 Comp. R 1 R 2 R 3 MIC [ g/mL] MBC [ g/mL] 1 2 3 4 1 2 3 4 1a 5-Cl 4-CH 3 (S)-CH 3 >256 >256 >256 >256 >256 >256 >256 >256 1b 5-Cl 4-CH 3 (S)-CH(CH 3 ) 2 >256 >256 32 32 >256 >256 128 >256 1c 5-Cl 4-CH 3 (S)-benzyl >256 >256 >256 >256 >256 >256 >256 >256 1d 5-Cl 4-CH 3 (R)-CH 2 -indolyl >256 >256 >256 >256 >256 >256 >256 >256 1e 5-Cl 4-OCH 3 (S)-CH(CH 3 ) 2 >256 >256 >256 >256 >256 >256 >256 >256 1f 5-Cl 4-CF 3 (S)-CH(CH 3 ) 2 4 2 2 2 4 4 8 4 1g 4-Cl 4-Br (S)-CH(CH 3 ) 2 8 4 4 4 1 6 8 8 8 1h 4-Cl 3,4-Cl (S)-CH(CH 3 ) 2 2 1 1 1 4 1 4 2 1i 4-Cl 3,4-Cl (S)-benzyl 1 1 0.5 0.5 8 1 8 1 AMP - - - >16 >16 >16 0.25 >16 >16 >16 0.25 CPX - - - >16 >16 >16 0.5 >16 >16 >16 0.5 VAN - - - 2 1 1 1 2 1 1 1", "Time-kill assays were performed by the broth macrodilution method according to previously described methodology [30] with some modifications. Briefly, flasks containing sterile fresh Mueller-Hinton broth (MHB) with the appropriate antimicrobial agent were inoculated with the test organism in logarithmic growth phase to obtain the starting inoculum with the concentration of approximately 7.5 × 10 6 CFU/mL (actual inoculum concentrations ranged from 0.9 × 10 5 to 2.9 × 10 6 CFU/mL) and a final concentration of the antibiotic equal to 1x, 2x, and 4x MIC in 10 mL volume. For the determination of viable counts, aliquots were removed at 0, 4, 6, 8, and 24 h time points after inoculation, serially diluted in sterile phosphate buffered saline, and aliquots (20 L) were plated on MHA plates in duplicate. Colony counts were performed on plates yielding 6 to 60 colonies, and the mean was calculated. Antimicrobial carry-over was controlled by dilution and visual inspection of the distribution of", "colonies on the plates with observation of possible inhibition of growth at the site of the initial streaks. The plates were incubated at 37 ∘ C for 24 to 48 h, and the number of colonies was determined. To ensure reproducibility, each time-kill experiment was carried out in duplicate on separate occasions with results presented as the mean of all experiments. The growth control without the addition of antimicrobial agents and the control containing DMSO without any antimicrobial agent to exclude antibacterial activity of this solvent were included. Time-kill curves were constructed by plotting the log 10 CFU per millilitre versus time (over 24 h), and the change in bacterial concentration was determined. The results were analysed by evaluating the numbers of strains that yielded Δ(log 10 CFU/mL) values of −1 (corresponding to 90% killing), −2 (99% killing), and −3 (99.9% killing) at 4, 6, 8, and 24 h compared to counts at 0 h. Bactericidal activity was defined as a reduction of at", "least 99.9% (≥3 log 10 ) of the total count of CFU/mL in the original inoculum.", "Diamides seem to be promising candidates for antibacterial agents with very strong anti-MRSA activity, as it was published recently [13] . In the present study the series of nine newly synthesized diamides was evaluated as prospective bactericidal agents against representatives of multidrugresistant bacteria, three clinical isolates of MRSA, and Staphylococcus aureus ATCC 29213 (methicillin-susceptible) as the reference and quality control strain. Since SALs and their analogues are known as compounds with bacteriostatic effect [31] , this is the first study where SAL-like compounds were considered as prospective bactericidal agents and the dependence of bactericidal effect of these compounds on time and concentration was evaluated. Thus, absolutely novel microbiological characteristics of these compounds were revealed in the present study.", "Recently MIC values of diamides expressed as molar concentrations in mol/L were published [13] . To allow comparison with MBC values of the present study, MICs in g/mL were calculated and are recorded in Table 1 along with the activity of reference antibacterial drugs, ampicillin, ciprofloxacin, and vancomycin. Potential bactericidal activity of diamides was assessed using MBC assay [26] . MBC values of all tested compounds are recorded in Table 1 as well.\n\nBased on the obtained results, all compounds assessed as active according to MIC values in our previous study (1f-i) showed low or moderate MBC values against all four strains. The MBC values of these compounds did not exceed the highest tested drug concentration and ranged from 1 to 16 g/mL. In all cases, there were comparable MBC values for the clinical isolates of MRSA and the S. aureus reference strain.\n\nBactericidal activity is defined as a ratio of MBC to MIC of ≤4 [32] . Table 1 bactericidal activity is expressed in bold.", "As mentioned above, SALs are known to exhibit a bacteriostatic effect [31] , so it was very interesting to discover that diamides possess bactericidal activity. The amide bond (-CONH-) can cause interactions with a variety of enzymes [33] ; therefore the presence of two amide bonds could be responsible for the bactericidal effect of diamides against MRSA. The activity of SALs and their analogues results from multiple mechanisms, which are still under investigation; for example, it was found that SALs are capable of inhibiting transglycosylases in later stages of S. aureus (including MRSA) cell wall biosynthesis [12] . These enzymes catalyse the step prior to the transpeptidation in the peptidoglycan biosynthesis and are responsible for polymerization of lipid II, which occurs at the outer face of the membrane [12] . Since antibacterial agents targeting cell wall biosynthesis act as bactericidal agents [30, 34] , the failure in the cell wall biosynthesis due to the inhibition of", "transglycosylases could be responsible for bactericidal activity of diamides against MRSA.", "Based on these findings, antibacterial active diamides with bactericidal effect against all four tested strains as prospective bactericidal agents were chosen for subsequent timekill curve studies to determine the real dependence of bactericidal effect on concentration over time.", "1-oxobutan-2-yl}-2-hydroxybenzamide (1h) were tested in time-kill studies at 1x, 2x, and 4x MIC against all MRSA isolates and the S. aureus reference strain. The antibacterial effect of DMSO [35] used as the solvent of the tested compounds was excluded in this assay, as time-kill curves of this solvent were identical or very similar to those of the growth control. The extent of bacterial killing was estimated by the number of these strains showing a decrease ranging from 1 to 3 log 10 CFU/mL in viable cell count at different times after incubation. A summary of these data is presented in Table 2 . Based on these data it can be concluded that the bactericidal potency of tested diamides against all four strains decreased as follows: 1f > 1h > 1g. No bactericidal activity (i.e., ≥3 log 10 CFU/mL decrease) was observed at 1x MIC for any strain and time after incubation tested. At 4x MIC from the four strains, compounds 1f, 1 g, and 1h killed 2, 1, and 2 strains, respectively, at 8 h after", "incubation and 4, 2, and 2 strains, respectively, at 24 h after incubation.", "The findings of time-kill studies for each of the four staphylococci strains at exposure to compounds 1f, 1g, and 1h are summarized in Table 3 . Bactericidal activity (i.e., ≥3 log 10 CFU/mL decrease) is expressed in bold.", "For compound 1f rapid concentration-dependent antibacterial effect was recorded against clinical isolate of MRSA 63718. Time was not the predictive factor influencing the antibacterial activity because log 10 differences in CFU/mL from the starting inoculum were the same for 4x MIC (with the highest efficiency with a reduction in bacterial count of 5.30 log 10 CFU/mL) or very similar for 2x MIC (with a moderate regrowth after 24 h causing a loss of bactericidal activity) over 24 h. The bactericidal effect was maintained even at 2x MIC at 4 h after incubation for this strain (reduction of 3.08 log 10 CFU/mL). For the remaining strains, clinical isolates of MRSA SA 630, MRSA SA 3202, and S. aureus ATCC 29213, reliable bactericidal effect was recorded at 4x MIC at 24 h after incubation for all these strains with a reduction in bacterial count of 3.22, 3.30, and 3.65 log 10 CFU/mL, respectively.", "For compound 1g bactericidal effect against MRSA 63718 was noticed at 2x MIC at 6 and 8 h after incubation and at 4x MIC at 4, 6, and 8 h after incubation with a reduction in bacterial count ranging from 3.10 to 3.58 log 10 CFU/mL. The most effective killing was achieved at 6 h for both concentrations. As in the case of compound 1f, a regrowth was observed after 24 h after incubation. For the remaining isolates of MRSA, SA 630 and SA 3202, bactericidal effect occurred only at 4x MIC at 24 h after incubation with a reduction in bacterial count of 3.38 and 4.01 log 10 CFU/mL, respectively. The highest bactericidal effect was recorded for MRSA SA 3202 at 4x MIC at 24 h after incubation. A reduction consistent with bacteriostatic effect (0.03 to 2.37 log 10 CFU/mL) was observed at other concentrations over time for both isolates. No bactericidal effect was observed for the S. aureus reference strain; compound 1g demonstrated a pattern of bacteriostatic activity against this strain with a", "reduction in bacterial count ranging from 0.07 to 2.33 log 10 CFU/mL at 4x MIC over time. In other cases, a slight increase in bacterial counts (i.e., overgrowth) compared with the starting inoculum was observed with values ranging from 0.10 to 1.57 log 10 CFU/mL for this reference strain.", "For compound 1h bactericidal effect against MRSA 63718 was maintained at 4x MIC at 6 and 8 h after incubation with a reduction in bacterial count of 3.54 and 3.31 log 10 CFU/mL, respectively. The same as for 1g, the most potent bactericidal effect was maintained at 6 h after incubation. Regrowth at 24 h after incubation causing a loss of bactericidal activity was recorded similarly as with previous compounds. The reason for regrowth of the test organism at 24 h in the experiment is unknown. Most probably, selection of resistant mutants is responsible for this phenomenon [30] ; degradation of the drug in the growth medium is not assumed, as regrowth was", "Number of strains showing the following log 10 CFU/mL decrease a at the designated incubation time not observed for any other tested strain. For MRSA SA 630 concentration-dependent killing was recorded at 4x MIC at 6, 8, and 24 h after incubation with log 10 differences in CFU/mL from the starting inoculum being very similar over time (ranging from 3.18 to 3.39 log 10 CFU/mL). For MRSA SA 3202 reliable bactericidal effect was maintained only at 4x MIC at 24 h after incubation with a reduction in bacterial count of 3.02 log 10 CFU/mL. As for compound 1g, bacteriostatic activity against S. aureus reference strain was observed with a reduction in bacterial count ranging from 0.34 to 2.62 log 10 CFU/mL at 2x and 4x MIC. Overgrowth (values ranging from 0.04 to 1.43 log 10 CFU/mL) was recorded at 1x MIC for this strain. It is of note that in all staphylococci strains with similar MICs and MBCs for compounds 1g and 1h the responsiveness to antibacterial activity of these compounds varied", "with clinical strains of MRSA being effectively killed and the reference strain remaining unaffected at 4x MIC.", "There is a discrepancy between bactericidal results of MBC assay compared with time-kill kinetics. This difference could be caused by comparing microtiter (MBC assay) to macrobroth (time-kill assay) dilutions [36] . Moreover, although time-kill assays are more labour intensive and time consuming than MBC assays, they are recognised to provide a greater degree of characterisation of the cell eradication potential of antibacterial agents [37] .", "Concerning antibacterial effect, it is not generally important if the antibacterial agent is also bactericidal at higher concentrations, because the inhibition of bacterial proliferation usually achieves a therapeutic effect; the patient's immune system is capable of coping with the infection then [34] . However, bactericidal therapy could produce a better treatment result by rapid reduction of the bacterial load [38] . Moreover, in the case of an immune system disorder (e.g., immunosuppressive therapy, AIDS patients, etc.) bactericidal agents are unequivocally indicated. Considering steadily escalating numbers of immunocompromised patients with endocarditis, meningitis, or osteomyelitis in recent years, it is necessary to achieve bacterial killing and broaden the spectrum of antimicrobial agents with bactericidal active compounds [30] .", "The clinical outcome of MRSA bacteraemia is significantly influenced by vancomycin MIC. Treatment failure exceeding 60% for S. aureus with vancomycin MIC of 4 g/mL resulted in the change of susceptibility breakpoint from 4 g/mL to 2 g/mL by the Clinical and Laboratory Standards Institute (CLSI) in 2006 [23] as well as by the US Food and Drug Administration (FDA) in 2008 [39] . It has been recommended that for infections caused by MRSA strains with elevated vancomycin MICs (2 g/mL), alternative therapy should be considered [40] . It is of note that based on time-kill assays in the present study, all tested diamides (particularly compound 1f exhibiting rapid bactericidal concentration-dependent effect even at 2x MIC) were most effective against isolate MRSA 63718, which is the strain with elevated vancomycin MIC of 2 g/mL. The activity against the remaining isolates with vancomycin MIC of 1 g/mL was lower.", "Considering the emergence of decreasing vancomycin susceptibility of MRSA isolates and thus the therapeutic efficacy of vancomycin therapy, our aim was to determine the potential bactericidal role of novel antibacterial compounds against MRSA in vitro. Based on the obtained results, diamides can be suitable candidates for such novel bactericidal active compounds presenting a promising starting point for further investigations to ascertain real in vivo activity and the exact mechanism of action. \n\nThe present study is the first evidence of bactericidal effect of SAL analogues. Against other strains, reliable bactericidal effect was maintained at 4x MIC at 24 h after incubation. Considering the necessity to broaden the spectrum of bactericidal agents, diamides from the current study with a novel mechanism of action could present a very promising and interesting solution to this challenge for the future." ]

[ 6 ]

[ "to assess the overall in vitro bactericidal activity of nine newly synthesized diamides" ]

[ "In Vitro Bactericidal Activity of 4- and 5-Chloro-2-hydroxy-N-[1-oxo-1-(phenylamino)alkan-2-yl]benzamides against MRSA\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4321674/\n\nSHA: f0e6cef57dbae030aea2f324e21e00945ac659cf\n\nAuthors: Zadrazilova, Iveta; Pospisilova, Sarka; Pauk, Karel; Imramovsky, Ales; Vinsova, Jarmila; Cizek, Alois; Jampilek, Josef\nDate: 2015-01-15\nDOI: 10.1155/2015/349534\nLicense: cc-by", "Abstract: A series of nine substituted 2-hydroxy-N-[1-oxo-1-(phenylamino)alkan-2-yl]benzamides was assessed as prospective bactericidal agents against three clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and S. aureus ATCC 29213 as the reference and quality control strain. The minimum bactericidal concentration was determined by subculturing aliquots from MIC determination onto substance-free agar plates. The bactericidal kinetics of compounds 5-chloro-2-hydroxy-N-[(2S)-3-methyl-1-oxo-1-{[4-(trifluoromethyl)phenyl]amino}butan-2-yl]benzamide (1f), N-{(2S)-1-[(4-bromophenyl)amino]-3-methyl-1-oxobutan-2-yl}-4-chloro-2-hydroxybenzamide (1g), and 4-chloro-N-{(2S)-1-[(3,4-dichlorophenyl)amino]-3-methyl-1-oxobutan-2-yl}-2-hydroxybenzamide (1h) was established by time-kill assay with a final concentration of the compound equal to 1x, 2x, and 4x MIC; aliquots were removed at 0, 4, 6, 8, and 24 h time points. The most potent bactericidal agent was compound 1f exhibiting", "remarkable rapid concentration-dependent bactericidal effect even at 2x MIC at 4, 6, and 8 h (with a reduction in bacterial count ranging from 3.08 to 3.75 log(10) CFU/mL) and at 4x MIC at 4, 6, 8, and 24 h (5.30 log(10) CFU/mL reduction in bacterial count) after incubation against MRSA 63718. Reliable bactericidal effect against other strains was maintained at 4x MIC at 24 h.", "Text: The antibiotic resistance of invasive pathogens has become one of the most challenging and persistent health problems [1] . Methicillin-resistant Staphylococcus aureus (MRSA) has become the most common clinically relevant multiresistant pathogen [2] causing both healthcare-associated and community-acquired bloodstream infections with mortality rates up to 40% [3] .\n\nThe prevalence of MRSA is increasing worldwide and, according to the latest information of the European Centre for Disease Prevention and Control from 2012 [4] , can be considered alarming in some European countries, especially in Portugal and Romania, where ≥50% of all S. aureus isolates from invasive infections were identified as MRSA in 2012 (although, e.g., in Romania the prevalence of MRSA was 25-50% in 2010), followed by Italy, Greece, and Poland with 25-50% isolates being MRSA in 2012 (for comparison, in Poland MRSA isolates constituted 10-25% from all S. aureus isolates in 2010).", "The treatment failure of vancomycin, the therapeutic anti-MRSA agent of choice, due to the strains with elevated vancomycin minimum inhibitory concentration (MIC) values (i.e., the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism) within the susceptible range was described previously [5, 6] . Thus, the emergence of MRSA (and vancomycin-resistant S. aureus in the recent years as well [7] ) makes the discovery of new molecular scaffolds a priority, and the current situation even necessitates the reengineering and repositioning of some old drug families to achieve adequate control of these bacteria [8] . However, for the treatment of S. aureus bloodstream infections, bactericidal antimicrobial agents are considered to be superior to bacteriostatic drugs [9] . This fact should be considered during the development of effective and safe treatment options for MRSA infections.", "The history of clinical usage of salicylanilides (2-hydroxy-N-phenylbenzamides) dates back to the 1940s in therapy of tinea capitis, followed by the discovery of their anthelmintic properties in the mid 1950s [10] . Nowadays, salicylanilides (SALs) are a class of aromatic compounds possessing a wide range of interesting pharmacological activities, such as anthelmintic [11] , antibacterial [12, 13] , antimycobacterial [13] , antifungal [14] , and antiviral [15, 16] , among others. Despite being studied since the 1960s, the mechanism of action responsible for biological activities of these compounds has not been explained so far. SALs have been found to inhibit the two-component regulatory systems (TCS) of bacteria [17] . The latest studies specified them also as selective inhibitors of interleukin-12p40 production that plays a specific role in initiation, expansion, and control of cellular response to tuberculosis [18] . Furthermore, salicylanilides have been recognised as inhibitors", "of some bacterial enzymes, such as sortase A from S. aureus [19] , d-alanine-d-alanine ligase [20] , or transglycosylases from S. aureus (but not from M. tuberculosis) [12] . These enzymes participate in secretion of various proteins or in biosynthesis of bacterial cell wall. Recently, salicylanilides-like derivatives were described to inhibit two enzymes essential for mycobacteria: (i) methionine aminopeptidase, catalyzing a key step of the posttranslational modification of nascent proteins, and (ii) isocitrate lyase, which is essential for the metabolism of fatty acids [21] . Thus, SALs seem to be promising candidates for development of new antibacterial agents with a novel mechanism of action. Such new agents could be a solution to the resistance challenges.", "This study is a follow-up paper to a recently published article [13] . The synthesis of the series of novel derivatives of salicylamides, 4-and 5-chloro-2-hydroxy-N-[1-oxo-1-(phenylamino)alkan-2-yl]benzamides, called diamides due to their skeleton (for general structure see Table 1 ), was described previously [13, 22] , and their antimycobacterial and antibacterial activities against various bacterial species were reported [13] . As these compounds expressed very significant antibacterial activity with low MIC values against clinical isolates of MRSA as representatives of multidrugresistant bacteria, we decided to extend the knowledge about the antibacterial properties of these compounds against MRSA.", "The aim of the current study was to assess the overall in vitro bactericidal activity of nine newly synthesized diamides in dependence on time and concentration against clinical isolates of MRSA as representatives of multidrug-resistant bacteria. To the best of our knowledge, this is the first study dealing with the evaluation of novel microbiological characteristics of SAL analogues and revealing their bactericidal effect.", "The synthetic pathway of the series of novel diamides was described recently [13, 22] , and their structures (see Table 1 ) were confirmed by IR, NMR, and MS spectrometry, and the purity of the compounds was checked by CHN analysis [13, 22] . [27] ; and MRSA SA 3202 [27] (National Institute of Public Health, Prague, Czech Republic) both of human origin. Suspected colonies were confirmed by PCR; a 108 bp fragment specific for S. aureus was detected [28] . All isolates were tested for the presence of the mecA gene encoding methicillin resistance [29] . These three clinical isolates were classified as vancomycin-susceptible (but with higher MIC of vancomycin equal to 2 g/mL (VA2-MRSA) within the susceptible range for MRSA 63718) methicillinresistant S. aureus (VS-MRSA). For the MICs of vancomycin, see Table 1 . Vancomycin-susceptible methicillin-susceptible Staphylococcus aureus (VS-MSSA) ATCC 29213, obtained from the American Type Culture Collection, was used as the reference and", "quality control strain. The bacteria were stored at −80 ∘ C and were kept on blood agar plates (Columbia agar base with 5% ovine blood) between experiments. (MBCs) . The MBCs (i.e., the lowest concentrations of antibacterial agents required to kill a particular bacterium) were determined by subculturing aliquots (20 L) from wells with no visible bacterial growth and from control wells of MIC determination onto substance-free Mueller-Hinton agar (MHA) plates. The plates were incubated aerobically at 37 ∘ C for 24 h for colony count. The MBC was defined as the lowest concentration of substance, which produced ≥99.9% killing Table 1 : Chemical structures and in vitro MIC and MBC [ g/mL] values of tested 5-and 4-chloro-2-hydroxy-N-[1-oxo-1-(phenylamino)alkan-2-yl]benzamides (bactericidal effect of individual compounds against particular strains marked in bold). after 24 h of incubation as compared to the colony count of the starting inoculum [30] . To ensure reproducibility, each MBC", "assay was performed in at least triplicate on separate occasions.", "N H O H N O OH 1 2 R 1 R 3 R 2 Comp. R 1 R 2 R 3 MIC [ g/mL] MBC [ g/mL] 1 2 3 4 1 2 3 4 1a 5-Cl 4-CH 3 (S)-CH 3 >256 >256 >256 >256 >256 >256 >256 >256 1b 5-Cl 4-CH 3 (S)-CH(CH 3 ) 2 >256 >256 32 32 >256 >256 128 >256 1c 5-Cl 4-CH 3 (S)-benzyl >256 >256 >256 >256 >256 >256 >256 >256 1d 5-Cl 4-CH 3 (R)-CH 2 -indolyl >256 >256 >256 >256 >256 >256 >256 >256 1e 5-Cl 4-OCH 3 (S)-CH(CH 3 ) 2 >256 >256 >256 >256 >256 >256 >256 >256 1f 5-Cl 4-CF 3 (S)-CH(CH 3 ) 2 4 2 2 2 4 4 8 4 1g 4-Cl 4-Br (S)-CH(CH 3 ) 2 8 4 4 4 1 6 8 8 8 1h 4-Cl 3,4-Cl (S)-CH(CH 3 ) 2 2 1 1 1 4 1 4 2 1i 4-Cl 3,4-Cl (S)-benzyl 1 1 0.5 0.5 8 1 8 1 AMP - - - >16 >16 >16 0.25 >16 >16 >16 0.25 CPX - - - >16 >16 >16 0.5 >16 >16 >16 0.5 VAN - - - 2 1 1 1 2 1 1 1", "Time-kill assays were performed by the broth macrodilution method according to previously described methodology [30] with some modifications. Briefly, flasks containing sterile fresh Mueller-Hinton broth (MHB) with the appropriate antimicrobial agent were inoculated with the test organism in logarithmic growth phase to obtain the starting inoculum with the concentration of approximately 7.5 × 10 6 CFU/mL (actual inoculum concentrations ranged from 0.9 × 10 5 to 2.9 × 10 6 CFU/mL) and a final concentration of the antibiotic equal to 1x, 2x, and 4x MIC in 10 mL volume. For the determination of viable counts, aliquots were removed at 0, 4, 6, 8, and 24 h time points after inoculation, serially diluted in sterile phosphate buffered saline, and aliquots (20 L) were plated on MHA plates in duplicate. Colony counts were performed on plates yielding 6 to 60 colonies, and the mean was calculated. Antimicrobial carry-over was controlled by dilution and visual inspection of the distribution of", "colonies on the plates with observation of possible inhibition of growth at the site of the initial streaks. The plates were incubated at 37 ∘ C for 24 to 48 h, and the number of colonies was determined. To ensure reproducibility, each time-kill experiment was carried out in duplicate on separate occasions with results presented as the mean of all experiments. The growth control without the addition of antimicrobial agents and the control containing DMSO without any antimicrobial agent to exclude antibacterial activity of this solvent were included. Time-kill curves were constructed by plotting the log 10 CFU per millilitre versus time (over 24 h), and the change in bacterial concentration was determined. The results were analysed by evaluating the numbers of strains that yielded Δ(log 10 CFU/mL) values of −1 (corresponding to 90% killing), −2 (99% killing), and −3 (99.9% killing) at 4, 6, 8, and 24 h compared to counts at 0 h. Bactericidal activity was defined as a reduction of at", "least 99.9% (≥3 log 10 ) of the total count of CFU/mL in the original inoculum.", "Diamides seem to be promising candidates for antibacterial agents with very strong anti-MRSA activity, as it was published recently [13] . In the present study the series of nine newly synthesized diamides was evaluated as prospective bactericidal agents against representatives of multidrugresistant bacteria, three clinical isolates of MRSA, and Staphylococcus aureus ATCC 29213 (methicillin-susceptible) as the reference and quality control strain. Since SALs and their analogues are known as compounds with bacteriostatic effect [31] , this is the first study where SAL-like compounds were considered as prospective bactericidal agents and the dependence of bactericidal effect of these compounds on time and concentration was evaluated. Thus, absolutely novel microbiological characteristics of these compounds were revealed in the present study.", "Recently MIC values of diamides expressed as molar concentrations in mol/L were published [13] . To allow comparison with MBC values of the present study, MICs in g/mL were calculated and are recorded in Table 1 along with the activity of reference antibacterial drugs, ampicillin, ciprofloxacin, and vancomycin. Potential bactericidal activity of diamides was assessed using MBC assay [26] . MBC values of all tested compounds are recorded in Table 1 as well.\n\nBased on the obtained results, all compounds assessed as active according to MIC values in our previous study (1f-i) showed low or moderate MBC values against all four strains. The MBC values of these compounds did not exceed the highest tested drug concentration and ranged from 1 to 16 g/mL. In all cases, there were comparable MBC values for the clinical isolates of MRSA and the S. aureus reference strain.\n\nBactericidal activity is defined as a ratio of MBC to MIC of ≤4 [32] . Table 1 bactericidal activity is expressed in bold.", "As mentioned above, SALs are known to exhibit a bacteriostatic effect [31] , so it was very interesting to discover that diamides possess bactericidal activity. The amide bond (-CONH-) can cause interactions with a variety of enzymes [33] ; therefore the presence of two amide bonds could be responsible for the bactericidal effect of diamides against MRSA. The activity of SALs and their analogues results from multiple mechanisms, which are still under investigation; for example, it was found that SALs are capable of inhibiting transglycosylases in later stages of S. aureus (including MRSA) cell wall biosynthesis [12] . These enzymes catalyse the step prior to the transpeptidation in the peptidoglycan biosynthesis and are responsible for polymerization of lipid II, which occurs at the outer face of the membrane [12] . Since antibacterial agents targeting cell wall biosynthesis act as bactericidal agents [30, 34] , the failure in the cell wall biosynthesis due to the inhibition of", "transglycosylases could be responsible for bactericidal activity of diamides against MRSA.", "Based on these findings, antibacterial active diamides with bactericidal effect against all four tested strains as prospective bactericidal agents were chosen for subsequent timekill curve studies to determine the real dependence of bactericidal effect on concentration over time.", "1-oxobutan-2-yl}-2-hydroxybenzamide (1h) were tested in time-kill studies at 1x, 2x, and 4x MIC against all MRSA isolates and the S. aureus reference strain. The antibacterial effect of DMSO [35] used as the solvent of the tested compounds was excluded in this assay, as time-kill curves of this solvent were identical or very similar to those of the growth control. The extent of bacterial killing was estimated by the number of these strains showing a decrease ranging from 1 to 3 log 10 CFU/mL in viable cell count at different times after incubation. A summary of these data is presented in Table 2 . Based on these data it can be concluded that the bactericidal potency of tested diamides against all four strains decreased as follows: 1f > 1h > 1g. No bactericidal activity (i.e., ≥3 log 10 CFU/mL decrease) was observed at 1x MIC for any strain and time after incubation tested. At 4x MIC from the four strains, compounds 1f, 1 g, and 1h killed 2, 1, and 2 strains, respectively, at 8 h after", "incubation and 4, 2, and 2 strains, respectively, at 24 h after incubation.", "The findings of time-kill studies for each of the four staphylococci strains at exposure to compounds 1f, 1g, and 1h are summarized in Table 3 . Bactericidal activity (i.e., ≥3 log 10 CFU/mL decrease) is expressed in bold.", "For compound 1f rapid concentration-dependent antibacterial effect was recorded against clinical isolate of MRSA 63718. Time was not the predictive factor influencing the antibacterial activity because log 10 differences in CFU/mL from the starting inoculum were the same for 4x MIC (with the highest efficiency with a reduction in bacterial count of 5.30 log 10 CFU/mL) or very similar for 2x MIC (with a moderate regrowth after 24 h causing a loss of bactericidal activity) over 24 h. The bactericidal effect was maintained even at 2x MIC at 4 h after incubation for this strain (reduction of 3.08 log 10 CFU/mL). For the remaining strains, clinical isolates of MRSA SA 630, MRSA SA 3202, and S. aureus ATCC 29213, reliable bactericidal effect was recorded at 4x MIC at 24 h after incubation for all these strains with a reduction in bacterial count of 3.22, 3.30, and 3.65 log 10 CFU/mL, respectively.", "For compound 1g bactericidal effect against MRSA 63718 was noticed at 2x MIC at 6 and 8 h after incubation and at 4x MIC at 4, 6, and 8 h after incubation with a reduction in bacterial count ranging from 3.10 to 3.58 log 10 CFU/mL. The most effective killing was achieved at 6 h for both concentrations. As in the case of compound 1f, a regrowth was observed after 24 h after incubation. For the remaining isolates of MRSA, SA 630 and SA 3202, bactericidal effect occurred only at 4x MIC at 24 h after incubation with a reduction in bacterial count of 3.38 and 4.01 log 10 CFU/mL, respectively. The highest bactericidal effect was recorded for MRSA SA 3202 at 4x MIC at 24 h after incubation. A reduction consistent with bacteriostatic effect (0.03 to 2.37 log 10 CFU/mL) was observed at other concentrations over time for both isolates. No bactericidal effect was observed for the S. aureus reference strain; compound 1g demonstrated a pattern of bacteriostatic activity against this strain with a", "reduction in bacterial count ranging from 0.07 to 2.33 log 10 CFU/mL at 4x MIC over time. In other cases, a slight increase in bacterial counts (i.e., overgrowth) compared with the starting inoculum was observed with values ranging from 0.10 to 1.57 log 10 CFU/mL for this reference strain.", "For compound 1h bactericidal effect against MRSA 63718 was maintained at 4x MIC at 6 and 8 h after incubation with a reduction in bacterial count of 3.54 and 3.31 log 10 CFU/mL, respectively. The same as for 1g, the most potent bactericidal effect was maintained at 6 h after incubation. Regrowth at 24 h after incubation causing a loss of bactericidal activity was recorded similarly as with previous compounds. The reason for regrowth of the test organism at 24 h in the experiment is unknown. Most probably, selection of resistant mutants is responsible for this phenomenon [30] ; degradation of the drug in the growth medium is not assumed, as regrowth was", "Number of strains showing the following log 10 CFU/mL decrease a at the designated incubation time not observed for any other tested strain. For MRSA SA 630 concentration-dependent killing was recorded at 4x MIC at 6, 8, and 24 h after incubation with log 10 differences in CFU/mL from the starting inoculum being very similar over time (ranging from 3.18 to 3.39 log 10 CFU/mL). For MRSA SA 3202 reliable bactericidal effect was maintained only at 4x MIC at 24 h after incubation with a reduction in bacterial count of 3.02 log 10 CFU/mL. As for compound 1g, bacteriostatic activity against S. aureus reference strain was observed with a reduction in bacterial count ranging from 0.34 to 2.62 log 10 CFU/mL at 2x and 4x MIC. Overgrowth (values ranging from 0.04 to 1.43 log 10 CFU/mL) was recorded at 1x MIC for this strain. It is of note that in all staphylococci strains with similar MICs and MBCs for compounds 1g and 1h the responsiveness to antibacterial activity of these compounds varied", "with clinical strains of MRSA being effectively killed and the reference strain remaining unaffected at 4x MIC.", "There is a discrepancy between bactericidal results of MBC assay compared with time-kill kinetics. This difference could be caused by comparing microtiter (MBC assay) to macrobroth (time-kill assay) dilutions [36] . Moreover, although time-kill assays are more labour intensive and time consuming than MBC assays, they are recognised to provide a greater degree of characterisation of the cell eradication potential of antibacterial agents [37] .", "Concerning antibacterial effect, it is not generally important if the antibacterial agent is also bactericidal at higher concentrations, because the inhibition of bacterial proliferation usually achieves a therapeutic effect; the patient's immune system is capable of coping with the infection then [34] . However, bactericidal therapy could produce a better treatment result by rapid reduction of the bacterial load [38] . Moreover, in the case of an immune system disorder (e.g., immunosuppressive therapy, AIDS patients, etc.) bactericidal agents are unequivocally indicated. Considering steadily escalating numbers of immunocompromised patients with endocarditis, meningitis, or osteomyelitis in recent years, it is necessary to achieve bacterial killing and broaden the spectrum of antimicrobial agents with bactericidal active compounds [30] .", "The clinical outcome of MRSA bacteraemia is significantly influenced by vancomycin MIC. Treatment failure exceeding 60% for S. aureus with vancomycin MIC of 4 g/mL resulted in the change of susceptibility breakpoint from 4 g/mL to 2 g/mL by the Clinical and Laboratory Standards Institute (CLSI) in 2006 [23] as well as by the US Food and Drug Administration (FDA) in 2008 [39] . It has been recommended that for infections caused by MRSA strains with elevated vancomycin MICs (2 g/mL), alternative therapy should be considered [40] . It is of note that based on time-kill assays in the present study, all tested diamides (particularly compound 1f exhibiting rapid bactericidal concentration-dependent effect even at 2x MIC) were most effective against isolate MRSA 63718, which is the strain with elevated vancomycin MIC of 2 g/mL. The activity against the remaining isolates with vancomycin MIC of 1 g/mL was lower.", "Considering the emergence of decreasing vancomycin susceptibility of MRSA isolates and thus the therapeutic efficacy of vancomycin therapy, our aim was to determine the potential bactericidal role of novel antibacterial compounds against MRSA in vitro. Based on the obtained results, diamides can be suitable candidates for such novel bactericidal active compounds presenting a promising starting point for further investigations to ascertain real in vivo activity and the exact mechanism of action. \n\nThe present study is the first evidence of bactericidal effect of SAL analogues. Against other strains, reliable bactericidal effect was maintained at 4x MIC at 24 h after incubation. Considering the necessity to broaden the spectrum of bactericidal agents, diamides from the current study with a novel mechanism of action could present a very promising and interesting solution to this challenge for the future." ]

[ 8 ]

[ "the marmoset an appropriate animal model for biodefense-related pathogens" ]

[ "Exploring the Innate Immunological Response of an Alternative Nonhuman Primate Model of Infectious Disease; the Common Marmoset\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4129158/\n\nSHA: f4c43e4ae49ca69dbac32620bd0a73ecbb683b91\n\nAuthors: Nelson, M.; Loveday, M.\nDate: 2014-07-22\nDOI: 10.1155/2014/913632\nLicense: cc-by", "Abstract: The common marmoset (Callithrix jacchus) is increasingly being utilised as a nonhuman primate model for human disease, ranging from autoimmune to infectious disease. In order to fully exploit these models, meaningful comparison to the human host response is necessary. Commercially available reagents, primarily targeted to human cells, were utilised to assess the phenotype and activation status of key immune cell types and cytokines in naive and infected animals. Single cell suspensions of blood, spleen, and lung were examined. Generally, the phenotype of cells was comparable between humans and marmosets, with approximately 63% of all lymphocytes in the blood of marmosets being T cells, 25% B-cells, and 12% NK cells. The percentage of neutrophils in marmoset blood were more similar to human values than mouse values. Comparison of the activation status of cells following experimental systemic or inhalational infection exhibited different trends in different tissues, most", "obvious in cell types active in the innate immune response. This work significantly enhances the ability to understand the immune response in these animals and fortifies their use as models of infectious disease.", "Text: The common marmoset (Callithrix jacchus), a New World monkey (NWM) species is a small, arboreal nonhuman primate (NHP), native to the Atlantic Coastal Forest in Northeast Brazil and parts of South East Brazil. In recent years the common marmoset has become more widely used in applied biomedical research, and an increasing body of evidence suggests the physiological and immunological responses to biological insults are similar between marmosets and humans [1] . In the field of infectious disease, the marmoset is primarily being investigated as an alternative NHP model to complement the more traditionally used Old World monkeys (OWM) (e.g., rhesus and cynomolgus macaques). Evolutionarily, both NWM and OWM sit within the simiiformes infraorder of the suborder Haplorhini of primates [2] . Marmosets sit within the family Callitrichidae of the Platyrrhini parvorder, while OWM sit within the Cercopithecidae family of the Catarrhini Parvorder. Marmosets therefore are separated from Old", "World monkeys by one ancestral step and are a lower order primate.", "Marmosets have been used to model the infection syndrome caused by a number of public health pathogens including Lassa virus [3] , Hepatitis C virus [4] , Dengue virus [5] , Herpesvirus [6] , Junin virus [7] Rift Valley Fever [8] , and SARS [9] . Marmosets have also been used to model a number of biodefense pathogens including Eastern Equine Encephalitis virus [10] , Bacillus anthracis [11] , Francisella tularensis [12, 13] , Burkholderia pseudomallei [14] , Marburg haemorrhagic fever virus [15, 16] , Ebola haemorrhagic fever virus [16] , and Variola virus [17] . The utility of marmosets to assess medical countermeasures has also been demonstrated; a vaccine has been tested for Lassa fever [18] and the efficacy of ciprofloxacin and levofloxacin has been tested as postexposure therapies for anthrax and tularemia, respectively [19, 20] .", "In order to exploit these models fully and to allow meaningful comparison with the human condition, the response of the immune system to infection/therapy needs to be 2 Journal of Immunology Research characterised and understood. Generally, NHPs have a close molecular, immunological, reproductive, and neurological similarity with humans making them ideal surrogates for humans and the study of infectious diseases. There is a high level of gene homology between humans and NHPs which underlies physiological and biochemical similarities. Similarities at the genetic level extend to the phenotypical level making NHPs well suited to modelling pathophysiological responses in man [21] . Immunologically, there is a high degree of homology between humans and marmosets [22] . The similarity of various immunological factors produced by humans and marmosets has been investigated at both the genetic and protein levels. There is at least 95% homology between human costimulatory molecules (e.g., CD80,", "CD86 etc.) and those of marmosets [23] . Also the immunoglobulin and T-cell receptor repertoire of humans and marmosets show at least 80% homology [24, 25] .", "Currently, the availability of commercial reagents specifically designed for the marmoset is limited although a number of antibodies designed for use with human samples have been shown to cross-react with leucocytes from marmoset blood [26] [27] [28] . However, these reagents have not been exploited to investigate the immune response to infectious disease. To date, investigation of the immune response in marmosets has primarily been achieved using pathogen-specific antibodies to determine the serological response using ELISA such as in the smallpox, Dengue, Rift Valley Fever, and Herpes models [5, 6, 8, 17] or by immunohistochemistry to identify, for example, CD8+, CD3+, CD20+ cells, and IL-6 in the smallpox model [17] ; neutrophils and macrophages in the Herpes model [6] ; or CD3+ and CD20+ cells in the Lassa model [3] .", "The work presented here focuses on understanding the immune profile of the naive marmoset as well as identifying and quantifying the immune response to infectious disease. The aim of this work is to determine key changes and identify correlates of infection or protection.", "Healthy sexually mature common marmosets (C. jacchus) were obtained from the Dstl Porton Down breeding colony and housed in vasectomized male and female pairs. The Dstl colony was established during the 1970s and is a closed colony with a stable genotype. Animals included in these studies were mixed sex pairs, between 18 months and 5 years old and weighing between 320 g to 500 g. All animals were allowed free access to food and water as well as environmental enrichment. All animal studies were carried out in accordance with the UK Animals (Scientific Procedures) Act of 1986 and the Codes of Practice for the Housing and Care of Animals used in Scientific Procedures 1989. Animals were challenged with an intracellular pathogen by either the subcutaneous or inhalational route and were humanely killed at various time points after challenge. Prior to the infection study, animals were bled to determine baseline immunological parameters. Studies were performed to establish infection models in", "order to evaluate the efficacy of suitable therapies for transition ultimately to the clinic.", "Populations. Blood and tissue samples were homogenised to provide single cell suspensions [12] . Red blood cells were lysed, and the mixed leucocyte population was washed and stained with various combinations of the following fluorescent antibody stains: CD3 (SP34-2), CD8 (LT8), CD11c (SHCL3), CD14 (M5E2), CD16 (3G8), CD20 (Bly1), CD45RA (5H9), CD54 (HCD54), CD56 (B159), CD69 (FN50), CD163 (GHI/61), and MCHII (L243) (BD Bioscience, Insight Bioscience, AbD serotec). Samples were fixed in 4% paraformaldehyde for 48 hrs at 4 ∘ C and analysed by flow cytometry (FACScanto II BD) within 72 hours of staining.", "Levels of circulating cytokines and chemokines were also quantified in the blood of marmosets from the Dstl colony using human multiplex kits available commercially (BD cytokine flex beads and the Luminex system). These systems show significant cross-reactivity with the marmoset suggesting a high degree of conservation between the two species for IL-6, MIP-1 , MIP-1 , and MCP-1 [29] . However, for other cytokines that are pivotal in the innate response, TNF and IFN reagents were obtained from U-CyTech Biosciences and Mabtech AB, respectively, due to a lack of cross-reactivity observed within the kit obtained from BD [13] .", "In order to fully characterise the immune response to infectious agent in the marmoset, single cell suspensions of lung and spleen tissue were also examined in conjunction with the traditionally used blood cells. These tissue homogenates are of particular interest in relation to target sites of infection: the lung as the site of initial infection following an inhalational challenge and the spleen as a representative organ following a parental challenge. Cell types targeted during this analysis include cells important in the innate response (e.g., neutrophils, macrophages, and NK cells) and the adaptive response (T and B cells) with a view to determine the response to infection and vaccination and to derive immune correlates of infection/protection. Dapi was included as a nuclear marker to ensure that the initial gating included only intact cells. Basic cell types in blood were easily identified by measuring size (forward) and granularity (side) scatter (Figure 1(a) ). Identification", "of cell types in tissue samples was more difficult as the scatter profiles are less clearly compartmentalized. The common leukocyte antigen (CD45) normally used to locate all leukocytes in human samples also worked well in marmoset blood but failed to provide relevant information in the tissue samples. Confirmation of neutrophil identification was done by nuclear morphology and macrophages were identified by their adherent nature in initial experiments (data not shown). Neutrophils were stained as CD11c dim CD14− and macrophages as CD11c + CD14+ regardless of tissue origin (Figure 1(b) ). Figure 1 shows the basic division of lymphocytes between T, B, and NK cells from a healthy blood sample.", "Using this approach, the percentage of NK cells, B-cells, total T-cells, CD8+ T-cells, neutrophils, and monocytes was determined in the blood of naive marmosets (Figure 2 (a), Table 1 ); approximately 63% of all lymphocytes were T cells, 25% B cells, and 12% NK cells. The variability of the data is depicted in Figure 2 (a) with the greatest variability observed in the proportion of neutrophils. There were no obvious differences attributable to age or sex of the animals. This analysis was also applied to lung and spleen homogenates from naive marmosets (Figures 2(b) and 2(c) ). Greater variability was observed in the data relating to the identification of cell types in tissue samples, attributed to the inherent difficulties in identifying cell types in tissue homogenates by size and granularity and also the smaller cohort of animals. As expected, low numbers of neutrophils are found in naive spleen or lung tissue (8% both). Healthy mouse spleens typically have approximately 1-2%", "granulocytes [30] .", "Understandably, there are few reports on the typical cell percentages expected in healthy human individuals for these tissues. However, it is reported that B cells are more prevalent in the spleens of humans at a ratio of 5 to 4 B to T cells than in the lungs which have a ratio of 1 to 8 B to T cells [34] . In marmoset data reported here, a ratio of 2 to 3 B to T cells in the spleen and 1 to 6 B to T-cells in the lungs was observed compared to a ratio of 3 to 2 B to T cells in mouse spleens [30] .", "Upon comparison, the marmoset data is generally consistent with previously reported data which is only available for marmoset blood samples [27] and information available for human blood [32, 33] (Table 1 ). However, one report found the proportion of CD8+ T-cells was almost three times greater in marmosets than humans, 61% to 21% respectively [35] compared to the 30% observed in this study and the work previously reported by Brok et al. [27] . Brok's study involved a small number of animals (eight) and also used a different CD8+ clone to identify cells. Contrastingly, in mice, differences are observed in the proportion of both B cells and neutrophils [31] , although these differences are highly strain specific. C57BL/6J mice are reported to have 67% B cells and BALB/C mice 46%; both of which are consistently higher than the percentage found in marmosets and humans of approximately 25% (Table 1 ) [27, 31] . The proportion of neutrophils found in the blood of C57BL/6J mice at 13% is", "lower than the 35% found in marmosets and the 40-75% expected for healthy human blood. This is encouraging as neutrophils play a pivotal role in the innate response to infection [36] . A cross-species comparison suggests that monocytes comprise 3% of leukocytes ( Table 1) . Levels of circulating cytokines and chemokines (IL-6, IL-1 , MIP-1 , MCP-1, Rantes, TNF , and IFN ) were also quantified in the blood, lung, and spleen of naïve marmosets from the Dstl colony. None of these cytokines were detected in blood samples from uninfected animals; however low levels of MIP-1 , MCP-1, and Rantes were found in spleen and lung tissue.", "Preliminary investigation of the immune response has supported the development of marmoset model of infection at Dstl. The levels of different cell types were measured at specific times after challenge with inhalational F. tularensis, B. pseudomallei, and Marburg virus [13] [14] [15] . Following challenge with F. tularensis, increasing levels of NK cells, neutrophils, T cells, and macrophages were observed, peaking at 48 hours after challenge before rapidly declining. This study also demonstrated the importance of investigating the immunological response in key target organs, as an increase in CD8+ T cells and T cells was observed in the spleen and lungs but not in the blood. Increasing levels of various cytokines, MCP-1, MIP-1 , MIP-1 , IL-6, and IL-1 , were observed in Table 1 : Comparison of the percentages of different cell types observed in the blood from healthy marmosets, mice, and humans.\n\nIdentification markers Marmoset (present data)", "Marmoset [27] Mouse 4 [30, 31] Human Asian [32] Human Caucasian [33] Number the lungs, spleen, and blood as the disease progressed (TNF and IFN were not measured in this study). Following inhalational challenge of marmosets with B. pseudomallei, an increase in the number of neutrophils was observed in the blood at 36 hours after challenge, followed by a rapid decline that was associated with an influx of neutrophils into the lung at 46 hours after challenge. A subsequent decline in the number of neutrophils in the lung was associated with the increased number in the spleen of animals that exhibited severe disease and were humanely killed. There was a gradual increase in the number of macrophages in the spleen as the disease progressed with numbers of macrophages peaking in the blood and lungs at 36 hours after challenge. A rapid decline in the number of macrophages in the lungs and blood was observed by 46 hours after challenge.", "The levels of various cell types and cytokines were also measured in the blood of animals following inhalational challenge with Marburg virus [15] . In these animals a general increase in the numbers of T cells, NK cells, macrophages IFN-, IL-1 , and MCP-1 was observed with time (TNF was not measured).", "In order to gain more information from these acute bacterial infection models, we have sought out other markers from the literature. Primarily this was from marmoset models of autoimmune disorders such as rheumatoid arthritis and multiple sclerosis where the cross-reactivity of human antibodies was investigated, as well as the functionality of cells [37] [38] [39] [40] . More recent work at Dstl has reported further cross-reactivity between marmoset cells and human cytokines to induce activity in marmoset T cells [36, 41] . These studies, combined with increasing information available on the cross-reactivity of human antibodies to various NHPs (e.g., NIH NHP reagent resource, http://www.nhpreagents.org/NHP/default.aspx), has expanded the ability to assess activation markers for disease. Detection of the following cell surface markers with human antibodies was trialed: CD54 (ICAM-1) associated with cellular adhesion, inflammation, and leukocyte extravasation; CD69 the early activation", "marker; CD16 as a macrophage activation marker; CD163 the alternative macrophage activation marker; and MHC class II (HLA-DR). CD56 was originally included to identify NK cells; however, it was noted that its expression on T cells was upregulated during disease and that cells defined as CD3+ CD16− CD56+ have been shown to be functionally cytotoxic in marmosets [37, 42] .", "These markers have been used to expand on our previously published work to determine changes in the activation status of basic cell types in response to an acute bacterial infection. Animals were challenged with bacteria at a comparable dose either by inhalation ( = 22) or by a systemic route ( = 12) and humanely killed once they had reached a humane endpoint (between day 4 and day 5 after challenge). Figure 3 illustrates the cellular activity in representative tissues following inhalational (Figures 3(b) and 3(e)) or systemic challenge (Figures 3(c) and 3(f)) and in naïve samples (Figures 3(a) and 3(d) ). Naïve T and NK cells appear to have similar resting activation states regardless of origin, whereas neutrophils and macrophages have differential expression of activation, for example, CD16. In response to disease, the proportions of the cell types appear to remain relativity constant; however, the activation markers provide more detailed information and show involvement of all the", "cell types explored. Extensive activation was to be expected considering that the samples were taken at the humane endpoint. There is also extensive variation between The response to infection within the lungs has similarities across disease routes in terms of neutrophil reduced expression of CD16 and CD54 and macrophage increased expression of CD16 and reduction in MHCII. Unexpectedly, the T and NK cells appear to be more actively involved in systemic disease, indicating that the disease develops a pneumonic element regardless of initial route of infection.", "Levels of circulating cytokines and chemokines (IL-6, IL-1 , MIP-1 , MCP-1, Rantes, TNF , and IFN ) were also quantified in the lung and spleen samples. All of the cytokines (with the exception of Rantes) were expressed at high levels (ng/mg) in all samples, which was expected as the animals had succumbed to terminal disease.", "The work presented here adds significant relevant information to the marmoset models of infection and to the understanding of the immune response in these animals. This work extends marmoset immunology from autoimmune disorders into the field of infectious diseases; this coupled with an increase in the information available on crossreactivity of human reagents to a variety of NHPs increases the utility/application of marmosets as models of human disease. In conclusion, the immune response in marmosets to infectious disease can be characterised in terms of the phenotype and activation status of all the major immune cells and key cytokine and chemokine expression. This can aid in the identification of correlates of infection or protection in medical countermeasures assessment studies. This information can also potentially be used for pivotal studies to support licensure of products under the FDA Animal Rule.", "This, in conjunction with the small size of marmosets, their immune response to infection that is comparable to humans, and the ability to house more statistically relevant numbers within high containment, makes the marmoset an appropriate animal model for biodefense-related pathogens." ]

[ 30 ]

[ "the small size of marmosets, their immune response to infection that is comparable to humans, and the ability to house more statistically relevant numbers within high containment" ]

[ "Exploring the Innate Immunological Response of an Alternative Nonhuman Primate Model of Infectious Disease; the Common Marmoset\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4129158/\n\nSHA: f4c43e4ae49ca69dbac32620bd0a73ecbb683b91\n\nAuthors: Nelson, M.; Loveday, M.\nDate: 2014-07-22\nDOI: 10.1155/2014/913632\nLicense: cc-by", "Abstract: The common marmoset (Callithrix jacchus) is increasingly being utilised as a nonhuman primate model for human disease, ranging from autoimmune to infectious disease. In order to fully exploit these models, meaningful comparison to the human host response is necessary. Commercially available reagents, primarily targeted to human cells, were utilised to assess the phenotype and activation status of key immune cell types and cytokines in naive and infected animals. Single cell suspensions of blood, spleen, and lung were examined. Generally, the phenotype of cells was comparable between humans and marmosets, with approximately 63% of all lymphocytes in the blood of marmosets being T cells, 25% B-cells, and 12% NK cells. The percentage of neutrophils in marmoset blood were more similar to human values than mouse values. Comparison of the activation status of cells following experimental systemic or inhalational infection exhibited different trends in different tissues, most", "obvious in cell types active in the innate immune response. This work significantly enhances the ability to understand the immune response in these animals and fortifies their use as models of infectious disease.", "Text: The common marmoset (Callithrix jacchus), a New World monkey (NWM) species is a small, arboreal nonhuman primate (NHP), native to the Atlantic Coastal Forest in Northeast Brazil and parts of South East Brazil. In recent years the common marmoset has become more widely used in applied biomedical research, and an increasing body of evidence suggests the physiological and immunological responses to biological insults are similar between marmosets and humans [1] . In the field of infectious disease, the marmoset is primarily being investigated as an alternative NHP model to complement the more traditionally used Old World monkeys (OWM) (e.g., rhesus and cynomolgus macaques). Evolutionarily, both NWM and OWM sit within the simiiformes infraorder of the suborder Haplorhini of primates [2] . Marmosets sit within the family Callitrichidae of the Platyrrhini parvorder, while OWM sit within the Cercopithecidae family of the Catarrhini Parvorder. Marmosets therefore are separated from Old", "World monkeys by one ancestral step and are a lower order primate.", "Marmosets have been used to model the infection syndrome caused by a number of public health pathogens including Lassa virus [3] , Hepatitis C virus [4] , Dengue virus [5] , Herpesvirus [6] , Junin virus [7] Rift Valley Fever [8] , and SARS [9] . Marmosets have also been used to model a number of biodefense pathogens including Eastern Equine Encephalitis virus [10] , Bacillus anthracis [11] , Francisella tularensis [12, 13] , Burkholderia pseudomallei [14] , Marburg haemorrhagic fever virus [15, 16] , Ebola haemorrhagic fever virus [16] , and Variola virus [17] . The utility of marmosets to assess medical countermeasures has also been demonstrated; a vaccine has been tested for Lassa fever [18] and the efficacy of ciprofloxacin and levofloxacin has been tested as postexposure therapies for anthrax and tularemia, respectively [19, 20] .", "In order to exploit these models fully and to allow meaningful comparison with the human condition, the response of the immune system to infection/therapy needs to be 2 Journal of Immunology Research characterised and understood. Generally, NHPs have a close molecular, immunological, reproductive, and neurological similarity with humans making them ideal surrogates for humans and the study of infectious diseases. There is a high level of gene homology between humans and NHPs which underlies physiological and biochemical similarities. Similarities at the genetic level extend to the phenotypical level making NHPs well suited to modelling pathophysiological responses in man [21] . Immunologically, there is a high degree of homology between humans and marmosets [22] . The similarity of various immunological factors produced by humans and marmosets has been investigated at both the genetic and protein levels. There is at least 95% homology between human costimulatory molecules (e.g., CD80,", "CD86 etc.) and those of marmosets [23] . Also the immunoglobulin and T-cell receptor repertoire of humans and marmosets show at least 80% homology [24, 25] .", "Currently, the availability of commercial reagents specifically designed for the marmoset is limited although a number of antibodies designed for use with human samples have been shown to cross-react with leucocytes from marmoset blood [26] [27] [28] . However, these reagents have not been exploited to investigate the immune response to infectious disease. To date, investigation of the immune response in marmosets has primarily been achieved using pathogen-specific antibodies to determine the serological response using ELISA such as in the smallpox, Dengue, Rift Valley Fever, and Herpes models [5, 6, 8, 17] or by immunohistochemistry to identify, for example, CD8+, CD3+, CD20+ cells, and IL-6 in the smallpox model [17] ; neutrophils and macrophages in the Herpes model [6] ; or CD3+ and CD20+ cells in the Lassa model [3] .", "The work presented here focuses on understanding the immune profile of the naive marmoset as well as identifying and quantifying the immune response to infectious disease. The aim of this work is to determine key changes and identify correlates of infection or protection.", "Healthy sexually mature common marmosets (C. jacchus) were obtained from the Dstl Porton Down breeding colony and housed in vasectomized male and female pairs. The Dstl colony was established during the 1970s and is a closed colony with a stable genotype. Animals included in these studies were mixed sex pairs, between 18 months and 5 years old and weighing between 320 g to 500 g. All animals were allowed free access to food and water as well as environmental enrichment. All animal studies were carried out in accordance with the UK Animals (Scientific Procedures) Act of 1986 and the Codes of Practice for the Housing and Care of Animals used in Scientific Procedures 1989. Animals were challenged with an intracellular pathogen by either the subcutaneous or inhalational route and were humanely killed at various time points after challenge. Prior to the infection study, animals were bled to determine baseline immunological parameters. Studies were performed to establish infection models in", "order to evaluate the efficacy of suitable therapies for transition ultimately to the clinic.", "Populations. Blood and tissue samples were homogenised to provide single cell suspensions [12] . Red blood cells were lysed, and the mixed leucocyte population was washed and stained with various combinations of the following fluorescent antibody stains: CD3 (SP34-2), CD8 (LT8), CD11c (SHCL3), CD14 (M5E2), CD16 (3G8), CD20 (Bly1), CD45RA (5H9), CD54 (HCD54), CD56 (B159), CD69 (FN50), CD163 (GHI/61), and MCHII (L243) (BD Bioscience, Insight Bioscience, AbD serotec). Samples were fixed in 4% paraformaldehyde for 48 hrs at 4 ∘ C and analysed by flow cytometry (FACScanto II BD) within 72 hours of staining.", "Levels of circulating cytokines and chemokines were also quantified in the blood of marmosets from the Dstl colony using human multiplex kits available commercially (BD cytokine flex beads and the Luminex system). These systems show significant cross-reactivity with the marmoset suggesting a high degree of conservation between the two species for IL-6, MIP-1 , MIP-1 , and MCP-1 [29] . However, for other cytokines that are pivotal in the innate response, TNF and IFN reagents were obtained from U-CyTech Biosciences and Mabtech AB, respectively, due to a lack of cross-reactivity observed within the kit obtained from BD [13] .", "In order to fully characterise the immune response to infectious agent in the marmoset, single cell suspensions of lung and spleen tissue were also examined in conjunction with the traditionally used blood cells. These tissue homogenates are of particular interest in relation to target sites of infection: the lung as the site of initial infection following an inhalational challenge and the spleen as a representative organ following a parental challenge. Cell types targeted during this analysis include cells important in the innate response (e.g., neutrophils, macrophages, and NK cells) and the adaptive response (T and B cells) with a view to determine the response to infection and vaccination and to derive immune correlates of infection/protection. Dapi was included as a nuclear marker to ensure that the initial gating included only intact cells. Basic cell types in blood were easily identified by measuring size (forward) and granularity (side) scatter (Figure 1(a) ). Identification", "of cell types in tissue samples was more difficult as the scatter profiles are less clearly compartmentalized. The common leukocyte antigen (CD45) normally used to locate all leukocytes in human samples also worked well in marmoset blood but failed to provide relevant information in the tissue samples. Confirmation of neutrophil identification was done by nuclear morphology and macrophages were identified by their adherent nature in initial experiments (data not shown). Neutrophils were stained as CD11c dim CD14− and macrophages as CD11c + CD14+ regardless of tissue origin (Figure 1(b) ). Figure 1 shows the basic division of lymphocytes between T, B, and NK cells from a healthy blood sample.", "Using this approach, the percentage of NK cells, B-cells, total T-cells, CD8+ T-cells, neutrophils, and monocytes was determined in the blood of naive marmosets (Figure 2 (a), Table 1 ); approximately 63% of all lymphocytes were T cells, 25% B cells, and 12% NK cells. The variability of the data is depicted in Figure 2 (a) with the greatest variability observed in the proportion of neutrophils. There were no obvious differences attributable to age or sex of the animals. This analysis was also applied to lung and spleen homogenates from naive marmosets (Figures 2(b) and 2(c) ). Greater variability was observed in the data relating to the identification of cell types in tissue samples, attributed to the inherent difficulties in identifying cell types in tissue homogenates by size and granularity and also the smaller cohort of animals. As expected, low numbers of neutrophils are found in naive spleen or lung tissue (8% both). Healthy mouse spleens typically have approximately 1-2%", "granulocytes [30] .", "Understandably, there are few reports on the typical cell percentages expected in healthy human individuals for these tissues. However, it is reported that B cells are more prevalent in the spleens of humans at a ratio of 5 to 4 B to T cells than in the lungs which have a ratio of 1 to 8 B to T cells [34] . In marmoset data reported here, a ratio of 2 to 3 B to T cells in the spleen and 1 to 6 B to T-cells in the lungs was observed compared to a ratio of 3 to 2 B to T cells in mouse spleens [30] .", "Upon comparison, the marmoset data is generally consistent with previously reported data which is only available for marmoset blood samples [27] and information available for human blood [32, 33] (Table 1 ). However, one report found the proportion of CD8+ T-cells was almost three times greater in marmosets than humans, 61% to 21% respectively [35] compared to the 30% observed in this study and the work previously reported by Brok et al. [27] . Brok's study involved a small number of animals (eight) and also used a different CD8+ clone to identify cells. Contrastingly, in mice, differences are observed in the proportion of both B cells and neutrophils [31] , although these differences are highly strain specific. C57BL/6J mice are reported to have 67% B cells and BALB/C mice 46%; both of which are consistently higher than the percentage found in marmosets and humans of approximately 25% (Table 1 ) [27, 31] . The proportion of neutrophils found in the blood of C57BL/6J mice at 13% is", "lower than the 35% found in marmosets and the 40-75% expected for healthy human blood. This is encouraging as neutrophils play a pivotal role in the innate response to infection [36] . A cross-species comparison suggests that monocytes comprise 3% of leukocytes ( Table 1) . Levels of circulating cytokines and chemokines (IL-6, IL-1 , MIP-1 , MCP-1, Rantes, TNF , and IFN ) were also quantified in the blood, lung, and spleen of naïve marmosets from the Dstl colony. None of these cytokines were detected in blood samples from uninfected animals; however low levels of MIP-1 , MCP-1, and Rantes were found in spleen and lung tissue.", "Preliminary investigation of the immune response has supported the development of marmoset model of infection at Dstl. The levels of different cell types were measured at specific times after challenge with inhalational F. tularensis, B. pseudomallei, and Marburg virus [13] [14] [15] . Following challenge with F. tularensis, increasing levels of NK cells, neutrophils, T cells, and macrophages were observed, peaking at 48 hours after challenge before rapidly declining. This study also demonstrated the importance of investigating the immunological response in key target organs, as an increase in CD8+ T cells and T cells was observed in the spleen and lungs but not in the blood. Increasing levels of various cytokines, MCP-1, MIP-1 , MIP-1 , IL-6, and IL-1 , were observed in Table 1 : Comparison of the percentages of different cell types observed in the blood from healthy marmosets, mice, and humans.\n\nIdentification markers Marmoset (present data)", "Marmoset [27] Mouse 4 [30, 31] Human Asian [32] Human Caucasian [33] Number the lungs, spleen, and blood as the disease progressed (TNF and IFN were not measured in this study). Following inhalational challenge of marmosets with B. pseudomallei, an increase in the number of neutrophils was observed in the blood at 36 hours after challenge, followed by a rapid decline that was associated with an influx of neutrophils into the lung at 46 hours after challenge. A subsequent decline in the number of neutrophils in the lung was associated with the increased number in the spleen of animals that exhibited severe disease and were humanely killed. There was a gradual increase in the number of macrophages in the spleen as the disease progressed with numbers of macrophages peaking in the blood and lungs at 36 hours after challenge. A rapid decline in the number of macrophages in the lungs and blood was observed by 46 hours after challenge.", "The levels of various cell types and cytokines were also measured in the blood of animals following inhalational challenge with Marburg virus [15] . In these animals a general increase in the numbers of T cells, NK cells, macrophages IFN-, IL-1 , and MCP-1 was observed with time (TNF was not measured).", "In order to gain more information from these acute bacterial infection models, we have sought out other markers from the literature. Primarily this was from marmoset models of autoimmune disorders such as rheumatoid arthritis and multiple sclerosis where the cross-reactivity of human antibodies was investigated, as well as the functionality of cells [37] [38] [39] [40] . More recent work at Dstl has reported further cross-reactivity between marmoset cells and human cytokines to induce activity in marmoset T cells [36, 41] . These studies, combined with increasing information available on the cross-reactivity of human antibodies to various NHPs (e.g., NIH NHP reagent resource, http://www.nhpreagents.org/NHP/default.aspx), has expanded the ability to assess activation markers for disease. Detection of the following cell surface markers with human antibodies was trialed: CD54 (ICAM-1) associated with cellular adhesion, inflammation, and leukocyte extravasation; CD69 the early activation", "marker; CD16 as a macrophage activation marker; CD163 the alternative macrophage activation marker; and MHC class II (HLA-DR). CD56 was originally included to identify NK cells; however, it was noted that its expression on T cells was upregulated during disease and that cells defined as CD3+ CD16− CD56+ have been shown to be functionally cytotoxic in marmosets [37, 42] .", "These markers have been used to expand on our previously published work to determine changes in the activation status of basic cell types in response to an acute bacterial infection. Animals were challenged with bacteria at a comparable dose either by inhalation ( = 22) or by a systemic route ( = 12) and humanely killed once they had reached a humane endpoint (between day 4 and day 5 after challenge). Figure 3 illustrates the cellular activity in representative tissues following inhalational (Figures 3(b) and 3(e)) or systemic challenge (Figures 3(c) and 3(f)) and in naïve samples (Figures 3(a) and 3(d) ). Naïve T and NK cells appear to have similar resting activation states regardless of origin, whereas neutrophils and macrophages have differential expression of activation, for example, CD16. In response to disease, the proportions of the cell types appear to remain relativity constant; however, the activation markers provide more detailed information and show involvement of all the", "cell types explored. Extensive activation was to be expected considering that the samples were taken at the humane endpoint. There is also extensive variation between The response to infection within the lungs has similarities across disease routes in terms of neutrophil reduced expression of CD16 and CD54 and macrophage increased expression of CD16 and reduction in MHCII. Unexpectedly, the T and NK cells appear to be more actively involved in systemic disease, indicating that the disease develops a pneumonic element regardless of initial route of infection.", "Levels of circulating cytokines and chemokines (IL-6, IL-1 , MIP-1 , MCP-1, Rantes, TNF , and IFN ) were also quantified in the lung and spleen samples. All of the cytokines (with the exception of Rantes) were expressed at high levels (ng/mg) in all samples, which was expected as the animals had succumbed to terminal disease.", "The work presented here adds significant relevant information to the marmoset models of infection and to the understanding of the immune response in these animals. This work extends marmoset immunology from autoimmune disorders into the field of infectious diseases; this coupled with an increase in the information available on crossreactivity of human reagents to a variety of NHPs increases the utility/application of marmosets as models of human disease. In conclusion, the immune response in marmosets to infectious disease can be characterised in terms of the phenotype and activation status of all the major immune cells and key cytokine and chemokine expression. This can aid in the identification of correlates of infection or protection in medical countermeasures assessment studies. This information can also potentially be used for pivotal studies to support licensure of products under the FDA Animal Rule.", "This, in conjunction with the small size of marmosets, their immune response to infection that is comparable to humans, and the ability to house more statistically relevant numbers within high containment, makes the marmoset an appropriate animal model for biodefense-related pathogens." ]

[ 30 ]

[ "the presence of resistance-associated substitutions" ]

[ "Pretreatment Hepatitis C Virus NS5A/NS5B Resistance-Associated Substitutions in Genotype 1 Uruguayan Infected Patients\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6112080/\n\nSHA: f01ad3545245b4f884b48aa2b69c9deb942c3e77\n\nAuthors: Aldunate, Fabián; Echeverría, Natalia; Chiodi, Daniela; López, Pablo; Sánchez-Cicerón, Adriana; Fajardo, Alvaro; Soñora, Martín; Cristina, Juan; Hernández, Nelia; Moreno, Pilar\nDate: 2018-08-14\nDOI: 10.1155/2018/2514901\nLicense: cc-by", "Abstract: Hepatitis C Virus (HCV) infection treatment has dramatically changed with the advent of direct-acting antiviral agents (DAAs). However, the efficacy of DAAs can be attenuated by the presence of resistance-associated substitutions (RASs) before and after treatment. Indeed, RASs detected in DAA treatment-naïve HCV-infected patients could be useful for clinical management and outcome prediction. Although the frequency of naturally occurring HCV NS5A and NS5B RASs has been addressed in many countries, there are only a few reports on their prevalence in the South American region. The aim of this study was to investigate the presence of RASs to NS5A and NS5B inhibitors in a DAA treatment naïve cohort of Uruguayan patients infected with chronic hepatitis C and compare them with reports from other South American countries. Here, we found that naturally occurring substitutions conferring resistance to NS5A and NS5B inhibitors were present in 8% and 19.2%, respectively, of", "treatment-naïve HCV genotype 1 infected patients. Importantly, the baseline substitutions in NS5A and NS5B herein identified differ from the studies previously reported in Brazil. Furthermore, Uruguayan strains subtype 1a clustered within all major world clades, showing that HCV variants currently circulating in this country are characterized by a remarkable genetic diversity.", "Text: Hepatitis C Virus (HCV) infection treatment has dramatically improved thanks to the introduction of direct-acting antiviral agents (DAAs). These antivirals have significantly increased response rates (up to 98%) and greatly reduced treatment duration [1] . Currently available DAAs are classified into four categories given their molecular targets in the HCV replication cycle: (1) NS3/4A protease inhibitors (PIs) bind to the active site of the NS3/4A protease; (2) NS5A inhibitors interact with domain 1 of the NS5A dimer, although the exact mechanism of NS5A inhibition remains to be fully elucidated; (3) nucleos(t)ide analog NS5B polymerase inhibitors are incorporated into the nascent RNA chain resulting in chain termination by compromising the binding of the incoming nucleotide; (4) nonnucleoside NS5B polymerase inhibitors interact with either the thumb 1, thumb 2, palm 1, or palm 2 domain of NS5B and inhibit polymerase activity by allosteric mechanisms [2] [3] [4] . However, the", "extreme mutation and high replication rates of HCV, together with the immune system pressure, lead to a remarkable genetic variability that can compromise the high response rates to DAAs due to the preexistence of resistanceassociated substitutions (RASs) [5, 6] .", "Each drug or class of DAA is characterized by specific resistance profiles. The likelihood that a DAA will select for and allow outgrowth of viral populations carrying RASs depends on the DAA's genetic barrier to resistance (the number and type of mutations needed to generate an amino acid substitution that confers resistance), the viral fitness (replicative capacity) of the resistant variant, and viral genotypes and subtypes [7, 8] .\n\nThe prevalence of RASs in treatment-naïve patients has been broadly reported worldwide [9] [10] [11] [12] [13] [14] [15] [16] . However, apart from Brazil and Argentina, this issue has not been fully addressed in other South American countries yet [9, [17] [18] [19] . The lack of information in relation to preexisting baseline RASs, added to the high cost of these new drugs, are the major limiting factors for the broad implementation of these new therapies in Uruguay as well as in other Latin American countries (low-or lower-middle income) [20] .", "In this study, we explored the presence of resistance variants to NS5A and NS5B inhibitors in a DAA treatment naïve cohort of Uruguayan patients chronically infected with hepatitis C. Here, we aimed to contribute to the knowledge of the circulation of HCV resistant variants in the South American region.", "Samples. Serum samples were obtained from 31 patients with serological markers for HCV, which were recruited between 2015 and 2017 at the Gastroenterology Clinic from Hospital de Clínicas, Montevideo, Uruguay. HCV infection was confirmed by Abbott realtime HCV (Abbott Molecular Inc., Des Plaines, USA). Patients selected for this study were both chronically infected with HCV genotype 1 and DAA treatment-naïve at the time of blood extraction. Written informed consent was obtained from all patients. The studies have been performed according to the World Medical Association Declaration of Helsinki and approved by the appropriate institutional board (Hospital de Clínicas ethical committee).", "2.2. RNA Extraction, cDNA Synthesis, and NS5A and NS5B Amplification. Viral RNA was extracted from 140 μl of serum using the QIAamp Viral RNA mini kit (QIAgen, Hilden, Germany) according to the manufacturer's protocol. The viral RNA was heated at 65°C for 5 min and used as a template for a reverse transcription reaction. The reverse transcription reaction mixture contained 5 μl of the RNA template, 1 μl of random hexamer 100 ng/μl (Invitrogen Life Technologies, Carlsbad, CA, USA), 1 μl of dNTP mix (10 mM each), 4 μl of 5X first-strand buffer, 2 μl of 0.1 M DTT, 1 μl of SuperScript II reverse transcriptase (200 U/μl) (Invitrogen Life Technologies, Carlsbad, CA, USA), and 1 μl (40 U/μl) RNaseOUT (Invitrogen Life Technologies, Carlsbad, CA, USA). The reverse transcription was performed at 42°C for 50 min, and then the reverse transcriptase enzyme was inactivated at 70°C for 15 min. PCR amplification of NS5A and NS5B genome regions was performed using primers and conditions previously", "described [10] . Amplicons were purified using the Illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare Life Science, Buckinghamshire, UK) according to the manufacturer's protocol.", "2.3. NS5A and NS5B Sequencing. The purified product was then sequenced using the same sets of primers used for PCR amplification. Bidirectional Sanger sequencing was performed by Macrogen Korea (http://www.macrogen.com).", "2.4. NS5A and NS5B Genotype Determination. HCV NS5A and NS5B consensus sequences obtained from Uruguayan patients were aligned with sequences from HCV representing all genotypes and main subtypes isolated in different geographic regions of the world. These sequences were obtained from Los Alamos HCV sequence database and from the NIAID Virus Pathogen Database and Analysis Resource (ViPR) [21, 22] . For strains included in these studies, see Supplementary Material Table S1 . Sequences were aligned using the CLUSTAL W software [23] . Once aligned, the best evolutionary model that described our sequence data was assessed using ModelGenerator program [24] . Using the GTR + G + I model (General time reversible + gamma + invariant sites), maximum likelihood phylogenetic trees were constructed for both NS5A and NS5B using the MEGA 5.0 software [25] . For NS5A, 953 nucleotides (positions 6367 to 7319, relative to HCV 1a reference strain, H77 NC_004102) were included in the phylogenetic", "analysis, whereas for NS5B, only 361 nucleotides corresponding to the Okamoto region (positions 8265 to 8625, relative to strain H77 NC_004102) were included. As a measure of the robustness of each node, we employed the bootstrapping method (1000 pseudoreplicates).", "For NS5A 1a Uruguayan sequences (n = 20), a second alignment and maximum likelihood phylogenetic tree was generated in order to analyze HCV evolutionary relationships between Uruguayan, Brazilian, and worldwide strains. For non-Uruguayan strains included in this analysis, see Supplementary Material Table S2. 2.5. NS5A and NS5B Sequence Analysis. In order to properly identify substitution changes in NS5A and NS5B regions from HCV strains circulating in Uruguayan patients, we generated world consensus sequences for 1a and 1b subtypes using a wide range of NS5A and NS5B sequences from HCV strains isolated worldwide. For this purpose, NS5A gene sequences corresponding to subtypes 1a (n = 160) and 1b (n = 88) were retrieved from Los Alamos HCV sequence database and from the NIAID ViPR [21, 22] . Likewise, datasets of 150 and 124 NS5B sequences were generated for subtypes 1a and 1b, respectively. Using Seqman program, implemented in DNAStar 5.01 package (DNASTAR, Madison, USA), a world", "consensus nucleotide sequences were generated for each gene and subtype. Each Uruguayan sequence was subsequently aligned to the corresponding reference sequences, and then in silico translated. The amino acid sequences obtained were compared in order to explore the presence of RASs as well as the presence of polymorphisms at a RAS position (RAPs) in Uruguayan HCV strains. RAPs are defined as any change from reference sequence for a specific genotype at a position associated with NS5A resistance [26] .", "To study the genetic variability of NS5A and NS5B regions of HCV strains circulating in Uruguayan patients, sequences of these regions (accession numbers MH070029-MH070090) were aligned with corresponding sequences from 59 HCV strains isolated elsewhere, representing all genotypes and main subtypes (for strains included in these analyses, see Supplementary Material Table S1 ). Therefore, maximum likelihood phylogenetic trees were constructed. The results of these studies are shown in Figure 1 All strains in the phylogenies were assigned according to their genotype, and each cluster was supported by very high bootstrap values for both analyzed regions. Strains isolated from Uruguayan patients (n = 31) were assigned to genotype 1, 20 of which corresponded to subtype 1a and 11 to subtype 1b. The results of NS5A (Figure 1 (a)) and NS5B (Figure 1", "Genotype 1b phylogenetic analyses were concordant for both genomic regions in all 31 sequences, suggesting no recombination events between these regions.\n\nTo further analyze the evolutionary relationships between the Uruguayan strains and those circulating in Brazil and elsewhere, a second maximum likelihood phylogenetic tree of HCV-1a sequences of NS5A partial region was built ( Figure 2 ). As was previously described, two distinct 1a clades (clades 1 and 2) were observed. Brazilian sequences clustered in a large group of related sequences inside clade 1 [9] . Whereas NS5A Uruguayan strains (in red) did not cluster in a particular clade, rather, they grouped dispersedly within all major world clades.", "With the purpose of studying the amino acid (AA) substitutions along the NS5A protein, Uruguayan HCV AA sequences were aligned with NS5A world consensus sequences (residues 23 to 354 relative to NS5A protein sequence). AA substitutions at positions previously found to be potentially associated with resistance to NS5A inhibitors, as well as polymorphisms at a RAS position, were identified. These results are summarized in Table 1 .", "RASs to NS5A inhibitors (L31M and L31V) were identified in 2 strains out of 25 (8%) fully sequenced samples. RAPs were found in 3 strains (subtype 1a): 2 exhibited the substitution H58P and 1 the substitution K24Q. Although these substitutions were not reported as resistant, some changes at these positions were previously described as RASs in subtype 1a, namely H58D and K24R [27, 28] . Finally, substitution E62D was found in one subtype 1a strain. This change is considered as a secondary substitution because, although it does not confer resistance by itself, when combined with a known RAS it does. In fact, it confers a higher level of resistance than the one achieved by the RAS alone [26] . In addition, several polymorphisms that have not been previously reported to be associated with a resistant phenotype were also detected (see Supplementary Material Table S3 ).", "In order to study substitutions along NS5B protein, Uruguayan HCV AA sequences were aligned to the NS5B world consensus sequences. Almost full-length AA sequences were obtained in 26 out of 31 analyzed strains. 23 sequences span residues 36 to 539 whereas the remaining 3 span residues 36 to 557 of NS5B protein.\n\nThis issue limited our studies, since many of the described RASs are observed as of residue 553.\n\nImportantly, RASs to NS5B inhibitors ( Table 2) were observed in 5 strains out of 26 sequenced samples (19.2%). C451R was found in two isolates while A421V was found in only one. In 2 of the 3 strains for which we were able to obtain longer sequences, RASs S556G (subtype 1a) and Q556R (subtype 1b) were observed.", "Finally, we found two RAPs: A421V (in 2 subtype 1b strains) and A553G (in 1 subtype 1a strain). Although A421V has been associated with resistance to beclabuvir (BCV) in patients infected with HCV subtype 1a, this resistant phenotype has not been proven in strains subtype 1b [29] . In position 553, the substitution reported as resistant was A553T [8] .\n\nAs was the case for NS5A, different polymorphisms not previously associated with a resistant phenotype were also detected in NS5B (see Supplementary Material Table S4 ).", "The advent of DAAs therapies constitutes one of the major breakthroughs in HCV infected patients management. However, these new treatment options are far from being universally available, in particular for HCV infected patients relying on Latin American public healthcare systems. The main limiting factors for worldwide access to DAAs in our region concern the high cost, the inadequate management of public healthcare systems, the limited access of low-income or uninsured populations to healthcare providers, and the lack of accurate epidemiological information [20, [30] [31] [32] . In Uruguay, these therapies became recently available, and although some have been approved for their use by the public health authorities (Viekira pak and sofosbuvir/ledipasvir therapies), they are not currently financially covered, except in specific cases. Despite the high rates of viral response achieved with DAA-based treatments, still 1 to10% of the patients fails to eliminate infection, and in these", "cases, baseline and emergent resistance variants turn out to be key factors contributing to treatment failure [5, 17, 33] .", "Unfortunately, we are currently unable to properly assess the number of HCV infected people in Uruguay and even more to figure out the frequency and type of RASs circulating. These facts could compromise the effectiveness of these new therapies in our country.", "We have previously reported that naturally occurring substitutions conferring resistance to NS3 inhibitors exist in a significant proportion of Uruguayan patients infected with HCV genotype 1, and we showed that this frequency seemed to be higher than in other South American countries (Brazil and Argentina) [34] . The present study describes the prevalence of baseline NS5A and NS5B RASs in HCV genotype 1 infected DAA-naïve patients in a Uruguayan cohort. The presence of substitutions conferring resistance to NS5A inhibitors has been widely reported both in therapynaïve and in relapser patients from Europe [10, 33, [35] [36] [37] [38] , USA [37, 39, 40] , and Asia [41] [42] [43] . However, NS5A sequences from South America are poorly analyzed yet [9, 44] . Recent studies have revealed that the mean prevalence of NS5A genotype 1 baseline RASs to different inhibitors ranges from 6% to 16% using population sequencing or deep sequencing [27, 37, 45, 46] . Importantly, the prevalence and", "type of baseline NS5A RASs varies slightly by geographic regions. For instance, L31M was found in 2.2% of genotype 1a infected patients in Europe, in 4.1% of those in Oceania, and strikingly in no patient from the USA [27] . For this reason, we believe that there is a need to contribute data from our region, for which we still do not have enough information, apart from Brazil [9, 44] . The results of this study indicate the presence of DAA NS5A RASs in 2 HCV strains (8% of the patients enrolled in this study), with baseline RASs detected at position 31 (see Table 1 ). L31M substitution confers resistance to daclatasvir (DCV), ledipasvir (LDV), and elbasvir (EBV) in both 1a and 1b subtypes [5, 6, 8, 28, 47, 48] , whereas substitution L31V does it to DCV in subtypes 1a and 1b, to LDV in subtype 1b, and to EBV in subtype 1a [5, 6, 28] . Given that both L31V and L31M are clinically relevant RASs, their detection at baseline may influence the choice of first-line treatment regimens [28] .", "The substitutions H58P and K24Q found in two patients are considered as resistance-associated polymorphisms (RAPs). The RASs characterized at these positions were H58D and K24G/N/R [5, 6, 27, 28, 49, 50] . The substitution H58P was found as a baseline RAP in relapsers to LDV (HARVONI prescription, https://www.gilead.com/-/ media/files/pdfs/medicines/liver-disease/harvoni/harvoni_pi. pdf?la=en). However, it is sometimes regarded as a RAS [10, 51] , despite conferring only 1.2 fold change in resistance in in vitro studies using the 1a replicon system [39] .\n\nWe did not find M28T/V, Q30R/H, or Y93H substitutions as there were previously reported in Brazil and worldwide [9, 27, 44] . The amino acid substitution E62H was found in one Uruguayan patient. Although this change does not confer resistance by itself but in combination with Q30R, it generates a high resistance level to DCV [52] .", "The presence of baseline NS5A RASs impacts treatment outcome in some patient groups by affecting SVR rates. The detection of NS5A preexistent RASs may play a relevant role in the choice of first-line treatment regimens or in the simplification/shortening of recommended regimens, in order to bring SVR rates close to the highest achievable [27, 38, 41, 53] , in particular in countries such as Uruguay, where only two different DAA-containing treatment regimens are approved for their use.", "Regarding NS5B gene, global analysis (with the exception of South America [17, 19] ) revealed that NS5B DAA resistance substitutions are infrequent [14] . Our study showed the presence of NS5B inhibitors RASs in 5 out of 26 analyzed HCV infected Uruguayan patients naïve to treatment (19.2%). Substitutions found in this work were A421V and S556G associated in subtype 1a with resistance to BCV and dasabuvir (DSV), respectively [8, 28, 29, 54, 55] , and Q556R associated with resistance to DSV both in genotype 1a and 1b [12, 28] . Substitution C451R, observed in two Uruguayan patients, was reported previously in patients who failed to clear the infection after treatment with OBV/PTV/r + DSV ± RBV. In these cases, it appeared in combination with G558R (Trial Coral I-Cohort 2: http:// www.hcv-trials.com/showStudy.asp?Study=86). RAPs in positions 421 and 553 (A421V in two subtype 1b isolates and A553G in one subtype 1b isolate) were also found. Although A421V has been associated with", "resistance to BCV in patients with subtype 1a, this phenotype has not been proven in strains of subtype 1b [29] . In position 553, the substitutions reported as resistant are A553T in subtype 1a [8] and A553V in subtype 1b [54] , conferring resistance to DSV.", "In contrast to our results, Noble and coworkers (2016) reported the presence of V321A, A421G, M414V, Y448H, L159F, and C316N in Brazilian isolates [17] , yet none of these mutations were found in this study, probably due to the diversity found between Uruguayan and Brazilian strains ( Figure 2 ). Nevertheless, substitution A421V was found in Brazil [17] , Argentina [19] , and Uruguay. The RAS S282T was detected neither in Brazilian reports nor in this current work (Uruguay) [17, 18, 56] . Our findings further confirm and complement previous studies which evidenced a low prevalence of this substitution in vivo, probably due to its low replicative fitness [14, 18, 57] . Despite our results, it is worth mentioning that the presence of baseline NS5B RASs conferring resistance to nucleotide or nonnucleoside NS5B inhibitors has not been shown to have any impact on virologic responses thus far [53, 58] .", "These results show both diversity in the baseline polymorphisms found in different Latin American countries and in the evolutionary relationships of Uruguayan isolates ( Figure 2 ). This fact could be linked not only to the isolates' geographic region and viral intrinsic characteristics but also to the genetic background of the host. It is worth mentioning that we live in a vast continent inhabited by populations with different genotypic characteristics that might, depending on the situation, require different approaches to treatment. Indeed, we have recently found that allele and genotype frequencies at IL28B locus of Uruguayan individuals closely resemble those of an admixed population rather than a uniformly European-descendant one [59] . Altogether, we believe that it could be important to carry out studies throughout the South American region in order to establish the prevalence of RASs in NS5A and NS5B in different countries. In fact, this will aid in understanding that not", "every treatment regimen might be adequate for every patient and country. The data we presented here might guide not only physicians in making therapeutic decisions but also public health authorities in approving more diverse treatment combinations. These treatment formulations would cover most of the circulating strains in our region, a region with an extremely diverse genetic background population.", "To our knowledge, the present study revealed for the first time the presence of RASs in the NS5A and NS5B regions of HCV genotype 1 Uruguayan strains from patients who have not been previously treated with DAAs and is one of the few South American countries to report on this matter. It is currently unclear if preexisting viral variants with reduced susceptibility to DAAs are clinically relevant for the prediction of virologic treatment failure. However, individualized DAA therapy based on baseline resistance analysis may be beneficial for optimizing treatment efficacy in patients with HCV genotype 1 infection and risk factors for treatment failure. Therefore, the potential role of baseline resistance testing remains an area of critical research and clinical questions.\n\nThe data used to support the findings of this study are included within the article.\n\nThe authors declare that they have no conflicts of interest.", "Fabián Aldunate and Natalia Echeverría contributed equally to this work.\n\nSupplementary Material Table S1 : hepatitis C Virus NS5A and NS5B sequences used as representatives of each genotype to perform the phylogenetic analysis. Their corresponding genotype, country of isolation, and GenBank accession number are indicated. Supplementary Material Table S2 : hepatitis C Virus NS5A subtype 1a sequences used to reveal evolutionary relationships between Uruguayan strains and others isolated elsewhere. Their corresponding country of isolation and GenBank accession number are indicated. Supplementary Material Table S3 : amino acid substitutions in NS5A protein not previously associated with resistance to NS5A inhibitors. Supplementary Material Table S4 : amino acid substitutions in NS5B protein not previously associated with resistance to polymerase inhibitors. (Supplementary Materials)" ]

[ 1 ]

[ "up to 98%" ]

[ "Pretreatment Hepatitis C Virus NS5A/NS5B Resistance-Associated Substitutions in Genotype 1 Uruguayan Infected Patients\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6112080/\n\nSHA: f01ad3545245b4f884b48aa2b69c9deb942c3e77\n\nAuthors: Aldunate, Fabián; Echeverría, Natalia; Chiodi, Daniela; López, Pablo; Sánchez-Cicerón, Adriana; Fajardo, Alvaro; Soñora, Martín; Cristina, Juan; Hernández, Nelia; Moreno, Pilar\nDate: 2018-08-14\nDOI: 10.1155/2018/2514901\nLicense: cc-by", "Abstract: Hepatitis C Virus (HCV) infection treatment has dramatically changed with the advent of direct-acting antiviral agents (DAAs). However, the efficacy of DAAs can be attenuated by the presence of resistance-associated substitutions (RASs) before and after treatment. Indeed, RASs detected in DAA treatment-naïve HCV-infected patients could be useful for clinical management and outcome prediction. Although the frequency of naturally occurring HCV NS5A and NS5B RASs has been addressed in many countries, there are only a few reports on their prevalence in the South American region. The aim of this study was to investigate the presence of RASs to NS5A and NS5B inhibitors in a DAA treatment naïve cohort of Uruguayan patients infected with chronic hepatitis C and compare them with reports from other South American countries. Here, we found that naturally occurring substitutions conferring resistance to NS5A and NS5B inhibitors were present in 8% and 19.2%, respectively, of", "treatment-naïve HCV genotype 1 infected patients. Importantly, the baseline substitutions in NS5A and NS5B herein identified differ from the studies previously reported in Brazil. Furthermore, Uruguayan strains subtype 1a clustered within all major world clades, showing that HCV variants currently circulating in this country are characterized by a remarkable genetic diversity.", "Text: Hepatitis C Virus (HCV) infection treatment has dramatically improved thanks to the introduction of direct-acting antiviral agents (DAAs). These antivirals have significantly increased response rates (up to 98%) and greatly reduced treatment duration [1] . Currently available DAAs are classified into four categories given their molecular targets in the HCV replication cycle: (1) NS3/4A protease inhibitors (PIs) bind to the active site of the NS3/4A protease; (2) NS5A inhibitors interact with domain 1 of the NS5A dimer, although the exact mechanism of NS5A inhibition remains to be fully elucidated; (3) nucleos(t)ide analog NS5B polymerase inhibitors are incorporated into the nascent RNA chain resulting in chain termination by compromising the binding of the incoming nucleotide; (4) nonnucleoside NS5B polymerase inhibitors interact with either the thumb 1, thumb 2, palm 1, or palm 2 domain of NS5B and inhibit polymerase activity by allosteric mechanisms [2] [3] [4] . However, the", "extreme mutation and high replication rates of HCV, together with the immune system pressure, lead to a remarkable genetic variability that can compromise the high response rates to DAAs due to the preexistence of resistanceassociated substitutions (RASs) [5, 6] .", "Each drug or class of DAA is characterized by specific resistance profiles. The likelihood that a DAA will select for and allow outgrowth of viral populations carrying RASs depends on the DAA's genetic barrier to resistance (the number and type of mutations needed to generate an amino acid substitution that confers resistance), the viral fitness (replicative capacity) of the resistant variant, and viral genotypes and subtypes [7, 8] .\n\nThe prevalence of RASs in treatment-naïve patients has been broadly reported worldwide [9] [10] [11] [12] [13] [14] [15] [16] . However, apart from Brazil and Argentina, this issue has not been fully addressed in other South American countries yet [9, [17] [18] [19] . The lack of information in relation to preexisting baseline RASs, added to the high cost of these new drugs, are the major limiting factors for the broad implementation of these new therapies in Uruguay as well as in other Latin American countries (low-or lower-middle income) [20] .", "In this study, we explored the presence of resistance variants to NS5A and NS5B inhibitors in a DAA treatment naïve cohort of Uruguayan patients chronically infected with hepatitis C. Here, we aimed to contribute to the knowledge of the circulation of HCV resistant variants in the South American region.", "Samples. Serum samples were obtained from 31 patients with serological markers for HCV, which were recruited between 2015 and 2017 at the Gastroenterology Clinic from Hospital de Clínicas, Montevideo, Uruguay. HCV infection was confirmed by Abbott realtime HCV (Abbott Molecular Inc., Des Plaines, USA). Patients selected for this study were both chronically infected with HCV genotype 1 and DAA treatment-naïve at the time of blood extraction. Written informed consent was obtained from all patients. The studies have been performed according to the World Medical Association Declaration of Helsinki and approved by the appropriate institutional board (Hospital de Clínicas ethical committee).", "2.2. RNA Extraction, cDNA Synthesis, and NS5A and NS5B Amplification. Viral RNA was extracted from 140 μl of serum using the QIAamp Viral RNA mini kit (QIAgen, Hilden, Germany) according to the manufacturer's protocol. The viral RNA was heated at 65°C for 5 min and used as a template for a reverse transcription reaction. The reverse transcription reaction mixture contained 5 μl of the RNA template, 1 μl of random hexamer 100 ng/μl (Invitrogen Life Technologies, Carlsbad, CA, USA), 1 μl of dNTP mix (10 mM each), 4 μl of 5X first-strand buffer, 2 μl of 0.1 M DTT, 1 μl of SuperScript II reverse transcriptase (200 U/μl) (Invitrogen Life Technologies, Carlsbad, CA, USA), and 1 μl (40 U/μl) RNaseOUT (Invitrogen Life Technologies, Carlsbad, CA, USA). The reverse transcription was performed at 42°C for 50 min, and then the reverse transcriptase enzyme was inactivated at 70°C for 15 min. PCR amplification of NS5A and NS5B genome regions was performed using primers and conditions previously", "described [10] . Amplicons were purified using the Illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare Life Science, Buckinghamshire, UK) according to the manufacturer's protocol.", "2.3. NS5A and NS5B Sequencing. The purified product was then sequenced using the same sets of primers used for PCR amplification. Bidirectional Sanger sequencing was performed by Macrogen Korea (http://www.macrogen.com).", "2.4. NS5A and NS5B Genotype Determination. HCV NS5A and NS5B consensus sequences obtained from Uruguayan patients were aligned with sequences from HCV representing all genotypes and main subtypes isolated in different geographic regions of the world. These sequences were obtained from Los Alamos HCV sequence database and from the NIAID Virus Pathogen Database and Analysis Resource (ViPR) [21, 22] . For strains included in these studies, see Supplementary Material Table S1 . Sequences were aligned using the CLUSTAL W software [23] . Once aligned, the best evolutionary model that described our sequence data was assessed using ModelGenerator program [24] . Using the GTR + G + I model (General time reversible + gamma + invariant sites), maximum likelihood phylogenetic trees were constructed for both NS5A and NS5B using the MEGA 5.0 software [25] . For NS5A, 953 nucleotides (positions 6367 to 7319, relative to HCV 1a reference strain, H77 NC_004102) were included in the phylogenetic", "analysis, whereas for NS5B, only 361 nucleotides corresponding to the Okamoto region (positions 8265 to 8625, relative to strain H77 NC_004102) were included. As a measure of the robustness of each node, we employed the bootstrapping method (1000 pseudoreplicates).", "For NS5A 1a Uruguayan sequences (n = 20), a second alignment and maximum likelihood phylogenetic tree was generated in order to analyze HCV evolutionary relationships between Uruguayan, Brazilian, and worldwide strains. For non-Uruguayan strains included in this analysis, see Supplementary Material Table S2. 2.5. NS5A and NS5B Sequence Analysis. In order to properly identify substitution changes in NS5A and NS5B regions from HCV strains circulating in Uruguayan patients, we generated world consensus sequences for 1a and 1b subtypes using a wide range of NS5A and NS5B sequences from HCV strains isolated worldwide. For this purpose, NS5A gene sequences corresponding to subtypes 1a (n = 160) and 1b (n = 88) were retrieved from Los Alamos HCV sequence database and from the NIAID ViPR [21, 22] . Likewise, datasets of 150 and 124 NS5B sequences were generated for subtypes 1a and 1b, respectively. Using Seqman program, implemented in DNAStar 5.01 package (DNASTAR, Madison, USA), a world", "consensus nucleotide sequences were generated for each gene and subtype. Each Uruguayan sequence was subsequently aligned to the corresponding reference sequences, and then in silico translated. The amino acid sequences obtained were compared in order to explore the presence of RASs as well as the presence of polymorphisms at a RAS position (RAPs) in Uruguayan HCV strains. RAPs are defined as any change from reference sequence for a specific genotype at a position associated with NS5A resistance [26] .", "To study the genetic variability of NS5A and NS5B regions of HCV strains circulating in Uruguayan patients, sequences of these regions (accession numbers MH070029-MH070090) were aligned with corresponding sequences from 59 HCV strains isolated elsewhere, representing all genotypes and main subtypes (for strains included in these analyses, see Supplementary Material Table S1 ). Therefore, maximum likelihood phylogenetic trees were constructed. The results of these studies are shown in Figure 1 All strains in the phylogenies were assigned according to their genotype, and each cluster was supported by very high bootstrap values for both analyzed regions. Strains isolated from Uruguayan patients (n = 31) were assigned to genotype 1, 20 of which corresponded to subtype 1a and 11 to subtype 1b. The results of NS5A (Figure 1 (a)) and NS5B (Figure 1", "Genotype 1b phylogenetic analyses were concordant for both genomic regions in all 31 sequences, suggesting no recombination events between these regions.\n\nTo further analyze the evolutionary relationships between the Uruguayan strains and those circulating in Brazil and elsewhere, a second maximum likelihood phylogenetic tree of HCV-1a sequences of NS5A partial region was built ( Figure 2 ). As was previously described, two distinct 1a clades (clades 1 and 2) were observed. Brazilian sequences clustered in a large group of related sequences inside clade 1 [9] . Whereas NS5A Uruguayan strains (in red) did not cluster in a particular clade, rather, they grouped dispersedly within all major world clades.", "With the purpose of studying the amino acid (AA) substitutions along the NS5A protein, Uruguayan HCV AA sequences were aligned with NS5A world consensus sequences (residues 23 to 354 relative to NS5A protein sequence). AA substitutions at positions previously found to be potentially associated with resistance to NS5A inhibitors, as well as polymorphisms at a RAS position, were identified. These results are summarized in Table 1 .", "RASs to NS5A inhibitors (L31M and L31V) were identified in 2 strains out of 25 (8%) fully sequenced samples. RAPs were found in 3 strains (subtype 1a): 2 exhibited the substitution H58P and 1 the substitution K24Q. Although these substitutions were not reported as resistant, some changes at these positions were previously described as RASs in subtype 1a, namely H58D and K24R [27, 28] . Finally, substitution E62D was found in one subtype 1a strain. This change is considered as a secondary substitution because, although it does not confer resistance by itself, when combined with a known RAS it does. In fact, it confers a higher level of resistance than the one achieved by the RAS alone [26] . In addition, several polymorphisms that have not been previously reported to be associated with a resistant phenotype were also detected (see Supplementary Material Table S3 ).", "In order to study substitutions along NS5B protein, Uruguayan HCV AA sequences were aligned to the NS5B world consensus sequences. Almost full-length AA sequences were obtained in 26 out of 31 analyzed strains. 23 sequences span residues 36 to 539 whereas the remaining 3 span residues 36 to 557 of NS5B protein.\n\nThis issue limited our studies, since many of the described RASs are observed as of residue 553.\n\nImportantly, RASs to NS5B inhibitors ( Table 2) were observed in 5 strains out of 26 sequenced samples (19.2%). C451R was found in two isolates while A421V was found in only one. In 2 of the 3 strains for which we were able to obtain longer sequences, RASs S556G (subtype 1a) and Q556R (subtype 1b) were observed.", "Finally, we found two RAPs: A421V (in 2 subtype 1b strains) and A553G (in 1 subtype 1a strain). Although A421V has been associated with resistance to beclabuvir (BCV) in patients infected with HCV subtype 1a, this resistant phenotype has not been proven in strains subtype 1b [29] . In position 553, the substitution reported as resistant was A553T [8] .\n\nAs was the case for NS5A, different polymorphisms not previously associated with a resistant phenotype were also detected in NS5B (see Supplementary Material Table S4 ).", "The advent of DAAs therapies constitutes one of the major breakthroughs in HCV infected patients management. However, these new treatment options are far from being universally available, in particular for HCV infected patients relying on Latin American public healthcare systems. The main limiting factors for worldwide access to DAAs in our region concern the high cost, the inadequate management of public healthcare systems, the limited access of low-income or uninsured populations to healthcare providers, and the lack of accurate epidemiological information [20, [30] [31] [32] . In Uruguay, these therapies became recently available, and although some have been approved for their use by the public health authorities (Viekira pak and sofosbuvir/ledipasvir therapies), they are not currently financially covered, except in specific cases. Despite the high rates of viral response achieved with DAA-based treatments, still 1 to10% of the patients fails to eliminate infection, and in these", "cases, baseline and emergent resistance variants turn out to be key factors contributing to treatment failure [5, 17, 33] .", "Unfortunately, we are currently unable to properly assess the number of HCV infected people in Uruguay and even more to figure out the frequency and type of RASs circulating. These facts could compromise the effectiveness of these new therapies in our country.", "We have previously reported that naturally occurring substitutions conferring resistance to NS3 inhibitors exist in a significant proportion of Uruguayan patients infected with HCV genotype 1, and we showed that this frequency seemed to be higher than in other South American countries (Brazil and Argentina) [34] . The present study describes the prevalence of baseline NS5A and NS5B RASs in HCV genotype 1 infected DAA-naïve patients in a Uruguayan cohort. The presence of substitutions conferring resistance to NS5A inhibitors has been widely reported both in therapynaïve and in relapser patients from Europe [10, 33, [35] [36] [37] [38] , USA [37, 39, 40] , and Asia [41] [42] [43] . However, NS5A sequences from South America are poorly analyzed yet [9, 44] . Recent studies have revealed that the mean prevalence of NS5A genotype 1 baseline RASs to different inhibitors ranges from 6% to 16% using population sequencing or deep sequencing [27, 37, 45, 46] . Importantly, the prevalence and", "type of baseline NS5A RASs varies slightly by geographic regions. For instance, L31M was found in 2.2% of genotype 1a infected patients in Europe, in 4.1% of those in Oceania, and strikingly in no patient from the USA [27] . For this reason, we believe that there is a need to contribute data from our region, for which we still do not have enough information, apart from Brazil [9, 44] . The results of this study indicate the presence of DAA NS5A RASs in 2 HCV strains (8% of the patients enrolled in this study), with baseline RASs detected at position 31 (see Table 1 ). L31M substitution confers resistance to daclatasvir (DCV), ledipasvir (LDV), and elbasvir (EBV) in both 1a and 1b subtypes [5, 6, 8, 28, 47, 48] , whereas substitution L31V does it to DCV in subtypes 1a and 1b, to LDV in subtype 1b, and to EBV in subtype 1a [5, 6, 28] . Given that both L31V and L31M are clinically relevant RASs, their detection at baseline may influence the choice of first-line treatment regimens [28] .", "The substitutions H58P and K24Q found in two patients are considered as resistance-associated polymorphisms (RAPs). The RASs characterized at these positions were H58D and K24G/N/R [5, 6, 27, 28, 49, 50] . The substitution H58P was found as a baseline RAP in relapsers to LDV (HARVONI prescription, https://www.gilead.com/-/ media/files/pdfs/medicines/liver-disease/harvoni/harvoni_pi. pdf?la=en). However, it is sometimes regarded as a RAS [10, 51] , despite conferring only 1.2 fold change in resistance in in vitro studies using the 1a replicon system [39] .\n\nWe did not find M28T/V, Q30R/H, or Y93H substitutions as there were previously reported in Brazil and worldwide [9, 27, 44] . The amino acid substitution E62H was found in one Uruguayan patient. Although this change does not confer resistance by itself but in combination with Q30R, it generates a high resistance level to DCV [52] .", "The presence of baseline NS5A RASs impacts treatment outcome in some patient groups by affecting SVR rates. The detection of NS5A preexistent RASs may play a relevant role in the choice of first-line treatment regimens or in the simplification/shortening of recommended regimens, in order to bring SVR rates close to the highest achievable [27, 38, 41, 53] , in particular in countries such as Uruguay, where only two different DAA-containing treatment regimens are approved for their use.", "Regarding NS5B gene, global analysis (with the exception of South America [17, 19] ) revealed that NS5B DAA resistance substitutions are infrequent [14] . Our study showed the presence of NS5B inhibitors RASs in 5 out of 26 analyzed HCV infected Uruguayan patients naïve to treatment (19.2%). Substitutions found in this work were A421V and S556G associated in subtype 1a with resistance to BCV and dasabuvir (DSV), respectively [8, 28, 29, 54, 55] , and Q556R associated with resistance to DSV both in genotype 1a and 1b [12, 28] . Substitution C451R, observed in two Uruguayan patients, was reported previously in patients who failed to clear the infection after treatment with OBV/PTV/r + DSV ± RBV. In these cases, it appeared in combination with G558R (Trial Coral I-Cohort 2: http:// www.hcv-trials.com/showStudy.asp?Study=86). RAPs in positions 421 and 553 (A421V in two subtype 1b isolates and A553G in one subtype 1b isolate) were also found. Although A421V has been associated with", "resistance to BCV in patients with subtype 1a, this phenotype has not been proven in strains of subtype 1b [29] . In position 553, the substitutions reported as resistant are A553T in subtype 1a [8] and A553V in subtype 1b [54] , conferring resistance to DSV.", "In contrast to our results, Noble and coworkers (2016) reported the presence of V321A, A421G, M414V, Y448H, L159F, and C316N in Brazilian isolates [17] , yet none of these mutations were found in this study, probably due to the diversity found between Uruguayan and Brazilian strains ( Figure 2 ). Nevertheless, substitution A421V was found in Brazil [17] , Argentina [19] , and Uruguay. The RAS S282T was detected neither in Brazilian reports nor in this current work (Uruguay) [17, 18, 56] . Our findings further confirm and complement previous studies which evidenced a low prevalence of this substitution in vivo, probably due to its low replicative fitness [14, 18, 57] . Despite our results, it is worth mentioning that the presence of baseline NS5B RASs conferring resistance to nucleotide or nonnucleoside NS5B inhibitors has not been shown to have any impact on virologic responses thus far [53, 58] .", "These results show both diversity in the baseline polymorphisms found in different Latin American countries and in the evolutionary relationships of Uruguayan isolates ( Figure 2 ). This fact could be linked not only to the isolates' geographic region and viral intrinsic characteristics but also to the genetic background of the host. It is worth mentioning that we live in a vast continent inhabited by populations with different genotypic characteristics that might, depending on the situation, require different approaches to treatment. Indeed, we have recently found that allele and genotype frequencies at IL28B locus of Uruguayan individuals closely resemble those of an admixed population rather than a uniformly European-descendant one [59] . Altogether, we believe that it could be important to carry out studies throughout the South American region in order to establish the prevalence of RASs in NS5A and NS5B in different countries. In fact, this will aid in understanding that not", "every treatment regimen might be adequate for every patient and country. The data we presented here might guide not only physicians in making therapeutic decisions but also public health authorities in approving more diverse treatment combinations. These treatment formulations would cover most of the circulating strains in our region, a region with an extremely diverse genetic background population.", "To our knowledge, the present study revealed for the first time the presence of RASs in the NS5A and NS5B regions of HCV genotype 1 Uruguayan strains from patients who have not been previously treated with DAAs and is one of the few South American countries to report on this matter. It is currently unclear if preexisting viral variants with reduced susceptibility to DAAs are clinically relevant for the prediction of virologic treatment failure. However, individualized DAA therapy based on baseline resistance analysis may be beneficial for optimizing treatment efficacy in patients with HCV genotype 1 infection and risk factors for treatment failure. Therefore, the potential role of baseline resistance testing remains an area of critical research and clinical questions.\n\nThe data used to support the findings of this study are included within the article.\n\nThe authors declare that they have no conflicts of interest.", "Fabián Aldunate and Natalia Echeverría contributed equally to this work.\n\nSupplementary Material Table S1 : hepatitis C Virus NS5A and NS5B sequences used as representatives of each genotype to perform the phylogenetic analysis. Their corresponding genotype, country of isolation, and GenBank accession number are indicated. Supplementary Material Table S2 : hepatitis C Virus NS5A subtype 1a sequences used to reveal evolutionary relationships between Uruguayan strains and others isolated elsewhere. Their corresponding country of isolation and GenBank accession number are indicated. Supplementary Material Table S3 : amino acid substitutions in NS5A protein not previously associated with resistance to NS5A inhibitors. Supplementary Material Table S4 : amino acid substitutions in NS5B protein not previously associated with resistance to polymerase inhibitors. (Supplementary Materials)" ]

[ 3 ]

[ "inhibit polymerase activity by allosteric mechanisms" ]

[ "Pretreatment Hepatitis C Virus NS5A/NS5B Resistance-Associated Substitutions in Genotype 1 Uruguayan Infected Patients\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6112080/\n\nSHA: f01ad3545245b4f884b48aa2b69c9deb942c3e77\n\nAuthors: Aldunate, Fabián; Echeverría, Natalia; Chiodi, Daniela; López, Pablo; Sánchez-Cicerón, Adriana; Fajardo, Alvaro; Soñora, Martín; Cristina, Juan; Hernández, Nelia; Moreno, Pilar\nDate: 2018-08-14\nDOI: 10.1155/2018/2514901\nLicense: cc-by", "Abstract: Hepatitis C Virus (HCV) infection treatment has dramatically changed with the advent of direct-acting antiviral agents (DAAs). However, the efficacy of DAAs can be attenuated by the presence of resistance-associated substitutions (RASs) before and after treatment. Indeed, RASs detected in DAA treatment-naïve HCV-infected patients could be useful for clinical management and outcome prediction. Although the frequency of naturally occurring HCV NS5A and NS5B RASs has been addressed in many countries, there are only a few reports on their prevalence in the South American region. The aim of this study was to investigate the presence of RASs to NS5A and NS5B inhibitors in a DAA treatment naïve cohort of Uruguayan patients infected with chronic hepatitis C and compare them with reports from other South American countries. Here, we found that naturally occurring substitutions conferring resistance to NS5A and NS5B inhibitors were present in 8% and 19.2%, respectively, of", "treatment-naïve HCV genotype 1 infected patients. Importantly, the baseline substitutions in NS5A and NS5B herein identified differ from the studies previously reported in Brazil. Furthermore, Uruguayan strains subtype 1a clustered within all major world clades, showing that HCV variants currently circulating in this country are characterized by a remarkable genetic diversity.", "Text: Hepatitis C Virus (HCV) infection treatment has dramatically improved thanks to the introduction of direct-acting antiviral agents (DAAs). These antivirals have significantly increased response rates (up to 98%) and greatly reduced treatment duration [1] . Currently available DAAs are classified into four categories given their molecular targets in the HCV replication cycle: (1) NS3/4A protease inhibitors (PIs) bind to the active site of the NS3/4A protease; (2) NS5A inhibitors interact with domain 1 of the NS5A dimer, although the exact mechanism of NS5A inhibition remains to be fully elucidated; (3) nucleos(t)ide analog NS5B polymerase inhibitors are incorporated into the nascent RNA chain resulting in chain termination by compromising the binding of the incoming nucleotide; (4) nonnucleoside NS5B polymerase inhibitors interact with either the thumb 1, thumb 2, palm 1, or palm 2 domain of NS5B and inhibit polymerase activity by allosteric mechanisms [2] [3] [4] . However, the", "extreme mutation and high replication rates of HCV, together with the immune system pressure, lead to a remarkable genetic variability that can compromise the high response rates to DAAs due to the preexistence of resistanceassociated substitutions (RASs) [5, 6] .", "Each drug or class of DAA is characterized by specific resistance profiles. The likelihood that a DAA will select for and allow outgrowth of viral populations carrying RASs depends on the DAA's genetic barrier to resistance (the number and type of mutations needed to generate an amino acid substitution that confers resistance), the viral fitness (replicative capacity) of the resistant variant, and viral genotypes and subtypes [7, 8] .\n\nThe prevalence of RASs in treatment-naïve patients has been broadly reported worldwide [9] [10] [11] [12] [13] [14] [15] [16] . However, apart from Brazil and Argentina, this issue has not been fully addressed in other South American countries yet [9, [17] [18] [19] . The lack of information in relation to preexisting baseline RASs, added to the high cost of these new drugs, are the major limiting factors for the broad implementation of these new therapies in Uruguay as well as in other Latin American countries (low-or lower-middle income) [20] .", "In this study, we explored the presence of resistance variants to NS5A and NS5B inhibitors in a DAA treatment naïve cohort of Uruguayan patients chronically infected with hepatitis C. Here, we aimed to contribute to the knowledge of the circulation of HCV resistant variants in the South American region.", "Samples. Serum samples were obtained from 31 patients with serological markers for HCV, which were recruited between 2015 and 2017 at the Gastroenterology Clinic from Hospital de Clínicas, Montevideo, Uruguay. HCV infection was confirmed by Abbott realtime HCV (Abbott Molecular Inc., Des Plaines, USA). Patients selected for this study were both chronically infected with HCV genotype 1 and DAA treatment-naïve at the time of blood extraction. Written informed consent was obtained from all patients. The studies have been performed according to the World Medical Association Declaration of Helsinki and approved by the appropriate institutional board (Hospital de Clínicas ethical committee).", "2.2. RNA Extraction, cDNA Synthesis, and NS5A and NS5B Amplification. Viral RNA was extracted from 140 μl of serum using the QIAamp Viral RNA mini kit (QIAgen, Hilden, Germany) according to the manufacturer's protocol. The viral RNA was heated at 65°C for 5 min and used as a template for a reverse transcription reaction. The reverse transcription reaction mixture contained 5 μl of the RNA template, 1 μl of random hexamer 100 ng/μl (Invitrogen Life Technologies, Carlsbad, CA, USA), 1 μl of dNTP mix (10 mM each), 4 μl of 5X first-strand buffer, 2 μl of 0.1 M DTT, 1 μl of SuperScript II reverse transcriptase (200 U/μl) (Invitrogen Life Technologies, Carlsbad, CA, USA), and 1 μl (40 U/μl) RNaseOUT (Invitrogen Life Technologies, Carlsbad, CA, USA). The reverse transcription was performed at 42°C for 50 min, and then the reverse transcriptase enzyme was inactivated at 70°C for 15 min. PCR amplification of NS5A and NS5B genome regions was performed using primers and conditions previously", "described [10] . Amplicons were purified using the Illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare Life Science, Buckinghamshire, UK) according to the manufacturer's protocol.", "2.3. NS5A and NS5B Sequencing. The purified product was then sequenced using the same sets of primers used for PCR amplification. Bidirectional Sanger sequencing was performed by Macrogen Korea (http://www.macrogen.com).", "2.4. NS5A and NS5B Genotype Determination. HCV NS5A and NS5B consensus sequences obtained from Uruguayan patients were aligned with sequences from HCV representing all genotypes and main subtypes isolated in different geographic regions of the world. These sequences were obtained from Los Alamos HCV sequence database and from the NIAID Virus Pathogen Database and Analysis Resource (ViPR) [21, 22] . For strains included in these studies, see Supplementary Material Table S1 . Sequences were aligned using the CLUSTAL W software [23] . Once aligned, the best evolutionary model that described our sequence data was assessed using ModelGenerator program [24] . Using the GTR + G + I model (General time reversible + gamma + invariant sites), maximum likelihood phylogenetic trees were constructed for both NS5A and NS5B using the MEGA 5.0 software [25] . For NS5A, 953 nucleotides (positions 6367 to 7319, relative to HCV 1a reference strain, H77 NC_004102) were included in the phylogenetic", "analysis, whereas for NS5B, only 361 nucleotides corresponding to the Okamoto region (positions 8265 to 8625, relative to strain H77 NC_004102) were included. As a measure of the robustness of each node, we employed the bootstrapping method (1000 pseudoreplicates).", "For NS5A 1a Uruguayan sequences (n = 20), a second alignment and maximum likelihood phylogenetic tree was generated in order to analyze HCV evolutionary relationships between Uruguayan, Brazilian, and worldwide strains. For non-Uruguayan strains included in this analysis, see Supplementary Material Table S2. 2.5. NS5A and NS5B Sequence Analysis. In order to properly identify substitution changes in NS5A and NS5B regions from HCV strains circulating in Uruguayan patients, we generated world consensus sequences for 1a and 1b subtypes using a wide range of NS5A and NS5B sequences from HCV strains isolated worldwide. For this purpose, NS5A gene sequences corresponding to subtypes 1a (n = 160) and 1b (n = 88) were retrieved from Los Alamos HCV sequence database and from the NIAID ViPR [21, 22] . Likewise, datasets of 150 and 124 NS5B sequences were generated for subtypes 1a and 1b, respectively. Using Seqman program, implemented in DNAStar 5.01 package (DNASTAR, Madison, USA), a world", "consensus nucleotide sequences were generated for each gene and subtype. Each Uruguayan sequence was subsequently aligned to the corresponding reference sequences, and then in silico translated. The amino acid sequences obtained were compared in order to explore the presence of RASs as well as the presence of polymorphisms at a RAS position (RAPs) in Uruguayan HCV strains. RAPs are defined as any change from reference sequence for a specific genotype at a position associated with NS5A resistance [26] .", "To study the genetic variability of NS5A and NS5B regions of HCV strains circulating in Uruguayan patients, sequences of these regions (accession numbers MH070029-MH070090) were aligned with corresponding sequences from 59 HCV strains isolated elsewhere, representing all genotypes and main subtypes (for strains included in these analyses, see Supplementary Material Table S1 ). Therefore, maximum likelihood phylogenetic trees were constructed. The results of these studies are shown in Figure 1 All strains in the phylogenies were assigned according to their genotype, and each cluster was supported by very high bootstrap values for both analyzed regions. Strains isolated from Uruguayan patients (n = 31) were assigned to genotype 1, 20 of which corresponded to subtype 1a and 11 to subtype 1b. The results of NS5A (Figure 1 (a)) and NS5B (Figure 1", "Genotype 1b phylogenetic analyses were concordant for both genomic regions in all 31 sequences, suggesting no recombination events between these regions.\n\nTo further analyze the evolutionary relationships between the Uruguayan strains and those circulating in Brazil and elsewhere, a second maximum likelihood phylogenetic tree of HCV-1a sequences of NS5A partial region was built ( Figure 2 ). As was previously described, two distinct 1a clades (clades 1 and 2) were observed. Brazilian sequences clustered in a large group of related sequences inside clade 1 [9] . Whereas NS5A Uruguayan strains (in red) did not cluster in a particular clade, rather, they grouped dispersedly within all major world clades.", "With the purpose of studying the amino acid (AA) substitutions along the NS5A protein, Uruguayan HCV AA sequences were aligned with NS5A world consensus sequences (residues 23 to 354 relative to NS5A protein sequence). AA substitutions at positions previously found to be potentially associated with resistance to NS5A inhibitors, as well as polymorphisms at a RAS position, were identified. These results are summarized in Table 1 .", "RASs to NS5A inhibitors (L31M and L31V) were identified in 2 strains out of 25 (8%) fully sequenced samples. RAPs were found in 3 strains (subtype 1a): 2 exhibited the substitution H58P and 1 the substitution K24Q. Although these substitutions were not reported as resistant, some changes at these positions were previously described as RASs in subtype 1a, namely H58D and K24R [27, 28] . Finally, substitution E62D was found in one subtype 1a strain. This change is considered as a secondary substitution because, although it does not confer resistance by itself, when combined with a known RAS it does. In fact, it confers a higher level of resistance than the one achieved by the RAS alone [26] . In addition, several polymorphisms that have not been previously reported to be associated with a resistant phenotype were also detected (see Supplementary Material Table S3 ).", "In order to study substitutions along NS5B protein, Uruguayan HCV AA sequences were aligned to the NS5B world consensus sequences. Almost full-length AA sequences were obtained in 26 out of 31 analyzed strains. 23 sequences span residues 36 to 539 whereas the remaining 3 span residues 36 to 557 of NS5B protein.\n\nThis issue limited our studies, since many of the described RASs are observed as of residue 553.\n\nImportantly, RASs to NS5B inhibitors ( Table 2) were observed in 5 strains out of 26 sequenced samples (19.2%). C451R was found in two isolates while A421V was found in only one. In 2 of the 3 strains for which we were able to obtain longer sequences, RASs S556G (subtype 1a) and Q556R (subtype 1b) were observed.", "Finally, we found two RAPs: A421V (in 2 subtype 1b strains) and A553G (in 1 subtype 1a strain). Although A421V has been associated with resistance to beclabuvir (BCV) in patients infected with HCV subtype 1a, this resistant phenotype has not been proven in strains subtype 1b [29] . In position 553, the substitution reported as resistant was A553T [8] .\n\nAs was the case for NS5A, different polymorphisms not previously associated with a resistant phenotype were also detected in NS5B (see Supplementary Material Table S4 ).", "The advent of DAAs therapies constitutes one of the major breakthroughs in HCV infected patients management. However, these new treatment options are far from being universally available, in particular for HCV infected patients relying on Latin American public healthcare systems. The main limiting factors for worldwide access to DAAs in our region concern the high cost, the inadequate management of public healthcare systems, the limited access of low-income or uninsured populations to healthcare providers, and the lack of accurate epidemiological information [20, [30] [31] [32] . In Uruguay, these therapies became recently available, and although some have been approved for their use by the public health authorities (Viekira pak and sofosbuvir/ledipasvir therapies), they are not currently financially covered, except in specific cases. Despite the high rates of viral response achieved with DAA-based treatments, still 1 to10% of the patients fails to eliminate infection, and in these", "cases, baseline and emergent resistance variants turn out to be key factors contributing to treatment failure [5, 17, 33] .", "Unfortunately, we are currently unable to properly assess the number of HCV infected people in Uruguay and even more to figure out the frequency and type of RASs circulating. These facts could compromise the effectiveness of these new therapies in our country.", "We have previously reported that naturally occurring substitutions conferring resistance to NS3 inhibitors exist in a significant proportion of Uruguayan patients infected with HCV genotype 1, and we showed that this frequency seemed to be higher than in other South American countries (Brazil and Argentina) [34] . The present study describes the prevalence of baseline NS5A and NS5B RASs in HCV genotype 1 infected DAA-naïve patients in a Uruguayan cohort. The presence of substitutions conferring resistance to NS5A inhibitors has been widely reported both in therapynaïve and in relapser patients from Europe [10, 33, [35] [36] [37] [38] , USA [37, 39, 40] , and Asia [41] [42] [43] . However, NS5A sequences from South America are poorly analyzed yet [9, 44] . Recent studies have revealed that the mean prevalence of NS5A genotype 1 baseline RASs to different inhibitors ranges from 6% to 16% using population sequencing or deep sequencing [27, 37, 45, 46] . Importantly, the prevalence and", "type of baseline NS5A RASs varies slightly by geographic regions. For instance, L31M was found in 2.2% of genotype 1a infected patients in Europe, in 4.1% of those in Oceania, and strikingly in no patient from the USA [27] . For this reason, we believe that there is a need to contribute data from our region, for which we still do not have enough information, apart from Brazil [9, 44] . The results of this study indicate the presence of DAA NS5A RASs in 2 HCV strains (8% of the patients enrolled in this study), with baseline RASs detected at position 31 (see Table 1 ). L31M substitution confers resistance to daclatasvir (DCV), ledipasvir (LDV), and elbasvir (EBV) in both 1a and 1b subtypes [5, 6, 8, 28, 47, 48] , whereas substitution L31V does it to DCV in subtypes 1a and 1b, to LDV in subtype 1b, and to EBV in subtype 1a [5, 6, 28] . Given that both L31V and L31M are clinically relevant RASs, their detection at baseline may influence the choice of first-line treatment regimens [28] .", "The substitutions H58P and K24Q found in two patients are considered as resistance-associated polymorphisms (RAPs). The RASs characterized at these positions were H58D and K24G/N/R [5, 6, 27, 28, 49, 50] . The substitution H58P was found as a baseline RAP in relapsers to LDV (HARVONI prescription, https://www.gilead.com/-/ media/files/pdfs/medicines/liver-disease/harvoni/harvoni_pi. pdf?la=en). However, it is sometimes regarded as a RAS [10, 51] , despite conferring only 1.2 fold change in resistance in in vitro studies using the 1a replicon system [39] .\n\nWe did not find M28T/V, Q30R/H, or Y93H substitutions as there were previously reported in Brazil and worldwide [9, 27, 44] . The amino acid substitution E62H was found in one Uruguayan patient. Although this change does not confer resistance by itself but in combination with Q30R, it generates a high resistance level to DCV [52] .", "The presence of baseline NS5A RASs impacts treatment outcome in some patient groups by affecting SVR rates. The detection of NS5A preexistent RASs may play a relevant role in the choice of first-line treatment regimens or in the simplification/shortening of recommended regimens, in order to bring SVR rates close to the highest achievable [27, 38, 41, 53] , in particular in countries such as Uruguay, where only two different DAA-containing treatment regimens are approved for their use.", "Regarding NS5B gene, global analysis (with the exception of South America [17, 19] ) revealed that NS5B DAA resistance substitutions are infrequent [14] . Our study showed the presence of NS5B inhibitors RASs in 5 out of 26 analyzed HCV infected Uruguayan patients naïve to treatment (19.2%). Substitutions found in this work were A421V and S556G associated in subtype 1a with resistance to BCV and dasabuvir (DSV), respectively [8, 28, 29, 54, 55] , and Q556R associated with resistance to DSV both in genotype 1a and 1b [12, 28] . Substitution C451R, observed in two Uruguayan patients, was reported previously in patients who failed to clear the infection after treatment with OBV/PTV/r + DSV ± RBV. In these cases, it appeared in combination with G558R (Trial Coral I-Cohort 2: http:// www.hcv-trials.com/showStudy.asp?Study=86). RAPs in positions 421 and 553 (A421V in two subtype 1b isolates and A553G in one subtype 1b isolate) were also found. Although A421V has been associated with", "resistance to BCV in patients with subtype 1a, this phenotype has not been proven in strains of subtype 1b [29] . In position 553, the substitutions reported as resistant are A553T in subtype 1a [8] and A553V in subtype 1b [54] , conferring resistance to DSV.", "In contrast to our results, Noble and coworkers (2016) reported the presence of V321A, A421G, M414V, Y448H, L159F, and C316N in Brazilian isolates [17] , yet none of these mutations were found in this study, probably due to the diversity found between Uruguayan and Brazilian strains ( Figure 2 ). Nevertheless, substitution A421V was found in Brazil [17] , Argentina [19] , and Uruguay. The RAS S282T was detected neither in Brazilian reports nor in this current work (Uruguay) [17, 18, 56] . Our findings further confirm and complement previous studies which evidenced a low prevalence of this substitution in vivo, probably due to its low replicative fitness [14, 18, 57] . Despite our results, it is worth mentioning that the presence of baseline NS5B RASs conferring resistance to nucleotide or nonnucleoside NS5B inhibitors has not been shown to have any impact on virologic responses thus far [53, 58] .", "These results show both diversity in the baseline polymorphisms found in different Latin American countries and in the evolutionary relationships of Uruguayan isolates ( Figure 2 ). This fact could be linked not only to the isolates' geographic region and viral intrinsic characteristics but also to the genetic background of the host. It is worth mentioning that we live in a vast continent inhabited by populations with different genotypic characteristics that might, depending on the situation, require different approaches to treatment. Indeed, we have recently found that allele and genotype frequencies at IL28B locus of Uruguayan individuals closely resemble those of an admixed population rather than a uniformly European-descendant one [59] . Altogether, we believe that it could be important to carry out studies throughout the South American region in order to establish the prevalence of RASs in NS5A and NS5B in different countries. In fact, this will aid in understanding that not", "every treatment regimen might be adequate for every patient and country. The data we presented here might guide not only physicians in making therapeutic decisions but also public health authorities in approving more diverse treatment combinations. These treatment formulations would cover most of the circulating strains in our region, a region with an extremely diverse genetic background population.", "To our knowledge, the present study revealed for the first time the presence of RASs in the NS5A and NS5B regions of HCV genotype 1 Uruguayan strains from patients who have not been previously treated with DAAs and is one of the few South American countries to report on this matter. It is currently unclear if preexisting viral variants with reduced susceptibility to DAAs are clinically relevant for the prediction of virologic treatment failure. However, individualized DAA therapy based on baseline resistance analysis may be beneficial for optimizing treatment efficacy in patients with HCV genotype 1 infection and risk factors for treatment failure. Therefore, the potential role of baseline resistance testing remains an area of critical research and clinical questions.\n\nThe data used to support the findings of this study are included within the article.\n\nThe authors declare that they have no conflicts of interest.", "Fabián Aldunate and Natalia Echeverría contributed equally to this work.\n\nSupplementary Material Table S1 : hepatitis C Virus NS5A and NS5B sequences used as representatives of each genotype to perform the phylogenetic analysis. Their corresponding genotype, country of isolation, and GenBank accession number are indicated. Supplementary Material Table S2 : hepatitis C Virus NS5A subtype 1a sequences used to reveal evolutionary relationships between Uruguayan strains and others isolated elsewhere. Their corresponding country of isolation and GenBank accession number are indicated. Supplementary Material Table S3 : amino acid substitutions in NS5A protein not previously associated with resistance to NS5A inhibitors. Supplementary Material Table S4 : amino acid substitutions in NS5B protein not previously associated with resistance to polymerase inhibitors. (Supplementary Materials)" ]

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[ "Pretreatment Hepatitis C Virus NS5A/NS5B Resistance-Associated Substitutions in Genotype 1 Uruguayan Infected Patients\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6112080/\n\nSHA: f01ad3545245b4f884b48aa2b69c9deb942c3e77\n\nAuthors: Aldunate, Fabián; Echeverría, Natalia; Chiodi, Daniela; López, Pablo; Sánchez-Cicerón, Adriana; Fajardo, Alvaro; Soñora, Martín; Cristina, Juan; Hernández, Nelia; Moreno, Pilar\nDate: 2018-08-14\nDOI: 10.1155/2018/2514901\nLicense: cc-by", "Abstract: Hepatitis C Virus (HCV) infection treatment has dramatically changed with the advent of direct-acting antiviral agents (DAAs). However, the efficacy of DAAs can be attenuated by the presence of resistance-associated substitutions (RASs) before and after treatment. Indeed, RASs detected in DAA treatment-naïve HCV-infected patients could be useful for clinical management and outcome prediction. Although the frequency of naturally occurring HCV NS5A and NS5B RASs has been addressed in many countries, there are only a few reports on their prevalence in the South American region. The aim of this study was to investigate the presence of RASs to NS5A and NS5B inhibitors in a DAA treatment naïve cohort of Uruguayan patients infected with chronic hepatitis C and compare them with reports from other South American countries. Here, we found that naturally occurring substitutions conferring resistance to NS5A and NS5B inhibitors were present in 8% and 19.2%, respectively, of", "treatment-naïve HCV genotype 1 infected patients. Importantly, the baseline substitutions in NS5A and NS5B herein identified differ from the studies previously reported in Brazil. Furthermore, Uruguayan strains subtype 1a clustered within all major world clades, showing that HCV variants currently circulating in this country are characterized by a remarkable genetic diversity.", "Text: Hepatitis C Virus (HCV) infection treatment has dramatically improved thanks to the introduction of direct-acting antiviral agents (DAAs). These antivirals have significantly increased response rates (up to 98%) and greatly reduced treatment duration [1] . Currently available DAAs are classified into four categories given their molecular targets in the HCV replication cycle: (1) NS3/4A protease inhibitors (PIs) bind to the active site of the NS3/4A protease; (2) NS5A inhibitors interact with domain 1 of the NS5A dimer, although the exact mechanism of NS5A inhibition remains to be fully elucidated; (3) nucleos(t)ide analog NS5B polymerase inhibitors are incorporated into the nascent RNA chain resulting in chain termination by compromising the binding of the incoming nucleotide; (4) nonnucleoside NS5B polymerase inhibitors interact with either the thumb 1, thumb 2, palm 1, or palm 2 domain of NS5B and inhibit polymerase activity by allosteric mechanisms [2] [3] [4] . However, the", "extreme mutation and high replication rates of HCV, together with the immune system pressure, lead to a remarkable genetic variability that can compromise the high response rates to DAAs due to the preexistence of resistanceassociated substitutions (RASs) [5, 6] .", "Each drug or class of DAA is characterized by specific resistance profiles. The likelihood that a DAA will select for and allow outgrowth of viral populations carrying RASs depends on the DAA's genetic barrier to resistance (the number and type of mutations needed to generate an amino acid substitution that confers resistance), the viral fitness (replicative capacity) of the resistant variant, and viral genotypes and subtypes [7, 8] .\n\nThe prevalence of RASs in treatment-naïve patients has been broadly reported worldwide [9] [10] [11] [12] [13] [14] [15] [16] . However, apart from Brazil and Argentina, this issue has not been fully addressed in other South American countries yet [9, [17] [18] [19] . The lack of information in relation to preexisting baseline RASs, added to the high cost of these new drugs, are the major limiting factors for the broad implementation of these new therapies in Uruguay as well as in other Latin American countries (low-or lower-middle income) [20] .", "In this study, we explored the presence of resistance variants to NS5A and NS5B inhibitors in a DAA treatment naïve cohort of Uruguayan patients chronically infected with hepatitis C. Here, we aimed to contribute to the knowledge of the circulation of HCV resistant variants in the South American region.", "Samples. Serum samples were obtained from 31 patients with serological markers for HCV, which were recruited between 2015 and 2017 at the Gastroenterology Clinic from Hospital de Clínicas, Montevideo, Uruguay. HCV infection was confirmed by Abbott realtime HCV (Abbott Molecular Inc., Des Plaines, USA). Patients selected for this study were both chronically infected with HCV genotype 1 and DAA treatment-naïve at the time of blood extraction. Written informed consent was obtained from all patients. The studies have been performed according to the World Medical Association Declaration of Helsinki and approved by the appropriate institutional board (Hospital de Clínicas ethical committee).", "2.2. RNA Extraction, cDNA Synthesis, and NS5A and NS5B Amplification. Viral RNA was extracted from 140 μl of serum using the QIAamp Viral RNA mini kit (QIAgen, Hilden, Germany) according to the manufacturer's protocol. The viral RNA was heated at 65°C for 5 min and used as a template for a reverse transcription reaction. The reverse transcription reaction mixture contained 5 μl of the RNA template, 1 μl of random hexamer 100 ng/μl (Invitrogen Life Technologies, Carlsbad, CA, USA), 1 μl of dNTP mix (10 mM each), 4 μl of 5X first-strand buffer, 2 μl of 0.1 M DTT, 1 μl of SuperScript II reverse transcriptase (200 U/μl) (Invitrogen Life Technologies, Carlsbad, CA, USA), and 1 μl (40 U/μl) RNaseOUT (Invitrogen Life Technologies, Carlsbad, CA, USA). The reverse transcription was performed at 42°C for 50 min, and then the reverse transcriptase enzyme was inactivated at 70°C for 15 min. PCR amplification of NS5A and NS5B genome regions was performed using primers and conditions previously", "described [10] . Amplicons were purified using the Illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare Life Science, Buckinghamshire, UK) according to the manufacturer's protocol.", "2.3. NS5A and NS5B Sequencing. The purified product was then sequenced using the same sets of primers used for PCR amplification. Bidirectional Sanger sequencing was performed by Macrogen Korea (http://www.macrogen.com).", "2.4. NS5A and NS5B Genotype Determination. HCV NS5A and NS5B consensus sequences obtained from Uruguayan patients were aligned with sequences from HCV representing all genotypes and main subtypes isolated in different geographic regions of the world. These sequences were obtained from Los Alamos HCV sequence database and from the NIAID Virus Pathogen Database and Analysis Resource (ViPR) [21, 22] . For strains included in these studies, see Supplementary Material Table S1 . Sequences were aligned using the CLUSTAL W software [23] . Once aligned, the best evolutionary model that described our sequence data was assessed using ModelGenerator program [24] . Using the GTR + G + I model (General time reversible + gamma + invariant sites), maximum likelihood phylogenetic trees were constructed for both NS5A and NS5B using the MEGA 5.0 software [25] . For NS5A, 953 nucleotides (positions 6367 to 7319, relative to HCV 1a reference strain, H77 NC_004102) were included in the phylogenetic", "analysis, whereas for NS5B, only 361 nucleotides corresponding to the Okamoto region (positions 8265 to 8625, relative to strain H77 NC_004102) were included. As a measure of the robustness of each node, we employed the bootstrapping method (1000 pseudoreplicates).", "For NS5A 1a Uruguayan sequences (n = 20), a second alignment and maximum likelihood phylogenetic tree was generated in order to analyze HCV evolutionary relationships between Uruguayan, Brazilian, and worldwide strains. For non-Uruguayan strains included in this analysis, see Supplementary Material Table S2. 2.5. NS5A and NS5B Sequence Analysis. In order to properly identify substitution changes in NS5A and NS5B regions from HCV strains circulating in Uruguayan patients, we generated world consensus sequences for 1a and 1b subtypes using a wide range of NS5A and NS5B sequences from HCV strains isolated worldwide. For this purpose, NS5A gene sequences corresponding to subtypes 1a (n = 160) and 1b (n = 88) were retrieved from Los Alamos HCV sequence database and from the NIAID ViPR [21, 22] . Likewise, datasets of 150 and 124 NS5B sequences were generated for subtypes 1a and 1b, respectively. Using Seqman program, implemented in DNAStar 5.01 package (DNASTAR, Madison, USA), a world", "consensus nucleotide sequences were generated for each gene and subtype. Each Uruguayan sequence was subsequently aligned to the corresponding reference sequences, and then in silico translated. The amino acid sequences obtained were compared in order to explore the presence of RASs as well as the presence of polymorphisms at a RAS position (RAPs) in Uruguayan HCV strains. RAPs are defined as any change from reference sequence for a specific genotype at a position associated with NS5A resistance [26] .", "To study the genetic variability of NS5A and NS5B regions of HCV strains circulating in Uruguayan patients, sequences of these regions (accession numbers MH070029-MH070090) were aligned with corresponding sequences from 59 HCV strains isolated elsewhere, representing all genotypes and main subtypes (for strains included in these analyses, see Supplementary Material Table S1 ). Therefore, maximum likelihood phylogenetic trees were constructed. The results of these studies are shown in Figure 1 All strains in the phylogenies were assigned according to their genotype, and each cluster was supported by very high bootstrap values for both analyzed regions. Strains isolated from Uruguayan patients (n = 31) were assigned to genotype 1, 20 of which corresponded to subtype 1a and 11 to subtype 1b. The results of NS5A (Figure 1 (a)) and NS5B (Figure 1", "Genotype 1b phylogenetic analyses were concordant for both genomic regions in all 31 sequences, suggesting no recombination events between these regions.\n\nTo further analyze the evolutionary relationships between the Uruguayan strains and those circulating in Brazil and elsewhere, a second maximum likelihood phylogenetic tree of HCV-1a sequences of NS5A partial region was built ( Figure 2 ). As was previously described, two distinct 1a clades (clades 1 and 2) were observed. Brazilian sequences clustered in a large group of related sequences inside clade 1 [9] . Whereas NS5A Uruguayan strains (in red) did not cluster in a particular clade, rather, they grouped dispersedly within all major world clades.", "With the purpose of studying the amino acid (AA) substitutions along the NS5A protein, Uruguayan HCV AA sequences were aligned with NS5A world consensus sequences (residues 23 to 354 relative to NS5A protein sequence). AA substitutions at positions previously found to be potentially associated with resistance to NS5A inhibitors, as well as polymorphisms at a RAS position, were identified. These results are summarized in Table 1 .", "RASs to NS5A inhibitors (L31M and L31V) were identified in 2 strains out of 25 (8%) fully sequenced samples. RAPs were found in 3 strains (subtype 1a): 2 exhibited the substitution H58P and 1 the substitution K24Q. Although these substitutions were not reported as resistant, some changes at these positions were previously described as RASs in subtype 1a, namely H58D and K24R [27, 28] . Finally, substitution E62D was found in one subtype 1a strain. This change is considered as a secondary substitution because, although it does not confer resistance by itself, when combined with a known RAS it does. In fact, it confers a higher level of resistance than the one achieved by the RAS alone [26] . In addition, several polymorphisms that have not been previously reported to be associated with a resistant phenotype were also detected (see Supplementary Material Table S3 ).", "In order to study substitutions along NS5B protein, Uruguayan HCV AA sequences were aligned to the NS5B world consensus sequences. Almost full-length AA sequences were obtained in 26 out of 31 analyzed strains. 23 sequences span residues 36 to 539 whereas the remaining 3 span residues 36 to 557 of NS5B protein.\n\nThis issue limited our studies, since many of the described RASs are observed as of residue 553.\n\nImportantly, RASs to NS5B inhibitors ( Table 2) were observed in 5 strains out of 26 sequenced samples (19.2%). C451R was found in two isolates while A421V was found in only one. In 2 of the 3 strains for which we were able to obtain longer sequences, RASs S556G (subtype 1a) and Q556R (subtype 1b) were observed.", "Finally, we found two RAPs: A421V (in 2 subtype 1b strains) and A553G (in 1 subtype 1a strain). Although A421V has been associated with resistance to beclabuvir (BCV) in patients infected with HCV subtype 1a, this resistant phenotype has not been proven in strains subtype 1b [29] . In position 553, the substitution reported as resistant was A553T [8] .\n\nAs was the case for NS5A, different polymorphisms not previously associated with a resistant phenotype were also detected in NS5B (see Supplementary Material Table S4 ).", "The advent of DAAs therapies constitutes one of the major breakthroughs in HCV infected patients management. However, these new treatment options are far from being universally available, in particular for HCV infected patients relying on Latin American public healthcare systems. The main limiting factors for worldwide access to DAAs in our region concern the high cost, the inadequate management of public healthcare systems, the limited access of low-income or uninsured populations to healthcare providers, and the lack of accurate epidemiological information [20, [30] [31] [32] . In Uruguay, these therapies became recently available, and although some have been approved for their use by the public health authorities (Viekira pak and sofosbuvir/ledipasvir therapies), they are not currently financially covered, except in specific cases. Despite the high rates of viral response achieved with DAA-based treatments, still 1 to10% of the patients fails to eliminate infection, and in these", "cases, baseline and emergent resistance variants turn out to be key factors contributing to treatment failure [5, 17, 33] .", "Unfortunately, we are currently unable to properly assess the number of HCV infected people in Uruguay and even more to figure out the frequency and type of RASs circulating. These facts could compromise the effectiveness of these new therapies in our country.", "We have previously reported that naturally occurring substitutions conferring resistance to NS3 inhibitors exist in a significant proportion of Uruguayan patients infected with HCV genotype 1, and we showed that this frequency seemed to be higher than in other South American countries (Brazil and Argentina) [34] . The present study describes the prevalence of baseline NS5A and NS5B RASs in HCV genotype 1 infected DAA-naïve patients in a Uruguayan cohort. The presence of substitutions conferring resistance to NS5A inhibitors has been widely reported both in therapynaïve and in relapser patients from Europe [10, 33, [35] [36] [37] [38] , USA [37, 39, 40] , and Asia [41] [42] [43] . However, NS5A sequences from South America are poorly analyzed yet [9, 44] . Recent studies have revealed that the mean prevalence of NS5A genotype 1 baseline RASs to different inhibitors ranges from 6% to 16% using population sequencing or deep sequencing [27, 37, 45, 46] . Importantly, the prevalence and", "type of baseline NS5A RASs varies slightly by geographic regions. For instance, L31M was found in 2.2% of genotype 1a infected patients in Europe, in 4.1% of those in Oceania, and strikingly in no patient from the USA [27] . For this reason, we believe that there is a need to contribute data from our region, for which we still do not have enough information, apart from Brazil [9, 44] . The results of this study indicate the presence of DAA NS5A RASs in 2 HCV strains (8% of the patients enrolled in this study), with baseline RASs detected at position 31 (see Table 1 ). L31M substitution confers resistance to daclatasvir (DCV), ledipasvir (LDV), and elbasvir (EBV) in both 1a and 1b subtypes [5, 6, 8, 28, 47, 48] , whereas substitution L31V does it to DCV in subtypes 1a and 1b, to LDV in subtype 1b, and to EBV in subtype 1a [5, 6, 28] . Given that both L31V and L31M are clinically relevant RASs, their detection at baseline may influence the choice of first-line treatment regimens [28] .", "The substitutions H58P and K24Q found in two patients are considered as resistance-associated polymorphisms (RAPs). The RASs characterized at these positions were H58D and K24G/N/R [5, 6, 27, 28, 49, 50] . The substitution H58P was found as a baseline RAP in relapsers to LDV (HARVONI prescription, https://www.gilead.com/-/ media/files/pdfs/medicines/liver-disease/harvoni/harvoni_pi. pdf?la=en). However, it is sometimes regarded as a RAS [10, 51] , despite conferring only 1.2 fold change in resistance in in vitro studies using the 1a replicon system [39] .\n\nWe did not find M28T/V, Q30R/H, or Y93H substitutions as there were previously reported in Brazil and worldwide [9, 27, 44] . The amino acid substitution E62H was found in one Uruguayan patient. Although this change does not confer resistance by itself but in combination with Q30R, it generates a high resistance level to DCV [52] .", "The presence of baseline NS5A RASs impacts treatment outcome in some patient groups by affecting SVR rates. The detection of NS5A preexistent RASs may play a relevant role in the choice of first-line treatment regimens or in the simplification/shortening of recommended regimens, in order to bring SVR rates close to the highest achievable [27, 38, 41, 53] , in particular in countries such as Uruguay, where only two different DAA-containing treatment regimens are approved for their use.", "Regarding NS5B gene, global analysis (with the exception of South America [17, 19] ) revealed that NS5B DAA resistance substitutions are infrequent [14] . Our study showed the presence of NS5B inhibitors RASs in 5 out of 26 analyzed HCV infected Uruguayan patients naïve to treatment (19.2%). Substitutions found in this work were A421V and S556G associated in subtype 1a with resistance to BCV and dasabuvir (DSV), respectively [8, 28, 29, 54, 55] , and Q556R associated with resistance to DSV both in genotype 1a and 1b [12, 28] . Substitution C451R, observed in two Uruguayan patients, was reported previously in patients who failed to clear the infection after treatment with OBV/PTV/r + DSV ± RBV. In these cases, it appeared in combination with G558R (Trial Coral I-Cohort 2: http:// www.hcv-trials.com/showStudy.asp?Study=86). RAPs in positions 421 and 553 (A421V in two subtype 1b isolates and A553G in one subtype 1b isolate) were also found. Although A421V has been associated with", "resistance to BCV in patients with subtype 1a, this phenotype has not been proven in strains of subtype 1b [29] . In position 553, the substitutions reported as resistant are A553T in subtype 1a [8] and A553V in subtype 1b [54] , conferring resistance to DSV.", "In contrast to our results, Noble and coworkers (2016) reported the presence of V321A, A421G, M414V, Y448H, L159F, and C316N in Brazilian isolates [17] , yet none of these mutations were found in this study, probably due to the diversity found between Uruguayan and Brazilian strains ( Figure 2 ). Nevertheless, substitution A421V was found in Brazil [17] , Argentina [19] , and Uruguay. The RAS S282T was detected neither in Brazilian reports nor in this current work (Uruguay) [17, 18, 56] . Our findings further confirm and complement previous studies which evidenced a low prevalence of this substitution in vivo, probably due to its low replicative fitness [14, 18, 57] . Despite our results, it is worth mentioning that the presence of baseline NS5B RASs conferring resistance to nucleotide or nonnucleoside NS5B inhibitors has not been shown to have any impact on virologic responses thus far [53, 58] .", "These results show both diversity in the baseline polymorphisms found in different Latin American countries and in the evolutionary relationships of Uruguayan isolates ( Figure 2 ). This fact could be linked not only to the isolates' geographic region and viral intrinsic characteristics but also to the genetic background of the host. It is worth mentioning that we live in a vast continent inhabited by populations with different genotypic characteristics that might, depending on the situation, require different approaches to treatment. Indeed, we have recently found that allele and genotype frequencies at IL28B locus of Uruguayan individuals closely resemble those of an admixed population rather than a uniformly European-descendant one [59] . Altogether, we believe that it could be important to carry out studies throughout the South American region in order to establish the prevalence of RASs in NS5A and NS5B in different countries. In fact, this will aid in understanding that not", "every treatment regimen might be adequate for every patient and country. The data we presented here might guide not only physicians in making therapeutic decisions but also public health authorities in approving more diverse treatment combinations. These treatment formulations would cover most of the circulating strains in our region, a region with an extremely diverse genetic background population.", "To our knowledge, the present study revealed for the first time the presence of RASs in the NS5A and NS5B regions of HCV genotype 1 Uruguayan strains from patients who have not been previously treated with DAAs and is one of the few South American countries to report on this matter. It is currently unclear if preexisting viral variants with reduced susceptibility to DAAs are clinically relevant for the prediction of virologic treatment failure. However, individualized DAA therapy based on baseline resistance analysis may be beneficial for optimizing treatment efficacy in patients with HCV genotype 1 infection and risk factors for treatment failure. Therefore, the potential role of baseline resistance testing remains an area of critical research and clinical questions.\n\nThe data used to support the findings of this study are included within the article.\n\nThe authors declare that they have no conflicts of interest.", "Fabián Aldunate and Natalia Echeverría contributed equally to this work.\n\nSupplementary Material Table S1 : hepatitis C Virus NS5A and NS5B sequences used as representatives of each genotype to perform the phylogenetic analysis. Their corresponding genotype, country of isolation, and GenBank accession number are indicated. Supplementary Material Table S2 : hepatitis C Virus NS5A subtype 1a sequences used to reveal evolutionary relationships between Uruguayan strains and others isolated elsewhere. Their corresponding country of isolation and GenBank accession number are indicated. Supplementary Material Table S3 : amino acid substitutions in NS5A protein not previously associated with resistance to NS5A inhibitors. Supplementary Material Table S4 : amino acid substitutions in NS5B protein not previously associated with resistance to polymerase inhibitors. (Supplementary Materials)" ]

[ 7 ]

[ "was obtained" ]

[ "Pretreatment Hepatitis C Virus NS5A/NS5B Resistance-Associated Substitutions in Genotype 1 Uruguayan Infected Patients\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6112080/\n\nSHA: f01ad3545245b4f884b48aa2b69c9deb942c3e77\n\nAuthors: Aldunate, Fabián; Echeverría, Natalia; Chiodi, Daniela; López, Pablo; Sánchez-Cicerón, Adriana; Fajardo, Alvaro; Soñora, Martín; Cristina, Juan; Hernández, Nelia; Moreno, Pilar\nDate: 2018-08-14\nDOI: 10.1155/2018/2514901\nLicense: cc-by", "Abstract: Hepatitis C Virus (HCV) infection treatment has dramatically changed with the advent of direct-acting antiviral agents (DAAs). However, the efficacy of DAAs can be attenuated by the presence of resistance-associated substitutions (RASs) before and after treatment. Indeed, RASs detected in DAA treatment-naïve HCV-infected patients could be useful for clinical management and outcome prediction. Although the frequency of naturally occurring HCV NS5A and NS5B RASs has been addressed in many countries, there are only a few reports on their prevalence in the South American region. The aim of this study was to investigate the presence of RASs to NS5A and NS5B inhibitors in a DAA treatment naïve cohort of Uruguayan patients infected with chronic hepatitis C and compare them with reports from other South American countries. Here, we found that naturally occurring substitutions conferring resistance to NS5A and NS5B inhibitors were present in 8% and 19.2%, respectively, of", "treatment-naïve HCV genotype 1 infected patients. Importantly, the baseline substitutions in NS5A and NS5B herein identified differ from the studies previously reported in Brazil. Furthermore, Uruguayan strains subtype 1a clustered within all major world clades, showing that HCV variants currently circulating in this country are characterized by a remarkable genetic diversity.", "Text: Hepatitis C Virus (HCV) infection treatment has dramatically improved thanks to the introduction of direct-acting antiviral agents (DAAs). These antivirals have significantly increased response rates (up to 98%) and greatly reduced treatment duration [1] . Currently available DAAs are classified into four categories given their molecular targets in the HCV replication cycle: (1) NS3/4A protease inhibitors (PIs) bind to the active site of the NS3/4A protease; (2) NS5A inhibitors interact with domain 1 of the NS5A dimer, although the exact mechanism of NS5A inhibition remains to be fully elucidated; (3) nucleos(t)ide analog NS5B polymerase inhibitors are incorporated into the nascent RNA chain resulting in chain termination by compromising the binding of the incoming nucleotide; (4) nonnucleoside NS5B polymerase inhibitors interact with either the thumb 1, thumb 2, palm 1, or palm 2 domain of NS5B and inhibit polymerase activity by allosteric mechanisms [2] [3] [4] . However, the", "extreme mutation and high replication rates of HCV, together with the immune system pressure, lead to a remarkable genetic variability that can compromise the high response rates to DAAs due to the preexistence of resistanceassociated substitutions (RASs) [5, 6] .", "Each drug or class of DAA is characterized by specific resistance profiles. The likelihood that a DAA will select for and allow outgrowth of viral populations carrying RASs depends on the DAA's genetic barrier to resistance (the number and type of mutations needed to generate an amino acid substitution that confers resistance), the viral fitness (replicative capacity) of the resistant variant, and viral genotypes and subtypes [7, 8] .\n\nThe prevalence of RASs in treatment-naïve patients has been broadly reported worldwide [9] [10] [11] [12] [13] [14] [15] [16] . However, apart from Brazil and Argentina, this issue has not been fully addressed in other South American countries yet [9, [17] [18] [19] . The lack of information in relation to preexisting baseline RASs, added to the high cost of these new drugs, are the major limiting factors for the broad implementation of these new therapies in Uruguay as well as in other Latin American countries (low-or lower-middle income) [20] .", "In this study, we explored the presence of resistance variants to NS5A and NS5B inhibitors in a DAA treatment naïve cohort of Uruguayan patients chronically infected with hepatitis C. Here, we aimed to contribute to the knowledge of the circulation of HCV resistant variants in the South American region.", "Samples. Serum samples were obtained from 31 patients with serological markers for HCV, which were recruited between 2015 and 2017 at the Gastroenterology Clinic from Hospital de Clínicas, Montevideo, Uruguay. HCV infection was confirmed by Abbott realtime HCV (Abbott Molecular Inc., Des Plaines, USA). Patients selected for this study were both chronically infected with HCV genotype 1 and DAA treatment-naïve at the time of blood extraction. Written informed consent was obtained from all patients. The studies have been performed according to the World Medical Association Declaration of Helsinki and approved by the appropriate institutional board (Hospital de Clínicas ethical committee).", "2.2. RNA Extraction, cDNA Synthesis, and NS5A and NS5B Amplification. Viral RNA was extracted from 140 μl of serum using the QIAamp Viral RNA mini kit (QIAgen, Hilden, Germany) according to the manufacturer's protocol. The viral RNA was heated at 65°C for 5 min and used as a template for a reverse transcription reaction. The reverse transcription reaction mixture contained 5 μl of the RNA template, 1 μl of random hexamer 100 ng/μl (Invitrogen Life Technologies, Carlsbad, CA, USA), 1 μl of dNTP mix (10 mM each), 4 μl of 5X first-strand buffer, 2 μl of 0.1 M DTT, 1 μl of SuperScript II reverse transcriptase (200 U/μl) (Invitrogen Life Technologies, Carlsbad, CA, USA), and 1 μl (40 U/μl) RNaseOUT (Invitrogen Life Technologies, Carlsbad, CA, USA). The reverse transcription was performed at 42°C for 50 min, and then the reverse transcriptase enzyme was inactivated at 70°C for 15 min. PCR amplification of NS5A and NS5B genome regions was performed using primers and conditions previously", "described [10] . Amplicons were purified using the Illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare Life Science, Buckinghamshire, UK) according to the manufacturer's protocol.", "2.3. NS5A and NS5B Sequencing. The purified product was then sequenced using the same sets of primers used for PCR amplification. Bidirectional Sanger sequencing was performed by Macrogen Korea (http://www.macrogen.com).", "2.4. NS5A and NS5B Genotype Determination. HCV NS5A and NS5B consensus sequences obtained from Uruguayan patients were aligned with sequences from HCV representing all genotypes and main subtypes isolated in different geographic regions of the world. These sequences were obtained from Los Alamos HCV sequence database and from the NIAID Virus Pathogen Database and Analysis Resource (ViPR) [21, 22] . For strains included in these studies, see Supplementary Material Table S1 . Sequences were aligned using the CLUSTAL W software [23] . Once aligned, the best evolutionary model that described our sequence data was assessed using ModelGenerator program [24] . Using the GTR + G + I model (General time reversible + gamma + invariant sites), maximum likelihood phylogenetic trees were constructed for both NS5A and NS5B using the MEGA 5.0 software [25] . For NS5A, 953 nucleotides (positions 6367 to 7319, relative to HCV 1a reference strain, H77 NC_004102) were included in the phylogenetic", "analysis, whereas for NS5B, only 361 nucleotides corresponding to the Okamoto region (positions 8265 to 8625, relative to strain H77 NC_004102) were included. As a measure of the robustness of each node, we employed the bootstrapping method (1000 pseudoreplicates).", "For NS5A 1a Uruguayan sequences (n = 20), a second alignment and maximum likelihood phylogenetic tree was generated in order to analyze HCV evolutionary relationships between Uruguayan, Brazilian, and worldwide strains. For non-Uruguayan strains included in this analysis, see Supplementary Material Table S2. 2.5. NS5A and NS5B Sequence Analysis. In order to properly identify substitution changes in NS5A and NS5B regions from HCV strains circulating in Uruguayan patients, we generated world consensus sequences for 1a and 1b subtypes using a wide range of NS5A and NS5B sequences from HCV strains isolated worldwide. For this purpose, NS5A gene sequences corresponding to subtypes 1a (n = 160) and 1b (n = 88) were retrieved from Los Alamos HCV sequence database and from the NIAID ViPR [21, 22] . Likewise, datasets of 150 and 124 NS5B sequences were generated for subtypes 1a and 1b, respectively. Using Seqman program, implemented in DNAStar 5.01 package (DNASTAR, Madison, USA), a world", "consensus nucleotide sequences were generated for each gene and subtype. Each Uruguayan sequence was subsequently aligned to the corresponding reference sequences, and then in silico translated. The amino acid sequences obtained were compared in order to explore the presence of RASs as well as the presence of polymorphisms at a RAS position (RAPs) in Uruguayan HCV strains. RAPs are defined as any change from reference sequence for a specific genotype at a position associated with NS5A resistance [26] .", "To study the genetic variability of NS5A and NS5B regions of HCV strains circulating in Uruguayan patients, sequences of these regions (accession numbers MH070029-MH070090) were aligned with corresponding sequences from 59 HCV strains isolated elsewhere, representing all genotypes and main subtypes (for strains included in these analyses, see Supplementary Material Table S1 ). Therefore, maximum likelihood phylogenetic trees were constructed. The results of these studies are shown in Figure 1 All strains in the phylogenies were assigned according to their genotype, and each cluster was supported by very high bootstrap values for both analyzed regions. Strains isolated from Uruguayan patients (n = 31) were assigned to genotype 1, 20 of which corresponded to subtype 1a and 11 to subtype 1b. The results of NS5A (Figure 1 (a)) and NS5B (Figure 1", "Genotype 1b phylogenetic analyses were concordant for both genomic regions in all 31 sequences, suggesting no recombination events between these regions.\n\nTo further analyze the evolutionary relationships between the Uruguayan strains and those circulating in Brazil and elsewhere, a second maximum likelihood phylogenetic tree of HCV-1a sequences of NS5A partial region was built ( Figure 2 ). As was previously described, two distinct 1a clades (clades 1 and 2) were observed. Brazilian sequences clustered in a large group of related sequences inside clade 1 [9] . Whereas NS5A Uruguayan strains (in red) did not cluster in a particular clade, rather, they grouped dispersedly within all major world clades.", "With the purpose of studying the amino acid (AA) substitutions along the NS5A protein, Uruguayan HCV AA sequences were aligned with NS5A world consensus sequences (residues 23 to 354 relative to NS5A protein sequence). AA substitutions at positions previously found to be potentially associated with resistance to NS5A inhibitors, as well as polymorphisms at a RAS position, were identified. These results are summarized in Table 1 .", "RASs to NS5A inhibitors (L31M and L31V) were identified in 2 strains out of 25 (8%) fully sequenced samples. RAPs were found in 3 strains (subtype 1a): 2 exhibited the substitution H58P and 1 the substitution K24Q. Although these substitutions were not reported as resistant, some changes at these positions were previously described as RASs in subtype 1a, namely H58D and K24R [27, 28] . Finally, substitution E62D was found in one subtype 1a strain. This change is considered as a secondary substitution because, although it does not confer resistance by itself, when combined with a known RAS it does. In fact, it confers a higher level of resistance than the one achieved by the RAS alone [26] . In addition, several polymorphisms that have not been previously reported to be associated with a resistant phenotype were also detected (see Supplementary Material Table S3 ).", "In order to study substitutions along NS5B protein, Uruguayan HCV AA sequences were aligned to the NS5B world consensus sequences. Almost full-length AA sequences were obtained in 26 out of 31 analyzed strains. 23 sequences span residues 36 to 539 whereas the remaining 3 span residues 36 to 557 of NS5B protein.\n\nThis issue limited our studies, since many of the described RASs are observed as of residue 553.\n\nImportantly, RASs to NS5B inhibitors ( Table 2) were observed in 5 strains out of 26 sequenced samples (19.2%). C451R was found in two isolates while A421V was found in only one. In 2 of the 3 strains for which we were able to obtain longer sequences, RASs S556G (subtype 1a) and Q556R (subtype 1b) were observed.", "Finally, we found two RAPs: A421V (in 2 subtype 1b strains) and A553G (in 1 subtype 1a strain). Although A421V has been associated with resistance to beclabuvir (BCV) in patients infected with HCV subtype 1a, this resistant phenotype has not been proven in strains subtype 1b [29] . In position 553, the substitution reported as resistant was A553T [8] .\n\nAs was the case for NS5A, different polymorphisms not previously associated with a resistant phenotype were also detected in NS5B (see Supplementary Material Table S4 ).", "The advent of DAAs therapies constitutes one of the major breakthroughs in HCV infected patients management. However, these new treatment options are far from being universally available, in particular for HCV infected patients relying on Latin American public healthcare systems. The main limiting factors for worldwide access to DAAs in our region concern the high cost, the inadequate management of public healthcare systems, the limited access of low-income or uninsured populations to healthcare providers, and the lack of accurate epidemiological information [20, [30] [31] [32] . In Uruguay, these therapies became recently available, and although some have been approved for their use by the public health authorities (Viekira pak and sofosbuvir/ledipasvir therapies), they are not currently financially covered, except in specific cases. Despite the high rates of viral response achieved with DAA-based treatments, still 1 to10% of the patients fails to eliminate infection, and in these", "cases, baseline and emergent resistance variants turn out to be key factors contributing to treatment failure [5, 17, 33] .", "Unfortunately, we are currently unable to properly assess the number of HCV infected people in Uruguay and even more to figure out the frequency and type of RASs circulating. These facts could compromise the effectiveness of these new therapies in our country.", "We have previously reported that naturally occurring substitutions conferring resistance to NS3 inhibitors exist in a significant proportion of Uruguayan patients infected with HCV genotype 1, and we showed that this frequency seemed to be higher than in other South American countries (Brazil and Argentina) [34] . The present study describes the prevalence of baseline NS5A and NS5B RASs in HCV genotype 1 infected DAA-naïve patients in a Uruguayan cohort. The presence of substitutions conferring resistance to NS5A inhibitors has been widely reported both in therapynaïve and in relapser patients from Europe [10, 33, [35] [36] [37] [38] , USA [37, 39, 40] , and Asia [41] [42] [43] . However, NS5A sequences from South America are poorly analyzed yet [9, 44] . Recent studies have revealed that the mean prevalence of NS5A genotype 1 baseline RASs to different inhibitors ranges from 6% to 16% using population sequencing or deep sequencing [27, 37, 45, 46] . Importantly, the prevalence and", "type of baseline NS5A RASs varies slightly by geographic regions. For instance, L31M was found in 2.2% of genotype 1a infected patients in Europe, in 4.1% of those in Oceania, and strikingly in no patient from the USA [27] . For this reason, we believe that there is a need to contribute data from our region, for which we still do not have enough information, apart from Brazil [9, 44] . The results of this study indicate the presence of DAA NS5A RASs in 2 HCV strains (8% of the patients enrolled in this study), with baseline RASs detected at position 31 (see Table 1 ). L31M substitution confers resistance to daclatasvir (DCV), ledipasvir (LDV), and elbasvir (EBV) in both 1a and 1b subtypes [5, 6, 8, 28, 47, 48] , whereas substitution L31V does it to DCV in subtypes 1a and 1b, to LDV in subtype 1b, and to EBV in subtype 1a [5, 6, 28] . Given that both L31V and L31M are clinically relevant RASs, their detection at baseline may influence the choice of first-line treatment regimens [28] .", "The substitutions H58P and K24Q found in two patients are considered as resistance-associated polymorphisms (RAPs). The RASs characterized at these positions were H58D and K24G/N/R [5, 6, 27, 28, 49, 50] . The substitution H58P was found as a baseline RAP in relapsers to LDV (HARVONI prescription, https://www.gilead.com/-/ media/files/pdfs/medicines/liver-disease/harvoni/harvoni_pi. pdf?la=en). However, it is sometimes regarded as a RAS [10, 51] , despite conferring only 1.2 fold change in resistance in in vitro studies using the 1a replicon system [39] .\n\nWe did not find M28T/V, Q30R/H, or Y93H substitutions as there were previously reported in Brazil and worldwide [9, 27, 44] . The amino acid substitution E62H was found in one Uruguayan patient. Although this change does not confer resistance by itself but in combination with Q30R, it generates a high resistance level to DCV [52] .", "The presence of baseline NS5A RASs impacts treatment outcome in some patient groups by affecting SVR rates. The detection of NS5A preexistent RASs may play a relevant role in the choice of first-line treatment regimens or in the simplification/shortening of recommended regimens, in order to bring SVR rates close to the highest achievable [27, 38, 41, 53] , in particular in countries such as Uruguay, where only two different DAA-containing treatment regimens are approved for their use.", "Regarding NS5B gene, global analysis (with the exception of South America [17, 19] ) revealed that NS5B DAA resistance substitutions are infrequent [14] . Our study showed the presence of NS5B inhibitors RASs in 5 out of 26 analyzed HCV infected Uruguayan patients naïve to treatment (19.2%). Substitutions found in this work were A421V and S556G associated in subtype 1a with resistance to BCV and dasabuvir (DSV), respectively [8, 28, 29, 54, 55] , and Q556R associated with resistance to DSV both in genotype 1a and 1b [12, 28] . Substitution C451R, observed in two Uruguayan patients, was reported previously in patients who failed to clear the infection after treatment with OBV/PTV/r + DSV ± RBV. In these cases, it appeared in combination with G558R (Trial Coral I-Cohort 2: http:// www.hcv-trials.com/showStudy.asp?Study=86). RAPs in positions 421 and 553 (A421V in two subtype 1b isolates and A553G in one subtype 1b isolate) were also found. Although A421V has been associated with", "resistance to BCV in patients with subtype 1a, this phenotype has not been proven in strains of subtype 1b [29] . In position 553, the substitutions reported as resistant are A553T in subtype 1a [8] and A553V in subtype 1b [54] , conferring resistance to DSV.", "In contrast to our results, Noble and coworkers (2016) reported the presence of V321A, A421G, M414V, Y448H, L159F, and C316N in Brazilian isolates [17] , yet none of these mutations were found in this study, probably due to the diversity found between Uruguayan and Brazilian strains ( Figure 2 ). Nevertheless, substitution A421V was found in Brazil [17] , Argentina [19] , and Uruguay. The RAS S282T was detected neither in Brazilian reports nor in this current work (Uruguay) [17, 18, 56] . Our findings further confirm and complement previous studies which evidenced a low prevalence of this substitution in vivo, probably due to its low replicative fitness [14, 18, 57] . Despite our results, it is worth mentioning that the presence of baseline NS5B RASs conferring resistance to nucleotide or nonnucleoside NS5B inhibitors has not been shown to have any impact on virologic responses thus far [53, 58] .", "These results show both diversity in the baseline polymorphisms found in different Latin American countries and in the evolutionary relationships of Uruguayan isolates ( Figure 2 ). This fact could be linked not only to the isolates' geographic region and viral intrinsic characteristics but also to the genetic background of the host. It is worth mentioning that we live in a vast continent inhabited by populations with different genotypic characteristics that might, depending on the situation, require different approaches to treatment. Indeed, we have recently found that allele and genotype frequencies at IL28B locus of Uruguayan individuals closely resemble those of an admixed population rather than a uniformly European-descendant one [59] . Altogether, we believe that it could be important to carry out studies throughout the South American region in order to establish the prevalence of RASs in NS5A and NS5B in different countries. In fact, this will aid in understanding that not", "every treatment regimen might be adequate for every patient and country. The data we presented here might guide not only physicians in making therapeutic decisions but also public health authorities in approving more diverse treatment combinations. These treatment formulations would cover most of the circulating strains in our region, a region with an extremely diverse genetic background population.", "To our knowledge, the present study revealed for the first time the presence of RASs in the NS5A and NS5B regions of HCV genotype 1 Uruguayan strains from patients who have not been previously treated with DAAs and is one of the few South American countries to report on this matter. It is currently unclear if preexisting viral variants with reduced susceptibility to DAAs are clinically relevant for the prediction of virologic treatment failure. However, individualized DAA therapy based on baseline resistance analysis may be beneficial for optimizing treatment efficacy in patients with HCV genotype 1 infection and risk factors for treatment failure. Therefore, the potential role of baseline resistance testing remains an area of critical research and clinical questions.\n\nThe data used to support the findings of this study are included within the article.\n\nThe authors declare that they have no conflicts of interest.", "Fabián Aldunate and Natalia Echeverría contributed equally to this work.\n\nSupplementary Material Table S1 : hepatitis C Virus NS5A and NS5B sequences used as representatives of each genotype to perform the phylogenetic analysis. Their corresponding genotype, country of isolation, and GenBank accession number are indicated. Supplementary Material Table S2 : hepatitis C Virus NS5A subtype 1a sequences used to reveal evolutionary relationships between Uruguayan strains and others isolated elsewhere. Their corresponding country of isolation and GenBank accession number are indicated. Supplementary Material Table S3 : amino acid substitutions in NS5A protein not previously associated with resistance to NS5A inhibitors. Supplementary Material Table S4 : amino acid substitutions in NS5B protein not previously associated with resistance to polymerase inhibitors. (Supplementary Materials)" ]

[ 7 ]

[ "5 μl" ]

[ "Pretreatment Hepatitis C Virus NS5A/NS5B Resistance-Associated Substitutions in Genotype 1 Uruguayan Infected Patients\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6112080/\n\nSHA: f01ad3545245b4f884b48aa2b69c9deb942c3e77\n\nAuthors: Aldunate, Fabián; Echeverría, Natalia; Chiodi, Daniela; López, Pablo; Sánchez-Cicerón, Adriana; Fajardo, Alvaro; Soñora, Martín; Cristina, Juan; Hernández, Nelia; Moreno, Pilar\nDate: 2018-08-14\nDOI: 10.1155/2018/2514901\nLicense: cc-by", "Abstract: Hepatitis C Virus (HCV) infection treatment has dramatically changed with the advent of direct-acting antiviral agents (DAAs). However, the efficacy of DAAs can be attenuated by the presence of resistance-associated substitutions (RASs) before and after treatment. Indeed, RASs detected in DAA treatment-naïve HCV-infected patients could be useful for clinical management and outcome prediction. Although the frequency of naturally occurring HCV NS5A and NS5B RASs has been addressed in many countries, there are only a few reports on their prevalence in the South American region. The aim of this study was to investigate the presence of RASs to NS5A and NS5B inhibitors in a DAA treatment naïve cohort of Uruguayan patients infected with chronic hepatitis C and compare them with reports from other South American countries. Here, we found that naturally occurring substitutions conferring resistance to NS5A and NS5B inhibitors were present in 8% and 19.2%, respectively, of", "treatment-naïve HCV genotype 1 infected patients. Importantly, the baseline substitutions in NS5A and NS5B herein identified differ from the studies previously reported in Brazil. Furthermore, Uruguayan strains subtype 1a clustered within all major world clades, showing that HCV variants currently circulating in this country are characterized by a remarkable genetic diversity.", "Text: Hepatitis C Virus (HCV) infection treatment has dramatically improved thanks to the introduction of direct-acting antiviral agents (DAAs). These antivirals have significantly increased response rates (up to 98%) and greatly reduced treatment duration [1] . Currently available DAAs are classified into four categories given their molecular targets in the HCV replication cycle: (1) NS3/4A protease inhibitors (PIs) bind to the active site of the NS3/4A protease; (2) NS5A inhibitors interact with domain 1 of the NS5A dimer, although the exact mechanism of NS5A inhibition remains to be fully elucidated; (3) nucleos(t)ide analog NS5B polymerase inhibitors are incorporated into the nascent RNA chain resulting in chain termination by compromising the binding of the incoming nucleotide; (4) nonnucleoside NS5B polymerase inhibitors interact with either the thumb 1, thumb 2, palm 1, or palm 2 domain of NS5B and inhibit polymerase activity by allosteric mechanisms [2] [3] [4] . However, the", "extreme mutation and high replication rates of HCV, together with the immune system pressure, lead to a remarkable genetic variability that can compromise the high response rates to DAAs due to the preexistence of resistanceassociated substitutions (RASs) [5, 6] .", "Each drug or class of DAA is characterized by specific resistance profiles. The likelihood that a DAA will select for and allow outgrowth of viral populations carrying RASs depends on the DAA's genetic barrier to resistance (the number and type of mutations needed to generate an amino acid substitution that confers resistance), the viral fitness (replicative capacity) of the resistant variant, and viral genotypes and subtypes [7, 8] .\n\nThe prevalence of RASs in treatment-naïve patients has been broadly reported worldwide [9] [10] [11] [12] [13] [14] [15] [16] . However, apart from Brazil and Argentina, this issue has not been fully addressed in other South American countries yet [9, [17] [18] [19] . The lack of information in relation to preexisting baseline RASs, added to the high cost of these new drugs, are the major limiting factors for the broad implementation of these new therapies in Uruguay as well as in other Latin American countries (low-or lower-middle income) [20] .", "In this study, we explored the presence of resistance variants to NS5A and NS5B inhibitors in a DAA treatment naïve cohort of Uruguayan patients chronically infected with hepatitis C. Here, we aimed to contribute to the knowledge of the circulation of HCV resistant variants in the South American region.", "Samples. Serum samples were obtained from 31 patients with serological markers for HCV, which were recruited between 2015 and 2017 at the Gastroenterology Clinic from Hospital de Clínicas, Montevideo, Uruguay. HCV infection was confirmed by Abbott realtime HCV (Abbott Molecular Inc., Des Plaines, USA). Patients selected for this study were both chronically infected with HCV genotype 1 and DAA treatment-naïve at the time of blood extraction. Written informed consent was obtained from all patients. The studies have been performed according to the World Medical Association Declaration of Helsinki and approved by the appropriate institutional board (Hospital de Clínicas ethical committee).", "2.2. RNA Extraction, cDNA Synthesis, and NS5A and NS5B Amplification. Viral RNA was extracted from 140 μl of serum using the QIAamp Viral RNA mini kit (QIAgen, Hilden, Germany) according to the manufacturer's protocol. The viral RNA was heated at 65°C for 5 min and used as a template for a reverse transcription reaction. The reverse transcription reaction mixture contained 5 μl of the RNA template, 1 μl of random hexamer 100 ng/μl (Invitrogen Life Technologies, Carlsbad, CA, USA), 1 μl of dNTP mix (10 mM each), 4 μl of 5X first-strand buffer, 2 μl of 0.1 M DTT, 1 μl of SuperScript II reverse transcriptase (200 U/μl) (Invitrogen Life Technologies, Carlsbad, CA, USA), and 1 μl (40 U/μl) RNaseOUT (Invitrogen Life Technologies, Carlsbad, CA, USA). The reverse transcription was performed at 42°C for 50 min, and then the reverse transcriptase enzyme was inactivated at 70°C for 15 min. PCR amplification of NS5A and NS5B genome regions was performed using primers and conditions previously", "described [10] . Amplicons were purified using the Illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare Life Science, Buckinghamshire, UK) according to the manufacturer's protocol.", "2.3. NS5A and NS5B Sequencing. The purified product was then sequenced using the same sets of primers used for PCR amplification. Bidirectional Sanger sequencing was performed by Macrogen Korea (http://www.macrogen.com).", "2.4. NS5A and NS5B Genotype Determination. HCV NS5A and NS5B consensus sequences obtained from Uruguayan patients were aligned with sequences from HCV representing all genotypes and main subtypes isolated in different geographic regions of the world. These sequences were obtained from Los Alamos HCV sequence database and from the NIAID Virus Pathogen Database and Analysis Resource (ViPR) [21, 22] . For strains included in these studies, see Supplementary Material Table S1 . Sequences were aligned using the CLUSTAL W software [23] . Once aligned, the best evolutionary model that described our sequence data was assessed using ModelGenerator program [24] . Using the GTR + G + I model (General time reversible + gamma + invariant sites), maximum likelihood phylogenetic trees were constructed for both NS5A and NS5B using the MEGA 5.0 software [25] . For NS5A, 953 nucleotides (positions 6367 to 7319, relative to HCV 1a reference strain, H77 NC_004102) were included in the phylogenetic", "analysis, whereas for NS5B, only 361 nucleotides corresponding to the Okamoto region (positions 8265 to 8625, relative to strain H77 NC_004102) were included. As a measure of the robustness of each node, we employed the bootstrapping method (1000 pseudoreplicates).", "For NS5A 1a Uruguayan sequences (n = 20), a second alignment and maximum likelihood phylogenetic tree was generated in order to analyze HCV evolutionary relationships between Uruguayan, Brazilian, and worldwide strains. For non-Uruguayan strains included in this analysis, see Supplementary Material Table S2. 2.5. NS5A and NS5B Sequence Analysis. In order to properly identify substitution changes in NS5A and NS5B regions from HCV strains circulating in Uruguayan patients, we generated world consensus sequences for 1a and 1b subtypes using a wide range of NS5A and NS5B sequences from HCV strains isolated worldwide. For this purpose, NS5A gene sequences corresponding to subtypes 1a (n = 160) and 1b (n = 88) were retrieved from Los Alamos HCV sequence database and from the NIAID ViPR [21, 22] . Likewise, datasets of 150 and 124 NS5B sequences were generated for subtypes 1a and 1b, respectively. Using Seqman program, implemented in DNAStar 5.01 package (DNASTAR, Madison, USA), a world", "consensus nucleotide sequences were generated for each gene and subtype. Each Uruguayan sequence was subsequently aligned to the corresponding reference sequences, and then in silico translated. The amino acid sequences obtained were compared in order to explore the presence of RASs as well as the presence of polymorphisms at a RAS position (RAPs) in Uruguayan HCV strains. RAPs are defined as any change from reference sequence for a specific genotype at a position associated with NS5A resistance [26] .", "To study the genetic variability of NS5A and NS5B regions of HCV strains circulating in Uruguayan patients, sequences of these regions (accession numbers MH070029-MH070090) were aligned with corresponding sequences from 59 HCV strains isolated elsewhere, representing all genotypes and main subtypes (for strains included in these analyses, see Supplementary Material Table S1 ). Therefore, maximum likelihood phylogenetic trees were constructed. The results of these studies are shown in Figure 1 All strains in the phylogenies were assigned according to their genotype, and each cluster was supported by very high bootstrap values for both analyzed regions. Strains isolated from Uruguayan patients (n = 31) were assigned to genotype 1, 20 of which corresponded to subtype 1a and 11 to subtype 1b. The results of NS5A (Figure 1 (a)) and NS5B (Figure 1", "Genotype 1b phylogenetic analyses were concordant for both genomic regions in all 31 sequences, suggesting no recombination events between these regions.\n\nTo further analyze the evolutionary relationships between the Uruguayan strains and those circulating in Brazil and elsewhere, a second maximum likelihood phylogenetic tree of HCV-1a sequences of NS5A partial region was built ( Figure 2 ). As was previously described, two distinct 1a clades (clades 1 and 2) were observed. Brazilian sequences clustered in a large group of related sequences inside clade 1 [9] . Whereas NS5A Uruguayan strains (in red) did not cluster in a particular clade, rather, they grouped dispersedly within all major world clades.", "With the purpose of studying the amino acid (AA) substitutions along the NS5A protein, Uruguayan HCV AA sequences were aligned with NS5A world consensus sequences (residues 23 to 354 relative to NS5A protein sequence). AA substitutions at positions previously found to be potentially associated with resistance to NS5A inhibitors, as well as polymorphisms at a RAS position, were identified. These results are summarized in Table 1 .", "RASs to NS5A inhibitors (L31M and L31V) were identified in 2 strains out of 25 (8%) fully sequenced samples. RAPs were found in 3 strains (subtype 1a): 2 exhibited the substitution H58P and 1 the substitution K24Q. Although these substitutions were not reported as resistant, some changes at these positions were previously described as RASs in subtype 1a, namely H58D and K24R [27, 28] . Finally, substitution E62D was found in one subtype 1a strain. This change is considered as a secondary substitution because, although it does not confer resistance by itself, when combined with a known RAS it does. In fact, it confers a higher level of resistance than the one achieved by the RAS alone [26] . In addition, several polymorphisms that have not been previously reported to be associated with a resistant phenotype were also detected (see Supplementary Material Table S3 ).", "In order to study substitutions along NS5B protein, Uruguayan HCV AA sequences were aligned to the NS5B world consensus sequences. Almost full-length AA sequences were obtained in 26 out of 31 analyzed strains. 23 sequences span residues 36 to 539 whereas the remaining 3 span residues 36 to 557 of NS5B protein.\n\nThis issue limited our studies, since many of the described RASs are observed as of residue 553.\n\nImportantly, RASs to NS5B inhibitors ( Table 2) were observed in 5 strains out of 26 sequenced samples (19.2%). C451R was found in two isolates while A421V was found in only one. In 2 of the 3 strains for which we were able to obtain longer sequences, RASs S556G (subtype 1a) and Q556R (subtype 1b) were observed.", "Finally, we found two RAPs: A421V (in 2 subtype 1b strains) and A553G (in 1 subtype 1a strain). Although A421V has been associated with resistance to beclabuvir (BCV) in patients infected with HCV subtype 1a, this resistant phenotype has not been proven in strains subtype 1b [29] . In position 553, the substitution reported as resistant was A553T [8] .\n\nAs was the case for NS5A, different polymorphisms not previously associated with a resistant phenotype were also detected in NS5B (see Supplementary Material Table S4 ).", "The advent of DAAs therapies constitutes one of the major breakthroughs in HCV infected patients management. However, these new treatment options are far from being universally available, in particular for HCV infected patients relying on Latin American public healthcare systems. The main limiting factors for worldwide access to DAAs in our region concern the high cost, the inadequate management of public healthcare systems, the limited access of low-income or uninsured populations to healthcare providers, and the lack of accurate epidemiological information [20, [30] [31] [32] . In Uruguay, these therapies became recently available, and although some have been approved for their use by the public health authorities (Viekira pak and sofosbuvir/ledipasvir therapies), they are not currently financially covered, except in specific cases. Despite the high rates of viral response achieved with DAA-based treatments, still 1 to10% of the patients fails to eliminate infection, and in these", "cases, baseline and emergent resistance variants turn out to be key factors contributing to treatment failure [5, 17, 33] .", "Unfortunately, we are currently unable to properly assess the number of HCV infected people in Uruguay and even more to figure out the frequency and type of RASs circulating. These facts could compromise the effectiveness of these new therapies in our country.", "We have previously reported that naturally occurring substitutions conferring resistance to NS3 inhibitors exist in a significant proportion of Uruguayan patients infected with HCV genotype 1, and we showed that this frequency seemed to be higher than in other South American countries (Brazil and Argentina) [34] . The present study describes the prevalence of baseline NS5A and NS5B RASs in HCV genotype 1 infected DAA-naïve patients in a Uruguayan cohort. The presence of substitutions conferring resistance to NS5A inhibitors has been widely reported both in therapynaïve and in relapser patients from Europe [10, 33, [35] [36] [37] [38] , USA [37, 39, 40] , and Asia [41] [42] [43] . However, NS5A sequences from South America are poorly analyzed yet [9, 44] . Recent studies have revealed that the mean prevalence of NS5A genotype 1 baseline RASs to different inhibitors ranges from 6% to 16% using population sequencing or deep sequencing [27, 37, 45, 46] . Importantly, the prevalence and", "type of baseline NS5A RASs varies slightly by geographic regions. For instance, L31M was found in 2.2% of genotype 1a infected patients in Europe, in 4.1% of those in Oceania, and strikingly in no patient from the USA [27] . For this reason, we believe that there is a need to contribute data from our region, for which we still do not have enough information, apart from Brazil [9, 44] . The results of this study indicate the presence of DAA NS5A RASs in 2 HCV strains (8% of the patients enrolled in this study), with baseline RASs detected at position 31 (see Table 1 ). L31M substitution confers resistance to daclatasvir (DCV), ledipasvir (LDV), and elbasvir (EBV) in both 1a and 1b subtypes [5, 6, 8, 28, 47, 48] , whereas substitution L31V does it to DCV in subtypes 1a and 1b, to LDV in subtype 1b, and to EBV in subtype 1a [5, 6, 28] . Given that both L31V and L31M are clinically relevant RASs, their detection at baseline may influence the choice of first-line treatment regimens [28] .", "The substitutions H58P and K24Q found in two patients are considered as resistance-associated polymorphisms (RAPs). The RASs characterized at these positions were H58D and K24G/N/R [5, 6, 27, 28, 49, 50] . The substitution H58P was found as a baseline RAP in relapsers to LDV (HARVONI prescription, https://www.gilead.com/-/ media/files/pdfs/medicines/liver-disease/harvoni/harvoni_pi. pdf?la=en). However, it is sometimes regarded as a RAS [10, 51] , despite conferring only 1.2 fold change in resistance in in vitro studies using the 1a replicon system [39] .\n\nWe did not find M28T/V, Q30R/H, or Y93H substitutions as there were previously reported in Brazil and worldwide [9, 27, 44] . The amino acid substitution E62H was found in one Uruguayan patient. Although this change does not confer resistance by itself but in combination with Q30R, it generates a high resistance level to DCV [52] .", "The presence of baseline NS5A RASs impacts treatment outcome in some patient groups by affecting SVR rates. The detection of NS5A preexistent RASs may play a relevant role in the choice of first-line treatment regimens or in the simplification/shortening of recommended regimens, in order to bring SVR rates close to the highest achievable [27, 38, 41, 53] , in particular in countries such as Uruguay, where only two different DAA-containing treatment regimens are approved for their use.", "Regarding NS5B gene, global analysis (with the exception of South America [17, 19] ) revealed that NS5B DAA resistance substitutions are infrequent [14] . Our study showed the presence of NS5B inhibitors RASs in 5 out of 26 analyzed HCV infected Uruguayan patients naïve to treatment (19.2%). Substitutions found in this work were A421V and S556G associated in subtype 1a with resistance to BCV and dasabuvir (DSV), respectively [8, 28, 29, 54, 55] , and Q556R associated with resistance to DSV both in genotype 1a and 1b [12, 28] . Substitution C451R, observed in two Uruguayan patients, was reported previously in patients who failed to clear the infection after treatment with OBV/PTV/r + DSV ± RBV. In these cases, it appeared in combination with G558R (Trial Coral I-Cohort 2: http:// www.hcv-trials.com/showStudy.asp?Study=86). RAPs in positions 421 and 553 (A421V in two subtype 1b isolates and A553G in one subtype 1b isolate) were also found. Although A421V has been associated with", "resistance to BCV in patients with subtype 1a, this phenotype has not been proven in strains of subtype 1b [29] . In position 553, the substitutions reported as resistant are A553T in subtype 1a [8] and A553V in subtype 1b [54] , conferring resistance to DSV.", "In contrast to our results, Noble and coworkers (2016) reported the presence of V321A, A421G, M414V, Y448H, L159F, and C316N in Brazilian isolates [17] , yet none of these mutations were found in this study, probably due to the diversity found between Uruguayan and Brazilian strains ( Figure 2 ). Nevertheless, substitution A421V was found in Brazil [17] , Argentina [19] , and Uruguay. The RAS S282T was detected neither in Brazilian reports nor in this current work (Uruguay) [17, 18, 56] . Our findings further confirm and complement previous studies which evidenced a low prevalence of this substitution in vivo, probably due to its low replicative fitness [14, 18, 57] . Despite our results, it is worth mentioning that the presence of baseline NS5B RASs conferring resistance to nucleotide or nonnucleoside NS5B inhibitors has not been shown to have any impact on virologic responses thus far [53, 58] .", "These results show both diversity in the baseline polymorphisms found in different Latin American countries and in the evolutionary relationships of Uruguayan isolates ( Figure 2 ). This fact could be linked not only to the isolates' geographic region and viral intrinsic characteristics but also to the genetic background of the host. It is worth mentioning that we live in a vast continent inhabited by populations with different genotypic characteristics that might, depending on the situation, require different approaches to treatment. Indeed, we have recently found that allele and genotype frequencies at IL28B locus of Uruguayan individuals closely resemble those of an admixed population rather than a uniformly European-descendant one [59] . Altogether, we believe that it could be important to carry out studies throughout the South American region in order to establish the prevalence of RASs in NS5A and NS5B in different countries. In fact, this will aid in understanding that not", "every treatment regimen might be adequate for every patient and country. The data we presented here might guide not only physicians in making therapeutic decisions but also public health authorities in approving more diverse treatment combinations. These treatment formulations would cover most of the circulating strains in our region, a region with an extremely diverse genetic background population.", "To our knowledge, the present study revealed for the first time the presence of RASs in the NS5A and NS5B regions of HCV genotype 1 Uruguayan strains from patients who have not been previously treated with DAAs and is one of the few South American countries to report on this matter. It is currently unclear if preexisting viral variants with reduced susceptibility to DAAs are clinically relevant for the prediction of virologic treatment failure. However, individualized DAA therapy based on baseline resistance analysis may be beneficial for optimizing treatment efficacy in patients with HCV genotype 1 infection and risk factors for treatment failure. Therefore, the potential role of baseline resistance testing remains an area of critical research and clinical questions.\n\nThe data used to support the findings of this study are included within the article.\n\nThe authors declare that they have no conflicts of interest.", "Fabián Aldunate and Natalia Echeverría contributed equally to this work.\n\nSupplementary Material Table S1 : hepatitis C Virus NS5A and NS5B sequences used as representatives of each genotype to perform the phylogenetic analysis. Their corresponding genotype, country of isolation, and GenBank accession number are indicated. Supplementary Material Table S2 : hepatitis C Virus NS5A subtype 1a sequences used to reveal evolutionary relationships between Uruguayan strains and others isolated elsewhere. Their corresponding country of isolation and GenBank accession number are indicated. Supplementary Material Table S3 : amino acid substitutions in NS5A protein not previously associated with resistance to NS5A inhibitors. Supplementary Material Table S4 : amino acid substitutions in NS5B protein not previously associated with resistance to polymerase inhibitors. (Supplementary Materials)" ]

[ 8 ]

[ "2 strains out of 25 (8%)" ]

[ "Pretreatment Hepatitis C Virus NS5A/NS5B Resistance-Associated Substitutions in Genotype 1 Uruguayan Infected Patients\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6112080/\n\nSHA: f01ad3545245b4f884b48aa2b69c9deb942c3e77\n\nAuthors: Aldunate, Fabián; Echeverría, Natalia; Chiodi, Daniela; López, Pablo; Sánchez-Cicerón, Adriana; Fajardo, Alvaro; Soñora, Martín; Cristina, Juan; Hernández, Nelia; Moreno, Pilar\nDate: 2018-08-14\nDOI: 10.1155/2018/2514901\nLicense: cc-by", "Abstract: Hepatitis C Virus (HCV) infection treatment has dramatically changed with the advent of direct-acting antiviral agents (DAAs). However, the efficacy of DAAs can be attenuated by the presence of resistance-associated substitutions (RASs) before and after treatment. Indeed, RASs detected in DAA treatment-naïve HCV-infected patients could be useful for clinical management and outcome prediction. Although the frequency of naturally occurring HCV NS5A and NS5B RASs has been addressed in many countries, there are only a few reports on their prevalence in the South American region. The aim of this study was to investigate the presence of RASs to NS5A and NS5B inhibitors in a DAA treatment naïve cohort of Uruguayan patients infected with chronic hepatitis C and compare them with reports from other South American countries. Here, we found that naturally occurring substitutions conferring resistance to NS5A and NS5B inhibitors were present in 8% and 19.2%, respectively, of", "treatment-naïve HCV genotype 1 infected patients. Importantly, the baseline substitutions in NS5A and NS5B herein identified differ from the studies previously reported in Brazil. Furthermore, Uruguayan strains subtype 1a clustered within all major world clades, showing that HCV variants currently circulating in this country are characterized by a remarkable genetic diversity.", "Text: Hepatitis C Virus (HCV) infection treatment has dramatically improved thanks to the introduction of direct-acting antiviral agents (DAAs). These antivirals have significantly increased response rates (up to 98%) and greatly reduced treatment duration [1] . Currently available DAAs are classified into four categories given their molecular targets in the HCV replication cycle: (1) NS3/4A protease inhibitors (PIs) bind to the active site of the NS3/4A protease; (2) NS5A inhibitors interact with domain 1 of the NS5A dimer, although the exact mechanism of NS5A inhibition remains to be fully elucidated; (3) nucleos(t)ide analog NS5B polymerase inhibitors are incorporated into the nascent RNA chain resulting in chain termination by compromising the binding of the incoming nucleotide; (4) nonnucleoside NS5B polymerase inhibitors interact with either the thumb 1, thumb 2, palm 1, or palm 2 domain of NS5B and inhibit polymerase activity by allosteric mechanisms [2] [3] [4] . However, the", "extreme mutation and high replication rates of HCV, together with the immune system pressure, lead to a remarkable genetic variability that can compromise the high response rates to DAAs due to the preexistence of resistanceassociated substitutions (RASs) [5, 6] .", "Each drug or class of DAA is characterized by specific resistance profiles. The likelihood that a DAA will select for and allow outgrowth of viral populations carrying RASs depends on the DAA's genetic barrier to resistance (the number and type of mutations needed to generate an amino acid substitution that confers resistance), the viral fitness (replicative capacity) of the resistant variant, and viral genotypes and subtypes [7, 8] .\n\nThe prevalence of RASs in treatment-naïve patients has been broadly reported worldwide [9] [10] [11] [12] [13] [14] [15] [16] . However, apart from Brazil and Argentina, this issue has not been fully addressed in other South American countries yet [9, [17] [18] [19] . The lack of information in relation to preexisting baseline RASs, added to the high cost of these new drugs, are the major limiting factors for the broad implementation of these new therapies in Uruguay as well as in other Latin American countries (low-or lower-middle income) [20] .", "In this study, we explored the presence of resistance variants to NS5A and NS5B inhibitors in a DAA treatment naïve cohort of Uruguayan patients chronically infected with hepatitis C. Here, we aimed to contribute to the knowledge of the circulation of HCV resistant variants in the South American region.", "Samples. Serum samples were obtained from 31 patients with serological markers for HCV, which were recruited between 2015 and 2017 at the Gastroenterology Clinic from Hospital de Clínicas, Montevideo, Uruguay. HCV infection was confirmed by Abbott realtime HCV (Abbott Molecular Inc., Des Plaines, USA). Patients selected for this study were both chronically infected with HCV genotype 1 and DAA treatment-naïve at the time of blood extraction. Written informed consent was obtained from all patients. The studies have been performed according to the World Medical Association Declaration of Helsinki and approved by the appropriate institutional board (Hospital de Clínicas ethical committee).", "2.2. RNA Extraction, cDNA Synthesis, and NS5A and NS5B Amplification. Viral RNA was extracted from 140 μl of serum using the QIAamp Viral RNA mini kit (QIAgen, Hilden, Germany) according to the manufacturer's protocol. The viral RNA was heated at 65°C for 5 min and used as a template for a reverse transcription reaction. The reverse transcription reaction mixture contained 5 μl of the RNA template, 1 μl of random hexamer 100 ng/μl (Invitrogen Life Technologies, Carlsbad, CA, USA), 1 μl of dNTP mix (10 mM each), 4 μl of 5X first-strand buffer, 2 μl of 0.1 M DTT, 1 μl of SuperScript II reverse transcriptase (200 U/μl) (Invitrogen Life Technologies, Carlsbad, CA, USA), and 1 μl (40 U/μl) RNaseOUT (Invitrogen Life Technologies, Carlsbad, CA, USA). The reverse transcription was performed at 42°C for 50 min, and then the reverse transcriptase enzyme was inactivated at 70°C for 15 min. PCR amplification of NS5A and NS5B genome regions was performed using primers and conditions previously", "described [10] . Amplicons were purified using the Illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare Life Science, Buckinghamshire, UK) according to the manufacturer's protocol.", "2.3. NS5A and NS5B Sequencing. The purified product was then sequenced using the same sets of primers used for PCR amplification. Bidirectional Sanger sequencing was performed by Macrogen Korea (http://www.macrogen.com).", "2.4. NS5A and NS5B Genotype Determination. HCV NS5A and NS5B consensus sequences obtained from Uruguayan patients were aligned with sequences from HCV representing all genotypes and main subtypes isolated in different geographic regions of the world. These sequences were obtained from Los Alamos HCV sequence database and from the NIAID Virus Pathogen Database and Analysis Resource (ViPR) [21, 22] . For strains included in these studies, see Supplementary Material Table S1 . Sequences were aligned using the CLUSTAL W software [23] . Once aligned, the best evolutionary model that described our sequence data was assessed using ModelGenerator program [24] . Using the GTR + G + I model (General time reversible + gamma + invariant sites), maximum likelihood phylogenetic trees were constructed for both NS5A and NS5B using the MEGA 5.0 software [25] . For NS5A, 953 nucleotides (positions 6367 to 7319, relative to HCV 1a reference strain, H77 NC_004102) were included in the phylogenetic", "analysis, whereas for NS5B, only 361 nucleotides corresponding to the Okamoto region (positions 8265 to 8625, relative to strain H77 NC_004102) were included. As a measure of the robustness of each node, we employed the bootstrapping method (1000 pseudoreplicates).", "For NS5A 1a Uruguayan sequences (n = 20), a second alignment and maximum likelihood phylogenetic tree was generated in order to analyze HCV evolutionary relationships between Uruguayan, Brazilian, and worldwide strains. For non-Uruguayan strains included in this analysis, see Supplementary Material Table S2. 2.5. NS5A and NS5B Sequence Analysis. In order to properly identify substitution changes in NS5A and NS5B regions from HCV strains circulating in Uruguayan patients, we generated world consensus sequences for 1a and 1b subtypes using a wide range of NS5A and NS5B sequences from HCV strains isolated worldwide. For this purpose, NS5A gene sequences corresponding to subtypes 1a (n = 160) and 1b (n = 88) were retrieved from Los Alamos HCV sequence database and from the NIAID ViPR [21, 22] . Likewise, datasets of 150 and 124 NS5B sequences were generated for subtypes 1a and 1b, respectively. Using Seqman program, implemented in DNAStar 5.01 package (DNASTAR, Madison, USA), a world", "consensus nucleotide sequences were generated for each gene and subtype. Each Uruguayan sequence was subsequently aligned to the corresponding reference sequences, and then in silico translated. The amino acid sequences obtained were compared in order to explore the presence of RASs as well as the presence of polymorphisms at a RAS position (RAPs) in Uruguayan HCV strains. RAPs are defined as any change from reference sequence for a specific genotype at a position associated with NS5A resistance [26] .", "To study the genetic variability of NS5A and NS5B regions of HCV strains circulating in Uruguayan patients, sequences of these regions (accession numbers MH070029-MH070090) were aligned with corresponding sequences from 59 HCV strains isolated elsewhere, representing all genotypes and main subtypes (for strains included in these analyses, see Supplementary Material Table S1 ). Therefore, maximum likelihood phylogenetic trees were constructed. The results of these studies are shown in Figure 1 All strains in the phylogenies were assigned according to their genotype, and each cluster was supported by very high bootstrap values for both analyzed regions. Strains isolated from Uruguayan patients (n = 31) were assigned to genotype 1, 20 of which corresponded to subtype 1a and 11 to subtype 1b. The results of NS5A (Figure 1 (a)) and NS5B (Figure 1", "Genotype 1b phylogenetic analyses were concordant for both genomic regions in all 31 sequences, suggesting no recombination events between these regions.\n\nTo further analyze the evolutionary relationships between the Uruguayan strains and those circulating in Brazil and elsewhere, a second maximum likelihood phylogenetic tree of HCV-1a sequences of NS5A partial region was built ( Figure 2 ). As was previously described, two distinct 1a clades (clades 1 and 2) were observed. Brazilian sequences clustered in a large group of related sequences inside clade 1 [9] . Whereas NS5A Uruguayan strains (in red) did not cluster in a particular clade, rather, they grouped dispersedly within all major world clades.", "With the purpose of studying the amino acid (AA) substitutions along the NS5A protein, Uruguayan HCV AA sequences were aligned with NS5A world consensus sequences (residues 23 to 354 relative to NS5A protein sequence). AA substitutions at positions previously found to be potentially associated with resistance to NS5A inhibitors, as well as polymorphisms at a RAS position, were identified. These results are summarized in Table 1 .", "RASs to NS5A inhibitors (L31M and L31V) were identified in 2 strains out of 25 (8%) fully sequenced samples. RAPs were found in 3 strains (subtype 1a): 2 exhibited the substitution H58P and 1 the substitution K24Q. Although these substitutions were not reported as resistant, some changes at these positions were previously described as RASs in subtype 1a, namely H58D and K24R [27, 28] . Finally, substitution E62D was found in one subtype 1a strain. This change is considered as a secondary substitution because, although it does not confer resistance by itself, when combined with a known RAS it does. In fact, it confers a higher level of resistance than the one achieved by the RAS alone [26] . In addition, several polymorphisms that have not been previously reported to be associated with a resistant phenotype were also detected (see Supplementary Material Table S3 ).", "In order to study substitutions along NS5B protein, Uruguayan HCV AA sequences were aligned to the NS5B world consensus sequences. Almost full-length AA sequences were obtained in 26 out of 31 analyzed strains. 23 sequences span residues 36 to 539 whereas the remaining 3 span residues 36 to 557 of NS5B protein.\n\nThis issue limited our studies, since many of the described RASs are observed as of residue 553.\n\nImportantly, RASs to NS5B inhibitors ( Table 2) were observed in 5 strains out of 26 sequenced samples (19.2%). C451R was found in two isolates while A421V was found in only one. In 2 of the 3 strains for which we were able to obtain longer sequences, RASs S556G (subtype 1a) and Q556R (subtype 1b) were observed.", "Finally, we found two RAPs: A421V (in 2 subtype 1b strains) and A553G (in 1 subtype 1a strain). Although A421V has been associated with resistance to beclabuvir (BCV) in patients infected with HCV subtype 1a, this resistant phenotype has not been proven in strains subtype 1b [29] . In position 553, the substitution reported as resistant was A553T [8] .\n\nAs was the case for NS5A, different polymorphisms not previously associated with a resistant phenotype were also detected in NS5B (see Supplementary Material Table S4 ).", "The advent of DAAs therapies constitutes one of the major breakthroughs in HCV infected patients management. However, these new treatment options are far from being universally available, in particular for HCV infected patients relying on Latin American public healthcare systems. The main limiting factors for worldwide access to DAAs in our region concern the high cost, the inadequate management of public healthcare systems, the limited access of low-income or uninsured populations to healthcare providers, and the lack of accurate epidemiological information [20, [30] [31] [32] . In Uruguay, these therapies became recently available, and although some have been approved for their use by the public health authorities (Viekira pak and sofosbuvir/ledipasvir therapies), they are not currently financially covered, except in specific cases. Despite the high rates of viral response achieved with DAA-based treatments, still 1 to10% of the patients fails to eliminate infection, and in these", "cases, baseline and emergent resistance variants turn out to be key factors contributing to treatment failure [5, 17, 33] .", "Unfortunately, we are currently unable to properly assess the number of HCV infected people in Uruguay and even more to figure out the frequency and type of RASs circulating. These facts could compromise the effectiveness of these new therapies in our country.", "We have previously reported that naturally occurring substitutions conferring resistance to NS3 inhibitors exist in a significant proportion of Uruguayan patients infected with HCV genotype 1, and we showed that this frequency seemed to be higher than in other South American countries (Brazil and Argentina) [34] . The present study describes the prevalence of baseline NS5A and NS5B RASs in HCV genotype 1 infected DAA-naïve patients in a Uruguayan cohort. The presence of substitutions conferring resistance to NS5A inhibitors has been widely reported both in therapynaïve and in relapser patients from Europe [10, 33, [35] [36] [37] [38] , USA [37, 39, 40] , and Asia [41] [42] [43] . However, NS5A sequences from South America are poorly analyzed yet [9, 44] . Recent studies have revealed that the mean prevalence of NS5A genotype 1 baseline RASs to different inhibitors ranges from 6% to 16% using population sequencing or deep sequencing [27, 37, 45, 46] . Importantly, the prevalence and", "type of baseline NS5A RASs varies slightly by geographic regions. For instance, L31M was found in 2.2% of genotype 1a infected patients in Europe, in 4.1% of those in Oceania, and strikingly in no patient from the USA [27] . For this reason, we believe that there is a need to contribute data from our region, for which we still do not have enough information, apart from Brazil [9, 44] . The results of this study indicate the presence of DAA NS5A RASs in 2 HCV strains (8% of the patients enrolled in this study), with baseline RASs detected at position 31 (see Table 1 ). L31M substitution confers resistance to daclatasvir (DCV), ledipasvir (LDV), and elbasvir (EBV) in both 1a and 1b subtypes [5, 6, 8, 28, 47, 48] , whereas substitution L31V does it to DCV in subtypes 1a and 1b, to LDV in subtype 1b, and to EBV in subtype 1a [5, 6, 28] . Given that both L31V and L31M are clinically relevant RASs, their detection at baseline may influence the choice of first-line treatment regimens [28] .", "The substitutions H58P and K24Q found in two patients are considered as resistance-associated polymorphisms (RAPs). The RASs characterized at these positions were H58D and K24G/N/R [5, 6, 27, 28, 49, 50] . The substitution H58P was found as a baseline RAP in relapsers to LDV (HARVONI prescription, https://www.gilead.com/-/ media/files/pdfs/medicines/liver-disease/harvoni/harvoni_pi. pdf?la=en). However, it is sometimes regarded as a RAS [10, 51] , despite conferring only 1.2 fold change in resistance in in vitro studies using the 1a replicon system [39] .\n\nWe did not find M28T/V, Q30R/H, or Y93H substitutions as there were previously reported in Brazil and worldwide [9, 27, 44] . The amino acid substitution E62H was found in one Uruguayan patient. Although this change does not confer resistance by itself but in combination with Q30R, it generates a high resistance level to DCV [52] .", "The presence of baseline NS5A RASs impacts treatment outcome in some patient groups by affecting SVR rates. The detection of NS5A preexistent RASs may play a relevant role in the choice of first-line treatment regimens or in the simplification/shortening of recommended regimens, in order to bring SVR rates close to the highest achievable [27, 38, 41, 53] , in particular in countries such as Uruguay, where only two different DAA-containing treatment regimens are approved for their use.", "Regarding NS5B gene, global analysis (with the exception of South America [17, 19] ) revealed that NS5B DAA resistance substitutions are infrequent [14] . Our study showed the presence of NS5B inhibitors RASs in 5 out of 26 analyzed HCV infected Uruguayan patients naïve to treatment (19.2%). Substitutions found in this work were A421V and S556G associated in subtype 1a with resistance to BCV and dasabuvir (DSV), respectively [8, 28, 29, 54, 55] , and Q556R associated with resistance to DSV both in genotype 1a and 1b [12, 28] . Substitution C451R, observed in two Uruguayan patients, was reported previously in patients who failed to clear the infection after treatment with OBV/PTV/r + DSV ± RBV. In these cases, it appeared in combination with G558R (Trial Coral I-Cohort 2: http:// www.hcv-trials.com/showStudy.asp?Study=86). RAPs in positions 421 and 553 (A421V in two subtype 1b isolates and A553G in one subtype 1b isolate) were also found. Although A421V has been associated with", "resistance to BCV in patients with subtype 1a, this phenotype has not been proven in strains of subtype 1b [29] . In position 553, the substitutions reported as resistant are A553T in subtype 1a [8] and A553V in subtype 1b [54] , conferring resistance to DSV.", "In contrast to our results, Noble and coworkers (2016) reported the presence of V321A, A421G, M414V, Y448H, L159F, and C316N in Brazilian isolates [17] , yet none of these mutations were found in this study, probably due to the diversity found between Uruguayan and Brazilian strains ( Figure 2 ). Nevertheless, substitution A421V was found in Brazil [17] , Argentina [19] , and Uruguay. The RAS S282T was detected neither in Brazilian reports nor in this current work (Uruguay) [17, 18, 56] . Our findings further confirm and complement previous studies which evidenced a low prevalence of this substitution in vivo, probably due to its low replicative fitness [14, 18, 57] . Despite our results, it is worth mentioning that the presence of baseline NS5B RASs conferring resistance to nucleotide or nonnucleoside NS5B inhibitors has not been shown to have any impact on virologic responses thus far [53, 58] .", "These results show both diversity in the baseline polymorphisms found in different Latin American countries and in the evolutionary relationships of Uruguayan isolates ( Figure 2 ). This fact could be linked not only to the isolates' geographic region and viral intrinsic characteristics but also to the genetic background of the host. It is worth mentioning that we live in a vast continent inhabited by populations with different genotypic characteristics that might, depending on the situation, require different approaches to treatment. Indeed, we have recently found that allele and genotype frequencies at IL28B locus of Uruguayan individuals closely resemble those of an admixed population rather than a uniformly European-descendant one [59] . Altogether, we believe that it could be important to carry out studies throughout the South American region in order to establish the prevalence of RASs in NS5A and NS5B in different countries. In fact, this will aid in understanding that not", "every treatment regimen might be adequate for every patient and country. The data we presented here might guide not only physicians in making therapeutic decisions but also public health authorities in approving more diverse treatment combinations. These treatment formulations would cover most of the circulating strains in our region, a region with an extremely diverse genetic background population.", "To our knowledge, the present study revealed for the first time the presence of RASs in the NS5A and NS5B regions of HCV genotype 1 Uruguayan strains from patients who have not been previously treated with DAAs and is one of the few South American countries to report on this matter. It is currently unclear if preexisting viral variants with reduced susceptibility to DAAs are clinically relevant for the prediction of virologic treatment failure. However, individualized DAA therapy based on baseline resistance analysis may be beneficial for optimizing treatment efficacy in patients with HCV genotype 1 infection and risk factors for treatment failure. Therefore, the potential role of baseline resistance testing remains an area of critical research and clinical questions.\n\nThe data used to support the findings of this study are included within the article.\n\nThe authors declare that they have no conflicts of interest.", "Fabián Aldunate and Natalia Echeverría contributed equally to this work.\n\nSupplementary Material Table S1 : hepatitis C Virus NS5A and NS5B sequences used as representatives of each genotype to perform the phylogenetic analysis. Their corresponding genotype, country of isolation, and GenBank accession number are indicated. Supplementary Material Table S2 : hepatitis C Virus NS5A subtype 1a sequences used to reveal evolutionary relationships between Uruguayan strains and others isolated elsewhere. Their corresponding country of isolation and GenBank accession number are indicated. Supplementary Material Table S3 : amino acid substitutions in NS5A protein not previously associated with resistance to NS5A inhibitors. Supplementary Material Table S4 : amino acid substitutions in NS5B protein not previously associated with resistance to polymerase inhibitors. (Supplementary Materials)" ]

[ 18 ]

[ "confers a higher level of resistance than the one achieved by the RAS alone" ]

[ "Pretreatment Hepatitis C Virus NS5A/NS5B Resistance-Associated Substitutions in Genotype 1 Uruguayan Infected Patients\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6112080/\n\nSHA: f01ad3545245b4f884b48aa2b69c9deb942c3e77\n\nAuthors: Aldunate, Fabián; Echeverría, Natalia; Chiodi, Daniela; López, Pablo; Sánchez-Cicerón, Adriana; Fajardo, Alvaro; Soñora, Martín; Cristina, Juan; Hernández, Nelia; Moreno, Pilar\nDate: 2018-08-14\nDOI: 10.1155/2018/2514901\nLicense: cc-by", "Abstract: Hepatitis C Virus (HCV) infection treatment has dramatically changed with the advent of direct-acting antiviral agents (DAAs). However, the efficacy of DAAs can be attenuated by the presence of resistance-associated substitutions (RASs) before and after treatment. Indeed, RASs detected in DAA treatment-naïve HCV-infected patients could be useful for clinical management and outcome prediction. Although the frequency of naturally occurring HCV NS5A and NS5B RASs has been addressed in many countries, there are only a few reports on their prevalence in the South American region. The aim of this study was to investigate the presence of RASs to NS5A and NS5B inhibitors in a DAA treatment naïve cohort of Uruguayan patients infected with chronic hepatitis C and compare them with reports from other South American countries. Here, we found that naturally occurring substitutions conferring resistance to NS5A and NS5B inhibitors were present in 8% and 19.2%, respectively, of", "treatment-naïve HCV genotype 1 infected patients. Importantly, the baseline substitutions in NS5A and NS5B herein identified differ from the studies previously reported in Brazil. Furthermore, Uruguayan strains subtype 1a clustered within all major world clades, showing that HCV variants currently circulating in this country are characterized by a remarkable genetic diversity.", "Text: Hepatitis C Virus (HCV) infection treatment has dramatically improved thanks to the introduction of direct-acting antiviral agents (DAAs). These antivirals have significantly increased response rates (up to 98%) and greatly reduced treatment duration [1] . Currently available DAAs are classified into four categories given their molecular targets in the HCV replication cycle: (1) NS3/4A protease inhibitors (PIs) bind to the active site of the NS3/4A protease; (2) NS5A inhibitors interact with domain 1 of the NS5A dimer, although the exact mechanism of NS5A inhibition remains to be fully elucidated; (3) nucleos(t)ide analog NS5B polymerase inhibitors are incorporated into the nascent RNA chain resulting in chain termination by compromising the binding of the incoming nucleotide; (4) nonnucleoside NS5B polymerase inhibitors interact with either the thumb 1, thumb 2, palm 1, or palm 2 domain of NS5B and inhibit polymerase activity by allosteric mechanisms [2] [3] [4] . However, the", "extreme mutation and high replication rates of HCV, together with the immune system pressure, lead to a remarkable genetic variability that can compromise the high response rates to DAAs due to the preexistence of resistanceassociated substitutions (RASs) [5, 6] .", "Each drug or class of DAA is characterized by specific resistance profiles. The likelihood that a DAA will select for and allow outgrowth of viral populations carrying RASs depends on the DAA's genetic barrier to resistance (the number and type of mutations needed to generate an amino acid substitution that confers resistance), the viral fitness (replicative capacity) of the resistant variant, and viral genotypes and subtypes [7, 8] .\n\nThe prevalence of RASs in treatment-naïve patients has been broadly reported worldwide [9] [10] [11] [12] [13] [14] [15] [16] . However, apart from Brazil and Argentina, this issue has not been fully addressed in other South American countries yet [9, [17] [18] [19] . The lack of information in relation to preexisting baseline RASs, added to the high cost of these new drugs, are the major limiting factors for the broad implementation of these new therapies in Uruguay as well as in other Latin American countries (low-or lower-middle income) [20] .", "In this study, we explored the presence of resistance variants to NS5A and NS5B inhibitors in a DAA treatment naïve cohort of Uruguayan patients chronically infected with hepatitis C. Here, we aimed to contribute to the knowledge of the circulation of HCV resistant variants in the South American region.", "Samples. Serum samples were obtained from 31 patients with serological markers for HCV, which were recruited between 2015 and 2017 at the Gastroenterology Clinic from Hospital de Clínicas, Montevideo, Uruguay. HCV infection was confirmed by Abbott realtime HCV (Abbott Molecular Inc., Des Plaines, USA). Patients selected for this study were both chronically infected with HCV genotype 1 and DAA treatment-naïve at the time of blood extraction. Written informed consent was obtained from all patients. The studies have been performed according to the World Medical Association Declaration of Helsinki and approved by the appropriate institutional board (Hospital de Clínicas ethical committee).", "2.2. RNA Extraction, cDNA Synthesis, and NS5A and NS5B Amplification. Viral RNA was extracted from 140 μl of serum using the QIAamp Viral RNA mini kit (QIAgen, Hilden, Germany) according to the manufacturer's protocol. The viral RNA was heated at 65°C for 5 min and used as a template for a reverse transcription reaction. The reverse transcription reaction mixture contained 5 μl of the RNA template, 1 μl of random hexamer 100 ng/μl (Invitrogen Life Technologies, Carlsbad, CA, USA), 1 μl of dNTP mix (10 mM each), 4 μl of 5X first-strand buffer, 2 μl of 0.1 M DTT, 1 μl of SuperScript II reverse transcriptase (200 U/μl) (Invitrogen Life Technologies, Carlsbad, CA, USA), and 1 μl (40 U/μl) RNaseOUT (Invitrogen Life Technologies, Carlsbad, CA, USA). The reverse transcription was performed at 42°C for 50 min, and then the reverse transcriptase enzyme was inactivated at 70°C for 15 min. PCR amplification of NS5A and NS5B genome regions was performed using primers and conditions previously", "described [10] . Amplicons were purified using the Illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare Life Science, Buckinghamshire, UK) according to the manufacturer's protocol.", "2.3. NS5A and NS5B Sequencing. The purified product was then sequenced using the same sets of primers used for PCR amplification. Bidirectional Sanger sequencing was performed by Macrogen Korea (http://www.macrogen.com).", "2.4. NS5A and NS5B Genotype Determination. HCV NS5A and NS5B consensus sequences obtained from Uruguayan patients were aligned with sequences from HCV representing all genotypes and main subtypes isolated in different geographic regions of the world. These sequences were obtained from Los Alamos HCV sequence database and from the NIAID Virus Pathogen Database and Analysis Resource (ViPR) [21, 22] . For strains included in these studies, see Supplementary Material Table S1 . Sequences were aligned using the CLUSTAL W software [23] . Once aligned, the best evolutionary model that described our sequence data was assessed using ModelGenerator program [24] . Using the GTR + G + I model (General time reversible + gamma + invariant sites), maximum likelihood phylogenetic trees were constructed for both NS5A and NS5B using the MEGA 5.0 software [25] . For NS5A, 953 nucleotides (positions 6367 to 7319, relative to HCV 1a reference strain, H77 NC_004102) were included in the phylogenetic", "analysis, whereas for NS5B, only 361 nucleotides corresponding to the Okamoto region (positions 8265 to 8625, relative to strain H77 NC_004102) were included. As a measure of the robustness of each node, we employed the bootstrapping method (1000 pseudoreplicates).", "For NS5A 1a Uruguayan sequences (n = 20), a second alignment and maximum likelihood phylogenetic tree was generated in order to analyze HCV evolutionary relationships between Uruguayan, Brazilian, and worldwide strains. For non-Uruguayan strains included in this analysis, see Supplementary Material Table S2. 2.5. NS5A and NS5B Sequence Analysis. In order to properly identify substitution changes in NS5A and NS5B regions from HCV strains circulating in Uruguayan patients, we generated world consensus sequences for 1a and 1b subtypes using a wide range of NS5A and NS5B sequences from HCV strains isolated worldwide. For this purpose, NS5A gene sequences corresponding to subtypes 1a (n = 160) and 1b (n = 88) were retrieved from Los Alamos HCV sequence database and from the NIAID ViPR [21, 22] . Likewise, datasets of 150 and 124 NS5B sequences were generated for subtypes 1a and 1b, respectively. Using Seqman program, implemented in DNAStar 5.01 package (DNASTAR, Madison, USA), a world", "consensus nucleotide sequences were generated for each gene and subtype. Each Uruguayan sequence was subsequently aligned to the corresponding reference sequences, and then in silico translated. The amino acid sequences obtained were compared in order to explore the presence of RASs as well as the presence of polymorphisms at a RAS position (RAPs) in Uruguayan HCV strains. RAPs are defined as any change from reference sequence for a specific genotype at a position associated with NS5A resistance [26] .", "To study the genetic variability of NS5A and NS5B regions of HCV strains circulating in Uruguayan patients, sequences of these regions (accession numbers MH070029-MH070090) were aligned with corresponding sequences from 59 HCV strains isolated elsewhere, representing all genotypes and main subtypes (for strains included in these analyses, see Supplementary Material Table S1 ). Therefore, maximum likelihood phylogenetic trees were constructed. The results of these studies are shown in Figure 1 All strains in the phylogenies were assigned according to their genotype, and each cluster was supported by very high bootstrap values for both analyzed regions. Strains isolated from Uruguayan patients (n = 31) were assigned to genotype 1, 20 of which corresponded to subtype 1a and 11 to subtype 1b. The results of NS5A (Figure 1 (a)) and NS5B (Figure 1", "Genotype 1b phylogenetic analyses were concordant for both genomic regions in all 31 sequences, suggesting no recombination events between these regions.\n\nTo further analyze the evolutionary relationships between the Uruguayan strains and those circulating in Brazil and elsewhere, a second maximum likelihood phylogenetic tree of HCV-1a sequences of NS5A partial region was built ( Figure 2 ). As was previously described, two distinct 1a clades (clades 1 and 2) were observed. Brazilian sequences clustered in a large group of related sequences inside clade 1 [9] . Whereas NS5A Uruguayan strains (in red) did not cluster in a particular clade, rather, they grouped dispersedly within all major world clades.", "With the purpose of studying the amino acid (AA) substitutions along the NS5A protein, Uruguayan HCV AA sequences were aligned with NS5A world consensus sequences (residues 23 to 354 relative to NS5A protein sequence). AA substitutions at positions previously found to be potentially associated with resistance to NS5A inhibitors, as well as polymorphisms at a RAS position, were identified. These results are summarized in Table 1 .", "RASs to NS5A inhibitors (L31M and L31V) were identified in 2 strains out of 25 (8%) fully sequenced samples. RAPs were found in 3 strains (subtype 1a): 2 exhibited the substitution H58P and 1 the substitution K24Q. Although these substitutions were not reported as resistant, some changes at these positions were previously described as RASs in subtype 1a, namely H58D and K24R [27, 28] . Finally, substitution E62D was found in one subtype 1a strain. This change is considered as a secondary substitution because, although it does not confer resistance by itself, when combined with a known RAS it does. In fact, it confers a higher level of resistance than the one achieved by the RAS alone [26] . In addition, several polymorphisms that have not been previously reported to be associated with a resistant phenotype were also detected (see Supplementary Material Table S3 ).", "In order to study substitutions along NS5B protein, Uruguayan HCV AA sequences were aligned to the NS5B world consensus sequences. Almost full-length AA sequences were obtained in 26 out of 31 analyzed strains. 23 sequences span residues 36 to 539 whereas the remaining 3 span residues 36 to 557 of NS5B protein.\n\nThis issue limited our studies, since many of the described RASs are observed as of residue 553.\n\nImportantly, RASs to NS5B inhibitors ( Table 2) were observed in 5 strains out of 26 sequenced samples (19.2%). C451R was found in two isolates while A421V was found in only one. In 2 of the 3 strains for which we were able to obtain longer sequences, RASs S556G (subtype 1a) and Q556R (subtype 1b) were observed.", "Finally, we found two RAPs: A421V (in 2 subtype 1b strains) and A553G (in 1 subtype 1a strain). Although A421V has been associated with resistance to beclabuvir (BCV) in patients infected with HCV subtype 1a, this resistant phenotype has not been proven in strains subtype 1b [29] . In position 553, the substitution reported as resistant was A553T [8] .\n\nAs was the case for NS5A, different polymorphisms not previously associated with a resistant phenotype were also detected in NS5B (see Supplementary Material Table S4 ).", "The advent of DAAs therapies constitutes one of the major breakthroughs in HCV infected patients management. However, these new treatment options are far from being universally available, in particular for HCV infected patients relying on Latin American public healthcare systems. The main limiting factors for worldwide access to DAAs in our region concern the high cost, the inadequate management of public healthcare systems, the limited access of low-income or uninsured populations to healthcare providers, and the lack of accurate epidemiological information [20, [30] [31] [32] . In Uruguay, these therapies became recently available, and although some have been approved for their use by the public health authorities (Viekira pak and sofosbuvir/ledipasvir therapies), they are not currently financially covered, except in specific cases. Despite the high rates of viral response achieved with DAA-based treatments, still 1 to10% of the patients fails to eliminate infection, and in these", "cases, baseline and emergent resistance variants turn out to be key factors contributing to treatment failure [5, 17, 33] .", "Unfortunately, we are currently unable to properly assess the number of HCV infected people in Uruguay and even more to figure out the frequency and type of RASs circulating. These facts could compromise the effectiveness of these new therapies in our country.", "We have previously reported that naturally occurring substitutions conferring resistance to NS3 inhibitors exist in a significant proportion of Uruguayan patients infected with HCV genotype 1, and we showed that this frequency seemed to be higher than in other South American countries (Brazil and Argentina) [34] . The present study describes the prevalence of baseline NS5A and NS5B RASs in HCV genotype 1 infected DAA-naïve patients in a Uruguayan cohort. The presence of substitutions conferring resistance to NS5A inhibitors has been widely reported both in therapynaïve and in relapser patients from Europe [10, 33, [35] [36] [37] [38] , USA [37, 39, 40] , and Asia [41] [42] [43] . However, NS5A sequences from South America are poorly analyzed yet [9, 44] . Recent studies have revealed that the mean prevalence of NS5A genotype 1 baseline RASs to different inhibitors ranges from 6% to 16% using population sequencing or deep sequencing [27, 37, 45, 46] . Importantly, the prevalence and", "type of baseline NS5A RASs varies slightly by geographic regions. For instance, L31M was found in 2.2% of genotype 1a infected patients in Europe, in 4.1% of those in Oceania, and strikingly in no patient from the USA [27] . For this reason, we believe that there is a need to contribute data from our region, for which we still do not have enough information, apart from Brazil [9, 44] . The results of this study indicate the presence of DAA NS5A RASs in 2 HCV strains (8% of the patients enrolled in this study), with baseline RASs detected at position 31 (see Table 1 ). L31M substitution confers resistance to daclatasvir (DCV), ledipasvir (LDV), and elbasvir (EBV) in both 1a and 1b subtypes [5, 6, 8, 28, 47, 48] , whereas substitution L31V does it to DCV in subtypes 1a and 1b, to LDV in subtype 1b, and to EBV in subtype 1a [5, 6, 28] . Given that both L31V and L31M are clinically relevant RASs, their detection at baseline may influence the choice of first-line treatment regimens [28] .", "The substitutions H58P and K24Q found in two patients are considered as resistance-associated polymorphisms (RAPs). The RASs characterized at these positions were H58D and K24G/N/R [5, 6, 27, 28, 49, 50] . The substitution H58P was found as a baseline RAP in relapsers to LDV (HARVONI prescription, https://www.gilead.com/-/ media/files/pdfs/medicines/liver-disease/harvoni/harvoni_pi. pdf?la=en). However, it is sometimes regarded as a RAS [10, 51] , despite conferring only 1.2 fold change in resistance in in vitro studies using the 1a replicon system [39] .\n\nWe did not find M28T/V, Q30R/H, or Y93H substitutions as there were previously reported in Brazil and worldwide [9, 27, 44] . The amino acid substitution E62H was found in one Uruguayan patient. Although this change does not confer resistance by itself but in combination with Q30R, it generates a high resistance level to DCV [52] .", "The presence of baseline NS5A RASs impacts treatment outcome in some patient groups by affecting SVR rates. The detection of NS5A preexistent RASs may play a relevant role in the choice of first-line treatment regimens or in the simplification/shortening of recommended regimens, in order to bring SVR rates close to the highest achievable [27, 38, 41, 53] , in particular in countries such as Uruguay, where only two different DAA-containing treatment regimens are approved for their use.", "Regarding NS5B gene, global analysis (with the exception of South America [17, 19] ) revealed that NS5B DAA resistance substitutions are infrequent [14] . Our study showed the presence of NS5B inhibitors RASs in 5 out of 26 analyzed HCV infected Uruguayan patients naïve to treatment (19.2%). Substitutions found in this work were A421V and S556G associated in subtype 1a with resistance to BCV and dasabuvir (DSV), respectively [8, 28, 29, 54, 55] , and Q556R associated with resistance to DSV both in genotype 1a and 1b [12, 28] . Substitution C451R, observed in two Uruguayan patients, was reported previously in patients who failed to clear the infection after treatment with OBV/PTV/r + DSV ± RBV. In these cases, it appeared in combination with G558R (Trial Coral I-Cohort 2: http:// www.hcv-trials.com/showStudy.asp?Study=86). RAPs in positions 421 and 553 (A421V in two subtype 1b isolates and A553G in one subtype 1b isolate) were also found. Although A421V has been associated with", "resistance to BCV in patients with subtype 1a, this phenotype has not been proven in strains of subtype 1b [29] . In position 553, the substitutions reported as resistant are A553T in subtype 1a [8] and A553V in subtype 1b [54] , conferring resistance to DSV.", "In contrast to our results, Noble and coworkers (2016) reported the presence of V321A, A421G, M414V, Y448H, L159F, and C316N in Brazilian isolates [17] , yet none of these mutations were found in this study, probably due to the diversity found between Uruguayan and Brazilian strains ( Figure 2 ). Nevertheless, substitution A421V was found in Brazil [17] , Argentina [19] , and Uruguay. The RAS S282T was detected neither in Brazilian reports nor in this current work (Uruguay) [17, 18, 56] . Our findings further confirm and complement previous studies which evidenced a low prevalence of this substitution in vivo, probably due to its low replicative fitness [14, 18, 57] . Despite our results, it is worth mentioning that the presence of baseline NS5B RASs conferring resistance to nucleotide or nonnucleoside NS5B inhibitors has not been shown to have any impact on virologic responses thus far [53, 58] .", "These results show both diversity in the baseline polymorphisms found in different Latin American countries and in the evolutionary relationships of Uruguayan isolates ( Figure 2 ). This fact could be linked not only to the isolates' geographic region and viral intrinsic characteristics but also to the genetic background of the host. It is worth mentioning that we live in a vast continent inhabited by populations with different genotypic characteristics that might, depending on the situation, require different approaches to treatment. Indeed, we have recently found that allele and genotype frequencies at IL28B locus of Uruguayan individuals closely resemble those of an admixed population rather than a uniformly European-descendant one [59] . Altogether, we believe that it could be important to carry out studies throughout the South American region in order to establish the prevalence of RASs in NS5A and NS5B in different countries. In fact, this will aid in understanding that not", "every treatment regimen might be adequate for every patient and country. The data we presented here might guide not only physicians in making therapeutic decisions but also public health authorities in approving more diverse treatment combinations. These treatment formulations would cover most of the circulating strains in our region, a region with an extremely diverse genetic background population.", "To our knowledge, the present study revealed for the first time the presence of RASs in the NS5A and NS5B regions of HCV genotype 1 Uruguayan strains from patients who have not been previously treated with DAAs and is one of the few South American countries to report on this matter. It is currently unclear if preexisting viral variants with reduced susceptibility to DAAs are clinically relevant for the prediction of virologic treatment failure. However, individualized DAA therapy based on baseline resistance analysis may be beneficial for optimizing treatment efficacy in patients with HCV genotype 1 infection and risk factors for treatment failure. Therefore, the potential role of baseline resistance testing remains an area of critical research and clinical questions.\n\nThe data used to support the findings of this study are included within the article.\n\nThe authors declare that they have no conflicts of interest.", "Fabián Aldunate and Natalia Echeverría contributed equally to this work.\n\nSupplementary Material Table S1 : hepatitis C Virus NS5A and NS5B sequences used as representatives of each genotype to perform the phylogenetic analysis. Their corresponding genotype, country of isolation, and GenBank accession number are indicated. Supplementary Material Table S2 : hepatitis C Virus NS5A subtype 1a sequences used to reveal evolutionary relationships between Uruguayan strains and others isolated elsewhere. Their corresponding country of isolation and GenBank accession number are indicated. Supplementary Material Table S3 : amino acid substitutions in NS5A protein not previously associated with resistance to NS5A inhibitors. Supplementary Material Table S4 : amino acid substitutions in NS5B protein not previously associated with resistance to polymerase inhibitors. (Supplementary Materials)" ]

[ 18 ]

[ "baseline and emergent resistance variants" ]

[ "Pretreatment Hepatitis C Virus NS5A/NS5B Resistance-Associated Substitutions in Genotype 1 Uruguayan Infected Patients\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6112080/\n\nSHA: f01ad3545245b4f884b48aa2b69c9deb942c3e77\n\nAuthors: Aldunate, Fabián; Echeverría, Natalia; Chiodi, Daniela; López, Pablo; Sánchez-Cicerón, Adriana; Fajardo, Alvaro; Soñora, Martín; Cristina, Juan; Hernández, Nelia; Moreno, Pilar\nDate: 2018-08-14\nDOI: 10.1155/2018/2514901\nLicense: cc-by", "Abstract: Hepatitis C Virus (HCV) infection treatment has dramatically changed with the advent of direct-acting antiviral agents (DAAs). However, the efficacy of DAAs can be attenuated by the presence of resistance-associated substitutions (RASs) before and after treatment. Indeed, RASs detected in DAA treatment-naïve HCV-infected patients could be useful for clinical management and outcome prediction. Although the frequency of naturally occurring HCV NS5A and NS5B RASs has been addressed in many countries, there are only a few reports on their prevalence in the South American region. The aim of this study was to investigate the presence of RASs to NS5A and NS5B inhibitors in a DAA treatment naïve cohort of Uruguayan patients infected with chronic hepatitis C and compare them with reports from other South American countries. Here, we found that naturally occurring substitutions conferring resistance to NS5A and NS5B inhibitors were present in 8% and 19.2%, respectively, of", "treatment-naïve HCV genotype 1 infected patients. Importantly, the baseline substitutions in NS5A and NS5B herein identified differ from the studies previously reported in Brazil. Furthermore, Uruguayan strains subtype 1a clustered within all major world clades, showing that HCV variants currently circulating in this country are characterized by a remarkable genetic diversity.", "Text: Hepatitis C Virus (HCV) infection treatment has dramatically improved thanks to the introduction of direct-acting antiviral agents (DAAs). These antivirals have significantly increased response rates (up to 98%) and greatly reduced treatment duration [1] . Currently available DAAs are classified into four categories given their molecular targets in the HCV replication cycle: (1) NS3/4A protease inhibitors (PIs) bind to the active site of the NS3/4A protease; (2) NS5A inhibitors interact with domain 1 of the NS5A dimer, although the exact mechanism of NS5A inhibition remains to be fully elucidated; (3) nucleos(t)ide analog NS5B polymerase inhibitors are incorporated into the nascent RNA chain resulting in chain termination by compromising the binding of the incoming nucleotide; (4) nonnucleoside NS5B polymerase inhibitors interact with either the thumb 1, thumb 2, palm 1, or palm 2 domain of NS5B and inhibit polymerase activity by allosteric mechanisms [2] [3] [4] . However, the", "extreme mutation and high replication rates of HCV, together with the immune system pressure, lead to a remarkable genetic variability that can compromise the high response rates to DAAs due to the preexistence of resistanceassociated substitutions (RASs) [5, 6] .", "Each drug or class of DAA is characterized by specific resistance profiles. The likelihood that a DAA will select for and allow outgrowth of viral populations carrying RASs depends on the DAA's genetic barrier to resistance (the number and type of mutations needed to generate an amino acid substitution that confers resistance), the viral fitness (replicative capacity) of the resistant variant, and viral genotypes and subtypes [7, 8] .\n\nThe prevalence of RASs in treatment-naïve patients has been broadly reported worldwide [9] [10] [11] [12] [13] [14] [15] [16] . However, apart from Brazil and Argentina, this issue has not been fully addressed in other South American countries yet [9, [17] [18] [19] . The lack of information in relation to preexisting baseline RASs, added to the high cost of these new drugs, are the major limiting factors for the broad implementation of these new therapies in Uruguay as well as in other Latin American countries (low-or lower-middle income) [20] .", "In this study, we explored the presence of resistance variants to NS5A and NS5B inhibitors in a DAA treatment naïve cohort of Uruguayan patients chronically infected with hepatitis C. Here, we aimed to contribute to the knowledge of the circulation of HCV resistant variants in the South American region.", "Samples. Serum samples were obtained from 31 patients with serological markers for HCV, which were recruited between 2015 and 2017 at the Gastroenterology Clinic from Hospital de Clínicas, Montevideo, Uruguay. HCV infection was confirmed by Abbott realtime HCV (Abbott Molecular Inc., Des Plaines, USA). Patients selected for this study were both chronically infected with HCV genotype 1 and DAA treatment-naïve at the time of blood extraction. Written informed consent was obtained from all patients. The studies have been performed according to the World Medical Association Declaration of Helsinki and approved by the appropriate institutional board (Hospital de Clínicas ethical committee).", "2.2. RNA Extraction, cDNA Synthesis, and NS5A and NS5B Amplification. Viral RNA was extracted from 140 μl of serum using the QIAamp Viral RNA mini kit (QIAgen, Hilden, Germany) according to the manufacturer's protocol. The viral RNA was heated at 65°C for 5 min and used as a template for a reverse transcription reaction. The reverse transcription reaction mixture contained 5 μl of the RNA template, 1 μl of random hexamer 100 ng/μl (Invitrogen Life Technologies, Carlsbad, CA, USA), 1 μl of dNTP mix (10 mM each), 4 μl of 5X first-strand buffer, 2 μl of 0.1 M DTT, 1 μl of SuperScript II reverse transcriptase (200 U/μl) (Invitrogen Life Technologies, Carlsbad, CA, USA), and 1 μl (40 U/μl) RNaseOUT (Invitrogen Life Technologies, Carlsbad, CA, USA). The reverse transcription was performed at 42°C for 50 min, and then the reverse transcriptase enzyme was inactivated at 70°C for 15 min. PCR amplification of NS5A and NS5B genome regions was performed using primers and conditions previously", "described [10] . Amplicons were purified using the Illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare Life Science, Buckinghamshire, UK) according to the manufacturer's protocol.", "2.3. NS5A and NS5B Sequencing. The purified product was then sequenced using the same sets of primers used for PCR amplification. Bidirectional Sanger sequencing was performed by Macrogen Korea (http://www.macrogen.com).", "2.4. NS5A and NS5B Genotype Determination. HCV NS5A and NS5B consensus sequences obtained from Uruguayan patients were aligned with sequences from HCV representing all genotypes and main subtypes isolated in different geographic regions of the world. These sequences were obtained from Los Alamos HCV sequence database and from the NIAID Virus Pathogen Database and Analysis Resource (ViPR) [21, 22] . For strains included in these studies, see Supplementary Material Table S1 . Sequences were aligned using the CLUSTAL W software [23] . Once aligned, the best evolutionary model that described our sequence data was assessed using ModelGenerator program [24] . Using the GTR + G + I model (General time reversible + gamma + invariant sites), maximum likelihood phylogenetic trees were constructed for both NS5A and NS5B using the MEGA 5.0 software [25] . For NS5A, 953 nucleotides (positions 6367 to 7319, relative to HCV 1a reference strain, H77 NC_004102) were included in the phylogenetic", "analysis, whereas for NS5B, only 361 nucleotides corresponding to the Okamoto region (positions 8265 to 8625, relative to strain H77 NC_004102) were included. As a measure of the robustness of each node, we employed the bootstrapping method (1000 pseudoreplicates).", "For NS5A 1a Uruguayan sequences (n = 20), a second alignment and maximum likelihood phylogenetic tree was generated in order to analyze HCV evolutionary relationships between Uruguayan, Brazilian, and worldwide strains. For non-Uruguayan strains included in this analysis, see Supplementary Material Table S2. 2.5. NS5A and NS5B Sequence Analysis. In order to properly identify substitution changes in NS5A and NS5B regions from HCV strains circulating in Uruguayan patients, we generated world consensus sequences for 1a and 1b subtypes using a wide range of NS5A and NS5B sequences from HCV strains isolated worldwide. For this purpose, NS5A gene sequences corresponding to subtypes 1a (n = 160) and 1b (n = 88) were retrieved from Los Alamos HCV sequence database and from the NIAID ViPR [21, 22] . Likewise, datasets of 150 and 124 NS5B sequences were generated for subtypes 1a and 1b, respectively. Using Seqman program, implemented in DNAStar 5.01 package (DNASTAR, Madison, USA), a world", "consensus nucleotide sequences were generated for each gene and subtype. Each Uruguayan sequence was subsequently aligned to the corresponding reference sequences, and then in silico translated. The amino acid sequences obtained were compared in order to explore the presence of RASs as well as the presence of polymorphisms at a RAS position (RAPs) in Uruguayan HCV strains. RAPs are defined as any change from reference sequence for a specific genotype at a position associated with NS5A resistance [26] .", "To study the genetic variability of NS5A and NS5B regions of HCV strains circulating in Uruguayan patients, sequences of these regions (accession numbers MH070029-MH070090) were aligned with corresponding sequences from 59 HCV strains isolated elsewhere, representing all genotypes and main subtypes (for strains included in these analyses, see Supplementary Material Table S1 ). Therefore, maximum likelihood phylogenetic trees were constructed. The results of these studies are shown in Figure 1 All strains in the phylogenies were assigned according to their genotype, and each cluster was supported by very high bootstrap values for both analyzed regions. Strains isolated from Uruguayan patients (n = 31) were assigned to genotype 1, 20 of which corresponded to subtype 1a and 11 to subtype 1b. The results of NS5A (Figure 1 (a)) and NS5B (Figure 1", "Genotype 1b phylogenetic analyses were concordant for both genomic regions in all 31 sequences, suggesting no recombination events between these regions.\n\nTo further analyze the evolutionary relationships between the Uruguayan strains and those circulating in Brazil and elsewhere, a second maximum likelihood phylogenetic tree of HCV-1a sequences of NS5A partial region was built ( Figure 2 ). As was previously described, two distinct 1a clades (clades 1 and 2) were observed. Brazilian sequences clustered in a large group of related sequences inside clade 1 [9] . Whereas NS5A Uruguayan strains (in red) did not cluster in a particular clade, rather, they grouped dispersedly within all major world clades.", "With the purpose of studying the amino acid (AA) substitutions along the NS5A protein, Uruguayan HCV AA sequences were aligned with NS5A world consensus sequences (residues 23 to 354 relative to NS5A protein sequence). AA substitutions at positions previously found to be potentially associated with resistance to NS5A inhibitors, as well as polymorphisms at a RAS position, were identified. These results are summarized in Table 1 .", "RASs to NS5A inhibitors (L31M and L31V) were identified in 2 strains out of 25 (8%) fully sequenced samples. RAPs were found in 3 strains (subtype 1a): 2 exhibited the substitution H58P and 1 the substitution K24Q. Although these substitutions were not reported as resistant, some changes at these positions were previously described as RASs in subtype 1a, namely H58D and K24R [27, 28] . Finally, substitution E62D was found in one subtype 1a strain. This change is considered as a secondary substitution because, although it does not confer resistance by itself, when combined with a known RAS it does. In fact, it confers a higher level of resistance than the one achieved by the RAS alone [26] . In addition, several polymorphisms that have not been previously reported to be associated with a resistant phenotype were also detected (see Supplementary Material Table S3 ).", "In order to study substitutions along NS5B protein, Uruguayan HCV AA sequences were aligned to the NS5B world consensus sequences. Almost full-length AA sequences were obtained in 26 out of 31 analyzed strains. 23 sequences span residues 36 to 539 whereas the remaining 3 span residues 36 to 557 of NS5B protein.\n\nThis issue limited our studies, since many of the described RASs are observed as of residue 553.\n\nImportantly, RASs to NS5B inhibitors ( Table 2) were observed in 5 strains out of 26 sequenced samples (19.2%). C451R was found in two isolates while A421V was found in only one. In 2 of the 3 strains for which we were able to obtain longer sequences, RASs S556G (subtype 1a) and Q556R (subtype 1b) were observed.", "Finally, we found two RAPs: A421V (in 2 subtype 1b strains) and A553G (in 1 subtype 1a strain). Although A421V has been associated with resistance to beclabuvir (BCV) in patients infected with HCV subtype 1a, this resistant phenotype has not been proven in strains subtype 1b [29] . In position 553, the substitution reported as resistant was A553T [8] .\n\nAs was the case for NS5A, different polymorphisms not previously associated with a resistant phenotype were also detected in NS5B (see Supplementary Material Table S4 ).", "The advent of DAAs therapies constitutes one of the major breakthroughs in HCV infected patients management. However, these new treatment options are far from being universally available, in particular for HCV infected patients relying on Latin American public healthcare systems. The main limiting factors for worldwide access to DAAs in our region concern the high cost, the inadequate management of public healthcare systems, the limited access of low-income or uninsured populations to healthcare providers, and the lack of accurate epidemiological information [20, [30] [31] [32] . In Uruguay, these therapies became recently available, and although some have been approved for their use by the public health authorities (Viekira pak and sofosbuvir/ledipasvir therapies), they are not currently financially covered, except in specific cases. Despite the high rates of viral response achieved with DAA-based treatments, still 1 to10% of the patients fails to eliminate infection, and in these", "cases, baseline and emergent resistance variants turn out to be key factors contributing to treatment failure [5, 17, 33] .", "Unfortunately, we are currently unable to properly assess the number of HCV infected people in Uruguay and even more to figure out the frequency and type of RASs circulating. These facts could compromise the effectiveness of these new therapies in our country.", "We have previously reported that naturally occurring substitutions conferring resistance to NS3 inhibitors exist in a significant proportion of Uruguayan patients infected with HCV genotype 1, and we showed that this frequency seemed to be higher than in other South American countries (Brazil and Argentina) [34] . The present study describes the prevalence of baseline NS5A and NS5B RASs in HCV genotype 1 infected DAA-naïve patients in a Uruguayan cohort. The presence of substitutions conferring resistance to NS5A inhibitors has been widely reported both in therapynaïve and in relapser patients from Europe [10, 33, [35] [36] [37] [38] , USA [37, 39, 40] , and Asia [41] [42] [43] . However, NS5A sequences from South America are poorly analyzed yet [9, 44] . Recent studies have revealed that the mean prevalence of NS5A genotype 1 baseline RASs to different inhibitors ranges from 6% to 16% using population sequencing or deep sequencing [27, 37, 45, 46] . Importantly, the prevalence and", "type of baseline NS5A RASs varies slightly by geographic regions. For instance, L31M was found in 2.2% of genotype 1a infected patients in Europe, in 4.1% of those in Oceania, and strikingly in no patient from the USA [27] . For this reason, we believe that there is a need to contribute data from our region, for which we still do not have enough information, apart from Brazil [9, 44] . The results of this study indicate the presence of DAA NS5A RASs in 2 HCV strains (8% of the patients enrolled in this study), with baseline RASs detected at position 31 (see Table 1 ). L31M substitution confers resistance to daclatasvir (DCV), ledipasvir (LDV), and elbasvir (EBV) in both 1a and 1b subtypes [5, 6, 8, 28, 47, 48] , whereas substitution L31V does it to DCV in subtypes 1a and 1b, to LDV in subtype 1b, and to EBV in subtype 1a [5, 6, 28] . Given that both L31V and L31M are clinically relevant RASs, their detection at baseline may influence the choice of first-line treatment regimens [28] .", "The substitutions H58P and K24Q found in two patients are considered as resistance-associated polymorphisms (RAPs). The RASs characterized at these positions were H58D and K24G/N/R [5, 6, 27, 28, 49, 50] . The substitution H58P was found as a baseline RAP in relapsers to LDV (HARVONI prescription, https://www.gilead.com/-/ media/files/pdfs/medicines/liver-disease/harvoni/harvoni_pi. pdf?la=en). However, it is sometimes regarded as a RAS [10, 51] , despite conferring only 1.2 fold change in resistance in in vitro studies using the 1a replicon system [39] .\n\nWe did not find M28T/V, Q30R/H, or Y93H substitutions as there were previously reported in Brazil and worldwide [9, 27, 44] . The amino acid substitution E62H was found in one Uruguayan patient. Although this change does not confer resistance by itself but in combination with Q30R, it generates a high resistance level to DCV [52] .", "The presence of baseline NS5A RASs impacts treatment outcome in some patient groups by affecting SVR rates. The detection of NS5A preexistent RASs may play a relevant role in the choice of first-line treatment regimens or in the simplification/shortening of recommended regimens, in order to bring SVR rates close to the highest achievable [27, 38, 41, 53] , in particular in countries such as Uruguay, where only two different DAA-containing treatment regimens are approved for their use.", "Regarding NS5B gene, global analysis (with the exception of South America [17, 19] ) revealed that NS5B DAA resistance substitutions are infrequent [14] . Our study showed the presence of NS5B inhibitors RASs in 5 out of 26 analyzed HCV infected Uruguayan patients naïve to treatment (19.2%). Substitutions found in this work were A421V and S556G associated in subtype 1a with resistance to BCV and dasabuvir (DSV), respectively [8, 28, 29, 54, 55] , and Q556R associated with resistance to DSV both in genotype 1a and 1b [12, 28] . Substitution C451R, observed in two Uruguayan patients, was reported previously in patients who failed to clear the infection after treatment with OBV/PTV/r + DSV ± RBV. In these cases, it appeared in combination with G558R (Trial Coral I-Cohort 2: http:// www.hcv-trials.com/showStudy.asp?Study=86). RAPs in positions 421 and 553 (A421V in two subtype 1b isolates and A553G in one subtype 1b isolate) were also found. Although A421V has been associated with", "resistance to BCV in patients with subtype 1a, this phenotype has not been proven in strains of subtype 1b [29] . In position 553, the substitutions reported as resistant are A553T in subtype 1a [8] and A553V in subtype 1b [54] , conferring resistance to DSV.", "In contrast to our results, Noble and coworkers (2016) reported the presence of V321A, A421G, M414V, Y448H, L159F, and C316N in Brazilian isolates [17] , yet none of these mutations were found in this study, probably due to the diversity found between Uruguayan and Brazilian strains ( Figure 2 ). Nevertheless, substitution A421V was found in Brazil [17] , Argentina [19] , and Uruguay. The RAS S282T was detected neither in Brazilian reports nor in this current work (Uruguay) [17, 18, 56] . Our findings further confirm and complement previous studies which evidenced a low prevalence of this substitution in vivo, probably due to its low replicative fitness [14, 18, 57] . Despite our results, it is worth mentioning that the presence of baseline NS5B RASs conferring resistance to nucleotide or nonnucleoside NS5B inhibitors has not been shown to have any impact on virologic responses thus far [53, 58] .", "These results show both diversity in the baseline polymorphisms found in different Latin American countries and in the evolutionary relationships of Uruguayan isolates ( Figure 2 ). This fact could be linked not only to the isolates' geographic region and viral intrinsic characteristics but also to the genetic background of the host. It is worth mentioning that we live in a vast continent inhabited by populations with different genotypic characteristics that might, depending on the situation, require different approaches to treatment. Indeed, we have recently found that allele and genotype frequencies at IL28B locus of Uruguayan individuals closely resemble those of an admixed population rather than a uniformly European-descendant one [59] . Altogether, we believe that it could be important to carry out studies throughout the South American region in order to establish the prevalence of RASs in NS5A and NS5B in different countries. In fact, this will aid in understanding that not", "every treatment regimen might be adequate for every patient and country. The data we presented here might guide not only physicians in making therapeutic decisions but also public health authorities in approving more diverse treatment combinations. These treatment formulations would cover most of the circulating strains in our region, a region with an extremely diverse genetic background population.", "To our knowledge, the present study revealed for the first time the presence of RASs in the NS5A and NS5B regions of HCV genotype 1 Uruguayan strains from patients who have not been previously treated with DAAs and is one of the few South American countries to report on this matter. It is currently unclear if preexisting viral variants with reduced susceptibility to DAAs are clinically relevant for the prediction of virologic treatment failure. However, individualized DAA therapy based on baseline resistance analysis may be beneficial for optimizing treatment efficacy in patients with HCV genotype 1 infection and risk factors for treatment failure. Therefore, the potential role of baseline resistance testing remains an area of critical research and clinical questions.\n\nThe data used to support the findings of this study are included within the article.\n\nThe authors declare that they have no conflicts of interest.", "Fabián Aldunate and Natalia Echeverría contributed equally to this work.\n\nSupplementary Material Table S1 : hepatitis C Virus NS5A and NS5B sequences used as representatives of each genotype to perform the phylogenetic analysis. Their corresponding genotype, country of isolation, and GenBank accession number are indicated. Supplementary Material Table S2 : hepatitis C Virus NS5A subtype 1a sequences used to reveal evolutionary relationships between Uruguayan strains and others isolated elsewhere. Their corresponding country of isolation and GenBank accession number are indicated. Supplementary Material Table S3 : amino acid substitutions in NS5A protein not previously associated with resistance to NS5A inhibitors. Supplementary Material Table S4 : amino acid substitutions in NS5B protein not previously associated with resistance to polymerase inhibitors. (Supplementary Materials)" ]

[ 22 ]

[ "Infectious cDNA clones" ]

[ "Efficient generation of recombinant RNA viruses using targeted recombination-mediated mutagenesis of bacterial artificial chromosomes containing full-length cDNA\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3840674/\n\nSHA: ef38ed2f4cc96e16ce011623cc5d15d2d8ca58c3\n\nAuthors: Rasmussen, Thomas Bruun; Risager, Peter Christian; Fahnøe, Ulrik; Friis, Martin Barfred; Belsham, Graham J; Höper, Dirk; Reimann, Ilona; Beer, Martin\nDate: 2013-11-22\nDOI: 10.1186/1471-2164-14-819\nLicense: cc-by", "Abstract: BACKGROUND: Infectious cDNA clones are a prerequisite for directed genetic manipulation of RNA viruses. Here, a strategy to facilitate manipulation and rescue of classical swine fever viruses (CSFVs) from full-length cDNAs present within bacterial artificial chromosomes (BACs) is described. This strategy allows manipulation of viral cDNA by targeted recombination-mediated mutagenesis within bacteria. RESULTS: A new CSFV-BAC (pBeloR26) derived from the Riems vaccine strain has been constructed and subsequently modified in the E2 coding sequence, using the targeted recombination strategy to enable rescue of chimeric pestiviruses (vR26_E2gif and vR26_TAV) with potential as new marker vaccine candidates. Sequencing of the BACs revealed a high genetic stability during passages within bacteria. The complete genome sequences of rescued viruses, after extensive passages in mammalian cells showed that modifications in the E2 protein coding sequence were stably maintained. A single", "amino acid substitution (D3431G) in the RNA dependent RNA polymerase was observed in the rescued viruses vR26_E2gif and vR26, which was reversion to the parental Riems sequence. CONCLUSIONS: These results show that targeted recombination-mediated mutagenesis provides a powerful tool for expediting the construction of novel RNA genomes and should be applicable to the manipulation of other RNA viruses.", "Text: Bacterial artificial chromosomes (BACs) are ideally suited for the stable maintenance of large DNA sequences derived from viral genomes [1] . A considerable number of BAC systems have been established for large DNA viruses; in particular many different herpesvirus genomes have been cloned into BACs (for review see [2] ). The first BAC systems using RNA virus cDNAs were described for coronaviruses [3] [4] [5] [6] and recently the first BAC containing a full-length cDNA for a negative-stranded RNA virus was described [7] . Similarly, cDNAs corresponding to the full-length genomes of members of the Flaviviridae family (Japanese encephalitis virus [8] and Dengue virus [9] ) have been inserted into BACs.", "BACs containing full-length cDNAs of pestiviruses (also within the Flaviviridae), including bovine viral diarrhea virus (BVDV) and classical swine fever virus (CSFV) have recently been established [10, 11] . Infectious pestiviruses can be rescued using RNA transcripts derived from these BACs. The pestiviruses have single stranded positive sense RNA genomes, about 12.3 kb in length, which includes a single long open reading frame, encoding a large polyprotein, flanked by 5′ and 3′ untranslated regions (UTRs) that are critical for autonomous replication of the genome [12, 13] . The polyprotein is cleaved by cellular and viral proteases into four structural proteins (nucleocapsid protein C, envelope glycoproteins E rns , E1 and E2) and eight nonstructural proteins (N pro , p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B). The availability of genetically defined and stable pestivirus BACs facilitates the functional study of viral proteins or RNA structures and also the development of new marker", "vaccine candidates. Several CSFV vaccines with marker properties based on chimeric pestiviruses have been developed over the years [14] . In particular, chimeric pestiviruses with substitution of the entire E2 protein have been described [15] [16] [17] but also mutants with more subtle modifications, such as the modification of the important TAV-epitope [18] within the CSFV-E2 protein [19, 20] are promising marker vaccine candidates.", "Manipulation of BACs using traditional cloning procedures can be difficult (e.g. because of a lack of convenient restriction enzyme sites) and thus a range of methodologies that apply bacterial genetics, including homologous recombination (e.g. Red/ET homologous recombineering) within the E. coli host, have been developed (for review, see [21] ). The use of homologous recombination allows site-directed mutagenesis of BACs [22] and, by employing a counterselection scheme, specific modifications can be obtained without leaving residual \"foreign\" sequences [23] . The main advantage of this method is that there are no target limitations (e.g. based on size or location) and no need for suitable restriction sites. The integration of the modified sequence is performed in vivo (within E. coli) thereby potentially being more accurate than in vitro approaches like PCR-based methods. Although in vitro cloning approaches based on the use of high-fidelity polymerases for PCR amplification have", "significantly improved in recent years, the use of in vivo approaches should allow a more accurate method of mutagenesis due to the use of the cells own high-fidelity replication system which includes proof reading. Whereas BAC recombination has been commonly used for modifying DNA viruses, there are only very few reports about the use of this technology for RNA viruses [7, 24, 25] .", "Here, a generally applicable strategy for the manipulation and rescue of chimeric pestiviruses from BACs is described as a model, and the flexibility of this approach is demonstrated by generating different modifications in the viral cDNA of the new CSFV-BAC, pBeloR26, derived from the modified live vaccine strain \"C-strain Riems\". The targeted recombination-mediated mutagenesis described here includes the substitution of the 9 amino acid (aa) linear TAV-epitope (TAVSPTTLR) present in the E2 protein with the corresponding region (TTVSTSTLA) of a heterologous pestivirus (border disease virus, BDV, strain \"Gifhorn\") and also the replacement of the entire CSFV E2 protein coding region with the whole E2 coding region from the same BDV, to generate marked vaccine viruses that can be discriminated using specific anti-E2 monoclonal antibodies. The genetic stabilities of both the BAC constructs (within E. coli) and the rescued viruses have also been assessed.", "Porcine kidney (PK15) and sheep fetal thymoid (SFT-R) cells were grown at 37°C (with 5% (v/v) CO 2 ) in Dulbecco's minimal essential medium (DMEM) supplemented with 5% (v/v) pestivirus-free fetal calf serum. Virus from a bait containing the modified live vaccine CSFV \"C-strain Riems\" (Riemser Arzneimittel AG, Germany) was propagated once in PK15 cells and termed vRiemser. RNA obtained from BDV strain \"Gifhorn\" [26] was used for amplification of the Gifhorn E2-coding sequence.\n\nOligonucleotide primers used are listed in Additional file 1: Table S1 .", "The BAC construct, pBeloR26, was constructed using the long RT-PCR method as previously described [11] using RNA derived from the \"C-strain Riems\". Briefly, full-length viral cDNAs flanked by NotI sites were amplified by long RT-PCR using primers 5′Cstrain_T7_Not1 (which includes a T7 promotor for in vitro transcription, a NotI site and a region corresponding to the first 44 nt of the genome) and 3′CSFV_Not1 (that contains a NotI site and sequence complementary to the 3′-terminal 35 nt of the genome that are conserved among many CSFVs including the Cstrain). The product (ca. 12.3 kbp) was digested with NotI and inserted into similarly digested pBeloBAC11 (New England Biolabs, GenBank accession U51113). All BACs were modified and maintained in E. coli DH10B cells (Invitrogen) grown at 37°C in LB medium containing chloramphenicol (Cam, 15 μg/ml). The electroporation of bacteria was performed in 0.1 cm cuvettes using 1 pulse at 1800 V, 25 μF and 200 Ω in a Gene Pulser Xcell (Bio-Rad).", "BACs to be used as templates for long PCR or for screening by restriction enzyme digestion were purified from 4 ml overnight cultures of E. coli DH10B using the ZR BAC DNA Miniprep Kit (Zymo Research). BACs required for direct genome sequencing were purified from 500 ml cultures using the Large-construct kit (Qiagen).", "Modifications to the full-length CSFV cDNA were accomplished in E. coli DH10B (streptomycin resistant, Strep R ) using the Counter Selection BAC Modification Kit (Gene Bridges, Heidelberg, Germany).\n\nThe Red/ET recombination involved three steps (i-iii).", "Step i) the temperature-sensitive pRedET expression plasmid (Gene Bridges) was introduced into electroporationcompetent E.coli DH10B cells containing the parental BAC (phenotype Cam R , Strep R ). The pRedET expresses the phage lambda proteins redα, redβ and redγ, under control of the arabinose-inducible pBAD promoter, allowing homologous recombination to occur. Immediately after electroporation, pre-warmed LB medium without antibiotics (1 ml) was added to the cells which were then incubated at 30°C for 1 hour, prior to spreading onto agar plates containing Cam (15 μg/ml) and tetracycline (Tet) (3 μg/ml) and then incubated at 30°C overnight to maintain the pRedET. The presence of the pRedET plasmid (conferring Tet R ) was verified by visual inspection of BAC-DNA preparations from the Cam R /Tet R colonies using agarose gel electrophoresis.", "Step ii) counter-selection marker cassettes with an extra NotI site for screening purposes (rpsL-neo, 1325 bp) were amplified by PCR using primers with 30 nt or 50 nt extensions that were homologous to the target site in the BAC using the rpsL-neo plasmid (Gene Bridges) as template and the Phusion hot start II HF DNA polymerase (Thermo Scientific) with cycling conditions as follows: 98°C for 30s, followed by 35 cycles of 98°C for 10s, 60°C for 20s, 72°C for 60s, and 1 cycle at 72°C for 4 min. The PCR products (ca. 1400 bp) were isolated on 1% (w/v) TBE agarose gels and purified using a GeneJET gel extraction kit (Thermo Scientific). Samples (30 μl), from an E. coli culture containing pRedET and the parental BAC grown overnight at 30°C in LB media (Cam, Tet), were used to inoculate 1.4 ml of fresh LB media with the same antibiotics to obtain exponentially growing bacteria at 30°C. Red/ET recombination proteins were induced by adding 50 μl of 10% (w/v) L-arabinose (Sigma). The PCR", "product (200 ng) containing the rpsL-neo cassette was introduced into these bacteria using electroporation (as above). Following electroporation, the cells were grown at 37°C for 70 min (to allow recombination) and then selected on plates containing Cam (15 μg/ml), Tet (3 μg/ml) and kanamycin (Kan, 15 μg/ml) overnight at 30°C to maintain the pRedET. Note, the rpsL cassette confers Streptomycin sensitivity (Strep S ) onto the resistant DH10B strain and the neo confers Kanamycin resistance (Kan R ). The correct phenotype (Cam R , Kan R , Tet R , Strep S ) of the resulting colonies was confirmed by streaking the colonies onto plates containing Cam (15 μg/ml), Tet (3 μg/ml) and Kan (15 μg/ml) and grown at 30°C. Importantly, for the third step, the replacement of the rpsL-neo cassette (using counter-selection), the selected colonies were also streaked onto plates containing Cam (15 μg/ml) plus Strep (50 μg/ml) and shown to be Strep S indicating incorporation of a functional rpsL gene. The", "structures of the intermediate BACs were verified by restriction enzyme analysis and sequencing around the inserts.", "Step iii) the replacement of the rpsL-neo selection cassettes from the intermediate constructs using linear DNA fragments was achieved through counter-selection and Red/ET recombination. Again, the homologous sequences at the ends of the DNA fragment were used for Red/ET mediated recombination events to replace the rpsL-neo cassette with the sequence of interest. Counterselection against the rpsL-neo cassette (phenotype Cam R , Kan R , Tet R , Strep S ) was employed using media containing Cam (15 μg/ml) and Strep (50 μg/ml) to isolate the required derivatives (phenotype Cam R and Strep R ).", "Initially, the intermediate construct, pBeloR26_E2rpsLneo ( Figure 1 ), was generated using Red/ET recombination by insertion of the rpsL-neo cassette with an extra NotI site for screening purposes which was amplified using primers Criems-TAVfor and Criems-TAVrev (Additional file 1: Table S1 ) in place of the TAVSPTTLR coding sequence (27 nt) . Secondly, the rpsL-neo cassette in this intermediate construct was then replaced using counter-selection Red/ ET recombination using a single-stranded oligonucleotide, Riems_TAV_Gifhorn (Additional file 1: Table S1 ) with the same homology arms as used for the rpsL-neo cassette, to introduce the coding sequence for the BDV \"Gifhorn\" epitope sequence (TTVSTSTLA). The resulting construct was named pBeloR26_TAV (Figure 1 ). The initial intermediate construct (with rpsL-neo) was then used to produce the pBeloR26_E2gif construct ( Figure 1 ). For this, the E2 coding sequence was amplified from cDNA prepared from BDV \"Gifhorn\" RNA using two different", "primer pairs, one set with 50 nt homology arms (Criems_E2_gifFlong/Criems_ E2_gifRlong) and another with 30 nt homologous sequences (Criems_E2_gifF/Criems_E2_gifR).", "For generation of BACs with substitution of the entire E2 coding sequences, PCR products consisting of the sequence of interest flanked with homology arms identical to the target area were generated by PCR (as for the rpsLneo cassette). For making constructs with substitution of shorter sequences (e.g. the TAV-epitope), the recombination was achieved using synthetic single stranded oligonucleotides rather than PCR products. Pre-heating of single stranded oligonucleotides at 95°C for 2 min followed by snap-freezing, prior to electroporation, empirically showed the best results. In each case, the DNA molecules were introduced into E. coli containing the BAC derivatives including the rpsL-neo cassettes together with the pRedET plasmid by electroporation as described above. The structures of the modified BACs were verified by restriction enzyme analysis and subsequent full-genome sequencing (see below).", "BAC DNA (1 μg) was linearized with NotI or 1 μl BAC DNA was used as template for long PCR amplification using primers 5′C-strain_T7_Not1 and 3′CSFV (Additional file 1: Table S1 ). Linearized BACs or PCR products were purified with the GeneJet PCR purification kit (Thermo Scientific) and transcribed in vitro using a Megascript T7 kit (Invitrogen). Viruses were rescued from RNA transcripts (1 to 5 μg) by electroporation of porcine (PK15) or ovine (SFT-R) cells essentially as described previously [24] . Cells were analysed using immunofluorescence microscopy (typically after 3 days) for the expression of NS3 and E2 proteins using specific monoclonal antibodies (mAbs), these were anti-NS3 (WB103/105, pan-pestivirus), anti-CSFV E2 (WH211, WH303, both CSFV specific) and anti-BDV E2 (WB166, BVDV/BDV specific) (AHVLA Scientific, United Kingdom) together with Alexa 488 conjugated goat antimouse IgG antibody (Molecular Probes, Invitrogen). The nuclei of cells were visualized using DAPI (Vector", "Laboratories) and images were recorded using a BX63 fluorescence microscope (Olympus). For peroxidase staining, cells were fixed and stained for the presence of pestivirus antigens using biotinylated pig anti-CSFV/BVDV polyclonal IgG followed by avidin-conjugated horseradish peroxidase (eBioscience) as previously described [27] . The same staining procedure was also performed using the anti-E2 mAbs. Samples containing virus-positive cells were passaged onto new cells. Virus growth curves were generated as previously described [24] . Briefly, PK15 or SFT-R cells were infected at a multiplicity of infection (MOI) of 0.1 pfu/cell and grown for three days.", "BAC DNAs (5 μg), purified using the Large-construct kit (Qiagen), or PCR products (1 μg) amplified from viral cDNA or from BACs using the long PCR method (as above) were consensus sequenced using a 454 FLX (Roche) or an Ion PGM (Life Technologies). Both Newbler (Roche) and the bwa.bwasw alignment algorithm [28] were used for mapping the reads to the expected sequence. A combination of Samtools [29] and LoFreq SNV-caller [30] was used for downstream single nucleotide variant (SNV) analysis. Finally, clone consensus sequences were aligned using MAFFT in the Geneious software platform (Biomatters).\n\nGeneration of a BAC containing full-length cDNA corresponding to the modified live vaccine \"C-strain Riems\"", "BACs containing the full-length cDNA corresponding to the parental vRiemser (\"C-strain Riems\") were constructed according to the method described previously for the \"Paderborn\" strain of CSFV [11] . BACs containing the complete CSFV cDNAs were identified by restriction Figure 1 Schematic representation of the CSFV genome organization and the BACs constructed and used in this study. Nucleotide (nt) and amino acid (aa) positions within R26 for the 5′ and 3′ termini together with the translational start and stop codons of the polyprotein coding region plus cleavage sites used to make the individual proteins (N pro , C, E rns , E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B) are indicated. Insertion of the rpsL-neo in place of the TAV-epitope within CSFV E2 for the intermediate construct (R26_rpsLneo) and the subsequent replacement with the TTVSTSTLA sequence (R26_TAV) and the complete substitution of the E2 sequence (R26_E2gif) are shown. Names of BAC constructs begin with \"pBelo\" and", "rescued viruses with \"v\" (e.g. pBeloR26 and vR26). Cell culture passage no. of virus is indicated with \"/P\" (e.g. vR26/P-4).", "digest analysis and following linearization by NotI, RNA transcripts were produced and electroporated into PK15 cells. This screening resulted in the identification of a BAC containing a cDNA insert of 12316 nt, pBeloR26 (Figure 1) , which yielded infectious virus, termed vR26, that could be propagated in SFT-R cells (Figure 2 , upper panels) and in PK15 cells (Figure 3 ). The rescued vR26 displayed higher growth rate at the early stage (about 10fold difference in virus yield at 24 h) compared to the parental vaccine virus, but after 48 hours similar virus titres were obtained (Figure 3 ). Full-genome sequencing of the cloned BAC template, pBeloR26, revealed a number of differences throughout the genome when compared to the full-length consensus sequence of the cDNA used for the cloning procedure (see Table 1 ). These differences are non-representative variants within the cDNA. Overall, the BAC sequence differed from the cDNA sequence in 18 positions, 9 of these lead to predicted", "amino acid substitutions within the polyprotein; one in each of N pro , E rns , E1, E2 and NS3 and four amino acid substitutions in NS5B (Table 1) . When compared to the published reference sequence (GenBank accession AY259122.1), the pBeloR26 BAC sequence differed at an additional 11 positions, 1 of these lead to a predicted amino acid substitution and there was one large insertion (27 nt) in the hypervariable region of the 3′-UTR (Additional file 2: Table S2 ).", "To determine the utility of the targeted recombinationmediated mutagenesis system for pestiviruses, two different modifications of the E2 protein coding sequence within pBeloR26 were generated using the Red/ET recombination methodology. Initially, the sequence encoding the linear TAV-epitope (TAVSPTTLR) within the CSFV-E2 was substituted with the sequence encoding the corresponding region (encoding TTVSTSTLA) from the BDV strain \"Gifhorn\" as described in the Materials and Methods section. More than 90% of the colonies obtained using this procedure contained the required BAC", "Anti-CSFV E2 (WH211) Figure 2 Antibody reaction patterns of pestivirus infected cells. SFT-R cells were infected with vR26 and its two derivatives vR26_E2gif and vR26_TAV plus vGifhorn [26] . After 72 h, the cells were fixed and stained with monoclonal antibodies against the NS3 protein (WB103/105, left column), the CSFV E2 protein (WH303 and WH211, middle columns) and the BDV E2 protein (WB166, right column) as indicated and viewed using a fluorescence microscope. structure as determined by NotI digestions. The complete genome sequences of the CSFV cDNA within two selected BACs, designated pBeloR26_TAV have been verified (data not shown). In addition, the complete coding sequence (1119 nt) for the CSFV-E2 protein was substituted by the corresponding sequence from BDV \"Gifhorn\". Again more than 90% of the colonies obtained contained the required BAC and the same proportion of correctly recombined BACs was obtained using either 30 nt or 50 nt homology arms. The chimeric BAC was", "designated, pBeloR26_E2gif and the complete virus genome sequence (cDNA) was verified (data not shown).", "After electroporation with RNA transcripts derived from either pBeloR26_TAV or pBeloR26_E2gif a large number of CSFV NS3-positive cells could be observed (data not shown) and chimeric virus stocks, termed vR26_TAV and vR26_E2gif, were generated after further passages in cells. Cells infected with these viruses and with the parental vR26 and vGifhorn strains were all stained with mAbs directed against the NS3 protein ( Figure 2 ). However, in contrast to the parental vR26 virus, the chimeric viruses rescued from the recombined BACs were not recognized by anti-E2 mAbs specific for the CSFV-E2 proteins ( Figure 2 ) and thus, consistent with their structure, displayed the same antibody reaction pattern as vGifhorn. Two different anti-CSFV E2 mAbs, WH211 and WH303, were used for the staining and the latter has been shown previously to target the TAV-epitope [18] . As anticipated, cells infected with either the vGifhorn or with the chimeric vR26_E2gif could be shown to express the \"Gifhorn\"", "E2 protein using staining with an anti-BDV mAb ( Figure 2 ). The presence of the BDV epitope TTVSTSTLA in vR26_ TAV was insufficient to permit efficient recognition by this anti-BDV mab, although a weak signal was observed in some cells.", "The BAC constructs pBeloR26 and pBeloR26_E2gif were analysed for the genetic stability of the cDNA to determine the suitability of the BAC vector for maintaining full-length pestivirus cDNAs. E. coli DH10B cells containing the BACs were passaged 15 times, by overnight growth, and the complete viral cDNAs within the BACs were sequenced after the 1st and the 15th passage. No mutations were observed within the 12316 nt virus cDNA sequences after this extensive propagation of the BACs in the bacterial host, indicating a highly stable system for the maintenance of complete pestivirus cDNA sequences.", "The viruses, vR26 and vR26_E2gif, rescued from their respective BAC constructs, were also tested for their genetic stability within mammalian cells. Linearized BAC DNA was transcribed in vitro and the RNA was electroporated into PK15 cells. Three days after electroporation the cells were stained with the anti-NS3 antibody to detect the presence of replicating virus. Samples containing virus positive cells were passaged onto new cells, this process \n\n*Nt position 10665 in vR26/P-12 is reverted from A to G as in the parental cDNA.", "was repeated for 12 separate passages (each of three days). The virus titre (as TCID 50 /ml) was determined for each passage. Passage of the rescued vR26_E2gif chimeric virus in PK15 cells resulted in rapidly decreasing virus titres and was discontinued after the 2nd passage ( Figure 4A ). Instead, further passage of this chimeric virus was performed in ovine SFT-R cells (the preferred cell type for BDV) and resulted in much higher titers of the chimeric virus. Virus titers reached more than 10 6 TCID 50 /ml after the 1st passage and remained stable for 12 passages ( Figure 4A ). The rescued vR26 was also efficiently propagated on the SFT-R cells but maintained a slightly lower titer than the vR26_E2gif chimeric virus ( Figure 4A ).", "To check that the viruses retained their antibody reaction properties ( Figure 2 ) after these passages, cells were infected with viruses from the 12th SFT-R cell culture passage (termed vR26/P-12 and vR26_E2gif/P-12) and stained with a polyclonal anti-pestivirus serum and with specific mAbs directed against the CSFV-E2 and BDV-E2 proteins ( Figure 4B ). Cells infected with either the vR26/P-12 or the chimeric vR26_E2gif/P-12 were each detected by the polyclonal anti-pestivirus serum as expected. The anti-CSFV-E2 mAb specifically detected cells infected with vR26/P-12 but not cells infected by the chimeric virus containing the BDV-E2 protein (consistent with the results shown in Figure 2 ). In contrast, the anti-BDV-E2 mAb specifically detected infection by the vR26_E2gif/P-12 and did not recognize cells infected with vR26/P-12. Each result is in accord with the structure of the viruses. The 4th passage of vR26 (vR26/P-4) displayed a slower growth rate than the virus obtained after 12", "passages (see Figure 5A ). It also had a reduced growth rate compared to both the vR26_E2gif/P-4 and vR26_E2gif/P-12. The fulllength sequence of pBeloR26 had revealed ten non-silent mutations compared to the reference sequence (AY25 9122.1) for this virus (Additional file 2: Table S2 ). Any of these mutations could be responsible for the impaired growth acting alone or in concert. For further investigation of this issue, full length cDNAs prepared from vR26/ P-4, vR26/P-12, vR26_E2gif/P-4 and vR26_E2gif/P-12 were deep-sequenced using both the 454 FLX and Ion PGM platforms for comparison and to determine the quasispecies distribution (Additional file 3: Figure S1 and Additional file 4: Figure S2 ). Sequencing data from both platforms revealed that both the vR26/P-12 and vR26_E2gif/P-12 were close to 100% changed at nt position A10665G compared to the BAC clones (resulting in the predicted amino acid substitution D3431G within the NS5B protein, the RNAdependent RNA polymerase, see", "Figure 5B ). This adaptation is a reversion back to the consensus cDNA sequence of the parental vaccine virus, vRiemser (Additional file 2: Table S2 ). Additionally, vR26/P-4 and vR26_E2gif/P-4 already showed evidence for this reversion being present within the population. For vR26/P-4, the level of reversion was 57%, while for vR26_E2gif/P-4 the extent of change was 73% (see Figure 5B ).", "In this study, we have established the first BAC containing the full-length cDNA of a CSFV vaccine strain. The BAC differed from the parental cDNA sequence in 18 positions leading to 9 aa substitutions ( Table 1 ). The method that has been used for the generation of pBeloR26 is based on full genome amplification of cDNA followed by direct cloning to obtain the BACs [11] . This approach results in cDNA clones that reflect the quasispecies composition of the parental viral RNA and thus it is not guaranteed to obtain cDNA clones corresponding to the consensus sequence of the cDNA used. However, it is possible to correct the mutations using the BAC recombination approach if a consensus clone is needed. To demonstrate the utility of the Red/ET mediated recombination method we have generated a series of modified BACs derived from this CSFV full-length cDNA. These include BACs with substitution of the linear TAV-epitope present in the E2 protein and also BACs with substitution of the", "complete E2 protein with heterologous pestivirus sequences. We have also used the same approach for a range of different targeted modifications within CSFV BACs including specific deletions and substitutions in the 5′UTR of CSFV [24] and for insertions of heterologous reporter sequences into CSFV replicons [25] . Using Red/ET recombinationmediated mutagenesis for the targeted design, the work can be expedited and focused, in principal, on any sequence within the viral genome and is not dependent on the use of internal restriction sites. The results demonstrate that Red/ ET recombination-mediated mutagenesis of pestivirus BAC cDNAs provides a useful tool for advancing the construction of modified pestiviruses. Cells infected with the parental vR26 virus were recognized by the two anti-E2 mAbs (WH211 and WH303) specific for the CSFV-E2 proteins, in contrast cells infected with the modified viruses vR26_TAV and vR26_E2gif, rescued from the recombined BACs, were not detected by these", "mAbs. Furthermore, as expected, cells infected with the vR26_E2gif were recognized by the anti-BDV mAb (WB166) whereas no staining was observed with this antibody in vR26 infected cells or in cells with vR26_TAV. The mAb WH303 recognizes the CSFV TAV-epitope [18] and the difference in 4 aa between the TAV-epitope and the corresponding sequence from BDV strain \"Gifhorn\" is enough to completely abolish the recognition by this mAb. The lack of staining of vR26_TAV infected cells by the WH211 indicated that the TAV-sequence is also important for the epitope recognized by this mAb. Thus, the chimeric pestiviruses, vR26_TAV and vR26_E2gif, containing heterologous E2 sequences can be readily discriminated from the vR26 using specific anti-E2 monoclonal antibodies. These new chimeric pestiviruses represents Cstrain based marked vaccine candidates with the characteristics desired for safe and efficacious DIVA vaccines against CSFV. Indeed, vR26_E2gif vaccinated pigs could be efficiently", "discriminated from C-strain vaccinated pigs and from CSFV infected pigs using CSFV-E2 specific antibody ELISAs (Rasmussen et al., unpublished results).", "Nucleotide sequence data for the pBeloR26 showed a number of changes from the published reference sequence for \"C-strain Riems\". Some of these differences are present in the cDNA derived from the vaccine stock at a detectable level whereas others may represent low-level variants within the cDNA or errors introduced by the RT-PCR amplification. Full-length sequencing revealed that no changes occurred in the cDNA during extensive propagation in E. coli DH10B of the pBeloR26 and the E2chimeric derivative, pBeloR26_E2gif, indicating a very high stability of these BAC-cloned CSFV cDNAs. This is essential if this system is to be useful for cloning and sequence manipulation, and contrasts with stability problems encountered with conventional plasmids containing fulllength pestivirus cDNAs [31] . The stability of these BACs is consistent with previous reports on the stability of BACs containing other viruses of the family Flaviviridae in E. coli [8, 10] .", "Extensive passaging of the rescued vR26 and the chimeric virus derivative, vR26_E2gif, resulted in a change at nucleotide position A10665G (resulting in the predicted aa" ]

[ 1 ]

[ "single stranded positive sense RNA genomes" ]

[ "Efficient generation of recombinant RNA viruses using targeted recombination-mediated mutagenesis of bacterial artificial chromosomes containing full-length cDNA\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3840674/\n\nSHA: ef38ed2f4cc96e16ce011623cc5d15d2d8ca58c3\n\nAuthors: Rasmussen, Thomas Bruun; Risager, Peter Christian; Fahnøe, Ulrik; Friis, Martin Barfred; Belsham, Graham J; Höper, Dirk; Reimann, Ilona; Beer, Martin\nDate: 2013-11-22\nDOI: 10.1186/1471-2164-14-819\nLicense: cc-by", "Abstract: BACKGROUND: Infectious cDNA clones are a prerequisite for directed genetic manipulation of RNA viruses. Here, a strategy to facilitate manipulation and rescue of classical swine fever viruses (CSFVs) from full-length cDNAs present within bacterial artificial chromosomes (BACs) is described. This strategy allows manipulation of viral cDNA by targeted recombination-mediated mutagenesis within bacteria. RESULTS: A new CSFV-BAC (pBeloR26) derived from the Riems vaccine strain has been constructed and subsequently modified in the E2 coding sequence, using the targeted recombination strategy to enable rescue of chimeric pestiviruses (vR26_E2gif and vR26_TAV) with potential as new marker vaccine candidates. Sequencing of the BACs revealed a high genetic stability during passages within bacteria. The complete genome sequences of rescued viruses, after extensive passages in mammalian cells showed that modifications in the E2 protein coding sequence were stably maintained. A single", "amino acid substitution (D3431G) in the RNA dependent RNA polymerase was observed in the rescued viruses vR26_E2gif and vR26, which was reversion to the parental Riems sequence. CONCLUSIONS: These results show that targeted recombination-mediated mutagenesis provides a powerful tool for expediting the construction of novel RNA genomes and should be applicable to the manipulation of other RNA viruses.", "Text: Bacterial artificial chromosomes (BACs) are ideally suited for the stable maintenance of large DNA sequences derived from viral genomes [1] . A considerable number of BAC systems have been established for large DNA viruses; in particular many different herpesvirus genomes have been cloned into BACs (for review see [2] ). The first BAC systems using RNA virus cDNAs were described for coronaviruses [3] [4] [5] [6] and recently the first BAC containing a full-length cDNA for a negative-stranded RNA virus was described [7] . Similarly, cDNAs corresponding to the full-length genomes of members of the Flaviviridae family (Japanese encephalitis virus [8] and Dengue virus [9] ) have been inserted into BACs.", "BACs containing full-length cDNAs of pestiviruses (also within the Flaviviridae), including bovine viral diarrhea virus (BVDV) and classical swine fever virus (CSFV) have recently been established [10, 11] . Infectious pestiviruses can be rescued using RNA transcripts derived from these BACs. The pestiviruses have single stranded positive sense RNA genomes, about 12.3 kb in length, which includes a single long open reading frame, encoding a large polyprotein, flanked by 5′ and 3′ untranslated regions (UTRs) that are critical for autonomous replication of the genome [12, 13] . The polyprotein is cleaved by cellular and viral proteases into four structural proteins (nucleocapsid protein C, envelope glycoproteins E rns , E1 and E2) and eight nonstructural proteins (N pro , p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B). The availability of genetically defined and stable pestivirus BACs facilitates the functional study of viral proteins or RNA structures and also the development of new marker", "vaccine candidates. Several CSFV vaccines with marker properties based on chimeric pestiviruses have been developed over the years [14] . In particular, chimeric pestiviruses with substitution of the entire E2 protein have been described [15] [16] [17] but also mutants with more subtle modifications, such as the modification of the important TAV-epitope [18] within the CSFV-E2 protein [19, 20] are promising marker vaccine candidates.", "Manipulation of BACs using traditional cloning procedures can be difficult (e.g. because of a lack of convenient restriction enzyme sites) and thus a range of methodologies that apply bacterial genetics, including homologous recombination (e.g. Red/ET homologous recombineering) within the E. coli host, have been developed (for review, see [21] ). The use of homologous recombination allows site-directed mutagenesis of BACs [22] and, by employing a counterselection scheme, specific modifications can be obtained without leaving residual \"foreign\" sequences [23] . The main advantage of this method is that there are no target limitations (e.g. based on size or location) and no need for suitable restriction sites. The integration of the modified sequence is performed in vivo (within E. coli) thereby potentially being more accurate than in vitro approaches like PCR-based methods. Although in vitro cloning approaches based on the use of high-fidelity polymerases for PCR amplification have", "significantly improved in recent years, the use of in vivo approaches should allow a more accurate method of mutagenesis due to the use of the cells own high-fidelity replication system which includes proof reading. Whereas BAC recombination has been commonly used for modifying DNA viruses, there are only very few reports about the use of this technology for RNA viruses [7, 24, 25] .", "Here, a generally applicable strategy for the manipulation and rescue of chimeric pestiviruses from BACs is described as a model, and the flexibility of this approach is demonstrated by generating different modifications in the viral cDNA of the new CSFV-BAC, pBeloR26, derived from the modified live vaccine strain \"C-strain Riems\". The targeted recombination-mediated mutagenesis described here includes the substitution of the 9 amino acid (aa) linear TAV-epitope (TAVSPTTLR) present in the E2 protein with the corresponding region (TTVSTSTLA) of a heterologous pestivirus (border disease virus, BDV, strain \"Gifhorn\") and also the replacement of the entire CSFV E2 protein coding region with the whole E2 coding region from the same BDV, to generate marked vaccine viruses that can be discriminated using specific anti-E2 monoclonal antibodies. The genetic stabilities of both the BAC constructs (within E. coli) and the rescued viruses have also been assessed.", "Porcine kidney (PK15) and sheep fetal thymoid (SFT-R) cells were grown at 37°C (with 5% (v/v) CO 2 ) in Dulbecco's minimal essential medium (DMEM) supplemented with 5% (v/v) pestivirus-free fetal calf serum. Virus from a bait containing the modified live vaccine CSFV \"C-strain Riems\" (Riemser Arzneimittel AG, Germany) was propagated once in PK15 cells and termed vRiemser. RNA obtained from BDV strain \"Gifhorn\" [26] was used for amplification of the Gifhorn E2-coding sequence.\n\nOligonucleotide primers used are listed in Additional file 1: Table S1 .", "The BAC construct, pBeloR26, was constructed using the long RT-PCR method as previously described [11] using RNA derived from the \"C-strain Riems\". Briefly, full-length viral cDNAs flanked by NotI sites were amplified by long RT-PCR using primers 5′Cstrain_T7_Not1 (which includes a T7 promotor for in vitro transcription, a NotI site and a region corresponding to the first 44 nt of the genome) and 3′CSFV_Not1 (that contains a NotI site and sequence complementary to the 3′-terminal 35 nt of the genome that are conserved among many CSFVs including the Cstrain). The product (ca. 12.3 kbp) was digested with NotI and inserted into similarly digested pBeloBAC11 (New England Biolabs, GenBank accession U51113). All BACs were modified and maintained in E. coli DH10B cells (Invitrogen) grown at 37°C in LB medium containing chloramphenicol (Cam, 15 μg/ml). The electroporation of bacteria was performed in 0.1 cm cuvettes using 1 pulse at 1800 V, 25 μF and 200 Ω in a Gene Pulser Xcell (Bio-Rad).", "BACs to be used as templates for long PCR or for screening by restriction enzyme digestion were purified from 4 ml overnight cultures of E. coli DH10B using the ZR BAC DNA Miniprep Kit (Zymo Research). BACs required for direct genome sequencing were purified from 500 ml cultures using the Large-construct kit (Qiagen).", "Modifications to the full-length CSFV cDNA were accomplished in E. coli DH10B (streptomycin resistant, Strep R ) using the Counter Selection BAC Modification Kit (Gene Bridges, Heidelberg, Germany).\n\nThe Red/ET recombination involved three steps (i-iii).", "Step i) the temperature-sensitive pRedET expression plasmid (Gene Bridges) was introduced into electroporationcompetent E.coli DH10B cells containing the parental BAC (phenotype Cam R , Strep R ). The pRedET expresses the phage lambda proteins redα, redβ and redγ, under control of the arabinose-inducible pBAD promoter, allowing homologous recombination to occur. Immediately after electroporation, pre-warmed LB medium without antibiotics (1 ml) was added to the cells which were then incubated at 30°C for 1 hour, prior to spreading onto agar plates containing Cam (15 μg/ml) and tetracycline (Tet) (3 μg/ml) and then incubated at 30°C overnight to maintain the pRedET. The presence of the pRedET plasmid (conferring Tet R ) was verified by visual inspection of BAC-DNA preparations from the Cam R /Tet R colonies using agarose gel electrophoresis.", "Step ii) counter-selection marker cassettes with an extra NotI site for screening purposes (rpsL-neo, 1325 bp) were amplified by PCR using primers with 30 nt or 50 nt extensions that were homologous to the target site in the BAC using the rpsL-neo plasmid (Gene Bridges) as template and the Phusion hot start II HF DNA polymerase (Thermo Scientific) with cycling conditions as follows: 98°C for 30s, followed by 35 cycles of 98°C for 10s, 60°C for 20s, 72°C for 60s, and 1 cycle at 72°C for 4 min. The PCR products (ca. 1400 bp) were isolated on 1% (w/v) TBE agarose gels and purified using a GeneJET gel extraction kit (Thermo Scientific). Samples (30 μl), from an E. coli culture containing pRedET and the parental BAC grown overnight at 30°C in LB media (Cam, Tet), were used to inoculate 1.4 ml of fresh LB media with the same antibiotics to obtain exponentially growing bacteria at 30°C. Red/ET recombination proteins were induced by adding 50 μl of 10% (w/v) L-arabinose (Sigma). The PCR", "product (200 ng) containing the rpsL-neo cassette was introduced into these bacteria using electroporation (as above). Following electroporation, the cells were grown at 37°C for 70 min (to allow recombination) and then selected on plates containing Cam (15 μg/ml), Tet (3 μg/ml) and kanamycin (Kan, 15 μg/ml) overnight at 30°C to maintain the pRedET. Note, the rpsL cassette confers Streptomycin sensitivity (Strep S ) onto the resistant DH10B strain and the neo confers Kanamycin resistance (Kan R ). The correct phenotype (Cam R , Kan R , Tet R , Strep S ) of the resulting colonies was confirmed by streaking the colonies onto plates containing Cam (15 μg/ml), Tet (3 μg/ml) and Kan (15 μg/ml) and grown at 30°C. Importantly, for the third step, the replacement of the rpsL-neo cassette (using counter-selection), the selected colonies were also streaked onto plates containing Cam (15 μg/ml) plus Strep (50 μg/ml) and shown to be Strep S indicating incorporation of a functional rpsL gene. The", "structures of the intermediate BACs were verified by restriction enzyme analysis and sequencing around the inserts.", "Step iii) the replacement of the rpsL-neo selection cassettes from the intermediate constructs using linear DNA fragments was achieved through counter-selection and Red/ET recombination. Again, the homologous sequences at the ends of the DNA fragment were used for Red/ET mediated recombination events to replace the rpsL-neo cassette with the sequence of interest. Counterselection against the rpsL-neo cassette (phenotype Cam R , Kan R , Tet R , Strep S ) was employed using media containing Cam (15 μg/ml) and Strep (50 μg/ml) to isolate the required derivatives (phenotype Cam R and Strep R ).", "Initially, the intermediate construct, pBeloR26_E2rpsLneo ( Figure 1 ), was generated using Red/ET recombination by insertion of the rpsL-neo cassette with an extra NotI site for screening purposes which was amplified using primers Criems-TAVfor and Criems-TAVrev (Additional file 1: Table S1 ) in place of the TAVSPTTLR coding sequence (27 nt) . Secondly, the rpsL-neo cassette in this intermediate construct was then replaced using counter-selection Red/ ET recombination using a single-stranded oligonucleotide, Riems_TAV_Gifhorn (Additional file 1: Table S1 ) with the same homology arms as used for the rpsL-neo cassette, to introduce the coding sequence for the BDV \"Gifhorn\" epitope sequence (TTVSTSTLA). The resulting construct was named pBeloR26_TAV (Figure 1 ). The initial intermediate construct (with rpsL-neo) was then used to produce the pBeloR26_E2gif construct ( Figure 1 ). For this, the E2 coding sequence was amplified from cDNA prepared from BDV \"Gifhorn\" RNA using two different", "primer pairs, one set with 50 nt homology arms (Criems_E2_gifFlong/Criems_ E2_gifRlong) and another with 30 nt homologous sequences (Criems_E2_gifF/Criems_E2_gifR).", "For generation of BACs with substitution of the entire E2 coding sequences, PCR products consisting of the sequence of interest flanked with homology arms identical to the target area were generated by PCR (as for the rpsLneo cassette). For making constructs with substitution of shorter sequences (e.g. the TAV-epitope), the recombination was achieved using synthetic single stranded oligonucleotides rather than PCR products. Pre-heating of single stranded oligonucleotides at 95°C for 2 min followed by snap-freezing, prior to electroporation, empirically showed the best results. In each case, the DNA molecules were introduced into E. coli containing the BAC derivatives including the rpsL-neo cassettes together with the pRedET plasmid by electroporation as described above. The structures of the modified BACs were verified by restriction enzyme analysis and subsequent full-genome sequencing (see below).", "BAC DNA (1 μg) was linearized with NotI or 1 μl BAC DNA was used as template for long PCR amplification using primers 5′C-strain_T7_Not1 and 3′CSFV (Additional file 1: Table S1 ). Linearized BACs or PCR products were purified with the GeneJet PCR purification kit (Thermo Scientific) and transcribed in vitro using a Megascript T7 kit (Invitrogen). Viruses were rescued from RNA transcripts (1 to 5 μg) by electroporation of porcine (PK15) or ovine (SFT-R) cells essentially as described previously [24] . Cells were analysed using immunofluorescence microscopy (typically after 3 days) for the expression of NS3 and E2 proteins using specific monoclonal antibodies (mAbs), these were anti-NS3 (WB103/105, pan-pestivirus), anti-CSFV E2 (WH211, WH303, both CSFV specific) and anti-BDV E2 (WB166, BVDV/BDV specific) (AHVLA Scientific, United Kingdom) together with Alexa 488 conjugated goat antimouse IgG antibody (Molecular Probes, Invitrogen). The nuclei of cells were visualized using DAPI (Vector", "Laboratories) and images were recorded using a BX63 fluorescence microscope (Olympus). For peroxidase staining, cells were fixed and stained for the presence of pestivirus antigens using biotinylated pig anti-CSFV/BVDV polyclonal IgG followed by avidin-conjugated horseradish peroxidase (eBioscience) as previously described [27] . The same staining procedure was also performed using the anti-E2 mAbs. Samples containing virus-positive cells were passaged onto new cells. Virus growth curves were generated as previously described [24] . Briefly, PK15 or SFT-R cells were infected at a multiplicity of infection (MOI) of 0.1 pfu/cell and grown for three days.", "BAC DNAs (5 μg), purified using the Large-construct kit (Qiagen), or PCR products (1 μg) amplified from viral cDNA or from BACs using the long PCR method (as above) were consensus sequenced using a 454 FLX (Roche) or an Ion PGM (Life Technologies). Both Newbler (Roche) and the bwa.bwasw alignment algorithm [28] were used for mapping the reads to the expected sequence. A combination of Samtools [29] and LoFreq SNV-caller [30] was used for downstream single nucleotide variant (SNV) analysis. Finally, clone consensus sequences were aligned using MAFFT in the Geneious software platform (Biomatters).\n\nGeneration of a BAC containing full-length cDNA corresponding to the modified live vaccine \"C-strain Riems\"", "BACs containing the full-length cDNA corresponding to the parental vRiemser (\"C-strain Riems\") were constructed according to the method described previously for the \"Paderborn\" strain of CSFV [11] . BACs containing the complete CSFV cDNAs were identified by restriction Figure 1 Schematic representation of the CSFV genome organization and the BACs constructed and used in this study. Nucleotide (nt) and amino acid (aa) positions within R26 for the 5′ and 3′ termini together with the translational start and stop codons of the polyprotein coding region plus cleavage sites used to make the individual proteins (N pro , C, E rns , E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B) are indicated. Insertion of the rpsL-neo in place of the TAV-epitope within CSFV E2 for the intermediate construct (R26_rpsLneo) and the subsequent replacement with the TTVSTSTLA sequence (R26_TAV) and the complete substitution of the E2 sequence (R26_E2gif) are shown. Names of BAC constructs begin with \"pBelo\" and", "rescued viruses with \"v\" (e.g. pBeloR26 and vR26). Cell culture passage no. of virus is indicated with \"/P\" (e.g. vR26/P-4).", "digest analysis and following linearization by NotI, RNA transcripts were produced and electroporated into PK15 cells. This screening resulted in the identification of a BAC containing a cDNA insert of 12316 nt, pBeloR26 (Figure 1) , which yielded infectious virus, termed vR26, that could be propagated in SFT-R cells (Figure 2 , upper panels) and in PK15 cells (Figure 3 ). The rescued vR26 displayed higher growth rate at the early stage (about 10fold difference in virus yield at 24 h) compared to the parental vaccine virus, but after 48 hours similar virus titres were obtained (Figure 3 ). Full-genome sequencing of the cloned BAC template, pBeloR26, revealed a number of differences throughout the genome when compared to the full-length consensus sequence of the cDNA used for the cloning procedure (see Table 1 ). These differences are non-representative variants within the cDNA. Overall, the BAC sequence differed from the cDNA sequence in 18 positions, 9 of these lead to predicted", "amino acid substitutions within the polyprotein; one in each of N pro , E rns , E1, E2 and NS3 and four amino acid substitutions in NS5B (Table 1) . When compared to the published reference sequence (GenBank accession AY259122.1), the pBeloR26 BAC sequence differed at an additional 11 positions, 1 of these lead to a predicted amino acid substitution and there was one large insertion (27 nt) in the hypervariable region of the 3′-UTR (Additional file 2: Table S2 ).", "To determine the utility of the targeted recombinationmediated mutagenesis system for pestiviruses, two different modifications of the E2 protein coding sequence within pBeloR26 were generated using the Red/ET recombination methodology. Initially, the sequence encoding the linear TAV-epitope (TAVSPTTLR) within the CSFV-E2 was substituted with the sequence encoding the corresponding region (encoding TTVSTSTLA) from the BDV strain \"Gifhorn\" as described in the Materials and Methods section. More than 90% of the colonies obtained using this procedure contained the required BAC", "Anti-CSFV E2 (WH211) Figure 2 Antibody reaction patterns of pestivirus infected cells. SFT-R cells were infected with vR26 and its two derivatives vR26_E2gif and vR26_TAV plus vGifhorn [26] . After 72 h, the cells were fixed and stained with monoclonal antibodies against the NS3 protein (WB103/105, left column), the CSFV E2 protein (WH303 and WH211, middle columns) and the BDV E2 protein (WB166, right column) as indicated and viewed using a fluorescence microscope. structure as determined by NotI digestions. The complete genome sequences of the CSFV cDNA within two selected BACs, designated pBeloR26_TAV have been verified (data not shown). In addition, the complete coding sequence (1119 nt) for the CSFV-E2 protein was substituted by the corresponding sequence from BDV \"Gifhorn\". Again more than 90% of the colonies obtained contained the required BAC and the same proportion of correctly recombined BACs was obtained using either 30 nt or 50 nt homology arms. The chimeric BAC was", "designated, pBeloR26_E2gif and the complete virus genome sequence (cDNA) was verified (data not shown).", "After electroporation with RNA transcripts derived from either pBeloR26_TAV or pBeloR26_E2gif a large number of CSFV NS3-positive cells could be observed (data not shown) and chimeric virus stocks, termed vR26_TAV and vR26_E2gif, were generated after further passages in cells. Cells infected with these viruses and with the parental vR26 and vGifhorn strains were all stained with mAbs directed against the NS3 protein ( Figure 2 ). However, in contrast to the parental vR26 virus, the chimeric viruses rescued from the recombined BACs were not recognized by anti-E2 mAbs specific for the CSFV-E2 proteins ( Figure 2 ) and thus, consistent with their structure, displayed the same antibody reaction pattern as vGifhorn. Two different anti-CSFV E2 mAbs, WH211 and WH303, were used for the staining and the latter has been shown previously to target the TAV-epitope [18] . As anticipated, cells infected with either the vGifhorn or with the chimeric vR26_E2gif could be shown to express the \"Gifhorn\"", "E2 protein using staining with an anti-BDV mAb ( Figure 2 ). The presence of the BDV epitope TTVSTSTLA in vR26_ TAV was insufficient to permit efficient recognition by this anti-BDV mab, although a weak signal was observed in some cells.", "The BAC constructs pBeloR26 and pBeloR26_E2gif were analysed for the genetic stability of the cDNA to determine the suitability of the BAC vector for maintaining full-length pestivirus cDNAs. E. coli DH10B cells containing the BACs were passaged 15 times, by overnight growth, and the complete viral cDNAs within the BACs were sequenced after the 1st and the 15th passage. No mutations were observed within the 12316 nt virus cDNA sequences after this extensive propagation of the BACs in the bacterial host, indicating a highly stable system for the maintenance of complete pestivirus cDNA sequences.", "The viruses, vR26 and vR26_E2gif, rescued from their respective BAC constructs, were also tested for their genetic stability within mammalian cells. Linearized BAC DNA was transcribed in vitro and the RNA was electroporated into PK15 cells. Three days after electroporation the cells were stained with the anti-NS3 antibody to detect the presence of replicating virus. Samples containing virus positive cells were passaged onto new cells, this process \n\n*Nt position 10665 in vR26/P-12 is reverted from A to G as in the parental cDNA.", "was repeated for 12 separate passages (each of three days). The virus titre (as TCID 50 /ml) was determined for each passage. Passage of the rescued vR26_E2gif chimeric virus in PK15 cells resulted in rapidly decreasing virus titres and was discontinued after the 2nd passage ( Figure 4A ). Instead, further passage of this chimeric virus was performed in ovine SFT-R cells (the preferred cell type for BDV) and resulted in much higher titers of the chimeric virus. Virus titers reached more than 10 6 TCID 50 /ml after the 1st passage and remained stable for 12 passages ( Figure 4A ). The rescued vR26 was also efficiently propagated on the SFT-R cells but maintained a slightly lower titer than the vR26_E2gif chimeric virus ( Figure 4A ).", "To check that the viruses retained their antibody reaction properties ( Figure 2 ) after these passages, cells were infected with viruses from the 12th SFT-R cell culture passage (termed vR26/P-12 and vR26_E2gif/P-12) and stained with a polyclonal anti-pestivirus serum and with specific mAbs directed against the CSFV-E2 and BDV-E2 proteins ( Figure 4B ). Cells infected with either the vR26/P-12 or the chimeric vR26_E2gif/P-12 were each detected by the polyclonal anti-pestivirus serum as expected. The anti-CSFV-E2 mAb specifically detected cells infected with vR26/P-12 but not cells infected by the chimeric virus containing the BDV-E2 protein (consistent with the results shown in Figure 2 ). In contrast, the anti-BDV-E2 mAb specifically detected infection by the vR26_E2gif/P-12 and did not recognize cells infected with vR26/P-12. Each result is in accord with the structure of the viruses. The 4th passage of vR26 (vR26/P-4) displayed a slower growth rate than the virus obtained after 12", "passages (see Figure 5A ). It also had a reduced growth rate compared to both the vR26_E2gif/P-4 and vR26_E2gif/P-12. The fulllength sequence of pBeloR26 had revealed ten non-silent mutations compared to the reference sequence (AY25 9122.1) for this virus (Additional file 2: Table S2 ). Any of these mutations could be responsible for the impaired growth acting alone or in concert. For further investigation of this issue, full length cDNAs prepared from vR26/ P-4, vR26/P-12, vR26_E2gif/P-4 and vR26_E2gif/P-12 were deep-sequenced using both the 454 FLX and Ion PGM platforms for comparison and to determine the quasispecies distribution (Additional file 3: Figure S1 and Additional file 4: Figure S2 ). Sequencing data from both platforms revealed that both the vR26/P-12 and vR26_E2gif/P-12 were close to 100% changed at nt position A10665G compared to the BAC clones (resulting in the predicted amino acid substitution D3431G within the NS5B protein, the RNAdependent RNA polymerase, see", "Figure 5B ). This adaptation is a reversion back to the consensus cDNA sequence of the parental vaccine virus, vRiemser (Additional file 2: Table S2 ). Additionally, vR26/P-4 and vR26_E2gif/P-4 already showed evidence for this reversion being present within the population. For vR26/P-4, the level of reversion was 57%, while for vR26_E2gif/P-4 the extent of change was 73% (see Figure 5B ).", "In this study, we have established the first BAC containing the full-length cDNA of a CSFV vaccine strain. The BAC differed from the parental cDNA sequence in 18 positions leading to 9 aa substitutions ( Table 1 ). The method that has been used for the generation of pBeloR26 is based on full genome amplification of cDNA followed by direct cloning to obtain the BACs [11] . This approach results in cDNA clones that reflect the quasispecies composition of the parental viral RNA and thus it is not guaranteed to obtain cDNA clones corresponding to the consensus sequence of the cDNA used. However, it is possible to correct the mutations using the BAC recombination approach if a consensus clone is needed. To demonstrate the utility of the Red/ET mediated recombination method we have generated a series of modified BACs derived from this CSFV full-length cDNA. These include BACs with substitution of the linear TAV-epitope present in the E2 protein and also BACs with substitution of the", "complete E2 protein with heterologous pestivirus sequences. We have also used the same approach for a range of different targeted modifications within CSFV BACs including specific deletions and substitutions in the 5′UTR of CSFV [24] and for insertions of heterologous reporter sequences into CSFV replicons [25] . Using Red/ET recombinationmediated mutagenesis for the targeted design, the work can be expedited and focused, in principal, on any sequence within the viral genome and is not dependent on the use of internal restriction sites. The results demonstrate that Red/ ET recombination-mediated mutagenesis of pestivirus BAC cDNAs provides a useful tool for advancing the construction of modified pestiviruses. Cells infected with the parental vR26 virus were recognized by the two anti-E2 mAbs (WH211 and WH303) specific for the CSFV-E2 proteins, in contrast cells infected with the modified viruses vR26_TAV and vR26_E2gif, rescued from the recombined BACs, were not detected by these", "mAbs. Furthermore, as expected, cells infected with the vR26_E2gif were recognized by the anti-BDV mAb (WB166) whereas no staining was observed with this antibody in vR26 infected cells or in cells with vR26_TAV. The mAb WH303 recognizes the CSFV TAV-epitope [18] and the difference in 4 aa between the TAV-epitope and the corresponding sequence from BDV strain \"Gifhorn\" is enough to completely abolish the recognition by this mAb. The lack of staining of vR26_TAV infected cells by the WH211 indicated that the TAV-sequence is also important for the epitope recognized by this mAb. Thus, the chimeric pestiviruses, vR26_TAV and vR26_E2gif, containing heterologous E2 sequences can be readily discriminated from the vR26 using specific anti-E2 monoclonal antibodies. These new chimeric pestiviruses represents Cstrain based marked vaccine candidates with the characteristics desired for safe and efficacious DIVA vaccines against CSFV. Indeed, vR26_E2gif vaccinated pigs could be efficiently", "discriminated from C-strain vaccinated pigs and from CSFV infected pigs using CSFV-E2 specific antibody ELISAs (Rasmussen et al., unpublished results).", "Nucleotide sequence data for the pBeloR26 showed a number of changes from the published reference sequence for \"C-strain Riems\". Some of these differences are present in the cDNA derived from the vaccine stock at a detectable level whereas others may represent low-level variants within the cDNA or errors introduced by the RT-PCR amplification. Full-length sequencing revealed that no changes occurred in the cDNA during extensive propagation in E. coli DH10B of the pBeloR26 and the E2chimeric derivative, pBeloR26_E2gif, indicating a very high stability of these BAC-cloned CSFV cDNAs. This is essential if this system is to be useful for cloning and sequence manipulation, and contrasts with stability problems encountered with conventional plasmids containing fulllength pestivirus cDNAs [31] . The stability of these BACs is consistent with previous reports on the stability of BACs containing other viruses of the family Flaviviridae in E. coli [8, 10] .", "Extensive passaging of the rescued vR26 and the chimeric virus derivative, vR26_E2gif, resulted in a change at nucleotide position A10665G (resulting in the predicted aa" ]

[ 4 ]

[ "5′ and 3′ untranslated regions (UTRs)" ]

[ "Efficient generation of recombinant RNA viruses using targeted recombination-mediated mutagenesis of bacterial artificial chromosomes containing full-length cDNA\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3840674/\n\nSHA: ef38ed2f4cc96e16ce011623cc5d15d2d8ca58c3\n\nAuthors: Rasmussen, Thomas Bruun; Risager, Peter Christian; Fahnøe, Ulrik; Friis, Martin Barfred; Belsham, Graham J; Höper, Dirk; Reimann, Ilona; Beer, Martin\nDate: 2013-11-22\nDOI: 10.1186/1471-2164-14-819\nLicense: cc-by", "Abstract: BACKGROUND: Infectious cDNA clones are a prerequisite for directed genetic manipulation of RNA viruses. Here, a strategy to facilitate manipulation and rescue of classical swine fever viruses (CSFVs) from full-length cDNAs present within bacterial artificial chromosomes (BACs) is described. This strategy allows manipulation of viral cDNA by targeted recombination-mediated mutagenesis within bacteria. RESULTS: A new CSFV-BAC (pBeloR26) derived from the Riems vaccine strain has been constructed and subsequently modified in the E2 coding sequence, using the targeted recombination strategy to enable rescue of chimeric pestiviruses (vR26_E2gif and vR26_TAV) with potential as new marker vaccine candidates. Sequencing of the BACs revealed a high genetic stability during passages within bacteria. The complete genome sequences of rescued viruses, after extensive passages in mammalian cells showed that modifications in the E2 protein coding sequence were stably maintained. A single", "amino acid substitution (D3431G) in the RNA dependent RNA polymerase was observed in the rescued viruses vR26_E2gif and vR26, which was reversion to the parental Riems sequence. CONCLUSIONS: These results show that targeted recombination-mediated mutagenesis provides a powerful tool for expediting the construction of novel RNA genomes and should be applicable to the manipulation of other RNA viruses.", "Text: Bacterial artificial chromosomes (BACs) are ideally suited for the stable maintenance of large DNA sequences derived from viral genomes [1] . A considerable number of BAC systems have been established for large DNA viruses; in particular many different herpesvirus genomes have been cloned into BACs (for review see [2] ). The first BAC systems using RNA virus cDNAs were described for coronaviruses [3] [4] [5] [6] and recently the first BAC containing a full-length cDNA for a negative-stranded RNA virus was described [7] . Similarly, cDNAs corresponding to the full-length genomes of members of the Flaviviridae family (Japanese encephalitis virus [8] and Dengue virus [9] ) have been inserted into BACs.", "BACs containing full-length cDNAs of pestiviruses (also within the Flaviviridae), including bovine viral diarrhea virus (BVDV) and classical swine fever virus (CSFV) have recently been established [10, 11] . Infectious pestiviruses can be rescued using RNA transcripts derived from these BACs. The pestiviruses have single stranded positive sense RNA genomes, about 12.3 kb in length, which includes a single long open reading frame, encoding a large polyprotein, flanked by 5′ and 3′ untranslated regions (UTRs) that are critical for autonomous replication of the genome [12, 13] . The polyprotein is cleaved by cellular and viral proteases into four structural proteins (nucleocapsid protein C, envelope glycoproteins E rns , E1 and E2) and eight nonstructural proteins (N pro , p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B). The availability of genetically defined and stable pestivirus BACs facilitates the functional study of viral proteins or RNA structures and also the development of new marker", "vaccine candidates. Several CSFV vaccines with marker properties based on chimeric pestiviruses have been developed over the years [14] . In particular, chimeric pestiviruses with substitution of the entire E2 protein have been described [15] [16] [17] but also mutants with more subtle modifications, such as the modification of the important TAV-epitope [18] within the CSFV-E2 protein [19, 20] are promising marker vaccine candidates.", "Manipulation of BACs using traditional cloning procedures can be difficult (e.g. because of a lack of convenient restriction enzyme sites) and thus a range of methodologies that apply bacterial genetics, including homologous recombination (e.g. Red/ET homologous recombineering) within the E. coli host, have been developed (for review, see [21] ). The use of homologous recombination allows site-directed mutagenesis of BACs [22] and, by employing a counterselection scheme, specific modifications can be obtained without leaving residual \"foreign\" sequences [23] . The main advantage of this method is that there are no target limitations (e.g. based on size or location) and no need for suitable restriction sites. The integration of the modified sequence is performed in vivo (within E. coli) thereby potentially being more accurate than in vitro approaches like PCR-based methods. Although in vitro cloning approaches based on the use of high-fidelity polymerases for PCR amplification have", "significantly improved in recent years, the use of in vivo approaches should allow a more accurate method of mutagenesis due to the use of the cells own high-fidelity replication system which includes proof reading. Whereas BAC recombination has been commonly used for modifying DNA viruses, there are only very few reports about the use of this technology for RNA viruses [7, 24, 25] .", "Here, a generally applicable strategy for the manipulation and rescue of chimeric pestiviruses from BACs is described as a model, and the flexibility of this approach is demonstrated by generating different modifications in the viral cDNA of the new CSFV-BAC, pBeloR26, derived from the modified live vaccine strain \"C-strain Riems\". The targeted recombination-mediated mutagenesis described here includes the substitution of the 9 amino acid (aa) linear TAV-epitope (TAVSPTTLR) present in the E2 protein with the corresponding region (TTVSTSTLA) of a heterologous pestivirus (border disease virus, BDV, strain \"Gifhorn\") and also the replacement of the entire CSFV E2 protein coding region with the whole E2 coding region from the same BDV, to generate marked vaccine viruses that can be discriminated using specific anti-E2 monoclonal antibodies. The genetic stabilities of both the BAC constructs (within E. coli) and the rescued viruses have also been assessed.", "Porcine kidney (PK15) and sheep fetal thymoid (SFT-R) cells were grown at 37°C (with 5% (v/v) CO 2 ) in Dulbecco's minimal essential medium (DMEM) supplemented with 5% (v/v) pestivirus-free fetal calf serum. Virus from a bait containing the modified live vaccine CSFV \"C-strain Riems\" (Riemser Arzneimittel AG, Germany) was propagated once in PK15 cells and termed vRiemser. RNA obtained from BDV strain \"Gifhorn\" [26] was used for amplification of the Gifhorn E2-coding sequence.\n\nOligonucleotide primers used are listed in Additional file 1: Table S1 .", "The BAC construct, pBeloR26, was constructed using the long RT-PCR method as previously described [11] using RNA derived from the \"C-strain Riems\". Briefly, full-length viral cDNAs flanked by NotI sites were amplified by long RT-PCR using primers 5′Cstrain_T7_Not1 (which includes a T7 promotor for in vitro transcription, a NotI site and a region corresponding to the first 44 nt of the genome) and 3′CSFV_Not1 (that contains a NotI site and sequence complementary to the 3′-terminal 35 nt of the genome that are conserved among many CSFVs including the Cstrain). The product (ca. 12.3 kbp) was digested with NotI and inserted into similarly digested pBeloBAC11 (New England Biolabs, GenBank accession U51113). All BACs were modified and maintained in E. coli DH10B cells (Invitrogen) grown at 37°C in LB medium containing chloramphenicol (Cam, 15 μg/ml). The electroporation of bacteria was performed in 0.1 cm cuvettes using 1 pulse at 1800 V, 25 μF and 200 Ω in a Gene Pulser Xcell (Bio-Rad).", "BACs to be used as templates for long PCR or for screening by restriction enzyme digestion were purified from 4 ml overnight cultures of E. coli DH10B using the ZR BAC DNA Miniprep Kit (Zymo Research). BACs required for direct genome sequencing were purified from 500 ml cultures using the Large-construct kit (Qiagen).", "Modifications to the full-length CSFV cDNA were accomplished in E. coli DH10B (streptomycin resistant, Strep R ) using the Counter Selection BAC Modification Kit (Gene Bridges, Heidelberg, Germany).\n\nThe Red/ET recombination involved three steps (i-iii).", "Step i) the temperature-sensitive pRedET expression plasmid (Gene Bridges) was introduced into electroporationcompetent E.coli DH10B cells containing the parental BAC (phenotype Cam R , Strep R ). The pRedET expresses the phage lambda proteins redα, redβ and redγ, under control of the arabinose-inducible pBAD promoter, allowing homologous recombination to occur. Immediately after electroporation, pre-warmed LB medium without antibiotics (1 ml) was added to the cells which were then incubated at 30°C for 1 hour, prior to spreading onto agar plates containing Cam (15 μg/ml) and tetracycline (Tet) (3 μg/ml) and then incubated at 30°C overnight to maintain the pRedET. The presence of the pRedET plasmid (conferring Tet R ) was verified by visual inspection of BAC-DNA preparations from the Cam R /Tet R colonies using agarose gel electrophoresis.", "Step ii) counter-selection marker cassettes with an extra NotI site for screening purposes (rpsL-neo, 1325 bp) were amplified by PCR using primers with 30 nt or 50 nt extensions that were homologous to the target site in the BAC using the rpsL-neo plasmid (Gene Bridges) as template and the Phusion hot start II HF DNA polymerase (Thermo Scientific) with cycling conditions as follows: 98°C for 30s, followed by 35 cycles of 98°C for 10s, 60°C for 20s, 72°C for 60s, and 1 cycle at 72°C for 4 min. The PCR products (ca. 1400 bp) were isolated on 1% (w/v) TBE agarose gels and purified using a GeneJET gel extraction kit (Thermo Scientific). Samples (30 μl), from an E. coli culture containing pRedET and the parental BAC grown overnight at 30°C in LB media (Cam, Tet), were used to inoculate 1.4 ml of fresh LB media with the same antibiotics to obtain exponentially growing bacteria at 30°C. Red/ET recombination proteins were induced by adding 50 μl of 10% (w/v) L-arabinose (Sigma). The PCR", "product (200 ng) containing the rpsL-neo cassette was introduced into these bacteria using electroporation (as above). Following electroporation, the cells were grown at 37°C for 70 min (to allow recombination) and then selected on plates containing Cam (15 μg/ml), Tet (3 μg/ml) and kanamycin (Kan, 15 μg/ml) overnight at 30°C to maintain the pRedET. Note, the rpsL cassette confers Streptomycin sensitivity (Strep S ) onto the resistant DH10B strain and the neo confers Kanamycin resistance (Kan R ). The correct phenotype (Cam R , Kan R , Tet R , Strep S ) of the resulting colonies was confirmed by streaking the colonies onto plates containing Cam (15 μg/ml), Tet (3 μg/ml) and Kan (15 μg/ml) and grown at 30°C. Importantly, for the third step, the replacement of the rpsL-neo cassette (using counter-selection), the selected colonies were also streaked onto plates containing Cam (15 μg/ml) plus Strep (50 μg/ml) and shown to be Strep S indicating incorporation of a functional rpsL gene. The", "structures of the intermediate BACs were verified by restriction enzyme analysis and sequencing around the inserts.", "Step iii) the replacement of the rpsL-neo selection cassettes from the intermediate constructs using linear DNA fragments was achieved through counter-selection and Red/ET recombination. Again, the homologous sequences at the ends of the DNA fragment were used for Red/ET mediated recombination events to replace the rpsL-neo cassette with the sequence of interest. Counterselection against the rpsL-neo cassette (phenotype Cam R , Kan R , Tet R , Strep S ) was employed using media containing Cam (15 μg/ml) and Strep (50 μg/ml) to isolate the required derivatives (phenotype Cam R and Strep R ).", "Initially, the intermediate construct, pBeloR26_E2rpsLneo ( Figure 1 ), was generated using Red/ET recombination by insertion of the rpsL-neo cassette with an extra NotI site for screening purposes which was amplified using primers Criems-TAVfor and Criems-TAVrev (Additional file 1: Table S1 ) in place of the TAVSPTTLR coding sequence (27 nt) . Secondly, the rpsL-neo cassette in this intermediate construct was then replaced using counter-selection Red/ ET recombination using a single-stranded oligonucleotide, Riems_TAV_Gifhorn (Additional file 1: Table S1 ) with the same homology arms as used for the rpsL-neo cassette, to introduce the coding sequence for the BDV \"Gifhorn\" epitope sequence (TTVSTSTLA). The resulting construct was named pBeloR26_TAV (Figure 1 ). The initial intermediate construct (with rpsL-neo) was then used to produce the pBeloR26_E2gif construct ( Figure 1 ). For this, the E2 coding sequence was amplified from cDNA prepared from BDV \"Gifhorn\" RNA using two different", "primer pairs, one set with 50 nt homology arms (Criems_E2_gifFlong/Criems_ E2_gifRlong) and another with 30 nt homologous sequences (Criems_E2_gifF/Criems_E2_gifR).", "For generation of BACs with substitution of the entire E2 coding sequences, PCR products consisting of the sequence of interest flanked with homology arms identical to the target area were generated by PCR (as for the rpsLneo cassette). For making constructs with substitution of shorter sequences (e.g. the TAV-epitope), the recombination was achieved using synthetic single stranded oligonucleotides rather than PCR products. Pre-heating of single stranded oligonucleotides at 95°C for 2 min followed by snap-freezing, prior to electroporation, empirically showed the best results. In each case, the DNA molecules were introduced into E. coli containing the BAC derivatives including the rpsL-neo cassettes together with the pRedET plasmid by electroporation as described above. The structures of the modified BACs were verified by restriction enzyme analysis and subsequent full-genome sequencing (see below).", "BAC DNA (1 μg) was linearized with NotI or 1 μl BAC DNA was used as template for long PCR amplification using primers 5′C-strain_T7_Not1 and 3′CSFV (Additional file 1: Table S1 ). Linearized BACs or PCR products were purified with the GeneJet PCR purification kit (Thermo Scientific) and transcribed in vitro using a Megascript T7 kit (Invitrogen). Viruses were rescued from RNA transcripts (1 to 5 μg) by electroporation of porcine (PK15) or ovine (SFT-R) cells essentially as described previously [24] . Cells were analysed using immunofluorescence microscopy (typically after 3 days) for the expression of NS3 and E2 proteins using specific monoclonal antibodies (mAbs), these were anti-NS3 (WB103/105, pan-pestivirus), anti-CSFV E2 (WH211, WH303, both CSFV specific) and anti-BDV E2 (WB166, BVDV/BDV specific) (AHVLA Scientific, United Kingdom) together with Alexa 488 conjugated goat antimouse IgG antibody (Molecular Probes, Invitrogen). The nuclei of cells were visualized using DAPI (Vector", "Laboratories) and images were recorded using a BX63 fluorescence microscope (Olympus). For peroxidase staining, cells were fixed and stained for the presence of pestivirus antigens using biotinylated pig anti-CSFV/BVDV polyclonal IgG followed by avidin-conjugated horseradish peroxidase (eBioscience) as previously described [27] . The same staining procedure was also performed using the anti-E2 mAbs. Samples containing virus-positive cells were passaged onto new cells. Virus growth curves were generated as previously described [24] . Briefly, PK15 or SFT-R cells were infected at a multiplicity of infection (MOI) of 0.1 pfu/cell and grown for three days.", "BAC DNAs (5 μg), purified using the Large-construct kit (Qiagen), or PCR products (1 μg) amplified from viral cDNA or from BACs using the long PCR method (as above) were consensus sequenced using a 454 FLX (Roche) or an Ion PGM (Life Technologies). Both Newbler (Roche) and the bwa.bwasw alignment algorithm [28] were used for mapping the reads to the expected sequence. A combination of Samtools [29] and LoFreq SNV-caller [30] was used for downstream single nucleotide variant (SNV) analysis. Finally, clone consensus sequences were aligned using MAFFT in the Geneious software platform (Biomatters).\n\nGeneration of a BAC containing full-length cDNA corresponding to the modified live vaccine \"C-strain Riems\"", "BACs containing the full-length cDNA corresponding to the parental vRiemser (\"C-strain Riems\") were constructed according to the method described previously for the \"Paderborn\" strain of CSFV [11] . BACs containing the complete CSFV cDNAs were identified by restriction Figure 1 Schematic representation of the CSFV genome organization and the BACs constructed and used in this study. Nucleotide (nt) and amino acid (aa) positions within R26 for the 5′ and 3′ termini together with the translational start and stop codons of the polyprotein coding region plus cleavage sites used to make the individual proteins (N pro , C, E rns , E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B) are indicated. Insertion of the rpsL-neo in place of the TAV-epitope within CSFV E2 for the intermediate construct (R26_rpsLneo) and the subsequent replacement with the TTVSTSTLA sequence (R26_TAV) and the complete substitution of the E2 sequence (R26_E2gif) are shown. Names of BAC constructs begin with \"pBelo\" and", "rescued viruses with \"v\" (e.g. pBeloR26 and vR26). Cell culture passage no. of virus is indicated with \"/P\" (e.g. vR26/P-4).", "digest analysis and following linearization by NotI, RNA transcripts were produced and electroporated into PK15 cells. This screening resulted in the identification of a BAC containing a cDNA insert of 12316 nt, pBeloR26 (Figure 1) , which yielded infectious virus, termed vR26, that could be propagated in SFT-R cells (Figure 2 , upper panels) and in PK15 cells (Figure 3 ). The rescued vR26 displayed higher growth rate at the early stage (about 10fold difference in virus yield at 24 h) compared to the parental vaccine virus, but after 48 hours similar virus titres were obtained (Figure 3 ). Full-genome sequencing of the cloned BAC template, pBeloR26, revealed a number of differences throughout the genome when compared to the full-length consensus sequence of the cDNA used for the cloning procedure (see Table 1 ). These differences are non-representative variants within the cDNA. Overall, the BAC sequence differed from the cDNA sequence in 18 positions, 9 of these lead to predicted", "amino acid substitutions within the polyprotein; one in each of N pro , E rns , E1, E2 and NS3 and four amino acid substitutions in NS5B (Table 1) . When compared to the published reference sequence (GenBank accession AY259122.1), the pBeloR26 BAC sequence differed at an additional 11 positions, 1 of these lead to a predicted amino acid substitution and there was one large insertion (27 nt) in the hypervariable region of the 3′-UTR (Additional file 2: Table S2 ).", "To determine the utility of the targeted recombinationmediated mutagenesis system for pestiviruses, two different modifications of the E2 protein coding sequence within pBeloR26 were generated using the Red/ET recombination methodology. Initially, the sequence encoding the linear TAV-epitope (TAVSPTTLR) within the CSFV-E2 was substituted with the sequence encoding the corresponding region (encoding TTVSTSTLA) from the BDV strain \"Gifhorn\" as described in the Materials and Methods section. More than 90% of the colonies obtained using this procedure contained the required BAC", "Anti-CSFV E2 (WH211) Figure 2 Antibody reaction patterns of pestivirus infected cells. SFT-R cells were infected with vR26 and its two derivatives vR26_E2gif and vR26_TAV plus vGifhorn [26] . After 72 h, the cells were fixed and stained with monoclonal antibodies against the NS3 protein (WB103/105, left column), the CSFV E2 protein (WH303 and WH211, middle columns) and the BDV E2 protein (WB166, right column) as indicated and viewed using a fluorescence microscope. structure as determined by NotI digestions. The complete genome sequences of the CSFV cDNA within two selected BACs, designated pBeloR26_TAV have been verified (data not shown). In addition, the complete coding sequence (1119 nt) for the CSFV-E2 protein was substituted by the corresponding sequence from BDV \"Gifhorn\". Again more than 90% of the colonies obtained contained the required BAC and the same proportion of correctly recombined BACs was obtained using either 30 nt or 50 nt homology arms. The chimeric BAC was", "designated, pBeloR26_E2gif and the complete virus genome sequence (cDNA) was verified (data not shown).", "After electroporation with RNA transcripts derived from either pBeloR26_TAV or pBeloR26_E2gif a large number of CSFV NS3-positive cells could be observed (data not shown) and chimeric virus stocks, termed vR26_TAV and vR26_E2gif, were generated after further passages in cells. Cells infected with these viruses and with the parental vR26 and vGifhorn strains were all stained with mAbs directed against the NS3 protein ( Figure 2 ). However, in contrast to the parental vR26 virus, the chimeric viruses rescued from the recombined BACs were not recognized by anti-E2 mAbs specific for the CSFV-E2 proteins ( Figure 2 ) and thus, consistent with their structure, displayed the same antibody reaction pattern as vGifhorn. Two different anti-CSFV E2 mAbs, WH211 and WH303, were used for the staining and the latter has been shown previously to target the TAV-epitope [18] . As anticipated, cells infected with either the vGifhorn or with the chimeric vR26_E2gif could be shown to express the \"Gifhorn\"", "E2 protein using staining with an anti-BDV mAb ( Figure 2 ). The presence of the BDV epitope TTVSTSTLA in vR26_ TAV was insufficient to permit efficient recognition by this anti-BDV mab, although a weak signal was observed in some cells.", "The BAC constructs pBeloR26 and pBeloR26_E2gif were analysed for the genetic stability of the cDNA to determine the suitability of the BAC vector for maintaining full-length pestivirus cDNAs. E. coli DH10B cells containing the BACs were passaged 15 times, by overnight growth, and the complete viral cDNAs within the BACs were sequenced after the 1st and the 15th passage. No mutations were observed within the 12316 nt virus cDNA sequences after this extensive propagation of the BACs in the bacterial host, indicating a highly stable system for the maintenance of complete pestivirus cDNA sequences.", "The viruses, vR26 and vR26_E2gif, rescued from their respective BAC constructs, were also tested for their genetic stability within mammalian cells. Linearized BAC DNA was transcribed in vitro and the RNA was electroporated into PK15 cells. Three days after electroporation the cells were stained with the anti-NS3 antibody to detect the presence of replicating virus. Samples containing virus positive cells were passaged onto new cells, this process \n\n*Nt position 10665 in vR26/P-12 is reverted from A to G as in the parental cDNA.", "was repeated for 12 separate passages (each of three days). The virus titre (as TCID 50 /ml) was determined for each passage. Passage of the rescued vR26_E2gif chimeric virus in PK15 cells resulted in rapidly decreasing virus titres and was discontinued after the 2nd passage ( Figure 4A ). Instead, further passage of this chimeric virus was performed in ovine SFT-R cells (the preferred cell type for BDV) and resulted in much higher titers of the chimeric virus. Virus titers reached more than 10 6 TCID 50 /ml after the 1st passage and remained stable for 12 passages ( Figure 4A ). The rescued vR26 was also efficiently propagated on the SFT-R cells but maintained a slightly lower titer than the vR26_E2gif chimeric virus ( Figure 4A ).", "To check that the viruses retained their antibody reaction properties ( Figure 2 ) after these passages, cells were infected with viruses from the 12th SFT-R cell culture passage (termed vR26/P-12 and vR26_E2gif/P-12) and stained with a polyclonal anti-pestivirus serum and with specific mAbs directed against the CSFV-E2 and BDV-E2 proteins ( Figure 4B ). Cells infected with either the vR26/P-12 or the chimeric vR26_E2gif/P-12 were each detected by the polyclonal anti-pestivirus serum as expected. The anti-CSFV-E2 mAb specifically detected cells infected with vR26/P-12 but not cells infected by the chimeric virus containing the BDV-E2 protein (consistent with the results shown in Figure 2 ). In contrast, the anti-BDV-E2 mAb specifically detected infection by the vR26_E2gif/P-12 and did not recognize cells infected with vR26/P-12. Each result is in accord with the structure of the viruses. The 4th passage of vR26 (vR26/P-4) displayed a slower growth rate than the virus obtained after 12", "passages (see Figure 5A ). It also had a reduced growth rate compared to both the vR26_E2gif/P-4 and vR26_E2gif/P-12. The fulllength sequence of pBeloR26 had revealed ten non-silent mutations compared to the reference sequence (AY25 9122.1) for this virus (Additional file 2: Table S2 ). Any of these mutations could be responsible for the impaired growth acting alone or in concert. For further investigation of this issue, full length cDNAs prepared from vR26/ P-4, vR26/P-12, vR26_E2gif/P-4 and vR26_E2gif/P-12 were deep-sequenced using both the 454 FLX and Ion PGM platforms for comparison and to determine the quasispecies distribution (Additional file 3: Figure S1 and Additional file 4: Figure S2 ). Sequencing data from both platforms revealed that both the vR26/P-12 and vR26_E2gif/P-12 were close to 100% changed at nt position A10665G compared to the BAC clones (resulting in the predicted amino acid substitution D3431G within the NS5B protein, the RNAdependent RNA polymerase, see", "Figure 5B ). This adaptation is a reversion back to the consensus cDNA sequence of the parental vaccine virus, vRiemser (Additional file 2: Table S2 ). Additionally, vR26/P-4 and vR26_E2gif/P-4 already showed evidence for this reversion being present within the population. For vR26/P-4, the level of reversion was 57%, while for vR26_E2gif/P-4 the extent of change was 73% (see Figure 5B ).", "In this study, we have established the first BAC containing the full-length cDNA of a CSFV vaccine strain. The BAC differed from the parental cDNA sequence in 18 positions leading to 9 aa substitutions ( Table 1 ). The method that has been used for the generation of pBeloR26 is based on full genome amplification of cDNA followed by direct cloning to obtain the BACs [11] . This approach results in cDNA clones that reflect the quasispecies composition of the parental viral RNA and thus it is not guaranteed to obtain cDNA clones corresponding to the consensus sequence of the cDNA used. However, it is possible to correct the mutations using the BAC recombination approach if a consensus clone is needed. To demonstrate the utility of the Red/ET mediated recombination method we have generated a series of modified BACs derived from this CSFV full-length cDNA. These include BACs with substitution of the linear TAV-epitope present in the E2 protein and also BACs with substitution of the", "complete E2 protein with heterologous pestivirus sequences. We have also used the same approach for a range of different targeted modifications within CSFV BACs including specific deletions and substitutions in the 5′UTR of CSFV [24] and for insertions of heterologous reporter sequences into CSFV replicons [25] . Using Red/ET recombinationmediated mutagenesis for the targeted design, the work can be expedited and focused, in principal, on any sequence within the viral genome and is not dependent on the use of internal restriction sites. The results demonstrate that Red/ ET recombination-mediated mutagenesis of pestivirus BAC cDNAs provides a useful tool for advancing the construction of modified pestiviruses. Cells infected with the parental vR26 virus were recognized by the two anti-E2 mAbs (WH211 and WH303) specific for the CSFV-E2 proteins, in contrast cells infected with the modified viruses vR26_TAV and vR26_E2gif, rescued from the recombined BACs, were not detected by these", "mAbs. Furthermore, as expected, cells infected with the vR26_E2gif were recognized by the anti-BDV mAb (WB166) whereas no staining was observed with this antibody in vR26 infected cells or in cells with vR26_TAV. The mAb WH303 recognizes the CSFV TAV-epitope [18] and the difference in 4 aa between the TAV-epitope and the corresponding sequence from BDV strain \"Gifhorn\" is enough to completely abolish the recognition by this mAb. The lack of staining of vR26_TAV infected cells by the WH211 indicated that the TAV-sequence is also important for the epitope recognized by this mAb. Thus, the chimeric pestiviruses, vR26_TAV and vR26_E2gif, containing heterologous E2 sequences can be readily discriminated from the vR26 using specific anti-E2 monoclonal antibodies. These new chimeric pestiviruses represents Cstrain based marked vaccine candidates with the characteristics desired for safe and efficacious DIVA vaccines against CSFV. Indeed, vR26_E2gif vaccinated pigs could be efficiently", "discriminated from C-strain vaccinated pigs and from CSFV infected pigs using CSFV-E2 specific antibody ELISAs (Rasmussen et al., unpublished results).", "Nucleotide sequence data for the pBeloR26 showed a number of changes from the published reference sequence for \"C-strain Riems\". Some of these differences are present in the cDNA derived from the vaccine stock at a detectable level whereas others may represent low-level variants within the cDNA or errors introduced by the RT-PCR amplification. Full-length sequencing revealed that no changes occurred in the cDNA during extensive propagation in E. coli DH10B of the pBeloR26 and the E2chimeric derivative, pBeloR26_E2gif, indicating a very high stability of these BAC-cloned CSFV cDNAs. This is essential if this system is to be useful for cloning and sequence manipulation, and contrasts with stability problems encountered with conventional plasmids containing fulllength pestivirus cDNAs [31] . The stability of these BACs is consistent with previous reports on the stability of BACs containing other viruses of the family Flaviviridae in E. coli [8, 10] .", "Extensive passaging of the rescued vR26 and the chimeric virus derivative, vR26_E2gif, resulted in a change at nucleotide position A10665G (resulting in the predicted aa" ]

[ 4 ]

[ "nucleocapsid protein C, envelope glycoproteins E rns , E1 and E2" ]

[ "Efficient generation of recombinant RNA viruses using targeted recombination-mediated mutagenesis of bacterial artificial chromosomes containing full-length cDNA\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3840674/\n\nSHA: ef38ed2f4cc96e16ce011623cc5d15d2d8ca58c3\n\nAuthors: Rasmussen, Thomas Bruun; Risager, Peter Christian; Fahnøe, Ulrik; Friis, Martin Barfred; Belsham, Graham J; Höper, Dirk; Reimann, Ilona; Beer, Martin\nDate: 2013-11-22\nDOI: 10.1186/1471-2164-14-819\nLicense: cc-by", "Abstract: BACKGROUND: Infectious cDNA clones are a prerequisite for directed genetic manipulation of RNA viruses. Here, a strategy to facilitate manipulation and rescue of classical swine fever viruses (CSFVs) from full-length cDNAs present within bacterial artificial chromosomes (BACs) is described. This strategy allows manipulation of viral cDNA by targeted recombination-mediated mutagenesis within bacteria. RESULTS: A new CSFV-BAC (pBeloR26) derived from the Riems vaccine strain has been constructed and subsequently modified in the E2 coding sequence, using the targeted recombination strategy to enable rescue of chimeric pestiviruses (vR26_E2gif and vR26_TAV) with potential as new marker vaccine candidates. Sequencing of the BACs revealed a high genetic stability during passages within bacteria. The complete genome sequences of rescued viruses, after extensive passages in mammalian cells showed that modifications in the E2 protein coding sequence were stably maintained. A single", "amino acid substitution (D3431G) in the RNA dependent RNA polymerase was observed in the rescued viruses vR26_E2gif and vR26, which was reversion to the parental Riems sequence. CONCLUSIONS: These results show that targeted recombination-mediated mutagenesis provides a powerful tool for expediting the construction of novel RNA genomes and should be applicable to the manipulation of other RNA viruses.", "Text: Bacterial artificial chromosomes (BACs) are ideally suited for the stable maintenance of large DNA sequences derived from viral genomes [1] . A considerable number of BAC systems have been established for large DNA viruses; in particular many different herpesvirus genomes have been cloned into BACs (for review see [2] ). The first BAC systems using RNA virus cDNAs were described for coronaviruses [3] [4] [5] [6] and recently the first BAC containing a full-length cDNA for a negative-stranded RNA virus was described [7] . Similarly, cDNAs corresponding to the full-length genomes of members of the Flaviviridae family (Japanese encephalitis virus [8] and Dengue virus [9] ) have been inserted into BACs.", "BACs containing full-length cDNAs of pestiviruses (also within the Flaviviridae), including bovine viral diarrhea virus (BVDV) and classical swine fever virus (CSFV) have recently been established [10, 11] . Infectious pestiviruses can be rescued using RNA transcripts derived from these BACs. The pestiviruses have single stranded positive sense RNA genomes, about 12.3 kb in length, which includes a single long open reading frame, encoding a large polyprotein, flanked by 5′ and 3′ untranslated regions (UTRs) that are critical for autonomous replication of the genome [12, 13] . The polyprotein is cleaved by cellular and viral proteases into four structural proteins (nucleocapsid protein C, envelope glycoproteins E rns , E1 and E2) and eight nonstructural proteins (N pro , p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B). The availability of genetically defined and stable pestivirus BACs facilitates the functional study of viral proteins or RNA structures and also the development of new marker", "vaccine candidates. Several CSFV vaccines with marker properties based on chimeric pestiviruses have been developed over the years [14] . In particular, chimeric pestiviruses with substitution of the entire E2 protein have been described [15] [16] [17] but also mutants with more subtle modifications, such as the modification of the important TAV-epitope [18] within the CSFV-E2 protein [19, 20] are promising marker vaccine candidates.", "Manipulation of BACs using traditional cloning procedures can be difficult (e.g. because of a lack of convenient restriction enzyme sites) and thus a range of methodologies that apply bacterial genetics, including homologous recombination (e.g. Red/ET homologous recombineering) within the E. coli host, have been developed (for review, see [21] ). The use of homologous recombination allows site-directed mutagenesis of BACs [22] and, by employing a counterselection scheme, specific modifications can be obtained without leaving residual \"foreign\" sequences [23] . The main advantage of this method is that there are no target limitations (e.g. based on size or location) and no need for suitable restriction sites. The integration of the modified sequence is performed in vivo (within E. coli) thereby potentially being more accurate than in vitro approaches like PCR-based methods. Although in vitro cloning approaches based on the use of high-fidelity polymerases for PCR amplification have", "significantly improved in recent years, the use of in vivo approaches should allow a more accurate method of mutagenesis due to the use of the cells own high-fidelity replication system which includes proof reading. Whereas BAC recombination has been commonly used for modifying DNA viruses, there are only very few reports about the use of this technology for RNA viruses [7, 24, 25] .", "Here, a generally applicable strategy for the manipulation and rescue of chimeric pestiviruses from BACs is described as a model, and the flexibility of this approach is demonstrated by generating different modifications in the viral cDNA of the new CSFV-BAC, pBeloR26, derived from the modified live vaccine strain \"C-strain Riems\". The targeted recombination-mediated mutagenesis described here includes the substitution of the 9 amino acid (aa) linear TAV-epitope (TAVSPTTLR) present in the E2 protein with the corresponding region (TTVSTSTLA) of a heterologous pestivirus (border disease virus, BDV, strain \"Gifhorn\") and also the replacement of the entire CSFV E2 protein coding region with the whole E2 coding region from the same BDV, to generate marked vaccine viruses that can be discriminated using specific anti-E2 monoclonal antibodies. The genetic stabilities of both the BAC constructs (within E. coli) and the rescued viruses have also been assessed.", "Porcine kidney (PK15) and sheep fetal thymoid (SFT-R) cells were grown at 37°C (with 5% (v/v) CO 2 ) in Dulbecco's minimal essential medium (DMEM) supplemented with 5% (v/v) pestivirus-free fetal calf serum. Virus from a bait containing the modified live vaccine CSFV \"C-strain Riems\" (Riemser Arzneimittel AG, Germany) was propagated once in PK15 cells and termed vRiemser. RNA obtained from BDV strain \"Gifhorn\" [26] was used for amplification of the Gifhorn E2-coding sequence.\n\nOligonucleotide primers used are listed in Additional file 1: Table S1 .", "The BAC construct, pBeloR26, was constructed using the long RT-PCR method as previously described [11] using RNA derived from the \"C-strain Riems\". Briefly, full-length viral cDNAs flanked by NotI sites were amplified by long RT-PCR using primers 5′Cstrain_T7_Not1 (which includes a T7 promotor for in vitro transcription, a NotI site and a region corresponding to the first 44 nt of the genome) and 3′CSFV_Not1 (that contains a NotI site and sequence complementary to the 3′-terminal 35 nt of the genome that are conserved among many CSFVs including the Cstrain). The product (ca. 12.3 kbp) was digested with NotI and inserted into similarly digested pBeloBAC11 (New England Biolabs, GenBank accession U51113). All BACs were modified and maintained in E. coli DH10B cells (Invitrogen) grown at 37°C in LB medium containing chloramphenicol (Cam, 15 μg/ml). The electroporation of bacteria was performed in 0.1 cm cuvettes using 1 pulse at 1800 V, 25 μF and 200 Ω in a Gene Pulser Xcell (Bio-Rad).", "BACs to be used as templates for long PCR or for screening by restriction enzyme digestion were purified from 4 ml overnight cultures of E. coli DH10B using the ZR BAC DNA Miniprep Kit (Zymo Research). BACs required for direct genome sequencing were purified from 500 ml cultures using the Large-construct kit (Qiagen).", "Modifications to the full-length CSFV cDNA were accomplished in E. coli DH10B (streptomycin resistant, Strep R ) using the Counter Selection BAC Modification Kit (Gene Bridges, Heidelberg, Germany).\n\nThe Red/ET recombination involved three steps (i-iii).", "Step i) the temperature-sensitive pRedET expression plasmid (Gene Bridges) was introduced into electroporationcompetent E.coli DH10B cells containing the parental BAC (phenotype Cam R , Strep R ). The pRedET expresses the phage lambda proteins redα, redβ and redγ, under control of the arabinose-inducible pBAD promoter, allowing homologous recombination to occur. Immediately after electroporation, pre-warmed LB medium without antibiotics (1 ml) was added to the cells which were then incubated at 30°C for 1 hour, prior to spreading onto agar plates containing Cam (15 μg/ml) and tetracycline (Tet) (3 μg/ml) and then incubated at 30°C overnight to maintain the pRedET. The presence of the pRedET plasmid (conferring Tet R ) was verified by visual inspection of BAC-DNA preparations from the Cam R /Tet R colonies using agarose gel electrophoresis.", "Step ii) counter-selection marker cassettes with an extra NotI site for screening purposes (rpsL-neo, 1325 bp) were amplified by PCR using primers with 30 nt or 50 nt extensions that were homologous to the target site in the BAC using the rpsL-neo plasmid (Gene Bridges) as template and the Phusion hot start II HF DNA polymerase (Thermo Scientific) with cycling conditions as follows: 98°C for 30s, followed by 35 cycles of 98°C for 10s, 60°C for 20s, 72°C for 60s, and 1 cycle at 72°C for 4 min. The PCR products (ca. 1400 bp) were isolated on 1% (w/v) TBE agarose gels and purified using a GeneJET gel extraction kit (Thermo Scientific). Samples (30 μl), from an E. coli culture containing pRedET and the parental BAC grown overnight at 30°C in LB media (Cam, Tet), were used to inoculate 1.4 ml of fresh LB media with the same antibiotics to obtain exponentially growing bacteria at 30°C. Red/ET recombination proteins were induced by adding 50 μl of 10% (w/v) L-arabinose (Sigma). The PCR", "product (200 ng) containing the rpsL-neo cassette was introduced into these bacteria using electroporation (as above). Following electroporation, the cells were grown at 37°C for 70 min (to allow recombination) and then selected on plates containing Cam (15 μg/ml), Tet (3 μg/ml) and kanamycin (Kan, 15 μg/ml) overnight at 30°C to maintain the pRedET. Note, the rpsL cassette confers Streptomycin sensitivity (Strep S ) onto the resistant DH10B strain and the neo confers Kanamycin resistance (Kan R ). The correct phenotype (Cam R , Kan R , Tet R , Strep S ) of the resulting colonies was confirmed by streaking the colonies onto plates containing Cam (15 μg/ml), Tet (3 μg/ml) and Kan (15 μg/ml) and grown at 30°C. Importantly, for the third step, the replacement of the rpsL-neo cassette (using counter-selection), the selected colonies were also streaked onto plates containing Cam (15 μg/ml) plus Strep (50 μg/ml) and shown to be Strep S indicating incorporation of a functional rpsL gene. The", "structures of the intermediate BACs were verified by restriction enzyme analysis and sequencing around the inserts.", "Step iii) the replacement of the rpsL-neo selection cassettes from the intermediate constructs using linear DNA fragments was achieved through counter-selection and Red/ET recombination. Again, the homologous sequences at the ends of the DNA fragment were used for Red/ET mediated recombination events to replace the rpsL-neo cassette with the sequence of interest. Counterselection against the rpsL-neo cassette (phenotype Cam R , Kan R , Tet R , Strep S ) was employed using media containing Cam (15 μg/ml) and Strep (50 μg/ml) to isolate the required derivatives (phenotype Cam R and Strep R ).", "Initially, the intermediate construct, pBeloR26_E2rpsLneo ( Figure 1 ), was generated using Red/ET recombination by insertion of the rpsL-neo cassette with an extra NotI site for screening purposes which was amplified using primers Criems-TAVfor and Criems-TAVrev (Additional file 1: Table S1 ) in place of the TAVSPTTLR coding sequence (27 nt) . Secondly, the rpsL-neo cassette in this intermediate construct was then replaced using counter-selection Red/ ET recombination using a single-stranded oligonucleotide, Riems_TAV_Gifhorn (Additional file 1: Table S1 ) with the same homology arms as used for the rpsL-neo cassette, to introduce the coding sequence for the BDV \"Gifhorn\" epitope sequence (TTVSTSTLA). The resulting construct was named pBeloR26_TAV (Figure 1 ). The initial intermediate construct (with rpsL-neo) was then used to produce the pBeloR26_E2gif construct ( Figure 1 ). For this, the E2 coding sequence was amplified from cDNA prepared from BDV \"Gifhorn\" RNA using two different", "primer pairs, one set with 50 nt homology arms (Criems_E2_gifFlong/Criems_ E2_gifRlong) and another with 30 nt homologous sequences (Criems_E2_gifF/Criems_E2_gifR).", "For generation of BACs with substitution of the entire E2 coding sequences, PCR products consisting of the sequence of interest flanked with homology arms identical to the target area were generated by PCR (as for the rpsLneo cassette). For making constructs with substitution of shorter sequences (e.g. the TAV-epitope), the recombination was achieved using synthetic single stranded oligonucleotides rather than PCR products. Pre-heating of single stranded oligonucleotides at 95°C for 2 min followed by snap-freezing, prior to electroporation, empirically showed the best results. In each case, the DNA molecules were introduced into E. coli containing the BAC derivatives including the rpsL-neo cassettes together with the pRedET plasmid by electroporation as described above. The structures of the modified BACs were verified by restriction enzyme analysis and subsequent full-genome sequencing (see below).", "BAC DNA (1 μg) was linearized with NotI or 1 μl BAC DNA was used as template for long PCR amplification using primers 5′C-strain_T7_Not1 and 3′CSFV (Additional file 1: Table S1 ). Linearized BACs or PCR products were purified with the GeneJet PCR purification kit (Thermo Scientific) and transcribed in vitro using a Megascript T7 kit (Invitrogen). Viruses were rescued from RNA transcripts (1 to 5 μg) by electroporation of porcine (PK15) or ovine (SFT-R) cells essentially as described previously [24] . Cells were analysed using immunofluorescence microscopy (typically after 3 days) for the expression of NS3 and E2 proteins using specific monoclonal antibodies (mAbs), these were anti-NS3 (WB103/105, pan-pestivirus), anti-CSFV E2 (WH211, WH303, both CSFV specific) and anti-BDV E2 (WB166, BVDV/BDV specific) (AHVLA Scientific, United Kingdom) together with Alexa 488 conjugated goat antimouse IgG antibody (Molecular Probes, Invitrogen). The nuclei of cells were visualized using DAPI (Vector", "Laboratories) and images were recorded using a BX63 fluorescence microscope (Olympus). For peroxidase staining, cells were fixed and stained for the presence of pestivirus antigens using biotinylated pig anti-CSFV/BVDV polyclonal IgG followed by avidin-conjugated horseradish peroxidase (eBioscience) as previously described [27] . The same staining procedure was also performed using the anti-E2 mAbs. Samples containing virus-positive cells were passaged onto new cells. Virus growth curves were generated as previously described [24] . Briefly, PK15 or SFT-R cells were infected at a multiplicity of infection (MOI) of 0.1 pfu/cell and grown for three days.", "BAC DNAs (5 μg), purified using the Large-construct kit (Qiagen), or PCR products (1 μg) amplified from viral cDNA or from BACs using the long PCR method (as above) were consensus sequenced using a 454 FLX (Roche) or an Ion PGM (Life Technologies). Both Newbler (Roche) and the bwa.bwasw alignment algorithm [28] were used for mapping the reads to the expected sequence. A combination of Samtools [29] and LoFreq SNV-caller [30] was used for downstream single nucleotide variant (SNV) analysis. Finally, clone consensus sequences were aligned using MAFFT in the Geneious software platform (Biomatters).\n\nGeneration of a BAC containing full-length cDNA corresponding to the modified live vaccine \"C-strain Riems\"", "BACs containing the full-length cDNA corresponding to the parental vRiemser (\"C-strain Riems\") were constructed according to the method described previously for the \"Paderborn\" strain of CSFV [11] . BACs containing the complete CSFV cDNAs were identified by restriction Figure 1 Schematic representation of the CSFV genome organization and the BACs constructed and used in this study. Nucleotide (nt) and amino acid (aa) positions within R26 for the 5′ and 3′ termini together with the translational start and stop codons of the polyprotein coding region plus cleavage sites used to make the individual proteins (N pro , C, E rns , E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B) are indicated. Insertion of the rpsL-neo in place of the TAV-epitope within CSFV E2 for the intermediate construct (R26_rpsLneo) and the subsequent replacement with the TTVSTSTLA sequence (R26_TAV) and the complete substitution of the E2 sequence (R26_E2gif) are shown. Names of BAC constructs begin with \"pBelo\" and", "rescued viruses with \"v\" (e.g. pBeloR26 and vR26). Cell culture passage no. of virus is indicated with \"/P\" (e.g. vR26/P-4).", "digest analysis and following linearization by NotI, RNA transcripts were produced and electroporated into PK15 cells. This screening resulted in the identification of a BAC containing a cDNA insert of 12316 nt, pBeloR26 (Figure 1) , which yielded infectious virus, termed vR26, that could be propagated in SFT-R cells (Figure 2 , upper panels) and in PK15 cells (Figure 3 ). The rescued vR26 displayed higher growth rate at the early stage (about 10fold difference in virus yield at 24 h) compared to the parental vaccine virus, but after 48 hours similar virus titres were obtained (Figure 3 ). Full-genome sequencing of the cloned BAC template, pBeloR26, revealed a number of differences throughout the genome when compared to the full-length consensus sequence of the cDNA used for the cloning procedure (see Table 1 ). These differences are non-representative variants within the cDNA. Overall, the BAC sequence differed from the cDNA sequence in 18 positions, 9 of these lead to predicted", "amino acid substitutions within the polyprotein; one in each of N pro , E rns , E1, E2 and NS3 and four amino acid substitutions in NS5B (Table 1) . When compared to the published reference sequence (GenBank accession AY259122.1), the pBeloR26 BAC sequence differed at an additional 11 positions, 1 of these lead to a predicted amino acid substitution and there was one large insertion (27 nt) in the hypervariable region of the 3′-UTR (Additional file 2: Table S2 ).", "To determine the utility of the targeted recombinationmediated mutagenesis system for pestiviruses, two different modifications of the E2 protein coding sequence within pBeloR26 were generated using the Red/ET recombination methodology. Initially, the sequence encoding the linear TAV-epitope (TAVSPTTLR) within the CSFV-E2 was substituted with the sequence encoding the corresponding region (encoding TTVSTSTLA) from the BDV strain \"Gifhorn\" as described in the Materials and Methods section. More than 90% of the colonies obtained using this procedure contained the required BAC", "Anti-CSFV E2 (WH211) Figure 2 Antibody reaction patterns of pestivirus infected cells. SFT-R cells were infected with vR26 and its two derivatives vR26_E2gif and vR26_TAV plus vGifhorn [26] . After 72 h, the cells were fixed and stained with monoclonal antibodies against the NS3 protein (WB103/105, left column), the CSFV E2 protein (WH303 and WH211, middle columns) and the BDV E2 protein (WB166, right column) as indicated and viewed using a fluorescence microscope. structure as determined by NotI digestions. The complete genome sequences of the CSFV cDNA within two selected BACs, designated pBeloR26_TAV have been verified (data not shown). In addition, the complete coding sequence (1119 nt) for the CSFV-E2 protein was substituted by the corresponding sequence from BDV \"Gifhorn\". Again more than 90% of the colonies obtained contained the required BAC and the same proportion of correctly recombined BACs was obtained using either 30 nt or 50 nt homology arms. The chimeric BAC was", "designated, pBeloR26_E2gif and the complete virus genome sequence (cDNA) was verified (data not shown).", "After electroporation with RNA transcripts derived from either pBeloR26_TAV or pBeloR26_E2gif a large number of CSFV NS3-positive cells could be observed (data not shown) and chimeric virus stocks, termed vR26_TAV and vR26_E2gif, were generated after further passages in cells. Cells infected with these viruses and with the parental vR26 and vGifhorn strains were all stained with mAbs directed against the NS3 protein ( Figure 2 ). However, in contrast to the parental vR26 virus, the chimeric viruses rescued from the recombined BACs were not recognized by anti-E2 mAbs specific for the CSFV-E2 proteins ( Figure 2 ) and thus, consistent with their structure, displayed the same antibody reaction pattern as vGifhorn. Two different anti-CSFV E2 mAbs, WH211 and WH303, were used for the staining and the latter has been shown previously to target the TAV-epitope [18] . As anticipated, cells infected with either the vGifhorn or with the chimeric vR26_E2gif could be shown to express the \"Gifhorn\"", "E2 protein using staining with an anti-BDV mAb ( Figure 2 ). The presence of the BDV epitope TTVSTSTLA in vR26_ TAV was insufficient to permit efficient recognition by this anti-BDV mab, although a weak signal was observed in some cells.", "The BAC constructs pBeloR26 and pBeloR26_E2gif were analysed for the genetic stability of the cDNA to determine the suitability of the BAC vector for maintaining full-length pestivirus cDNAs. E. coli DH10B cells containing the BACs were passaged 15 times, by overnight growth, and the complete viral cDNAs within the BACs were sequenced after the 1st and the 15th passage. No mutations were observed within the 12316 nt virus cDNA sequences after this extensive propagation of the BACs in the bacterial host, indicating a highly stable system for the maintenance of complete pestivirus cDNA sequences.", "The viruses, vR26 and vR26_E2gif, rescued from their respective BAC constructs, were also tested for their genetic stability within mammalian cells. Linearized BAC DNA was transcribed in vitro and the RNA was electroporated into PK15 cells. Three days after electroporation the cells were stained with the anti-NS3 antibody to detect the presence of replicating virus. Samples containing virus positive cells were passaged onto new cells, this process \n\n*Nt position 10665 in vR26/P-12 is reverted from A to G as in the parental cDNA.", "was repeated for 12 separate passages (each of three days). The virus titre (as TCID 50 /ml) was determined for each passage. Passage of the rescued vR26_E2gif chimeric virus in PK15 cells resulted in rapidly decreasing virus titres and was discontinued after the 2nd passage ( Figure 4A ). Instead, further passage of this chimeric virus was performed in ovine SFT-R cells (the preferred cell type for BDV) and resulted in much higher titers of the chimeric virus. Virus titers reached more than 10 6 TCID 50 /ml after the 1st passage and remained stable for 12 passages ( Figure 4A ). The rescued vR26 was also efficiently propagated on the SFT-R cells but maintained a slightly lower titer than the vR26_E2gif chimeric virus ( Figure 4A ).", "To check that the viruses retained their antibody reaction properties ( Figure 2 ) after these passages, cells were infected with viruses from the 12th SFT-R cell culture passage (termed vR26/P-12 and vR26_E2gif/P-12) and stained with a polyclonal anti-pestivirus serum and with specific mAbs directed against the CSFV-E2 and BDV-E2 proteins ( Figure 4B ). Cells infected with either the vR26/P-12 or the chimeric vR26_E2gif/P-12 were each detected by the polyclonal anti-pestivirus serum as expected. The anti-CSFV-E2 mAb specifically detected cells infected with vR26/P-12 but not cells infected by the chimeric virus containing the BDV-E2 protein (consistent with the results shown in Figure 2 ). In contrast, the anti-BDV-E2 mAb specifically detected infection by the vR26_E2gif/P-12 and did not recognize cells infected with vR26/P-12. Each result is in accord with the structure of the viruses. The 4th passage of vR26 (vR26/P-4) displayed a slower growth rate than the virus obtained after 12", "passages (see Figure 5A ). It also had a reduced growth rate compared to both the vR26_E2gif/P-4 and vR26_E2gif/P-12. The fulllength sequence of pBeloR26 had revealed ten non-silent mutations compared to the reference sequence (AY25 9122.1) for this virus (Additional file 2: Table S2 ). Any of these mutations could be responsible for the impaired growth acting alone or in concert. For further investigation of this issue, full length cDNAs prepared from vR26/ P-4, vR26/P-12, vR26_E2gif/P-4 and vR26_E2gif/P-12 were deep-sequenced using both the 454 FLX and Ion PGM platforms for comparison and to determine the quasispecies distribution (Additional file 3: Figure S1 and Additional file 4: Figure S2 ). Sequencing data from both platforms revealed that both the vR26/P-12 and vR26_E2gif/P-12 were close to 100% changed at nt position A10665G compared to the BAC clones (resulting in the predicted amino acid substitution D3431G within the NS5B protein, the RNAdependent RNA polymerase, see", "Figure 5B ). This adaptation is a reversion back to the consensus cDNA sequence of the parental vaccine virus, vRiemser (Additional file 2: Table S2 ). Additionally, vR26/P-4 and vR26_E2gif/P-4 already showed evidence for this reversion being present within the population. For vR26/P-4, the level of reversion was 57%, while for vR26_E2gif/P-4 the extent of change was 73% (see Figure 5B ).", "In this study, we have established the first BAC containing the full-length cDNA of a CSFV vaccine strain. The BAC differed from the parental cDNA sequence in 18 positions leading to 9 aa substitutions ( Table 1 ). The method that has been used for the generation of pBeloR26 is based on full genome amplification of cDNA followed by direct cloning to obtain the BACs [11] . This approach results in cDNA clones that reflect the quasispecies composition of the parental viral RNA and thus it is not guaranteed to obtain cDNA clones corresponding to the consensus sequence of the cDNA used. However, it is possible to correct the mutations using the BAC recombination approach if a consensus clone is needed. To demonstrate the utility of the Red/ET mediated recombination method we have generated a series of modified BACs derived from this CSFV full-length cDNA. These include BACs with substitution of the linear TAV-epitope present in the E2 protein and also BACs with substitution of the", "complete E2 protein with heterologous pestivirus sequences. We have also used the same approach for a range of different targeted modifications within CSFV BACs including specific deletions and substitutions in the 5′UTR of CSFV [24] and for insertions of heterologous reporter sequences into CSFV replicons [25] . Using Red/ET recombinationmediated mutagenesis for the targeted design, the work can be expedited and focused, in principal, on any sequence within the viral genome and is not dependent on the use of internal restriction sites. The results demonstrate that Red/ ET recombination-mediated mutagenesis of pestivirus BAC cDNAs provides a useful tool for advancing the construction of modified pestiviruses. Cells infected with the parental vR26 virus were recognized by the two anti-E2 mAbs (WH211 and WH303) specific for the CSFV-E2 proteins, in contrast cells infected with the modified viruses vR26_TAV and vR26_E2gif, rescued from the recombined BACs, were not detected by these", "mAbs. Furthermore, as expected, cells infected with the vR26_E2gif were recognized by the anti-BDV mAb (WB166) whereas no staining was observed with this antibody in vR26 infected cells or in cells with vR26_TAV. The mAb WH303 recognizes the CSFV TAV-epitope [18] and the difference in 4 aa between the TAV-epitope and the corresponding sequence from BDV strain \"Gifhorn\" is enough to completely abolish the recognition by this mAb. The lack of staining of vR26_TAV infected cells by the WH211 indicated that the TAV-sequence is also important for the epitope recognized by this mAb. Thus, the chimeric pestiviruses, vR26_TAV and vR26_E2gif, containing heterologous E2 sequences can be readily discriminated from the vR26 using specific anti-E2 monoclonal antibodies. These new chimeric pestiviruses represents Cstrain based marked vaccine candidates with the characteristics desired for safe and efficacious DIVA vaccines against CSFV. Indeed, vR26_E2gif vaccinated pigs could be efficiently", "discriminated from C-strain vaccinated pigs and from CSFV infected pigs using CSFV-E2 specific antibody ELISAs (Rasmussen et al., unpublished results).", "Nucleotide sequence data for the pBeloR26 showed a number of changes from the published reference sequence for \"C-strain Riems\". Some of these differences are present in the cDNA derived from the vaccine stock at a detectable level whereas others may represent low-level variants within the cDNA or errors introduced by the RT-PCR amplification. Full-length sequencing revealed that no changes occurred in the cDNA during extensive propagation in E. coli DH10B of the pBeloR26 and the E2chimeric derivative, pBeloR26_E2gif, indicating a very high stability of these BAC-cloned CSFV cDNAs. This is essential if this system is to be useful for cloning and sequence manipulation, and contrasts with stability problems encountered with conventional plasmids containing fulllength pestivirus cDNAs [31] . The stability of these BACs is consistent with previous reports on the stability of BACs containing other viruses of the family Flaviviridae in E. coli [8, 10] .", "Extensive passaging of the rescued vR26 and the chimeric virus derivative, vR26_E2gif, resulted in a change at nucleotide position A10665G (resulting in the predicted aa" ]

[ 4 ]

[ "aa" ]

[ "Efficient generation of recombinant RNA viruses using targeted recombination-mediated mutagenesis of bacterial artificial chromosomes containing full-length cDNA\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3840674/\n\nSHA: ef38ed2f4cc96e16ce011623cc5d15d2d8ca58c3\n\nAuthors: Rasmussen, Thomas Bruun; Risager, Peter Christian; Fahnøe, Ulrik; Friis, Martin Barfred; Belsham, Graham J; Höper, Dirk; Reimann, Ilona; Beer, Martin\nDate: 2013-11-22\nDOI: 10.1186/1471-2164-14-819\nLicense: cc-by", "Abstract: BACKGROUND: Infectious cDNA clones are a prerequisite for directed genetic manipulation of RNA viruses. Here, a strategy to facilitate manipulation and rescue of classical swine fever viruses (CSFVs) from full-length cDNAs present within bacterial artificial chromosomes (BACs) is described. This strategy allows manipulation of viral cDNA by targeted recombination-mediated mutagenesis within bacteria. RESULTS: A new CSFV-BAC (pBeloR26) derived from the Riems vaccine strain has been constructed and subsequently modified in the E2 coding sequence, using the targeted recombination strategy to enable rescue of chimeric pestiviruses (vR26_E2gif and vR26_TAV) with potential as new marker vaccine candidates. Sequencing of the BACs revealed a high genetic stability during passages within bacteria. The complete genome sequences of rescued viruses, after extensive passages in mammalian cells showed that modifications in the E2 protein coding sequence were stably maintained. A single", "amino acid substitution (D3431G) in the RNA dependent RNA polymerase was observed in the rescued viruses vR26_E2gif and vR26, which was reversion to the parental Riems sequence. CONCLUSIONS: These results show that targeted recombination-mediated mutagenesis provides a powerful tool for expediting the construction of novel RNA genomes and should be applicable to the manipulation of other RNA viruses.", "Text: Bacterial artificial chromosomes (BACs) are ideally suited for the stable maintenance of large DNA sequences derived from viral genomes [1] . A considerable number of BAC systems have been established for large DNA viruses; in particular many different herpesvirus genomes have been cloned into BACs (for review see [2] ). The first BAC systems using RNA virus cDNAs were described for coronaviruses [3] [4] [5] [6] and recently the first BAC containing a full-length cDNA for a negative-stranded RNA virus was described [7] . Similarly, cDNAs corresponding to the full-length genomes of members of the Flaviviridae family (Japanese encephalitis virus [8] and Dengue virus [9] ) have been inserted into BACs.", "BACs containing full-length cDNAs of pestiviruses (also within the Flaviviridae), including bovine viral diarrhea virus (BVDV) and classical swine fever virus (CSFV) have recently been established [10, 11] . Infectious pestiviruses can be rescued using RNA transcripts derived from these BACs. The pestiviruses have single stranded positive sense RNA genomes, about 12.3 kb in length, which includes a single long open reading frame, encoding a large polyprotein, flanked by 5′ and 3′ untranslated regions (UTRs) that are critical for autonomous replication of the genome [12, 13] . The polyprotein is cleaved by cellular and viral proteases into four structural proteins (nucleocapsid protein C, envelope glycoproteins E rns , E1 and E2) and eight nonstructural proteins (N pro , p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B). The availability of genetically defined and stable pestivirus BACs facilitates the functional study of viral proteins or RNA structures and also the development of new marker", "vaccine candidates. Several CSFV vaccines with marker properties based on chimeric pestiviruses have been developed over the years [14] . In particular, chimeric pestiviruses with substitution of the entire E2 protein have been described [15] [16] [17] but also mutants with more subtle modifications, such as the modification of the important TAV-epitope [18] within the CSFV-E2 protein [19, 20] are promising marker vaccine candidates.", "Manipulation of BACs using traditional cloning procedures can be difficult (e.g. because of a lack of convenient restriction enzyme sites) and thus a range of methodologies that apply bacterial genetics, including homologous recombination (e.g. Red/ET homologous recombineering) within the E. coli host, have been developed (for review, see [21] ). The use of homologous recombination allows site-directed mutagenesis of BACs [22] and, by employing a counterselection scheme, specific modifications can be obtained without leaving residual \"foreign\" sequences [23] . The main advantage of this method is that there are no target limitations (e.g. based on size or location) and no need for suitable restriction sites. The integration of the modified sequence is performed in vivo (within E. coli) thereby potentially being more accurate than in vitro approaches like PCR-based methods. Although in vitro cloning approaches based on the use of high-fidelity polymerases for PCR amplification have", "significantly improved in recent years, the use of in vivo approaches should allow a more accurate method of mutagenesis due to the use of the cells own high-fidelity replication system which includes proof reading. Whereas BAC recombination has been commonly used for modifying DNA viruses, there are only very few reports about the use of this technology for RNA viruses [7, 24, 25] .", "Here, a generally applicable strategy for the manipulation and rescue of chimeric pestiviruses from BACs is described as a model, and the flexibility of this approach is demonstrated by generating different modifications in the viral cDNA of the new CSFV-BAC, pBeloR26, derived from the modified live vaccine strain \"C-strain Riems\". The targeted recombination-mediated mutagenesis described here includes the substitution of the 9 amino acid (aa) linear TAV-epitope (TAVSPTTLR) present in the E2 protein with the corresponding region (TTVSTSTLA) of a heterologous pestivirus (border disease virus, BDV, strain \"Gifhorn\") and also the replacement of the entire CSFV E2 protein coding region with the whole E2 coding region from the same BDV, to generate marked vaccine viruses that can be discriminated using specific anti-E2 monoclonal antibodies. The genetic stabilities of both the BAC constructs (within E. coli) and the rescued viruses have also been assessed.", "Porcine kidney (PK15) and sheep fetal thymoid (SFT-R) cells were grown at 37°C (with 5% (v/v) CO 2 ) in Dulbecco's minimal essential medium (DMEM) supplemented with 5% (v/v) pestivirus-free fetal calf serum. Virus from a bait containing the modified live vaccine CSFV \"C-strain Riems\" (Riemser Arzneimittel AG, Germany) was propagated once in PK15 cells and termed vRiemser. RNA obtained from BDV strain \"Gifhorn\" [26] was used for amplification of the Gifhorn E2-coding sequence.\n\nOligonucleotide primers used are listed in Additional file 1: Table S1 .", "The BAC construct, pBeloR26, was constructed using the long RT-PCR method as previously described [11] using RNA derived from the \"C-strain Riems\". Briefly, full-length viral cDNAs flanked by NotI sites were amplified by long RT-PCR using primers 5′Cstrain_T7_Not1 (which includes a T7 promotor for in vitro transcription, a NotI site and a region corresponding to the first 44 nt of the genome) and 3′CSFV_Not1 (that contains a NotI site and sequence complementary to the 3′-terminal 35 nt of the genome that are conserved among many CSFVs including the Cstrain). The product (ca. 12.3 kbp) was digested with NotI and inserted into similarly digested pBeloBAC11 (New England Biolabs, GenBank accession U51113). All BACs were modified and maintained in E. coli DH10B cells (Invitrogen) grown at 37°C in LB medium containing chloramphenicol (Cam, 15 μg/ml). The electroporation of bacteria was performed in 0.1 cm cuvettes using 1 pulse at 1800 V, 25 μF and 200 Ω in a Gene Pulser Xcell (Bio-Rad).", "BACs to be used as templates for long PCR or for screening by restriction enzyme digestion were purified from 4 ml overnight cultures of E. coli DH10B using the ZR BAC DNA Miniprep Kit (Zymo Research). BACs required for direct genome sequencing were purified from 500 ml cultures using the Large-construct kit (Qiagen).", "Modifications to the full-length CSFV cDNA were accomplished in E. coli DH10B (streptomycin resistant, Strep R ) using the Counter Selection BAC Modification Kit (Gene Bridges, Heidelberg, Germany).\n\nThe Red/ET recombination involved three steps (i-iii).", "Step i) the temperature-sensitive pRedET expression plasmid (Gene Bridges) was introduced into electroporationcompetent E.coli DH10B cells containing the parental BAC (phenotype Cam R , Strep R ). The pRedET expresses the phage lambda proteins redα, redβ and redγ, under control of the arabinose-inducible pBAD promoter, allowing homologous recombination to occur. Immediately after electroporation, pre-warmed LB medium without antibiotics (1 ml) was added to the cells which were then incubated at 30°C for 1 hour, prior to spreading onto agar plates containing Cam (15 μg/ml) and tetracycline (Tet) (3 μg/ml) and then incubated at 30°C overnight to maintain the pRedET. The presence of the pRedET plasmid (conferring Tet R ) was verified by visual inspection of BAC-DNA preparations from the Cam R /Tet R colonies using agarose gel electrophoresis.", "Step ii) counter-selection marker cassettes with an extra NotI site for screening purposes (rpsL-neo, 1325 bp) were amplified by PCR using primers with 30 nt or 50 nt extensions that were homologous to the target site in the BAC using the rpsL-neo plasmid (Gene Bridges) as template and the Phusion hot start II HF DNA polymerase (Thermo Scientific) with cycling conditions as follows: 98°C for 30s, followed by 35 cycles of 98°C for 10s, 60°C for 20s, 72°C for 60s, and 1 cycle at 72°C for 4 min. The PCR products (ca. 1400 bp) were isolated on 1% (w/v) TBE agarose gels and purified using a GeneJET gel extraction kit (Thermo Scientific). Samples (30 μl), from an E. coli culture containing pRedET and the parental BAC grown overnight at 30°C in LB media (Cam, Tet), were used to inoculate 1.4 ml of fresh LB media with the same antibiotics to obtain exponentially growing bacteria at 30°C. Red/ET recombination proteins were induced by adding 50 μl of 10% (w/v) L-arabinose (Sigma). The PCR", "product (200 ng) containing the rpsL-neo cassette was introduced into these bacteria using electroporation (as above). Following electroporation, the cells were grown at 37°C for 70 min (to allow recombination) and then selected on plates containing Cam (15 μg/ml), Tet (3 μg/ml) and kanamycin (Kan, 15 μg/ml) overnight at 30°C to maintain the pRedET. Note, the rpsL cassette confers Streptomycin sensitivity (Strep S ) onto the resistant DH10B strain and the neo confers Kanamycin resistance (Kan R ). The correct phenotype (Cam R , Kan R , Tet R , Strep S ) of the resulting colonies was confirmed by streaking the colonies onto plates containing Cam (15 μg/ml), Tet (3 μg/ml) and Kan (15 μg/ml) and grown at 30°C. Importantly, for the third step, the replacement of the rpsL-neo cassette (using counter-selection), the selected colonies were also streaked onto plates containing Cam (15 μg/ml) plus Strep (50 μg/ml) and shown to be Strep S indicating incorporation of a functional rpsL gene. The", "structures of the intermediate BACs were verified by restriction enzyme analysis and sequencing around the inserts.", "Step iii) the replacement of the rpsL-neo selection cassettes from the intermediate constructs using linear DNA fragments was achieved through counter-selection and Red/ET recombination. Again, the homologous sequences at the ends of the DNA fragment were used for Red/ET mediated recombination events to replace the rpsL-neo cassette with the sequence of interest. Counterselection against the rpsL-neo cassette (phenotype Cam R , Kan R , Tet R , Strep S ) was employed using media containing Cam (15 μg/ml) and Strep (50 μg/ml) to isolate the required derivatives (phenotype Cam R and Strep R ).", "Initially, the intermediate construct, pBeloR26_E2rpsLneo ( Figure 1 ), was generated using Red/ET recombination by insertion of the rpsL-neo cassette with an extra NotI site for screening purposes which was amplified using primers Criems-TAVfor and Criems-TAVrev (Additional file 1: Table S1 ) in place of the TAVSPTTLR coding sequence (27 nt) . Secondly, the rpsL-neo cassette in this intermediate construct was then replaced using counter-selection Red/ ET recombination using a single-stranded oligonucleotide, Riems_TAV_Gifhorn (Additional file 1: Table S1 ) with the same homology arms as used for the rpsL-neo cassette, to introduce the coding sequence for the BDV \"Gifhorn\" epitope sequence (TTVSTSTLA). The resulting construct was named pBeloR26_TAV (Figure 1 ). The initial intermediate construct (with rpsL-neo) was then used to produce the pBeloR26_E2gif construct ( Figure 1 ). For this, the E2 coding sequence was amplified from cDNA prepared from BDV \"Gifhorn\" RNA using two different", "primer pairs, one set with 50 nt homology arms (Criems_E2_gifFlong/Criems_ E2_gifRlong) and another with 30 nt homologous sequences (Criems_E2_gifF/Criems_E2_gifR).", "For generation of BACs with substitution of the entire E2 coding sequences, PCR products consisting of the sequence of interest flanked with homology arms identical to the target area were generated by PCR (as for the rpsLneo cassette). For making constructs with substitution of shorter sequences (e.g. the TAV-epitope), the recombination was achieved using synthetic single stranded oligonucleotides rather than PCR products. Pre-heating of single stranded oligonucleotides at 95°C for 2 min followed by snap-freezing, prior to electroporation, empirically showed the best results. In each case, the DNA molecules were introduced into E. coli containing the BAC derivatives including the rpsL-neo cassettes together with the pRedET plasmid by electroporation as described above. The structures of the modified BACs were verified by restriction enzyme analysis and subsequent full-genome sequencing (see below).", "BAC DNA (1 μg) was linearized with NotI or 1 μl BAC DNA was used as template for long PCR amplification using primers 5′C-strain_T7_Not1 and 3′CSFV (Additional file 1: Table S1 ). Linearized BACs or PCR products were purified with the GeneJet PCR purification kit (Thermo Scientific) and transcribed in vitro using a Megascript T7 kit (Invitrogen). Viruses were rescued from RNA transcripts (1 to 5 μg) by electroporation of porcine (PK15) or ovine (SFT-R) cells essentially as described previously [24] . Cells were analysed using immunofluorescence microscopy (typically after 3 days) for the expression of NS3 and E2 proteins using specific monoclonal antibodies (mAbs), these were anti-NS3 (WB103/105, pan-pestivirus), anti-CSFV E2 (WH211, WH303, both CSFV specific) and anti-BDV E2 (WB166, BVDV/BDV specific) (AHVLA Scientific, United Kingdom) together with Alexa 488 conjugated goat antimouse IgG antibody (Molecular Probes, Invitrogen). The nuclei of cells were visualized using DAPI (Vector", "Laboratories) and images were recorded using a BX63 fluorescence microscope (Olympus). For peroxidase staining, cells were fixed and stained for the presence of pestivirus antigens using biotinylated pig anti-CSFV/BVDV polyclonal IgG followed by avidin-conjugated horseradish peroxidase (eBioscience) as previously described [27] . The same staining procedure was also performed using the anti-E2 mAbs. Samples containing virus-positive cells were passaged onto new cells. Virus growth curves were generated as previously described [24] . Briefly, PK15 or SFT-R cells were infected at a multiplicity of infection (MOI) of 0.1 pfu/cell and grown for three days.", "BAC DNAs (5 μg), purified using the Large-construct kit (Qiagen), or PCR products (1 μg) amplified from viral cDNA or from BACs using the long PCR method (as above) were consensus sequenced using a 454 FLX (Roche) or an Ion PGM (Life Technologies). Both Newbler (Roche) and the bwa.bwasw alignment algorithm [28] were used for mapping the reads to the expected sequence. A combination of Samtools [29] and LoFreq SNV-caller [30] was used for downstream single nucleotide variant (SNV) analysis. Finally, clone consensus sequences were aligned using MAFFT in the Geneious software platform (Biomatters).\n\nGeneration of a BAC containing full-length cDNA corresponding to the modified live vaccine \"C-strain Riems\"", "BACs containing the full-length cDNA corresponding to the parental vRiemser (\"C-strain Riems\") were constructed according to the method described previously for the \"Paderborn\" strain of CSFV [11] . BACs containing the complete CSFV cDNAs were identified by restriction Figure 1 Schematic representation of the CSFV genome organization and the BACs constructed and used in this study. Nucleotide (nt) and amino acid (aa) positions within R26 for the 5′ and 3′ termini together with the translational start and stop codons of the polyprotein coding region plus cleavage sites used to make the individual proteins (N pro , C, E rns , E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B) are indicated. Insertion of the rpsL-neo in place of the TAV-epitope within CSFV E2 for the intermediate construct (R26_rpsLneo) and the subsequent replacement with the TTVSTSTLA sequence (R26_TAV) and the complete substitution of the E2 sequence (R26_E2gif) are shown. Names of BAC constructs begin with \"pBelo\" and", "rescued viruses with \"v\" (e.g. pBeloR26 and vR26). Cell culture passage no. of virus is indicated with \"/P\" (e.g. vR26/P-4).", "digest analysis and following linearization by NotI, RNA transcripts were produced and electroporated into PK15 cells. This screening resulted in the identification of a BAC containing a cDNA insert of 12316 nt, pBeloR26 (Figure 1) , which yielded infectious virus, termed vR26, that could be propagated in SFT-R cells (Figure 2 , upper panels) and in PK15 cells (Figure 3 ). The rescued vR26 displayed higher growth rate at the early stage (about 10fold difference in virus yield at 24 h) compared to the parental vaccine virus, but after 48 hours similar virus titres were obtained (Figure 3 ). Full-genome sequencing of the cloned BAC template, pBeloR26, revealed a number of differences throughout the genome when compared to the full-length consensus sequence of the cDNA used for the cloning procedure (see Table 1 ). These differences are non-representative variants within the cDNA. Overall, the BAC sequence differed from the cDNA sequence in 18 positions, 9 of these lead to predicted", "amino acid substitutions within the polyprotein; one in each of N pro , E rns , E1, E2 and NS3 and four amino acid substitutions in NS5B (Table 1) . When compared to the published reference sequence (GenBank accession AY259122.1), the pBeloR26 BAC sequence differed at an additional 11 positions, 1 of these lead to a predicted amino acid substitution and there was one large insertion (27 nt) in the hypervariable region of the 3′-UTR (Additional file 2: Table S2 ).", "To determine the utility of the targeted recombinationmediated mutagenesis system for pestiviruses, two different modifications of the E2 protein coding sequence within pBeloR26 were generated using the Red/ET recombination methodology. Initially, the sequence encoding the linear TAV-epitope (TAVSPTTLR) within the CSFV-E2 was substituted with the sequence encoding the corresponding region (encoding TTVSTSTLA) from the BDV strain \"Gifhorn\" as described in the Materials and Methods section. More than 90% of the colonies obtained using this procedure contained the required BAC", "Anti-CSFV E2 (WH211) Figure 2 Antibody reaction patterns of pestivirus infected cells. SFT-R cells were infected with vR26 and its two derivatives vR26_E2gif and vR26_TAV plus vGifhorn [26] . After 72 h, the cells were fixed and stained with monoclonal antibodies against the NS3 protein (WB103/105, left column), the CSFV E2 protein (WH303 and WH211, middle columns) and the BDV E2 protein (WB166, right column) as indicated and viewed using a fluorescence microscope. structure as determined by NotI digestions. The complete genome sequences of the CSFV cDNA within two selected BACs, designated pBeloR26_TAV have been verified (data not shown). In addition, the complete coding sequence (1119 nt) for the CSFV-E2 protein was substituted by the corresponding sequence from BDV \"Gifhorn\". Again more than 90% of the colonies obtained contained the required BAC and the same proportion of correctly recombined BACs was obtained using either 30 nt or 50 nt homology arms. The chimeric BAC was", "designated, pBeloR26_E2gif and the complete virus genome sequence (cDNA) was verified (data not shown).", "After electroporation with RNA transcripts derived from either pBeloR26_TAV or pBeloR26_E2gif a large number of CSFV NS3-positive cells could be observed (data not shown) and chimeric virus stocks, termed vR26_TAV and vR26_E2gif, were generated after further passages in cells. Cells infected with these viruses and with the parental vR26 and vGifhorn strains were all stained with mAbs directed against the NS3 protein ( Figure 2 ). However, in contrast to the parental vR26 virus, the chimeric viruses rescued from the recombined BACs were not recognized by anti-E2 mAbs specific for the CSFV-E2 proteins ( Figure 2 ) and thus, consistent with their structure, displayed the same antibody reaction pattern as vGifhorn. Two different anti-CSFV E2 mAbs, WH211 and WH303, were used for the staining and the latter has been shown previously to target the TAV-epitope [18] . As anticipated, cells infected with either the vGifhorn or with the chimeric vR26_E2gif could be shown to express the \"Gifhorn\"", "E2 protein using staining with an anti-BDV mAb ( Figure 2 ). The presence of the BDV epitope TTVSTSTLA in vR26_ TAV was insufficient to permit efficient recognition by this anti-BDV mab, although a weak signal was observed in some cells.", "The BAC constructs pBeloR26 and pBeloR26_E2gif were analysed for the genetic stability of the cDNA to determine the suitability of the BAC vector for maintaining full-length pestivirus cDNAs. E. coli DH10B cells containing the BACs were passaged 15 times, by overnight growth, and the complete viral cDNAs within the BACs were sequenced after the 1st and the 15th passage. No mutations were observed within the 12316 nt virus cDNA sequences after this extensive propagation of the BACs in the bacterial host, indicating a highly stable system for the maintenance of complete pestivirus cDNA sequences.", "The viruses, vR26 and vR26_E2gif, rescued from their respective BAC constructs, were also tested for their genetic stability within mammalian cells. Linearized BAC DNA was transcribed in vitro and the RNA was electroporated into PK15 cells. Three days after electroporation the cells were stained with the anti-NS3 antibody to detect the presence of replicating virus. Samples containing virus positive cells were passaged onto new cells, this process \n\n*Nt position 10665 in vR26/P-12 is reverted from A to G as in the parental cDNA.", "was repeated for 12 separate passages (each of three days). The virus titre (as TCID 50 /ml) was determined for each passage. Passage of the rescued vR26_E2gif chimeric virus in PK15 cells resulted in rapidly decreasing virus titres and was discontinued after the 2nd passage ( Figure 4A ). Instead, further passage of this chimeric virus was performed in ovine SFT-R cells (the preferred cell type for BDV) and resulted in much higher titers of the chimeric virus. Virus titers reached more than 10 6 TCID 50 /ml after the 1st passage and remained stable for 12 passages ( Figure 4A ). The rescued vR26 was also efficiently propagated on the SFT-R cells but maintained a slightly lower titer than the vR26_E2gif chimeric virus ( Figure 4A ).", "To check that the viruses retained their antibody reaction properties ( Figure 2 ) after these passages, cells were infected with viruses from the 12th SFT-R cell culture passage (termed vR26/P-12 and vR26_E2gif/P-12) and stained with a polyclonal anti-pestivirus serum and with specific mAbs directed against the CSFV-E2 and BDV-E2 proteins ( Figure 4B ). Cells infected with either the vR26/P-12 or the chimeric vR26_E2gif/P-12 were each detected by the polyclonal anti-pestivirus serum as expected. The anti-CSFV-E2 mAb specifically detected cells infected with vR26/P-12 but not cells infected by the chimeric virus containing the BDV-E2 protein (consistent with the results shown in Figure 2 ). In contrast, the anti-BDV-E2 mAb specifically detected infection by the vR26_E2gif/P-12 and did not recognize cells infected with vR26/P-12. Each result is in accord with the structure of the viruses. The 4th passage of vR26 (vR26/P-4) displayed a slower growth rate than the virus obtained after 12", "passages (see Figure 5A ). It also had a reduced growth rate compared to both the vR26_E2gif/P-4 and vR26_E2gif/P-12. The fulllength sequence of pBeloR26 had revealed ten non-silent mutations compared to the reference sequence (AY25 9122.1) for this virus (Additional file 2: Table S2 ). Any of these mutations could be responsible for the impaired growth acting alone or in concert. For further investigation of this issue, full length cDNAs prepared from vR26/ P-4, vR26/P-12, vR26_E2gif/P-4 and vR26_E2gif/P-12 were deep-sequenced using both the 454 FLX and Ion PGM platforms for comparison and to determine the quasispecies distribution (Additional file 3: Figure S1 and Additional file 4: Figure S2 ). Sequencing data from both platforms revealed that both the vR26/P-12 and vR26_E2gif/P-12 were close to 100% changed at nt position A10665G compared to the BAC clones (resulting in the predicted amino acid substitution D3431G within the NS5B protein, the RNAdependent RNA polymerase, see", "Figure 5B ). This adaptation is a reversion back to the consensus cDNA sequence of the parental vaccine virus, vRiemser (Additional file 2: Table S2 ). Additionally, vR26/P-4 and vR26_E2gif/P-4 already showed evidence for this reversion being present within the population. For vR26/P-4, the level of reversion was 57%, while for vR26_E2gif/P-4 the extent of change was 73% (see Figure 5B ).", "In this study, we have established the first BAC containing the full-length cDNA of a CSFV vaccine strain. The BAC differed from the parental cDNA sequence in 18 positions leading to 9 aa substitutions ( Table 1 ). The method that has been used for the generation of pBeloR26 is based on full genome amplification of cDNA followed by direct cloning to obtain the BACs [11] . This approach results in cDNA clones that reflect the quasispecies composition of the parental viral RNA and thus it is not guaranteed to obtain cDNA clones corresponding to the consensus sequence of the cDNA used. However, it is possible to correct the mutations using the BAC recombination approach if a consensus clone is needed. To demonstrate the utility of the Red/ET mediated recombination method we have generated a series of modified BACs derived from this CSFV full-length cDNA. These include BACs with substitution of the linear TAV-epitope present in the E2 protein and also BACs with substitution of the", "complete E2 protein with heterologous pestivirus sequences. We have also used the same approach for a range of different targeted modifications within CSFV BACs including specific deletions and substitutions in the 5′UTR of CSFV [24] and for insertions of heterologous reporter sequences into CSFV replicons [25] . Using Red/ET recombinationmediated mutagenesis for the targeted design, the work can be expedited and focused, in principal, on any sequence within the viral genome and is not dependent on the use of internal restriction sites. The results demonstrate that Red/ ET recombination-mediated mutagenesis of pestivirus BAC cDNAs provides a useful tool for advancing the construction of modified pestiviruses. Cells infected with the parental vR26 virus were recognized by the two anti-E2 mAbs (WH211 and WH303) specific for the CSFV-E2 proteins, in contrast cells infected with the modified viruses vR26_TAV and vR26_E2gif, rescued from the recombined BACs, were not detected by these", "mAbs. Furthermore, as expected, cells infected with the vR26_E2gif were recognized by the anti-BDV mAb (WB166) whereas no staining was observed with this antibody in vR26 infected cells or in cells with vR26_TAV. The mAb WH303 recognizes the CSFV TAV-epitope [18] and the difference in 4 aa between the TAV-epitope and the corresponding sequence from BDV strain \"Gifhorn\" is enough to completely abolish the recognition by this mAb. The lack of staining of vR26_TAV infected cells by the WH211 indicated that the TAV-sequence is also important for the epitope recognized by this mAb. Thus, the chimeric pestiviruses, vR26_TAV and vR26_E2gif, containing heterologous E2 sequences can be readily discriminated from the vR26 using specific anti-E2 monoclonal antibodies. These new chimeric pestiviruses represents Cstrain based marked vaccine candidates with the characteristics desired for safe and efficacious DIVA vaccines against CSFV. Indeed, vR26_E2gif vaccinated pigs could be efficiently", "discriminated from C-strain vaccinated pigs and from CSFV infected pigs using CSFV-E2 specific antibody ELISAs (Rasmussen et al., unpublished results).", "Nucleotide sequence data for the pBeloR26 showed a number of changes from the published reference sequence for \"C-strain Riems\". Some of these differences are present in the cDNA derived from the vaccine stock at a detectable level whereas others may represent low-level variants within the cDNA or errors introduced by the RT-PCR amplification. Full-length sequencing revealed that no changes occurred in the cDNA during extensive propagation in E. coli DH10B of the pBeloR26 and the E2chimeric derivative, pBeloR26_E2gif, indicating a very high stability of these BAC-cloned CSFV cDNAs. This is essential if this system is to be useful for cloning and sequence manipulation, and contrasts with stability problems encountered with conventional plasmids containing fulllength pestivirus cDNAs [31] . The stability of these BACs is consistent with previous reports on the stability of BACs containing other viruses of the family Flaviviridae in E. coli [8, 10] .", "Extensive passaging of the rescued vR26 and the chimeric virus derivative, vR26_E2gif, resulted in a change at nucleotide position A10665G (resulting in the predicted aa" ]

[ 8 ]

[ "to examine whether a relationship exists between operation volumes and SSIs" ]

[ "Which Kind of Provider’s Operation Volumes Matters? Associations between CABG Surgical Site Infection Risk and Hospital and Surgeon Operation Volumes among Medical Centers in Taiwan\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4459823/\n\nSHA: f3cbc0503581249a834895fc94cd3bae24714a0d\n\nAuthors: Yu, Tsung-Hsien; Tung, Yu-Chi; Chung, Kuo-Piao\nDate: 2015-06-08\nDOI: 10.1371/journal.pone.0129178\nLicense: cc-by", "Abstract: BACKGROUND: Volume-infection relationships have been examined for high-risk surgical procedures, but the conclusions remain controversial. The inconsistency might be due to inaccurate identification of cases of infection and different methods of categorizing service volumes. This study takes coronary artery bypass graft (CABG) surgical site infections (SSIs) as an example to examine whether a relationship exists between operation volumes and SSIs, when different SSIs case identification, definitions and categorization methods of operation volumes were implemented. METHODS: A population-based cross-sectional multilevel study was conducted. A total of 7,007 patients who received CABG surgery between 2006 and 2008 from19 medical centers in Taiwan were recruited. SSIs associated with CABG surgery were identified using International Classification of Diseases, 9th Revision, Clinical Modification (ICD-9 CM) codes and a Classification and Regression Trees (CART) model. Two", "definitions of surgeon and hospital operation volumes were used: (1) the cumulative CABG operation volumes within the study period; and (2) the cumulative CABG operation volumes in the previous one year before each CABG surgery. Operation volumes were further treated in three different ways: (1) a continuous variable; (2) a categorical variable based on the quartile; and (3) a data-driven categorical variable based on k-means clustering algorithm. Furthermore, subgroup analysis for comorbidities was also conducted. RESULTS: This study showed that hospital volumes were not significantly associated with SSIs, no matter which definitions or categorization methods of operation volume, or SSIs case identification approaches were used. On the contrary, the relationships between surgeon’s volumes varied. Most of the models demonstrated that the low-volume surgeons had higher risk than high-volume surgeons. CONCLUSION: Surgeon volumes were more important than hospital volumes in exploring the", "relationship between CABG operation volumes and SSIs in Taiwan. However, the relationships were not robust. Definitions and categorization methods of operation volume and correct identification of SSIs are important issues for future research.", "Text: data, which should use hierarchical models, may result in biased estimation of the variation and also lead to incorrect conclusions.\n\nSSIs following coronary artery bypass graft (CABG) procedures place a heavy burden on patients and healthcare systems. The total length of stay and expenditure for patients with SSIs after CABG surgery is significantly longer and higher than those without SSIs. [20, 21] In 2008, the Centers for Medicare & Medicaid of the United States of America implemented the \"Never Event\" policy, where hospitals would no longer receive higher payments for the additional costs associated with treating patients for certain healthcare-acquired infections, including those related to CABG.", "In view of the accuracy of SSIs identification and the heterogeneity of definition and categorization methods, no existing studies have used different infection case identification nor definitions and categorization methods of operation volume simultaneously to explore the relationship between operation volumes and infection. The current study takes CABG SSIs as an example to examine whether a relationship exists between operation volumes and SSIs, given different SSI cases identification, operation volume definitions and categorization methods.\n\nThis retrospective and cross-sectional study adopted a multilevel design to examine the relationships between provider volumes and SSIs after adjusting for patient-, surgeon-, and hospital-level covariates.", "We used data from the Taiwan National Health Insurance Research Database (NHIRD) from 2005 and 2008. The NHIRD, published by the Taiwan National Health Research Institute, includes all the original claims data and registration files for beneficiaries enrolled under the National Health Insurance (NHI) program. The database covers the 23 million Taiwanese enrollees (approximately 98% of the population) in the NHI program. It is a de-identified secondary database containing patient-level demographic and administrative information; however, treatment items are aggregated and without time-related and clinical information. The data is released for research purposes.\n\nThe protocol for the study was approved by the Institutional Review Board of the National Taiwan University Hospital (protocol #201001027R). The dataset we used in this study was secondary data; all information was de-identified by data owners.", "In this study, we adopted the ICD-9-CM SSI codes (hereafter referred to as the ICD-9-CM based model) and the Classification and Regression Trees (CART) model, which was developed in our previous work [11] to identify SSI cases. As we mentioned above, the ICD-9-CM SSI codes were the most popular tool to identify the SSI cases in claims data. In the ICD-9-CM based model, SSI cases were divided into two categories: index hospitalization events and post-discharge events (i.e., SSIs that occurred within 1 year after discharge and required readmission to a hospital and/ or the use of ambulatory services). Following Wu et al [13] , this study adopted the secondary ICD-9-CM diagnosis codes for index hospitalization events (ICD-9-CM code: 996.03, 996.61, 996.72, and 998.5), and the primary and secondary diagnosis codes for post-discharge events (ICD-9-CM code: 038.0-038. 4 ) as the criteria for SSI identification, in order to avoid cases in which infection existed prior to hospitalization. If", "a case had an index hospitalization event or a post-discharge event, then he/ she will be identified as SSIs by the ICD-9-CM based model. In the CART model, we adopted the type of antibiotics, dose of cefazolin, length of stay, and number of vessels obstructed (as a proxy indicator of duration of operation) as the parameters to identify the SSIs, according to our previous findings. [11] In our previous work, we used the 2005-2008 National Health Insurance claims data and healthcare-associated infection surveillance data from two medical centers for model development and model verification. Infection cases based on surveillance were identified by infection control personnel if the patient met the Taiwan CDC's criteria, which are the same as those adopted in the U.S. CDC. They manually review medical records of all patients at risk for the specified healthcare-associated infection.", "The classification algorithms, the multivariable regression model, and the data mining model were adopted to develop alternative models based on surrogate indicators to identify cases of CABG SSIs and to compare the performance among these models and the ICD-9-CMbased model. For the classification algorithms, researchers build up several criteria, and if a case satisfies (or exceeds) a specific number of criteria, then it will be identified as a case of infection. For the multivariable regression model, researchers usually calculated a risk score by the logistic regression model, and the optimal cutoff point was determined according to the resulting receiver operating characteristic curve.", "Concerning the data mining approach, which is widely used for predicting and classifying objects, the characteristics are: automatic discovery of patterns, prediction of likely outcomes, creation of actionable information, and focus on large data sets and databases. The classification and regression tree (CART) model, which is the most popular approach as applied in our work, and the growing, stopping, and pruning of the tree were determined by Gini improvement measures. [22, 23] After referring to the literature and conferring with infectious disease specialists, we adopted the following seven parameters: type of antibiotic, doses of antibiotic, doses of cefazolin, use of second-line antibiotics, length of stay, and number of vessels obstructed. Additionally, cross-validation was also employed, where data from one medical center was used for model development, and another one was used for model validation.", "The results of our previous work revealed that the CART model offered better performance than that of the other identification models or the ICD-9-CM based model, especially in the positive predictive value (>70%), which was only found to be 20% in the ICD-9-CM based model. (Table 1 ) The findings also implied that the CART was a decidedly better tool for identifying cases of SSI in the Taiwan National Health Insurance database. Therefore, this study also adopted the CART model for identifying CABG SSIs.\n\nTo ensure homogeneity, current study analyzed 7,007 patients from 19 medical centers in Taiwan who underwent CABG surgery (ICD-9-CM procedure codes 36.1x-36.2x) between 2006 and 2008. CABG patients under the age of 18 years or over 85 years were excluded in this study. A total of 302 cases were identified as SSIs by ICD-9-CM based model, and a total of 107 cases were identified as SSIs by CART model.", "In this study, we used the following two definitions to define operation volumes: (1) the cumulative operation volumes by each surgeon and hospital within the study period, which was the most common definition in the literature; and (2) following Yasunaga et al.'s study, [24] cumulative operation volumes by each surgeon and hospital in the previous one year for each surgery. However, our data was skewed, which did not follow a normal distribution. Therefore, we conducted the log transformations on operation volumes.", "The current work treated operation volumes in three different ways: (1) a continuous variable; (2) a categorical variable based on the first and the third quartile as cutoff points (the most common method to categorize service/ operation volumes) [25] [26] [27] [28] ; and (3) a data-driven categorical variable based on k-means clustering algorithm. This study categorized surgeon and hospital volumes into low, medium, and high volume groups by quartile method and kmeans clustering algorithm.", "In the quartile method, the cut-off value (transformed by logarithm) of the first quartile (<25%) for hospital volumes was 5.65, and the third quartile (>75%) was 6.43. In terms of surgeon volumes, the first quartile was 4.38, and the third was 5.35, when we used the cumulative operation volumes within the study period as the definition. While the definition changed, first quartile (<25%) for hospital volumes was 4.66, and the third quartile (>75%) was 5.31. In terms of surgeon volumes, the first quartile was 3.40, and the third was 4.32.", "K-means clustering is an unsupervised machine-learning algorithm introduced by MacQueen in 1960s. This method is not only a simple and very reliable method in categorization/ classification, but is also recognized as one of the top 10 algorithms in data mining. [29] This method has often been applied in many fields. [30] [31] [32] Yu and his colleagues even applied it to define the quality of CABG care, and to explore the relationship among patient's income status, the level of quality of care, and inpatient mortality. [33] The main idea of this method is to partition observed data points into k non-overlapping clusters by minimizing the within-group sum of squares. Each point is assigned to the mean of its cluster using the Euclidian distance. Firstly, k cluster centers were randomly generated. Previous studies usually divided surgeons and hospitals into low-, medium-, and high-volume groups; therefore, we also predetermined the surgeon and hospital service volumes into 3 groups (k =", "3). Then, participants were assigned to the cluster with the shortest distance to these cluster centers. Finally, the cluster centers were recomputed using the new cluster assignment and these steps would be iterated until convergence was achieved. [34] The cut-off values of hospital volumes were 5.21 and 5.69, and for surgeon's volumes were 2.40 and 4.38 respectively, when cumulative operation volumes within the study period was used as the definition. Likewise, when cumulative operation volumes before each surgery was used as definition, the cut-off values were 4.11 and 4.89 for hospital volumes, and 2.64 and 3.91 for surgeon's volumes. All cutoff values were transformed by logarithm. The results of k-means clustering are demonstrated in Figs 1-4. As the results show, the operation volumes were divided into three groups separately. In addition to surgeon and hospital volumes and SSI, we collected patient-, surgeon-, and hospital-level data. Firstly, patient-level variables included", "age, gender, length of ICU stay, number of vessels obstructed that were involved in the surgical operation, and the presence of important underlying diseases (e.g. diabetes mellitus, chronic obstructive pulmonary disease (COPD), heart failure, renal failure and renal insufficiency, which were associated with SSI).", "[13] Secondly, the surgeon-level variables included age and gender. Thirdly, the hospital-level variables included hospital ownership and geographic location.", "All statistical analyses of volume-infection relationship were performed using SAS (version 9.2, SAS Institution Inc., Cary, NC, USA). In statistical testing, a two-sided p value 0.05 was considered statistically significant. The distributional properties of continuous variables were expressed by mean ± standard deviation (SD), whereas categorical variables were presented by frequency and percentage. In univariate analysis, the potential three-level predictors of SSI were examined using chi-square test or two-sample t-test as appropriate. Next, to account for the correlations within surgeon (level-2) and hospital (level-3), multivariate analysis was conducted by fitting mixed-effects logistic regression models to each patient's data for estimating the effects of three-level predictors on the probability of post-operational SSI. Furthermore, subgroup analysis for comorbidities was also conducted. Table 2 shows that there were 7,007 patients with CABG performed by 199 surgeons in 19", "hospitals during 2006-2008 in Taiwan. The majority of patients were male (77.5%), and the mean age of patients was 65.3 years. The average ICU stay was 6.05 days, the mean level of number of vessels obstructed was around 1.6, while 51.8% of patients had diabetes mellitus, 33.3% had heart failure, 14.1% had renal failure and renal insufficiency, and 22.0% had COPD. Three hundred and two patients (4.31%) were identified as having the ICD-9-CM SSI codes. However, identification by the CART model only revealed 107 infection cases, and 94 cases were identified in both models. Most cases received CABG surgery by male surgeons, with a mean age of 45.0 years, and the surgeon's average operation volumes within the study period was 151.64, while the average operation volumes before surgery was 52.18. More than half of the cases were performed with CABG in not-for-profit hospitals, and the hospitals' average operation volumes within the study period was 473.60, while the average operation", "volumes before each surgery was 158.79. Moreover, most of patients received their surgeries by high-volume surgeons and hospitals, when k-means algorithm was used for categorization, regardless of which definition of operation volumes were used. Table 3 shows the results of multilevel mixed-effect models, with the SSIs being identified by ICD-9-CM codes, and the operation volumes defined as the cumulative volumes within the study period. The results of Model 1 (continuous) reveal that the surgeon's volumes were negatively associated with SSIs, while hospital's volumes were not associated with surgical site infection SSIs. Model 2 (quartile) suggests that low-volume surgeons had higher SSI risk (OR = 2.220, p-value = 0.022) than high-volume surgeons. There were also no associations between hospital's operation volumes and SSIs. Model 3 (k-means) shows that the association did not exist between hospital's/ surgeon's volumes and SSIs. Table 4 displays the results of multilevel", "mixed-effect models, in which the SSIs were identified by the CART model, and the operation volumes were also defined as the cumulative volumes within the study period. Model 1 again indicated a negative association between surgeon's volumes and SSIs, and hospital's volumes were not found to be associated with SSIs. In Model 2, the results showed that the relationship between hospital's/ surgeon's volumes and SSIs did not exist. In Model 3, results revealed low-volume surgeons had higher risk (OR = 1.691, p = 0.002) than high-volume surgeons. Table 5 displays the results of multilevel mixed-effect models, in which the SSIs were identified by ICD-9-CM codes, but the operation volumes were defined as the cumulative volume in the previous one year for each surgery. Model 1 also indicated a negative association between surgeon's volumes and SSIs, and hospital's volumes were not found to be associated with SSIs. In Model 2, the results showed that the relationship between hospital's/", "surgeon's volumes and SSIs did not exist. In Model 3, results also revealed low-volume surgeons had higher risk (OR = 1.642, p = 0.040) than high-volume surgeons. Table 6 displays the results of multilevel mixed-effect models, in which the SSIs were identified by the CART model, and the operation volumes were also defined as the cumulative volume in previous one year for each surgery. In Model 1, different to the above findings, there was no association between hospital's/ surgeon's volumes and SSIs. In Model 2, the results showed that the relationship between hospital's/ surgeon's volumes and SSIs did not exist. In Model 3, results also revealed low-volume surgeons had higher risk (OR = 1.163, p = 0.020) than high-volume surgeons.", "We further examined the associations of surgeon and hospital volumes with SSIs in stratification analyses by underlying diseases. When the operation volumes were defined as the cumulative operation volume within the study period, no relationships existed between hospital/ surgeon operation volumes and SSIs. (Table 7 ) However, when the operation volumes were defined as the cumulative operation volumes in the previous one year for each surgery, the results suggested that there was a negative association between surgeon volumes and SSIs in the diabetes group, except that the volumes were treated as continuous variable and the infection cases were identified by ICD-9 codes. In terms of hospital operation volumes, the association did not exist. (Table 8 )", "No studies have evaluated how different service/ operation volumes definitions and categorization methods affect volume-infection relationships. Moreover, several studies have pointed out the inappropriateness of identifying infection cases using the ICD-9-CM codes in claims data. Given these reasons, this study adopted two approaches to identifying SSIs, two definitions of operation volumes, and three methods for categorizing operation volumes to examine the relationships between operation volumes and SSIs. Our findings showed that the relationships between hospital volumes and SSIs did not exist, no matter which definitions, categorization mehods, or SSIs case identification approaches were used. On the contrary, the relationships between surgeon volumes and SSIs were not robust in our data. It might be affected by different definitions and categorization methods of operation volumes, and also by different SSI cases identification approaches. In summary, most of the models", "demonstrated that the low-volume surgeons had higher risk than high-volume surgeons, and they also showed the risks were similar between medium-volume and high-volume surgeons. However, why did surgeon volume relate to SSIs, but hospital volume did not? Except for those issues we were concerned about in this study, there are some disagreements in the literature. Such as \"Does provider volume really represent quality of care?\" [12, 35] Or \"Is provider volume the only one predictor for outcome of care?\" [35, 36] These issues are worthy of further discussion, but are out of the scope of this study.", "Service/ operation volumes are treated as a proxy indicator for experiences; previous studies used it to examine whether practice makes perfect or not. But, except for provider's experiences, SSIs are also impacted by many factors, such as environmental and clinical factors. Wu et al once used Taiwan 2001 NHI claims data to explore the relationship between provider CABG operation volumes and SSIs. [13] They found that hospital volumes had a greater effect than surgeon volumes and claimed that this may imply that hospital teamwork is more important than individual surgeon. However, our findings demonstrated that there was no relationship between hospital volumes and SSIs. Wu et al. adopted the cumulative operation volumes within the study period as the definition, and identified SSIs by ICD-9-CM codes. Except, there were two differences between our work and Wu et al., which were the length and year of the data; our data was longer and more updated than theirs. Moreover, it is worth", "noting that there was an outbreak of severe acute respiratory syndrome (SARS) in Taiwan in 2003, after which the hospital infection control system in Taiwan was reviewed and re-designed. Wu et al data was before SARS, so these efforts may also have improved the level of SSIs control in hospitals, leading to different findings in this study.", "In addition, although most models revealed that there were negative relationships between surgeon's volumes and surgical site infection, the relationships were not robust. The results varied between different definitions and categorization method of operation volumes, and between SSIs identification approaches. Researchers need to consider how to identify SSIs correctly, how to choose optimal cut-off values, and how to decide on which definition is appropriate.\n\nFinally, the results of stratification analyses showed that low-volume surgeon had higher risk than high-volume surgeon in the diabetes mellitus group, when the cumulative operation in the previous one year before surgery was used as definition. A large number of studies have indicated diabetes mellitus is associated with a higher risk of SSIs, [37] [38] [39] and the findings of this study suggest that CABG patients with diabetes mellitus should be cared for by experienced surgeons.", "A multilevel analysis was applied to manage the nested factors, and two definitions of operation volume along with three different operation volume categorization methods were adopted to examine the relationship between volume and SSIs under two kinds of SSIs identification approaches. Nevertheless, the study suffered from several major limitations. First, the accuracy of SSIs identification was still an issue. Although the performance of the CART model to identify CABG SSIs was better than ICD-9-CM codes in Taiwan NHI claims data, it did not reach the perfect scenario. The accuracy of SSIs identification was still a challenge in our work. The second limitation relates to unmeasured variables, such as length of stay before operation, infection condition, hair removal, clinical information (e.g. blood glucose level, causative microorganism), time-related information (e.g. the duration of operation), the environment, surgical skills, use of post-operative drains, number of operations", "involved, and surgical site and wound care, etc. [40] Furthermore, information about type (elective or urgent) and incision site for surgery was not available in the Taiwan NHI claims data.", "In conclusion, the findings of this study suggest that different definitions and categorization methods of operation volumes, and different SSIs identification approaches might lead to different findings, although surgeon volumes were more important than hospital volumes in exploring the relationships between CABG operation volumes and SSIs in Taiwan, but they were still not robust. Definitions and categorization methods of operation volumes, and correct identification of SSIs are important issues for future research." ]

[ 1 ]

[ "The total length of stay and expenditure for patients with SSIs after CABG surgery is significantly longer and higher than those without SSIs" ]

[ "Which Kind of Provider’s Operation Volumes Matters? Associations between CABG Surgical Site Infection Risk and Hospital and Surgeon Operation Volumes among Medical Centers in Taiwan\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4459823/\n\nSHA: f3cbc0503581249a834895fc94cd3bae24714a0d\n\nAuthors: Yu, Tsung-Hsien; Tung, Yu-Chi; Chung, Kuo-Piao\nDate: 2015-06-08\nDOI: 10.1371/journal.pone.0129178\nLicense: cc-by", "Abstract: BACKGROUND: Volume-infection relationships have been examined for high-risk surgical procedures, but the conclusions remain controversial. The inconsistency might be due to inaccurate identification of cases of infection and different methods of categorizing service volumes. This study takes coronary artery bypass graft (CABG) surgical site infections (SSIs) as an example to examine whether a relationship exists between operation volumes and SSIs, when different SSIs case identification, definitions and categorization methods of operation volumes were implemented. METHODS: A population-based cross-sectional multilevel study was conducted. A total of 7,007 patients who received CABG surgery between 2006 and 2008 from19 medical centers in Taiwan were recruited. SSIs associated with CABG surgery were identified using International Classification of Diseases, 9th Revision, Clinical Modification (ICD-9 CM) codes and a Classification and Regression Trees (CART) model. Two", "definitions of surgeon and hospital operation volumes were used: (1) the cumulative CABG operation volumes within the study period; and (2) the cumulative CABG operation volumes in the previous one year before each CABG surgery. Operation volumes were further treated in three different ways: (1) a continuous variable; (2) a categorical variable based on the quartile; and (3) a data-driven categorical variable based on k-means clustering algorithm. Furthermore, subgroup analysis for comorbidities was also conducted. RESULTS: This study showed that hospital volumes were not significantly associated with SSIs, no matter which definitions or categorization methods of operation volume, or SSIs case identification approaches were used. On the contrary, the relationships between surgeon’s volumes varied. Most of the models demonstrated that the low-volume surgeons had higher risk than high-volume surgeons. CONCLUSION: Surgeon volumes were more important than hospital volumes in exploring the", "relationship between CABG operation volumes and SSIs in Taiwan. However, the relationships were not robust. Definitions and categorization methods of operation volume and correct identification of SSIs are important issues for future research.", "Text: data, which should use hierarchical models, may result in biased estimation of the variation and also lead to incorrect conclusions.\n\nSSIs following coronary artery bypass graft (CABG) procedures place a heavy burden on patients and healthcare systems. The total length of stay and expenditure for patients with SSIs after CABG surgery is significantly longer and higher than those without SSIs. [20, 21] In 2008, the Centers for Medicare & Medicaid of the United States of America implemented the \"Never Event\" policy, where hospitals would no longer receive higher payments for the additional costs associated with treating patients for certain healthcare-acquired infections, including those related to CABG.", "In view of the accuracy of SSIs identification and the heterogeneity of definition and categorization methods, no existing studies have used different infection case identification nor definitions and categorization methods of operation volume simultaneously to explore the relationship between operation volumes and infection. The current study takes CABG SSIs as an example to examine whether a relationship exists between operation volumes and SSIs, given different SSI cases identification, operation volume definitions and categorization methods.\n\nThis retrospective and cross-sectional study adopted a multilevel design to examine the relationships between provider volumes and SSIs after adjusting for patient-, surgeon-, and hospital-level covariates.", "We used data from the Taiwan National Health Insurance Research Database (NHIRD) from 2005 and 2008. The NHIRD, published by the Taiwan National Health Research Institute, includes all the original claims data and registration files for beneficiaries enrolled under the National Health Insurance (NHI) program. The database covers the 23 million Taiwanese enrollees (approximately 98% of the population) in the NHI program. It is a de-identified secondary database containing patient-level demographic and administrative information; however, treatment items are aggregated and without time-related and clinical information. The data is released for research purposes.\n\nThe protocol for the study was approved by the Institutional Review Board of the National Taiwan University Hospital (protocol #201001027R). The dataset we used in this study was secondary data; all information was de-identified by data owners.", "In this study, we adopted the ICD-9-CM SSI codes (hereafter referred to as the ICD-9-CM based model) and the Classification and Regression Trees (CART) model, which was developed in our previous work [11] to identify SSI cases. As we mentioned above, the ICD-9-CM SSI codes were the most popular tool to identify the SSI cases in claims data. In the ICD-9-CM based model, SSI cases were divided into two categories: index hospitalization events and post-discharge events (i.e., SSIs that occurred within 1 year after discharge and required readmission to a hospital and/ or the use of ambulatory services). Following Wu et al [13] , this study adopted the secondary ICD-9-CM diagnosis codes for index hospitalization events (ICD-9-CM code: 996.03, 996.61, 996.72, and 998.5), and the primary and secondary diagnosis codes for post-discharge events (ICD-9-CM code: 038.0-038. 4 ) as the criteria for SSI identification, in order to avoid cases in which infection existed prior to hospitalization. If", "a case had an index hospitalization event or a post-discharge event, then he/ she will be identified as SSIs by the ICD-9-CM based model. In the CART model, we adopted the type of antibiotics, dose of cefazolin, length of stay, and number of vessels obstructed (as a proxy indicator of duration of operation) as the parameters to identify the SSIs, according to our previous findings. [11] In our previous work, we used the 2005-2008 National Health Insurance claims data and healthcare-associated infection surveillance data from two medical centers for model development and model verification. Infection cases based on surveillance were identified by infection control personnel if the patient met the Taiwan CDC's criteria, which are the same as those adopted in the U.S. CDC. They manually review medical records of all patients at risk for the specified healthcare-associated infection.", "The classification algorithms, the multivariable regression model, and the data mining model were adopted to develop alternative models based on surrogate indicators to identify cases of CABG SSIs and to compare the performance among these models and the ICD-9-CMbased model. For the classification algorithms, researchers build up several criteria, and if a case satisfies (or exceeds) a specific number of criteria, then it will be identified as a case of infection. For the multivariable regression model, researchers usually calculated a risk score by the logistic regression model, and the optimal cutoff point was determined according to the resulting receiver operating characteristic curve.", "Concerning the data mining approach, which is widely used for predicting and classifying objects, the characteristics are: automatic discovery of patterns, prediction of likely outcomes, creation of actionable information, and focus on large data sets and databases. The classification and regression tree (CART) model, which is the most popular approach as applied in our work, and the growing, stopping, and pruning of the tree were determined by Gini improvement measures. [22, 23] After referring to the literature and conferring with infectious disease specialists, we adopted the following seven parameters: type of antibiotic, doses of antibiotic, doses of cefazolin, use of second-line antibiotics, length of stay, and number of vessels obstructed. Additionally, cross-validation was also employed, where data from one medical center was used for model development, and another one was used for model validation.", "The results of our previous work revealed that the CART model offered better performance than that of the other identification models or the ICD-9-CM based model, especially in the positive predictive value (>70%), which was only found to be 20% in the ICD-9-CM based model. (Table 1 ) The findings also implied that the CART was a decidedly better tool for identifying cases of SSI in the Taiwan National Health Insurance database. Therefore, this study also adopted the CART model for identifying CABG SSIs.\n\nTo ensure homogeneity, current study analyzed 7,007 patients from 19 medical centers in Taiwan who underwent CABG surgery (ICD-9-CM procedure codes 36.1x-36.2x) between 2006 and 2008. CABG patients under the age of 18 years or over 85 years were excluded in this study. A total of 302 cases were identified as SSIs by ICD-9-CM based model, and a total of 107 cases were identified as SSIs by CART model.", "In this study, we used the following two definitions to define operation volumes: (1) the cumulative operation volumes by each surgeon and hospital within the study period, which was the most common definition in the literature; and (2) following Yasunaga et al.'s study, [24] cumulative operation volumes by each surgeon and hospital in the previous one year for each surgery. However, our data was skewed, which did not follow a normal distribution. Therefore, we conducted the log transformations on operation volumes.", "The current work treated operation volumes in three different ways: (1) a continuous variable; (2) a categorical variable based on the first and the third quartile as cutoff points (the most common method to categorize service/ operation volumes) [25] [26] [27] [28] ; and (3) a data-driven categorical variable based on k-means clustering algorithm. This study categorized surgeon and hospital volumes into low, medium, and high volume groups by quartile method and kmeans clustering algorithm.", "In the quartile method, the cut-off value (transformed by logarithm) of the first quartile (<25%) for hospital volumes was 5.65, and the third quartile (>75%) was 6.43. In terms of surgeon volumes, the first quartile was 4.38, and the third was 5.35, when we used the cumulative operation volumes within the study period as the definition. While the definition changed, first quartile (<25%) for hospital volumes was 4.66, and the third quartile (>75%) was 5.31. In terms of surgeon volumes, the first quartile was 3.40, and the third was 4.32.", "K-means clustering is an unsupervised machine-learning algorithm introduced by MacQueen in 1960s. This method is not only a simple and very reliable method in categorization/ classification, but is also recognized as one of the top 10 algorithms in data mining. [29] This method has often been applied in many fields. [30] [31] [32] Yu and his colleagues even applied it to define the quality of CABG care, and to explore the relationship among patient's income status, the level of quality of care, and inpatient mortality. [33] The main idea of this method is to partition observed data points into k non-overlapping clusters by minimizing the within-group sum of squares. Each point is assigned to the mean of its cluster using the Euclidian distance. Firstly, k cluster centers were randomly generated. Previous studies usually divided surgeons and hospitals into low-, medium-, and high-volume groups; therefore, we also predetermined the surgeon and hospital service volumes into 3 groups (k =", "3). Then, participants were assigned to the cluster with the shortest distance to these cluster centers. Finally, the cluster centers were recomputed using the new cluster assignment and these steps would be iterated until convergence was achieved. [34] The cut-off values of hospital volumes were 5.21 and 5.69, and for surgeon's volumes were 2.40 and 4.38 respectively, when cumulative operation volumes within the study period was used as the definition. Likewise, when cumulative operation volumes before each surgery was used as definition, the cut-off values were 4.11 and 4.89 for hospital volumes, and 2.64 and 3.91 for surgeon's volumes. All cutoff values were transformed by logarithm. The results of k-means clustering are demonstrated in Figs 1-4. As the results show, the operation volumes were divided into three groups separately. In addition to surgeon and hospital volumes and SSI, we collected patient-, surgeon-, and hospital-level data. Firstly, patient-level variables included", "age, gender, length of ICU stay, number of vessels obstructed that were involved in the surgical operation, and the presence of important underlying diseases (e.g. diabetes mellitus, chronic obstructive pulmonary disease (COPD), heart failure, renal failure and renal insufficiency, which were associated with SSI).", "[13] Secondly, the surgeon-level variables included age and gender. Thirdly, the hospital-level variables included hospital ownership and geographic location.", "All statistical analyses of volume-infection relationship were performed using SAS (version 9.2, SAS Institution Inc., Cary, NC, USA). In statistical testing, a two-sided p value 0.05 was considered statistically significant. The distributional properties of continuous variables were expressed by mean ± standard deviation (SD), whereas categorical variables were presented by frequency and percentage. In univariate analysis, the potential three-level predictors of SSI were examined using chi-square test or two-sample t-test as appropriate. Next, to account for the correlations within surgeon (level-2) and hospital (level-3), multivariate analysis was conducted by fitting mixed-effects logistic regression models to each patient's data for estimating the effects of three-level predictors on the probability of post-operational SSI. Furthermore, subgroup analysis for comorbidities was also conducted. Table 2 shows that there were 7,007 patients with CABG performed by 199 surgeons in 19", "hospitals during 2006-2008 in Taiwan. The majority of patients were male (77.5%), and the mean age of patients was 65.3 years. The average ICU stay was 6.05 days, the mean level of number of vessels obstructed was around 1.6, while 51.8% of patients had diabetes mellitus, 33.3% had heart failure, 14.1% had renal failure and renal insufficiency, and 22.0% had COPD. Three hundred and two patients (4.31%) were identified as having the ICD-9-CM SSI codes. However, identification by the CART model only revealed 107 infection cases, and 94 cases were identified in both models. Most cases received CABG surgery by male surgeons, with a mean age of 45.0 years, and the surgeon's average operation volumes within the study period was 151.64, while the average operation volumes before surgery was 52.18. More than half of the cases were performed with CABG in not-for-profit hospitals, and the hospitals' average operation volumes within the study period was 473.60, while the average operation", "volumes before each surgery was 158.79. Moreover, most of patients received their surgeries by high-volume surgeons and hospitals, when k-means algorithm was used for categorization, regardless of which definition of operation volumes were used. Table 3 shows the results of multilevel mixed-effect models, with the SSIs being identified by ICD-9-CM codes, and the operation volumes defined as the cumulative volumes within the study period. The results of Model 1 (continuous) reveal that the surgeon's volumes were negatively associated with SSIs, while hospital's volumes were not associated with surgical site infection SSIs. Model 2 (quartile) suggests that low-volume surgeons had higher SSI risk (OR = 2.220, p-value = 0.022) than high-volume surgeons. There were also no associations between hospital's operation volumes and SSIs. Model 3 (k-means) shows that the association did not exist between hospital's/ surgeon's volumes and SSIs. Table 4 displays the results of multilevel", "mixed-effect models, in which the SSIs were identified by the CART model, and the operation volumes were also defined as the cumulative volumes within the study period. Model 1 again indicated a negative association between surgeon's volumes and SSIs, and hospital's volumes were not found to be associated with SSIs. In Model 2, the results showed that the relationship between hospital's/ surgeon's volumes and SSIs did not exist. In Model 3, results revealed low-volume surgeons had higher risk (OR = 1.691, p = 0.002) than high-volume surgeons. Table 5 displays the results of multilevel mixed-effect models, in which the SSIs were identified by ICD-9-CM codes, but the operation volumes were defined as the cumulative volume in the previous one year for each surgery. Model 1 also indicated a negative association between surgeon's volumes and SSIs, and hospital's volumes were not found to be associated with SSIs. In Model 2, the results showed that the relationship between hospital's/", "surgeon's volumes and SSIs did not exist. In Model 3, results also revealed low-volume surgeons had higher risk (OR = 1.642, p = 0.040) than high-volume surgeons. Table 6 displays the results of multilevel mixed-effect models, in which the SSIs were identified by the CART model, and the operation volumes were also defined as the cumulative volume in previous one year for each surgery. In Model 1, different to the above findings, there was no association between hospital's/ surgeon's volumes and SSIs. In Model 2, the results showed that the relationship between hospital's/ surgeon's volumes and SSIs did not exist. In Model 3, results also revealed low-volume surgeons had higher risk (OR = 1.163, p = 0.020) than high-volume surgeons.", "We further examined the associations of surgeon and hospital volumes with SSIs in stratification analyses by underlying diseases. When the operation volumes were defined as the cumulative operation volume within the study period, no relationships existed between hospital/ surgeon operation volumes and SSIs. (Table 7 ) However, when the operation volumes were defined as the cumulative operation volumes in the previous one year for each surgery, the results suggested that there was a negative association between surgeon volumes and SSIs in the diabetes group, except that the volumes were treated as continuous variable and the infection cases were identified by ICD-9 codes. In terms of hospital operation volumes, the association did not exist. (Table 8 )", "No studies have evaluated how different service/ operation volumes definitions and categorization methods affect volume-infection relationships. Moreover, several studies have pointed out the inappropriateness of identifying infection cases using the ICD-9-CM codes in claims data. Given these reasons, this study adopted two approaches to identifying SSIs, two definitions of operation volumes, and three methods for categorizing operation volumes to examine the relationships between operation volumes and SSIs. Our findings showed that the relationships between hospital volumes and SSIs did not exist, no matter which definitions, categorization mehods, or SSIs case identification approaches were used. On the contrary, the relationships between surgeon volumes and SSIs were not robust in our data. It might be affected by different definitions and categorization methods of operation volumes, and also by different SSI cases identification approaches. In summary, most of the models", "demonstrated that the low-volume surgeons had higher risk than high-volume surgeons, and they also showed the risks were similar between medium-volume and high-volume surgeons. However, why did surgeon volume relate to SSIs, but hospital volume did not? Except for those issues we were concerned about in this study, there are some disagreements in the literature. Such as \"Does provider volume really represent quality of care?\" [12, 35] Or \"Is provider volume the only one predictor for outcome of care?\" [35, 36] These issues are worthy of further discussion, but are out of the scope of this study.", "Service/ operation volumes are treated as a proxy indicator for experiences; previous studies used it to examine whether practice makes perfect or not. But, except for provider's experiences, SSIs are also impacted by many factors, such as environmental and clinical factors. Wu et al once used Taiwan 2001 NHI claims data to explore the relationship between provider CABG operation volumes and SSIs. [13] They found that hospital volumes had a greater effect than surgeon volumes and claimed that this may imply that hospital teamwork is more important than individual surgeon. However, our findings demonstrated that there was no relationship between hospital volumes and SSIs. Wu et al. adopted the cumulative operation volumes within the study period as the definition, and identified SSIs by ICD-9-CM codes. Except, there were two differences between our work and Wu et al., which were the length and year of the data; our data was longer and more updated than theirs. Moreover, it is worth", "noting that there was an outbreak of severe acute respiratory syndrome (SARS) in Taiwan in 2003, after which the hospital infection control system in Taiwan was reviewed and re-designed. Wu et al data was before SARS, so these efforts may also have improved the level of SSIs control in hospitals, leading to different findings in this study.", "In addition, although most models revealed that there were negative relationships between surgeon's volumes and surgical site infection, the relationships were not robust. The results varied between different definitions and categorization method of operation volumes, and between SSIs identification approaches. Researchers need to consider how to identify SSIs correctly, how to choose optimal cut-off values, and how to decide on which definition is appropriate.\n\nFinally, the results of stratification analyses showed that low-volume surgeon had higher risk than high-volume surgeon in the diabetes mellitus group, when the cumulative operation in the previous one year before surgery was used as definition. A large number of studies have indicated diabetes mellitus is associated with a higher risk of SSIs, [37] [38] [39] and the findings of this study suggest that CABG patients with diabetes mellitus should be cared for by experienced surgeons.", "A multilevel analysis was applied to manage the nested factors, and two definitions of operation volume along with three different operation volume categorization methods were adopted to examine the relationship between volume and SSIs under two kinds of SSIs identification approaches. Nevertheless, the study suffered from several major limitations. First, the accuracy of SSIs identification was still an issue. Although the performance of the CART model to identify CABG SSIs was better than ICD-9-CM codes in Taiwan NHI claims data, it did not reach the perfect scenario. The accuracy of SSIs identification was still a challenge in our work. The second limitation relates to unmeasured variables, such as length of stay before operation, infection condition, hair removal, clinical information (e.g. blood glucose level, causative microorganism), time-related information (e.g. the duration of operation), the environment, surgical skills, use of post-operative drains, number of operations", "involved, and surgical site and wound care, etc. [40] Furthermore, information about type (elective or urgent) and incision site for surgery was not available in the Taiwan NHI claims data.", "In conclusion, the findings of this study suggest that different definitions and categorization methods of operation volumes, and different SSIs identification approaches might lead to different findings, although surgeon volumes were more important than hospital volumes in exploring the relationships between CABG operation volumes and SSIs in Taiwan, but they were still not robust. Definitions and categorization methods of operation volumes, and correct identification of SSIs are important issues for future research." ]

[ 4 ]

[ "hospitals would no longer receive higher payments for the additional costs associated with treating patients for certain healthcare-acquired infections" ]

[ "Which Kind of Provider’s Operation Volumes Matters? Associations between CABG Surgical Site Infection Risk and Hospital and Surgeon Operation Volumes among Medical Centers in Taiwan\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4459823/\n\nSHA: f3cbc0503581249a834895fc94cd3bae24714a0d\n\nAuthors: Yu, Tsung-Hsien; Tung, Yu-Chi; Chung, Kuo-Piao\nDate: 2015-06-08\nDOI: 10.1371/journal.pone.0129178\nLicense: cc-by", "Abstract: BACKGROUND: Volume-infection relationships have been examined for high-risk surgical procedures, but the conclusions remain controversial. The inconsistency might be due to inaccurate identification of cases of infection and different methods of categorizing service volumes. This study takes coronary artery bypass graft (CABG) surgical site infections (SSIs) as an example to examine whether a relationship exists between operation volumes and SSIs, when different SSIs case identification, definitions and categorization methods of operation volumes were implemented. METHODS: A population-based cross-sectional multilevel study was conducted. A total of 7,007 patients who received CABG surgery between 2006 and 2008 from19 medical centers in Taiwan were recruited. SSIs associated with CABG surgery were identified using International Classification of Diseases, 9th Revision, Clinical Modification (ICD-9 CM) codes and a Classification and Regression Trees (CART) model. Two", "definitions of surgeon and hospital operation volumes were used: (1) the cumulative CABG operation volumes within the study period; and (2) the cumulative CABG operation volumes in the previous one year before each CABG surgery. Operation volumes were further treated in three different ways: (1) a continuous variable; (2) a categorical variable based on the quartile; and (3) a data-driven categorical variable based on k-means clustering algorithm. Furthermore, subgroup analysis for comorbidities was also conducted. RESULTS: This study showed that hospital volumes were not significantly associated with SSIs, no matter which definitions or categorization methods of operation volume, or SSIs case identification approaches were used. On the contrary, the relationships between surgeon’s volumes varied. Most of the models demonstrated that the low-volume surgeons had higher risk than high-volume surgeons. CONCLUSION: Surgeon volumes were more important than hospital volumes in exploring the", "relationship between CABG operation volumes and SSIs in Taiwan. However, the relationships were not robust. Definitions and categorization methods of operation volume and correct identification of SSIs are important issues for future research.", "Text: data, which should use hierarchical models, may result in biased estimation of the variation and also lead to incorrect conclusions.\n\nSSIs following coronary artery bypass graft (CABG) procedures place a heavy burden on patients and healthcare systems. The total length of stay and expenditure for patients with SSIs after CABG surgery is significantly longer and higher than those without SSIs. [20, 21] In 2008, the Centers for Medicare & Medicaid of the United States of America implemented the \"Never Event\" policy, where hospitals would no longer receive higher payments for the additional costs associated with treating patients for certain healthcare-acquired infections, including those related to CABG.", "In view of the accuracy of SSIs identification and the heterogeneity of definition and categorization methods, no existing studies have used different infection case identification nor definitions and categorization methods of operation volume simultaneously to explore the relationship between operation volumes and infection. The current study takes CABG SSIs as an example to examine whether a relationship exists between operation volumes and SSIs, given different SSI cases identification, operation volume definitions and categorization methods.\n\nThis retrospective and cross-sectional study adopted a multilevel design to examine the relationships between provider volumes and SSIs after adjusting for patient-, surgeon-, and hospital-level covariates.", "We used data from the Taiwan National Health Insurance Research Database (NHIRD) from 2005 and 2008. The NHIRD, published by the Taiwan National Health Research Institute, includes all the original claims data and registration files for beneficiaries enrolled under the National Health Insurance (NHI) program. The database covers the 23 million Taiwanese enrollees (approximately 98% of the population) in the NHI program. It is a de-identified secondary database containing patient-level demographic and administrative information; however, treatment items are aggregated and without time-related and clinical information. The data is released for research purposes.\n\nThe protocol for the study was approved by the Institutional Review Board of the National Taiwan University Hospital (protocol #201001027R). The dataset we used in this study was secondary data; all information was de-identified by data owners.", "In this study, we adopted the ICD-9-CM SSI codes (hereafter referred to as the ICD-9-CM based model) and the Classification and Regression Trees (CART) model, which was developed in our previous work [11] to identify SSI cases. As we mentioned above, the ICD-9-CM SSI codes were the most popular tool to identify the SSI cases in claims data. In the ICD-9-CM based model, SSI cases were divided into two categories: index hospitalization events and post-discharge events (i.e., SSIs that occurred within 1 year after discharge and required readmission to a hospital and/ or the use of ambulatory services). Following Wu et al [13] , this study adopted the secondary ICD-9-CM diagnosis codes for index hospitalization events (ICD-9-CM code: 996.03, 996.61, 996.72, and 998.5), and the primary and secondary diagnosis codes for post-discharge events (ICD-9-CM code: 038.0-038. 4 ) as the criteria for SSI identification, in order to avoid cases in which infection existed prior to hospitalization. If", "a case had an index hospitalization event or a post-discharge event, then he/ she will be identified as SSIs by the ICD-9-CM based model. In the CART model, we adopted the type of antibiotics, dose of cefazolin, length of stay, and number of vessels obstructed (as a proxy indicator of duration of operation) as the parameters to identify the SSIs, according to our previous findings. [11] In our previous work, we used the 2005-2008 National Health Insurance claims data and healthcare-associated infection surveillance data from two medical centers for model development and model verification. Infection cases based on surveillance were identified by infection control personnel if the patient met the Taiwan CDC's criteria, which are the same as those adopted in the U.S. CDC. They manually review medical records of all patients at risk for the specified healthcare-associated infection.", "The classification algorithms, the multivariable regression model, and the data mining model were adopted to develop alternative models based on surrogate indicators to identify cases of CABG SSIs and to compare the performance among these models and the ICD-9-CMbased model. For the classification algorithms, researchers build up several criteria, and if a case satisfies (or exceeds) a specific number of criteria, then it will be identified as a case of infection. For the multivariable regression model, researchers usually calculated a risk score by the logistic regression model, and the optimal cutoff point was determined according to the resulting receiver operating characteristic curve.", "Concerning the data mining approach, which is widely used for predicting and classifying objects, the characteristics are: automatic discovery of patterns, prediction of likely outcomes, creation of actionable information, and focus on large data sets and databases. The classification and regression tree (CART) model, which is the most popular approach as applied in our work, and the growing, stopping, and pruning of the tree were determined by Gini improvement measures. [22, 23] After referring to the literature and conferring with infectious disease specialists, we adopted the following seven parameters: type of antibiotic, doses of antibiotic, doses of cefazolin, use of second-line antibiotics, length of stay, and number of vessels obstructed. Additionally, cross-validation was also employed, where data from one medical center was used for model development, and another one was used for model validation.", "The results of our previous work revealed that the CART model offered better performance than that of the other identification models or the ICD-9-CM based model, especially in the positive predictive value (>70%), which was only found to be 20% in the ICD-9-CM based model. (Table 1 ) The findings also implied that the CART was a decidedly better tool for identifying cases of SSI in the Taiwan National Health Insurance database. Therefore, this study also adopted the CART model for identifying CABG SSIs.\n\nTo ensure homogeneity, current study analyzed 7,007 patients from 19 medical centers in Taiwan who underwent CABG surgery (ICD-9-CM procedure codes 36.1x-36.2x) between 2006 and 2008. CABG patients under the age of 18 years or over 85 years were excluded in this study. A total of 302 cases were identified as SSIs by ICD-9-CM based model, and a total of 107 cases were identified as SSIs by CART model.", "In this study, we used the following two definitions to define operation volumes: (1) the cumulative operation volumes by each surgeon and hospital within the study period, which was the most common definition in the literature; and (2) following Yasunaga et al.'s study, [24] cumulative operation volumes by each surgeon and hospital in the previous one year for each surgery. However, our data was skewed, which did not follow a normal distribution. Therefore, we conducted the log transformations on operation volumes.", "The current work treated operation volumes in three different ways: (1) a continuous variable; (2) a categorical variable based on the first and the third quartile as cutoff points (the most common method to categorize service/ operation volumes) [25] [26] [27] [28] ; and (3) a data-driven categorical variable based on k-means clustering algorithm. This study categorized surgeon and hospital volumes into low, medium, and high volume groups by quartile method and kmeans clustering algorithm.", "In the quartile method, the cut-off value (transformed by logarithm) of the first quartile (<25%) for hospital volumes was 5.65, and the third quartile (>75%) was 6.43. In terms of surgeon volumes, the first quartile was 4.38, and the third was 5.35, when we used the cumulative operation volumes within the study period as the definition. While the definition changed, first quartile (<25%) for hospital volumes was 4.66, and the third quartile (>75%) was 5.31. In terms of surgeon volumes, the first quartile was 3.40, and the third was 4.32.", "K-means clustering is an unsupervised machine-learning algorithm introduced by MacQueen in 1960s. This method is not only a simple and very reliable method in categorization/ classification, but is also recognized as one of the top 10 algorithms in data mining. [29] This method has often been applied in many fields. [30] [31] [32] Yu and his colleagues even applied it to define the quality of CABG care, and to explore the relationship among patient's income status, the level of quality of care, and inpatient mortality. [33] The main idea of this method is to partition observed data points into k non-overlapping clusters by minimizing the within-group sum of squares. Each point is assigned to the mean of its cluster using the Euclidian distance. Firstly, k cluster centers were randomly generated. Previous studies usually divided surgeons and hospitals into low-, medium-, and high-volume groups; therefore, we also predetermined the surgeon and hospital service volumes into 3 groups (k =", "3). Then, participants were assigned to the cluster with the shortest distance to these cluster centers. Finally, the cluster centers were recomputed using the new cluster assignment and these steps would be iterated until convergence was achieved. [34] The cut-off values of hospital volumes were 5.21 and 5.69, and for surgeon's volumes were 2.40 and 4.38 respectively, when cumulative operation volumes within the study period was used as the definition. Likewise, when cumulative operation volumes before each surgery was used as definition, the cut-off values were 4.11 and 4.89 for hospital volumes, and 2.64 and 3.91 for surgeon's volumes. All cutoff values were transformed by logarithm. The results of k-means clustering are demonstrated in Figs 1-4. As the results show, the operation volumes were divided into three groups separately. In addition to surgeon and hospital volumes and SSI, we collected patient-, surgeon-, and hospital-level data. Firstly, patient-level variables included", "age, gender, length of ICU stay, number of vessels obstructed that were involved in the surgical operation, and the presence of important underlying diseases (e.g. diabetes mellitus, chronic obstructive pulmonary disease (COPD), heart failure, renal failure and renal insufficiency, which were associated with SSI).", "[13] Secondly, the surgeon-level variables included age and gender. Thirdly, the hospital-level variables included hospital ownership and geographic location.", "All statistical analyses of volume-infection relationship were performed using SAS (version 9.2, SAS Institution Inc., Cary, NC, USA). In statistical testing, a two-sided p value 0.05 was considered statistically significant. The distributional properties of continuous variables were expressed by mean ± standard deviation (SD), whereas categorical variables were presented by frequency and percentage. In univariate analysis, the potential three-level predictors of SSI were examined using chi-square test or two-sample t-test as appropriate. Next, to account for the correlations within surgeon (level-2) and hospital (level-3), multivariate analysis was conducted by fitting mixed-effects logistic regression models to each patient's data for estimating the effects of three-level predictors on the probability of post-operational SSI. Furthermore, subgroup analysis for comorbidities was also conducted. Table 2 shows that there were 7,007 patients with CABG performed by 199 surgeons in 19", "hospitals during 2006-2008 in Taiwan. The majority of patients were male (77.5%), and the mean age of patients was 65.3 years. The average ICU stay was 6.05 days, the mean level of number of vessels obstructed was around 1.6, while 51.8% of patients had diabetes mellitus, 33.3% had heart failure, 14.1% had renal failure and renal insufficiency, and 22.0% had COPD. Three hundred and two patients (4.31%) were identified as having the ICD-9-CM SSI codes. However, identification by the CART model only revealed 107 infection cases, and 94 cases were identified in both models. Most cases received CABG surgery by male surgeons, with a mean age of 45.0 years, and the surgeon's average operation volumes within the study period was 151.64, while the average operation volumes before surgery was 52.18. More than half of the cases were performed with CABG in not-for-profit hospitals, and the hospitals' average operation volumes within the study period was 473.60, while the average operation", "volumes before each surgery was 158.79. Moreover, most of patients received their surgeries by high-volume surgeons and hospitals, when k-means algorithm was used for categorization, regardless of which definition of operation volumes were used. Table 3 shows the results of multilevel mixed-effect models, with the SSIs being identified by ICD-9-CM codes, and the operation volumes defined as the cumulative volumes within the study period. The results of Model 1 (continuous) reveal that the surgeon's volumes were negatively associated with SSIs, while hospital's volumes were not associated with surgical site infection SSIs. Model 2 (quartile) suggests that low-volume surgeons had higher SSI risk (OR = 2.220, p-value = 0.022) than high-volume surgeons. There were also no associations between hospital's operation volumes and SSIs. Model 3 (k-means) shows that the association did not exist between hospital's/ surgeon's volumes and SSIs. Table 4 displays the results of multilevel", "mixed-effect models, in which the SSIs were identified by the CART model, and the operation volumes were also defined as the cumulative volumes within the study period. Model 1 again indicated a negative association between surgeon's volumes and SSIs, and hospital's volumes were not found to be associated with SSIs. In Model 2, the results showed that the relationship between hospital's/ surgeon's volumes and SSIs did not exist. In Model 3, results revealed low-volume surgeons had higher risk (OR = 1.691, p = 0.002) than high-volume surgeons. Table 5 displays the results of multilevel mixed-effect models, in which the SSIs were identified by ICD-9-CM codes, but the operation volumes were defined as the cumulative volume in the previous one year for each surgery. Model 1 also indicated a negative association between surgeon's volumes and SSIs, and hospital's volumes were not found to be associated with SSIs. In Model 2, the results showed that the relationship between hospital's/", "surgeon's volumes and SSIs did not exist. In Model 3, results also revealed low-volume surgeons had higher risk (OR = 1.642, p = 0.040) than high-volume surgeons. Table 6 displays the results of multilevel mixed-effect models, in which the SSIs were identified by the CART model, and the operation volumes were also defined as the cumulative volume in previous one year for each surgery. In Model 1, different to the above findings, there was no association between hospital's/ surgeon's volumes and SSIs. In Model 2, the results showed that the relationship between hospital's/ surgeon's volumes and SSIs did not exist. In Model 3, results also revealed low-volume surgeons had higher risk (OR = 1.163, p = 0.020) than high-volume surgeons.", "We further examined the associations of surgeon and hospital volumes with SSIs in stratification analyses by underlying diseases. When the operation volumes were defined as the cumulative operation volume within the study period, no relationships existed between hospital/ surgeon operation volumes and SSIs. (Table 7 ) However, when the operation volumes were defined as the cumulative operation volumes in the previous one year for each surgery, the results suggested that there was a negative association between surgeon volumes and SSIs in the diabetes group, except that the volumes were treated as continuous variable and the infection cases were identified by ICD-9 codes. In terms of hospital operation volumes, the association did not exist. (Table 8 )", "No studies have evaluated how different service/ operation volumes definitions and categorization methods affect volume-infection relationships. Moreover, several studies have pointed out the inappropriateness of identifying infection cases using the ICD-9-CM codes in claims data. Given these reasons, this study adopted two approaches to identifying SSIs, two definitions of operation volumes, and three methods for categorizing operation volumes to examine the relationships between operation volumes and SSIs. Our findings showed that the relationships between hospital volumes and SSIs did not exist, no matter which definitions, categorization mehods, or SSIs case identification approaches were used. On the contrary, the relationships between surgeon volumes and SSIs were not robust in our data. It might be affected by different definitions and categorization methods of operation volumes, and also by different SSI cases identification approaches. In summary, most of the models", "demonstrated that the low-volume surgeons had higher risk than high-volume surgeons, and they also showed the risks were similar between medium-volume and high-volume surgeons. However, why did surgeon volume relate to SSIs, but hospital volume did not? Except for those issues we were concerned about in this study, there are some disagreements in the literature. Such as \"Does provider volume really represent quality of care?\" [12, 35] Or \"Is provider volume the only one predictor for outcome of care?\" [35, 36] These issues are worthy of further discussion, but are out of the scope of this study.", "Service/ operation volumes are treated as a proxy indicator for experiences; previous studies used it to examine whether practice makes perfect or not. But, except for provider's experiences, SSIs are also impacted by many factors, such as environmental and clinical factors. Wu et al once used Taiwan 2001 NHI claims data to explore the relationship between provider CABG operation volumes and SSIs. [13] They found that hospital volumes had a greater effect than surgeon volumes and claimed that this may imply that hospital teamwork is more important than individual surgeon. However, our findings demonstrated that there was no relationship between hospital volumes and SSIs. Wu et al. adopted the cumulative operation volumes within the study period as the definition, and identified SSIs by ICD-9-CM codes. Except, there were two differences between our work and Wu et al., which were the length and year of the data; our data was longer and more updated than theirs. Moreover, it is worth", "noting that there was an outbreak of severe acute respiratory syndrome (SARS) in Taiwan in 2003, after which the hospital infection control system in Taiwan was reviewed and re-designed. Wu et al data was before SARS, so these efforts may also have improved the level of SSIs control in hospitals, leading to different findings in this study.", "In addition, although most models revealed that there were negative relationships between surgeon's volumes and surgical site infection, the relationships were not robust. The results varied between different definitions and categorization method of operation volumes, and between SSIs identification approaches. Researchers need to consider how to identify SSIs correctly, how to choose optimal cut-off values, and how to decide on which definition is appropriate.\n\nFinally, the results of stratification analyses showed that low-volume surgeon had higher risk than high-volume surgeon in the diabetes mellitus group, when the cumulative operation in the previous one year before surgery was used as definition. A large number of studies have indicated diabetes mellitus is associated with a higher risk of SSIs, [37] [38] [39] and the findings of this study suggest that CABG patients with diabetes mellitus should be cared for by experienced surgeons.", "A multilevel analysis was applied to manage the nested factors, and two definitions of operation volume along with three different operation volume categorization methods were adopted to examine the relationship between volume and SSIs under two kinds of SSIs identification approaches. Nevertheless, the study suffered from several major limitations. First, the accuracy of SSIs identification was still an issue. Although the performance of the CART model to identify CABG SSIs was better than ICD-9-CM codes in Taiwan NHI claims data, it did not reach the perfect scenario. The accuracy of SSIs identification was still a challenge in our work. The second limitation relates to unmeasured variables, such as length of stay before operation, infection condition, hair removal, clinical information (e.g. blood glucose level, causative microorganism), time-related information (e.g. the duration of operation), the environment, surgical skills, use of post-operative drains, number of operations", "involved, and surgical site and wound care, etc. [40] Furthermore, information about type (elective or urgent) and incision site for surgery was not available in the Taiwan NHI claims data.", "In conclusion, the findings of this study suggest that different definitions and categorization methods of operation volumes, and different SSIs identification approaches might lead to different findings, although surgeon volumes were more important than hospital volumes in exploring the relationships between CABG operation volumes and SSIs in Taiwan, but they were still not robust. Definitions and categorization methods of operation volumes, and correct identification of SSIs are important issues for future research." ]

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[ "19" ]

[ "Which Kind of Provider’s Operation Volumes Matters? Associations between CABG Surgical Site Infection Risk and Hospital and Surgeon Operation Volumes among Medical Centers in Taiwan\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4459823/\n\nSHA: f3cbc0503581249a834895fc94cd3bae24714a0d\n\nAuthors: Yu, Tsung-Hsien; Tung, Yu-Chi; Chung, Kuo-Piao\nDate: 2015-06-08\nDOI: 10.1371/journal.pone.0129178\nLicense: cc-by", "Abstract: BACKGROUND: Volume-infection relationships have been examined for high-risk surgical procedures, but the conclusions remain controversial. The inconsistency might be due to inaccurate identification of cases of infection and different methods of categorizing service volumes. This study takes coronary artery bypass graft (CABG) surgical site infections (SSIs) as an example to examine whether a relationship exists between operation volumes and SSIs, when different SSIs case identification, definitions and categorization methods of operation volumes were implemented. METHODS: A population-based cross-sectional multilevel study was conducted. A total of 7,007 patients who received CABG surgery between 2006 and 2008 from19 medical centers in Taiwan were recruited. SSIs associated with CABG surgery were identified using International Classification of Diseases, 9th Revision, Clinical Modification (ICD-9 CM) codes and a Classification and Regression Trees (CART) model. Two", "definitions of surgeon and hospital operation volumes were used: (1) the cumulative CABG operation volumes within the study period; and (2) the cumulative CABG operation volumes in the previous one year before each CABG surgery. Operation volumes were further treated in three different ways: (1) a continuous variable; (2) a categorical variable based on the quartile; and (3) a data-driven categorical variable based on k-means clustering algorithm. Furthermore, subgroup analysis for comorbidities was also conducted. RESULTS: This study showed that hospital volumes were not significantly associated with SSIs, no matter which definitions or categorization methods of operation volume, or SSIs case identification approaches were used. On the contrary, the relationships between surgeon’s volumes varied. Most of the models demonstrated that the low-volume surgeons had higher risk than high-volume surgeons. CONCLUSION: Surgeon volumes were more important than hospital volumes in exploring the", "relationship between CABG operation volumes and SSIs in Taiwan. However, the relationships were not robust. Definitions and categorization methods of operation volume and correct identification of SSIs are important issues for future research.", "Text: data, which should use hierarchical models, may result in biased estimation of the variation and also lead to incorrect conclusions.\n\nSSIs following coronary artery bypass graft (CABG) procedures place a heavy burden on patients and healthcare systems. The total length of stay and expenditure for patients with SSIs after CABG surgery is significantly longer and higher than those without SSIs. [20, 21] In 2008, the Centers for Medicare & Medicaid of the United States of America implemented the \"Never Event\" policy, where hospitals would no longer receive higher payments for the additional costs associated with treating patients for certain healthcare-acquired infections, including those related to CABG.", "In view of the accuracy of SSIs identification and the heterogeneity of definition and categorization methods, no existing studies have used different infection case identification nor definitions and categorization methods of operation volume simultaneously to explore the relationship between operation volumes and infection. The current study takes CABG SSIs as an example to examine whether a relationship exists between operation volumes and SSIs, given different SSI cases identification, operation volume definitions and categorization methods.\n\nThis retrospective and cross-sectional study adopted a multilevel design to examine the relationships between provider volumes and SSIs after adjusting for patient-, surgeon-, and hospital-level covariates.", "We used data from the Taiwan National Health Insurance Research Database (NHIRD) from 2005 and 2008. The NHIRD, published by the Taiwan National Health Research Institute, includes all the original claims data and registration files for beneficiaries enrolled under the National Health Insurance (NHI) program. The database covers the 23 million Taiwanese enrollees (approximately 98% of the population) in the NHI program. It is a de-identified secondary database containing patient-level demographic and administrative information; however, treatment items are aggregated and without time-related and clinical information. The data is released for research purposes.\n\nThe protocol for the study was approved by the Institutional Review Board of the National Taiwan University Hospital (protocol #201001027R). The dataset we used in this study was secondary data; all information was de-identified by data owners.", "In this study, we adopted the ICD-9-CM SSI codes (hereafter referred to as the ICD-9-CM based model) and the Classification and Regression Trees (CART) model, which was developed in our previous work [11] to identify SSI cases. As we mentioned above, the ICD-9-CM SSI codes were the most popular tool to identify the SSI cases in claims data. In the ICD-9-CM based model, SSI cases were divided into two categories: index hospitalization events and post-discharge events (i.e., SSIs that occurred within 1 year after discharge and required readmission to a hospital and/ or the use of ambulatory services). Following Wu et al [13] , this study adopted the secondary ICD-9-CM diagnosis codes for index hospitalization events (ICD-9-CM code: 996.03, 996.61, 996.72, and 998.5), and the primary and secondary diagnosis codes for post-discharge events (ICD-9-CM code: 038.0-038. 4 ) as the criteria for SSI identification, in order to avoid cases in which infection existed prior to hospitalization. If", "a case had an index hospitalization event or a post-discharge event, then he/ she will be identified as SSIs by the ICD-9-CM based model. In the CART model, we adopted the type of antibiotics, dose of cefazolin, length of stay, and number of vessels obstructed (as a proxy indicator of duration of operation) as the parameters to identify the SSIs, according to our previous findings. [11] In our previous work, we used the 2005-2008 National Health Insurance claims data and healthcare-associated infection surveillance data from two medical centers for model development and model verification. Infection cases based on surveillance were identified by infection control personnel if the patient met the Taiwan CDC's criteria, which are the same as those adopted in the U.S. CDC. They manually review medical records of all patients at risk for the specified healthcare-associated infection.", "The classification algorithms, the multivariable regression model, and the data mining model were adopted to develop alternative models based on surrogate indicators to identify cases of CABG SSIs and to compare the performance among these models and the ICD-9-CMbased model. For the classification algorithms, researchers build up several criteria, and if a case satisfies (or exceeds) a specific number of criteria, then it will be identified as a case of infection. For the multivariable regression model, researchers usually calculated a risk score by the logistic regression model, and the optimal cutoff point was determined according to the resulting receiver operating characteristic curve.", "Concerning the data mining approach, which is widely used for predicting and classifying objects, the characteristics are: automatic discovery of patterns, prediction of likely outcomes, creation of actionable information, and focus on large data sets and databases. The classification and regression tree (CART) model, which is the most popular approach as applied in our work, and the growing, stopping, and pruning of the tree were determined by Gini improvement measures. [22, 23] After referring to the literature and conferring with infectious disease specialists, we adopted the following seven parameters: type of antibiotic, doses of antibiotic, doses of cefazolin, use of second-line antibiotics, length of stay, and number of vessels obstructed. Additionally, cross-validation was also employed, where data from one medical center was used for model development, and another one was used for model validation.", "The results of our previous work revealed that the CART model offered better performance than that of the other identification models or the ICD-9-CM based model, especially in the positive predictive value (>70%), which was only found to be 20% in the ICD-9-CM based model. (Table 1 ) The findings also implied that the CART was a decidedly better tool for identifying cases of SSI in the Taiwan National Health Insurance database. Therefore, this study also adopted the CART model for identifying CABG SSIs.\n\nTo ensure homogeneity, current study analyzed 7,007 patients from 19 medical centers in Taiwan who underwent CABG surgery (ICD-9-CM procedure codes 36.1x-36.2x) between 2006 and 2008. CABG patients under the age of 18 years or over 85 years were excluded in this study. A total of 302 cases were identified as SSIs by ICD-9-CM based model, and a total of 107 cases were identified as SSIs by CART model.", "In this study, we used the following two definitions to define operation volumes: (1) the cumulative operation volumes by each surgeon and hospital within the study period, which was the most common definition in the literature; and (2) following Yasunaga et al.'s study, [24] cumulative operation volumes by each surgeon and hospital in the previous one year for each surgery. However, our data was skewed, which did not follow a normal distribution. Therefore, we conducted the log transformations on operation volumes.", "The current work treated operation volumes in three different ways: (1) a continuous variable; (2) a categorical variable based on the first and the third quartile as cutoff points (the most common method to categorize service/ operation volumes) [25] [26] [27] [28] ; and (3) a data-driven categorical variable based on k-means clustering algorithm. This study categorized surgeon and hospital volumes into low, medium, and high volume groups by quartile method and kmeans clustering algorithm.", "In the quartile method, the cut-off value (transformed by logarithm) of the first quartile (<25%) for hospital volumes was 5.65, and the third quartile (>75%) was 6.43. In terms of surgeon volumes, the first quartile was 4.38, and the third was 5.35, when we used the cumulative operation volumes within the study period as the definition. While the definition changed, first quartile (<25%) for hospital volumes was 4.66, and the third quartile (>75%) was 5.31. In terms of surgeon volumes, the first quartile was 3.40, and the third was 4.32.", "K-means clustering is an unsupervised machine-learning algorithm introduced by MacQueen in 1960s. This method is not only a simple and very reliable method in categorization/ classification, but is also recognized as one of the top 10 algorithms in data mining. [29] This method has often been applied in many fields. [30] [31] [32] Yu and his colleagues even applied it to define the quality of CABG care, and to explore the relationship among patient's income status, the level of quality of care, and inpatient mortality. [33] The main idea of this method is to partition observed data points into k non-overlapping clusters by minimizing the within-group sum of squares. Each point is assigned to the mean of its cluster using the Euclidian distance. Firstly, k cluster centers were randomly generated. Previous studies usually divided surgeons and hospitals into low-, medium-, and high-volume groups; therefore, we also predetermined the surgeon and hospital service volumes into 3 groups (k =", "3). Then, participants were assigned to the cluster with the shortest distance to these cluster centers. Finally, the cluster centers were recomputed using the new cluster assignment and these steps would be iterated until convergence was achieved. [34] The cut-off values of hospital volumes were 5.21 and 5.69, and for surgeon's volumes were 2.40 and 4.38 respectively, when cumulative operation volumes within the study period was used as the definition. Likewise, when cumulative operation volumes before each surgery was used as definition, the cut-off values were 4.11 and 4.89 for hospital volumes, and 2.64 and 3.91 for surgeon's volumes. All cutoff values were transformed by logarithm. The results of k-means clustering are demonstrated in Figs 1-4. As the results show, the operation volumes were divided into three groups separately. In addition to surgeon and hospital volumes and SSI, we collected patient-, surgeon-, and hospital-level data. Firstly, patient-level variables included", "age, gender, length of ICU stay, number of vessels obstructed that were involved in the surgical operation, and the presence of important underlying diseases (e.g. diabetes mellitus, chronic obstructive pulmonary disease (COPD), heart failure, renal failure and renal insufficiency, which were associated with SSI).", "[13] Secondly, the surgeon-level variables included age and gender. Thirdly, the hospital-level variables included hospital ownership and geographic location.", "All statistical analyses of volume-infection relationship were performed using SAS (version 9.2, SAS Institution Inc., Cary, NC, USA). In statistical testing, a two-sided p value 0.05 was considered statistically significant. The distributional properties of continuous variables were expressed by mean ± standard deviation (SD), whereas categorical variables were presented by frequency and percentage. In univariate analysis, the potential three-level predictors of SSI were examined using chi-square test or two-sample t-test as appropriate. Next, to account for the correlations within surgeon (level-2) and hospital (level-3), multivariate analysis was conducted by fitting mixed-effects logistic regression models to each patient's data for estimating the effects of three-level predictors on the probability of post-operational SSI. Furthermore, subgroup analysis for comorbidities was also conducted. Table 2 shows that there were 7,007 patients with CABG performed by 199 surgeons in 19", "hospitals during 2006-2008 in Taiwan. The majority of patients were male (77.5%), and the mean age of patients was 65.3 years. The average ICU stay was 6.05 days, the mean level of number of vessels obstructed was around 1.6, while 51.8% of patients had diabetes mellitus, 33.3% had heart failure, 14.1% had renal failure and renal insufficiency, and 22.0% had COPD. Three hundred and two patients (4.31%) were identified as having the ICD-9-CM SSI codes. However, identification by the CART model only revealed 107 infection cases, and 94 cases were identified in both models. Most cases received CABG surgery by male surgeons, with a mean age of 45.0 years, and the surgeon's average operation volumes within the study period was 151.64, while the average operation volumes before surgery was 52.18. More than half of the cases were performed with CABG in not-for-profit hospitals, and the hospitals' average operation volumes within the study period was 473.60, while the average operation", "volumes before each surgery was 158.79. Moreover, most of patients received their surgeries by high-volume surgeons and hospitals, when k-means algorithm was used for categorization, regardless of which definition of operation volumes were used. Table 3 shows the results of multilevel mixed-effect models, with the SSIs being identified by ICD-9-CM codes, and the operation volumes defined as the cumulative volumes within the study period. The results of Model 1 (continuous) reveal that the surgeon's volumes were negatively associated with SSIs, while hospital's volumes were not associated with surgical site infection SSIs. Model 2 (quartile) suggests that low-volume surgeons had higher SSI risk (OR = 2.220, p-value = 0.022) than high-volume surgeons. There were also no associations between hospital's operation volumes and SSIs. Model 3 (k-means) shows that the association did not exist between hospital's/ surgeon's volumes and SSIs. Table 4 displays the results of multilevel", "mixed-effect models, in which the SSIs were identified by the CART model, and the operation volumes were also defined as the cumulative volumes within the study period. Model 1 again indicated a negative association between surgeon's volumes and SSIs, and hospital's volumes were not found to be associated with SSIs. In Model 2, the results showed that the relationship between hospital's/ surgeon's volumes and SSIs did not exist. In Model 3, results revealed low-volume surgeons had higher risk (OR = 1.691, p = 0.002) than high-volume surgeons. Table 5 displays the results of multilevel mixed-effect models, in which the SSIs were identified by ICD-9-CM codes, but the operation volumes were defined as the cumulative volume in the previous one year for each surgery. Model 1 also indicated a negative association between surgeon's volumes and SSIs, and hospital's volumes were not found to be associated with SSIs. In Model 2, the results showed that the relationship between hospital's/", "surgeon's volumes and SSIs did not exist. In Model 3, results also revealed low-volume surgeons had higher risk (OR = 1.642, p = 0.040) than high-volume surgeons. Table 6 displays the results of multilevel mixed-effect models, in which the SSIs were identified by the CART model, and the operation volumes were also defined as the cumulative volume in previous one year for each surgery. In Model 1, different to the above findings, there was no association between hospital's/ surgeon's volumes and SSIs. In Model 2, the results showed that the relationship between hospital's/ surgeon's volumes and SSIs did not exist. In Model 3, results also revealed low-volume surgeons had higher risk (OR = 1.163, p = 0.020) than high-volume surgeons.", "We further examined the associations of surgeon and hospital volumes with SSIs in stratification analyses by underlying diseases. When the operation volumes were defined as the cumulative operation volume within the study period, no relationships existed between hospital/ surgeon operation volumes and SSIs. (Table 7 ) However, when the operation volumes were defined as the cumulative operation volumes in the previous one year for each surgery, the results suggested that there was a negative association between surgeon volumes and SSIs in the diabetes group, except that the volumes were treated as continuous variable and the infection cases were identified by ICD-9 codes. In terms of hospital operation volumes, the association did not exist. (Table 8 )", "No studies have evaluated how different service/ operation volumes definitions and categorization methods affect volume-infection relationships. Moreover, several studies have pointed out the inappropriateness of identifying infection cases using the ICD-9-CM codes in claims data. Given these reasons, this study adopted two approaches to identifying SSIs, two definitions of operation volumes, and three methods for categorizing operation volumes to examine the relationships between operation volumes and SSIs. Our findings showed that the relationships between hospital volumes and SSIs did not exist, no matter which definitions, categorization mehods, or SSIs case identification approaches were used. On the contrary, the relationships between surgeon volumes and SSIs were not robust in our data. It might be affected by different definitions and categorization methods of operation volumes, and also by different SSI cases identification approaches. In summary, most of the models", "demonstrated that the low-volume surgeons had higher risk than high-volume surgeons, and they also showed the risks were similar between medium-volume and high-volume surgeons. However, why did surgeon volume relate to SSIs, but hospital volume did not? Except for those issues we were concerned about in this study, there are some disagreements in the literature. Such as \"Does provider volume really represent quality of care?\" [12, 35] Or \"Is provider volume the only one predictor for outcome of care?\" [35, 36] These issues are worthy of further discussion, but are out of the scope of this study.", "Service/ operation volumes are treated as a proxy indicator for experiences; previous studies used it to examine whether practice makes perfect or not. But, except for provider's experiences, SSIs are also impacted by many factors, such as environmental and clinical factors. Wu et al once used Taiwan 2001 NHI claims data to explore the relationship between provider CABG operation volumes and SSIs. [13] They found that hospital volumes had a greater effect than surgeon volumes and claimed that this may imply that hospital teamwork is more important than individual surgeon. However, our findings demonstrated that there was no relationship between hospital volumes and SSIs. Wu et al. adopted the cumulative operation volumes within the study period as the definition, and identified SSIs by ICD-9-CM codes. Except, there were two differences between our work and Wu et al., which were the length and year of the data; our data was longer and more updated than theirs. Moreover, it is worth", "noting that there was an outbreak of severe acute respiratory syndrome (SARS) in Taiwan in 2003, after which the hospital infection control system in Taiwan was reviewed and re-designed. Wu et al data was before SARS, so these efforts may also have improved the level of SSIs control in hospitals, leading to different findings in this study.", "In addition, although most models revealed that there were negative relationships between surgeon's volumes and surgical site infection, the relationships were not robust. The results varied between different definitions and categorization method of operation volumes, and between SSIs identification approaches. Researchers need to consider how to identify SSIs correctly, how to choose optimal cut-off values, and how to decide on which definition is appropriate.\n\nFinally, the results of stratification analyses showed that low-volume surgeon had higher risk than high-volume surgeon in the diabetes mellitus group, when the cumulative operation in the previous one year before surgery was used as definition. A large number of studies have indicated diabetes mellitus is associated with a higher risk of SSIs, [37] [38] [39] and the findings of this study suggest that CABG patients with diabetes mellitus should be cared for by experienced surgeons.", "A multilevel analysis was applied to manage the nested factors, and two definitions of operation volume along with three different operation volume categorization methods were adopted to examine the relationship between volume and SSIs under two kinds of SSIs identification approaches. Nevertheless, the study suffered from several major limitations. First, the accuracy of SSIs identification was still an issue. Although the performance of the CART model to identify CABG SSIs was better than ICD-9-CM codes in Taiwan NHI claims data, it did not reach the perfect scenario. The accuracy of SSIs identification was still a challenge in our work. The second limitation relates to unmeasured variables, such as length of stay before operation, infection condition, hair removal, clinical information (e.g. blood glucose level, causative microorganism), time-related information (e.g. the duration of operation), the environment, surgical skills, use of post-operative drains, number of operations", "involved, and surgical site and wound care, etc. [40] Furthermore, information about type (elective or urgent) and incision site for surgery was not available in the Taiwan NHI claims data.", "In conclusion, the findings of this study suggest that different definitions and categorization methods of operation volumes, and different SSIs identification approaches might lead to different findings, although surgeon volumes were more important than hospital volumes in exploring the relationships between CABG operation volumes and SSIs in Taiwan, but they were still not robust. Definitions and categorization methods of operation volumes, and correct identification of SSIs are important issues for future research." ]

[ 1 ]

[ "CABG patients under the age of 18 years or over 85 years" ]

[ "Which Kind of Provider’s Operation Volumes Matters? Associations between CABG Surgical Site Infection Risk and Hospital and Surgeon Operation Volumes among Medical Centers in Taiwan\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4459823/\n\nSHA: f3cbc0503581249a834895fc94cd3bae24714a0d\n\nAuthors: Yu, Tsung-Hsien; Tung, Yu-Chi; Chung, Kuo-Piao\nDate: 2015-06-08\nDOI: 10.1371/journal.pone.0129178\nLicense: cc-by", "Abstract: BACKGROUND: Volume-infection relationships have been examined for high-risk surgical procedures, but the conclusions remain controversial. The inconsistency might be due to inaccurate identification of cases of infection and different methods of categorizing service volumes. This study takes coronary artery bypass graft (CABG) surgical site infections (SSIs) as an example to examine whether a relationship exists between operation volumes and SSIs, when different SSIs case identification, definitions and categorization methods of operation volumes were implemented. METHODS: A population-based cross-sectional multilevel study was conducted. A total of 7,007 patients who received CABG surgery between 2006 and 2008 from19 medical centers in Taiwan were recruited. SSIs associated with CABG surgery were identified using International Classification of Diseases, 9th Revision, Clinical Modification (ICD-9 CM) codes and a Classification and Regression Trees (CART) model. Two", "definitions of surgeon and hospital operation volumes were used: (1) the cumulative CABG operation volumes within the study period; and (2) the cumulative CABG operation volumes in the previous one year before each CABG surgery. Operation volumes were further treated in three different ways: (1) a continuous variable; (2) a categorical variable based on the quartile; and (3) a data-driven categorical variable based on k-means clustering algorithm. Furthermore, subgroup analysis for comorbidities was also conducted. RESULTS: This study showed that hospital volumes were not significantly associated with SSIs, no matter which definitions or categorization methods of operation volume, or SSIs case identification approaches were used. On the contrary, the relationships between surgeon’s volumes varied. Most of the models demonstrated that the low-volume surgeons had higher risk than high-volume surgeons. CONCLUSION: Surgeon volumes were more important than hospital volumes in exploring the", "relationship between CABG operation volumes and SSIs in Taiwan. However, the relationships were not robust. Definitions and categorization methods of operation volume and correct identification of SSIs are important issues for future research.", "Text: data, which should use hierarchical models, may result in biased estimation of the variation and also lead to incorrect conclusions.\n\nSSIs following coronary artery bypass graft (CABG) procedures place a heavy burden on patients and healthcare systems. The total length of stay and expenditure for patients with SSIs after CABG surgery is significantly longer and higher than those without SSIs. [20, 21] In 2008, the Centers for Medicare & Medicaid of the United States of America implemented the \"Never Event\" policy, where hospitals would no longer receive higher payments for the additional costs associated with treating patients for certain healthcare-acquired infections, including those related to CABG.", "In view of the accuracy of SSIs identification and the heterogeneity of definition and categorization methods, no existing studies have used different infection case identification nor definitions and categorization methods of operation volume simultaneously to explore the relationship between operation volumes and infection. The current study takes CABG SSIs as an example to examine whether a relationship exists between operation volumes and SSIs, given different SSI cases identification, operation volume definitions and categorization methods.\n\nThis retrospective and cross-sectional study adopted a multilevel design to examine the relationships between provider volumes and SSIs after adjusting for patient-, surgeon-, and hospital-level covariates.", "We used data from the Taiwan National Health Insurance Research Database (NHIRD) from 2005 and 2008. The NHIRD, published by the Taiwan National Health Research Institute, includes all the original claims data and registration files for beneficiaries enrolled under the National Health Insurance (NHI) program. The database covers the 23 million Taiwanese enrollees (approximately 98% of the population) in the NHI program. It is a de-identified secondary database containing patient-level demographic and administrative information; however, treatment items are aggregated and without time-related and clinical information. The data is released for research purposes.\n\nThe protocol for the study was approved by the Institutional Review Board of the National Taiwan University Hospital (protocol #201001027R). The dataset we used in this study was secondary data; all information was de-identified by data owners.", "In this study, we adopted the ICD-9-CM SSI codes (hereafter referred to as the ICD-9-CM based model) and the Classification and Regression Trees (CART) model, which was developed in our previous work [11] to identify SSI cases. As we mentioned above, the ICD-9-CM SSI codes were the most popular tool to identify the SSI cases in claims data. In the ICD-9-CM based model, SSI cases were divided into two categories: index hospitalization events and post-discharge events (i.e., SSIs that occurred within 1 year after discharge and required readmission to a hospital and/ or the use of ambulatory services). Following Wu et al [13] , this study adopted the secondary ICD-9-CM diagnosis codes for index hospitalization events (ICD-9-CM code: 996.03, 996.61, 996.72, and 998.5), and the primary and secondary diagnosis codes for post-discharge events (ICD-9-CM code: 038.0-038. 4 ) as the criteria for SSI identification, in order to avoid cases in which infection existed prior to hospitalization. If", "a case had an index hospitalization event or a post-discharge event, then he/ she will be identified as SSIs by the ICD-9-CM based model. In the CART model, we adopted the type of antibiotics, dose of cefazolin, length of stay, and number of vessels obstructed (as a proxy indicator of duration of operation) as the parameters to identify the SSIs, according to our previous findings. [11] In our previous work, we used the 2005-2008 National Health Insurance claims data and healthcare-associated infection surveillance data from two medical centers for model development and model verification. Infection cases based on surveillance were identified by infection control personnel if the patient met the Taiwan CDC's criteria, which are the same as those adopted in the U.S. CDC. They manually review medical records of all patients at risk for the specified healthcare-associated infection.", "The classification algorithms, the multivariable regression model, and the data mining model were adopted to develop alternative models based on surrogate indicators to identify cases of CABG SSIs and to compare the performance among these models and the ICD-9-CMbased model. For the classification algorithms, researchers build up several criteria, and if a case satisfies (or exceeds) a specific number of criteria, then it will be identified as a case of infection. For the multivariable regression model, researchers usually calculated a risk score by the logistic regression model, and the optimal cutoff point was determined according to the resulting receiver operating characteristic curve.", "Concerning the data mining approach, which is widely used for predicting and classifying objects, the characteristics are: automatic discovery of patterns, prediction of likely outcomes, creation of actionable information, and focus on large data sets and databases. The classification and regression tree (CART) model, which is the most popular approach as applied in our work, and the growing, stopping, and pruning of the tree were determined by Gini improvement measures. [22, 23] After referring to the literature and conferring with infectious disease specialists, we adopted the following seven parameters: type of antibiotic, doses of antibiotic, doses of cefazolin, use of second-line antibiotics, length of stay, and number of vessels obstructed. Additionally, cross-validation was also employed, where data from one medical center was used for model development, and another one was used for model validation.", "The results of our previous work revealed that the CART model offered better performance than that of the other identification models or the ICD-9-CM based model, especially in the positive predictive value (>70%), which was only found to be 20% in the ICD-9-CM based model. (Table 1 ) The findings also implied that the CART was a decidedly better tool for identifying cases of SSI in the Taiwan National Health Insurance database. Therefore, this study also adopted the CART model for identifying CABG SSIs.\n\nTo ensure homogeneity, current study analyzed 7,007 patients from 19 medical centers in Taiwan who underwent CABG surgery (ICD-9-CM procedure codes 36.1x-36.2x) between 2006 and 2008. CABG patients under the age of 18 years or over 85 years were excluded in this study. A total of 302 cases were identified as SSIs by ICD-9-CM based model, and a total of 107 cases were identified as SSIs by CART model.", "In this study, we used the following two definitions to define operation volumes: (1) the cumulative operation volumes by each surgeon and hospital within the study period, which was the most common definition in the literature; and (2) following Yasunaga et al.'s study, [24] cumulative operation volumes by each surgeon and hospital in the previous one year for each surgery. However, our data was skewed, which did not follow a normal distribution. Therefore, we conducted the log transformations on operation volumes.", "The current work treated operation volumes in three different ways: (1) a continuous variable; (2) a categorical variable based on the first and the third quartile as cutoff points (the most common method to categorize service/ operation volumes) [25] [26] [27] [28] ; and (3) a data-driven categorical variable based on k-means clustering algorithm. This study categorized surgeon and hospital volumes into low, medium, and high volume groups by quartile method and kmeans clustering algorithm.", "In the quartile method, the cut-off value (transformed by logarithm) of the first quartile (<25%) for hospital volumes was 5.65, and the third quartile (>75%) was 6.43. In terms of surgeon volumes, the first quartile was 4.38, and the third was 5.35, when we used the cumulative operation volumes within the study period as the definition. While the definition changed, first quartile (<25%) for hospital volumes was 4.66, and the third quartile (>75%) was 5.31. In terms of surgeon volumes, the first quartile was 3.40, and the third was 4.32.", "K-means clustering is an unsupervised machine-learning algorithm introduced by MacQueen in 1960s. This method is not only a simple and very reliable method in categorization/ classification, but is also recognized as one of the top 10 algorithms in data mining. [29] This method has often been applied in many fields. [30] [31] [32] Yu and his colleagues even applied it to define the quality of CABG care, and to explore the relationship among patient's income status, the level of quality of care, and inpatient mortality. [33] The main idea of this method is to partition observed data points into k non-overlapping clusters by minimizing the within-group sum of squares. Each point is assigned to the mean of its cluster using the Euclidian distance. Firstly, k cluster centers were randomly generated. Previous studies usually divided surgeons and hospitals into low-, medium-, and high-volume groups; therefore, we also predetermined the surgeon and hospital service volumes into 3 groups (k =", "3). Then, participants were assigned to the cluster with the shortest distance to these cluster centers. Finally, the cluster centers were recomputed using the new cluster assignment and these steps would be iterated until convergence was achieved. [34] The cut-off values of hospital volumes were 5.21 and 5.69, and for surgeon's volumes were 2.40 and 4.38 respectively, when cumulative operation volumes within the study period was used as the definition. Likewise, when cumulative operation volumes before each surgery was used as definition, the cut-off values were 4.11 and 4.89 for hospital volumes, and 2.64 and 3.91 for surgeon's volumes. All cutoff values were transformed by logarithm. The results of k-means clustering are demonstrated in Figs 1-4. As the results show, the operation volumes were divided into three groups separately. In addition to surgeon and hospital volumes and SSI, we collected patient-, surgeon-, and hospital-level data. Firstly, patient-level variables included", "age, gender, length of ICU stay, number of vessels obstructed that were involved in the surgical operation, and the presence of important underlying diseases (e.g. diabetes mellitus, chronic obstructive pulmonary disease (COPD), heart failure, renal failure and renal insufficiency, which were associated with SSI).", "[13] Secondly, the surgeon-level variables included age and gender. Thirdly, the hospital-level variables included hospital ownership and geographic location.", "All statistical analyses of volume-infection relationship were performed using SAS (version 9.2, SAS Institution Inc., Cary, NC, USA). In statistical testing, a two-sided p value 0.05 was considered statistically significant. The distributional properties of continuous variables were expressed by mean ± standard deviation (SD), whereas categorical variables were presented by frequency and percentage. In univariate analysis, the potential three-level predictors of SSI were examined using chi-square test or two-sample t-test as appropriate. Next, to account for the correlations within surgeon (level-2) and hospital (level-3), multivariate analysis was conducted by fitting mixed-effects logistic regression models to each patient's data for estimating the effects of three-level predictors on the probability of post-operational SSI. Furthermore, subgroup analysis for comorbidities was also conducted. Table 2 shows that there were 7,007 patients with CABG performed by 199 surgeons in 19", "hospitals during 2006-2008 in Taiwan. The majority of patients were male (77.5%), and the mean age of patients was 65.3 years. The average ICU stay was 6.05 days, the mean level of number of vessels obstructed was around 1.6, while 51.8% of patients had diabetes mellitus, 33.3% had heart failure, 14.1% had renal failure and renal insufficiency, and 22.0% had COPD. Three hundred and two patients (4.31%) were identified as having the ICD-9-CM SSI codes. However, identification by the CART model only revealed 107 infection cases, and 94 cases were identified in both models. Most cases received CABG surgery by male surgeons, with a mean age of 45.0 years, and the surgeon's average operation volumes within the study period was 151.64, while the average operation volumes before surgery was 52.18. More than half of the cases were performed with CABG in not-for-profit hospitals, and the hospitals' average operation volumes within the study period was 473.60, while the average operation", "volumes before each surgery was 158.79. Moreover, most of patients received their surgeries by high-volume surgeons and hospitals, when k-means algorithm was used for categorization, regardless of which definition of operation volumes were used. Table 3 shows the results of multilevel mixed-effect models, with the SSIs being identified by ICD-9-CM codes, and the operation volumes defined as the cumulative volumes within the study period. The results of Model 1 (continuous) reveal that the surgeon's volumes were negatively associated with SSIs, while hospital's volumes were not associated with surgical site infection SSIs. Model 2 (quartile) suggests that low-volume surgeons had higher SSI risk (OR = 2.220, p-value = 0.022) than high-volume surgeons. There were also no associations between hospital's operation volumes and SSIs. Model 3 (k-means) shows that the association did not exist between hospital's/ surgeon's volumes and SSIs. Table 4 displays the results of multilevel", "mixed-effect models, in which the SSIs were identified by the CART model, and the operation volumes were also defined as the cumulative volumes within the study period. Model 1 again indicated a negative association between surgeon's volumes and SSIs, and hospital's volumes were not found to be associated with SSIs. In Model 2, the results showed that the relationship between hospital's/ surgeon's volumes and SSIs did not exist. In Model 3, results revealed low-volume surgeons had higher risk (OR = 1.691, p = 0.002) than high-volume surgeons. Table 5 displays the results of multilevel mixed-effect models, in which the SSIs were identified by ICD-9-CM codes, but the operation volumes were defined as the cumulative volume in the previous one year for each surgery. Model 1 also indicated a negative association between surgeon's volumes and SSIs, and hospital's volumes were not found to be associated with SSIs. In Model 2, the results showed that the relationship between hospital's/", "surgeon's volumes and SSIs did not exist. In Model 3, results also revealed low-volume surgeons had higher risk (OR = 1.642, p = 0.040) than high-volume surgeons. Table 6 displays the results of multilevel mixed-effect models, in which the SSIs were identified by the CART model, and the operation volumes were also defined as the cumulative volume in previous one year for each surgery. In Model 1, different to the above findings, there was no association between hospital's/ surgeon's volumes and SSIs. In Model 2, the results showed that the relationship between hospital's/ surgeon's volumes and SSIs did not exist. In Model 3, results also revealed low-volume surgeons had higher risk (OR = 1.163, p = 0.020) than high-volume surgeons.", "We further examined the associations of surgeon and hospital volumes with SSIs in stratification analyses by underlying diseases. When the operation volumes were defined as the cumulative operation volume within the study period, no relationships existed between hospital/ surgeon operation volumes and SSIs. (Table 7 ) However, when the operation volumes were defined as the cumulative operation volumes in the previous one year for each surgery, the results suggested that there was a negative association between surgeon volumes and SSIs in the diabetes group, except that the volumes were treated as continuous variable and the infection cases were identified by ICD-9 codes. In terms of hospital operation volumes, the association did not exist. (Table 8 )", "No studies have evaluated how different service/ operation volumes definitions and categorization methods affect volume-infection relationships. Moreover, several studies have pointed out the inappropriateness of identifying infection cases using the ICD-9-CM codes in claims data. Given these reasons, this study adopted two approaches to identifying SSIs, two definitions of operation volumes, and three methods for categorizing operation volumes to examine the relationships between operation volumes and SSIs. Our findings showed that the relationships between hospital volumes and SSIs did not exist, no matter which definitions, categorization mehods, or SSIs case identification approaches were used. On the contrary, the relationships between surgeon volumes and SSIs were not robust in our data. It might be affected by different definitions and categorization methods of operation volumes, and also by different SSI cases identification approaches. In summary, most of the models", "demonstrated that the low-volume surgeons had higher risk than high-volume surgeons, and they also showed the risks were similar between medium-volume and high-volume surgeons. However, why did surgeon volume relate to SSIs, but hospital volume did not? Except for those issues we were concerned about in this study, there are some disagreements in the literature. Such as \"Does provider volume really represent quality of care?\" [12, 35] Or \"Is provider volume the only one predictor for outcome of care?\" [35, 36] These issues are worthy of further discussion, but are out of the scope of this study.", "Service/ operation volumes are treated as a proxy indicator for experiences; previous studies used it to examine whether practice makes perfect or not. But, except for provider's experiences, SSIs are also impacted by many factors, such as environmental and clinical factors. Wu et al once used Taiwan 2001 NHI claims data to explore the relationship between provider CABG operation volumes and SSIs. [13] They found that hospital volumes had a greater effect than surgeon volumes and claimed that this may imply that hospital teamwork is more important than individual surgeon. However, our findings demonstrated that there was no relationship between hospital volumes and SSIs. Wu et al. adopted the cumulative operation volumes within the study period as the definition, and identified SSIs by ICD-9-CM codes. Except, there were two differences between our work and Wu et al., which were the length and year of the data; our data was longer and more updated than theirs. Moreover, it is worth", "noting that there was an outbreak of severe acute respiratory syndrome (SARS) in Taiwan in 2003, after which the hospital infection control system in Taiwan was reviewed and re-designed. Wu et al data was before SARS, so these efforts may also have improved the level of SSIs control in hospitals, leading to different findings in this study.", "In addition, although most models revealed that there were negative relationships between surgeon's volumes and surgical site infection, the relationships were not robust. The results varied between different definitions and categorization method of operation volumes, and between SSIs identification approaches. Researchers need to consider how to identify SSIs correctly, how to choose optimal cut-off values, and how to decide on which definition is appropriate.\n\nFinally, the results of stratification analyses showed that low-volume surgeon had higher risk than high-volume surgeon in the diabetes mellitus group, when the cumulative operation in the previous one year before surgery was used as definition. A large number of studies have indicated diabetes mellitus is associated with a higher risk of SSIs, [37] [38] [39] and the findings of this study suggest that CABG patients with diabetes mellitus should be cared for by experienced surgeons.", "A multilevel analysis was applied to manage the nested factors, and two definitions of operation volume along with three different operation volume categorization methods were adopted to examine the relationship between volume and SSIs under two kinds of SSIs identification approaches. Nevertheless, the study suffered from several major limitations. First, the accuracy of SSIs identification was still an issue. Although the performance of the CART model to identify CABG SSIs was better than ICD-9-CM codes in Taiwan NHI claims data, it did not reach the perfect scenario. The accuracy of SSIs identification was still a challenge in our work. The second limitation relates to unmeasured variables, such as length of stay before operation, infection condition, hair removal, clinical information (e.g. blood glucose level, causative microorganism), time-related information (e.g. the duration of operation), the environment, surgical skills, use of post-operative drains, number of operations", "involved, and surgical site and wound care, etc. [40] Furthermore, information about type (elective or urgent) and incision site for surgery was not available in the Taiwan NHI claims data.", "In conclusion, the findings of this study suggest that different definitions and categorization methods of operation volumes, and different SSIs identification approaches might lead to different findings, although surgeon volumes were more important than hospital volumes in exploring the relationships between CABG operation volumes and SSIs in Taiwan, but they were still not robust. Definitions and categorization methods of operation volumes, and correct identification of SSIs are important issues for future research." ]

[ 11 ]

[ "cannot reliably represent effects originating from stochasticity, from effects in small populations, or from heterogeneities" ]

[ "The influenza pandemic preparedness planning tool InfluSim\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC1832202/\n\nSHA: f3f471d10a36a7a28e9050c10bd4dfd680cba17b\n\nAuthors: Eichner, Martin; Schwehm, Markus; Duerr, Hans-Peter; Brockmann, Stefan O\nDate: 2007-03-13\nDOI: 10.1186/1471-2334-7-17\nLicense: cc-by", "Abstract: BACKGROUND: Planning public health responses against pandemic influenza relies on predictive models by which the impact of different intervention strategies can be evaluated. Research has to date rather focused on producing predictions for certain localities or under specific conditions, than on designing a publicly available planning tool which can be applied by public health administrations. Here, we provide such a tool which is reproducible by an explicitly formulated structure and designed to operate with an optimal combination of the competing requirements of precision, realism and generality. RESULTS: InfluSim is a deterministic compartment model based on a system of over 1,000 differential equations which extend the classic SEIR model by clinical and demographic parameters relevant for pandemic preparedness planning. It allows for producing time courses and cumulative numbers of influenza cases, outpatient visits, applied antiviral treatment doses, hospitalizations,", "deaths and work days lost due to sickness, all of which may be associated with economic aspects. The software is programmed in Java, operates platform independent and can be executed on regular desktop computers. CONCLUSION: InfluSim is an online available software which efficiently assists public health planners in designing optimal interventions against pandemic influenza. It can reproduce the infection dynamics of pandemic influenza like complex computer simulations while offering at the same time reproducibility, higher computational performance and better operability.", "Text: Preparedness against pandemic influenza has become a high priority public health issue and many countries that have pandemic preparedness plans [1] . For the design of such plans, mathematical models and computer simulations play an essential role because they allow to predict and compare the effects of different intervention strategies [2] . The outstanding significance of the tools for purposes of intervention optimization is limited by the fact that they cannot maximize realism, generality and precision at the same time [3] . Public health planners, on the other hand, wish to have an optimal combination of these properties, because they need to formulate intervention strategies which can be generalized into recommendations, but are sufficiently realistic and precise to satisfy public health requirements.", "Published influenza models which came into application, are represented by two extremes: generalized but oversimplified models without dynamic structure which are publicly available (e.g. [4] ), and complex computer simulations which are specifically adjusted to real conditions and/or are not publicly available (e.g. [5, 6] ). The complexity of the latter simulations, however, is not necessary for a reliable description of infection dynamics in large populations [7] . A minimum requirement for a pandemic influenza planning tool is a dynamic modelling structure which allows investigation of time-dependent variables like incidence, height of the epidemic peak, antiviral availability etc. The tool should, on the other hand, be adjustable to local conditions to adequately support the pandemic preparedness plans of different countries which involve considerably different assumptions (Table 1) .", "Here we describe a publicly available influenza pandemic preparedness planning tool [8] which is designed to meet the requirements in preparedness planning. It is based on an explicitly formulated dynamic system which allows addressing time-dependent factors. It is sufficiently flexible to evaluate the impact of most candidate interventions and to consider local conditions like demographic and economic factors, contact patterns or constraints within the public health system. In subsequent papers we will also provide examples and applications of this model for various interventions, like antiviral treatment and social distancing measures.", "The model is based on a system of 1,081 differential equations which extend the classic SEIR model. Demographic parameters reflect the situation in Germany in 2005, but can be adjusted to other countries. Epidemiologic and clinic values were taken from the literature (see Tables 1, 2 , 3, 4, 5, 6 and the sources quoted there). Pre-set values can be varied by sliders and input fields to make different assumptions on the transmissibility and clinical severity of a new pandemic strain, to change the costs connected to medical treatment or work loss, or to simply apply the simulation to different demographic settings. Model properties can be summarized as follows. The mathematical formulation of this model is presented in detail in the online supporting material. The corresponding source code, programmed in Java, and further information can be downloaded from [8] .", "According to the German National Pandemic Preparedness Plan [9] , the total population is divided in age classes, each of which is subdivided into individuals of low and high risk ( Table 2) . Transmission between these age classes is based on a contact matrix (Table 3) which is scaled such that the model with standard parameter values yields a given basic reproduction number R0. Values for the R0 associated with an influenza strain with pandemic potential are suggested to lie between 2 and 3 [10] . This value is higher than the effective reproduction number which has been estimated to be slightly lower than 2 [11, 12] . As a standard parameter, we use R0 = 2.5 which means that cases infect on average 2.5 individuals if everybody is susceptible and if no interventions are performed.", "Susceptible individuals who become infected, incubate the infection, then become fully contagious and finally develop protective immunity (Table 4) . A fraction of cases remains asymptomatic; others become moderately sick or clinically ill (i.e. they need medical help). Depending on the combination of age and risk group, a fraction of the clinically ill cases needs to be hospitalized, and an agedependent fraction of hospitalized cases may die from the disease ( Table 5 ). This partitioning of the cases into four categories allows combining the realistic description of the transmission dynamics with an easy calculation of the resources consumed during an outbreak. The degree and duration of contagiousness of a patient depend on the course of the disease; the latter furthermore depends on the age of the patient (Table 5) . Passing through the incubation and contagious period is modelled in several stages which allows for realistic distributions of the sojourn times ( Table 4 ). The last", "two stages of the incubation period are used as early infectious period during which the patient can already spread the disease. Infectiousness is highest after onset of symptoms and thereafter declines geometrically (Table 6 ). Clinically ill patients seek medical help on average one day after onset of symptoms. Very sick patients are advised to withdraw to their home until their disease is over, whereas extremely sick patients need to be hospitalized and may die from the disease (Table 4) . After the end of their contagious period, clinically ill patients go through a convalescent period before they can resume their ordinary life and go back to work (Table 4) .", "We provide some examples of model output of InfluSim [8] , version 2.0, by means of four sensitivity analyses; further investigations will be presented elsewhere. Figure 1 shows the graphical user interface of the software which is divided into input and output windows. The user may set new values in the input fields or move sliders to almost simultaneously obtain new results for the course of an epidemic in a given population. Figures 2A and 2B show pandemic waves which result from varying the basic reproduction number from 1.5 to 4.0. Using the standard parameter values as given in Tables 2, 3 , 4, 5, 6 and omitting all interventions in a town of 100,000 inhabitants results in a pandemic wave which lasts for about ten weeks (Figure 2A , with R 0 = 2.5). The peak of the pandemic wave is reached after six to seven weeks, with a daily incidence of up to 2,340 influenza patients seeking medical help, with up to 280 hospital beds occupied by influenza cases and with up to 14,000 out of", "60,000 working adults unable to go to work because of illness or convalescence. These results depend on the assumptions concerning the yet unknown contagiousness and pathogenicity of the virus. Figures 2C and 2D show how the shape of the curves depends on the course of contagiousness: the pandemic wave proceeds relative slowly if the contagiousness does not change during the infectious period (x 50 = 50%), but proceeds quickly if the contagiousness is highest after onset of symptoms and decreases thereafter (x 50 > 50%).", "The influenza pandemic preparedness planning tool InfluSim stands between simple spreadsheet models and sophisticated stochastic computer simulations. It describes a pandemic wave within a homogeneously mixing population like a town or city, but surprisingly produces the same dynamics as individual-based simulations which explicitly consider geographic spread through the US (cf. [6] and [5] with Figure 2 using R 0 = 2). Similar observations were made with a simple deterministic compartmental model [7] . Stochastic models are known to behave quasi-deterministically when the simulated population becomes very large.", "A further reason for the congruence of complex stochastic and simple deterministic models must lie in the incredi-bly quick way in which pandemic influenza spreads geographically. Unless being controlled at the place of origin [12, 13] , a pandemic starting in a far-off country will lead to multiple introductions [14] into the large industrialized nations where it can be expected to quickly spread to neighbouring towns and to rural areas. The large populations which have to be considered susceptible to a pandemic virus and the quick geographic spread tend to diminish the differences between the results of sophisticated individual-based and simple deterministic models.", "However, a deterministic model like InfluSim cannot reliably represent effects originating from stochasticity, from effects in small populations, or from heterogeneities. Examples are: (i) a geographically limited spread and fairly effective control measures can imply that the epidemic affects only a small population and thus, may be strongly influenced by stochastic events [15] [16] [17] ; (ii) transmission which predominantly occurs in households or hospitals, or which is driven by other substantial features of the contact network is not in agreement with the assumption of homogeneous mixing in the deterministic model cannot reliably predict the spread of infection [18] [19] [20] [21] [22] [23] . In particular, (iii) super-spreading events can substantially change the course of an epidemic compared to the deterministic prediction [24] [25] [26] [27] . Apart from such factors, the predictability of intervention success is generally subject to uncertainties in the choice of parameter", "values, Assumed scenarios and outcomes of pandemic preparedness plans. * Gross attack rate (i.e. clinically ill and moderately ill cases). A population of N = 100,000 inhabitants of Germany is subdivided according to age a and risk category r. We assume that all age groups are fully susceptible at begin of the outbreak. A fraction of F a = 6% of all children (age < 20 years) are regarded as being under high risk (r = r 1 ) after an influenza infection whereby the remaining 94% are under low risk (r = r 2 ). The high risk fractions of working adults (ages 20-59) and elderly (ages 60+) are F a = 14% and F a = 47%, respectively. Source: [9] demanding additional efforts like Bayesian approaches [28] to evaluate the reliability of predictions [29] .", "Pandemic preparedness plans must consider constraints and capacities of locally operating public health systems. The time-dependent solutions of InfluSim allow assessing peak values of the relevant variables, such as outpatients, hospitalizations and deaths. Various interventions may be combined to find optimal ways to reduce the total number of cases, to lower the peak values or to delay the peak, hoping that at least part of the population may benefit from a newly developed vaccine.", "Special care was taken when implementing a variety of pharmaceutical and non-pharmaceutical interventions which will be discussed in subsequent papers. Despite its comprehensible structure, the model does not suffer from over-simplifications common to usual compartment models. Instead of implicitly using exponentially distributed sojourn times, we have implemented realistically distributed delays. For example, the model considers that individuals may transmit infection before onset of symptoms, and that some cases may remain asymptomatic, but still infecting others. Such features have serious implications for the success of targeted control measures.", "InfluSim is freely accessible, runs on a regular desktop computer and produces results within a second after changing parameter values. The user-friendly interface and the ease at which results can be generated make this program a useful public health planning tool. Although we have taken care of providing a bug-free program, including the source code, the user is encouraged to treat results with due caution, to test it, and to participate in bug-reports and discussions on the open-source platform [30] which also provides regular updates of InfluSim. \n\nThe author(s) declare that they have no competing interests.\n\nME developed the model, MS designed the software, HPD wrote the manuscript and SOB formulated the public The who-acquires-infection-from-whom matrix shows the frequency of contacts (per week per person) between different age classes.", "Source: [38] . Distribution of sojourn times (the last two stages of the latent period are used as early infectious period with an average duration of D L = 0.5 days). Sources: A [11] , B [39, 40] , C assumed, D [41] health requirements of the software. All authors read and approved the final manuscript.\n\nSusceptible individuals S a, r are infected at a rate λ a (t) which depends on their age a and on time t. Infected individuals, E a, r , incubate the infection for a mean duration D E . To obtain a realistic distribution of this duration, the incubation period is modelled in n stages so that progression from one stage to the next one occurs at rate δ = n/D E .", "The last l incubation stages are regarded as early infectious period during which patients may already spread the infection (this accounts for an average time of lD E /n for the \"early infectious period\" which is about half a day for the standard set of parameters). After passing through the last incubation stage, infected individuals become fully contagious and a fraction of them develops clinical symptoms. The course of disease depends on the age a of the infected individual and on the risk category r to which he or she belongs: a fraction c a, r (A) becomes asymptomatic (A a ), a fraction c a, r (M) becomes moderately sick (M a ), a fraction c a, r (V) becomes very sick (V a ) and the remaining fraction c a, r (X) becomes extremely sick (X a ) and need hospitalization (i.e., c a, r (A) + c a, r (M) + c a, r (V) + c a, r (X) = 1 for each combination of a and r). ) . A fraction f V (t) of all severe and a fraction f X (t) of all extremely severe cases who visit the doctor within D T", "days after onset of symptoms are offered antiviral treatment, given that its supply has not yet been exhausted. As our model does not explicitly consider the age of the disease (which would demand partial differential equations), we use the contagious stages to measure time since onset and allow for treatment up to stage m a, T Sources: Contagiousness of asymptomatic cases: [11] ; degree of contagiousness during the early infectious period and equality of the contagiousness of moderately and severely sick cases: assumed. Independent of age a and risk group r, a fraction c a, r (A) = 33% of infections result in asymptomatic cases, a fraction c a, r (M) = 33.5% become moderately sick and the remaining fraction develops severe disease. An age-and risk-dependent fraction h a, r of untreated patients with severe disease needs hospitalization. An age-dependent fraction d a of hospitalized cases dies. Sources: fraction of asymptomatic cases: [11] ; 50% of symptomatic cases see a doctor: [9]", "; hospitalizations per severe case: [9] ; case fatality of hospitalized, but untreated patients calculated from [4] .", "(see below for details). This imposes some variability to the maximum time until which treatment can be given, which may even improve the realism of the model with respect to real-life scenarios. Antiviral treatment reduces the patients' contagiousness by f I percent and it reduces hospitalization and death by f H percent. Extremely sick patients, whose hospitalization is prevented by treatment, are sent home and join the group of treated very sick patients(W a, T ). The remaining duration of disease and contagiousness of treated cases is reduced by f D percent so that their rate of progressing from one stage to the next has To obtain a realistic distribution of this sojourn time, convalescence is modelled in j stages so that progression from one stage to the next occurs at rate ρ = j/D C . Fully recovered patients who have passed through their last stage of convalescence join the group of healthy immunes I; working adults will go back to work. Further interventions, describing the", "reduction of contacts, will be discussed after the presentation of the differential equations.", "InfluSim user interface Figure 1 InfluSim user interface. x 50 = 95% means that 95% of the cumulative contagiousness is concentrated during the first half of the contagious period, see Table 6 ). D: Cumulative number of deaths for values of x 50 as in C. All other parameters as listed in Tables 2-6 . Hospitalized, but untreated cases\n\nContact matrix For the mixing of the age classes, we employ a whoacquires-infection-from whom matrix which gives the relative frequency of contacts of infective individuals of age a i with other people of age a s . In this paper, we assume bi-directional contacts (e.g. children have the same total number of contacts with adults as adults with children). Multiplication of this matrix with an appropriate constant scaling factor κ (see below)\n\nresults in the matrix of crude contact rates .\n\nIn the absence of interventions, we have to multiply these contact rates with the contagiousness factors b L , b A , b M and b V to obtain the effective contact rates:", "during the early infectious period, of asymptomatic cases, of moderately sick cases, of (untreated) very sick cases.\n\nTo assess the effect of day care centre and school closing on the transmission of an infectious disease, we have to first make an assumption on what fraction r sch of the contacts among healthy children who are in the same age class occurs in day care centres and schools. The contact rates between very sick or hospitalized children (who do not attend day care centre or school) and other children need, therefore, be reduced to (contact rate between healthy and very sick children in the same age class, i.e. a i = a s ).\n\nAs very sick children have to be taken care of by adults at home or in hospital, their contact rate to adults increases by a factor F HC (contact rate between very sick children of age a i and adults of age a s ).", "Contacts between very sick children and other children in a higher or lower age class remain unchanged: (contact rate between healthy children of age a s and very sick children of a different age a i ).\n\nClosing day care centres and schools at time t will not necessarily prevent all the contacts that would have happened with other children. During the closing of schools and day care centres, the contact rates between susceptible children of age a s and infected children of age a i who are in their late incubation period ( ), who are asymptomatic ( ), or who are moderately sick ( ) are reduced by the factor r sch if the children are in the same age class:\n\nwhere 1 sch (t) is a function which indicates when schools and day care centres are opened or closed: \n\n,..., While day care centres and schools are closed, children (age a i ) need adult supervision at home. Their contact with susceptible adults (age a s ) increases by the \"child care factor\" F CC :", "Child care at home also increases the exposure of healthy children (age a s ) to contagious adults (age a i ):\n\nCancelling mass gathering events effects only the contacts of adults who are healthy enough to attend such events. Assuming that such an intervention at time t reduces contacts by a fraction r mass , we get for all contacts between susceptible adults of age a s and infectious adults of age a i the following contact rates:\n\nwhere 1 mass (t) is a function which indicates when mass gathering events are possible or when they are closed:\n\nAs contacts with adults who are too sick to attend such mass gathering events cannot be prevented by this measure it is .", "During some time in the epidemic, the general population may effectively reduce contacts which can be a result of wearing facial masks, increasing \"social distance\", adopting improved measures of \"respiratory hygiene\" or simply of a general change in behaviour. This will be implemented in the program by reducing the contacts of susceptible individuals at that time t by factor r gen (t \n\nwhile mass gathering events are forbidden while m mass gathering events are allowed. while the population reduces their contacts while the population behaves as usual.\n\nThe contact rates of cases in the late incubation period and that of asymptomatic cases remain unchanged:\n\nfor infected individuals in the late incubation period, for asymptomatic cases.\n\nTo allow for a contagiousness which changes over the course of disease, we multiply each contact rate with a weighting factor whereby k is the stage of contagiousness. This leads to the following contact rates:", "for asymptomatic cases in For x = 1, contagiousness is equally high in all stages; for x = 0, only the first stage is contagious; for 0 <x < 1, the contagiousness decreases in a geometric procession. We make the simplifying assumption that contagiousness does not change during the late incubation period for cases in stage k = n -l,..,n of the incubation period.\n\nAt time t = 0 and in the absence of interventions, the next generation matrix has the following elements where is the fraction of untreated extremely severe cases who die from the disease (see below for details). The dominant eigenvalue of this matrix is called the basic reproduction number R 0 . If κ (which determines the value of the contact rates ) is given, the eigenvectors of this matrix can numerically be calculated. The user-specified value of R 0 is now used to determine numerically the scaling factor κ. Let be the eigenvector which has the largest eigenvalue R 0 . \n\n) ) −", "Using the user-specified numbers of people N a in the age classes and the fractions F a of people under high risk within each age class (Table 2) , we obtain the initial population sizes according to age and risk class: Using these initial values, the set of differential equations is solved numerically with a Runge-Kutta method with step-size control. \n\nif and in treatment window otherwise 0 ⎩ ⎩" ]

[ 14 ]

[ "fecal-oral contact" ]

[ "Genome Sequences of Porcine Epidemic Diarrhea Virus: In Vivo and In Vitro Phenotypes\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4056290/\n\nSHA: f6d6d7efc1686a7d219ecfc55f9a48ce72d4fb00\n\nAuthors: Lawrence, Paulraj K.; Bumgardner, Eric; Bey, Russell F.; Stine, Douglas; Bumgarner, Roger E.\nDate: 2014-06-12\nDOI: 10.1128/genomea.00503-14\nLicense: cc-by", "Abstract: Since the outbreak of porcine epidemic diarrhea virus (PEDV) in May 2013, U.S. swine producers have lost almost five million baby pigs. In an attempt to understand the evolution of PEDV in the United States and possibly develop a control strategy, we compared the genome sequences of a PEDV strain isolated from an infected piglet against its in vitro adapted version. The original PEDV strain was grown in Vero cells and passed 10 times serially in a MARC145 cell line. The sequence analysis of the native PEDV strain and in vitro passaged virus shows that the cell culture adaptation specifically modifies PEDV spike protein whereas the open reading frame 1a/b (ORF1a/b)-encoded polyprotein, the nucleoprotein, NS3B (ORF3), and membrane and envelope proteins remain unchanged.", "Text: highly contagious swine disease. While older pigs have a chance of survival, 80 to 100 percent of PEDV-infected piglets die within 24 h of being infected. PEDV spreads primarily through fecal-oral contact (1, 2) . Once the virus is internalized, it destroys the lining of piglets' intestines, making them incapable of digesting and deriving nutrition from milk and feed (1) . The virus causes diarrhea, vomiting, and death from dehydration and starvation (2) .\n\nPEDV is a member of the Coronavirinae subfamily and belongs to the Alphacoronavirus genus. Its genomic size ranges from approximately 26 to 32 kb, which is relatively large for an RNA virus. Although vaccines for PEDV exist in China, Japan, and South Korea, there is no approved vaccine in the United States or Europe (3) . Furthermore, PEDV is still evolving within the U.S. swine population.", "This report briefly describes the comparison of genome sequences of a PEDV strain isolated from small intestine samples of an infected piglet and its in vitro adapted version. The original PEDV strain was dubbed NPL-PEDV/2013, grown in Vero cells, and passed 10 times in a MARC145 cell line. The serial in vitro passage strain was named NPL-PEDV/2013/P10. The total viral RNA was extracted by TRIzol LS reagent and sequenced by Sanger dideoxy sequencing using a primer walking technique. The raw sequences were imported into the Geneious assembler (Biomatters, CA), assembled, annotated, and compared against each other using USA/Colorado/2013 (GenBank accession no. KF272920) as a reference sequence.", "The whole-genome sequences of NPL-PEDV/2013 and NPL-PEDV/2013/P10 contain 28,038 and 28,025 nucleotides (nt), respectively, including the 5= and 3= untranslated regions (UTR). The NPL-PEDV/2013 genome shares 99% identity with all the U.S. isolates sequenced to date and many Chinese isolates as well. The top three BLAST hits were against U.S. isolates, USA/Colora-do/2013 (GenBank accession no. KF272920), IA1 (GenBank accession no. KF468753.1), and an isolate from Iowa, 13-019349 (GenBank accession no. KF267450.1). The NPL-PEDV/2013 isolate also shares 99% identity with the Chinese outbreak isolate AH2012 (GenBank accession no. KC210145).", "When the NPL-PEDV/2013/P10 strain was compared against NPL-PEDV/2013 , the open reading frame 1a/b (ORF1a/b) polyprotein, the nucleoprotein, NS3B, and membrane and envelope proteins were found to be 100% identical at the amino acid level. In contrast, the spike gene contains six nonsynonymous single nucleotide polymorphisms, resulting in amino acid (aa) substitutions in the following positions: 375 (F¡L), 486 (T¡P), 856 (D¡E), 1081 (A¡V), 1099 (A¡S), and 1253 (Y¡D). The S1 domain of spike protein contains 2 aa substitutions, whereas the S2 domain contains 4 aa substitutions. PEDV has been shown to use porcine aminopeptidase N (pAPN) as the major receptor for cell entry (4, 5) . However, Vero and MARC145 cells lack pAPN, clearly indicating that other receptors or receptor-independent pathways may be used for entry (6) . The spike protein in its trimeric conformation interacts with the cell receptor and contains numerous neutralizing antibody binding epitopes (7) . Analysis of the spike", "by PeptideCutter (http://web.expasy.org/ peptide_cutter/) shows that the native spike protein of NPL-PEDV/2013 has 63 trypsin and 2 chymotrypsin cleavage sites at 100% efficiency whereas NPL-PEDV/2013/P10 has lost one trypsin cleavage site but the number of chymotrypsin sites remain unchanged. This indicates that cell culture adaptation specifically modifies the PEDV spike protein; however, the immunological implications are unknown.", "Nucleotide sequence accession numbers. The whole-genome sequences of the NPL-PEDV/2013 and NPL-PEDV/2013/P10 strains have been deposited at DDBJ/EMBL/GenBank under accession no. KJ778615 and KJ778616." ]

[ 2 ]

[ "destroys the lining of piglets' intestines" ]

[ "Genome Sequences of Porcine Epidemic Diarrhea Virus: In Vivo and In Vitro Phenotypes\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4056290/\n\nSHA: f6d6d7efc1686a7d219ecfc55f9a48ce72d4fb00\n\nAuthors: Lawrence, Paulraj K.; Bumgardner, Eric; Bey, Russell F.; Stine, Douglas; Bumgarner, Roger E.\nDate: 2014-06-12\nDOI: 10.1128/genomea.00503-14\nLicense: cc-by", "Abstract: Since the outbreak of porcine epidemic diarrhea virus (PEDV) in May 2013, U.S. swine producers have lost almost five million baby pigs. In an attempt to understand the evolution of PEDV in the United States and possibly develop a control strategy, we compared the genome sequences of a PEDV strain isolated from an infected piglet against its in vitro adapted version. The original PEDV strain was grown in Vero cells and passed 10 times serially in a MARC145 cell line. The sequence analysis of the native PEDV strain and in vitro passaged virus shows that the cell culture adaptation specifically modifies PEDV spike protein whereas the open reading frame 1a/b (ORF1a/b)-encoded polyprotein, the nucleoprotein, NS3B (ORF3), and membrane and envelope proteins remain unchanged.", "Text: highly contagious swine disease. While older pigs have a chance of survival, 80 to 100 percent of PEDV-infected piglets die within 24 h of being infected. PEDV spreads primarily through fecal-oral contact (1, 2) . Once the virus is internalized, it destroys the lining of piglets' intestines, making them incapable of digesting and deriving nutrition from milk and feed (1) . The virus causes diarrhea, vomiting, and death from dehydration and starvation (2) .\n\nPEDV is a member of the Coronavirinae subfamily and belongs to the Alphacoronavirus genus. Its genomic size ranges from approximately 26 to 32 kb, which is relatively large for an RNA virus. Although vaccines for PEDV exist in China, Japan, and South Korea, there is no approved vaccine in the United States or Europe (3) . Furthermore, PEDV is still evolving within the U.S. swine population.", "This report briefly describes the comparison of genome sequences of a PEDV strain isolated from small intestine samples of an infected piglet and its in vitro adapted version. The original PEDV strain was dubbed NPL-PEDV/2013, grown in Vero cells, and passed 10 times in a MARC145 cell line. The serial in vitro passage strain was named NPL-PEDV/2013/P10. The total viral RNA was extracted by TRIzol LS reagent and sequenced by Sanger dideoxy sequencing using a primer walking technique. The raw sequences were imported into the Geneious assembler (Biomatters, CA), assembled, annotated, and compared against each other using USA/Colorado/2013 (GenBank accession no. KF272920) as a reference sequence.", "The whole-genome sequences of NPL-PEDV/2013 and NPL-PEDV/2013/P10 contain 28,038 and 28,025 nucleotides (nt), respectively, including the 5= and 3= untranslated regions (UTR). The NPL-PEDV/2013 genome shares 99% identity with all the U.S. isolates sequenced to date and many Chinese isolates as well. The top three BLAST hits were against U.S. isolates, USA/Colora-do/2013 (GenBank accession no. KF272920), IA1 (GenBank accession no. KF468753.1), and an isolate from Iowa, 13-019349 (GenBank accession no. KF267450.1). The NPL-PEDV/2013 isolate also shares 99% identity with the Chinese outbreak isolate AH2012 (GenBank accession no. KC210145).", "When the NPL-PEDV/2013/P10 strain was compared against NPL-PEDV/2013 , the open reading frame 1a/b (ORF1a/b) polyprotein, the nucleoprotein, NS3B, and membrane and envelope proteins were found to be 100% identical at the amino acid level. In contrast, the spike gene contains six nonsynonymous single nucleotide polymorphisms, resulting in amino acid (aa) substitutions in the following positions: 375 (F¡L), 486 (T¡P), 856 (D¡E), 1081 (A¡V), 1099 (A¡S), and 1253 (Y¡D). The S1 domain of spike protein contains 2 aa substitutions, whereas the S2 domain contains 4 aa substitutions. PEDV has been shown to use porcine aminopeptidase N (pAPN) as the major receptor for cell entry (4, 5) . However, Vero and MARC145 cells lack pAPN, clearly indicating that other receptors or receptor-independent pathways may be used for entry (6) . The spike protein in its trimeric conformation interacts with the cell receptor and contains numerous neutralizing antibody binding epitopes (7) . Analysis of the spike", "by PeptideCutter (http://web.expasy.org/ peptide_cutter/) shows that the native spike protein of NPL-PEDV/2013 has 63 trypsin and 2 chymotrypsin cleavage sites at 100% efficiency whereas NPL-PEDV/2013/P10 has lost one trypsin cleavage site but the number of chymotrypsin sites remain unchanged. This indicates that cell culture adaptation specifically modifies the PEDV spike protein; however, the immunological implications are unknown.", "Nucleotide sequence accession numbers. The whole-genome sequences of the NPL-PEDV/2013 and NPL-PEDV/2013/P10 strains have been deposited at DDBJ/EMBL/GenBank under accession no. KJ778615 and KJ778616." ]

[ 2 ]

[ "26 to 32 kb" ]

[ "Genome Sequences of Porcine Epidemic Diarrhea Virus: In Vivo and In Vitro Phenotypes\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4056290/\n\nSHA: f6d6d7efc1686a7d219ecfc55f9a48ce72d4fb00\n\nAuthors: Lawrence, Paulraj K.; Bumgardner, Eric; Bey, Russell F.; Stine, Douglas; Bumgarner, Roger E.\nDate: 2014-06-12\nDOI: 10.1128/genomea.00503-14\nLicense: cc-by", "Abstract: Since the outbreak of porcine epidemic diarrhea virus (PEDV) in May 2013, U.S. swine producers have lost almost five million baby pigs. In an attempt to understand the evolution of PEDV in the United States and possibly develop a control strategy, we compared the genome sequences of a PEDV strain isolated from an infected piglet against its in vitro adapted version. The original PEDV strain was grown in Vero cells and passed 10 times serially in a MARC145 cell line. The sequence analysis of the native PEDV strain and in vitro passaged virus shows that the cell culture adaptation specifically modifies PEDV spike protein whereas the open reading frame 1a/b (ORF1a/b)-encoded polyprotein, the nucleoprotein, NS3B (ORF3), and membrane and envelope proteins remain unchanged.", "Text: highly contagious swine disease. While older pigs have a chance of survival, 80 to 100 percent of PEDV-infected piglets die within 24 h of being infected. PEDV spreads primarily through fecal-oral contact (1, 2) . Once the virus is internalized, it destroys the lining of piglets' intestines, making them incapable of digesting and deriving nutrition from milk and feed (1) . The virus causes diarrhea, vomiting, and death from dehydration and starvation (2) .\n\nPEDV is a member of the Coronavirinae subfamily and belongs to the Alphacoronavirus genus. Its genomic size ranges from approximately 26 to 32 kb, which is relatively large for an RNA virus. Although vaccines for PEDV exist in China, Japan, and South Korea, there is no approved vaccine in the United States or Europe (3) . Furthermore, PEDV is still evolving within the U.S. swine population.", "This report briefly describes the comparison of genome sequences of a PEDV strain isolated from small intestine samples of an infected piglet and its in vitro adapted version. The original PEDV strain was dubbed NPL-PEDV/2013, grown in Vero cells, and passed 10 times in a MARC145 cell line. The serial in vitro passage strain was named NPL-PEDV/2013/P10. The total viral RNA was extracted by TRIzol LS reagent and sequenced by Sanger dideoxy sequencing using a primer walking technique. The raw sequences were imported into the Geneious assembler (Biomatters, CA), assembled, annotated, and compared against each other using USA/Colorado/2013 (GenBank accession no. KF272920) as a reference sequence.", "The whole-genome sequences of NPL-PEDV/2013 and NPL-PEDV/2013/P10 contain 28,038 and 28,025 nucleotides (nt), respectively, including the 5= and 3= untranslated regions (UTR). The NPL-PEDV/2013 genome shares 99% identity with all the U.S. isolates sequenced to date and many Chinese isolates as well. The top three BLAST hits were against U.S. isolates, USA/Colora-do/2013 (GenBank accession no. KF272920), IA1 (GenBank accession no. KF468753.1), and an isolate from Iowa, 13-019349 (GenBank accession no. KF267450.1). The NPL-PEDV/2013 isolate also shares 99% identity with the Chinese outbreak isolate AH2012 (GenBank accession no. KC210145).", "When the NPL-PEDV/2013/P10 strain was compared against NPL-PEDV/2013 , the open reading frame 1a/b (ORF1a/b) polyprotein, the nucleoprotein, NS3B, and membrane and envelope proteins were found to be 100% identical at the amino acid level. In contrast, the spike gene contains six nonsynonymous single nucleotide polymorphisms, resulting in amino acid (aa) substitutions in the following positions: 375 (F¡L), 486 (T¡P), 856 (D¡E), 1081 (A¡V), 1099 (A¡S), and 1253 (Y¡D). The S1 domain of spike protein contains 2 aa substitutions, whereas the S2 domain contains 4 aa substitutions. PEDV has been shown to use porcine aminopeptidase N (pAPN) as the major receptor for cell entry (4, 5) . However, Vero and MARC145 cells lack pAPN, clearly indicating that other receptors or receptor-independent pathways may be used for entry (6) . The spike protein in its trimeric conformation interacts with the cell receptor and contains numerous neutralizing antibody binding epitopes (7) . Analysis of the spike", "by PeptideCutter (http://web.expasy.org/ peptide_cutter/) shows that the native spike protein of NPL-PEDV/2013 has 63 trypsin and 2 chymotrypsin cleavage sites at 100% efficiency whereas NPL-PEDV/2013/P10 has lost one trypsin cleavage site but the number of chymotrypsin sites remain unchanged. This indicates that cell culture adaptation specifically modifies the PEDV spike protein; however, the immunological implications are unknown.", "Nucleotide sequence accession numbers. The whole-genome sequences of the NPL-PEDV/2013 and NPL-PEDV/2013/P10 strains have been deposited at DDBJ/EMBL/GenBank under accession no. KJ778615 and KJ778616." ]

[ 2 ]

[ "Lassa and Lujo viruses" ]

[ "Serological Assays Based on Recombinant Viral Proteins for the Diagnosis of Arenavirus Hemorrhagic Fevers\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3497043/\n\nSHA: f1d308db379b3c293bcfc8fe251c043fe8842358\n\nAuthors: Fukushi, Shuetsu; Tani, Hideki; Yoshikawa, Tomoki; Saijo, Masayuki; Morikawa, Shigeru\nDate: 2012-10-12\nDOI: 10.3390/v4102097\nLicense: cc-by", "Abstract: The family Arenaviridae, genus Arenavirus, consists of two phylogenetically independent groups: Old World (OW) and New World (NW) complexes. The Lassa and Lujo viruses in the OW complex and the Guanarito, Junin, Machupo, Sabia, and Chapare viruses in the NW complex cause viral hemorrhagic fever (VHF) in humans, leading to serious public health concerns. These viruses are also considered potential bioterrorism agents. Therefore, it is of great importance to detect these pathogens rapidly and specifically in order to minimize the risk and scale of arenavirus outbreaks. However, these arenaviruses are classified as BSL-4 pathogens, thus making it difficult to develop diagnostic techniques for these virus infections in institutes without BSL-4 facilities. To overcome these difficulties, antibody detection systems in the form of an enzyme-linked immunosorbent assay (ELISA) and an indirect immunofluorescence assay were developed using recombinant nucleoproteins (rNPs) derived from", "these viruses. Furthermore, several antigen-detection assays were developed. For example, novel monoclonal antibodies (mAbs) to the rNPs of Lassa and Junin viruses were generated. Sandwich antigen-capture (Ag-capture) ELISAs using these mAbs as capture antibodies were developed and confirmed to be sensitive and specific for detecting the respective arenavirus NPs. These rNP-based assays were proposed to be useful not only for an etiological diagnosis of VHFs, but also for seroepidemiological studies on VHFs. We recently developed arenavirus neutralization assays using vesicular stomatitis virus (VSV)-based pseudotypes bearing arenavirus recombinant glycoproteins. The goal of this article is to review the recent advances in developing laboratory diagnostic assays based on recombinant viral proteins for the diagnosis of VHFs and epidemiological studies on the VHFs caused by arenaviruses.", "Text: The virus family Arenaviridae consists of only one genus, but most viruses within this genus can be divided into two different groups: the Old World arenaviruses and the New World arenaviruses (also known as the Tacaribe complex) [1, 2] . The differences between the two groups have been established through the use of serological assays. Most of the arenaviruses cause persistent infection in rodents without any symptoms, and humans acquire a variety of diseases when zoonotically infected. Lymphocytic choriomeningitis virus (LCMV) is the only arenavirus to exhibit a worldwide distribution, and causes illnesses such as meningitis [3, 4] . Congenital LCMV infections have also been reported [4, 5] . Most importantly, viral hemorrhagic fever (VHF) can be caused by several arenaviruses. Lassa fever, caused by the Lassa virus (LASV), an Old World arenavirus, is one of the most devastating VHFs in humans [6] . Hemorrhaging and organ failure occur in a subset of patients infected with", "this virus, and it is associated with high mortality. Many cases of Lassa fever occur in Western Africa in countries such as Guinea, Sierra Leone, and Nigeria [7] [8] [9] [10] [11] [12] [13] . Tacaribe complex lineage B of the New World arenaviruses consists of the Junin virus (JUNV), Guanarito virus (GUNV), Sabia virus (SABV) and Machupo virus (MACV), the etiological agents of Argentine, Venezuelan, Brazilian, and Bolivian hemorrhagic fevers, respectively [14, 15] . Although genetically distinct from one another, they appear to produce similar symptoms, accompanied by hemorrhaging in humans [14, 15] . These pathogenic New World arenavirus species are closely associated with a specific rodent species [6] .", "Humans are usually infected with pathogenic arenaviruses through direct contact with tissue or blood, or after inhaling aerosolized particles from urine, feces, and saliva of infected rodents. After an incubation period of 1-3 weeks, infected individuals abruptly develop fever, retrosternal pain, sore throat, back pain, cough, abdominal pain, vomiting, diarrhea, conjunctivitis, facial swelling, proteinuria, and mucosal bleeding. Neurological problems have also been described, including hearing loss, tremors, and encephalitis. Because the symptoms of pathogenic arenavirus-related illness are varied and nonspecific, the clinical diagnosis is often difficult [14, 16] . Human-to-human transmission may occur via mucosal or cutaneous contact, or through nosocomial contamination [14, 16] . These viruses are also considered to be potential bioterrorism agents [2] .", "A number of arenavirus species have been recently discovered as a result of both rodent surveys and disease outbreaks [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] . A novel pathogenic New World arenavirus, Chapare virus (CHPV), has been isolated from a fatal case of VHF in Bolivia [20] . In addition, five cases of VHF have been reported in South Africa, and a novel arenavirus, named Lujo virus, was isolated from a patient [17] . The Lujo virus is most distantly related to the other Old World arenaviruses [17] . To date, there is no information concerning the vertebrate host for the Chapare and Lujo viruses.", "There is some evidence of endemicity of the Lassa virus in neighboring countries [27, 28] . However, as the magnitude of international trade and travel is continuously increasing, and the perturbation of the environment (due either to human activity or natural ecological changes) may result in behavioral changes of reservoir rodents, highly pathogenic arenaviruses could be introduced to virus-free countries from endemic areas. In fact, more than twenty cases of Lassa fever have been reported outside of the endemic region in areas such as the USA, Canada, Europe, and Japan [29] [30] [31] [32] [33] . It is of great importance to detect these pathogens rapidly and specifically in order to minimize the risk and scale of outbreaks of VHFs caused by arenaviruses. However, these arenaviruses are classified as biosafety level (BSL)-4 pathogens, making it difficult to develop diagnostic techniques for these virus infections in laboratories without BSL-4 facilities. To overcome these", "difficulties, we have established recombinant viral nucleoproteins (rNPs)-based serological assays, such as IgG-enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IFA), and antigen (Ag)-capture ELISA for the diagnosis of VHFs caused by highly pathogenic arenaviruses. Furthermore, virus neutralization assays using pseudotype virus-bearing arenavirus GPs have been developed. In this review, we describe the usefulness of such recombinant protein-based diagnostic assays for diagnosing VHFs caused by arenaviruses.", "In outbreaks of VHFs, infections are confirmed by various laboratory diagnostic methods. Virus detection is performed by virus isolation, reverse transcription-polymerase chain reaction (RT-PCR), and antigen-capture ELISA. It has been shown that monoclonal antibody panels against pathogenic arenaviruses are useful for detecting viral antigens on the virus-infected cells as well as for investigating of antigenic relationships of arenaviruses [34] [35] [36] . Detection of the virus genome is suitable for a rapid and sensitive diagnosis of VHF patients in the early stage of illness, and extensive reviews of such RT-PCR assays have been described [37, 38] . More recently, progress in the RT-PCR method covering genetic variations of the hemorrhagic fever viruses (HFVs) [39, 40] and a multiplexed oligonucleotide microarray for the differential diagnosis of VHFs have also been reported [41] . On the other hand, antibodies against these viruses can be detected by the indirect", "immunofluorescence assay (IFA), or IgG-and IgM-ELISA. An IFA detects the antibody in the serum, which is able to bind to the fixed monolayer of the virus-infected cells. Although the interpretation of immunofluorescence results requires experience, the assay has advantages over other methods, since each virus generates a characteristic fluorescence pattern that adds specificity to the assay compared to a simple ELISA readout. A serological diagnosis by the detection of specific IgM and IgG antibodies to the HFVs must be sensitive, specific and reliable, because a misdiagnosis can lead to panic in the general population. An IgM-specific ELISA is suitable for detecting recent infection, but the relevance of IgM testing for acute VHF depends on the virus and the duration of illness; specific IgM is not often present in the very early stage of illness, and patients who die of VHF often fail to seroconvert at all. An IgG-specific ELISA is efficacious, not only in the diagnosis of a large", "number of VHF cases, especially during convalescence, but also for epidemiological studies in the endemic regions. The detailed methods used for the IFA and IgG-and IgM-ELISAs for the diagnosis of VHF using authentic virus-antigens have been described in detail [42] [43] [44] [45] .", "Arenaviruses have a bisegmented, negative-sense, single stranded RNA genome with a unique ambisense coding strategy that produces just four known proteins: a glycoprotein, a nucleoprotein (NP), a matrix protein (Z), and a polymerase (L) [46] . Of these proteins, the NP is the most abundant in virus-infected cells. Recombinant protein technology could meet the demand for a simple and reliable VHF test system, and recombinant NP (rNP) has been shown to be useful for serological surveys of IgM-and IgG antibodies against arenaviruses [47] [48] [49] [50] .", "Recombinant baculoviruses that express the full-length rNP of arenaviruses have been generated [48, 50, 51] . The method used for the purification of arenavirus rNP from insect Tn5 cells infected with recombinant baculoviruses is effective and simple compared to those for Ebola, Marburg, and Crimean-Congo hemorrhagic fever virus rNPs [51] [52] [53] [54] [55] . Most of the arenavirus rNPs expressed in insect cells using the recombinant baculoviruses are crystallized [56] and are solubilized in PBS containing 8M urea. Since the majority of Tn5 cellular proteins are solubilized in PBS containing 2M urea, the arenavirus rNPs in the insoluble fraction in PBS containing 2M urea can be solubilized by sonication in PBS containing 8M urea. After a simple centrifugation of the lysates in PBS containing 8M urea, the supernatant fractions can be used as purified rNP antigens without further purification steps [51] . The control antigen is produced from Tn5 cells infected with baculovirus lacking", "the polyhedrin gene (ΔP) in the same manner as the arenavirus rNPs ( Figure 1 ).", "Purified rNPs. The expression and purification efficiency of arenavirus rNP were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) after staining the gels with Coomassie blue. Purified NP antigens with approximate molecular weights of 62 kDa from Luna, LCM, Lassa, Lujo, Junin, Machupo, Guanarito, Sabia, and Chapare viruses and the purified negative control antigen (ΔP) are shown.", "As described above, recombinant baculoviruses allow the delivery of rNP antigens without using infectious live arenaviruses. An ELISA plate coated with the predetermined optimal quantity of purified rNPs (approximately 100 ng/well) is used for the IgG-antibody detection assay. An advantage of using recombinant rNP for the IgG-ELISA is that it enables a direct comparison of antibody cross-reactivity among arenavirus rNPs, since antigen preparations of all arenavirus rNPs tested are performed using the same method [51] . Rabbit anti-sera raised against LCMV-rNP and LASV-rNP show cross-reactivity to LASV-rNP and LCMV-rNP, respectively, indicating that rabbit antibodies against rNPs of Old World arenaviruses cross-react with rNPs of other Old World arenaviruses (Table 1 ) [51] . Similarly, rabbit anti-sera generated against JUNV-NP show cross-reactivity to the LASV-rNP and LCMV-rNP, although the reaction is weak. However, rabbit anti-sera against LASV-NP and LCMV-NP show a negative", "reaction to the JUNV-rNP (Table 1 ) [51] , indicating that rabbit antibodies against JUNV (a pathogenic New World arenavirus) NP might cross-react with the Old World arenavirus NP, whereas antibodies against Old World arenavirus NPs may not be able to react with pathogenic New World arenavirus NPs.", "The rNP-based IgG-ELISA has also been used for the characterization of a mouse monoclonal antibody (MAb). Nakauchi et al. [50] have investigated the cross-reactivity of MAbs against JUNV rNP to pathogenic New World arenavirus rNPs, as well as LASV rNP. MAb C11-12 reacts at the same level with the rNPs of all of the pathogenic New World arenaviruses, including JUNV, GTOV, MACV, SABV, and CHPV, indicating that this MAb recognizes an epitope conserved among pathogenic New World arenaviruses. Another MAb, C6-9, reacts specifically with the rNP of JUNV, but does not react with those of the other pathogenic New World arenaviruses [50] . This indicates that MAb C6-9 recognizes a JUNV-specific epitope. None of these MAbs reacts with the rNP of the human pathogenic Old World arenavirus LASV. Thus, the MAb C11-12 is considered to be a broadly reactive MAb against New World arenaviruses, whereas MAb C6-9 is JUNV-specific. These findings have been confirmed by detailed epitope analyses using", "peptide mapping [50] . Similarly, the cross-reactivity of MAbs against LASV rNP has been analyzed [51] . MAb 4A5 cross-reacts with the Mopeia virus (MOPV) but not with the LCMV rNP. MAb 6C11 cross-reacts with LCMV rNP, while MAb 2-11 does not cross-react with LCMV rNP [51] . Table 1 . Anti-serum reactivity for rNPs of different arenaviruses in IgG ELISAs.", "Reactivity for rNP from LASV LCMV JUNV anti-LASV NP", "It is important to evaluate whether rNP-based ELISA is useful for the diagnosis of human VHF cases. The specificity of the LASV-rNP-based IgG ELISA has been confirmed by using sera obtained from Lassa fever patients [51] . The Lassa fever patients' sera show a highly positive reaction in the LASV-rNP-based IgG-ELISA, but sera from patients with Argentine hemorrhagic fever (AHF), which is caused by JUNV, do not. The serum from an AHF patient showed a highly positive reaction in the JUNV-rNP-based IgG-ELISA [49] . In addition, it was shown that, using sera obtained from AHF cases, the results of the JUNV rNP-based IgG ELISA correlate well with an authentic JUNV antigen-based IgG ELISA [49] . An IgM-capture ELISA using purified LASV-rNP as an antigen has been developed in the same way as in previous reports [54, 57] and detects an LASV-IgM antibody [58] . In addition, immunoblot assays based on N-terminally truncated LASV rNP have been developed for detecting IgG and IgM antibodies", "against LASV. These methods may provide a rapid and simple Lassa fever test for use under field conditions [47] .", "An IFA using virus-infected cells is a common antibody test for VHF viruses [59] [60] [61] [62] [63] . To avoid the use of highly pathogenic viruses for the antigen preparation, mammalian cells expressing recombinant rNP have been developed [51, 57, [64] [65] [66] [67] [68] . Lassa virus NP antigen for IFA can be prepared simply as described [51] . Briefly, the procedure involves (1) transfecting HeLa cells with a mammalian cell expression vector inserted with the cloned NP cDNA; (2) expanding the stable NP-expressing cells by antibiotic selection; (3) mixing the rNP-expressing cells with un-transfected HeLa cells (at a ratio of 1:1); (4) spotting the cell mixtures onto glass slides, then drying and fixing them in acetone.", "In the IFA specific for LASV-NP, antibody positive sera show characteristic granular staining patterns in the cytoplasm (Figure 2 ) [69] , thus making it easy to distinguish positive from negative samples. The specificity of the assay has also been confirmed by using sera obtained from Lassa fever patients [51] . In addition, an IFA using JUNV rNP-expressing HeLa cells has been developed to detect antibodies against JUNV, and the assay has been evaluated by using AHF patients' sera [70] . The LASV-rNP-based antibody detection systems such as ELISA and IFA are suggested to be useful not only for the diagnosis of Lassa fever, but also for seroepidemiological studies of LASV infection. In our preliminary study, approximately 15% of the sera collected from 334 Ghanaians and less than 3% of 280 Zambians showed positive reactions in the LASV-rNP-based IgG ELISA [58] . These results are in agreement with the fact that Lassa fever is endemic to the West African region, including Ghana, but", "less in the East African region.", "For the diagnosis of many viral infections, PCR assays have been shown to have an excellent analytical sensitivity, but the established techniques are limited by their requirement for expensive equipment and technical expertise. Moreover, the high degree of genetic variability of the RNA viruses, including arenavirus and bunyavirus, poses difficulties in selecting primers for RT-PCR assays that can detect all strains of the virus. Since the sensitivity of the Ag-capture ELISA is comparable to that of RT-PCR for several virus-mediated infectious diseases, including Lassa fever and filovirus hemorrhagic fever [51, [71] [72] [73] , the Ag-capture ELISA is a sophisticated approach that can be used for the diagnosis of viral infections. Ag-capture ELISAs detecting viral NP in viremic sera have been widely applied to detect various viruses, since they are the most abundant viral antigens and have highly conserved amino acid sequences [50, 51, 54, 71, 72, 74, 75] . Polyclonal anti-sera or a", "mixture of MAbs present in the ascetic fluids from animals immunized for HFVs have been used for capture-antibodies in the Ag-capture ELISA [36, [76] [77] [78] [79] . MAbs recognizing conserved epitopes of the rNP are also used as capture antibodies since they have a high specificity for the antigens, and an identification of the epitopes of these MAbs is of crucial importance for the assessment of the specificity and cross-reactivity of the assay system [50, 51, 53, 54, 71, 75] . In order to develop a sensitive diagnostic test for Lassa fever and AHF, rNPs of LASV and JUNV (see above) have been prepared, and newly established MAbs against them have been characterized and used for Ag-capture ELISAs [50, 51] . The Ag-capture ELISA using MAb 4A5 has been confirmed to be useful in the detection of authentic LASV antigen in sera serially collected from hamsters infected with LASV [51] . The sensitivity of the MAb 4A5-based Ag-capture ELISA was similar to that of conventional RT-PCR,", "suggesting that the Ag-capture ELISA can be efficiently used in the diagnosis of Lassa fever [51] . Therefore, the MAb 4A5-based Ag-capture ELISA is considered to be useful in the diagnosis of Lassa fever. Also, by using MAbs raised against the rNP of JUNV, Ag-capture ELISAs specific for JUNV and broadly reactive to human pathogenic New World arenaviruses have been developed [50] . The Ag-capture ELISA using MAb E4-2 and C11-12 detected the Ags of all of the pathogenic New World arenaviruses tested, including JUNV. On the other hand, the Ag-capture ELISA using MAb C6-9 detects only the JUNV Ag. Considering that the symptoms of JUNV infection in humans are indistinguishable from those due to other pathogenic New World arenaviruses, the Ag capture ELISA using MAb C6-9 may be a useful diagnostic tool, especially for AHF [50] .", "The virus neutralization assay is accepted as the \"gold standard\" serodiagnostic assay to quantify the antibody response to infection and vaccination of a wide variety of viruses associated with human diseases [80] [81] [82] [83] [84] [85] [86] . The presence of neutralizing antibodies is a reliable indicator of protective immunity against VHF [87] [88] [89] . The most direct method for detection of neutralizing antibodies against HFVs is by plaque reduction neutralization tests using infectious viruses. However, because of the high pathogenicity of HFVs to humans and the strict regulation of select agents, only a limited number of laboratories are able to perform such neutralization tests. For many HFVs, replication-incompetent pseudotyped virus particles bearing viral envelope protein (GP) have been shown to mimic the respective HFV infections, thus, neutralization assays using the pseudotypes may be advantageous in some laboratory settings for the detection of antibodies to HFVs", "without the need for heightened biocontainment requirements.", "The VSV-based vector has already been used to generate replication-competent recombinant VSVs to study of the role of GPs of various viruses [90] [91] [92] . Recent advances in producing pseudotype virus particles have enabled the investigation of the virus cell entry, viral tropism, and effect of entry inhibitors, as well as measurement of the neutralization titers, by using human immunodeficiency virus-, feline immunodeficiency virus-, murine leukemia virus-, or VSV-based vectors [86, [93] [94] [95] [96] [97] [98] [99] [100] [101] [102] [103] . Pseudotypes based on VSV have advantages compared with other pseudotypes based on retroviruses for the following reasons. First, the pseudotype virus titer obtained with the VSV system is generally higher than that of the pseudotyped retrovirus system [104] . Second, the infection of target cells with a VSV pseudotype can be readily detected as green fluorescent protein (GFP)-positive cells at 7-16 h post-infection because of the high level", "of GFP expression in the VSV system [104, 105] . In contrast, the time required for infection in the pseudotyped retrovirus system is 48 h [106, 107] , which is similar to the time required for infectious viruses to replicate to a level that results in plaque-forming or cytopathic effects in infected cells. A high-throughput assay for determining neutralizing antibody titers using VSV pseudotypes expressing secreted alkaline phosphatase [108, 109] or luciferase ( Figure 3 ) has also been developed. We have recently developed a VSV-based pseudotype bearing Lassa virus GP (VSV-LAS-GP) for the detection of neutralizing antibodies in the sera obtained from a Lassa fever patient. An example of the LASV neutralization assay using the VSV pseudotype is shown (Figure 4 ). In the presence of serum from Lassa fever patients, the number of GFP-positive cells (infectivity of VSV-LAS-GP) is significantly reduced compared with the number in the absence of the patient's serum ( Figure 4A ). The", "control VSV pseudotype bearing VSV GP (VSV-VSV-G) is not neutralized by any sera. When the cut-off serum dilution is set at 50% inhibition of infectivity compared with the infectivity in the absence of the test serum, the neutralization titer of this patient's serum for VSV-LAS-GP is calculated to be 75 ( Figure 4B ). Likewise, a VSV-based pseudotype bearing the Junin virus GP has been developed for the detection of neutralizing antibodies from AHF patients' sera. The accuracy of the results of VSV-based neutralization assays has been confirmed by comparison with the results of the neutralization assay using live Junin virus [70] . The Lujo virus is a new member of the hemorrhagic fever-associated arenavirus family from Zambia and southern Africa, and the virus is classified as a BSL-4 pathogen [17] . The genome sequence analysis of the Lujo virus suggests that the virus is genetically distinct from previously characterized arenaviruses. In order to study the infectivity of this newly", "identified arenavirus, we have recently developed a luciferase-expressing VSV pseudotype bearing Lujo virus GPC (VSV-Lujo-GP). As shown in Figure 3 , infection with VSV-Lujo-GPC is specifically neutralized by rabbit anti-Lujo GPC serum. Thus, the VSV-Lujo-GP may be a useful tool not only for determining the neutralizing antibody titer within the serum, but also for exploring yet-to-be-defined cellular receptor(s) for Lujo virus infection or for screening inhibitors of the Lujo virus GP-mediated cell entry.", "Hemorrhagic fever outbreaks caused by pathogenic arenaviruses result in high fatality rates. A rapid and accurate diagnosis is a critical first step in any outbreak. Serologic diagnostic methods for VHFs most often employ an ELISA, IFA, and/or virus neutralization assay. Diagnostic methods using recombinant viral proteins have been developed and their utilities for diagnosing of VHF have been reviewed. IgG-and IgM-ELISAs and IFAs using rNPs as antigens are useful for the detection of antibodies induced in the patients' sera. These methods are also useful for seroepidemiological surveys for HFVs. Ag-capture ELISAs using MAbs to the arenavirus rNPs are specific for the virus species or can be broadly reactive for New World arenaviruses, depending on the MAb used. Furthermore, the VSV-based pseudotype system provides a safe and rapid tool for measuring virus neutralizing antibody titers, as well as a model to analyze the entry of the respective arenavirus in susceptible cells without", "using live arenaviruses. Recent discoveries of novel arenavirus species [17, 26, 110] and their potential to evolve predominantly via host switching, rather than with their hosts [110, 111] , suggest that an unknown pathogenic arenavirus may emerge in the future, and that the diagnostic methods for VHF caused by arenaviruses should thus be further developed and improved." ]

[ 1 ]

[ "through the use of serological assays" ]

[ "Serological Assays Based on Recombinant Viral Proteins for the Diagnosis of Arenavirus Hemorrhagic Fevers\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3497043/\n\nSHA: f1d308db379b3c293bcfc8fe251c043fe8842358\n\nAuthors: Fukushi, Shuetsu; Tani, Hideki; Yoshikawa, Tomoki; Saijo, Masayuki; Morikawa, Shigeru\nDate: 2012-10-12\nDOI: 10.3390/v4102097\nLicense: cc-by", "Abstract: The family Arenaviridae, genus Arenavirus, consists of two phylogenetically independent groups: Old World (OW) and New World (NW) complexes. The Lassa and Lujo viruses in the OW complex and the Guanarito, Junin, Machupo, Sabia, and Chapare viruses in the NW complex cause viral hemorrhagic fever (VHF) in humans, leading to serious public health concerns. These viruses are also considered potential bioterrorism agents. Therefore, it is of great importance to detect these pathogens rapidly and specifically in order to minimize the risk and scale of arenavirus outbreaks. However, these arenaviruses are classified as BSL-4 pathogens, thus making it difficult to develop diagnostic techniques for these virus infections in institutes without BSL-4 facilities. To overcome these difficulties, antibody detection systems in the form of an enzyme-linked immunosorbent assay (ELISA) and an indirect immunofluorescence assay were developed using recombinant nucleoproteins (rNPs) derived from", "these viruses. Furthermore, several antigen-detection assays were developed. For example, novel monoclonal antibodies (mAbs) to the rNPs of Lassa and Junin viruses were generated. Sandwich antigen-capture (Ag-capture) ELISAs using these mAbs as capture antibodies were developed and confirmed to be sensitive and specific for detecting the respective arenavirus NPs. These rNP-based assays were proposed to be useful not only for an etiological diagnosis of VHFs, but also for seroepidemiological studies on VHFs. We recently developed arenavirus neutralization assays using vesicular stomatitis virus (VSV)-based pseudotypes bearing arenavirus recombinant glycoproteins. The goal of this article is to review the recent advances in developing laboratory diagnostic assays based on recombinant viral proteins for the diagnosis of VHFs and epidemiological studies on the VHFs caused by arenaviruses.", "Text: The virus family Arenaviridae consists of only one genus, but most viruses within this genus can be divided into two different groups: the Old World arenaviruses and the New World arenaviruses (also known as the Tacaribe complex) [1, 2] . The differences between the two groups have been established through the use of serological assays. Most of the arenaviruses cause persistent infection in rodents without any symptoms, and humans acquire a variety of diseases when zoonotically infected. Lymphocytic choriomeningitis virus (LCMV) is the only arenavirus to exhibit a worldwide distribution, and causes illnesses such as meningitis [3, 4] . Congenital LCMV infections have also been reported [4, 5] . Most importantly, viral hemorrhagic fever (VHF) can be caused by several arenaviruses. Lassa fever, caused by the Lassa virus (LASV), an Old World arenavirus, is one of the most devastating VHFs in humans [6] . Hemorrhaging and organ failure occur in a subset of patients infected with", "this virus, and it is associated with high mortality. Many cases of Lassa fever occur in Western Africa in countries such as Guinea, Sierra Leone, and Nigeria [7] [8] [9] [10] [11] [12] [13] . Tacaribe complex lineage B of the New World arenaviruses consists of the Junin virus (JUNV), Guanarito virus (GUNV), Sabia virus (SABV) and Machupo virus (MACV), the etiological agents of Argentine, Venezuelan, Brazilian, and Bolivian hemorrhagic fevers, respectively [14, 15] . Although genetically distinct from one another, they appear to produce similar symptoms, accompanied by hemorrhaging in humans [14, 15] . These pathogenic New World arenavirus species are closely associated with a specific rodent species [6] .", "Humans are usually infected with pathogenic arenaviruses through direct contact with tissue or blood, or after inhaling aerosolized particles from urine, feces, and saliva of infected rodents. After an incubation period of 1-3 weeks, infected individuals abruptly develop fever, retrosternal pain, sore throat, back pain, cough, abdominal pain, vomiting, diarrhea, conjunctivitis, facial swelling, proteinuria, and mucosal bleeding. Neurological problems have also been described, including hearing loss, tremors, and encephalitis. Because the symptoms of pathogenic arenavirus-related illness are varied and nonspecific, the clinical diagnosis is often difficult [14, 16] . Human-to-human transmission may occur via mucosal or cutaneous contact, or through nosocomial contamination [14, 16] . These viruses are also considered to be potential bioterrorism agents [2] .", "A number of arenavirus species have been recently discovered as a result of both rodent surveys and disease outbreaks [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] . A novel pathogenic New World arenavirus, Chapare virus (CHPV), has been isolated from a fatal case of VHF in Bolivia [20] . In addition, five cases of VHF have been reported in South Africa, and a novel arenavirus, named Lujo virus, was isolated from a patient [17] . The Lujo virus is most distantly related to the other Old World arenaviruses [17] . To date, there is no information concerning the vertebrate host for the Chapare and Lujo viruses.", "There is some evidence of endemicity of the Lassa virus in neighboring countries [27, 28] . However, as the magnitude of international trade and travel is continuously increasing, and the perturbation of the environment (due either to human activity or natural ecological changes) may result in behavioral changes of reservoir rodents, highly pathogenic arenaviruses could be introduced to virus-free countries from endemic areas. In fact, more than twenty cases of Lassa fever have been reported outside of the endemic region in areas such as the USA, Canada, Europe, and Japan [29] [30] [31] [32] [33] . It is of great importance to detect these pathogens rapidly and specifically in order to minimize the risk and scale of outbreaks of VHFs caused by arenaviruses. However, these arenaviruses are classified as biosafety level (BSL)-4 pathogens, making it difficult to develop diagnostic techniques for these virus infections in laboratories without BSL-4 facilities. To overcome these", "difficulties, we have established recombinant viral nucleoproteins (rNPs)-based serological assays, such as IgG-enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IFA), and antigen (Ag)-capture ELISA for the diagnosis of VHFs caused by highly pathogenic arenaviruses. Furthermore, virus neutralization assays using pseudotype virus-bearing arenavirus GPs have been developed. In this review, we describe the usefulness of such recombinant protein-based diagnostic assays for diagnosing VHFs caused by arenaviruses.", "In outbreaks of VHFs, infections are confirmed by various laboratory diagnostic methods. Virus detection is performed by virus isolation, reverse transcription-polymerase chain reaction (RT-PCR), and antigen-capture ELISA. It has been shown that monoclonal antibody panels against pathogenic arenaviruses are useful for detecting viral antigens on the virus-infected cells as well as for investigating of antigenic relationships of arenaviruses [34] [35] [36] . Detection of the virus genome is suitable for a rapid and sensitive diagnosis of VHF patients in the early stage of illness, and extensive reviews of such RT-PCR assays have been described [37, 38] . More recently, progress in the RT-PCR method covering genetic variations of the hemorrhagic fever viruses (HFVs) [39, 40] and a multiplexed oligonucleotide microarray for the differential diagnosis of VHFs have also been reported [41] . On the other hand, antibodies against these viruses can be detected by the indirect", "immunofluorescence assay (IFA), or IgG-and IgM-ELISA. An IFA detects the antibody in the serum, which is able to bind to the fixed monolayer of the virus-infected cells. Although the interpretation of immunofluorescence results requires experience, the assay has advantages over other methods, since each virus generates a characteristic fluorescence pattern that adds specificity to the assay compared to a simple ELISA readout. A serological diagnosis by the detection of specific IgM and IgG antibodies to the HFVs must be sensitive, specific and reliable, because a misdiagnosis can lead to panic in the general population. An IgM-specific ELISA is suitable for detecting recent infection, but the relevance of IgM testing for acute VHF depends on the virus and the duration of illness; specific IgM is not often present in the very early stage of illness, and patients who die of VHF often fail to seroconvert at all. An IgG-specific ELISA is efficacious, not only in the diagnosis of a large", "number of VHF cases, especially during convalescence, but also for epidemiological studies in the endemic regions. The detailed methods used for the IFA and IgG-and IgM-ELISAs for the diagnosis of VHF using authentic virus-antigens have been described in detail [42] [43] [44] [45] .", "Arenaviruses have a bisegmented, negative-sense, single stranded RNA genome with a unique ambisense coding strategy that produces just four known proteins: a glycoprotein, a nucleoprotein (NP), a matrix protein (Z), and a polymerase (L) [46] . Of these proteins, the NP is the most abundant in virus-infected cells. Recombinant protein technology could meet the demand for a simple and reliable VHF test system, and recombinant NP (rNP) has been shown to be useful for serological surveys of IgM-and IgG antibodies against arenaviruses [47] [48] [49] [50] .", "Recombinant baculoviruses that express the full-length rNP of arenaviruses have been generated [48, 50, 51] . The method used for the purification of arenavirus rNP from insect Tn5 cells infected with recombinant baculoviruses is effective and simple compared to those for Ebola, Marburg, and Crimean-Congo hemorrhagic fever virus rNPs [51] [52] [53] [54] [55] . Most of the arenavirus rNPs expressed in insect cells using the recombinant baculoviruses are crystallized [56] and are solubilized in PBS containing 8M urea. Since the majority of Tn5 cellular proteins are solubilized in PBS containing 2M urea, the arenavirus rNPs in the insoluble fraction in PBS containing 2M urea can be solubilized by sonication in PBS containing 8M urea. After a simple centrifugation of the lysates in PBS containing 8M urea, the supernatant fractions can be used as purified rNP antigens without further purification steps [51] . The control antigen is produced from Tn5 cells infected with baculovirus lacking", "the polyhedrin gene (ΔP) in the same manner as the arenavirus rNPs ( Figure 1 ).", "Purified rNPs. The expression and purification efficiency of arenavirus rNP were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) after staining the gels with Coomassie blue. Purified NP antigens with approximate molecular weights of 62 kDa from Luna, LCM, Lassa, Lujo, Junin, Machupo, Guanarito, Sabia, and Chapare viruses and the purified negative control antigen (ΔP) are shown.", "As described above, recombinant baculoviruses allow the delivery of rNP antigens without using infectious live arenaviruses. An ELISA plate coated with the predetermined optimal quantity of purified rNPs (approximately 100 ng/well) is used for the IgG-antibody detection assay. An advantage of using recombinant rNP for the IgG-ELISA is that it enables a direct comparison of antibody cross-reactivity among arenavirus rNPs, since antigen preparations of all arenavirus rNPs tested are performed using the same method [51] . Rabbit anti-sera raised against LCMV-rNP and LASV-rNP show cross-reactivity to LASV-rNP and LCMV-rNP, respectively, indicating that rabbit antibodies against rNPs of Old World arenaviruses cross-react with rNPs of other Old World arenaviruses (Table 1 ) [51] . Similarly, rabbit anti-sera generated against JUNV-NP show cross-reactivity to the LASV-rNP and LCMV-rNP, although the reaction is weak. However, rabbit anti-sera against LASV-NP and LCMV-NP show a negative", "reaction to the JUNV-rNP (Table 1 ) [51] , indicating that rabbit antibodies against JUNV (a pathogenic New World arenavirus) NP might cross-react with the Old World arenavirus NP, whereas antibodies against Old World arenavirus NPs may not be able to react with pathogenic New World arenavirus NPs.", "The rNP-based IgG-ELISA has also been used for the characterization of a mouse monoclonal antibody (MAb). Nakauchi et al. [50] have investigated the cross-reactivity of MAbs against JUNV rNP to pathogenic New World arenavirus rNPs, as well as LASV rNP. MAb C11-12 reacts at the same level with the rNPs of all of the pathogenic New World arenaviruses, including JUNV, GTOV, MACV, SABV, and CHPV, indicating that this MAb recognizes an epitope conserved among pathogenic New World arenaviruses. Another MAb, C6-9, reacts specifically with the rNP of JUNV, but does not react with those of the other pathogenic New World arenaviruses [50] . This indicates that MAb C6-9 recognizes a JUNV-specific epitope. None of these MAbs reacts with the rNP of the human pathogenic Old World arenavirus LASV. Thus, the MAb C11-12 is considered to be a broadly reactive MAb against New World arenaviruses, whereas MAb C6-9 is JUNV-specific. These findings have been confirmed by detailed epitope analyses using", "peptide mapping [50] . Similarly, the cross-reactivity of MAbs against LASV rNP has been analyzed [51] . MAb 4A5 cross-reacts with the Mopeia virus (MOPV) but not with the LCMV rNP. MAb 6C11 cross-reacts with LCMV rNP, while MAb 2-11 does not cross-react with LCMV rNP [51] . Table 1 . Anti-serum reactivity for rNPs of different arenaviruses in IgG ELISAs.", "Reactivity for rNP from LASV LCMV JUNV anti-LASV NP", "It is important to evaluate whether rNP-based ELISA is useful for the diagnosis of human VHF cases. The specificity of the LASV-rNP-based IgG ELISA has been confirmed by using sera obtained from Lassa fever patients [51] . The Lassa fever patients' sera show a highly positive reaction in the LASV-rNP-based IgG-ELISA, but sera from patients with Argentine hemorrhagic fever (AHF), which is caused by JUNV, do not. The serum from an AHF patient showed a highly positive reaction in the JUNV-rNP-based IgG-ELISA [49] . In addition, it was shown that, using sera obtained from AHF cases, the results of the JUNV rNP-based IgG ELISA correlate well with an authentic JUNV antigen-based IgG ELISA [49] . An IgM-capture ELISA using purified LASV-rNP as an antigen has been developed in the same way as in previous reports [54, 57] and detects an LASV-IgM antibody [58] . In addition, immunoblot assays based on N-terminally truncated LASV rNP have been developed for detecting IgG and IgM antibodies", "against LASV. These methods may provide a rapid and simple Lassa fever test for use under field conditions [47] .", "An IFA using virus-infected cells is a common antibody test for VHF viruses [59] [60] [61] [62] [63] . To avoid the use of highly pathogenic viruses for the antigen preparation, mammalian cells expressing recombinant rNP have been developed [51, 57, [64] [65] [66] [67] [68] . Lassa virus NP antigen for IFA can be prepared simply as described [51] . Briefly, the procedure involves (1) transfecting HeLa cells with a mammalian cell expression vector inserted with the cloned NP cDNA; (2) expanding the stable NP-expressing cells by antibiotic selection; (3) mixing the rNP-expressing cells with un-transfected HeLa cells (at a ratio of 1:1); (4) spotting the cell mixtures onto glass slides, then drying and fixing them in acetone.", "In the IFA specific for LASV-NP, antibody positive sera show characteristic granular staining patterns in the cytoplasm (Figure 2 ) [69] , thus making it easy to distinguish positive from negative samples. The specificity of the assay has also been confirmed by using sera obtained from Lassa fever patients [51] . In addition, an IFA using JUNV rNP-expressing HeLa cells has been developed to detect antibodies against JUNV, and the assay has been evaluated by using AHF patients' sera [70] . The LASV-rNP-based antibody detection systems such as ELISA and IFA are suggested to be useful not only for the diagnosis of Lassa fever, but also for seroepidemiological studies of LASV infection. In our preliminary study, approximately 15% of the sera collected from 334 Ghanaians and less than 3% of 280 Zambians showed positive reactions in the LASV-rNP-based IgG ELISA [58] . These results are in agreement with the fact that Lassa fever is endemic to the West African region, including Ghana, but", "less in the East African region.", "For the diagnosis of many viral infections, PCR assays have been shown to have an excellent analytical sensitivity, but the established techniques are limited by their requirement for expensive equipment and technical expertise. Moreover, the high degree of genetic variability of the RNA viruses, including arenavirus and bunyavirus, poses difficulties in selecting primers for RT-PCR assays that can detect all strains of the virus. Since the sensitivity of the Ag-capture ELISA is comparable to that of RT-PCR for several virus-mediated infectious diseases, including Lassa fever and filovirus hemorrhagic fever [51, [71] [72] [73] , the Ag-capture ELISA is a sophisticated approach that can be used for the diagnosis of viral infections. Ag-capture ELISAs detecting viral NP in viremic sera have been widely applied to detect various viruses, since they are the most abundant viral antigens and have highly conserved amino acid sequences [50, 51, 54, 71, 72, 74, 75] . Polyclonal anti-sera or a", "mixture of MAbs present in the ascetic fluids from animals immunized for HFVs have been used for capture-antibodies in the Ag-capture ELISA [36, [76] [77] [78] [79] . MAbs recognizing conserved epitopes of the rNP are also used as capture antibodies since they have a high specificity for the antigens, and an identification of the epitopes of these MAbs is of crucial importance for the assessment of the specificity and cross-reactivity of the assay system [50, 51, 53, 54, 71, 75] . In order to develop a sensitive diagnostic test for Lassa fever and AHF, rNPs of LASV and JUNV (see above) have been prepared, and newly established MAbs against them have been characterized and used for Ag-capture ELISAs [50, 51] . The Ag-capture ELISA using MAb 4A5 has been confirmed to be useful in the detection of authentic LASV antigen in sera serially collected from hamsters infected with LASV [51] . The sensitivity of the MAb 4A5-based Ag-capture ELISA was similar to that of conventional RT-PCR,", "suggesting that the Ag-capture ELISA can be efficiently used in the diagnosis of Lassa fever [51] . Therefore, the MAb 4A5-based Ag-capture ELISA is considered to be useful in the diagnosis of Lassa fever. Also, by using MAbs raised against the rNP of JUNV, Ag-capture ELISAs specific for JUNV and broadly reactive to human pathogenic New World arenaviruses have been developed [50] . The Ag-capture ELISA using MAb E4-2 and C11-12 detected the Ags of all of the pathogenic New World arenaviruses tested, including JUNV. On the other hand, the Ag-capture ELISA using MAb C6-9 detects only the JUNV Ag. Considering that the symptoms of JUNV infection in humans are indistinguishable from those due to other pathogenic New World arenaviruses, the Ag capture ELISA using MAb C6-9 may be a useful diagnostic tool, especially for AHF [50] .", "The virus neutralization assay is accepted as the \"gold standard\" serodiagnostic assay to quantify the antibody response to infection and vaccination of a wide variety of viruses associated with human diseases [80] [81] [82] [83] [84] [85] [86] . The presence of neutralizing antibodies is a reliable indicator of protective immunity against VHF [87] [88] [89] . The most direct method for detection of neutralizing antibodies against HFVs is by plaque reduction neutralization tests using infectious viruses. However, because of the high pathogenicity of HFVs to humans and the strict regulation of select agents, only a limited number of laboratories are able to perform such neutralization tests. For many HFVs, replication-incompetent pseudotyped virus particles bearing viral envelope protein (GP) have been shown to mimic the respective HFV infections, thus, neutralization assays using the pseudotypes may be advantageous in some laboratory settings for the detection of antibodies to HFVs", "without the need for heightened biocontainment requirements.", "The VSV-based vector has already been used to generate replication-competent recombinant VSVs to study of the role of GPs of various viruses [90] [91] [92] . Recent advances in producing pseudotype virus particles have enabled the investigation of the virus cell entry, viral tropism, and effect of entry inhibitors, as well as measurement of the neutralization titers, by using human immunodeficiency virus-, feline immunodeficiency virus-, murine leukemia virus-, or VSV-based vectors [86, [93] [94] [95] [96] [97] [98] [99] [100] [101] [102] [103] . Pseudotypes based on VSV have advantages compared with other pseudotypes based on retroviruses for the following reasons. First, the pseudotype virus titer obtained with the VSV system is generally higher than that of the pseudotyped retrovirus system [104] . Second, the infection of target cells with a VSV pseudotype can be readily detected as green fluorescent protein (GFP)-positive cells at 7-16 h post-infection because of the high level", "of GFP expression in the VSV system [104, 105] . In contrast, the time required for infection in the pseudotyped retrovirus system is 48 h [106, 107] , which is similar to the time required for infectious viruses to replicate to a level that results in plaque-forming or cytopathic effects in infected cells. A high-throughput assay for determining neutralizing antibody titers using VSV pseudotypes expressing secreted alkaline phosphatase [108, 109] or luciferase ( Figure 3 ) has also been developed. We have recently developed a VSV-based pseudotype bearing Lassa virus GP (VSV-LAS-GP) for the detection of neutralizing antibodies in the sera obtained from a Lassa fever patient. An example of the LASV neutralization assay using the VSV pseudotype is shown (Figure 4 ). In the presence of serum from Lassa fever patients, the number of GFP-positive cells (infectivity of VSV-LAS-GP) is significantly reduced compared with the number in the absence of the patient's serum ( Figure 4A ). The", "control VSV pseudotype bearing VSV GP (VSV-VSV-G) is not neutralized by any sera. When the cut-off serum dilution is set at 50% inhibition of infectivity compared with the infectivity in the absence of the test serum, the neutralization titer of this patient's serum for VSV-LAS-GP is calculated to be 75 ( Figure 4B ). Likewise, a VSV-based pseudotype bearing the Junin virus GP has been developed for the detection of neutralizing antibodies from AHF patients' sera. The accuracy of the results of VSV-based neutralization assays has been confirmed by comparison with the results of the neutralization assay using live Junin virus [70] . The Lujo virus is a new member of the hemorrhagic fever-associated arenavirus family from Zambia and southern Africa, and the virus is classified as a BSL-4 pathogen [17] . The genome sequence analysis of the Lujo virus suggests that the virus is genetically distinct from previously characterized arenaviruses. In order to study the infectivity of this newly", "identified arenavirus, we have recently developed a luciferase-expressing VSV pseudotype bearing Lujo virus GPC (VSV-Lujo-GP). As shown in Figure 3 , infection with VSV-Lujo-GPC is specifically neutralized by rabbit anti-Lujo GPC serum. Thus, the VSV-Lujo-GP may be a useful tool not only for determining the neutralizing antibody titer within the serum, but also for exploring yet-to-be-defined cellular receptor(s) for Lujo virus infection or for screening inhibitors of the Lujo virus GP-mediated cell entry.", "Hemorrhagic fever outbreaks caused by pathogenic arenaviruses result in high fatality rates. A rapid and accurate diagnosis is a critical first step in any outbreak. Serologic diagnostic methods for VHFs most often employ an ELISA, IFA, and/or virus neutralization assay. Diagnostic methods using recombinant viral proteins have been developed and their utilities for diagnosing of VHF have been reviewed. IgG-and IgM-ELISAs and IFAs using rNPs as antigens are useful for the detection of antibodies induced in the patients' sera. These methods are also useful for seroepidemiological surveys for HFVs. Ag-capture ELISAs using MAbs to the arenavirus rNPs are specific for the virus species or can be broadly reactive for New World arenaviruses, depending on the MAb used. Furthermore, the VSV-based pseudotype system provides a safe and rapid tool for measuring virus neutralizing antibody titers, as well as a model to analyze the entry of the respective arenavirus in susceptible cells without", "using live arenaviruses. Recent discoveries of novel arenavirus species [17, 26, 110] and their potential to evolve predominantly via host switching, rather than with their hosts [110, 111] , suggest that an unknown pathogenic arenavirus may emerge in the future, and that the diagnostic methods for VHF caused by arenaviruses should thus be further developed and improved." ]

[ 3 ]

[ "1-3 weeks" ]

[ "Serological Assays Based on Recombinant Viral Proteins for the Diagnosis of Arenavirus Hemorrhagic Fevers\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3497043/\n\nSHA: f1d308db379b3c293bcfc8fe251c043fe8842358\n\nAuthors: Fukushi, Shuetsu; Tani, Hideki; Yoshikawa, Tomoki; Saijo, Masayuki; Morikawa, Shigeru\nDate: 2012-10-12\nDOI: 10.3390/v4102097\nLicense: cc-by", "Abstract: The family Arenaviridae, genus Arenavirus, consists of two phylogenetically independent groups: Old World (OW) and New World (NW) complexes. The Lassa and Lujo viruses in the OW complex and the Guanarito, Junin, Machupo, Sabia, and Chapare viruses in the NW complex cause viral hemorrhagic fever (VHF) in humans, leading to serious public health concerns. These viruses are also considered potential bioterrorism agents. Therefore, it is of great importance to detect these pathogens rapidly and specifically in order to minimize the risk and scale of arenavirus outbreaks. However, these arenaviruses are classified as BSL-4 pathogens, thus making it difficult to develop diagnostic techniques for these virus infections in institutes without BSL-4 facilities. To overcome these difficulties, antibody detection systems in the form of an enzyme-linked immunosorbent assay (ELISA) and an indirect immunofluorescence assay were developed using recombinant nucleoproteins (rNPs) derived from", "these viruses. Furthermore, several antigen-detection assays were developed. For example, novel monoclonal antibodies (mAbs) to the rNPs of Lassa and Junin viruses were generated. Sandwich antigen-capture (Ag-capture) ELISAs using these mAbs as capture antibodies were developed and confirmed to be sensitive and specific for detecting the respective arenavirus NPs. These rNP-based assays were proposed to be useful not only for an etiological diagnosis of VHFs, but also for seroepidemiological studies on VHFs. We recently developed arenavirus neutralization assays using vesicular stomatitis virus (VSV)-based pseudotypes bearing arenavirus recombinant glycoproteins. The goal of this article is to review the recent advances in developing laboratory diagnostic assays based on recombinant viral proteins for the diagnosis of VHFs and epidemiological studies on the VHFs caused by arenaviruses.", "Text: The virus family Arenaviridae consists of only one genus, but most viruses within this genus can be divided into two different groups: the Old World arenaviruses and the New World arenaviruses (also known as the Tacaribe complex) [1, 2] . The differences between the two groups have been established through the use of serological assays. Most of the arenaviruses cause persistent infection in rodents without any symptoms, and humans acquire a variety of diseases when zoonotically infected. Lymphocytic choriomeningitis virus (LCMV) is the only arenavirus to exhibit a worldwide distribution, and causes illnesses such as meningitis [3, 4] . Congenital LCMV infections have also been reported [4, 5] . Most importantly, viral hemorrhagic fever (VHF) can be caused by several arenaviruses. Lassa fever, caused by the Lassa virus (LASV), an Old World arenavirus, is one of the most devastating VHFs in humans [6] . Hemorrhaging and organ failure occur in a subset of patients infected with", "this virus, and it is associated with high mortality. Many cases of Lassa fever occur in Western Africa in countries such as Guinea, Sierra Leone, and Nigeria [7] [8] [9] [10] [11] [12] [13] . Tacaribe complex lineage B of the New World arenaviruses consists of the Junin virus (JUNV), Guanarito virus (GUNV), Sabia virus (SABV) and Machupo virus (MACV), the etiological agents of Argentine, Venezuelan, Brazilian, and Bolivian hemorrhagic fevers, respectively [14, 15] . Although genetically distinct from one another, they appear to produce similar symptoms, accompanied by hemorrhaging in humans [14, 15] . These pathogenic New World arenavirus species are closely associated with a specific rodent species [6] .", "Humans are usually infected with pathogenic arenaviruses through direct contact with tissue or blood, or after inhaling aerosolized particles from urine, feces, and saliva of infected rodents. After an incubation period of 1-3 weeks, infected individuals abruptly develop fever, retrosternal pain, sore throat, back pain, cough, abdominal pain, vomiting, diarrhea, conjunctivitis, facial swelling, proteinuria, and mucosal bleeding. Neurological problems have also been described, including hearing loss, tremors, and encephalitis. Because the symptoms of pathogenic arenavirus-related illness are varied and nonspecific, the clinical diagnosis is often difficult [14, 16] . Human-to-human transmission may occur via mucosal or cutaneous contact, or through nosocomial contamination [14, 16] . These viruses are also considered to be potential bioterrorism agents [2] .", "A number of arenavirus species have been recently discovered as a result of both rodent surveys and disease outbreaks [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] . A novel pathogenic New World arenavirus, Chapare virus (CHPV), has been isolated from a fatal case of VHF in Bolivia [20] . In addition, five cases of VHF have been reported in South Africa, and a novel arenavirus, named Lujo virus, was isolated from a patient [17] . The Lujo virus is most distantly related to the other Old World arenaviruses [17] . To date, there is no information concerning the vertebrate host for the Chapare and Lujo viruses.", "There is some evidence of endemicity of the Lassa virus in neighboring countries [27, 28] . However, as the magnitude of international trade and travel is continuously increasing, and the perturbation of the environment (due either to human activity or natural ecological changes) may result in behavioral changes of reservoir rodents, highly pathogenic arenaviruses could be introduced to virus-free countries from endemic areas. In fact, more than twenty cases of Lassa fever have been reported outside of the endemic region in areas such as the USA, Canada, Europe, and Japan [29] [30] [31] [32] [33] . It is of great importance to detect these pathogens rapidly and specifically in order to minimize the risk and scale of outbreaks of VHFs caused by arenaviruses. However, these arenaviruses are classified as biosafety level (BSL)-4 pathogens, making it difficult to develop diagnostic techniques for these virus infections in laboratories without BSL-4 facilities. To overcome these", "difficulties, we have established recombinant viral nucleoproteins (rNPs)-based serological assays, such as IgG-enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IFA), and antigen (Ag)-capture ELISA for the diagnosis of VHFs caused by highly pathogenic arenaviruses. Furthermore, virus neutralization assays using pseudotype virus-bearing arenavirus GPs have been developed. In this review, we describe the usefulness of such recombinant protein-based diagnostic assays for diagnosing VHFs caused by arenaviruses.", "In outbreaks of VHFs, infections are confirmed by various laboratory diagnostic methods. Virus detection is performed by virus isolation, reverse transcription-polymerase chain reaction (RT-PCR), and antigen-capture ELISA. It has been shown that monoclonal antibody panels against pathogenic arenaviruses are useful for detecting viral antigens on the virus-infected cells as well as for investigating of antigenic relationships of arenaviruses [34] [35] [36] . Detection of the virus genome is suitable for a rapid and sensitive diagnosis of VHF patients in the early stage of illness, and extensive reviews of such RT-PCR assays have been described [37, 38] . More recently, progress in the RT-PCR method covering genetic variations of the hemorrhagic fever viruses (HFVs) [39, 40] and a multiplexed oligonucleotide microarray for the differential diagnosis of VHFs have also been reported [41] . On the other hand, antibodies against these viruses can be detected by the indirect", "immunofluorescence assay (IFA), or IgG-and IgM-ELISA. An IFA detects the antibody in the serum, which is able to bind to the fixed monolayer of the virus-infected cells. Although the interpretation of immunofluorescence results requires experience, the assay has advantages over other methods, since each virus generates a characteristic fluorescence pattern that adds specificity to the assay compared to a simple ELISA readout. A serological diagnosis by the detection of specific IgM and IgG antibodies to the HFVs must be sensitive, specific and reliable, because a misdiagnosis can lead to panic in the general population. An IgM-specific ELISA is suitable for detecting recent infection, but the relevance of IgM testing for acute VHF depends on the virus and the duration of illness; specific IgM is not often present in the very early stage of illness, and patients who die of VHF often fail to seroconvert at all. An IgG-specific ELISA is efficacious, not only in the diagnosis of a large", "number of VHF cases, especially during convalescence, but also for epidemiological studies in the endemic regions. The detailed methods used for the IFA and IgG-and IgM-ELISAs for the diagnosis of VHF using authentic virus-antigens have been described in detail [42] [43] [44] [45] .", "Arenaviruses have a bisegmented, negative-sense, single stranded RNA genome with a unique ambisense coding strategy that produces just four known proteins: a glycoprotein, a nucleoprotein (NP), a matrix protein (Z), and a polymerase (L) [46] . Of these proteins, the NP is the most abundant in virus-infected cells. Recombinant protein technology could meet the demand for a simple and reliable VHF test system, and recombinant NP (rNP) has been shown to be useful for serological surveys of IgM-and IgG antibodies against arenaviruses [47] [48] [49] [50] .", "Recombinant baculoviruses that express the full-length rNP of arenaviruses have been generated [48, 50, 51] . The method used for the purification of arenavirus rNP from insect Tn5 cells infected with recombinant baculoviruses is effective and simple compared to those for Ebola, Marburg, and Crimean-Congo hemorrhagic fever virus rNPs [51] [52] [53] [54] [55] . Most of the arenavirus rNPs expressed in insect cells using the recombinant baculoviruses are crystallized [56] and are solubilized in PBS containing 8M urea. Since the majority of Tn5 cellular proteins are solubilized in PBS containing 2M urea, the arenavirus rNPs in the insoluble fraction in PBS containing 2M urea can be solubilized by sonication in PBS containing 8M urea. After a simple centrifugation of the lysates in PBS containing 8M urea, the supernatant fractions can be used as purified rNP antigens without further purification steps [51] . The control antigen is produced from Tn5 cells infected with baculovirus lacking", "the polyhedrin gene (ΔP) in the same manner as the arenavirus rNPs ( Figure 1 ).", "Purified rNPs. The expression and purification efficiency of arenavirus rNP were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) after staining the gels with Coomassie blue. Purified NP antigens with approximate molecular weights of 62 kDa from Luna, LCM, Lassa, Lujo, Junin, Machupo, Guanarito, Sabia, and Chapare viruses and the purified negative control antigen (ΔP) are shown.", "As described above, recombinant baculoviruses allow the delivery of rNP antigens without using infectious live arenaviruses. An ELISA plate coated with the predetermined optimal quantity of purified rNPs (approximately 100 ng/well) is used for the IgG-antibody detection assay. An advantage of using recombinant rNP for the IgG-ELISA is that it enables a direct comparison of antibody cross-reactivity among arenavirus rNPs, since antigen preparations of all arenavirus rNPs tested are performed using the same method [51] . Rabbit anti-sera raised against LCMV-rNP and LASV-rNP show cross-reactivity to LASV-rNP and LCMV-rNP, respectively, indicating that rabbit antibodies against rNPs of Old World arenaviruses cross-react with rNPs of other Old World arenaviruses (Table 1 ) [51] . Similarly, rabbit anti-sera generated against JUNV-NP show cross-reactivity to the LASV-rNP and LCMV-rNP, although the reaction is weak. However, rabbit anti-sera against LASV-NP and LCMV-NP show a negative", "reaction to the JUNV-rNP (Table 1 ) [51] , indicating that rabbit antibodies against JUNV (a pathogenic New World arenavirus) NP might cross-react with the Old World arenavirus NP, whereas antibodies against Old World arenavirus NPs may not be able to react with pathogenic New World arenavirus NPs.", "The rNP-based IgG-ELISA has also been used for the characterization of a mouse monoclonal antibody (MAb). Nakauchi et al. [50] have investigated the cross-reactivity of MAbs against JUNV rNP to pathogenic New World arenavirus rNPs, as well as LASV rNP. MAb C11-12 reacts at the same level with the rNPs of all of the pathogenic New World arenaviruses, including JUNV, GTOV, MACV, SABV, and CHPV, indicating that this MAb recognizes an epitope conserved among pathogenic New World arenaviruses. Another MAb, C6-9, reacts specifically with the rNP of JUNV, but does not react with those of the other pathogenic New World arenaviruses [50] . This indicates that MAb C6-9 recognizes a JUNV-specific epitope. None of these MAbs reacts with the rNP of the human pathogenic Old World arenavirus LASV. Thus, the MAb C11-12 is considered to be a broadly reactive MAb against New World arenaviruses, whereas MAb C6-9 is JUNV-specific. These findings have been confirmed by detailed epitope analyses using", "peptide mapping [50] . Similarly, the cross-reactivity of MAbs against LASV rNP has been analyzed [51] . MAb 4A5 cross-reacts with the Mopeia virus (MOPV) but not with the LCMV rNP. MAb 6C11 cross-reacts with LCMV rNP, while MAb 2-11 does not cross-react with LCMV rNP [51] . Table 1 . Anti-serum reactivity for rNPs of different arenaviruses in IgG ELISAs.", "Reactivity for rNP from LASV LCMV JUNV anti-LASV NP", "It is important to evaluate whether rNP-based ELISA is useful for the diagnosis of human VHF cases. The specificity of the LASV-rNP-based IgG ELISA has been confirmed by using sera obtained from Lassa fever patients [51] . The Lassa fever patients' sera show a highly positive reaction in the LASV-rNP-based IgG-ELISA, but sera from patients with Argentine hemorrhagic fever (AHF), which is caused by JUNV, do not. The serum from an AHF patient showed a highly positive reaction in the JUNV-rNP-based IgG-ELISA [49] . In addition, it was shown that, using sera obtained from AHF cases, the results of the JUNV rNP-based IgG ELISA correlate well with an authentic JUNV antigen-based IgG ELISA [49] . An IgM-capture ELISA using purified LASV-rNP as an antigen has been developed in the same way as in previous reports [54, 57] and detects an LASV-IgM antibody [58] . In addition, immunoblot assays based on N-terminally truncated LASV rNP have been developed for detecting IgG and IgM antibodies", "against LASV. These methods may provide a rapid and simple Lassa fever test for use under field conditions [47] .", "An IFA using virus-infected cells is a common antibody test for VHF viruses [59] [60] [61] [62] [63] . To avoid the use of highly pathogenic viruses for the antigen preparation, mammalian cells expressing recombinant rNP have been developed [51, 57, [64] [65] [66] [67] [68] . Lassa virus NP antigen for IFA can be prepared simply as described [51] . Briefly, the procedure involves (1) transfecting HeLa cells with a mammalian cell expression vector inserted with the cloned NP cDNA; (2) expanding the stable NP-expressing cells by antibiotic selection; (3) mixing the rNP-expressing cells with un-transfected HeLa cells (at a ratio of 1:1); (4) spotting the cell mixtures onto glass slides, then drying and fixing them in acetone.", "In the IFA specific for LASV-NP, antibody positive sera show characteristic granular staining patterns in the cytoplasm (Figure 2 ) [69] , thus making it easy to distinguish positive from negative samples. The specificity of the assay has also been confirmed by using sera obtained from Lassa fever patients [51] . In addition, an IFA using JUNV rNP-expressing HeLa cells has been developed to detect antibodies against JUNV, and the assay has been evaluated by using AHF patients' sera [70] . The LASV-rNP-based antibody detection systems such as ELISA and IFA are suggested to be useful not only for the diagnosis of Lassa fever, but also for seroepidemiological studies of LASV infection. In our preliminary study, approximately 15% of the sera collected from 334 Ghanaians and less than 3% of 280 Zambians showed positive reactions in the LASV-rNP-based IgG ELISA [58] . These results are in agreement with the fact that Lassa fever is endemic to the West African region, including Ghana, but", "less in the East African region.", "For the diagnosis of many viral infections, PCR assays have been shown to have an excellent analytical sensitivity, but the established techniques are limited by their requirement for expensive equipment and technical expertise. Moreover, the high degree of genetic variability of the RNA viruses, including arenavirus and bunyavirus, poses difficulties in selecting primers for RT-PCR assays that can detect all strains of the virus. Since the sensitivity of the Ag-capture ELISA is comparable to that of RT-PCR for several virus-mediated infectious diseases, including Lassa fever and filovirus hemorrhagic fever [51, [71] [72] [73] , the Ag-capture ELISA is a sophisticated approach that can be used for the diagnosis of viral infections. Ag-capture ELISAs detecting viral NP in viremic sera have been widely applied to detect various viruses, since they are the most abundant viral antigens and have highly conserved amino acid sequences [50, 51, 54, 71, 72, 74, 75] . Polyclonal anti-sera or a", "mixture of MAbs present in the ascetic fluids from animals immunized for HFVs have been used for capture-antibodies in the Ag-capture ELISA [36, [76] [77] [78] [79] . MAbs recognizing conserved epitopes of the rNP are also used as capture antibodies since they have a high specificity for the antigens, and an identification of the epitopes of these MAbs is of crucial importance for the assessment of the specificity and cross-reactivity of the assay system [50, 51, 53, 54, 71, 75] . In order to develop a sensitive diagnostic test for Lassa fever and AHF, rNPs of LASV and JUNV (see above) have been prepared, and newly established MAbs against them have been characterized and used for Ag-capture ELISAs [50, 51] . The Ag-capture ELISA using MAb 4A5 has been confirmed to be useful in the detection of authentic LASV antigen in sera serially collected from hamsters infected with LASV [51] . The sensitivity of the MAb 4A5-based Ag-capture ELISA was similar to that of conventional RT-PCR,", "suggesting that the Ag-capture ELISA can be efficiently used in the diagnosis of Lassa fever [51] . Therefore, the MAb 4A5-based Ag-capture ELISA is considered to be useful in the diagnosis of Lassa fever. Also, by using MAbs raised against the rNP of JUNV, Ag-capture ELISAs specific for JUNV and broadly reactive to human pathogenic New World arenaviruses have been developed [50] . The Ag-capture ELISA using MAb E4-2 and C11-12 detected the Ags of all of the pathogenic New World arenaviruses tested, including JUNV. On the other hand, the Ag-capture ELISA using MAb C6-9 detects only the JUNV Ag. Considering that the symptoms of JUNV infection in humans are indistinguishable from those due to other pathogenic New World arenaviruses, the Ag capture ELISA using MAb C6-9 may be a useful diagnostic tool, especially for AHF [50] .", "The virus neutralization assay is accepted as the \"gold standard\" serodiagnostic assay to quantify the antibody response to infection and vaccination of a wide variety of viruses associated with human diseases [80] [81] [82] [83] [84] [85] [86] . The presence of neutralizing antibodies is a reliable indicator of protective immunity against VHF [87] [88] [89] . The most direct method for detection of neutralizing antibodies against HFVs is by plaque reduction neutralization tests using infectious viruses. However, because of the high pathogenicity of HFVs to humans and the strict regulation of select agents, only a limited number of laboratories are able to perform such neutralization tests. For many HFVs, replication-incompetent pseudotyped virus particles bearing viral envelope protein (GP) have been shown to mimic the respective HFV infections, thus, neutralization assays using the pseudotypes may be advantageous in some laboratory settings for the detection of antibodies to HFVs", "without the need for heightened biocontainment requirements.", "The VSV-based vector has already been used to generate replication-competent recombinant VSVs to study of the role of GPs of various viruses [90] [91] [92] . Recent advances in producing pseudotype virus particles have enabled the investigation of the virus cell entry, viral tropism, and effect of entry inhibitors, as well as measurement of the neutralization titers, by using human immunodeficiency virus-, feline immunodeficiency virus-, murine leukemia virus-, or VSV-based vectors [86, [93] [94] [95] [96] [97] [98] [99] [100] [101] [102] [103] . Pseudotypes based on VSV have advantages compared with other pseudotypes based on retroviruses for the following reasons. First, the pseudotype virus titer obtained with the VSV system is generally higher than that of the pseudotyped retrovirus system [104] . Second, the infection of target cells with a VSV pseudotype can be readily detected as green fluorescent protein (GFP)-positive cells at 7-16 h post-infection because of the high level", "of GFP expression in the VSV system [104, 105] . In contrast, the time required for infection in the pseudotyped retrovirus system is 48 h [106, 107] , which is similar to the time required for infectious viruses to replicate to a level that results in plaque-forming or cytopathic effects in infected cells. A high-throughput assay for determining neutralizing antibody titers using VSV pseudotypes expressing secreted alkaline phosphatase [108, 109] or luciferase ( Figure 3 ) has also been developed. We have recently developed a VSV-based pseudotype bearing Lassa virus GP (VSV-LAS-GP) for the detection of neutralizing antibodies in the sera obtained from a Lassa fever patient. An example of the LASV neutralization assay using the VSV pseudotype is shown (Figure 4 ). In the presence of serum from Lassa fever patients, the number of GFP-positive cells (infectivity of VSV-LAS-GP) is significantly reduced compared with the number in the absence of the patient's serum ( Figure 4A ). The", "control VSV pseudotype bearing VSV GP (VSV-VSV-G) is not neutralized by any sera. When the cut-off serum dilution is set at 50% inhibition of infectivity compared with the infectivity in the absence of the test serum, the neutralization titer of this patient's serum for VSV-LAS-GP is calculated to be 75 ( Figure 4B ). Likewise, a VSV-based pseudotype bearing the Junin virus GP has been developed for the detection of neutralizing antibodies from AHF patients' sera. The accuracy of the results of VSV-based neutralization assays has been confirmed by comparison with the results of the neutralization assay using live Junin virus [70] . The Lujo virus is a new member of the hemorrhagic fever-associated arenavirus family from Zambia and southern Africa, and the virus is classified as a BSL-4 pathogen [17] . The genome sequence analysis of the Lujo virus suggests that the virus is genetically distinct from previously characterized arenaviruses. In order to study the infectivity of this newly", "identified arenavirus, we have recently developed a luciferase-expressing VSV pseudotype bearing Lujo virus GPC (VSV-Lujo-GP). As shown in Figure 3 , infection with VSV-Lujo-GPC is specifically neutralized by rabbit anti-Lujo GPC serum. Thus, the VSV-Lujo-GP may be a useful tool not only for determining the neutralizing antibody titer within the serum, but also for exploring yet-to-be-defined cellular receptor(s) for Lujo virus infection or for screening inhibitors of the Lujo virus GP-mediated cell entry.", "Hemorrhagic fever outbreaks caused by pathogenic arenaviruses result in high fatality rates. A rapid and accurate diagnosis is a critical first step in any outbreak. Serologic diagnostic methods for VHFs most often employ an ELISA, IFA, and/or virus neutralization assay. Diagnostic methods using recombinant viral proteins have been developed and their utilities for diagnosing of VHF have been reviewed. IgG-and IgM-ELISAs and IFAs using rNPs as antigens are useful for the detection of antibodies induced in the patients' sera. These methods are also useful for seroepidemiological surveys for HFVs. Ag-capture ELISAs using MAbs to the arenavirus rNPs are specific for the virus species or can be broadly reactive for New World arenaviruses, depending on the MAb used. Furthermore, the VSV-based pseudotype system provides a safe and rapid tool for measuring virus neutralizing antibody titers, as well as a model to analyze the entry of the respective arenavirus in susceptible cells without", "using live arenaviruses. Recent discoveries of novel arenavirus species [17, 26, 110] and their potential to evolve predominantly via host switching, rather than with their hosts [110, 111] , suggest that an unknown pathogenic arenavirus may emerge in the future, and that the diagnostic methods for VHF caused by arenaviruses should thus be further developed and improved." ]

[ 5 ]

[ "bisegmented, negative-sense, single stranded RNA genome" ]

[ "Serological Assays Based on Recombinant Viral Proteins for the Diagnosis of Arenavirus Hemorrhagic Fevers\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3497043/\n\nSHA: f1d308db379b3c293bcfc8fe251c043fe8842358\n\nAuthors: Fukushi, Shuetsu; Tani, Hideki; Yoshikawa, Tomoki; Saijo, Masayuki; Morikawa, Shigeru\nDate: 2012-10-12\nDOI: 10.3390/v4102097\nLicense: cc-by", "Abstract: The family Arenaviridae, genus Arenavirus, consists of two phylogenetically independent groups: Old World (OW) and New World (NW) complexes. The Lassa and Lujo viruses in the OW complex and the Guanarito, Junin, Machupo, Sabia, and Chapare viruses in the NW complex cause viral hemorrhagic fever (VHF) in humans, leading to serious public health concerns. These viruses are also considered potential bioterrorism agents. Therefore, it is of great importance to detect these pathogens rapidly and specifically in order to minimize the risk and scale of arenavirus outbreaks. However, these arenaviruses are classified as BSL-4 pathogens, thus making it difficult to develop diagnostic techniques for these virus infections in institutes without BSL-4 facilities. To overcome these difficulties, antibody detection systems in the form of an enzyme-linked immunosorbent assay (ELISA) and an indirect immunofluorescence assay were developed using recombinant nucleoproteins (rNPs) derived from", "these viruses. Furthermore, several antigen-detection assays were developed. For example, novel monoclonal antibodies (mAbs) to the rNPs of Lassa and Junin viruses were generated. Sandwich antigen-capture (Ag-capture) ELISAs using these mAbs as capture antibodies were developed and confirmed to be sensitive and specific for detecting the respective arenavirus NPs. These rNP-based assays were proposed to be useful not only for an etiological diagnosis of VHFs, but also for seroepidemiological studies on VHFs. We recently developed arenavirus neutralization assays using vesicular stomatitis virus (VSV)-based pseudotypes bearing arenavirus recombinant glycoproteins. The goal of this article is to review the recent advances in developing laboratory diagnostic assays based on recombinant viral proteins for the diagnosis of VHFs and epidemiological studies on the VHFs caused by arenaviruses.", "Text: The virus family Arenaviridae consists of only one genus, but most viruses within this genus can be divided into two different groups: the Old World arenaviruses and the New World arenaviruses (also known as the Tacaribe complex) [1, 2] . The differences between the two groups have been established through the use of serological assays. Most of the arenaviruses cause persistent infection in rodents without any symptoms, and humans acquire a variety of diseases when zoonotically infected. Lymphocytic choriomeningitis virus (LCMV) is the only arenavirus to exhibit a worldwide distribution, and causes illnesses such as meningitis [3, 4] . Congenital LCMV infections have also been reported [4, 5] . Most importantly, viral hemorrhagic fever (VHF) can be caused by several arenaviruses. Lassa fever, caused by the Lassa virus (LASV), an Old World arenavirus, is one of the most devastating VHFs in humans [6] . Hemorrhaging and organ failure occur in a subset of patients infected with", "this virus, and it is associated with high mortality. Many cases of Lassa fever occur in Western Africa in countries such as Guinea, Sierra Leone, and Nigeria [7] [8] [9] [10] [11] [12] [13] . Tacaribe complex lineage B of the New World arenaviruses consists of the Junin virus (JUNV), Guanarito virus (GUNV), Sabia virus (SABV) and Machupo virus (MACV), the etiological agents of Argentine, Venezuelan, Brazilian, and Bolivian hemorrhagic fevers, respectively [14, 15] . Although genetically distinct from one another, they appear to produce similar symptoms, accompanied by hemorrhaging in humans [14, 15] . These pathogenic New World arenavirus species are closely associated with a specific rodent species [6] .", "Humans are usually infected with pathogenic arenaviruses through direct contact with tissue or blood, or after inhaling aerosolized particles from urine, feces, and saliva of infected rodents. After an incubation period of 1-3 weeks, infected individuals abruptly develop fever, retrosternal pain, sore throat, back pain, cough, abdominal pain, vomiting, diarrhea, conjunctivitis, facial swelling, proteinuria, and mucosal bleeding. Neurological problems have also been described, including hearing loss, tremors, and encephalitis. Because the symptoms of pathogenic arenavirus-related illness are varied and nonspecific, the clinical diagnosis is often difficult [14, 16] . Human-to-human transmission may occur via mucosal or cutaneous contact, or through nosocomial contamination [14, 16] . These viruses are also considered to be potential bioterrorism agents [2] .", "A number of arenavirus species have been recently discovered as a result of both rodent surveys and disease outbreaks [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] . A novel pathogenic New World arenavirus, Chapare virus (CHPV), has been isolated from a fatal case of VHF in Bolivia [20] . In addition, five cases of VHF have been reported in South Africa, and a novel arenavirus, named Lujo virus, was isolated from a patient [17] . The Lujo virus is most distantly related to the other Old World arenaviruses [17] . To date, there is no information concerning the vertebrate host for the Chapare and Lujo viruses.", "There is some evidence of endemicity of the Lassa virus in neighboring countries [27, 28] . However, as the magnitude of international trade and travel is continuously increasing, and the perturbation of the environment (due either to human activity or natural ecological changes) may result in behavioral changes of reservoir rodents, highly pathogenic arenaviruses could be introduced to virus-free countries from endemic areas. In fact, more than twenty cases of Lassa fever have been reported outside of the endemic region in areas such as the USA, Canada, Europe, and Japan [29] [30] [31] [32] [33] . It is of great importance to detect these pathogens rapidly and specifically in order to minimize the risk and scale of outbreaks of VHFs caused by arenaviruses. However, these arenaviruses are classified as biosafety level (BSL)-4 pathogens, making it difficult to develop diagnostic techniques for these virus infections in laboratories without BSL-4 facilities. To overcome these", "difficulties, we have established recombinant viral nucleoproteins (rNPs)-based serological assays, such as IgG-enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IFA), and antigen (Ag)-capture ELISA for the diagnosis of VHFs caused by highly pathogenic arenaviruses. Furthermore, virus neutralization assays using pseudotype virus-bearing arenavirus GPs have been developed. In this review, we describe the usefulness of such recombinant protein-based diagnostic assays for diagnosing VHFs caused by arenaviruses.", "In outbreaks of VHFs, infections are confirmed by various laboratory diagnostic methods. Virus detection is performed by virus isolation, reverse transcription-polymerase chain reaction (RT-PCR), and antigen-capture ELISA. It has been shown that monoclonal antibody panels against pathogenic arenaviruses are useful for detecting viral antigens on the virus-infected cells as well as for investigating of antigenic relationships of arenaviruses [34] [35] [36] . Detection of the virus genome is suitable for a rapid and sensitive diagnosis of VHF patients in the early stage of illness, and extensive reviews of such RT-PCR assays have been described [37, 38] . More recently, progress in the RT-PCR method covering genetic variations of the hemorrhagic fever viruses (HFVs) [39, 40] and a multiplexed oligonucleotide microarray for the differential diagnosis of VHFs have also been reported [41] . On the other hand, antibodies against these viruses can be detected by the indirect", "immunofluorescence assay (IFA), or IgG-and IgM-ELISA. An IFA detects the antibody in the serum, which is able to bind to the fixed monolayer of the virus-infected cells. Although the interpretation of immunofluorescence results requires experience, the assay has advantages over other methods, since each virus generates a characteristic fluorescence pattern that adds specificity to the assay compared to a simple ELISA readout. A serological diagnosis by the detection of specific IgM and IgG antibodies to the HFVs must be sensitive, specific and reliable, because a misdiagnosis can lead to panic in the general population. An IgM-specific ELISA is suitable for detecting recent infection, but the relevance of IgM testing for acute VHF depends on the virus and the duration of illness; specific IgM is not often present in the very early stage of illness, and patients who die of VHF often fail to seroconvert at all. An IgG-specific ELISA is efficacious, not only in the diagnosis of a large", "number of VHF cases, especially during convalescence, but also for epidemiological studies in the endemic regions. The detailed methods used for the IFA and IgG-and IgM-ELISAs for the diagnosis of VHF using authentic virus-antigens have been described in detail [42] [43] [44] [45] .", "Arenaviruses have a bisegmented, negative-sense, single stranded RNA genome with a unique ambisense coding strategy that produces just four known proteins: a glycoprotein, a nucleoprotein (NP), a matrix protein (Z), and a polymerase (L) [46] . Of these proteins, the NP is the most abundant in virus-infected cells. Recombinant protein technology could meet the demand for a simple and reliable VHF test system, and recombinant NP (rNP) has been shown to be useful for serological surveys of IgM-and IgG antibodies against arenaviruses [47] [48] [49] [50] .", "Recombinant baculoviruses that express the full-length rNP of arenaviruses have been generated [48, 50, 51] . The method used for the purification of arenavirus rNP from insect Tn5 cells infected with recombinant baculoviruses is effective and simple compared to those for Ebola, Marburg, and Crimean-Congo hemorrhagic fever virus rNPs [51] [52] [53] [54] [55] . Most of the arenavirus rNPs expressed in insect cells using the recombinant baculoviruses are crystallized [56] and are solubilized in PBS containing 8M urea. Since the majority of Tn5 cellular proteins are solubilized in PBS containing 2M urea, the arenavirus rNPs in the insoluble fraction in PBS containing 2M urea can be solubilized by sonication in PBS containing 8M urea. After a simple centrifugation of the lysates in PBS containing 8M urea, the supernatant fractions can be used as purified rNP antigens without further purification steps [51] . The control antigen is produced from Tn5 cells infected with baculovirus lacking", "the polyhedrin gene (ΔP) in the same manner as the arenavirus rNPs ( Figure 1 ).", "Purified rNPs. The expression and purification efficiency of arenavirus rNP were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) after staining the gels with Coomassie blue. Purified NP antigens with approximate molecular weights of 62 kDa from Luna, LCM, Lassa, Lujo, Junin, Machupo, Guanarito, Sabia, and Chapare viruses and the purified negative control antigen (ΔP) are shown.", "As described above, recombinant baculoviruses allow the delivery of rNP antigens without using infectious live arenaviruses. An ELISA plate coated with the predetermined optimal quantity of purified rNPs (approximately 100 ng/well) is used for the IgG-antibody detection assay. An advantage of using recombinant rNP for the IgG-ELISA is that it enables a direct comparison of antibody cross-reactivity among arenavirus rNPs, since antigen preparations of all arenavirus rNPs tested are performed using the same method [51] . Rabbit anti-sera raised against LCMV-rNP and LASV-rNP show cross-reactivity to LASV-rNP and LCMV-rNP, respectively, indicating that rabbit antibodies against rNPs of Old World arenaviruses cross-react with rNPs of other Old World arenaviruses (Table 1 ) [51] . Similarly, rabbit anti-sera generated against JUNV-NP show cross-reactivity to the LASV-rNP and LCMV-rNP, although the reaction is weak. However, rabbit anti-sera against LASV-NP and LCMV-NP show a negative", "reaction to the JUNV-rNP (Table 1 ) [51] , indicating that rabbit antibodies against JUNV (a pathogenic New World arenavirus) NP might cross-react with the Old World arenavirus NP, whereas antibodies against Old World arenavirus NPs may not be able to react with pathogenic New World arenavirus NPs.", "The rNP-based IgG-ELISA has also been used for the characterization of a mouse monoclonal antibody (MAb). Nakauchi et al. [50] have investigated the cross-reactivity of MAbs against JUNV rNP to pathogenic New World arenavirus rNPs, as well as LASV rNP. MAb C11-12 reacts at the same level with the rNPs of all of the pathogenic New World arenaviruses, including JUNV, GTOV, MACV, SABV, and CHPV, indicating that this MAb recognizes an epitope conserved among pathogenic New World arenaviruses. Another MAb, C6-9, reacts specifically with the rNP of JUNV, but does not react with those of the other pathogenic New World arenaviruses [50] . This indicates that MAb C6-9 recognizes a JUNV-specific epitope. None of these MAbs reacts with the rNP of the human pathogenic Old World arenavirus LASV. Thus, the MAb C11-12 is considered to be a broadly reactive MAb against New World arenaviruses, whereas MAb C6-9 is JUNV-specific. These findings have been confirmed by detailed epitope analyses using", "peptide mapping [50] . Similarly, the cross-reactivity of MAbs against LASV rNP has been analyzed [51] . MAb 4A5 cross-reacts with the Mopeia virus (MOPV) but not with the LCMV rNP. MAb 6C11 cross-reacts with LCMV rNP, while MAb 2-11 does not cross-react with LCMV rNP [51] . Table 1 . Anti-serum reactivity for rNPs of different arenaviruses in IgG ELISAs.", "Reactivity for rNP from LASV LCMV JUNV anti-LASV NP", "It is important to evaluate whether rNP-based ELISA is useful for the diagnosis of human VHF cases. The specificity of the LASV-rNP-based IgG ELISA has been confirmed by using sera obtained from Lassa fever patients [51] . The Lassa fever patients' sera show a highly positive reaction in the LASV-rNP-based IgG-ELISA, but sera from patients with Argentine hemorrhagic fever (AHF), which is caused by JUNV, do not. The serum from an AHF patient showed a highly positive reaction in the JUNV-rNP-based IgG-ELISA [49] . In addition, it was shown that, using sera obtained from AHF cases, the results of the JUNV rNP-based IgG ELISA correlate well with an authentic JUNV antigen-based IgG ELISA [49] . An IgM-capture ELISA using purified LASV-rNP as an antigen has been developed in the same way as in previous reports [54, 57] and detects an LASV-IgM antibody [58] . In addition, immunoblot assays based on N-terminally truncated LASV rNP have been developed for detecting IgG and IgM antibodies", "against LASV. These methods may provide a rapid and simple Lassa fever test for use under field conditions [47] .", "An IFA using virus-infected cells is a common antibody test for VHF viruses [59] [60] [61] [62] [63] . To avoid the use of highly pathogenic viruses for the antigen preparation, mammalian cells expressing recombinant rNP have been developed [51, 57, [64] [65] [66] [67] [68] . Lassa virus NP antigen for IFA can be prepared simply as described [51] . Briefly, the procedure involves (1) transfecting HeLa cells with a mammalian cell expression vector inserted with the cloned NP cDNA; (2) expanding the stable NP-expressing cells by antibiotic selection; (3) mixing the rNP-expressing cells with un-transfected HeLa cells (at a ratio of 1:1); (4) spotting the cell mixtures onto glass slides, then drying and fixing them in acetone.", "In the IFA specific for LASV-NP, antibody positive sera show characteristic granular staining patterns in the cytoplasm (Figure 2 ) [69] , thus making it easy to distinguish positive from negative samples. The specificity of the assay has also been confirmed by using sera obtained from Lassa fever patients [51] . In addition, an IFA using JUNV rNP-expressing HeLa cells has been developed to detect antibodies against JUNV, and the assay has been evaluated by using AHF patients' sera [70] . The LASV-rNP-based antibody detection systems such as ELISA and IFA are suggested to be useful not only for the diagnosis of Lassa fever, but also for seroepidemiological studies of LASV infection. In our preliminary study, approximately 15% of the sera collected from 334 Ghanaians and less than 3% of 280 Zambians showed positive reactions in the LASV-rNP-based IgG ELISA [58] . These results are in agreement with the fact that Lassa fever is endemic to the West African region, including Ghana, but", "less in the East African region.", "For the diagnosis of many viral infections, PCR assays have been shown to have an excellent analytical sensitivity, but the established techniques are limited by their requirement for expensive equipment and technical expertise. Moreover, the high degree of genetic variability of the RNA viruses, including arenavirus and bunyavirus, poses difficulties in selecting primers for RT-PCR assays that can detect all strains of the virus. Since the sensitivity of the Ag-capture ELISA is comparable to that of RT-PCR for several virus-mediated infectious diseases, including Lassa fever and filovirus hemorrhagic fever [51, [71] [72] [73] , the Ag-capture ELISA is a sophisticated approach that can be used for the diagnosis of viral infections. Ag-capture ELISAs detecting viral NP in viremic sera have been widely applied to detect various viruses, since they are the most abundant viral antigens and have highly conserved amino acid sequences [50, 51, 54, 71, 72, 74, 75] . Polyclonal anti-sera or a", "mixture of MAbs present in the ascetic fluids from animals immunized for HFVs have been used for capture-antibodies in the Ag-capture ELISA [36, [76] [77] [78] [79] . MAbs recognizing conserved epitopes of the rNP are also used as capture antibodies since they have a high specificity for the antigens, and an identification of the epitopes of these MAbs is of crucial importance for the assessment of the specificity and cross-reactivity of the assay system [50, 51, 53, 54, 71, 75] . In order to develop a sensitive diagnostic test for Lassa fever and AHF, rNPs of LASV and JUNV (see above) have been prepared, and newly established MAbs against them have been characterized and used for Ag-capture ELISAs [50, 51] . The Ag-capture ELISA using MAb 4A5 has been confirmed to be useful in the detection of authentic LASV antigen in sera serially collected from hamsters infected with LASV [51] . The sensitivity of the MAb 4A5-based Ag-capture ELISA was similar to that of conventional RT-PCR,", "suggesting that the Ag-capture ELISA can be efficiently used in the diagnosis of Lassa fever [51] . Therefore, the MAb 4A5-based Ag-capture ELISA is considered to be useful in the diagnosis of Lassa fever. Also, by using MAbs raised against the rNP of JUNV, Ag-capture ELISAs specific for JUNV and broadly reactive to human pathogenic New World arenaviruses have been developed [50] . The Ag-capture ELISA using MAb E4-2 and C11-12 detected the Ags of all of the pathogenic New World arenaviruses tested, including JUNV. On the other hand, the Ag-capture ELISA using MAb C6-9 detects only the JUNV Ag. Considering that the symptoms of JUNV infection in humans are indistinguishable from those due to other pathogenic New World arenaviruses, the Ag capture ELISA using MAb C6-9 may be a useful diagnostic tool, especially for AHF [50] .", "The virus neutralization assay is accepted as the \"gold standard\" serodiagnostic assay to quantify the antibody response to infection and vaccination of a wide variety of viruses associated with human diseases [80] [81] [82] [83] [84] [85] [86] . The presence of neutralizing antibodies is a reliable indicator of protective immunity against VHF [87] [88] [89] . The most direct method for detection of neutralizing antibodies against HFVs is by plaque reduction neutralization tests using infectious viruses. However, because of the high pathogenicity of HFVs to humans and the strict regulation of select agents, only a limited number of laboratories are able to perform such neutralization tests. For many HFVs, replication-incompetent pseudotyped virus particles bearing viral envelope protein (GP) have been shown to mimic the respective HFV infections, thus, neutralization assays using the pseudotypes may be advantageous in some laboratory settings for the detection of antibodies to HFVs", "without the need for heightened biocontainment requirements.", "The VSV-based vector has already been used to generate replication-competent recombinant VSVs to study of the role of GPs of various viruses [90] [91] [92] . Recent advances in producing pseudotype virus particles have enabled the investigation of the virus cell entry, viral tropism, and effect of entry inhibitors, as well as measurement of the neutralization titers, by using human immunodeficiency virus-, feline immunodeficiency virus-, murine leukemia virus-, or VSV-based vectors [86, [93] [94] [95] [96] [97] [98] [99] [100] [101] [102] [103] . Pseudotypes based on VSV have advantages compared with other pseudotypes based on retroviruses for the following reasons. First, the pseudotype virus titer obtained with the VSV system is generally higher than that of the pseudotyped retrovirus system [104] . Second, the infection of target cells with a VSV pseudotype can be readily detected as green fluorescent protein (GFP)-positive cells at 7-16 h post-infection because of the high level", "of GFP expression in the VSV system [104, 105] . In contrast, the time required for infection in the pseudotyped retrovirus system is 48 h [106, 107] , which is similar to the time required for infectious viruses to replicate to a level that results in plaque-forming or cytopathic effects in infected cells. A high-throughput assay for determining neutralizing antibody titers using VSV pseudotypes expressing secreted alkaline phosphatase [108, 109] or luciferase ( Figure 3 ) has also been developed. We have recently developed a VSV-based pseudotype bearing Lassa virus GP (VSV-LAS-GP) for the detection of neutralizing antibodies in the sera obtained from a Lassa fever patient. An example of the LASV neutralization assay using the VSV pseudotype is shown (Figure 4 ). In the presence of serum from Lassa fever patients, the number of GFP-positive cells (infectivity of VSV-LAS-GP) is significantly reduced compared with the number in the absence of the patient's serum ( Figure 4A ). The", "control VSV pseudotype bearing VSV GP (VSV-VSV-G) is not neutralized by any sera. When the cut-off serum dilution is set at 50% inhibition of infectivity compared with the infectivity in the absence of the test serum, the neutralization titer of this patient's serum for VSV-LAS-GP is calculated to be 75 ( Figure 4B ). Likewise, a VSV-based pseudotype bearing the Junin virus GP has been developed for the detection of neutralizing antibodies from AHF patients' sera. The accuracy of the results of VSV-based neutralization assays has been confirmed by comparison with the results of the neutralization assay using live Junin virus [70] . The Lujo virus is a new member of the hemorrhagic fever-associated arenavirus family from Zambia and southern Africa, and the virus is classified as a BSL-4 pathogen [17] . The genome sequence analysis of the Lujo virus suggests that the virus is genetically distinct from previously characterized arenaviruses. In order to study the infectivity of this newly", "identified arenavirus, we have recently developed a luciferase-expressing VSV pseudotype bearing Lujo virus GPC (VSV-Lujo-GP). As shown in Figure 3 , infection with VSV-Lujo-GPC is specifically neutralized by rabbit anti-Lujo GPC serum. Thus, the VSV-Lujo-GP may be a useful tool not only for determining the neutralizing antibody titer within the serum, but also for exploring yet-to-be-defined cellular receptor(s) for Lujo virus infection or for screening inhibitors of the Lujo virus GP-mediated cell entry.", "Hemorrhagic fever outbreaks caused by pathogenic arenaviruses result in high fatality rates. A rapid and accurate diagnosis is a critical first step in any outbreak. Serologic diagnostic methods for VHFs most often employ an ELISA, IFA, and/or virus neutralization assay. Diagnostic methods using recombinant viral proteins have been developed and their utilities for diagnosing of VHF have been reviewed. IgG-and IgM-ELISAs and IFAs using rNPs as antigens are useful for the detection of antibodies induced in the patients' sera. These methods are also useful for seroepidemiological surveys for HFVs. Ag-capture ELISAs using MAbs to the arenavirus rNPs are specific for the virus species or can be broadly reactive for New World arenaviruses, depending on the MAb used. Furthermore, the VSV-based pseudotype system provides a safe and rapid tool for measuring virus neutralizing antibody titers, as well as a model to analyze the entry of the respective arenavirus in susceptible cells without", "using live arenaviruses. Recent discoveries of novel arenavirus species [17, 26, 110] and their potential to evolve predominantly via host switching, rather than with their hosts [110, 111] , suggest that an unknown pathogenic arenavirus may emerge in the future, and that the diagnostic methods for VHF caused by arenaviruses should thus be further developed and improved." ]

[ 12 ]

[ "a glycoprotein, a nucleoprotein (NP), a matrix protein (Z), and a polymerase (L)" ]

[ "Serological Assays Based on Recombinant Viral Proteins for the Diagnosis of Arenavirus Hemorrhagic Fevers\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3497043/\n\nSHA: f1d308db379b3c293bcfc8fe251c043fe8842358\n\nAuthors: Fukushi, Shuetsu; Tani, Hideki; Yoshikawa, Tomoki; Saijo, Masayuki; Morikawa, Shigeru\nDate: 2012-10-12\nDOI: 10.3390/v4102097\nLicense: cc-by", "Abstract: The family Arenaviridae, genus Arenavirus, consists of two phylogenetically independent groups: Old World (OW) and New World (NW) complexes. The Lassa and Lujo viruses in the OW complex and the Guanarito, Junin, Machupo, Sabia, and Chapare viruses in the NW complex cause viral hemorrhagic fever (VHF) in humans, leading to serious public health concerns. These viruses are also considered potential bioterrorism agents. Therefore, it is of great importance to detect these pathogens rapidly and specifically in order to minimize the risk and scale of arenavirus outbreaks. However, these arenaviruses are classified as BSL-4 pathogens, thus making it difficult to develop diagnostic techniques for these virus infections in institutes without BSL-4 facilities. To overcome these difficulties, antibody detection systems in the form of an enzyme-linked immunosorbent assay (ELISA) and an indirect immunofluorescence assay were developed using recombinant nucleoproteins (rNPs) derived from", "these viruses. Furthermore, several antigen-detection assays were developed. For example, novel monoclonal antibodies (mAbs) to the rNPs of Lassa and Junin viruses were generated. Sandwich antigen-capture (Ag-capture) ELISAs using these mAbs as capture antibodies were developed and confirmed to be sensitive and specific for detecting the respective arenavirus NPs. These rNP-based assays were proposed to be useful not only for an etiological diagnosis of VHFs, but also for seroepidemiological studies on VHFs. We recently developed arenavirus neutralization assays using vesicular stomatitis virus (VSV)-based pseudotypes bearing arenavirus recombinant glycoproteins. The goal of this article is to review the recent advances in developing laboratory diagnostic assays based on recombinant viral proteins for the diagnosis of VHFs and epidemiological studies on the VHFs caused by arenaviruses.", "Text: The virus family Arenaviridae consists of only one genus, but most viruses within this genus can be divided into two different groups: the Old World arenaviruses and the New World arenaviruses (also known as the Tacaribe complex) [1, 2] . The differences between the two groups have been established through the use of serological assays. Most of the arenaviruses cause persistent infection in rodents without any symptoms, and humans acquire a variety of diseases when zoonotically infected. Lymphocytic choriomeningitis virus (LCMV) is the only arenavirus to exhibit a worldwide distribution, and causes illnesses such as meningitis [3, 4] . Congenital LCMV infections have also been reported [4, 5] . Most importantly, viral hemorrhagic fever (VHF) can be caused by several arenaviruses. Lassa fever, caused by the Lassa virus (LASV), an Old World arenavirus, is one of the most devastating VHFs in humans [6] . Hemorrhaging and organ failure occur in a subset of patients infected with", "this virus, and it is associated with high mortality. Many cases of Lassa fever occur in Western Africa in countries such as Guinea, Sierra Leone, and Nigeria [7] [8] [9] [10] [11] [12] [13] . Tacaribe complex lineage B of the New World arenaviruses consists of the Junin virus (JUNV), Guanarito virus (GUNV), Sabia virus (SABV) and Machupo virus (MACV), the etiological agents of Argentine, Venezuelan, Brazilian, and Bolivian hemorrhagic fevers, respectively [14, 15] . Although genetically distinct from one another, they appear to produce similar symptoms, accompanied by hemorrhaging in humans [14, 15] . These pathogenic New World arenavirus species are closely associated with a specific rodent species [6] .", "Humans are usually infected with pathogenic arenaviruses through direct contact with tissue or blood, or after inhaling aerosolized particles from urine, feces, and saliva of infected rodents. After an incubation period of 1-3 weeks, infected individuals abruptly develop fever, retrosternal pain, sore throat, back pain, cough, abdominal pain, vomiting, diarrhea, conjunctivitis, facial swelling, proteinuria, and mucosal bleeding. Neurological problems have also been described, including hearing loss, tremors, and encephalitis. Because the symptoms of pathogenic arenavirus-related illness are varied and nonspecific, the clinical diagnosis is often difficult [14, 16] . Human-to-human transmission may occur via mucosal or cutaneous contact, or through nosocomial contamination [14, 16] . These viruses are also considered to be potential bioterrorism agents [2] .", "A number of arenavirus species have been recently discovered as a result of both rodent surveys and disease outbreaks [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] . A novel pathogenic New World arenavirus, Chapare virus (CHPV), has been isolated from a fatal case of VHF in Bolivia [20] . In addition, five cases of VHF have been reported in South Africa, and a novel arenavirus, named Lujo virus, was isolated from a patient [17] . The Lujo virus is most distantly related to the other Old World arenaviruses [17] . To date, there is no information concerning the vertebrate host for the Chapare and Lujo viruses.", "There is some evidence of endemicity of the Lassa virus in neighboring countries [27, 28] . However, as the magnitude of international trade and travel is continuously increasing, and the perturbation of the environment (due either to human activity or natural ecological changes) may result in behavioral changes of reservoir rodents, highly pathogenic arenaviruses could be introduced to virus-free countries from endemic areas. In fact, more than twenty cases of Lassa fever have been reported outside of the endemic region in areas such as the USA, Canada, Europe, and Japan [29] [30] [31] [32] [33] . It is of great importance to detect these pathogens rapidly and specifically in order to minimize the risk and scale of outbreaks of VHFs caused by arenaviruses. However, these arenaviruses are classified as biosafety level (BSL)-4 pathogens, making it difficult to develop diagnostic techniques for these virus infections in laboratories without BSL-4 facilities. To overcome these", "difficulties, we have established recombinant viral nucleoproteins (rNPs)-based serological assays, such as IgG-enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IFA), and antigen (Ag)-capture ELISA for the diagnosis of VHFs caused by highly pathogenic arenaviruses. Furthermore, virus neutralization assays using pseudotype virus-bearing arenavirus GPs have been developed. In this review, we describe the usefulness of such recombinant protein-based diagnostic assays for diagnosing VHFs caused by arenaviruses.", "In outbreaks of VHFs, infections are confirmed by various laboratory diagnostic methods. Virus detection is performed by virus isolation, reverse transcription-polymerase chain reaction (RT-PCR), and antigen-capture ELISA. It has been shown that monoclonal antibody panels against pathogenic arenaviruses are useful for detecting viral antigens on the virus-infected cells as well as for investigating of antigenic relationships of arenaviruses [34] [35] [36] . Detection of the virus genome is suitable for a rapid and sensitive diagnosis of VHF patients in the early stage of illness, and extensive reviews of such RT-PCR assays have been described [37, 38] . More recently, progress in the RT-PCR method covering genetic variations of the hemorrhagic fever viruses (HFVs) [39, 40] and a multiplexed oligonucleotide microarray for the differential diagnosis of VHFs have also been reported [41] . On the other hand, antibodies against these viruses can be detected by the indirect", "immunofluorescence assay (IFA), or IgG-and IgM-ELISA. An IFA detects the antibody in the serum, which is able to bind to the fixed monolayer of the virus-infected cells. Although the interpretation of immunofluorescence results requires experience, the assay has advantages over other methods, since each virus generates a characteristic fluorescence pattern that adds specificity to the assay compared to a simple ELISA readout. A serological diagnosis by the detection of specific IgM and IgG antibodies to the HFVs must be sensitive, specific and reliable, because a misdiagnosis can lead to panic in the general population. An IgM-specific ELISA is suitable for detecting recent infection, but the relevance of IgM testing for acute VHF depends on the virus and the duration of illness; specific IgM is not often present in the very early stage of illness, and patients who die of VHF often fail to seroconvert at all. An IgG-specific ELISA is efficacious, not only in the diagnosis of a large", "number of VHF cases, especially during convalescence, but also for epidemiological studies in the endemic regions. The detailed methods used for the IFA and IgG-and IgM-ELISAs for the diagnosis of VHF using authentic virus-antigens have been described in detail [42] [43] [44] [45] .", "Arenaviruses have a bisegmented, negative-sense, single stranded RNA genome with a unique ambisense coding strategy that produces just four known proteins: a glycoprotein, a nucleoprotein (NP), a matrix protein (Z), and a polymerase (L) [46] . Of these proteins, the NP is the most abundant in virus-infected cells. Recombinant protein technology could meet the demand for a simple and reliable VHF test system, and recombinant NP (rNP) has been shown to be useful for serological surveys of IgM-and IgG antibodies against arenaviruses [47] [48] [49] [50] .", "Recombinant baculoviruses that express the full-length rNP of arenaviruses have been generated [48, 50, 51] . The method used for the purification of arenavirus rNP from insect Tn5 cells infected with recombinant baculoviruses is effective and simple compared to those for Ebola, Marburg, and Crimean-Congo hemorrhagic fever virus rNPs [51] [52] [53] [54] [55] . Most of the arenavirus rNPs expressed in insect cells using the recombinant baculoviruses are crystallized [56] and are solubilized in PBS containing 8M urea. Since the majority of Tn5 cellular proteins are solubilized in PBS containing 2M urea, the arenavirus rNPs in the insoluble fraction in PBS containing 2M urea can be solubilized by sonication in PBS containing 8M urea. After a simple centrifugation of the lysates in PBS containing 8M urea, the supernatant fractions can be used as purified rNP antigens without further purification steps [51] . The control antigen is produced from Tn5 cells infected with baculovirus lacking", "the polyhedrin gene (ΔP) in the same manner as the arenavirus rNPs ( Figure 1 ).", "Purified rNPs. The expression and purification efficiency of arenavirus rNP were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) after staining the gels with Coomassie blue. Purified NP antigens with approximate molecular weights of 62 kDa from Luna, LCM, Lassa, Lujo, Junin, Machupo, Guanarito, Sabia, and Chapare viruses and the purified negative control antigen (ΔP) are shown.", "As described above, recombinant baculoviruses allow the delivery of rNP antigens without using infectious live arenaviruses. An ELISA plate coated with the predetermined optimal quantity of purified rNPs (approximately 100 ng/well) is used for the IgG-antibody detection assay. An advantage of using recombinant rNP for the IgG-ELISA is that it enables a direct comparison of antibody cross-reactivity among arenavirus rNPs, since antigen preparations of all arenavirus rNPs tested are performed using the same method [51] . Rabbit anti-sera raised against LCMV-rNP and LASV-rNP show cross-reactivity to LASV-rNP and LCMV-rNP, respectively, indicating that rabbit antibodies against rNPs of Old World arenaviruses cross-react with rNPs of other Old World arenaviruses (Table 1 ) [51] . Similarly, rabbit anti-sera generated against JUNV-NP show cross-reactivity to the LASV-rNP and LCMV-rNP, although the reaction is weak. However, rabbit anti-sera against LASV-NP and LCMV-NP show a negative", "reaction to the JUNV-rNP (Table 1 ) [51] , indicating that rabbit antibodies against JUNV (a pathogenic New World arenavirus) NP might cross-react with the Old World arenavirus NP, whereas antibodies against Old World arenavirus NPs may not be able to react with pathogenic New World arenavirus NPs.", "The rNP-based IgG-ELISA has also been used for the characterization of a mouse monoclonal antibody (MAb). Nakauchi et al. [50] have investigated the cross-reactivity of MAbs against JUNV rNP to pathogenic New World arenavirus rNPs, as well as LASV rNP. MAb C11-12 reacts at the same level with the rNPs of all of the pathogenic New World arenaviruses, including JUNV, GTOV, MACV, SABV, and CHPV, indicating that this MAb recognizes an epitope conserved among pathogenic New World arenaviruses. Another MAb, C6-9, reacts specifically with the rNP of JUNV, but does not react with those of the other pathogenic New World arenaviruses [50] . This indicates that MAb C6-9 recognizes a JUNV-specific epitope. None of these MAbs reacts with the rNP of the human pathogenic Old World arenavirus LASV. Thus, the MAb C11-12 is considered to be a broadly reactive MAb against New World arenaviruses, whereas MAb C6-9 is JUNV-specific. These findings have been confirmed by detailed epitope analyses using", "peptide mapping [50] . Similarly, the cross-reactivity of MAbs against LASV rNP has been analyzed [51] . MAb 4A5 cross-reacts with the Mopeia virus (MOPV) but not with the LCMV rNP. MAb 6C11 cross-reacts with LCMV rNP, while MAb 2-11 does not cross-react with LCMV rNP [51] . Table 1 . Anti-serum reactivity for rNPs of different arenaviruses in IgG ELISAs.", "Reactivity for rNP from LASV LCMV JUNV anti-LASV NP", "It is important to evaluate whether rNP-based ELISA is useful for the diagnosis of human VHF cases. The specificity of the LASV-rNP-based IgG ELISA has been confirmed by using sera obtained from Lassa fever patients [51] . The Lassa fever patients' sera show a highly positive reaction in the LASV-rNP-based IgG-ELISA, but sera from patients with Argentine hemorrhagic fever (AHF), which is caused by JUNV, do not. The serum from an AHF patient showed a highly positive reaction in the JUNV-rNP-based IgG-ELISA [49] . In addition, it was shown that, using sera obtained from AHF cases, the results of the JUNV rNP-based IgG ELISA correlate well with an authentic JUNV antigen-based IgG ELISA [49] . An IgM-capture ELISA using purified LASV-rNP as an antigen has been developed in the same way as in previous reports [54, 57] and detects an LASV-IgM antibody [58] . In addition, immunoblot assays based on N-terminally truncated LASV rNP have been developed for detecting IgG and IgM antibodies", "against LASV. These methods may provide a rapid and simple Lassa fever test for use under field conditions [47] .", "An IFA using virus-infected cells is a common antibody test for VHF viruses [59] [60] [61] [62] [63] . To avoid the use of highly pathogenic viruses for the antigen preparation, mammalian cells expressing recombinant rNP have been developed [51, 57, [64] [65] [66] [67] [68] . Lassa virus NP antigen for IFA can be prepared simply as described [51] . Briefly, the procedure involves (1) transfecting HeLa cells with a mammalian cell expression vector inserted with the cloned NP cDNA; (2) expanding the stable NP-expressing cells by antibiotic selection; (3) mixing the rNP-expressing cells with un-transfected HeLa cells (at a ratio of 1:1); (4) spotting the cell mixtures onto glass slides, then drying and fixing them in acetone.", "In the IFA specific for LASV-NP, antibody positive sera show characteristic granular staining patterns in the cytoplasm (Figure 2 ) [69] , thus making it easy to distinguish positive from negative samples. The specificity of the assay has also been confirmed by using sera obtained from Lassa fever patients [51] . In addition, an IFA using JUNV rNP-expressing HeLa cells has been developed to detect antibodies against JUNV, and the assay has been evaluated by using AHF patients' sera [70] . The LASV-rNP-based antibody detection systems such as ELISA and IFA are suggested to be useful not only for the diagnosis of Lassa fever, but also for seroepidemiological studies of LASV infection. In our preliminary study, approximately 15% of the sera collected from 334 Ghanaians and less than 3% of 280 Zambians showed positive reactions in the LASV-rNP-based IgG ELISA [58] . These results are in agreement with the fact that Lassa fever is endemic to the West African region, including Ghana, but", "less in the East African region.", "For the diagnosis of many viral infections, PCR assays have been shown to have an excellent analytical sensitivity, but the established techniques are limited by their requirement for expensive equipment and technical expertise. Moreover, the high degree of genetic variability of the RNA viruses, including arenavirus and bunyavirus, poses difficulties in selecting primers for RT-PCR assays that can detect all strains of the virus. Since the sensitivity of the Ag-capture ELISA is comparable to that of RT-PCR for several virus-mediated infectious diseases, including Lassa fever and filovirus hemorrhagic fever [51, [71] [72] [73] , the Ag-capture ELISA is a sophisticated approach that can be used for the diagnosis of viral infections. Ag-capture ELISAs detecting viral NP in viremic sera have been widely applied to detect various viruses, since they are the most abundant viral antigens and have highly conserved amino acid sequences [50, 51, 54, 71, 72, 74, 75] . Polyclonal anti-sera or a", "mixture of MAbs present in the ascetic fluids from animals immunized for HFVs have been used for capture-antibodies in the Ag-capture ELISA [36, [76] [77] [78] [79] . MAbs recognizing conserved epitopes of the rNP are also used as capture antibodies since they have a high specificity for the antigens, and an identification of the epitopes of these MAbs is of crucial importance for the assessment of the specificity and cross-reactivity of the assay system [50, 51, 53, 54, 71, 75] . In order to develop a sensitive diagnostic test for Lassa fever and AHF, rNPs of LASV and JUNV (see above) have been prepared, and newly established MAbs against them have been characterized and used for Ag-capture ELISAs [50, 51] . The Ag-capture ELISA using MAb 4A5 has been confirmed to be useful in the detection of authentic LASV antigen in sera serially collected from hamsters infected with LASV [51] . The sensitivity of the MAb 4A5-based Ag-capture ELISA was similar to that of conventional RT-PCR,", "suggesting that the Ag-capture ELISA can be efficiently used in the diagnosis of Lassa fever [51] . Therefore, the MAb 4A5-based Ag-capture ELISA is considered to be useful in the diagnosis of Lassa fever. Also, by using MAbs raised against the rNP of JUNV, Ag-capture ELISAs specific for JUNV and broadly reactive to human pathogenic New World arenaviruses have been developed [50] . The Ag-capture ELISA using MAb E4-2 and C11-12 detected the Ags of all of the pathogenic New World arenaviruses tested, including JUNV. On the other hand, the Ag-capture ELISA using MAb C6-9 detects only the JUNV Ag. Considering that the symptoms of JUNV infection in humans are indistinguishable from those due to other pathogenic New World arenaviruses, the Ag capture ELISA using MAb C6-9 may be a useful diagnostic tool, especially for AHF [50] .", "The virus neutralization assay is accepted as the \"gold standard\" serodiagnostic assay to quantify the antibody response to infection and vaccination of a wide variety of viruses associated with human diseases [80] [81] [82] [83] [84] [85] [86] . The presence of neutralizing antibodies is a reliable indicator of protective immunity against VHF [87] [88] [89] . The most direct method for detection of neutralizing antibodies against HFVs is by plaque reduction neutralization tests using infectious viruses. However, because of the high pathogenicity of HFVs to humans and the strict regulation of select agents, only a limited number of laboratories are able to perform such neutralization tests. For many HFVs, replication-incompetent pseudotyped virus particles bearing viral envelope protein (GP) have been shown to mimic the respective HFV infections, thus, neutralization assays using the pseudotypes may be advantageous in some laboratory settings for the detection of antibodies to HFVs", "without the need for heightened biocontainment requirements.", "The VSV-based vector has already been used to generate replication-competent recombinant VSVs to study of the role of GPs of various viruses [90] [91] [92] . Recent advances in producing pseudotype virus particles have enabled the investigation of the virus cell entry, viral tropism, and effect of entry inhibitors, as well as measurement of the neutralization titers, by using human immunodeficiency virus-, feline immunodeficiency virus-, murine leukemia virus-, or VSV-based vectors [86, [93] [94] [95] [96] [97] [98] [99] [100] [101] [102] [103] . Pseudotypes based on VSV have advantages compared with other pseudotypes based on retroviruses for the following reasons. First, the pseudotype virus titer obtained with the VSV system is generally higher than that of the pseudotyped retrovirus system [104] . Second, the infection of target cells with a VSV pseudotype can be readily detected as green fluorescent protein (GFP)-positive cells at 7-16 h post-infection because of the high level", "of GFP expression in the VSV system [104, 105] . In contrast, the time required for infection in the pseudotyped retrovirus system is 48 h [106, 107] , which is similar to the time required for infectious viruses to replicate to a level that results in plaque-forming or cytopathic effects in infected cells. A high-throughput assay for determining neutralizing antibody titers using VSV pseudotypes expressing secreted alkaline phosphatase [108, 109] or luciferase ( Figure 3 ) has also been developed. We have recently developed a VSV-based pseudotype bearing Lassa virus GP (VSV-LAS-GP) for the detection of neutralizing antibodies in the sera obtained from a Lassa fever patient. An example of the LASV neutralization assay using the VSV pseudotype is shown (Figure 4 ). In the presence of serum from Lassa fever patients, the number of GFP-positive cells (infectivity of VSV-LAS-GP) is significantly reduced compared with the number in the absence of the patient's serum ( Figure 4A ). The", "control VSV pseudotype bearing VSV GP (VSV-VSV-G) is not neutralized by any sera. When the cut-off serum dilution is set at 50% inhibition of infectivity compared with the infectivity in the absence of the test serum, the neutralization titer of this patient's serum for VSV-LAS-GP is calculated to be 75 ( Figure 4B ). Likewise, a VSV-based pseudotype bearing the Junin virus GP has been developed for the detection of neutralizing antibodies from AHF patients' sera. The accuracy of the results of VSV-based neutralization assays has been confirmed by comparison with the results of the neutralization assay using live Junin virus [70] . The Lujo virus is a new member of the hemorrhagic fever-associated arenavirus family from Zambia and southern Africa, and the virus is classified as a BSL-4 pathogen [17] . The genome sequence analysis of the Lujo virus suggests that the virus is genetically distinct from previously characterized arenaviruses. In order to study the infectivity of this newly", "identified arenavirus, we have recently developed a luciferase-expressing VSV pseudotype bearing Lujo virus GPC (VSV-Lujo-GP). As shown in Figure 3 , infection with VSV-Lujo-GPC is specifically neutralized by rabbit anti-Lujo GPC serum. Thus, the VSV-Lujo-GP may be a useful tool not only for determining the neutralizing antibody titer within the serum, but also for exploring yet-to-be-defined cellular receptor(s) for Lujo virus infection or for screening inhibitors of the Lujo virus GP-mediated cell entry.", "Hemorrhagic fever outbreaks caused by pathogenic arenaviruses result in high fatality rates. A rapid and accurate diagnosis is a critical first step in any outbreak. Serologic diagnostic methods for VHFs most often employ an ELISA, IFA, and/or virus neutralization assay. Diagnostic methods using recombinant viral proteins have been developed and their utilities for diagnosing of VHF have been reviewed. IgG-and IgM-ELISAs and IFAs using rNPs as antigens are useful for the detection of antibodies induced in the patients' sera. These methods are also useful for seroepidemiological surveys for HFVs. Ag-capture ELISAs using MAbs to the arenavirus rNPs are specific for the virus species or can be broadly reactive for New World arenaviruses, depending on the MAb used. Furthermore, the VSV-based pseudotype system provides a safe and rapid tool for measuring virus neutralizing antibody titers, as well as a model to analyze the entry of the respective arenavirus in susceptible cells without", "using live arenaviruses. Recent discoveries of novel arenavirus species [17, 26, 110] and their potential to evolve predominantly via host switching, rather than with their hosts [110, 111] , suggest that an unknown pathogenic arenavirus may emerge in the future, and that the diagnostic methods for VHF caused by arenaviruses should thus be further developed and improved." ]

[ 12 ]

[ "PCR assays" ]

[ "Serological Assays Based on Recombinant Viral Proteins for the Diagnosis of Arenavirus Hemorrhagic Fevers\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3497043/\n\nSHA: f1d308db379b3c293bcfc8fe251c043fe8842358\n\nAuthors: Fukushi, Shuetsu; Tani, Hideki; Yoshikawa, Tomoki; Saijo, Masayuki; Morikawa, Shigeru\nDate: 2012-10-12\nDOI: 10.3390/v4102097\nLicense: cc-by", "Abstract: The family Arenaviridae, genus Arenavirus, consists of two phylogenetically independent groups: Old World (OW) and New World (NW) complexes. The Lassa and Lujo viruses in the OW complex and the Guanarito, Junin, Machupo, Sabia, and Chapare viruses in the NW complex cause viral hemorrhagic fever (VHF) in humans, leading to serious public health concerns. These viruses are also considered potential bioterrorism agents. Therefore, it is of great importance to detect these pathogens rapidly and specifically in order to minimize the risk and scale of arenavirus outbreaks. However, these arenaviruses are classified as BSL-4 pathogens, thus making it difficult to develop diagnostic techniques for these virus infections in institutes without BSL-4 facilities. To overcome these difficulties, antibody detection systems in the form of an enzyme-linked immunosorbent assay (ELISA) and an indirect immunofluorescence assay were developed using recombinant nucleoproteins (rNPs) derived from", "these viruses. Furthermore, several antigen-detection assays were developed. For example, novel monoclonal antibodies (mAbs) to the rNPs of Lassa and Junin viruses were generated. Sandwich antigen-capture (Ag-capture) ELISAs using these mAbs as capture antibodies were developed and confirmed to be sensitive and specific for detecting the respective arenavirus NPs. These rNP-based assays were proposed to be useful not only for an etiological diagnosis of VHFs, but also for seroepidemiological studies on VHFs. We recently developed arenavirus neutralization assays using vesicular stomatitis virus (VSV)-based pseudotypes bearing arenavirus recombinant glycoproteins. The goal of this article is to review the recent advances in developing laboratory diagnostic assays based on recombinant viral proteins for the diagnosis of VHFs and epidemiological studies on the VHFs caused by arenaviruses.", "Text: The virus family Arenaviridae consists of only one genus, but most viruses within this genus can be divided into two different groups: the Old World arenaviruses and the New World arenaviruses (also known as the Tacaribe complex) [1, 2] . The differences between the two groups have been established through the use of serological assays. Most of the arenaviruses cause persistent infection in rodents without any symptoms, and humans acquire a variety of diseases when zoonotically infected. Lymphocytic choriomeningitis virus (LCMV) is the only arenavirus to exhibit a worldwide distribution, and causes illnesses such as meningitis [3, 4] . Congenital LCMV infections have also been reported [4, 5] . Most importantly, viral hemorrhagic fever (VHF) can be caused by several arenaviruses. Lassa fever, caused by the Lassa virus (LASV), an Old World arenavirus, is one of the most devastating VHFs in humans [6] . Hemorrhaging and organ failure occur in a subset of patients infected with", "this virus, and it is associated with high mortality. Many cases of Lassa fever occur in Western Africa in countries such as Guinea, Sierra Leone, and Nigeria [7] [8] [9] [10] [11] [12] [13] . Tacaribe complex lineage B of the New World arenaviruses consists of the Junin virus (JUNV), Guanarito virus (GUNV), Sabia virus (SABV) and Machupo virus (MACV), the etiological agents of Argentine, Venezuelan, Brazilian, and Bolivian hemorrhagic fevers, respectively [14, 15] . Although genetically distinct from one another, they appear to produce similar symptoms, accompanied by hemorrhaging in humans [14, 15] . These pathogenic New World arenavirus species are closely associated with a specific rodent species [6] .", "Humans are usually infected with pathogenic arenaviruses through direct contact with tissue or blood, or after inhaling aerosolized particles from urine, feces, and saliva of infected rodents. After an incubation period of 1-3 weeks, infected individuals abruptly develop fever, retrosternal pain, sore throat, back pain, cough, abdominal pain, vomiting, diarrhea, conjunctivitis, facial swelling, proteinuria, and mucosal bleeding. Neurological problems have also been described, including hearing loss, tremors, and encephalitis. Because the symptoms of pathogenic arenavirus-related illness are varied and nonspecific, the clinical diagnosis is often difficult [14, 16] . Human-to-human transmission may occur via mucosal or cutaneous contact, or through nosocomial contamination [14, 16] . These viruses are also considered to be potential bioterrorism agents [2] .", "A number of arenavirus species have been recently discovered as a result of both rodent surveys and disease outbreaks [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] . A novel pathogenic New World arenavirus, Chapare virus (CHPV), has been isolated from a fatal case of VHF in Bolivia [20] . In addition, five cases of VHF have been reported in South Africa, and a novel arenavirus, named Lujo virus, was isolated from a patient [17] . The Lujo virus is most distantly related to the other Old World arenaviruses [17] . To date, there is no information concerning the vertebrate host for the Chapare and Lujo viruses.", "There is some evidence of endemicity of the Lassa virus in neighboring countries [27, 28] . However, as the magnitude of international trade and travel is continuously increasing, and the perturbation of the environment (due either to human activity or natural ecological changes) may result in behavioral changes of reservoir rodents, highly pathogenic arenaviruses could be introduced to virus-free countries from endemic areas. In fact, more than twenty cases of Lassa fever have been reported outside of the endemic region in areas such as the USA, Canada, Europe, and Japan [29] [30] [31] [32] [33] . It is of great importance to detect these pathogens rapidly and specifically in order to minimize the risk and scale of outbreaks of VHFs caused by arenaviruses. However, these arenaviruses are classified as biosafety level (BSL)-4 pathogens, making it difficult to develop diagnostic techniques for these virus infections in laboratories without BSL-4 facilities. To overcome these", "difficulties, we have established recombinant viral nucleoproteins (rNPs)-based serological assays, such as IgG-enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IFA), and antigen (Ag)-capture ELISA for the diagnosis of VHFs caused by highly pathogenic arenaviruses. Furthermore, virus neutralization assays using pseudotype virus-bearing arenavirus GPs have been developed. In this review, we describe the usefulness of such recombinant protein-based diagnostic assays for diagnosing VHFs caused by arenaviruses.", "In outbreaks of VHFs, infections are confirmed by various laboratory diagnostic methods. Virus detection is performed by virus isolation, reverse transcription-polymerase chain reaction (RT-PCR), and antigen-capture ELISA. It has been shown that monoclonal antibody panels against pathogenic arenaviruses are useful for detecting viral antigens on the virus-infected cells as well as for investigating of antigenic relationships of arenaviruses [34] [35] [36] . Detection of the virus genome is suitable for a rapid and sensitive diagnosis of VHF patients in the early stage of illness, and extensive reviews of such RT-PCR assays have been described [37, 38] . More recently, progress in the RT-PCR method covering genetic variations of the hemorrhagic fever viruses (HFVs) [39, 40] and a multiplexed oligonucleotide microarray for the differential diagnosis of VHFs have also been reported [41] . On the other hand, antibodies against these viruses can be detected by the indirect", "immunofluorescence assay (IFA), or IgG-and IgM-ELISA. An IFA detects the antibody in the serum, which is able to bind to the fixed monolayer of the virus-infected cells. Although the interpretation of immunofluorescence results requires experience, the assay has advantages over other methods, since each virus generates a characteristic fluorescence pattern that adds specificity to the assay compared to a simple ELISA readout. A serological diagnosis by the detection of specific IgM and IgG antibodies to the HFVs must be sensitive, specific and reliable, because a misdiagnosis can lead to panic in the general population. An IgM-specific ELISA is suitable for detecting recent infection, but the relevance of IgM testing for acute VHF depends on the virus and the duration of illness; specific IgM is not often present in the very early stage of illness, and patients who die of VHF often fail to seroconvert at all. An IgG-specific ELISA is efficacious, not only in the diagnosis of a large", "number of VHF cases, especially during convalescence, but also for epidemiological studies in the endemic regions. The detailed methods used for the IFA and IgG-and IgM-ELISAs for the diagnosis of VHF using authentic virus-antigens have been described in detail [42] [43] [44] [45] .", "Arenaviruses have a bisegmented, negative-sense, single stranded RNA genome with a unique ambisense coding strategy that produces just four known proteins: a glycoprotein, a nucleoprotein (NP), a matrix protein (Z), and a polymerase (L) [46] . Of these proteins, the NP is the most abundant in virus-infected cells. Recombinant protein technology could meet the demand for a simple and reliable VHF test system, and recombinant NP (rNP) has been shown to be useful for serological surveys of IgM-and IgG antibodies against arenaviruses [47] [48] [49] [50] .", "Recombinant baculoviruses that express the full-length rNP of arenaviruses have been generated [48, 50, 51] . The method used for the purification of arenavirus rNP from insect Tn5 cells infected with recombinant baculoviruses is effective and simple compared to those for Ebola, Marburg, and Crimean-Congo hemorrhagic fever virus rNPs [51] [52] [53] [54] [55] . Most of the arenavirus rNPs expressed in insect cells using the recombinant baculoviruses are crystallized [56] and are solubilized in PBS containing 8M urea. Since the majority of Tn5 cellular proteins are solubilized in PBS containing 2M urea, the arenavirus rNPs in the insoluble fraction in PBS containing 2M urea can be solubilized by sonication in PBS containing 8M urea. After a simple centrifugation of the lysates in PBS containing 8M urea, the supernatant fractions can be used as purified rNP antigens without further purification steps [51] . The control antigen is produced from Tn5 cells infected with baculovirus lacking", "the polyhedrin gene (ΔP) in the same manner as the arenavirus rNPs ( Figure 1 ).", "Purified rNPs. The expression and purification efficiency of arenavirus rNP were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) after staining the gels with Coomassie blue. Purified NP antigens with approximate molecular weights of 62 kDa from Luna, LCM, Lassa, Lujo, Junin, Machupo, Guanarito, Sabia, and Chapare viruses and the purified negative control antigen (ΔP) are shown.", "As described above, recombinant baculoviruses allow the delivery of rNP antigens without using infectious live arenaviruses. An ELISA plate coated with the predetermined optimal quantity of purified rNPs (approximately 100 ng/well) is used for the IgG-antibody detection assay. An advantage of using recombinant rNP for the IgG-ELISA is that it enables a direct comparison of antibody cross-reactivity among arenavirus rNPs, since antigen preparations of all arenavirus rNPs tested are performed using the same method [51] . Rabbit anti-sera raised against LCMV-rNP and LASV-rNP show cross-reactivity to LASV-rNP and LCMV-rNP, respectively, indicating that rabbit antibodies against rNPs of Old World arenaviruses cross-react with rNPs of other Old World arenaviruses (Table 1 ) [51] . Similarly, rabbit anti-sera generated against JUNV-NP show cross-reactivity to the LASV-rNP and LCMV-rNP, although the reaction is weak. However, rabbit anti-sera against LASV-NP and LCMV-NP show a negative", "reaction to the JUNV-rNP (Table 1 ) [51] , indicating that rabbit antibodies against JUNV (a pathogenic New World arenavirus) NP might cross-react with the Old World arenavirus NP, whereas antibodies against Old World arenavirus NPs may not be able to react with pathogenic New World arenavirus NPs.", "The rNP-based IgG-ELISA has also been used for the characterization of a mouse monoclonal antibody (MAb). Nakauchi et al. [50] have investigated the cross-reactivity of MAbs against JUNV rNP to pathogenic New World arenavirus rNPs, as well as LASV rNP. MAb C11-12 reacts at the same level with the rNPs of all of the pathogenic New World arenaviruses, including JUNV, GTOV, MACV, SABV, and CHPV, indicating that this MAb recognizes an epitope conserved among pathogenic New World arenaviruses. Another MAb, C6-9, reacts specifically with the rNP of JUNV, but does not react with those of the other pathogenic New World arenaviruses [50] . This indicates that MAb C6-9 recognizes a JUNV-specific epitope. None of these MAbs reacts with the rNP of the human pathogenic Old World arenavirus LASV. Thus, the MAb C11-12 is considered to be a broadly reactive MAb against New World arenaviruses, whereas MAb C6-9 is JUNV-specific. These findings have been confirmed by detailed epitope analyses using", "peptide mapping [50] . Similarly, the cross-reactivity of MAbs against LASV rNP has been analyzed [51] . MAb 4A5 cross-reacts with the Mopeia virus (MOPV) but not with the LCMV rNP. MAb 6C11 cross-reacts with LCMV rNP, while MAb 2-11 does not cross-react with LCMV rNP [51] . Table 1 . Anti-serum reactivity for rNPs of different arenaviruses in IgG ELISAs.", "Reactivity for rNP from LASV LCMV JUNV anti-LASV NP", "It is important to evaluate whether rNP-based ELISA is useful for the diagnosis of human VHF cases. The specificity of the LASV-rNP-based IgG ELISA has been confirmed by using sera obtained from Lassa fever patients [51] . The Lassa fever patients' sera show a highly positive reaction in the LASV-rNP-based IgG-ELISA, but sera from patients with Argentine hemorrhagic fever (AHF), which is caused by JUNV, do not. The serum from an AHF patient showed a highly positive reaction in the JUNV-rNP-based IgG-ELISA [49] . In addition, it was shown that, using sera obtained from AHF cases, the results of the JUNV rNP-based IgG ELISA correlate well with an authentic JUNV antigen-based IgG ELISA [49] . An IgM-capture ELISA using purified LASV-rNP as an antigen has been developed in the same way as in previous reports [54, 57] and detects an LASV-IgM antibody [58] . In addition, immunoblot assays based on N-terminally truncated LASV rNP have been developed for detecting IgG and IgM antibodies", "against LASV. These methods may provide a rapid and simple Lassa fever test for use under field conditions [47] .", "An IFA using virus-infected cells is a common antibody test for VHF viruses [59] [60] [61] [62] [63] . To avoid the use of highly pathogenic viruses for the antigen preparation, mammalian cells expressing recombinant rNP have been developed [51, 57, [64] [65] [66] [67] [68] . Lassa virus NP antigen for IFA can be prepared simply as described [51] . Briefly, the procedure involves (1) transfecting HeLa cells with a mammalian cell expression vector inserted with the cloned NP cDNA; (2) expanding the stable NP-expressing cells by antibiotic selection; (3) mixing the rNP-expressing cells with un-transfected HeLa cells (at a ratio of 1:1); (4) spotting the cell mixtures onto glass slides, then drying and fixing them in acetone.", "In the IFA specific for LASV-NP, antibody positive sera show characteristic granular staining patterns in the cytoplasm (Figure 2 ) [69] , thus making it easy to distinguish positive from negative samples. The specificity of the assay has also been confirmed by using sera obtained from Lassa fever patients [51] . In addition, an IFA using JUNV rNP-expressing HeLa cells has been developed to detect antibodies against JUNV, and the assay has been evaluated by using AHF patients' sera [70] . The LASV-rNP-based antibody detection systems such as ELISA and IFA are suggested to be useful not only for the diagnosis of Lassa fever, but also for seroepidemiological studies of LASV infection. In our preliminary study, approximately 15% of the sera collected from 334 Ghanaians and less than 3% of 280 Zambians showed positive reactions in the LASV-rNP-based IgG ELISA [58] . These results are in agreement with the fact that Lassa fever is endemic to the West African region, including Ghana, but", "less in the East African region.", "For the diagnosis of many viral infections, PCR assays have been shown to have an excellent analytical sensitivity, but the established techniques are limited by their requirement for expensive equipment and technical expertise. Moreover, the high degree of genetic variability of the RNA viruses, including arenavirus and bunyavirus, poses difficulties in selecting primers for RT-PCR assays that can detect all strains of the virus. Since the sensitivity of the Ag-capture ELISA is comparable to that of RT-PCR for several virus-mediated infectious diseases, including Lassa fever and filovirus hemorrhagic fever [51, [71] [72] [73] , the Ag-capture ELISA is a sophisticated approach that can be used for the diagnosis of viral infections. Ag-capture ELISAs detecting viral NP in viremic sera have been widely applied to detect various viruses, since they are the most abundant viral antigens and have highly conserved amino acid sequences [50, 51, 54, 71, 72, 74, 75] . Polyclonal anti-sera or a", "mixture of MAbs present in the ascetic fluids from animals immunized for HFVs have been used for capture-antibodies in the Ag-capture ELISA [36, [76] [77] [78] [79] . MAbs recognizing conserved epitopes of the rNP are also used as capture antibodies since they have a high specificity for the antigens, and an identification of the epitopes of these MAbs is of crucial importance for the assessment of the specificity and cross-reactivity of the assay system [50, 51, 53, 54, 71, 75] . In order to develop a sensitive diagnostic test for Lassa fever and AHF, rNPs of LASV and JUNV (see above) have been prepared, and newly established MAbs against them have been characterized and used for Ag-capture ELISAs [50, 51] . The Ag-capture ELISA using MAb 4A5 has been confirmed to be useful in the detection of authentic LASV antigen in sera serially collected from hamsters infected with LASV [51] . The sensitivity of the MAb 4A5-based Ag-capture ELISA was similar to that of conventional RT-PCR,", "suggesting that the Ag-capture ELISA can be efficiently used in the diagnosis of Lassa fever [51] . Therefore, the MAb 4A5-based Ag-capture ELISA is considered to be useful in the diagnosis of Lassa fever. Also, by using MAbs raised against the rNP of JUNV, Ag-capture ELISAs specific for JUNV and broadly reactive to human pathogenic New World arenaviruses have been developed [50] . The Ag-capture ELISA using MAb E4-2 and C11-12 detected the Ags of all of the pathogenic New World arenaviruses tested, including JUNV. On the other hand, the Ag-capture ELISA using MAb C6-9 detects only the JUNV Ag. Considering that the symptoms of JUNV infection in humans are indistinguishable from those due to other pathogenic New World arenaviruses, the Ag capture ELISA using MAb C6-9 may be a useful diagnostic tool, especially for AHF [50] .", "The virus neutralization assay is accepted as the \"gold standard\" serodiagnostic assay to quantify the antibody response to infection and vaccination of a wide variety of viruses associated with human diseases [80] [81] [82] [83] [84] [85] [86] . The presence of neutralizing antibodies is a reliable indicator of protective immunity against VHF [87] [88] [89] . The most direct method for detection of neutralizing antibodies against HFVs is by plaque reduction neutralization tests using infectious viruses. However, because of the high pathogenicity of HFVs to humans and the strict regulation of select agents, only a limited number of laboratories are able to perform such neutralization tests. For many HFVs, replication-incompetent pseudotyped virus particles bearing viral envelope protein (GP) have been shown to mimic the respective HFV infections, thus, neutralization assays using the pseudotypes may be advantageous in some laboratory settings for the detection of antibodies to HFVs", "without the need for heightened biocontainment requirements.", "The VSV-based vector has already been used to generate replication-competent recombinant VSVs to study of the role of GPs of various viruses [90] [91] [92] . Recent advances in producing pseudotype virus particles have enabled the investigation of the virus cell entry, viral tropism, and effect of entry inhibitors, as well as measurement of the neutralization titers, by using human immunodeficiency virus-, feline immunodeficiency virus-, murine leukemia virus-, or VSV-based vectors [86, [93] [94] [95] [96] [97] [98] [99] [100] [101] [102] [103] . Pseudotypes based on VSV have advantages compared with other pseudotypes based on retroviruses for the following reasons. First, the pseudotype virus titer obtained with the VSV system is generally higher than that of the pseudotyped retrovirus system [104] . Second, the infection of target cells with a VSV pseudotype can be readily detected as green fluorescent protein (GFP)-positive cells at 7-16 h post-infection because of the high level", "of GFP expression in the VSV system [104, 105] . In contrast, the time required for infection in the pseudotyped retrovirus system is 48 h [106, 107] , which is similar to the time required for infectious viruses to replicate to a level that results in plaque-forming or cytopathic effects in infected cells. A high-throughput assay for determining neutralizing antibody titers using VSV pseudotypes expressing secreted alkaline phosphatase [108, 109] or luciferase ( Figure 3 ) has also been developed. We have recently developed a VSV-based pseudotype bearing Lassa virus GP (VSV-LAS-GP) for the detection of neutralizing antibodies in the sera obtained from a Lassa fever patient. An example of the LASV neutralization assay using the VSV pseudotype is shown (Figure 4 ). In the presence of serum from Lassa fever patients, the number of GFP-positive cells (infectivity of VSV-LAS-GP) is significantly reduced compared with the number in the absence of the patient's serum ( Figure 4A ). The", "control VSV pseudotype bearing VSV GP (VSV-VSV-G) is not neutralized by any sera. When the cut-off serum dilution is set at 50% inhibition of infectivity compared with the infectivity in the absence of the test serum, the neutralization titer of this patient's serum for VSV-LAS-GP is calculated to be 75 ( Figure 4B ). Likewise, a VSV-based pseudotype bearing the Junin virus GP has been developed for the detection of neutralizing antibodies from AHF patients' sera. The accuracy of the results of VSV-based neutralization assays has been confirmed by comparison with the results of the neutralization assay using live Junin virus [70] . The Lujo virus is a new member of the hemorrhagic fever-associated arenavirus family from Zambia and southern Africa, and the virus is classified as a BSL-4 pathogen [17] . The genome sequence analysis of the Lujo virus suggests that the virus is genetically distinct from previously characterized arenaviruses. In order to study the infectivity of this newly", "identified arenavirus, we have recently developed a luciferase-expressing VSV pseudotype bearing Lujo virus GPC (VSV-Lujo-GP). As shown in Figure 3 , infection with VSV-Lujo-GPC is specifically neutralized by rabbit anti-Lujo GPC serum. Thus, the VSV-Lujo-GP may be a useful tool not only for determining the neutralizing antibody titer within the serum, but also for exploring yet-to-be-defined cellular receptor(s) for Lujo virus infection or for screening inhibitors of the Lujo virus GP-mediated cell entry.", "Hemorrhagic fever outbreaks caused by pathogenic arenaviruses result in high fatality rates. A rapid and accurate diagnosis is a critical first step in any outbreak. Serologic diagnostic methods for VHFs most often employ an ELISA, IFA, and/or virus neutralization assay. Diagnostic methods using recombinant viral proteins have been developed and their utilities for diagnosing of VHF have been reviewed. IgG-and IgM-ELISAs and IFAs using rNPs as antigens are useful for the detection of antibodies induced in the patients' sera. These methods are also useful for seroepidemiological surveys for HFVs. Ag-capture ELISAs using MAbs to the arenavirus rNPs are specific for the virus species or can be broadly reactive for New World arenaviruses, depending on the MAb used. Furthermore, the VSV-based pseudotype system provides a safe and rapid tool for measuring virus neutralizing antibody titers, as well as a model to analyze the entry of the respective arenavirus in susceptible cells without", "using live arenaviruses. Recent discoveries of novel arenavirus species [17, 26, 110] and their potential to evolve predominantly via host switching, rather than with their hosts [110, 111] , suggest that an unknown pathogenic arenavirus may emerge in the future, and that the diagnostic methods for VHF caused by arenaviruses should thus be further developed and improved." ]

[ 9 ]

[ "neuraminidase inhibitors" ]

[ "Glycyrrhizin Exerts Antioxidative Effects in H5N1 Influenza A Virus-Infected Cells and Inhibits Virus Replication and Pro-Inflammatory Gene Expression\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3096629/\n\nSHA: f3b7f4469ac01f1ce916d24172570c43c537627e\n\nAuthors: Michaelis, Martin; Geiler, Janina; Naczk, Patrizia; Sithisarn, Patchima; Leutz, Anke; Doerr, Hans Wilhelm; Cinatl, Jindrich\nDate: 2011-05-17\nDOI: 10.1371/journal.pone.0019705\nLicense: cc-by", "Abstract: Glycyrrhizin is known to exert antiviral and anti-inflammatory effects. Here, the effects of an approved parenteral glycyrrhizin preparation (Stronger Neo-Minophafen C) were investigated on highly pathogenic influenza A H5N1 virus replication, H5N1-induced apoptosis, and H5N1-induced pro-inflammatory responses in lung epithelial (A549) cells. Therapeutic glycyrrhizin concentrations substantially inhibited H5N1-induced expression of the pro-inflammatory molecules CXCL10, interleukin 6, CCL2, and CCL5 (effective glycyrrhizin concentrations 25 to 50 µg/ml) but interfered with H5N1 replication and H5N1-induced apoptosis to a lesser extent (effective glycyrrhizin concentrations 100 µg/ml or higher). Glycyrrhizin also diminished monocyte migration towards supernatants of H5N1-infected A549 cells. The mechanism by which glycyrrhizin interferes with H5N1 replication and H5N1-induced pro-inflammatory gene expression includes inhibition of H5N1-induced formation of reactive oxygen", "species and (in turn) reduced activation of NFκB, JNK, and p38, redox-sensitive signalling events known to be relevant for influenza A virus replication. Therefore, glycyrrhizin may complement the arsenal of potential drugs for the treatment of H5N1 disease.", "Text: Highly pathogenic H5N1 influenza A viruses are considered to be potential influenza pandemic progenitors [1] [2] [3] [4] [5] [6] . At least for the first wave of an H5N1 pandemic, no sufficient amounts of adequate vaccines will be available [1] [2] [3] [4] [6] [7] [8] . Therefore, antiviral therapy for influenza A viruses including highly pathogenic H5N1 virus strains remains of great importance for the first line defense against the virus [1] [2] [3] [4] 6, 9] .", "The neuraminidase inhibitors oseltamivir and zanamivir as well as the adamantanes amantadin and rimantadin that interfere with the influenza M2 protein are licensed for the treament of influenza [1] [2] [3] [4] 6] . However, the use of both drug classes is limited by the emergence of resistant virus strains. In seasonal influenza strains, the majority of H3N2 viruses and a great proportion of H1N1 viruses in humans are now considered to be amantadine-and rimantadine-resistant [10] [11] [12] [13] . Moreover, a drastic increase in oseltamivir-resistant H1N1 viruses has been reported during the 2007/2008 influenza season in the northern hemisphere [14] [15] [16] [17] . Preliminary data from the United States predict a further rise for the 2008/2009 season, possibly resulting in more than 90% of the circulating H1N1 strains to be oseltamivir resistant [14] .", "H5N1 virus strains appear to be generally less sensitive to antiviral treatment than seasonal influenza A virus strains and treatment-resistant H5N1 strains emerge [1] [2] [3] [4] 6, [18] [19] [20] [21] . More-over, parenteral agents for the treatment of seriously ill patients are missing. Glycyrrhizin, a triterpene saponine, is a constituent of licorice root. It has been found to interfere with replication and/or cytopathogenic effect (CPE) induction of many viruses including respiratory viruses such as respiratory syncytial virus, SARS coronavirus, HIV, and influenza viruses [22] [23] [24] [25] [26] [27] [28] . Moreover, antiinflammatory and immunomodulatory properties were attributed to glycyrrhizin [26] . The severity of human H5N1 disease has been associated with hypercytokinaemia (''cytokine storm'') [29, 30] . Delayed antiviral plus immunomodulator treatment reduced H5N1-induced mortality in mice [31] . Therefore, antiinflammatory and immunomodulatory effects exerted by", "glycyrrhizin may be beneficial for treatment of H5N1. Also, glycyrrhizin is a known antioxidant [26] and antioxidants were already shown to interfere with influenza A virus replication and virus-induced pro-inflammatory responses [32] [33] [34] .", "Stronger Neo-Minophagen C (SNMC) is a glycyrrhizin preparation (available as tablets or parenteral formulation) that is approved in Japan for the treatment of chronic hepatic diseases and is marketed in Japan, China, Korea, Taiwan, Indonesia, India, and Mongolia. Here, we investigated the influence of SNMC on H5N1 replication, on H5N1-induced cytokine expression, on H5N1-induced cellular oxidative stress, and on critical H5N1-induced cellular signalling events in human pneumocytes (A549 cell line).\n\nGlycyrrhizin (Stronger Neo Minophagen C) was obtained from Minophagen Pharmaceuticals Co., Ltd. (Tokyo, Japan).\n\nThe influenza strain A/Vietnam/1203/04 (H5N1) was received from the WHO Influenza Centre (National Institute for Medical Research, London, UK). The H5N1 influenza strain A/Thailand/ 1(Kan-1)/04 was obtained from Prof. Pilaipan Puthavathana (Mahidol University, Bangkok, Thailand).", "Virus stocks were prepared by infecting Vero cells (African green monkey kidney; ATCC, Manassas, VA) and aliquots were stored at 280uC. Virus titres were determined as 50% tissue culture infectious dose (TCID 50 /ml) in confluent Vero cells in 96-well microtiter plates.\n\nA549 cells (human lung carcinoma; ATCC: CCL-185, obtained from LGC Standards GmbH, Wesel, Germany) were grown at 37uC in minimal essential medium (MEM) supplemented with 10% FBS, 100 IU/ml of penicillin and 100 mg/ml streptomycin.", "Human monocytes were isolated from buffy coats of healthy donors, obtained from Institute of Transfusion Medicine and Immune Haematology, German Red Cross Blood Donor Center, Johann Wolfgang Goethe-University, Frankfurt am Main. After centrifugation on Ficoll (Biocoll)-Hypaque density gradient (Biochrom AG, Berlin, Germany), mononuclear cells were collected from the interface and washed with PBS. Then, monocytes were isolated using magnetically labeled CD14 MicroBeads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) following the manufacturer's instructions. Monocytes were cultivated in IMDM supplemented with 10% pooled human serum, 100 IU/ml of penicillin, and 100 mg/ml streptomycin.\n\nThe cellular viability was assessed on confluent cell layers with CellTiter-GloH Luminescent Cell Viability Assay (Promega GmbH, Mannheim, Germany) according to the manufacturers' protocol. Cell viability was expressed as percentage of non-treated control.", "To determine intracellular NP localisation, H5N1-infected A549 were fixed 8 hours p.i. for 15 min with ice-cold acetone/ methanol (40:60, Mallinckrodt Baker B.V., Deventer, The Netherlands) and stained with a mouse monoclonal antibody (1 h incubation, 1:1000 in PBS) directed against the influenza A virus nucleoprotein (NP) (Millipore, Molsheim, France). An Alexa Fluor 488 goat anti-mouse IgG (H&L) (Invitrogen, Eugene, Oregon, USA) was used (1 h incubation, 1:1000 in PBS) as secondary antibody. Nuclei were stained using 49,6-diamidino-2phenylindole (DAPI) (Sigma-Aldrich Chemie GmbH, Munich, Germany). Fluorescence was visualised using Olympus IX 1 fluorescence microscope (Olympus, Planegg, Germany).\n\nFor flow cytometric analysis, the same antibodies were used.", "The cytopathogenic effect (CPE) reduction assay was performed as described before [34] . Confluent A549 cell monolayers grown in 96-well microtitre plates were infected with influenza A strains at the indicated multiplicities of infection (MOIs). After a one hour adsorption period, cells were washed to remove non-detached virus. The virus-induced CPE was recorded at 24 h post infection (p.i.).\n\nUnless otherwise stated, A549 cells were continuously treated with glycyrrhizin starting with a 1 h pre-incubation period. For time-ofaddition experiments, glycyrrhizin was added exclusively during the 1 h pre-incubation period, exclusively during the 1 h adsorption period, or after exclusively after the wash-out of input virus.", "Total RNA was isolated from cell cultures using TRI reagent (Sigma-Aldrich, Munich, Germany). Real time PCR for H5 was performed using described methods [35] . The following primers were used: sense 59 acg tat gac tac ccg cag tat tca g 39; antisense 59 aga cca gcy acc atg att gc 39; probe 6-FAM-tca aca gtg gcg agt tcc cta gca-TAMRA.\n\nThe fraction of cells with fractional DNA content (''sub-G1'' cell subpopulation) indicates cytotoxicity. Sub-G1 cells are considered to be dead (usually apoptotic) cells. Cells were fixed with 70% ethanol for two hours at 220uC. The cellular DNA was stained using propidium iodide (20 mg/ml) and analysed by flow cytometry (FacsCalibur, BD Biosciences, Heidelberg, Germany).\n\nCaspase activation was measured using the Caspase-Glo 8, 9, or 3/7 Assays (Promega, Mannheim, Germany) following the manufacturer's instructions.", "Cell culture supernatants were collected and frozen at 280uC. Cytokines/chemokines were quantified by specific ELISA Duo Sets (R&D Systems GmbH, Wiesbaden, Germany) following the manufacturer's instructions.\n\nNFkB activity was investigated in H5N1 (MOI 0.01)-infected cells by quantification of the NFkB subunits Rel A (p65) and NFkB1 (p50) from nuclear extracts using the TransAM TM transcription factor DNA-binding ELISAs (Active Motif, Rixensart, Belgium). Nuclear extract were prepared using the Nuclear Extract Kit (Active Motif, Carlsbad, CA, USA) following the manufacturer's instruction.", "Cell culture supernatants were investigated for chemotactic activity by measurement of the activity to induce monocyte migration through membrane inserts in 24-well plates (pore size 8 mm; BD Biosciences, Heidelberg, Germany). Monocytes (1610 6 in 100 ml of IMDM with 10% pooled human serum) were added into the cell culture inserts (upper chamber) and cell culture supernatants (300 ml), were added to the lower chamber of the well. After a 48 h incubation period, cells were fixed with 4% paraformaldehyde and permeabilised with PBS containing 0.3% Tritron X-100. Then, nuclei were stained with 49,6-diamidino-2phenylindole (DAPI). The upper side of the membrane was wiped with a wet swab to remove the cells, while the lower side of the membrane was rinsed with PBS. The number of cells at the lower side of each membrane was quantified by counting of cells from three randomly chosen sections (3.7 mm 2 ) using an Olympus IX 1 fluorescence microscope (Olympus, Planegg, Germany).", "Cells were lysed in Triton X-sample buffer and separated by SDS-PAGE. Nuclear extract were prepared using the Nuclear Extract Kit (Active Motif, Carlsbad, CA, USA) following the manufacturer's instruction. Proteins were detected using specific antibodies against bactin (Sigma-Aldrich Chemie GmbH, Munich, Germany), JNK, phosphorylated JNK, p38, or phosphorylated p38, (all purchased from New England Biolabs GmbH, Frankfurt am Main, Germany) and were visualised by enhanced chemiluminescence using a commercially available kit (Amersham, Freiburg, Germany).\n\nReactive oxygen species (ROS) were detected using the Image-iT LIVE Green Reactive Oxygen Species Kit (Molecular Probes, distributed by Invitrogen, Karlsruhe, Germany).\n\nTwo groups were compared by t-test. More groups were compared by ANOVA with subsequent Student-Newman-Keuls test.", "The A549 cell line, derived from a human pulmonary adenocarcinoma, is an established model for type II pneumocytes [36] , and commonly used for the investigation of the effect of influenza viruses on this cell type [see e.g. 6,37,38]. If not otherwise stated, glycyrrhizin was continuously present in cell culture media starting with a 1 h preinfection period. Glycyrrhizin 200 mg/ml (the maximum tested concentration) did not affect A549 cell viability (data not shown) but clearly decreased CPE formation in A549 cells infected with the H5N1 influenza strain A/Thailand/1(Kan-1)/04 at MOIs of 0.01, 0.1 or 1 ( Figure 1A ). Similar results were obtained in A549 cells infected with strain A/Vietnam/1203/04 (H5N1) (Suppl. Figure 1A) . Staining of A549 cells for influenza A nucleoprotein 24 h after infection with strain H5N1 A/Thailand/1(Kan-1)/04 indicated that glycyrrhizin 200 mg/ml significantly reduces the number of influenza A nucleoprotein positive cells ( Figure 1B) .", "To examine the influence of glycyrrhizin on virus progeny, A549 cells were infected with the H5N1 influenza strain A/ Thailand/1(Kan-1)/04 at MOI 0.01 or MOI 1 and infectious virus titres were determined 24 h post infection ( Figure 1C ). While glycyrrhizin in concentrations up to 50 mg/ml did not affect H5N1 replication, moderate effects were exerted by glycyrrhizin 100 mg/ ml and more pronounced effects by glycyrrhizin 200 mg/ml (MOI 0.01: 13-fold reduction, MOI 1: 10-fold reduction). Next, influence of glycyrrhizin on H5N1 replication was confirmed by the detection of viral (H5) RNA using quantitative PCR. Only glycyrrhizin concentrations $100 mg/ml significantly reduced Figure 1B) or H5N1 A/Vietnam/1203/04-infected (Suppl. Figure 1C ) A549 cells (MOI 0.01) 24 h post infection.", "Time-of-addition experiments revealed that maximal effects were achieved when glycyrrhizin was continuously present starting with a 1 h pre-incubation period ( Figure 1D ). Addition of glycyrrhizin post infection showed reduced antiviral effects while pre-incubation alone or glycyrrhizin addition during the adsorption period did not significantly affect H5N1 replication.", "For investigation of H5N1-induced cytokine expression, five pro-inflammatory genes were chosen that had been correlated to severity of influenza disease: CXCL10 (also known as interferon-cinducible protein 10, IP-10), interleukin 6 (IL6), interleukin 8, (IL8; also known as CXCL8), CCL2 (also known as monocyte chemoattractant protein 1, MCP-1), and CCL5 (also known as RANTES). A549 cells were infected with H5N1 A/Thailand/ 1(Kan-1)/04 or H5N1 A/Vietnam/1203/04 at MOI 0.01, 0.1, or 1. Glycyrrhizin treatment was performed with 25, 50, 100, or 200 mg/ml. Cytokine expression was detected 24 h post infection by ELISA. Glycyrrhizin did not affect cytokine expression of noninfected cells (data not shown) but inhibited expression of all cytokines investigated in H5N1-infected cells in a dose-dependent manner (Figure 2, Figure 3A ). Effects were more pronounced at lower MOIs. Notably, expression of all cytokines except IL8 was significantly inhibited after treatment with glycyrrhizin 50 mg/ml", "Figure 3A ) although these glycyrrhizin concentrations had no effect on H5N1 replication in A549 cells (Figure 1, Figure S1 ).", "Cytokine expression by influenza A virus-infected respiratory cells causes recruitment of peripheral blood monocytes into the lungs of patients where they differentiate to macrophages which are thought to contribute to influenza A virus pathogenicity [5, 39] . In a chemotaxis assay, the influence of glycyrrhizin was investigated on migration of monocytes towards supernatants of H5N1 A/Thailand/1(Kan-1)/04 (MOI 0.1)-infected A549 cells through 8 mm filters. Monocyte migration towards supernatants of H5N1-infected cells was strongly increased relative to migration towards supernatants of non-infected cells. Treatment of H5N1- infected cells with glycyrrhizin 100 mg/ml clearly suppressed chemoattraction activity of supernatants ( Figure 3B ).", "Influenza viruses including H5N1 have been shown to induce caspase-dependent apoptosis in airway cells and this apoptosis has been correlated to the virus pathogenicity [40, 41] . Glycyrrhizin concentrations up to 200 mg/ml did not affect caspase activation in non-infected cells ( Figure 4A-C) . Glycyrrhizin concentrations $100 mg/ml inhibited H5N1 A/Thailand/1(Kan-1)/04 (MOI 0.01)-induced activation of the initiator caspases 8 and 9 as well as of the effector caspases 3/7 in A549 cells as determined 24 h post infection ( Figure 4A-C) . Lower glycyrrhizin concentrations did not affect H5N1-induced apoptosis. The detection of cells in sub-G1 phase resulted in similar findings ( Figure 4D ).", "Substances that inhibit H5N1-induced caspase 3 activation including caspase 3 inhibitors cause nuclear retention of RNP complexes [34, 42] . In accordance, glycyrrhizin also interfered with nuclear export RNP at MOI 1 ( Figure S2 ). Similar results were obtained in MOI 0.01 H5N1 A/Thailand/1(Kan-1)/04infected cells ( Figure S3 ).\n\nInfluence of glycyrrhizin on H5N1-induced activation of nuclear factor kB (NFkB), p38, and on H5N1-induced cellular reactive oxygen species (ROS) formation Activation of NFkB, p38, and JNK have been associated with influenza A virus replication and virus-induced pro-inflammatory gene expression [34, [43] [44] [45] [46] [47] . While glycyrrhizin did not influence NFkB activity in non-infected A549 cells in the tested concentra-tions (data not shown), glycyrrhizin inhibited NFkB activation in H5N1-infected cells ( Figure 5A ). Moreover, glycyrrhizin inhibited H5N1-induced phosphorylation of the MAPKs p38 and JNK ( Figure 5B ).", "In addition to their roles during influenza A virus replication and virus-induced cytokine/chemokine expression, NFkB, p38, and JNK are constituents of redox-sensitive signalling pathways [48] [49] [50] [51] . Antioxidants had been already found to interfere with influenza A virus-induced signalling through NFkB, p38, and JNK, with influenza A virus replication, and with influenza A virus-induced pro-inflammatory gene expression [32] [33] [34] . Since glycyrrhizin is known to exert antioxidative effects [26] we speculated that glycyrrhizin may interfere with H5N1-induced ROS formation. Indeed glycyrrhizin exerted clear antioxidative effects in H5N1 (MOI 0.01)-infected cells ( Figure 5C ) causing significant reduction of ROS formation already at a concentration of 25 mg/ml ( Figure 5D ).", "Here, we show that glycyrrhizin inhibits the replication of highly pathogenic H5N1 influenza A virus, H5N1-induced apoptosis, and H5N1-induced expression of pro-inflammatory cytokines in lung-derived A549 cells. After intravenous administration, achievable plasma concentrations of glycyrrhizin have been described to be about 100 mg/ml [52] . Therefore, the glycyrrhizin concentrations found to interfere with H5N1 replication and H5N1-induced pro-inflammatory gene expression in the present report are in the range of therapeutic plasma levels. Notably, although higher glycyrrhizin concentrations were needed to interfere with SARS coronavirus replication [22] than with H5N1 replication, beneficial results were reported in glycyrrhizin (SNMC)-treated SARS patients in comparison to SARS patients who did not receive glycyrrhizin [23] . Notably, investigation of different glycyrrhizin derivatives against SARS coronavirus led to the identification of compounds with enhanced antiviral activity", "[53] . Therefore, glycyrrhizin might also serve as lead structure for the development of novel anti-influenza drugs.", "Experimental results suggested that glycyrrhizin might be able to affect seasonal influenza A virus disease by antiviral and immunomodulatory effects [26, 27] . Mice were prevented from lethal H2N2 infection by glycyrrhizin although no influence on virus replication was detected. The mechanism was suggested to be induction of interferon-c in T-cells by glycyrrhizin [54] . Moreover, glycyrrhizin was shown to influence seasonal influenza A virus replication through interaction with the cell membrane [25, 28] . However, these effects were observed only in concentrations $200 mg/ml when glycyrrhizin was added during the virus adsorption period. Since glycyrrhizin addition during the adsorption period did not influence H5N1 replication in our experiments it appears not likely that membrane effects contribute to anti-H5N1 effects detected here in lower concentrations.", "Our results rather suggest that glycyrrhizin interferes with H5N1-induced oxidative stress. Influenza A virus (including H5N1) infection induces ROS formation. Antioxidants were found to inhibit influenza A virus replication and influenza A virus-induced pro-inflammatory gene expression [32] [33] [34] and glycyrrhizin is known to exert antioxidative effects [26] . Here, glycyrrhizin interfered with H5N1-induced activation of NFkB, p38, and JNK representing redox-sensitive signalling events [48] [49] [50] [51] involved in influenza A virus replication and influenza A virusinduced cellular cytokine/chemokine production [34, [43] [44] [45] [46] 55] . Glycyrrhizin 50 mg/ml significantly reduced H5N1-induced activation of NFkB. In addition, glycyrrhizin concentrations as low as 25 mg/ml effectively interfered with H5N1-induced ROS formation and with phosphorylation of the redox-sensitive MAPKs p38 and JNK. In our model, activation of p38 appears to be critical for H5N1-associated redox", "signalling since p38 inhibition had been shown before to mimick effects of the antioxidant N-acetyl-cysteine (NAC) [34] . Interestingly and in contrast to glycyrrhizin, NAC failed to inhibit H5N1 replication or H5N1-induced cytokine/chemokine expression in therapeutically relevant concentrations.", "Glycyrrhizin diminished H5N1-induced cellular cytokine/ chemokine production in concentrations (#50 mg/ml) that did not interfere with H5N1 replication although redox-sensitive signalling pathways have been described to be involved in both processes. Therefore, H5N1-induced proinflammatory gene expression appears to be more sensitive to inhibition of ROS formation than H5N1 replication. Indeed, influenza viruses had been shown to induce cellular pathways through replicationdependent and -independent events [56] . In a previous report, we could show that similar glycyrrhizin concentrations like those investigated here interfered with H5N1-induced pro-inflammatory gene expression but not with H5N1 replication in human monocyte-derived macrophages [57] . In addition, other immunomodulatory treatment regimens that did not influence H5N1 replication reduced mortality in H5N1-infected mice [31, 58] . Therefore, glycyrrhizin represents a potential additional treatment option that interfers", "with both H5N1 replication and H5N1induced expression of pro-inflammatory cytokines in lung cells.", "Interference with immune responses may also result in the loss of control of virus replication by cytotoxic immune cells including natural killer cells and cytotoxic CD8 + T-lymphocytes. Global immunosuppressants like corticosteroids failed to protect from lethal influenza virus infection [59] . Moreover, antiviral drugs may interfere with cytotoxic cells that control virus replication as demonstrated for ribavirin that was shown to hamper NK cell cytolytic activity [60] . In this context, glycyrrhizin had already been shown not to affect natural killer cell activity in the concentrations used here [57] .", "In conclusion, we show in this report that therapeutic concentrations of glycyrrhizin (used as clinically approved parenteral preparation SNMC) interfere with highly pathogenic H5N1 influenza A virus replication and H5N1-induced proinflammatory gene expression at least in part through interference with H5N1-induced ROS formation and in turn reduced activation of p38, JNK, and NFkB in lung cells. Since we used the clinical formulation SNMC effects of other ingredients like glycin or cystein cannot be excluded. Vaccines and antiviral agents will fail to meet global needs at least at the beginning of a severe influenza A virus pandemic [61] . Anti-inflammatory and immunomodulatory agents are considered to be important candidates as constituents of anti-influenza treatment strategies that may save lives in an influenza pandemic situation [61] . Therefore, glycyrrhizin may complement the arsenal of potential drugs for the treatment of H5N1-caused disease." ]

[ 4 ]

[ "a triterpene saponine" ]

[ "Glycyrrhizin Exerts Antioxidative Effects in H5N1 Influenza A Virus-Infected Cells and Inhibits Virus Replication and Pro-Inflammatory Gene Expression\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3096629/\n\nSHA: f3b7f4469ac01f1ce916d24172570c43c537627e\n\nAuthors: Michaelis, Martin; Geiler, Janina; Naczk, Patrizia; Sithisarn, Patchima; Leutz, Anke; Doerr, Hans Wilhelm; Cinatl, Jindrich\nDate: 2011-05-17\nDOI: 10.1371/journal.pone.0019705\nLicense: cc-by", "Abstract: Glycyrrhizin is known to exert antiviral and anti-inflammatory effects. Here, the effects of an approved parenteral glycyrrhizin preparation (Stronger Neo-Minophafen C) were investigated on highly pathogenic influenza A H5N1 virus replication, H5N1-induced apoptosis, and H5N1-induced pro-inflammatory responses in lung epithelial (A549) cells. Therapeutic glycyrrhizin concentrations substantially inhibited H5N1-induced expression of the pro-inflammatory molecules CXCL10, interleukin 6, CCL2, and CCL5 (effective glycyrrhizin concentrations 25 to 50 µg/ml) but interfered with H5N1 replication and H5N1-induced apoptosis to a lesser extent (effective glycyrrhizin concentrations 100 µg/ml or higher). Glycyrrhizin also diminished monocyte migration towards supernatants of H5N1-infected A549 cells. The mechanism by which glycyrrhizin interferes with H5N1 replication and H5N1-induced pro-inflammatory gene expression includes inhibition of H5N1-induced formation of reactive oxygen", "species and (in turn) reduced activation of NFκB, JNK, and p38, redox-sensitive signalling events known to be relevant for influenza A virus replication. Therefore, glycyrrhizin may complement the arsenal of potential drugs for the treatment of H5N1 disease.", "Text: Highly pathogenic H5N1 influenza A viruses are considered to be potential influenza pandemic progenitors [1] [2] [3] [4] [5] [6] . At least for the first wave of an H5N1 pandemic, no sufficient amounts of adequate vaccines will be available [1] [2] [3] [4] [6] [7] [8] . Therefore, antiviral therapy for influenza A viruses including highly pathogenic H5N1 virus strains remains of great importance for the first line defense against the virus [1] [2] [3] [4] 6, 9] .", "The neuraminidase inhibitors oseltamivir and zanamivir as well as the adamantanes amantadin and rimantadin that interfere with the influenza M2 protein are licensed for the treament of influenza [1] [2] [3] [4] 6] . However, the use of both drug classes is limited by the emergence of resistant virus strains. In seasonal influenza strains, the majority of H3N2 viruses and a great proportion of H1N1 viruses in humans are now considered to be amantadine-and rimantadine-resistant [10] [11] [12] [13] . Moreover, a drastic increase in oseltamivir-resistant H1N1 viruses has been reported during the 2007/2008 influenza season in the northern hemisphere [14] [15] [16] [17] . Preliminary data from the United States predict a further rise for the 2008/2009 season, possibly resulting in more than 90% of the circulating H1N1 strains to be oseltamivir resistant [14] .", "H5N1 virus strains appear to be generally less sensitive to antiviral treatment than seasonal influenza A virus strains and treatment-resistant H5N1 strains emerge [1] [2] [3] [4] 6, [18] [19] [20] [21] . More-over, parenteral agents for the treatment of seriously ill patients are missing. Glycyrrhizin, a triterpene saponine, is a constituent of licorice root. It has been found to interfere with replication and/or cytopathogenic effect (CPE) induction of many viruses including respiratory viruses such as respiratory syncytial virus, SARS coronavirus, HIV, and influenza viruses [22] [23] [24] [25] [26] [27] [28] . Moreover, antiinflammatory and immunomodulatory properties were attributed to glycyrrhizin [26] . The severity of human H5N1 disease has been associated with hypercytokinaemia (''cytokine storm'') [29, 30] . Delayed antiviral plus immunomodulator treatment reduced H5N1-induced mortality in mice [31] . Therefore, antiinflammatory and immunomodulatory effects exerted by", "glycyrrhizin may be beneficial for treatment of H5N1. Also, glycyrrhizin is a known antioxidant [26] and antioxidants were already shown to interfere with influenza A virus replication and virus-induced pro-inflammatory responses [32] [33] [34] .", "Stronger Neo-Minophagen C (SNMC) is a glycyrrhizin preparation (available as tablets or parenteral formulation) that is approved in Japan for the treatment of chronic hepatic diseases and is marketed in Japan, China, Korea, Taiwan, Indonesia, India, and Mongolia. Here, we investigated the influence of SNMC on H5N1 replication, on H5N1-induced cytokine expression, on H5N1-induced cellular oxidative stress, and on critical H5N1-induced cellular signalling events in human pneumocytes (A549 cell line).\n\nGlycyrrhizin (Stronger Neo Minophagen C) was obtained from Minophagen Pharmaceuticals Co., Ltd. (Tokyo, Japan).\n\nThe influenza strain A/Vietnam/1203/04 (H5N1) was received from the WHO Influenza Centre (National Institute for Medical Research, London, UK). The H5N1 influenza strain A/Thailand/ 1(Kan-1)/04 was obtained from Prof. Pilaipan Puthavathana (Mahidol University, Bangkok, Thailand).", "Virus stocks were prepared by infecting Vero cells (African green monkey kidney; ATCC, Manassas, VA) and aliquots were stored at 280uC. Virus titres were determined as 50% tissue culture infectious dose (TCID 50 /ml) in confluent Vero cells in 96-well microtiter plates.\n\nA549 cells (human lung carcinoma; ATCC: CCL-185, obtained from LGC Standards GmbH, Wesel, Germany) were grown at 37uC in minimal essential medium (MEM) supplemented with 10% FBS, 100 IU/ml of penicillin and 100 mg/ml streptomycin.", "Human monocytes were isolated from buffy coats of healthy donors, obtained from Institute of Transfusion Medicine and Immune Haematology, German Red Cross Blood Donor Center, Johann Wolfgang Goethe-University, Frankfurt am Main. After centrifugation on Ficoll (Biocoll)-Hypaque density gradient (Biochrom AG, Berlin, Germany), mononuclear cells were collected from the interface and washed with PBS. Then, monocytes were isolated using magnetically labeled CD14 MicroBeads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) following the manufacturer's instructions. Monocytes were cultivated in IMDM supplemented with 10% pooled human serum, 100 IU/ml of penicillin, and 100 mg/ml streptomycin.\n\nThe cellular viability was assessed on confluent cell layers with CellTiter-GloH Luminescent Cell Viability Assay (Promega GmbH, Mannheim, Germany) according to the manufacturers' protocol. Cell viability was expressed as percentage of non-treated control.", "To determine intracellular NP localisation, H5N1-infected A549 were fixed 8 hours p.i. for 15 min with ice-cold acetone/ methanol (40:60, Mallinckrodt Baker B.V., Deventer, The Netherlands) and stained with a mouse monoclonal antibody (1 h incubation, 1:1000 in PBS) directed against the influenza A virus nucleoprotein (NP) (Millipore, Molsheim, France). An Alexa Fluor 488 goat anti-mouse IgG (H&L) (Invitrogen, Eugene, Oregon, USA) was used (1 h incubation, 1:1000 in PBS) as secondary antibody. Nuclei were stained using 49,6-diamidino-2phenylindole (DAPI) (Sigma-Aldrich Chemie GmbH, Munich, Germany). Fluorescence was visualised using Olympus IX 1 fluorescence microscope (Olympus, Planegg, Germany).\n\nFor flow cytometric analysis, the same antibodies were used.", "The cytopathogenic effect (CPE) reduction assay was performed as described before [34] . Confluent A549 cell monolayers grown in 96-well microtitre plates were infected with influenza A strains at the indicated multiplicities of infection (MOIs). After a one hour adsorption period, cells were washed to remove non-detached virus. The virus-induced CPE was recorded at 24 h post infection (p.i.).\n\nUnless otherwise stated, A549 cells were continuously treated with glycyrrhizin starting with a 1 h pre-incubation period. For time-ofaddition experiments, glycyrrhizin was added exclusively during the 1 h pre-incubation period, exclusively during the 1 h adsorption period, or after exclusively after the wash-out of input virus.", "Total RNA was isolated from cell cultures using TRI reagent (Sigma-Aldrich, Munich, Germany). Real time PCR for H5 was performed using described methods [35] . The following primers were used: sense 59 acg tat gac tac ccg cag tat tca g 39; antisense 59 aga cca gcy acc atg att gc 39; probe 6-FAM-tca aca gtg gcg agt tcc cta gca-TAMRA.\n\nThe fraction of cells with fractional DNA content (''sub-G1'' cell subpopulation) indicates cytotoxicity. Sub-G1 cells are considered to be dead (usually apoptotic) cells. Cells were fixed with 70% ethanol for two hours at 220uC. The cellular DNA was stained using propidium iodide (20 mg/ml) and analysed by flow cytometry (FacsCalibur, BD Biosciences, Heidelberg, Germany).\n\nCaspase activation was measured using the Caspase-Glo 8, 9, or 3/7 Assays (Promega, Mannheim, Germany) following the manufacturer's instructions.", "Cell culture supernatants were collected and frozen at 280uC. Cytokines/chemokines were quantified by specific ELISA Duo Sets (R&D Systems GmbH, Wiesbaden, Germany) following the manufacturer's instructions.\n\nNFkB activity was investigated in H5N1 (MOI 0.01)-infected cells by quantification of the NFkB subunits Rel A (p65) and NFkB1 (p50) from nuclear extracts using the TransAM TM transcription factor DNA-binding ELISAs (Active Motif, Rixensart, Belgium). Nuclear extract were prepared using the Nuclear Extract Kit (Active Motif, Carlsbad, CA, USA) following the manufacturer's instruction.", "Cell culture supernatants were investigated for chemotactic activity by measurement of the activity to induce monocyte migration through membrane inserts in 24-well plates (pore size 8 mm; BD Biosciences, Heidelberg, Germany). Monocytes (1610 6 in 100 ml of IMDM with 10% pooled human serum) were added into the cell culture inserts (upper chamber) and cell culture supernatants (300 ml), were added to the lower chamber of the well. After a 48 h incubation period, cells were fixed with 4% paraformaldehyde and permeabilised with PBS containing 0.3% Tritron X-100. Then, nuclei were stained with 49,6-diamidino-2phenylindole (DAPI). The upper side of the membrane was wiped with a wet swab to remove the cells, while the lower side of the membrane was rinsed with PBS. The number of cells at the lower side of each membrane was quantified by counting of cells from three randomly chosen sections (3.7 mm 2 ) using an Olympus IX 1 fluorescence microscope (Olympus, Planegg, Germany).", "Cells were lysed in Triton X-sample buffer and separated by SDS-PAGE. Nuclear extract were prepared using the Nuclear Extract Kit (Active Motif, Carlsbad, CA, USA) following the manufacturer's instruction. Proteins were detected using specific antibodies against bactin (Sigma-Aldrich Chemie GmbH, Munich, Germany), JNK, phosphorylated JNK, p38, or phosphorylated p38, (all purchased from New England Biolabs GmbH, Frankfurt am Main, Germany) and were visualised by enhanced chemiluminescence using a commercially available kit (Amersham, Freiburg, Germany).\n\nReactive oxygen species (ROS) were detected using the Image-iT LIVE Green Reactive Oxygen Species Kit (Molecular Probes, distributed by Invitrogen, Karlsruhe, Germany).\n\nTwo groups were compared by t-test. More groups were compared by ANOVA with subsequent Student-Newman-Keuls test.", "The A549 cell line, derived from a human pulmonary adenocarcinoma, is an established model for type II pneumocytes [36] , and commonly used for the investigation of the effect of influenza viruses on this cell type [see e.g. 6,37,38]. If not otherwise stated, glycyrrhizin was continuously present in cell culture media starting with a 1 h preinfection period. Glycyrrhizin 200 mg/ml (the maximum tested concentration) did not affect A549 cell viability (data not shown) but clearly decreased CPE formation in A549 cells infected with the H5N1 influenza strain A/Thailand/1(Kan-1)/04 at MOIs of 0.01, 0.1 or 1 ( Figure 1A ). Similar results were obtained in A549 cells infected with strain A/Vietnam/1203/04 (H5N1) (Suppl. Figure 1A) . Staining of A549 cells for influenza A nucleoprotein 24 h after infection with strain H5N1 A/Thailand/1(Kan-1)/04 indicated that glycyrrhizin 200 mg/ml significantly reduces the number of influenza A nucleoprotein positive cells ( Figure 1B) .", "To examine the influence of glycyrrhizin on virus progeny, A549 cells were infected with the H5N1 influenza strain A/ Thailand/1(Kan-1)/04 at MOI 0.01 or MOI 1 and infectious virus titres were determined 24 h post infection ( Figure 1C ). While glycyrrhizin in concentrations up to 50 mg/ml did not affect H5N1 replication, moderate effects were exerted by glycyrrhizin 100 mg/ ml and more pronounced effects by glycyrrhizin 200 mg/ml (MOI 0.01: 13-fold reduction, MOI 1: 10-fold reduction). Next, influence of glycyrrhizin on H5N1 replication was confirmed by the detection of viral (H5) RNA using quantitative PCR. Only glycyrrhizin concentrations $100 mg/ml significantly reduced Figure 1B) or H5N1 A/Vietnam/1203/04-infected (Suppl. Figure 1C ) A549 cells (MOI 0.01) 24 h post infection.", "Time-of-addition experiments revealed that maximal effects were achieved when glycyrrhizin was continuously present starting with a 1 h pre-incubation period ( Figure 1D ). Addition of glycyrrhizin post infection showed reduced antiviral effects while pre-incubation alone or glycyrrhizin addition during the adsorption period did not significantly affect H5N1 replication.", "For investigation of H5N1-induced cytokine expression, five pro-inflammatory genes were chosen that had been correlated to severity of influenza disease: CXCL10 (also known as interferon-cinducible protein 10, IP-10), interleukin 6 (IL6), interleukin 8, (IL8; also known as CXCL8), CCL2 (also known as monocyte chemoattractant protein 1, MCP-1), and CCL5 (also known as RANTES). A549 cells were infected with H5N1 A/Thailand/ 1(Kan-1)/04 or H5N1 A/Vietnam/1203/04 at MOI 0.01, 0.1, or 1. Glycyrrhizin treatment was performed with 25, 50, 100, or 200 mg/ml. Cytokine expression was detected 24 h post infection by ELISA. Glycyrrhizin did not affect cytokine expression of noninfected cells (data not shown) but inhibited expression of all cytokines investigated in H5N1-infected cells in a dose-dependent manner (Figure 2, Figure 3A ). Effects were more pronounced at lower MOIs. Notably, expression of all cytokines except IL8 was significantly inhibited after treatment with glycyrrhizin 50 mg/ml", "Figure 3A ) although these glycyrrhizin concentrations had no effect on H5N1 replication in A549 cells (Figure 1, Figure S1 ).", "Cytokine expression by influenza A virus-infected respiratory cells causes recruitment of peripheral blood monocytes into the lungs of patients where they differentiate to macrophages which are thought to contribute to influenza A virus pathogenicity [5, 39] . In a chemotaxis assay, the influence of glycyrrhizin was investigated on migration of monocytes towards supernatants of H5N1 A/Thailand/1(Kan-1)/04 (MOI 0.1)-infected A549 cells through 8 mm filters. Monocyte migration towards supernatants of H5N1-infected cells was strongly increased relative to migration towards supernatants of non-infected cells. Treatment of H5N1- infected cells with glycyrrhizin 100 mg/ml clearly suppressed chemoattraction activity of supernatants ( Figure 3B ).", "Influenza viruses including H5N1 have been shown to induce caspase-dependent apoptosis in airway cells and this apoptosis has been correlated to the virus pathogenicity [40, 41] . Glycyrrhizin concentrations up to 200 mg/ml did not affect caspase activation in non-infected cells ( Figure 4A-C) . Glycyrrhizin concentrations $100 mg/ml inhibited H5N1 A/Thailand/1(Kan-1)/04 (MOI 0.01)-induced activation of the initiator caspases 8 and 9 as well as of the effector caspases 3/7 in A549 cells as determined 24 h post infection ( Figure 4A-C) . Lower glycyrrhizin concentrations did not affect H5N1-induced apoptosis. The detection of cells in sub-G1 phase resulted in similar findings ( Figure 4D ).", "Substances that inhibit H5N1-induced caspase 3 activation including caspase 3 inhibitors cause nuclear retention of RNP complexes [34, 42] . In accordance, glycyrrhizin also interfered with nuclear export RNP at MOI 1 ( Figure S2 ). Similar results were obtained in MOI 0.01 H5N1 A/Thailand/1(Kan-1)/04infected cells ( Figure S3 ).\n\nInfluence of glycyrrhizin on H5N1-induced activation of nuclear factor kB (NFkB), p38, and on H5N1-induced cellular reactive oxygen species (ROS) formation Activation of NFkB, p38, and JNK have been associated with influenza A virus replication and virus-induced pro-inflammatory gene expression [34, [43] [44] [45] [46] [47] . While glycyrrhizin did not influence NFkB activity in non-infected A549 cells in the tested concentra-tions (data not shown), glycyrrhizin inhibited NFkB activation in H5N1-infected cells ( Figure 5A ). Moreover, glycyrrhizin inhibited H5N1-induced phosphorylation of the MAPKs p38 and JNK ( Figure 5B ).", "In addition to their roles during influenza A virus replication and virus-induced cytokine/chemokine expression, NFkB, p38, and JNK are constituents of redox-sensitive signalling pathways [48] [49] [50] [51] . Antioxidants had been already found to interfere with influenza A virus-induced signalling through NFkB, p38, and JNK, with influenza A virus replication, and with influenza A virus-induced pro-inflammatory gene expression [32] [33] [34] . Since glycyrrhizin is known to exert antioxidative effects [26] we speculated that glycyrrhizin may interfere with H5N1-induced ROS formation. Indeed glycyrrhizin exerted clear antioxidative effects in H5N1 (MOI 0.01)-infected cells ( Figure 5C ) causing significant reduction of ROS formation already at a concentration of 25 mg/ml ( Figure 5D ).", "Here, we show that glycyrrhizin inhibits the replication of highly pathogenic H5N1 influenza A virus, H5N1-induced apoptosis, and H5N1-induced expression of pro-inflammatory cytokines in lung-derived A549 cells. After intravenous administration, achievable plasma concentrations of glycyrrhizin have been described to be about 100 mg/ml [52] . Therefore, the glycyrrhizin concentrations found to interfere with H5N1 replication and H5N1-induced pro-inflammatory gene expression in the present report are in the range of therapeutic plasma levels. Notably, although higher glycyrrhizin concentrations were needed to interfere with SARS coronavirus replication [22] than with H5N1 replication, beneficial results were reported in glycyrrhizin (SNMC)-treated SARS patients in comparison to SARS patients who did not receive glycyrrhizin [23] . Notably, investigation of different glycyrrhizin derivatives against SARS coronavirus led to the identification of compounds with enhanced antiviral activity", "[53] . Therefore, glycyrrhizin might also serve as lead structure for the development of novel anti-influenza drugs.", "Experimental results suggested that glycyrrhizin might be able to affect seasonal influenza A virus disease by antiviral and immunomodulatory effects [26, 27] . Mice were prevented from lethal H2N2 infection by glycyrrhizin although no influence on virus replication was detected. The mechanism was suggested to be induction of interferon-c in T-cells by glycyrrhizin [54] . Moreover, glycyrrhizin was shown to influence seasonal influenza A virus replication through interaction with the cell membrane [25, 28] . However, these effects were observed only in concentrations $200 mg/ml when glycyrrhizin was added during the virus adsorption period. Since glycyrrhizin addition during the adsorption period did not influence H5N1 replication in our experiments it appears not likely that membrane effects contribute to anti-H5N1 effects detected here in lower concentrations.", "Our results rather suggest that glycyrrhizin interferes with H5N1-induced oxidative stress. Influenza A virus (including H5N1) infection induces ROS formation. Antioxidants were found to inhibit influenza A virus replication and influenza A virus-induced pro-inflammatory gene expression [32] [33] [34] and glycyrrhizin is known to exert antioxidative effects [26] . Here, glycyrrhizin interfered with H5N1-induced activation of NFkB, p38, and JNK representing redox-sensitive signalling events [48] [49] [50] [51] involved in influenza A virus replication and influenza A virusinduced cellular cytokine/chemokine production [34, [43] [44] [45] [46] 55] . Glycyrrhizin 50 mg/ml significantly reduced H5N1-induced activation of NFkB. In addition, glycyrrhizin concentrations as low as 25 mg/ml effectively interfered with H5N1-induced ROS formation and with phosphorylation of the redox-sensitive MAPKs p38 and JNK. In our model, activation of p38 appears to be critical for H5N1-associated redox", "signalling since p38 inhibition had been shown before to mimick effects of the antioxidant N-acetyl-cysteine (NAC) [34] . Interestingly and in contrast to glycyrrhizin, NAC failed to inhibit H5N1 replication or H5N1-induced cytokine/chemokine expression in therapeutically relevant concentrations.", "Glycyrrhizin diminished H5N1-induced cellular cytokine/ chemokine production in concentrations (#50 mg/ml) that did not interfere with H5N1 replication although redox-sensitive signalling pathways have been described to be involved in both processes. Therefore, H5N1-induced proinflammatory gene expression appears to be more sensitive to inhibition of ROS formation than H5N1 replication. Indeed, influenza viruses had been shown to induce cellular pathways through replicationdependent and -independent events [56] . In a previous report, we could show that similar glycyrrhizin concentrations like those investigated here interfered with H5N1-induced pro-inflammatory gene expression but not with H5N1 replication in human monocyte-derived macrophages [57] . In addition, other immunomodulatory treatment regimens that did not influence H5N1 replication reduced mortality in H5N1-infected mice [31, 58] . Therefore, glycyrrhizin represents a potential additional treatment option that interfers", "with both H5N1 replication and H5N1induced expression of pro-inflammatory cytokines in lung cells.", "Interference with immune responses may also result in the loss of control of virus replication by cytotoxic immune cells including natural killer cells and cytotoxic CD8 + T-lymphocytes. Global immunosuppressants like corticosteroids failed to protect from lethal influenza virus infection [59] . Moreover, antiviral drugs may interfere with cytotoxic cells that control virus replication as demonstrated for ribavirin that was shown to hamper NK cell cytolytic activity [60] . In this context, glycyrrhizin had already been shown not to affect natural killer cell activity in the concentrations used here [57] .", "In conclusion, we show in this report that therapeutic concentrations of glycyrrhizin (used as clinically approved parenteral preparation SNMC) interfere with highly pathogenic H5N1 influenza A virus replication and H5N1-induced proinflammatory gene expression at least in part through interference with H5N1-induced ROS formation and in turn reduced activation of p38, JNK, and NFkB in lung cells. Since we used the clinical formulation SNMC effects of other ingredients like glycin or cystein cannot be excluded. Vaccines and antiviral agents will fail to meet global needs at least at the beginning of a severe influenza A virus pandemic [61] . Anti-inflammatory and immunomodulatory agents are considered to be important candidates as constituents of anti-influenza treatment strategies that may save lives in an influenza pandemic situation [61] . Therefore, glycyrrhizin may complement the arsenal of potential drugs for the treatment of H5N1-caused disease." ]

[ 5 ]

[ "interfere with replication and/or cytopathogenic effect (CPE) induction of many viruses" ]

[ "Glycyrrhizin Exerts Antioxidative Effects in H5N1 Influenza A Virus-Infected Cells and Inhibits Virus Replication and Pro-Inflammatory Gene Expression\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3096629/\n\nSHA: f3b7f4469ac01f1ce916d24172570c43c537627e\n\nAuthors: Michaelis, Martin; Geiler, Janina; Naczk, Patrizia; Sithisarn, Patchima; Leutz, Anke; Doerr, Hans Wilhelm; Cinatl, Jindrich\nDate: 2011-05-17\nDOI: 10.1371/journal.pone.0019705\nLicense: cc-by", "Abstract: Glycyrrhizin is known to exert antiviral and anti-inflammatory effects. Here, the effects of an approved parenteral glycyrrhizin preparation (Stronger Neo-Minophafen C) were investigated on highly pathogenic influenza A H5N1 virus replication, H5N1-induced apoptosis, and H5N1-induced pro-inflammatory responses in lung epithelial (A549) cells. Therapeutic glycyrrhizin concentrations substantially inhibited H5N1-induced expression of the pro-inflammatory molecules CXCL10, interleukin 6, CCL2, and CCL5 (effective glycyrrhizin concentrations 25 to 50 µg/ml) but interfered with H5N1 replication and H5N1-induced apoptosis to a lesser extent (effective glycyrrhizin concentrations 100 µg/ml or higher). Glycyrrhizin also diminished monocyte migration towards supernatants of H5N1-infected A549 cells. The mechanism by which glycyrrhizin interferes with H5N1 replication and H5N1-induced pro-inflammatory gene expression includes inhibition of H5N1-induced formation of reactive oxygen", "species and (in turn) reduced activation of NFκB, JNK, and p38, redox-sensitive signalling events known to be relevant for influenza A virus replication. Therefore, glycyrrhizin may complement the arsenal of potential drugs for the treatment of H5N1 disease.", "Text: Highly pathogenic H5N1 influenza A viruses are considered to be potential influenza pandemic progenitors [1] [2] [3] [4] [5] [6] . At least for the first wave of an H5N1 pandemic, no sufficient amounts of adequate vaccines will be available [1] [2] [3] [4] [6] [7] [8] . Therefore, antiviral therapy for influenza A viruses including highly pathogenic H5N1 virus strains remains of great importance for the first line defense against the virus [1] [2] [3] [4] 6, 9] .", "The neuraminidase inhibitors oseltamivir and zanamivir as well as the adamantanes amantadin and rimantadin that interfere with the influenza M2 protein are licensed for the treament of influenza [1] [2] [3] [4] 6] . However, the use of both drug classes is limited by the emergence of resistant virus strains. In seasonal influenza strains, the majority of H3N2 viruses and a great proportion of H1N1 viruses in humans are now considered to be amantadine-and rimantadine-resistant [10] [11] [12] [13] . Moreover, a drastic increase in oseltamivir-resistant H1N1 viruses has been reported during the 2007/2008 influenza season in the northern hemisphere [14] [15] [16] [17] . Preliminary data from the United States predict a further rise for the 2008/2009 season, possibly resulting in more than 90% of the circulating H1N1 strains to be oseltamivir resistant [14] .", "H5N1 virus strains appear to be generally less sensitive to antiviral treatment than seasonal influenza A virus strains and treatment-resistant H5N1 strains emerge [1] [2] [3] [4] 6, [18] [19] [20] [21] . More-over, parenteral agents for the treatment of seriously ill patients are missing. Glycyrrhizin, a triterpene saponine, is a constituent of licorice root. It has been found to interfere with replication and/or cytopathogenic effect (CPE) induction of many viruses including respiratory viruses such as respiratory syncytial virus, SARS coronavirus, HIV, and influenza viruses [22] [23] [24] [25] [26] [27] [28] . Moreover, antiinflammatory and immunomodulatory properties were attributed to glycyrrhizin [26] . The severity of human H5N1 disease has been associated with hypercytokinaemia (''cytokine storm'') [29, 30] . Delayed antiviral plus immunomodulator treatment reduced H5N1-induced mortality in mice [31] . Therefore, antiinflammatory and immunomodulatory effects exerted by", "glycyrrhizin may be beneficial for treatment of H5N1. Also, glycyrrhizin is a known antioxidant [26] and antioxidants were already shown to interfere with influenza A virus replication and virus-induced pro-inflammatory responses [32] [33] [34] .", "Stronger Neo-Minophagen C (SNMC) is a glycyrrhizin preparation (available as tablets or parenteral formulation) that is approved in Japan for the treatment of chronic hepatic diseases and is marketed in Japan, China, Korea, Taiwan, Indonesia, India, and Mongolia. Here, we investigated the influence of SNMC on H5N1 replication, on H5N1-induced cytokine expression, on H5N1-induced cellular oxidative stress, and on critical H5N1-induced cellular signalling events in human pneumocytes (A549 cell line).\n\nGlycyrrhizin (Stronger Neo Minophagen C) was obtained from Minophagen Pharmaceuticals Co., Ltd. (Tokyo, Japan).\n\nThe influenza strain A/Vietnam/1203/04 (H5N1) was received from the WHO Influenza Centre (National Institute for Medical Research, London, UK). The H5N1 influenza strain A/Thailand/ 1(Kan-1)/04 was obtained from Prof. Pilaipan Puthavathana (Mahidol University, Bangkok, Thailand).", "Virus stocks were prepared by infecting Vero cells (African green monkey kidney; ATCC, Manassas, VA) and aliquots were stored at 280uC. Virus titres were determined as 50% tissue culture infectious dose (TCID 50 /ml) in confluent Vero cells in 96-well microtiter plates.\n\nA549 cells (human lung carcinoma; ATCC: CCL-185, obtained from LGC Standards GmbH, Wesel, Germany) were grown at 37uC in minimal essential medium (MEM) supplemented with 10% FBS, 100 IU/ml of penicillin and 100 mg/ml streptomycin.", "Human monocytes were isolated from buffy coats of healthy donors, obtained from Institute of Transfusion Medicine and Immune Haematology, German Red Cross Blood Donor Center, Johann Wolfgang Goethe-University, Frankfurt am Main. After centrifugation on Ficoll (Biocoll)-Hypaque density gradient (Biochrom AG, Berlin, Germany), mononuclear cells were collected from the interface and washed with PBS. Then, monocytes were isolated using magnetically labeled CD14 MicroBeads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) following the manufacturer's instructions. Monocytes were cultivated in IMDM supplemented with 10% pooled human serum, 100 IU/ml of penicillin, and 100 mg/ml streptomycin.\n\nThe cellular viability was assessed on confluent cell layers with CellTiter-GloH Luminescent Cell Viability Assay (Promega GmbH, Mannheim, Germany) according to the manufacturers' protocol. Cell viability was expressed as percentage of non-treated control.", "To determine intracellular NP localisation, H5N1-infected A549 were fixed 8 hours p.i. for 15 min with ice-cold acetone/ methanol (40:60, Mallinckrodt Baker B.V., Deventer, The Netherlands) and stained with a mouse monoclonal antibody (1 h incubation, 1:1000 in PBS) directed against the influenza A virus nucleoprotein (NP) (Millipore, Molsheim, France). An Alexa Fluor 488 goat anti-mouse IgG (H&L) (Invitrogen, Eugene, Oregon, USA) was used (1 h incubation, 1:1000 in PBS) as secondary antibody. Nuclei were stained using 49,6-diamidino-2phenylindole (DAPI) (Sigma-Aldrich Chemie GmbH, Munich, Germany). Fluorescence was visualised using Olympus IX 1 fluorescence microscope (Olympus, Planegg, Germany).\n\nFor flow cytometric analysis, the same antibodies were used.", "The cytopathogenic effect (CPE) reduction assay was performed as described before [34] . Confluent A549 cell monolayers grown in 96-well microtitre plates were infected with influenza A strains at the indicated multiplicities of infection (MOIs). After a one hour adsorption period, cells were washed to remove non-detached virus. The virus-induced CPE was recorded at 24 h post infection (p.i.).\n\nUnless otherwise stated, A549 cells were continuously treated with glycyrrhizin starting with a 1 h pre-incubation period. For time-ofaddition experiments, glycyrrhizin was added exclusively during the 1 h pre-incubation period, exclusively during the 1 h adsorption period, or after exclusively after the wash-out of input virus.", "Total RNA was isolated from cell cultures using TRI reagent (Sigma-Aldrich, Munich, Germany). Real time PCR for H5 was performed using described methods [35] . The following primers were used: sense 59 acg tat gac tac ccg cag tat tca g 39; antisense 59 aga cca gcy acc atg att gc 39; probe 6-FAM-tca aca gtg gcg agt tcc cta gca-TAMRA.\n\nThe fraction of cells with fractional DNA content (''sub-G1'' cell subpopulation) indicates cytotoxicity. Sub-G1 cells are considered to be dead (usually apoptotic) cells. Cells were fixed with 70% ethanol for two hours at 220uC. The cellular DNA was stained using propidium iodide (20 mg/ml) and analysed by flow cytometry (FacsCalibur, BD Biosciences, Heidelberg, Germany).\n\nCaspase activation was measured using the Caspase-Glo 8, 9, or 3/7 Assays (Promega, Mannheim, Germany) following the manufacturer's instructions.", "Cell culture supernatants were collected and frozen at 280uC. Cytokines/chemokines were quantified by specific ELISA Duo Sets (R&D Systems GmbH, Wiesbaden, Germany) following the manufacturer's instructions.\n\nNFkB activity was investigated in H5N1 (MOI 0.01)-infected cells by quantification of the NFkB subunits Rel A (p65) and NFkB1 (p50) from nuclear extracts using the TransAM TM transcription factor DNA-binding ELISAs (Active Motif, Rixensart, Belgium). Nuclear extract were prepared using the Nuclear Extract Kit (Active Motif, Carlsbad, CA, USA) following the manufacturer's instruction.", "Cell culture supernatants were investigated for chemotactic activity by measurement of the activity to induce monocyte migration through membrane inserts in 24-well plates (pore size 8 mm; BD Biosciences, Heidelberg, Germany). Monocytes (1610 6 in 100 ml of IMDM with 10% pooled human serum) were added into the cell culture inserts (upper chamber) and cell culture supernatants (300 ml), were added to the lower chamber of the well. After a 48 h incubation period, cells were fixed with 4% paraformaldehyde and permeabilised with PBS containing 0.3% Tritron X-100. Then, nuclei were stained with 49,6-diamidino-2phenylindole (DAPI). The upper side of the membrane was wiped with a wet swab to remove the cells, while the lower side of the membrane was rinsed with PBS. The number of cells at the lower side of each membrane was quantified by counting of cells from three randomly chosen sections (3.7 mm 2 ) using an Olympus IX 1 fluorescence microscope (Olympus, Planegg, Germany).", "Cells were lysed in Triton X-sample buffer and separated by SDS-PAGE. Nuclear extract were prepared using the Nuclear Extract Kit (Active Motif, Carlsbad, CA, USA) following the manufacturer's instruction. Proteins were detected using specific antibodies against bactin (Sigma-Aldrich Chemie GmbH, Munich, Germany), JNK, phosphorylated JNK, p38, or phosphorylated p38, (all purchased from New England Biolabs GmbH, Frankfurt am Main, Germany) and were visualised by enhanced chemiluminescence using a commercially available kit (Amersham, Freiburg, Germany).\n\nReactive oxygen species (ROS) were detected using the Image-iT LIVE Green Reactive Oxygen Species Kit (Molecular Probes, distributed by Invitrogen, Karlsruhe, Germany).\n\nTwo groups were compared by t-test. More groups were compared by ANOVA with subsequent Student-Newman-Keuls test.", "The A549 cell line, derived from a human pulmonary adenocarcinoma, is an established model for type II pneumocytes [36] , and commonly used for the investigation of the effect of influenza viruses on this cell type [see e.g. 6,37,38]. If not otherwise stated, glycyrrhizin was continuously present in cell culture media starting with a 1 h preinfection period. Glycyrrhizin 200 mg/ml (the maximum tested concentration) did not affect A549 cell viability (data not shown) but clearly decreased CPE formation in A549 cells infected with the H5N1 influenza strain A/Thailand/1(Kan-1)/04 at MOIs of 0.01, 0.1 or 1 ( Figure 1A ). Similar results were obtained in A549 cells infected with strain A/Vietnam/1203/04 (H5N1) (Suppl. Figure 1A) . Staining of A549 cells for influenza A nucleoprotein 24 h after infection with strain H5N1 A/Thailand/1(Kan-1)/04 indicated that glycyrrhizin 200 mg/ml significantly reduces the number of influenza A nucleoprotein positive cells ( Figure 1B) .", "To examine the influence of glycyrrhizin on virus progeny, A549 cells were infected with the H5N1 influenza strain A/ Thailand/1(Kan-1)/04 at MOI 0.01 or MOI 1 and infectious virus titres were determined 24 h post infection ( Figure 1C ). While glycyrrhizin in concentrations up to 50 mg/ml did not affect H5N1 replication, moderate effects were exerted by glycyrrhizin 100 mg/ ml and more pronounced effects by glycyrrhizin 200 mg/ml (MOI 0.01: 13-fold reduction, MOI 1: 10-fold reduction). Next, influence of glycyrrhizin on H5N1 replication was confirmed by the detection of viral (H5) RNA using quantitative PCR. Only glycyrrhizin concentrations $100 mg/ml significantly reduced Figure 1B) or H5N1 A/Vietnam/1203/04-infected (Suppl. Figure 1C ) A549 cells (MOI 0.01) 24 h post infection.", "Time-of-addition experiments revealed that maximal effects were achieved when glycyrrhizin was continuously present starting with a 1 h pre-incubation period ( Figure 1D ). Addition of glycyrrhizin post infection showed reduced antiviral effects while pre-incubation alone or glycyrrhizin addition during the adsorption period did not significantly affect H5N1 replication.", "For investigation of H5N1-induced cytokine expression, five pro-inflammatory genes were chosen that had been correlated to severity of influenza disease: CXCL10 (also known as interferon-cinducible protein 10, IP-10), interleukin 6 (IL6), interleukin 8, (IL8; also known as CXCL8), CCL2 (also known as monocyte chemoattractant protein 1, MCP-1), and CCL5 (also known as RANTES). A549 cells were infected with H5N1 A/Thailand/ 1(Kan-1)/04 or H5N1 A/Vietnam/1203/04 at MOI 0.01, 0.1, or 1. Glycyrrhizin treatment was performed with 25, 50, 100, or 200 mg/ml. Cytokine expression was detected 24 h post infection by ELISA. Glycyrrhizin did not affect cytokine expression of noninfected cells (data not shown) but inhibited expression of all cytokines investigated in H5N1-infected cells in a dose-dependent manner (Figure 2, Figure 3A ). Effects were more pronounced at lower MOIs. Notably, expression of all cytokines except IL8 was significantly inhibited after treatment with glycyrrhizin 50 mg/ml", "Figure 3A ) although these glycyrrhizin concentrations had no effect on H5N1 replication in A549 cells (Figure 1, Figure S1 ).", "Cytokine expression by influenza A virus-infected respiratory cells causes recruitment of peripheral blood monocytes into the lungs of patients where they differentiate to macrophages which are thought to contribute to influenza A virus pathogenicity [5, 39] . In a chemotaxis assay, the influence of glycyrrhizin was investigated on migration of monocytes towards supernatants of H5N1 A/Thailand/1(Kan-1)/04 (MOI 0.1)-infected A549 cells through 8 mm filters. Monocyte migration towards supernatants of H5N1-infected cells was strongly increased relative to migration towards supernatants of non-infected cells. Treatment of H5N1- infected cells with glycyrrhizin 100 mg/ml clearly suppressed chemoattraction activity of supernatants ( Figure 3B ).", "Influenza viruses including H5N1 have been shown to induce caspase-dependent apoptosis in airway cells and this apoptosis has been correlated to the virus pathogenicity [40, 41] . Glycyrrhizin concentrations up to 200 mg/ml did not affect caspase activation in non-infected cells ( Figure 4A-C) . Glycyrrhizin concentrations $100 mg/ml inhibited H5N1 A/Thailand/1(Kan-1)/04 (MOI 0.01)-induced activation of the initiator caspases 8 and 9 as well as of the effector caspases 3/7 in A549 cells as determined 24 h post infection ( Figure 4A-C) . Lower glycyrrhizin concentrations did not affect H5N1-induced apoptosis. The detection of cells in sub-G1 phase resulted in similar findings ( Figure 4D ).", "Substances that inhibit H5N1-induced caspase 3 activation including caspase 3 inhibitors cause nuclear retention of RNP complexes [34, 42] . In accordance, glycyrrhizin also interfered with nuclear export RNP at MOI 1 ( Figure S2 ). Similar results were obtained in MOI 0.01 H5N1 A/Thailand/1(Kan-1)/04infected cells ( Figure S3 ).\n\nInfluence of glycyrrhizin on H5N1-induced activation of nuclear factor kB (NFkB), p38, and on H5N1-induced cellular reactive oxygen species (ROS) formation Activation of NFkB, p38, and JNK have been associated with influenza A virus replication and virus-induced pro-inflammatory gene expression [34, [43] [44] [45] [46] [47] . While glycyrrhizin did not influence NFkB activity in non-infected A549 cells in the tested concentra-tions (data not shown), glycyrrhizin inhibited NFkB activation in H5N1-infected cells ( Figure 5A ). Moreover, glycyrrhizin inhibited H5N1-induced phosphorylation of the MAPKs p38 and JNK ( Figure 5B ).", "In addition to their roles during influenza A virus replication and virus-induced cytokine/chemokine expression, NFkB, p38, and JNK are constituents of redox-sensitive signalling pathways [48] [49] [50] [51] . Antioxidants had been already found to interfere with influenza A virus-induced signalling through NFkB, p38, and JNK, with influenza A virus replication, and with influenza A virus-induced pro-inflammatory gene expression [32] [33] [34] . Since glycyrrhizin is known to exert antioxidative effects [26] we speculated that glycyrrhizin may interfere with H5N1-induced ROS formation. Indeed glycyrrhizin exerted clear antioxidative effects in H5N1 (MOI 0.01)-infected cells ( Figure 5C ) causing significant reduction of ROS formation already at a concentration of 25 mg/ml ( Figure 5D ).", "Here, we show that glycyrrhizin inhibits the replication of highly pathogenic H5N1 influenza A virus, H5N1-induced apoptosis, and H5N1-induced expression of pro-inflammatory cytokines in lung-derived A549 cells. After intravenous administration, achievable plasma concentrations of glycyrrhizin have been described to be about 100 mg/ml [52] . Therefore, the glycyrrhizin concentrations found to interfere with H5N1 replication and H5N1-induced pro-inflammatory gene expression in the present report are in the range of therapeutic plasma levels. Notably, although higher glycyrrhizin concentrations were needed to interfere with SARS coronavirus replication [22] than with H5N1 replication, beneficial results were reported in glycyrrhizin (SNMC)-treated SARS patients in comparison to SARS patients who did not receive glycyrrhizin [23] . Notably, investigation of different glycyrrhizin derivatives against SARS coronavirus led to the identification of compounds with enhanced antiviral activity", "[53] . Therefore, glycyrrhizin might also serve as lead structure for the development of novel anti-influenza drugs.", "Experimental results suggested that glycyrrhizin might be able to affect seasonal influenza A virus disease by antiviral and immunomodulatory effects [26, 27] . Mice were prevented from lethal H2N2 infection by glycyrrhizin although no influence on virus replication was detected. The mechanism was suggested to be induction of interferon-c in T-cells by glycyrrhizin [54] . Moreover, glycyrrhizin was shown to influence seasonal influenza A virus replication through interaction with the cell membrane [25, 28] . However, these effects were observed only in concentrations $200 mg/ml when glycyrrhizin was added during the virus adsorption period. Since glycyrrhizin addition during the adsorption period did not influence H5N1 replication in our experiments it appears not likely that membrane effects contribute to anti-H5N1 effects detected here in lower concentrations.", "Our results rather suggest that glycyrrhizin interferes with H5N1-induced oxidative stress. Influenza A virus (including H5N1) infection induces ROS formation. Antioxidants were found to inhibit influenza A virus replication and influenza A virus-induced pro-inflammatory gene expression [32] [33] [34] and glycyrrhizin is known to exert antioxidative effects [26] . Here, glycyrrhizin interfered with H5N1-induced activation of NFkB, p38, and JNK representing redox-sensitive signalling events [48] [49] [50] [51] involved in influenza A virus replication and influenza A virusinduced cellular cytokine/chemokine production [34, [43] [44] [45] [46] 55] . Glycyrrhizin 50 mg/ml significantly reduced H5N1-induced activation of NFkB. In addition, glycyrrhizin concentrations as low as 25 mg/ml effectively interfered with H5N1-induced ROS formation and with phosphorylation of the redox-sensitive MAPKs p38 and JNK. In our model, activation of p38 appears to be critical for H5N1-associated redox", "signalling since p38 inhibition had been shown before to mimick effects of the antioxidant N-acetyl-cysteine (NAC) [34] . Interestingly and in contrast to glycyrrhizin, NAC failed to inhibit H5N1 replication or H5N1-induced cytokine/chemokine expression in therapeutically relevant concentrations.", "Glycyrrhizin diminished H5N1-induced cellular cytokine/ chemokine production in concentrations (#50 mg/ml) that did not interfere with H5N1 replication although redox-sensitive signalling pathways have been described to be involved in both processes. Therefore, H5N1-induced proinflammatory gene expression appears to be more sensitive to inhibition of ROS formation than H5N1 replication. Indeed, influenza viruses had been shown to induce cellular pathways through replicationdependent and -independent events [56] . In a previous report, we could show that similar glycyrrhizin concentrations like those investigated here interfered with H5N1-induced pro-inflammatory gene expression but not with H5N1 replication in human monocyte-derived macrophages [57] . In addition, other immunomodulatory treatment regimens that did not influence H5N1 replication reduced mortality in H5N1-infected mice [31, 58] . Therefore, glycyrrhizin represents a potential additional treatment option that interfers", "with both H5N1 replication and H5N1induced expression of pro-inflammatory cytokines in lung cells.", "Interference with immune responses may also result in the loss of control of virus replication by cytotoxic immune cells including natural killer cells and cytotoxic CD8 + T-lymphocytes. Global immunosuppressants like corticosteroids failed to protect from lethal influenza virus infection [59] . Moreover, antiviral drugs may interfere with cytotoxic cells that control virus replication as demonstrated for ribavirin that was shown to hamper NK cell cytolytic activity [60] . In this context, glycyrrhizin had already been shown not to affect natural killer cell activity in the concentrations used here [57] .", "In conclusion, we show in this report that therapeutic concentrations of glycyrrhizin (used as clinically approved parenteral preparation SNMC) interfere with highly pathogenic H5N1 influenza A virus replication and H5N1-induced proinflammatory gene expression at least in part through interference with H5N1-induced ROS formation and in turn reduced activation of p38, JNK, and NFkB in lung cells. Since we used the clinical formulation SNMC effects of other ingredients like glycin or cystein cannot be excluded. Vaccines and antiviral agents will fail to meet global needs at least at the beginning of a severe influenza A virus pandemic [61] . Anti-inflammatory and immunomodulatory agents are considered to be important candidates as constituents of anti-influenza treatment strategies that may save lives in an influenza pandemic situation [61] . Therefore, glycyrrhizin may complement the arsenal of potential drugs for the treatment of H5N1-caused disease." ]

[ 5 ]

[ "cytokine storm'" ]

[ "Glycyrrhizin Exerts Antioxidative Effects in H5N1 Influenza A Virus-Infected Cells and Inhibits Virus Replication and Pro-Inflammatory Gene Expression\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3096629/\n\nSHA: f3b7f4469ac01f1ce916d24172570c43c537627e\n\nAuthors: Michaelis, Martin; Geiler, Janina; Naczk, Patrizia; Sithisarn, Patchima; Leutz, Anke; Doerr, Hans Wilhelm; Cinatl, Jindrich\nDate: 2011-05-17\nDOI: 10.1371/journal.pone.0019705\nLicense: cc-by", "Abstract: Glycyrrhizin is known to exert antiviral and anti-inflammatory effects. Here, the effects of an approved parenteral glycyrrhizin preparation (Stronger Neo-Minophafen C) were investigated on highly pathogenic influenza A H5N1 virus replication, H5N1-induced apoptosis, and H5N1-induced pro-inflammatory responses in lung epithelial (A549) cells. Therapeutic glycyrrhizin concentrations substantially inhibited H5N1-induced expression of the pro-inflammatory molecules CXCL10, interleukin 6, CCL2, and CCL5 (effective glycyrrhizin concentrations 25 to 50 µg/ml) but interfered with H5N1 replication and H5N1-induced apoptosis to a lesser extent (effective glycyrrhizin concentrations 100 µg/ml or higher). Glycyrrhizin also diminished monocyte migration towards supernatants of H5N1-infected A549 cells. The mechanism by which glycyrrhizin interferes with H5N1 replication and H5N1-induced pro-inflammatory gene expression includes inhibition of H5N1-induced formation of reactive oxygen", "species and (in turn) reduced activation of NFκB, JNK, and p38, redox-sensitive signalling events known to be relevant for influenza A virus replication. Therefore, glycyrrhizin may complement the arsenal of potential drugs for the treatment of H5N1 disease.", "Text: Highly pathogenic H5N1 influenza A viruses are considered to be potential influenza pandemic progenitors [1] [2] [3] [4] [5] [6] . At least for the first wave of an H5N1 pandemic, no sufficient amounts of adequate vaccines will be available [1] [2] [3] [4] [6] [7] [8] . Therefore, antiviral therapy for influenza A viruses including highly pathogenic H5N1 virus strains remains of great importance for the first line defense against the virus [1] [2] [3] [4] 6, 9] .", "The neuraminidase inhibitors oseltamivir and zanamivir as well as the adamantanes amantadin and rimantadin that interfere with the influenza M2 protein are licensed for the treament of influenza [1] [2] [3] [4] 6] . However, the use of both drug classes is limited by the emergence of resistant virus strains. In seasonal influenza strains, the majority of H3N2 viruses and a great proportion of H1N1 viruses in humans are now considered to be amantadine-and rimantadine-resistant [10] [11] [12] [13] . Moreover, a drastic increase in oseltamivir-resistant H1N1 viruses has been reported during the 2007/2008 influenza season in the northern hemisphere [14] [15] [16] [17] . Preliminary data from the United States predict a further rise for the 2008/2009 season, possibly resulting in more than 90% of the circulating H1N1 strains to be oseltamivir resistant [14] .", "H5N1 virus strains appear to be generally less sensitive to antiviral treatment than seasonal influenza A virus strains and treatment-resistant H5N1 strains emerge [1] [2] [3] [4] 6, [18] [19] [20] [21] . More-over, parenteral agents for the treatment of seriously ill patients are missing. Glycyrrhizin, a triterpene saponine, is a constituent of licorice root. It has been found to interfere with replication and/or cytopathogenic effect (CPE) induction of many viruses including respiratory viruses such as respiratory syncytial virus, SARS coronavirus, HIV, and influenza viruses [22] [23] [24] [25] [26] [27] [28] . Moreover, antiinflammatory and immunomodulatory properties were attributed to glycyrrhizin [26] . The severity of human H5N1 disease has been associated with hypercytokinaemia (''cytokine storm'') [29, 30] . Delayed antiviral plus immunomodulator treatment reduced H5N1-induced mortality in mice [31] . Therefore, antiinflammatory and immunomodulatory effects exerted by", "glycyrrhizin may be beneficial for treatment of H5N1. Also, glycyrrhizin is a known antioxidant [26] and antioxidants were already shown to interfere with influenza A virus replication and virus-induced pro-inflammatory responses [32] [33] [34] .", "Stronger Neo-Minophagen C (SNMC) is a glycyrrhizin preparation (available as tablets or parenteral formulation) that is approved in Japan for the treatment of chronic hepatic diseases and is marketed in Japan, China, Korea, Taiwan, Indonesia, India, and Mongolia. Here, we investigated the influence of SNMC on H5N1 replication, on H5N1-induced cytokine expression, on H5N1-induced cellular oxidative stress, and on critical H5N1-induced cellular signalling events in human pneumocytes (A549 cell line).\n\nGlycyrrhizin (Stronger Neo Minophagen C) was obtained from Minophagen Pharmaceuticals Co., Ltd. (Tokyo, Japan).\n\nThe influenza strain A/Vietnam/1203/04 (H5N1) was received from the WHO Influenza Centre (National Institute for Medical Research, London, UK). The H5N1 influenza strain A/Thailand/ 1(Kan-1)/04 was obtained from Prof. Pilaipan Puthavathana (Mahidol University, Bangkok, Thailand).", "Virus stocks were prepared by infecting Vero cells (African green monkey kidney; ATCC, Manassas, VA) and aliquots were stored at 280uC. Virus titres were determined as 50% tissue culture infectious dose (TCID 50 /ml) in confluent Vero cells in 96-well microtiter plates.\n\nA549 cells (human lung carcinoma; ATCC: CCL-185, obtained from LGC Standards GmbH, Wesel, Germany) were grown at 37uC in minimal essential medium (MEM) supplemented with 10% FBS, 100 IU/ml of penicillin and 100 mg/ml streptomycin.", "Human monocytes were isolated from buffy coats of healthy donors, obtained from Institute of Transfusion Medicine and Immune Haematology, German Red Cross Blood Donor Center, Johann Wolfgang Goethe-University, Frankfurt am Main. After centrifugation on Ficoll (Biocoll)-Hypaque density gradient (Biochrom AG, Berlin, Germany), mononuclear cells were collected from the interface and washed with PBS. Then, monocytes were isolated using magnetically labeled CD14 MicroBeads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) following the manufacturer's instructions. Monocytes were cultivated in IMDM supplemented with 10% pooled human serum, 100 IU/ml of penicillin, and 100 mg/ml streptomycin.\n\nThe cellular viability was assessed on confluent cell layers with CellTiter-GloH Luminescent Cell Viability Assay (Promega GmbH, Mannheim, Germany) according to the manufacturers' protocol. Cell viability was expressed as percentage of non-treated control.", "To determine intracellular NP localisation, H5N1-infected A549 were fixed 8 hours p.i. for 15 min with ice-cold acetone/ methanol (40:60, Mallinckrodt Baker B.V., Deventer, The Netherlands) and stained with a mouse monoclonal antibody (1 h incubation, 1:1000 in PBS) directed against the influenza A virus nucleoprotein (NP) (Millipore, Molsheim, France). An Alexa Fluor 488 goat anti-mouse IgG (H&L) (Invitrogen, Eugene, Oregon, USA) was used (1 h incubation, 1:1000 in PBS) as secondary antibody. Nuclei were stained using 49,6-diamidino-2phenylindole (DAPI) (Sigma-Aldrich Chemie GmbH, Munich, Germany). Fluorescence was visualised using Olympus IX 1 fluorescence microscope (Olympus, Planegg, Germany).\n\nFor flow cytometric analysis, the same antibodies were used.", "The cytopathogenic effect (CPE) reduction assay was performed as described before [34] . Confluent A549 cell monolayers grown in 96-well microtitre plates were infected with influenza A strains at the indicated multiplicities of infection (MOIs). After a one hour adsorption period, cells were washed to remove non-detached virus. The virus-induced CPE was recorded at 24 h post infection (p.i.).\n\nUnless otherwise stated, A549 cells were continuously treated with glycyrrhizin starting with a 1 h pre-incubation period. For time-ofaddition experiments, glycyrrhizin was added exclusively during the 1 h pre-incubation period, exclusively during the 1 h adsorption period, or after exclusively after the wash-out of input virus.", "Total RNA was isolated from cell cultures using TRI reagent (Sigma-Aldrich, Munich, Germany). Real time PCR for H5 was performed using described methods [35] . The following primers were used: sense 59 acg tat gac tac ccg cag tat tca g 39; antisense 59 aga cca gcy acc atg att gc 39; probe 6-FAM-tca aca gtg gcg agt tcc cta gca-TAMRA.\n\nThe fraction of cells with fractional DNA content (''sub-G1'' cell subpopulation) indicates cytotoxicity. Sub-G1 cells are considered to be dead (usually apoptotic) cells. Cells were fixed with 70% ethanol for two hours at 220uC. The cellular DNA was stained using propidium iodide (20 mg/ml) and analysed by flow cytometry (FacsCalibur, BD Biosciences, Heidelberg, Germany).\n\nCaspase activation was measured using the Caspase-Glo 8, 9, or 3/7 Assays (Promega, Mannheim, Germany) following the manufacturer's instructions.", "Cell culture supernatants were collected and frozen at 280uC. Cytokines/chemokines were quantified by specific ELISA Duo Sets (R&D Systems GmbH, Wiesbaden, Germany) following the manufacturer's instructions.\n\nNFkB activity was investigated in H5N1 (MOI 0.01)-infected cells by quantification of the NFkB subunits Rel A (p65) and NFkB1 (p50) from nuclear extracts using the TransAM TM transcription factor DNA-binding ELISAs (Active Motif, Rixensart, Belgium). Nuclear extract were prepared using the Nuclear Extract Kit (Active Motif, Carlsbad, CA, USA) following the manufacturer's instruction.", "Cell culture supernatants were investigated for chemotactic activity by measurement of the activity to induce monocyte migration through membrane inserts in 24-well plates (pore size 8 mm; BD Biosciences, Heidelberg, Germany). Monocytes (1610 6 in 100 ml of IMDM with 10% pooled human serum) were added into the cell culture inserts (upper chamber) and cell culture supernatants (300 ml), were added to the lower chamber of the well. After a 48 h incubation period, cells were fixed with 4% paraformaldehyde and permeabilised with PBS containing 0.3% Tritron X-100. Then, nuclei were stained with 49,6-diamidino-2phenylindole (DAPI). The upper side of the membrane was wiped with a wet swab to remove the cells, while the lower side of the membrane was rinsed with PBS. The number of cells at the lower side of each membrane was quantified by counting of cells from three randomly chosen sections (3.7 mm 2 ) using an Olympus IX 1 fluorescence microscope (Olympus, Planegg, Germany).", "Cells were lysed in Triton X-sample buffer and separated by SDS-PAGE. Nuclear extract were prepared using the Nuclear Extract Kit (Active Motif, Carlsbad, CA, USA) following the manufacturer's instruction. Proteins were detected using specific antibodies against bactin (Sigma-Aldrich Chemie GmbH, Munich, Germany), JNK, phosphorylated JNK, p38, or phosphorylated p38, (all purchased from New England Biolabs GmbH, Frankfurt am Main, Germany) and were visualised by enhanced chemiluminescence using a commercially available kit (Amersham, Freiburg, Germany).\n\nReactive oxygen species (ROS) were detected using the Image-iT LIVE Green Reactive Oxygen Species Kit (Molecular Probes, distributed by Invitrogen, Karlsruhe, Germany).\n\nTwo groups were compared by t-test. More groups were compared by ANOVA with subsequent Student-Newman-Keuls test.", "The A549 cell line, derived from a human pulmonary adenocarcinoma, is an established model for type II pneumocytes [36] , and commonly used for the investigation of the effect of influenza viruses on this cell type [see e.g. 6,37,38]. If not otherwise stated, glycyrrhizin was continuously present in cell culture media starting with a 1 h preinfection period. Glycyrrhizin 200 mg/ml (the maximum tested concentration) did not affect A549 cell viability (data not shown) but clearly decreased CPE formation in A549 cells infected with the H5N1 influenza strain A/Thailand/1(Kan-1)/04 at MOIs of 0.01, 0.1 or 1 ( Figure 1A ). Similar results were obtained in A549 cells infected with strain A/Vietnam/1203/04 (H5N1) (Suppl. Figure 1A) . Staining of A549 cells for influenza A nucleoprotein 24 h after infection with strain H5N1 A/Thailand/1(Kan-1)/04 indicated that glycyrrhizin 200 mg/ml significantly reduces the number of influenza A nucleoprotein positive cells ( Figure 1B) .", "To examine the influence of glycyrrhizin on virus progeny, A549 cells were infected with the H5N1 influenza strain A/ Thailand/1(Kan-1)/04 at MOI 0.01 or MOI 1 and infectious virus titres were determined 24 h post infection ( Figure 1C ). While glycyrrhizin in concentrations up to 50 mg/ml did not affect H5N1 replication, moderate effects were exerted by glycyrrhizin 100 mg/ ml and more pronounced effects by glycyrrhizin 200 mg/ml (MOI 0.01: 13-fold reduction, MOI 1: 10-fold reduction). Next, influence of glycyrrhizin on H5N1 replication was confirmed by the detection of viral (H5) RNA using quantitative PCR. Only glycyrrhizin concentrations $100 mg/ml significantly reduced Figure 1B) or H5N1 A/Vietnam/1203/04-infected (Suppl. Figure 1C ) A549 cells (MOI 0.01) 24 h post infection.", "Time-of-addition experiments revealed that maximal effects were achieved when glycyrrhizin was continuously present starting with a 1 h pre-incubation period ( Figure 1D ). Addition of glycyrrhizin post infection showed reduced antiviral effects while pre-incubation alone or glycyrrhizin addition during the adsorption period did not significantly affect H5N1 replication.", "For investigation of H5N1-induced cytokine expression, five pro-inflammatory genes were chosen that had been correlated to severity of influenza disease: CXCL10 (also known as interferon-cinducible protein 10, IP-10), interleukin 6 (IL6), interleukin 8, (IL8; also known as CXCL8), CCL2 (also known as monocyte chemoattractant protein 1, MCP-1), and CCL5 (also known as RANTES). A549 cells were infected with H5N1 A/Thailand/ 1(Kan-1)/04 or H5N1 A/Vietnam/1203/04 at MOI 0.01, 0.1, or 1. Glycyrrhizin treatment was performed with 25, 50, 100, or 200 mg/ml. Cytokine expression was detected 24 h post infection by ELISA. Glycyrrhizin did not affect cytokine expression of noninfected cells (data not shown) but inhibited expression of all cytokines investigated in H5N1-infected cells in a dose-dependent manner (Figure 2, Figure 3A ). Effects were more pronounced at lower MOIs. Notably, expression of all cytokines except IL8 was significantly inhibited after treatment with glycyrrhizin 50 mg/ml", "Figure 3A ) although these glycyrrhizin concentrations had no effect on H5N1 replication in A549 cells (Figure 1, Figure S1 ).", "Cytokine expression by influenza A virus-infected respiratory cells causes recruitment of peripheral blood monocytes into the lungs of patients where they differentiate to macrophages which are thought to contribute to influenza A virus pathogenicity [5, 39] . In a chemotaxis assay, the influence of glycyrrhizin was investigated on migration of monocytes towards supernatants of H5N1 A/Thailand/1(Kan-1)/04 (MOI 0.1)-infected A549 cells through 8 mm filters. Monocyte migration towards supernatants of H5N1-infected cells was strongly increased relative to migration towards supernatants of non-infected cells. Treatment of H5N1- infected cells with glycyrrhizin 100 mg/ml clearly suppressed chemoattraction activity of supernatants ( Figure 3B ).", "Influenza viruses including H5N1 have been shown to induce caspase-dependent apoptosis in airway cells and this apoptosis has been correlated to the virus pathogenicity [40, 41] . Glycyrrhizin concentrations up to 200 mg/ml did not affect caspase activation in non-infected cells ( Figure 4A-C) . Glycyrrhizin concentrations $100 mg/ml inhibited H5N1 A/Thailand/1(Kan-1)/04 (MOI 0.01)-induced activation of the initiator caspases 8 and 9 as well as of the effector caspases 3/7 in A549 cells as determined 24 h post infection ( Figure 4A-C) . Lower glycyrrhizin concentrations did not affect H5N1-induced apoptosis. The detection of cells in sub-G1 phase resulted in similar findings ( Figure 4D ).", "Substances that inhibit H5N1-induced caspase 3 activation including caspase 3 inhibitors cause nuclear retention of RNP complexes [34, 42] . In accordance, glycyrrhizin also interfered with nuclear export RNP at MOI 1 ( Figure S2 ). Similar results were obtained in MOI 0.01 H5N1 A/Thailand/1(Kan-1)/04infected cells ( Figure S3 ).\n\nInfluence of glycyrrhizin on H5N1-induced activation of nuclear factor kB (NFkB), p38, and on H5N1-induced cellular reactive oxygen species (ROS) formation Activation of NFkB, p38, and JNK have been associated with influenza A virus replication and virus-induced pro-inflammatory gene expression [34, [43] [44] [45] [46] [47] . While glycyrrhizin did not influence NFkB activity in non-infected A549 cells in the tested concentra-tions (data not shown), glycyrrhizin inhibited NFkB activation in H5N1-infected cells ( Figure 5A ). Moreover, glycyrrhizin inhibited H5N1-induced phosphorylation of the MAPKs p38 and JNK ( Figure 5B ).", "In addition to their roles during influenza A virus replication and virus-induced cytokine/chemokine expression, NFkB, p38, and JNK are constituents of redox-sensitive signalling pathways [48] [49] [50] [51] . Antioxidants had been already found to interfere with influenza A virus-induced signalling through NFkB, p38, and JNK, with influenza A virus replication, and with influenza A virus-induced pro-inflammatory gene expression [32] [33] [34] . Since glycyrrhizin is known to exert antioxidative effects [26] we speculated that glycyrrhizin may interfere with H5N1-induced ROS formation. Indeed glycyrrhizin exerted clear antioxidative effects in H5N1 (MOI 0.01)-infected cells ( Figure 5C ) causing significant reduction of ROS formation already at a concentration of 25 mg/ml ( Figure 5D ).", "Here, we show that glycyrrhizin inhibits the replication of highly pathogenic H5N1 influenza A virus, H5N1-induced apoptosis, and H5N1-induced expression of pro-inflammatory cytokines in lung-derived A549 cells. After intravenous administration, achievable plasma concentrations of glycyrrhizin have been described to be about 100 mg/ml [52] . Therefore, the glycyrrhizin concentrations found to interfere with H5N1 replication and H5N1-induced pro-inflammatory gene expression in the present report are in the range of therapeutic plasma levels. Notably, although higher glycyrrhizin concentrations were needed to interfere with SARS coronavirus replication [22] than with H5N1 replication, beneficial results were reported in glycyrrhizin (SNMC)-treated SARS patients in comparison to SARS patients who did not receive glycyrrhizin [23] . Notably, investigation of different glycyrrhizin derivatives against SARS coronavirus led to the identification of compounds with enhanced antiviral activity", "[53] . Therefore, glycyrrhizin might also serve as lead structure for the development of novel anti-influenza drugs.", "Experimental results suggested that glycyrrhizin might be able to affect seasonal influenza A virus disease by antiviral and immunomodulatory effects [26, 27] . Mice were prevented from lethal H2N2 infection by glycyrrhizin although no influence on virus replication was detected. The mechanism was suggested to be induction of interferon-c in T-cells by glycyrrhizin [54] . Moreover, glycyrrhizin was shown to influence seasonal influenza A virus replication through interaction with the cell membrane [25, 28] . However, these effects were observed only in concentrations $200 mg/ml when glycyrrhizin was added during the virus adsorption period. Since glycyrrhizin addition during the adsorption period did not influence H5N1 replication in our experiments it appears not likely that membrane effects contribute to anti-H5N1 effects detected here in lower concentrations.", "Our results rather suggest that glycyrrhizin interferes with H5N1-induced oxidative stress. Influenza A virus (including H5N1) infection induces ROS formation. Antioxidants were found to inhibit influenza A virus replication and influenza A virus-induced pro-inflammatory gene expression [32] [33] [34] and glycyrrhizin is known to exert antioxidative effects [26] . Here, glycyrrhizin interfered with H5N1-induced activation of NFkB, p38, and JNK representing redox-sensitive signalling events [48] [49] [50] [51] involved in influenza A virus replication and influenza A virusinduced cellular cytokine/chemokine production [34, [43] [44] [45] [46] 55] . Glycyrrhizin 50 mg/ml significantly reduced H5N1-induced activation of NFkB. In addition, glycyrrhizin concentrations as low as 25 mg/ml effectively interfered with H5N1-induced ROS formation and with phosphorylation of the redox-sensitive MAPKs p38 and JNK. In our model, activation of p38 appears to be critical for H5N1-associated redox", "signalling since p38 inhibition had been shown before to mimick effects of the antioxidant N-acetyl-cysteine (NAC) [34] . Interestingly and in contrast to glycyrrhizin, NAC failed to inhibit H5N1 replication or H5N1-induced cytokine/chemokine expression in therapeutically relevant concentrations.", "Glycyrrhizin diminished H5N1-induced cellular cytokine/ chemokine production in concentrations (#50 mg/ml) that did not interfere with H5N1 replication although redox-sensitive signalling pathways have been described to be involved in both processes. Therefore, H5N1-induced proinflammatory gene expression appears to be more sensitive to inhibition of ROS formation than H5N1 replication. Indeed, influenza viruses had been shown to induce cellular pathways through replicationdependent and -independent events [56] . In a previous report, we could show that similar glycyrrhizin concentrations like those investigated here interfered with H5N1-induced pro-inflammatory gene expression but not with H5N1 replication in human monocyte-derived macrophages [57] . In addition, other immunomodulatory treatment regimens that did not influence H5N1 replication reduced mortality in H5N1-infected mice [31, 58] . Therefore, glycyrrhizin represents a potential additional treatment option that interfers", "with both H5N1 replication and H5N1induced expression of pro-inflammatory cytokines in lung cells.", "Interference with immune responses may also result in the loss of control of virus replication by cytotoxic immune cells including natural killer cells and cytotoxic CD8 + T-lymphocytes. Global immunosuppressants like corticosteroids failed to protect from lethal influenza virus infection [59] . Moreover, antiviral drugs may interfere with cytotoxic cells that control virus replication as demonstrated for ribavirin that was shown to hamper NK cell cytolytic activity [60] . In this context, glycyrrhizin had already been shown not to affect natural killer cell activity in the concentrations used here [57] .", "In conclusion, we show in this report that therapeutic concentrations of glycyrrhizin (used as clinically approved parenteral preparation SNMC) interfere with highly pathogenic H5N1 influenza A virus replication and H5N1-induced proinflammatory gene expression at least in part through interference with H5N1-induced ROS formation and in turn reduced activation of p38, JNK, and NFkB in lung cells. Since we used the clinical formulation SNMC effects of other ingredients like glycin or cystein cannot be excluded. Vaccines and antiviral agents will fail to meet global needs at least at the beginning of a severe influenza A virus pandemic [61] . Anti-inflammatory and immunomodulatory agents are considered to be important candidates as constituents of anti-influenza treatment strategies that may save lives in an influenza pandemic situation [61] . Therefore, glycyrrhizin may complement the arsenal of potential drugs for the treatment of H5N1-caused disease." ]

[ 5 ]

[ "caspase-dependent apoptosis in airway cells" ]

[ "Glycyrrhizin Exerts Antioxidative Effects in H5N1 Influenza A Virus-Infected Cells and Inhibits Virus Replication and Pro-Inflammatory Gene Expression\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3096629/\n\nSHA: f3b7f4469ac01f1ce916d24172570c43c537627e\n\nAuthors: Michaelis, Martin; Geiler, Janina; Naczk, Patrizia; Sithisarn, Patchima; Leutz, Anke; Doerr, Hans Wilhelm; Cinatl, Jindrich\nDate: 2011-05-17\nDOI: 10.1371/journal.pone.0019705\nLicense: cc-by", "Abstract: Glycyrrhizin is known to exert antiviral and anti-inflammatory effects. Here, the effects of an approved parenteral glycyrrhizin preparation (Stronger Neo-Minophafen C) were investigated on highly pathogenic influenza A H5N1 virus replication, H5N1-induced apoptosis, and H5N1-induced pro-inflammatory responses in lung epithelial (A549) cells. Therapeutic glycyrrhizin concentrations substantially inhibited H5N1-induced expression of the pro-inflammatory molecules CXCL10, interleukin 6, CCL2, and CCL5 (effective glycyrrhizin concentrations 25 to 50 µg/ml) but interfered with H5N1 replication and H5N1-induced apoptosis to a lesser extent (effective glycyrrhizin concentrations 100 µg/ml or higher). Glycyrrhizin also diminished monocyte migration towards supernatants of H5N1-infected A549 cells. The mechanism by which glycyrrhizin interferes with H5N1 replication and H5N1-induced pro-inflammatory gene expression includes inhibition of H5N1-induced formation of reactive oxygen", "species and (in turn) reduced activation of NFκB, JNK, and p38, redox-sensitive signalling events known to be relevant for influenza A virus replication. Therefore, glycyrrhizin may complement the arsenal of potential drugs for the treatment of H5N1 disease.", "Text: Highly pathogenic H5N1 influenza A viruses are considered to be potential influenza pandemic progenitors [1] [2] [3] [4] [5] [6] . At least for the first wave of an H5N1 pandemic, no sufficient amounts of adequate vaccines will be available [1] [2] [3] [4] [6] [7] [8] . Therefore, antiviral therapy for influenza A viruses including highly pathogenic H5N1 virus strains remains of great importance for the first line defense against the virus [1] [2] [3] [4] 6, 9] .", "The neuraminidase inhibitors oseltamivir and zanamivir as well as the adamantanes amantadin and rimantadin that interfere with the influenza M2 protein are licensed for the treament of influenza [1] [2] [3] [4] 6] . However, the use of both drug classes is limited by the emergence of resistant virus strains. In seasonal influenza strains, the majority of H3N2 viruses and a great proportion of H1N1 viruses in humans are now considered to be amantadine-and rimantadine-resistant [10] [11] [12] [13] . Moreover, a drastic increase in oseltamivir-resistant H1N1 viruses has been reported during the 2007/2008 influenza season in the northern hemisphere [14] [15] [16] [17] . Preliminary data from the United States predict a further rise for the 2008/2009 season, possibly resulting in more than 90% of the circulating H1N1 strains to be oseltamivir resistant [14] .", "H5N1 virus strains appear to be generally less sensitive to antiviral treatment than seasonal influenza A virus strains and treatment-resistant H5N1 strains emerge [1] [2] [3] [4] 6, [18] [19] [20] [21] . More-over, parenteral agents for the treatment of seriously ill patients are missing. Glycyrrhizin, a triterpene saponine, is a constituent of licorice root. It has been found to interfere with replication and/or cytopathogenic effect (CPE) induction of many viruses including respiratory viruses such as respiratory syncytial virus, SARS coronavirus, HIV, and influenza viruses [22] [23] [24] [25] [26] [27] [28] . Moreover, antiinflammatory and immunomodulatory properties were attributed to glycyrrhizin [26] . The severity of human H5N1 disease has been associated with hypercytokinaemia (''cytokine storm'') [29, 30] . Delayed antiviral plus immunomodulator treatment reduced H5N1-induced mortality in mice [31] . Therefore, antiinflammatory and immunomodulatory effects exerted by", "glycyrrhizin may be beneficial for treatment of H5N1. Also, glycyrrhizin is a known antioxidant [26] and antioxidants were already shown to interfere with influenza A virus replication and virus-induced pro-inflammatory responses [32] [33] [34] .", "Stronger Neo-Minophagen C (SNMC) is a glycyrrhizin preparation (available as tablets or parenteral formulation) that is approved in Japan for the treatment of chronic hepatic diseases and is marketed in Japan, China, Korea, Taiwan, Indonesia, India, and Mongolia. Here, we investigated the influence of SNMC on H5N1 replication, on H5N1-induced cytokine expression, on H5N1-induced cellular oxidative stress, and on critical H5N1-induced cellular signalling events in human pneumocytes (A549 cell line).\n\nGlycyrrhizin (Stronger Neo Minophagen C) was obtained from Minophagen Pharmaceuticals Co., Ltd. (Tokyo, Japan).\n\nThe influenza strain A/Vietnam/1203/04 (H5N1) was received from the WHO Influenza Centre (National Institute for Medical Research, London, UK). The H5N1 influenza strain A/Thailand/ 1(Kan-1)/04 was obtained from Prof. Pilaipan Puthavathana (Mahidol University, Bangkok, Thailand).", "Virus stocks were prepared by infecting Vero cells (African green monkey kidney; ATCC, Manassas, VA) and aliquots were stored at 280uC. Virus titres were determined as 50% tissue culture infectious dose (TCID 50 /ml) in confluent Vero cells in 96-well microtiter plates.\n\nA549 cells (human lung carcinoma; ATCC: CCL-185, obtained from LGC Standards GmbH, Wesel, Germany) were grown at 37uC in minimal essential medium (MEM) supplemented with 10% FBS, 100 IU/ml of penicillin and 100 mg/ml streptomycin.", "Human monocytes were isolated from buffy coats of healthy donors, obtained from Institute of Transfusion Medicine and Immune Haematology, German Red Cross Blood Donor Center, Johann Wolfgang Goethe-University, Frankfurt am Main. After centrifugation on Ficoll (Biocoll)-Hypaque density gradient (Biochrom AG, Berlin, Germany), mononuclear cells were collected from the interface and washed with PBS. Then, monocytes were isolated using magnetically labeled CD14 MicroBeads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) following the manufacturer's instructions. Monocytes were cultivated in IMDM supplemented with 10% pooled human serum, 100 IU/ml of penicillin, and 100 mg/ml streptomycin.\n\nThe cellular viability was assessed on confluent cell layers with CellTiter-GloH Luminescent Cell Viability Assay (Promega GmbH, Mannheim, Germany) according to the manufacturers' protocol. Cell viability was expressed as percentage of non-treated control.", "To determine intracellular NP localisation, H5N1-infected A549 were fixed 8 hours p.i. for 15 min with ice-cold acetone/ methanol (40:60, Mallinckrodt Baker B.V., Deventer, The Netherlands) and stained with a mouse monoclonal antibody (1 h incubation, 1:1000 in PBS) directed against the influenza A virus nucleoprotein (NP) (Millipore, Molsheim, France). An Alexa Fluor 488 goat anti-mouse IgG (H&L) (Invitrogen, Eugene, Oregon, USA) was used (1 h incubation, 1:1000 in PBS) as secondary antibody. Nuclei were stained using 49,6-diamidino-2phenylindole (DAPI) (Sigma-Aldrich Chemie GmbH, Munich, Germany). Fluorescence was visualised using Olympus IX 1 fluorescence microscope (Olympus, Planegg, Germany).\n\nFor flow cytometric analysis, the same antibodies were used.", "The cytopathogenic effect (CPE) reduction assay was performed as described before [34] . Confluent A549 cell monolayers grown in 96-well microtitre plates were infected with influenza A strains at the indicated multiplicities of infection (MOIs). After a one hour adsorption period, cells were washed to remove non-detached virus. The virus-induced CPE was recorded at 24 h post infection (p.i.).\n\nUnless otherwise stated, A549 cells were continuously treated with glycyrrhizin starting with a 1 h pre-incubation period. For time-ofaddition experiments, glycyrrhizin was added exclusively during the 1 h pre-incubation period, exclusively during the 1 h adsorption period, or after exclusively after the wash-out of input virus.", "Total RNA was isolated from cell cultures using TRI reagent (Sigma-Aldrich, Munich, Germany). Real time PCR for H5 was performed using described methods [35] . The following primers were used: sense 59 acg tat gac tac ccg cag tat tca g 39; antisense 59 aga cca gcy acc atg att gc 39; probe 6-FAM-tca aca gtg gcg agt tcc cta gca-TAMRA.\n\nThe fraction of cells with fractional DNA content (''sub-G1'' cell subpopulation) indicates cytotoxicity. Sub-G1 cells are considered to be dead (usually apoptotic) cells. Cells were fixed with 70% ethanol for two hours at 220uC. The cellular DNA was stained using propidium iodide (20 mg/ml) and analysed by flow cytometry (FacsCalibur, BD Biosciences, Heidelberg, Germany).\n\nCaspase activation was measured using the Caspase-Glo 8, 9, or 3/7 Assays (Promega, Mannheim, Germany) following the manufacturer's instructions.", "Cell culture supernatants were collected and frozen at 280uC. Cytokines/chemokines were quantified by specific ELISA Duo Sets (R&D Systems GmbH, Wiesbaden, Germany) following the manufacturer's instructions.\n\nNFkB activity was investigated in H5N1 (MOI 0.01)-infected cells by quantification of the NFkB subunits Rel A (p65) and NFkB1 (p50) from nuclear extracts using the TransAM TM transcription factor DNA-binding ELISAs (Active Motif, Rixensart, Belgium). Nuclear extract were prepared using the Nuclear Extract Kit (Active Motif, Carlsbad, CA, USA) following the manufacturer's instruction.", "Cell culture supernatants were investigated for chemotactic activity by measurement of the activity to induce monocyte migration through membrane inserts in 24-well plates (pore size 8 mm; BD Biosciences, Heidelberg, Germany). Monocytes (1610 6 in 100 ml of IMDM with 10% pooled human serum) were added into the cell culture inserts (upper chamber) and cell culture supernatants (300 ml), were added to the lower chamber of the well. After a 48 h incubation period, cells were fixed with 4% paraformaldehyde and permeabilised with PBS containing 0.3% Tritron X-100. Then, nuclei were stained with 49,6-diamidino-2phenylindole (DAPI). The upper side of the membrane was wiped with a wet swab to remove the cells, while the lower side of the membrane was rinsed with PBS. The number of cells at the lower side of each membrane was quantified by counting of cells from three randomly chosen sections (3.7 mm 2 ) using an Olympus IX 1 fluorescence microscope (Olympus, Planegg, Germany).", "Cells were lysed in Triton X-sample buffer and separated by SDS-PAGE. Nuclear extract were prepared using the Nuclear Extract Kit (Active Motif, Carlsbad, CA, USA) following the manufacturer's instruction. Proteins were detected using specific antibodies against bactin (Sigma-Aldrich Chemie GmbH, Munich, Germany), JNK, phosphorylated JNK, p38, or phosphorylated p38, (all purchased from New England Biolabs GmbH, Frankfurt am Main, Germany) and were visualised by enhanced chemiluminescence using a commercially available kit (Amersham, Freiburg, Germany).\n\nReactive oxygen species (ROS) were detected using the Image-iT LIVE Green Reactive Oxygen Species Kit (Molecular Probes, distributed by Invitrogen, Karlsruhe, Germany).\n\nTwo groups were compared by t-test. More groups were compared by ANOVA with subsequent Student-Newman-Keuls test.", "The A549 cell line, derived from a human pulmonary adenocarcinoma, is an established model for type II pneumocytes [36] , and commonly used for the investigation of the effect of influenza viruses on this cell type [see e.g. 6,37,38]. If not otherwise stated, glycyrrhizin was continuously present in cell culture media starting with a 1 h preinfection period. Glycyrrhizin 200 mg/ml (the maximum tested concentration) did not affect A549 cell viability (data not shown) but clearly decreased CPE formation in A549 cells infected with the H5N1 influenza strain A/Thailand/1(Kan-1)/04 at MOIs of 0.01, 0.1 or 1 ( Figure 1A ). Similar results were obtained in A549 cells infected with strain A/Vietnam/1203/04 (H5N1) (Suppl. Figure 1A) . Staining of A549 cells for influenza A nucleoprotein 24 h after infection with strain H5N1 A/Thailand/1(Kan-1)/04 indicated that glycyrrhizin 200 mg/ml significantly reduces the number of influenza A nucleoprotein positive cells ( Figure 1B) .", "To examine the influence of glycyrrhizin on virus progeny, A549 cells were infected with the H5N1 influenza strain A/ Thailand/1(Kan-1)/04 at MOI 0.01 or MOI 1 and infectious virus titres were determined 24 h post infection ( Figure 1C ). While glycyrrhizin in concentrations up to 50 mg/ml did not affect H5N1 replication, moderate effects were exerted by glycyrrhizin 100 mg/ ml and more pronounced effects by glycyrrhizin 200 mg/ml (MOI 0.01: 13-fold reduction, MOI 1: 10-fold reduction). Next, influence of glycyrrhizin on H5N1 replication was confirmed by the detection of viral (H5) RNA using quantitative PCR. Only glycyrrhizin concentrations $100 mg/ml significantly reduced Figure 1B) or H5N1 A/Vietnam/1203/04-infected (Suppl. Figure 1C ) A549 cells (MOI 0.01) 24 h post infection.", "Time-of-addition experiments revealed that maximal effects were achieved when glycyrrhizin was continuously present starting with a 1 h pre-incubation period ( Figure 1D ). Addition of glycyrrhizin post infection showed reduced antiviral effects while pre-incubation alone or glycyrrhizin addition during the adsorption period did not significantly affect H5N1 replication.", "For investigation of H5N1-induced cytokine expression, five pro-inflammatory genes were chosen that had been correlated to severity of influenza disease: CXCL10 (also known as interferon-cinducible protein 10, IP-10), interleukin 6 (IL6), interleukin 8, (IL8; also known as CXCL8), CCL2 (also known as monocyte chemoattractant protein 1, MCP-1), and CCL5 (also known as RANTES). A549 cells were infected with H5N1 A/Thailand/ 1(Kan-1)/04 or H5N1 A/Vietnam/1203/04 at MOI 0.01, 0.1, or 1. Glycyrrhizin treatment was performed with 25, 50, 100, or 200 mg/ml. Cytokine expression was detected 24 h post infection by ELISA. Glycyrrhizin did not affect cytokine expression of noninfected cells (data not shown) but inhibited expression of all cytokines investigated in H5N1-infected cells in a dose-dependent manner (Figure 2, Figure 3A ). Effects were more pronounced at lower MOIs. Notably, expression of all cytokines except IL8 was significantly inhibited after treatment with glycyrrhizin 50 mg/ml", "Figure 3A ) although these glycyrrhizin concentrations had no effect on H5N1 replication in A549 cells (Figure 1, Figure S1 ).", "Cytokine expression by influenza A virus-infected respiratory cells causes recruitment of peripheral blood monocytes into the lungs of patients where they differentiate to macrophages which are thought to contribute to influenza A virus pathogenicity [5, 39] . In a chemotaxis assay, the influence of glycyrrhizin was investigated on migration of monocytes towards supernatants of H5N1 A/Thailand/1(Kan-1)/04 (MOI 0.1)-infected A549 cells through 8 mm filters. Monocyte migration towards supernatants of H5N1-infected cells was strongly increased relative to migration towards supernatants of non-infected cells. Treatment of H5N1- infected cells with glycyrrhizin 100 mg/ml clearly suppressed chemoattraction activity of supernatants ( Figure 3B ).", "Influenza viruses including H5N1 have been shown to induce caspase-dependent apoptosis in airway cells and this apoptosis has been correlated to the virus pathogenicity [40, 41] . Glycyrrhizin concentrations up to 200 mg/ml did not affect caspase activation in non-infected cells ( Figure 4A-C) . Glycyrrhizin concentrations $100 mg/ml inhibited H5N1 A/Thailand/1(Kan-1)/04 (MOI 0.01)-induced activation of the initiator caspases 8 and 9 as well as of the effector caspases 3/7 in A549 cells as determined 24 h post infection ( Figure 4A-C) . Lower glycyrrhizin concentrations did not affect H5N1-induced apoptosis. The detection of cells in sub-G1 phase resulted in similar findings ( Figure 4D ).", "Substances that inhibit H5N1-induced caspase 3 activation including caspase 3 inhibitors cause nuclear retention of RNP complexes [34, 42] . In accordance, glycyrrhizin also interfered with nuclear export RNP at MOI 1 ( Figure S2 ). Similar results were obtained in MOI 0.01 H5N1 A/Thailand/1(Kan-1)/04infected cells ( Figure S3 ).\n\nInfluence of glycyrrhizin on H5N1-induced activation of nuclear factor kB (NFkB), p38, and on H5N1-induced cellular reactive oxygen species (ROS) formation Activation of NFkB, p38, and JNK have been associated with influenza A virus replication and virus-induced pro-inflammatory gene expression [34, [43] [44] [45] [46] [47] . While glycyrrhizin did not influence NFkB activity in non-infected A549 cells in the tested concentra-tions (data not shown), glycyrrhizin inhibited NFkB activation in H5N1-infected cells ( Figure 5A ). Moreover, glycyrrhizin inhibited H5N1-induced phosphorylation of the MAPKs p38 and JNK ( Figure 5B ).", "In addition to their roles during influenza A virus replication and virus-induced cytokine/chemokine expression, NFkB, p38, and JNK are constituents of redox-sensitive signalling pathways [48] [49] [50] [51] . Antioxidants had been already found to interfere with influenza A virus-induced signalling through NFkB, p38, and JNK, with influenza A virus replication, and with influenza A virus-induced pro-inflammatory gene expression [32] [33] [34] . Since glycyrrhizin is known to exert antioxidative effects [26] we speculated that glycyrrhizin may interfere with H5N1-induced ROS formation. Indeed glycyrrhizin exerted clear antioxidative effects in H5N1 (MOI 0.01)-infected cells ( Figure 5C ) causing significant reduction of ROS formation already at a concentration of 25 mg/ml ( Figure 5D ).", "Here, we show that glycyrrhizin inhibits the replication of highly pathogenic H5N1 influenza A virus, H5N1-induced apoptosis, and H5N1-induced expression of pro-inflammatory cytokines in lung-derived A549 cells. After intravenous administration, achievable plasma concentrations of glycyrrhizin have been described to be about 100 mg/ml [52] . Therefore, the glycyrrhizin concentrations found to interfere with H5N1 replication and H5N1-induced pro-inflammatory gene expression in the present report are in the range of therapeutic plasma levels. Notably, although higher glycyrrhizin concentrations were needed to interfere with SARS coronavirus replication [22] than with H5N1 replication, beneficial results were reported in glycyrrhizin (SNMC)-treated SARS patients in comparison to SARS patients who did not receive glycyrrhizin [23] . Notably, investigation of different glycyrrhizin derivatives against SARS coronavirus led to the identification of compounds with enhanced antiviral activity", "[53] . Therefore, glycyrrhizin might also serve as lead structure for the development of novel anti-influenza drugs.", "Experimental results suggested that glycyrrhizin might be able to affect seasonal influenza A virus disease by antiviral and immunomodulatory effects [26, 27] . Mice were prevented from lethal H2N2 infection by glycyrrhizin although no influence on virus replication was detected. The mechanism was suggested to be induction of interferon-c in T-cells by glycyrrhizin [54] . Moreover, glycyrrhizin was shown to influence seasonal influenza A virus replication through interaction with the cell membrane [25, 28] . However, these effects were observed only in concentrations $200 mg/ml when glycyrrhizin was added during the virus adsorption period. Since glycyrrhizin addition during the adsorption period did not influence H5N1 replication in our experiments it appears not likely that membrane effects contribute to anti-H5N1 effects detected here in lower concentrations.", "Our results rather suggest that glycyrrhizin interferes with H5N1-induced oxidative stress. Influenza A virus (including H5N1) infection induces ROS formation. Antioxidants were found to inhibit influenza A virus replication and influenza A virus-induced pro-inflammatory gene expression [32] [33] [34] and glycyrrhizin is known to exert antioxidative effects [26] . Here, glycyrrhizin interfered with H5N1-induced activation of NFkB, p38, and JNK representing redox-sensitive signalling events [48] [49] [50] [51] involved in influenza A virus replication and influenza A virusinduced cellular cytokine/chemokine production [34, [43] [44] [45] [46] 55] . Glycyrrhizin 50 mg/ml significantly reduced H5N1-induced activation of NFkB. In addition, glycyrrhizin concentrations as low as 25 mg/ml effectively interfered with H5N1-induced ROS formation and with phosphorylation of the redox-sensitive MAPKs p38 and JNK. In our model, activation of p38 appears to be critical for H5N1-associated redox", "signalling since p38 inhibition had been shown before to mimick effects of the antioxidant N-acetyl-cysteine (NAC) [34] . Interestingly and in contrast to glycyrrhizin, NAC failed to inhibit H5N1 replication or H5N1-induced cytokine/chemokine expression in therapeutically relevant concentrations.", "Glycyrrhizin diminished H5N1-induced cellular cytokine/ chemokine production in concentrations (#50 mg/ml) that did not interfere with H5N1 replication although redox-sensitive signalling pathways have been described to be involved in both processes. Therefore, H5N1-induced proinflammatory gene expression appears to be more sensitive to inhibition of ROS formation than H5N1 replication. Indeed, influenza viruses had been shown to induce cellular pathways through replicationdependent and -independent events [56] . In a previous report, we could show that similar glycyrrhizin concentrations like those investigated here interfered with H5N1-induced pro-inflammatory gene expression but not with H5N1 replication in human monocyte-derived macrophages [57] . In addition, other immunomodulatory treatment regimens that did not influence H5N1 replication reduced mortality in H5N1-infected mice [31, 58] . Therefore, glycyrrhizin represents a potential additional treatment option that interfers", "with both H5N1 replication and H5N1induced expression of pro-inflammatory cytokines in lung cells.", "Interference with immune responses may also result in the loss of control of virus replication by cytotoxic immune cells including natural killer cells and cytotoxic CD8 + T-lymphocytes. Global immunosuppressants like corticosteroids failed to protect from lethal influenza virus infection [59] . Moreover, antiviral drugs may interfere with cytotoxic cells that control virus replication as demonstrated for ribavirin that was shown to hamper NK cell cytolytic activity [60] . In this context, glycyrrhizin had already been shown not to affect natural killer cell activity in the concentrations used here [57] .", "In conclusion, we show in this report that therapeutic concentrations of glycyrrhizin (used as clinically approved parenteral preparation SNMC) interfere with highly pathogenic H5N1 influenza A virus replication and H5N1-induced proinflammatory gene expression at least in part through interference with H5N1-induced ROS formation and in turn reduced activation of p38, JNK, and NFkB in lung cells. Since we used the clinical formulation SNMC effects of other ingredients like glycin or cystein cannot be excluded. Vaccines and antiviral agents will fail to meet global needs at least at the beginning of a severe influenza A virus pandemic [61] . Anti-inflammatory and immunomodulatory agents are considered to be important candidates as constituents of anti-influenza treatment strategies that may save lives in an influenza pandemic situation [61] . Therefore, glycyrrhizin may complement the arsenal of potential drugs for the treatment of H5N1-caused disease." ]

[ 22 ]

[ "glycyrrhizin might also serve as lead structure for the development of novel anti-influenza drugs" ]

[ "Glycyrrhizin Exerts Antioxidative Effects in H5N1 Influenza A Virus-Infected Cells and Inhibits Virus Replication and Pro-Inflammatory Gene Expression\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3096629/\n\nSHA: f3b7f4469ac01f1ce916d24172570c43c537627e\n\nAuthors: Michaelis, Martin; Geiler, Janina; Naczk, Patrizia; Sithisarn, Patchima; Leutz, Anke; Doerr, Hans Wilhelm; Cinatl, Jindrich\nDate: 2011-05-17\nDOI: 10.1371/journal.pone.0019705\nLicense: cc-by", "Abstract: Glycyrrhizin is known to exert antiviral and anti-inflammatory effects. Here, the effects of an approved parenteral glycyrrhizin preparation (Stronger Neo-Minophafen C) were investigated on highly pathogenic influenza A H5N1 virus replication, H5N1-induced apoptosis, and H5N1-induced pro-inflammatory responses in lung epithelial (A549) cells. Therapeutic glycyrrhizin concentrations substantially inhibited H5N1-induced expression of the pro-inflammatory molecules CXCL10, interleukin 6, CCL2, and CCL5 (effective glycyrrhizin concentrations 25 to 50 µg/ml) but interfered with H5N1 replication and H5N1-induced apoptosis to a lesser extent (effective glycyrrhizin concentrations 100 µg/ml or higher). Glycyrrhizin also diminished monocyte migration towards supernatants of H5N1-infected A549 cells. The mechanism by which glycyrrhizin interferes with H5N1 replication and H5N1-induced pro-inflammatory gene expression includes inhibition of H5N1-induced formation of reactive oxygen", "species and (in turn) reduced activation of NFκB, JNK, and p38, redox-sensitive signalling events known to be relevant for influenza A virus replication. Therefore, glycyrrhizin may complement the arsenal of potential drugs for the treatment of H5N1 disease.", "Text: Highly pathogenic H5N1 influenza A viruses are considered to be potential influenza pandemic progenitors [1] [2] [3] [4] [5] [6] . At least for the first wave of an H5N1 pandemic, no sufficient amounts of adequate vaccines will be available [1] [2] [3] [4] [6] [7] [8] . Therefore, antiviral therapy for influenza A viruses including highly pathogenic H5N1 virus strains remains of great importance for the first line defense against the virus [1] [2] [3] [4] 6, 9] .", "The neuraminidase inhibitors oseltamivir and zanamivir as well as the adamantanes amantadin and rimantadin that interfere with the influenza M2 protein are licensed for the treament of influenza [1] [2] [3] [4] 6] . However, the use of both drug classes is limited by the emergence of resistant virus strains. In seasonal influenza strains, the majority of H3N2 viruses and a great proportion of H1N1 viruses in humans are now considered to be amantadine-and rimantadine-resistant [10] [11] [12] [13] . Moreover, a drastic increase in oseltamivir-resistant H1N1 viruses has been reported during the 2007/2008 influenza season in the northern hemisphere [14] [15] [16] [17] . Preliminary data from the United States predict a further rise for the 2008/2009 season, possibly resulting in more than 90% of the circulating H1N1 strains to be oseltamivir resistant [14] .", "H5N1 virus strains appear to be generally less sensitive to antiviral treatment than seasonal influenza A virus strains and treatment-resistant H5N1 strains emerge [1] [2] [3] [4] 6, [18] [19] [20] [21] . More-over, parenteral agents for the treatment of seriously ill patients are missing. Glycyrrhizin, a triterpene saponine, is a constituent of licorice root. It has been found to interfere with replication and/or cytopathogenic effect (CPE) induction of many viruses including respiratory viruses such as respiratory syncytial virus, SARS coronavirus, HIV, and influenza viruses [22] [23] [24] [25] [26] [27] [28] . Moreover, antiinflammatory and immunomodulatory properties were attributed to glycyrrhizin [26] . The severity of human H5N1 disease has been associated with hypercytokinaemia (''cytokine storm'') [29, 30] . Delayed antiviral plus immunomodulator treatment reduced H5N1-induced mortality in mice [31] . Therefore, antiinflammatory and immunomodulatory effects exerted by", "glycyrrhizin may be beneficial for treatment of H5N1. Also, glycyrrhizin is a known antioxidant [26] and antioxidants were already shown to interfere with influenza A virus replication and virus-induced pro-inflammatory responses [32] [33] [34] .", "Stronger Neo-Minophagen C (SNMC) is a glycyrrhizin preparation (available as tablets or parenteral formulation) that is approved in Japan for the treatment of chronic hepatic diseases and is marketed in Japan, China, Korea, Taiwan, Indonesia, India, and Mongolia. Here, we investigated the influence of SNMC on H5N1 replication, on H5N1-induced cytokine expression, on H5N1-induced cellular oxidative stress, and on critical H5N1-induced cellular signalling events in human pneumocytes (A549 cell line).\n\nGlycyrrhizin (Stronger Neo Minophagen C) was obtained from Minophagen Pharmaceuticals Co., Ltd. (Tokyo, Japan).\n\nThe influenza strain A/Vietnam/1203/04 (H5N1) was received from the WHO Influenza Centre (National Institute for Medical Research, London, UK). The H5N1 influenza strain A/Thailand/ 1(Kan-1)/04 was obtained from Prof. Pilaipan Puthavathana (Mahidol University, Bangkok, Thailand).", "Virus stocks were prepared by infecting Vero cells (African green monkey kidney; ATCC, Manassas, VA) and aliquots were stored at 280uC. Virus titres were determined as 50% tissue culture infectious dose (TCID 50 /ml) in confluent Vero cells in 96-well microtiter plates.\n\nA549 cells (human lung carcinoma; ATCC: CCL-185, obtained from LGC Standards GmbH, Wesel, Germany) were grown at 37uC in minimal essential medium (MEM) supplemented with 10% FBS, 100 IU/ml of penicillin and 100 mg/ml streptomycin.", "Human monocytes were isolated from buffy coats of healthy donors, obtained from Institute of Transfusion Medicine and Immune Haematology, German Red Cross Blood Donor Center, Johann Wolfgang Goethe-University, Frankfurt am Main. After centrifugation on Ficoll (Biocoll)-Hypaque density gradient (Biochrom AG, Berlin, Germany), mononuclear cells were collected from the interface and washed with PBS. Then, monocytes were isolated using magnetically labeled CD14 MicroBeads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) following the manufacturer's instructions. Monocytes were cultivated in IMDM supplemented with 10% pooled human serum, 100 IU/ml of penicillin, and 100 mg/ml streptomycin.\n\nThe cellular viability was assessed on confluent cell layers with CellTiter-GloH Luminescent Cell Viability Assay (Promega GmbH, Mannheim, Germany) according to the manufacturers' protocol. Cell viability was expressed as percentage of non-treated control.", "To determine intracellular NP localisation, H5N1-infected A549 were fixed 8 hours p.i. for 15 min with ice-cold acetone/ methanol (40:60, Mallinckrodt Baker B.V., Deventer, The Netherlands) and stained with a mouse monoclonal antibody (1 h incubation, 1:1000 in PBS) directed against the influenza A virus nucleoprotein (NP) (Millipore, Molsheim, France). An Alexa Fluor 488 goat anti-mouse IgG (H&L) (Invitrogen, Eugene, Oregon, USA) was used (1 h incubation, 1:1000 in PBS) as secondary antibody. Nuclei were stained using 49,6-diamidino-2phenylindole (DAPI) (Sigma-Aldrich Chemie GmbH, Munich, Germany). Fluorescence was visualised using Olympus IX 1 fluorescence microscope (Olympus, Planegg, Germany).\n\nFor flow cytometric analysis, the same antibodies were used.", "The cytopathogenic effect (CPE) reduction assay was performed as described before [34] . Confluent A549 cell monolayers grown in 96-well microtitre plates were infected with influenza A strains at the indicated multiplicities of infection (MOIs). After a one hour adsorption period, cells were washed to remove non-detached virus. The virus-induced CPE was recorded at 24 h post infection (p.i.).\n\nUnless otherwise stated, A549 cells were continuously treated with glycyrrhizin starting with a 1 h pre-incubation period. For time-ofaddition experiments, glycyrrhizin was added exclusively during the 1 h pre-incubation period, exclusively during the 1 h adsorption period, or after exclusively after the wash-out of input virus.", "Total RNA was isolated from cell cultures using TRI reagent (Sigma-Aldrich, Munich, Germany). Real time PCR for H5 was performed using described methods [35] . The following primers were used: sense 59 acg tat gac tac ccg cag tat tca g 39; antisense 59 aga cca gcy acc atg att gc 39; probe 6-FAM-tca aca gtg gcg agt tcc cta gca-TAMRA.\n\nThe fraction of cells with fractional DNA content (''sub-G1'' cell subpopulation) indicates cytotoxicity. Sub-G1 cells are considered to be dead (usually apoptotic) cells. Cells were fixed with 70% ethanol for two hours at 220uC. The cellular DNA was stained using propidium iodide (20 mg/ml) and analysed by flow cytometry (FacsCalibur, BD Biosciences, Heidelberg, Germany).\n\nCaspase activation was measured using the Caspase-Glo 8, 9, or 3/7 Assays (Promega, Mannheim, Germany) following the manufacturer's instructions.", "Cell culture supernatants were collected and frozen at 280uC. Cytokines/chemokines were quantified by specific ELISA Duo Sets (R&D Systems GmbH, Wiesbaden, Germany) following the manufacturer's instructions.\n\nNFkB activity was investigated in H5N1 (MOI 0.01)-infected cells by quantification of the NFkB subunits Rel A (p65) and NFkB1 (p50) from nuclear extracts using the TransAM TM transcription factor DNA-binding ELISAs (Active Motif, Rixensart, Belgium). Nuclear extract were prepared using the Nuclear Extract Kit (Active Motif, Carlsbad, CA, USA) following the manufacturer's instruction.", "Cell culture supernatants were investigated for chemotactic activity by measurement of the activity to induce monocyte migration through membrane inserts in 24-well plates (pore size 8 mm; BD Biosciences, Heidelberg, Germany). Monocytes (1610 6 in 100 ml of IMDM with 10% pooled human serum) were added into the cell culture inserts (upper chamber) and cell culture supernatants (300 ml), were added to the lower chamber of the well. After a 48 h incubation period, cells were fixed with 4% paraformaldehyde and permeabilised with PBS containing 0.3% Tritron X-100. Then, nuclei were stained with 49,6-diamidino-2phenylindole (DAPI). The upper side of the membrane was wiped with a wet swab to remove the cells, while the lower side of the membrane was rinsed with PBS. The number of cells at the lower side of each membrane was quantified by counting of cells from three randomly chosen sections (3.7 mm 2 ) using an Olympus IX 1 fluorescence microscope (Olympus, Planegg, Germany).", "Cells were lysed in Triton X-sample buffer and separated by SDS-PAGE. Nuclear extract were prepared using the Nuclear Extract Kit (Active Motif, Carlsbad, CA, USA) following the manufacturer's instruction. Proteins were detected using specific antibodies against bactin (Sigma-Aldrich Chemie GmbH, Munich, Germany), JNK, phosphorylated JNK, p38, or phosphorylated p38, (all purchased from New England Biolabs GmbH, Frankfurt am Main, Germany) and were visualised by enhanced chemiluminescence using a commercially available kit (Amersham, Freiburg, Germany).\n\nReactive oxygen species (ROS) were detected using the Image-iT LIVE Green Reactive Oxygen Species Kit (Molecular Probes, distributed by Invitrogen, Karlsruhe, Germany).\n\nTwo groups were compared by t-test. More groups were compared by ANOVA with subsequent Student-Newman-Keuls test.", "The A549 cell line, derived from a human pulmonary adenocarcinoma, is an established model for type II pneumocytes [36] , and commonly used for the investigation of the effect of influenza viruses on this cell type [see e.g. 6,37,38]. If not otherwise stated, glycyrrhizin was continuously present in cell culture media starting with a 1 h preinfection period. Glycyrrhizin 200 mg/ml (the maximum tested concentration) did not affect A549 cell viability (data not shown) but clearly decreased CPE formation in A549 cells infected with the H5N1 influenza strain A/Thailand/1(Kan-1)/04 at MOIs of 0.01, 0.1 or 1 ( Figure 1A ). Similar results were obtained in A549 cells infected with strain A/Vietnam/1203/04 (H5N1) (Suppl. Figure 1A) . Staining of A549 cells for influenza A nucleoprotein 24 h after infection with strain H5N1 A/Thailand/1(Kan-1)/04 indicated that glycyrrhizin 200 mg/ml significantly reduces the number of influenza A nucleoprotein positive cells ( Figure 1B) .", "To examine the influence of glycyrrhizin on virus progeny, A549 cells were infected with the H5N1 influenza strain A/ Thailand/1(Kan-1)/04 at MOI 0.01 or MOI 1 and infectious virus titres were determined 24 h post infection ( Figure 1C ). While glycyrrhizin in concentrations up to 50 mg/ml did not affect H5N1 replication, moderate effects were exerted by glycyrrhizin 100 mg/ ml and more pronounced effects by glycyrrhizin 200 mg/ml (MOI 0.01: 13-fold reduction, MOI 1: 10-fold reduction). Next, influence of glycyrrhizin on H5N1 replication was confirmed by the detection of viral (H5) RNA using quantitative PCR. Only glycyrrhizin concentrations $100 mg/ml significantly reduced Figure 1B) or H5N1 A/Vietnam/1203/04-infected (Suppl. Figure 1C ) A549 cells (MOI 0.01) 24 h post infection.", "Time-of-addition experiments revealed that maximal effects were achieved when glycyrrhizin was continuously present starting with a 1 h pre-incubation period ( Figure 1D ). Addition of glycyrrhizin post infection showed reduced antiviral effects while pre-incubation alone or glycyrrhizin addition during the adsorption period did not significantly affect H5N1 replication.", "For investigation of H5N1-induced cytokine expression, five pro-inflammatory genes were chosen that had been correlated to severity of influenza disease: CXCL10 (also known as interferon-cinducible protein 10, IP-10), interleukin 6 (IL6), interleukin 8, (IL8; also known as CXCL8), CCL2 (also known as monocyte chemoattractant protein 1, MCP-1), and CCL5 (also known as RANTES). A549 cells were infected with H5N1 A/Thailand/ 1(Kan-1)/04 or H5N1 A/Vietnam/1203/04 at MOI 0.01, 0.1, or 1. Glycyrrhizin treatment was performed with 25, 50, 100, or 200 mg/ml. Cytokine expression was detected 24 h post infection by ELISA. Glycyrrhizin did not affect cytokine expression of noninfected cells (data not shown) but inhibited expression of all cytokines investigated in H5N1-infected cells in a dose-dependent manner (Figure 2, Figure 3A ). Effects were more pronounced at lower MOIs. Notably, expression of all cytokines except IL8 was significantly inhibited after treatment with glycyrrhizin 50 mg/ml", "Figure 3A ) although these glycyrrhizin concentrations had no effect on H5N1 replication in A549 cells (Figure 1, Figure S1 ).", "Cytokine expression by influenza A virus-infected respiratory cells causes recruitment of peripheral blood monocytes into the lungs of patients where they differentiate to macrophages which are thought to contribute to influenza A virus pathogenicity [5, 39] . In a chemotaxis assay, the influence of glycyrrhizin was investigated on migration of monocytes towards supernatants of H5N1 A/Thailand/1(Kan-1)/04 (MOI 0.1)-infected A549 cells through 8 mm filters. Monocyte migration towards supernatants of H5N1-infected cells was strongly increased relative to migration towards supernatants of non-infected cells. Treatment of H5N1- infected cells with glycyrrhizin 100 mg/ml clearly suppressed chemoattraction activity of supernatants ( Figure 3B ).", "Influenza viruses including H5N1 have been shown to induce caspase-dependent apoptosis in airway cells and this apoptosis has been correlated to the virus pathogenicity [40, 41] . Glycyrrhizin concentrations up to 200 mg/ml did not affect caspase activation in non-infected cells ( Figure 4A-C) . Glycyrrhizin concentrations $100 mg/ml inhibited H5N1 A/Thailand/1(Kan-1)/04 (MOI 0.01)-induced activation of the initiator caspases 8 and 9 as well as of the effector caspases 3/7 in A549 cells as determined 24 h post infection ( Figure 4A-C) . Lower glycyrrhizin concentrations did not affect H5N1-induced apoptosis. The detection of cells in sub-G1 phase resulted in similar findings ( Figure 4D ).", "Substances that inhibit H5N1-induced caspase 3 activation including caspase 3 inhibitors cause nuclear retention of RNP complexes [34, 42] . In accordance, glycyrrhizin also interfered with nuclear export RNP at MOI 1 ( Figure S2 ). Similar results were obtained in MOI 0.01 H5N1 A/Thailand/1(Kan-1)/04infected cells ( Figure S3 ).\n\nInfluence of glycyrrhizin on H5N1-induced activation of nuclear factor kB (NFkB), p38, and on H5N1-induced cellular reactive oxygen species (ROS) formation Activation of NFkB, p38, and JNK have been associated with influenza A virus replication and virus-induced pro-inflammatory gene expression [34, [43] [44] [45] [46] [47] . While glycyrrhizin did not influence NFkB activity in non-infected A549 cells in the tested concentra-tions (data not shown), glycyrrhizin inhibited NFkB activation in H5N1-infected cells ( Figure 5A ). Moreover, glycyrrhizin inhibited H5N1-induced phosphorylation of the MAPKs p38 and JNK ( Figure 5B ).", "In addition to their roles during influenza A virus replication and virus-induced cytokine/chemokine expression, NFkB, p38, and JNK are constituents of redox-sensitive signalling pathways [48] [49] [50] [51] . Antioxidants had been already found to interfere with influenza A virus-induced signalling through NFkB, p38, and JNK, with influenza A virus replication, and with influenza A virus-induced pro-inflammatory gene expression [32] [33] [34] . Since glycyrrhizin is known to exert antioxidative effects [26] we speculated that glycyrrhizin may interfere with H5N1-induced ROS formation. Indeed glycyrrhizin exerted clear antioxidative effects in H5N1 (MOI 0.01)-infected cells ( Figure 5C ) causing significant reduction of ROS formation already at a concentration of 25 mg/ml ( Figure 5D ).", "Here, we show that glycyrrhizin inhibits the replication of highly pathogenic H5N1 influenza A virus, H5N1-induced apoptosis, and H5N1-induced expression of pro-inflammatory cytokines in lung-derived A549 cells. After intravenous administration, achievable plasma concentrations of glycyrrhizin have been described to be about 100 mg/ml [52] . Therefore, the glycyrrhizin concentrations found to interfere with H5N1 replication and H5N1-induced pro-inflammatory gene expression in the present report are in the range of therapeutic plasma levels. Notably, although higher glycyrrhizin concentrations were needed to interfere with SARS coronavirus replication [22] than with H5N1 replication, beneficial results were reported in glycyrrhizin (SNMC)-treated SARS patients in comparison to SARS patients who did not receive glycyrrhizin [23] . Notably, investigation of different glycyrrhizin derivatives against SARS coronavirus led to the identification of compounds with enhanced antiviral activity", "[53] . Therefore, glycyrrhizin might also serve as lead structure for the development of novel anti-influenza drugs.", "Experimental results suggested that glycyrrhizin might be able to affect seasonal influenza A virus disease by antiviral and immunomodulatory effects [26, 27] . Mice were prevented from lethal H2N2 infection by glycyrrhizin although no influence on virus replication was detected. The mechanism was suggested to be induction of interferon-c in T-cells by glycyrrhizin [54] . Moreover, glycyrrhizin was shown to influence seasonal influenza A virus replication through interaction with the cell membrane [25, 28] . However, these effects were observed only in concentrations $200 mg/ml when glycyrrhizin was added during the virus adsorption period. Since glycyrrhizin addition during the adsorption period did not influence H5N1 replication in our experiments it appears not likely that membrane effects contribute to anti-H5N1 effects detected here in lower concentrations.", "Our results rather suggest that glycyrrhizin interferes with H5N1-induced oxidative stress. Influenza A virus (including H5N1) infection induces ROS formation. Antioxidants were found to inhibit influenza A virus replication and influenza A virus-induced pro-inflammatory gene expression [32] [33] [34] and glycyrrhizin is known to exert antioxidative effects [26] . Here, glycyrrhizin interfered with H5N1-induced activation of NFkB, p38, and JNK representing redox-sensitive signalling events [48] [49] [50] [51] involved in influenza A virus replication and influenza A virusinduced cellular cytokine/chemokine production [34, [43] [44] [45] [46] 55] . Glycyrrhizin 50 mg/ml significantly reduced H5N1-induced activation of NFkB. In addition, glycyrrhizin concentrations as low as 25 mg/ml effectively interfered with H5N1-induced ROS formation and with phosphorylation of the redox-sensitive MAPKs p38 and JNK. In our model, activation of p38 appears to be critical for H5N1-associated redox", "signalling since p38 inhibition had been shown before to mimick effects of the antioxidant N-acetyl-cysteine (NAC) [34] . Interestingly and in contrast to glycyrrhizin, NAC failed to inhibit H5N1 replication or H5N1-induced cytokine/chemokine expression in therapeutically relevant concentrations.", "Glycyrrhizin diminished H5N1-induced cellular cytokine/ chemokine production in concentrations (#50 mg/ml) that did not interfere with H5N1 replication although redox-sensitive signalling pathways have been described to be involved in both processes. Therefore, H5N1-induced proinflammatory gene expression appears to be more sensitive to inhibition of ROS formation than H5N1 replication. Indeed, influenza viruses had been shown to induce cellular pathways through replicationdependent and -independent events [56] . In a previous report, we could show that similar glycyrrhizin concentrations like those investigated here interfered with H5N1-induced pro-inflammatory gene expression but not with H5N1 replication in human monocyte-derived macrophages [57] . In addition, other immunomodulatory treatment regimens that did not influence H5N1 replication reduced mortality in H5N1-infected mice [31, 58] . Therefore, glycyrrhizin represents a potential additional treatment option that interfers", "with both H5N1 replication and H5N1induced expression of pro-inflammatory cytokines in lung cells.", "Interference with immune responses may also result in the loss of control of virus replication by cytotoxic immune cells including natural killer cells and cytotoxic CD8 + T-lymphocytes. Global immunosuppressants like corticosteroids failed to protect from lethal influenza virus infection [59] . Moreover, antiviral drugs may interfere with cytotoxic cells that control virus replication as demonstrated for ribavirin that was shown to hamper NK cell cytolytic activity [60] . In this context, glycyrrhizin had already been shown not to affect natural killer cell activity in the concentrations used here [57] .", "In conclusion, we show in this report that therapeutic concentrations of glycyrrhizin (used as clinically approved parenteral preparation SNMC) interfere with highly pathogenic H5N1 influenza A virus replication and H5N1-induced proinflammatory gene expression at least in part through interference with H5N1-induced ROS formation and in turn reduced activation of p38, JNK, and NFkB in lung cells. Since we used the clinical formulation SNMC effects of other ingredients like glycin or cystein cannot be excluded. Vaccines and antiviral agents will fail to meet global needs at least at the beginning of a severe influenza A virus pandemic [61] . Anti-inflammatory and immunomodulatory agents are considered to be important candidates as constituents of anti-influenza treatment strategies that may save lives in an influenza pandemic situation [61] . Therefore, glycyrrhizin may complement the arsenal of potential drugs for the treatment of H5N1-caused disease." ]

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[ "Emergent severe acute respiratory distress syndrome caused by adenovirus type 55 in immunocompetent adults in 2013: a prospective observational study\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4243941/\n\nSHA: f5b706d0529bfcf7e2d1dfc037df5b6f95fc5ec0\n\nAuthors: Sun, Bing; He, Hangyong; Wang, Zheng; Qu, Jiuxin; Li, Xuyan; Ban, Chengjun; Wan, Jun; Cao, Bin; Tong, Zhaohui; Wang, Chen\nDate: 2014-08-12\nDOI: 10.1186/s13054-014-0456-6\nLicense: cc-by", "Abstract: INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 (HAdV-55) in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome (ARDS) caused by HAdV-55 requiring invasive mechanical ventilation (IMV) and/or extracorporeal membrane oxygenation (ECMO) are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was", "5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10(8) copies in three patients and was 1 × 10(4) in one patient. It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation (NPPV) failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four (80%) of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most", "frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012", "Text: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients [1] [2] [3] [4] [5] . Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 [7] and in Singapore in 2005 [8] . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] . This pathogen was fully characterized by whole-genome sequencing [10] . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the", "serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes (HAdV-3, HAdV-7 and HAdV-14) [6] .", "Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU.", "Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation (NPPV) in ARDS patients was carried out in our center (ClinicalTrials.gov ID: NCT01585922). From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: (1) developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; (2) bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; (3) respiratory failure not fully explained by cardiac failure or fluid overload; (4) partial oxygen pressure/ fraction of inspired oxygen (PaO 2 /FiO 2 ) ≤100 mmHg with positive end-expiratory pressure (PEEP) ≥5 cmH 2 O; and (5) a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive", "mechanical ventilation (IMV) were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital (LLKYPJ2012031). Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication.", "Clinical information collected by investigators with a standardized data form included the following: demographic characteristics (age and sex), comorbidities, clinical symptoms (fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache), signs (body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs), laboratory tests (whole-blood cell count and blood chemistry) and microbiological findings and images of the lung (chest X-ray (CXR) and computed tomography). Concomitant medications, respiratory support, complications and outcomes were also recorded.", "Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay.\n\nViral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods.", "Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range).\n\nDuring the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs.", "All five patients were immunocompetent young men with a median age of 32 years (range, 28 to 40 years). All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay (Oxford Immunotec, Marlborough, MA, USA) and antibody of Mycobacterium tuberculosis, were negative.", "Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C (range, 39.0°C to 40.0°C), which persisted for 8 days (range, 6 to 11 days). Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days (range, 1 to 10 days). After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days (range, 8 to 11 days) ( Table 1) . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52). Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 (range = 49 to 62.5). White blood cell counts were low or in the normal range. All patients had elevated serum", "aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and hydroxybutyrate dehydrogenase (HBDH) ( Table 1) . At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range.", "Four patients had lower than normal T-cell subset counts (Table 2) .\n\nCXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ). Three patients were examined by highresolution computed tomography (HRCT). Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ). The mean duration from onset to a single-lobe consolidation on CXRs was 2 days (range = 1 to 5 days). The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days).", "All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days (Table 3) . Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy (Figure 3 ).", "Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours (range = 22 to 48 hours), and the mean time until IMV failure was 6.2 days (range 2 = to 13 days) ( Table 1) . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support.", "All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip). Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient (Table 3) . Four (80%) of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively.", "To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. (1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. (2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. (3) Viral load monitoring may help predict disease severity and patient outcome. (4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. (5) HAdV-55-induced severe ARDS", "has a very high mortality rate (80%) despite appropriate respiratory support.", "Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] . HAdV-55 was first completely characterized in Shaanxi, China [7] and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease [9] . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS.", "The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region.\n\nOur study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports [6, 9] .", "Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the", "reaction to therapy and patient outcome.", "The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation.\n\nOur study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response." ]

[ 2 ]

[ "4.8 days" ]

[ "Emergent severe acute respiratory distress syndrome caused by adenovirus type 55 in immunocompetent adults in 2013: a prospective observational study\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4243941/\n\nSHA: f5b706d0529bfcf7e2d1dfc037df5b6f95fc5ec0\n\nAuthors: Sun, Bing; He, Hangyong; Wang, Zheng; Qu, Jiuxin; Li, Xuyan; Ban, Chengjun; Wan, Jun; Cao, Bin; Tong, Zhaohui; Wang, Chen\nDate: 2014-08-12\nDOI: 10.1186/s13054-014-0456-6\nLicense: cc-by", "Abstract: INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 (HAdV-55) in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome (ARDS) caused by HAdV-55 requiring invasive mechanical ventilation (IMV) and/or extracorporeal membrane oxygenation (ECMO) are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was", "5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10(8) copies in three patients and was 1 × 10(4) in one patient. It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation (NPPV) failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four (80%) of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most", "frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012", "Text: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients [1] [2] [3] [4] [5] . Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 [7] and in Singapore in 2005 [8] . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] . This pathogen was fully characterized by whole-genome sequencing [10] . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the", "serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes (HAdV-3, HAdV-7 and HAdV-14) [6] .", "Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU.", "Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation (NPPV) in ARDS patients was carried out in our center (ClinicalTrials.gov ID: NCT01585922). From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: (1) developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; (2) bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; (3) respiratory failure not fully explained by cardiac failure or fluid overload; (4) partial oxygen pressure/ fraction of inspired oxygen (PaO 2 /FiO 2 ) ≤100 mmHg with positive end-expiratory pressure (PEEP) ≥5 cmH 2 O; and (5) a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive", "mechanical ventilation (IMV) were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital (LLKYPJ2012031). Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication.", "Clinical information collected by investigators with a standardized data form included the following: demographic characteristics (age and sex), comorbidities, clinical symptoms (fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache), signs (body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs), laboratory tests (whole-blood cell count and blood chemistry) and microbiological findings and images of the lung (chest X-ray (CXR) and computed tomography). Concomitant medications, respiratory support, complications and outcomes were also recorded.", "Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay.\n\nViral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods.", "Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range).\n\nDuring the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs.", "All five patients were immunocompetent young men with a median age of 32 years (range, 28 to 40 years). All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay (Oxford Immunotec, Marlborough, MA, USA) and antibody of Mycobacterium tuberculosis, were negative.", "Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C (range, 39.0°C to 40.0°C), which persisted for 8 days (range, 6 to 11 days). Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days (range, 1 to 10 days). After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days (range, 8 to 11 days) ( Table 1) . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52). Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 (range = 49 to 62.5). White blood cell counts were low or in the normal range. All patients had elevated serum", "aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and hydroxybutyrate dehydrogenase (HBDH) ( Table 1) . At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range.", "Four patients had lower than normal T-cell subset counts (Table 2) .\n\nCXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ). Three patients were examined by highresolution computed tomography (HRCT). Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ). The mean duration from onset to a single-lobe consolidation on CXRs was 2 days (range = 1 to 5 days). The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days).", "All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days (Table 3) . Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy (Figure 3 ).", "Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours (range = 22 to 48 hours), and the mean time until IMV failure was 6.2 days (range 2 = to 13 days) ( Table 1) . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support.", "All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip). Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient (Table 3) . Four (80%) of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively.", "To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. (1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. (2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. (3) Viral load monitoring may help predict disease severity and patient outcome. (4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. (5) HAdV-55-induced severe ARDS", "has a very high mortality rate (80%) despite appropriate respiratory support.", "Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] . HAdV-55 was first completely characterized in Shaanxi, China [7] and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease [9] . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS.", "The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region.\n\nOur study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports [6, 9] .", "Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the", "reaction to therapy and patient outcome.", "The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation.\n\nOur study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response." ]

[ 2 ]

[ "Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates" ]

[ "Emergent severe acute respiratory distress syndrome caused by adenovirus type 55 in immunocompetent adults in 2013: a prospective observational study\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4243941/\n\nSHA: f5b706d0529bfcf7e2d1dfc037df5b6f95fc5ec0\n\nAuthors: Sun, Bing; He, Hangyong; Wang, Zheng; Qu, Jiuxin; Li, Xuyan; Ban, Chengjun; Wan, Jun; Cao, Bin; Tong, Zhaohui; Wang, Chen\nDate: 2014-08-12\nDOI: 10.1186/s13054-014-0456-6\nLicense: cc-by", "Abstract: INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 (HAdV-55) in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome (ARDS) caused by HAdV-55 requiring invasive mechanical ventilation (IMV) and/or extracorporeal membrane oxygenation (ECMO) are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was", "5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10(8) copies in three patients and was 1 × 10(4) in one patient. It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation (NPPV) failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four (80%) of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most", "frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012", "Text: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients [1] [2] [3] [4] [5] . Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 [7] and in Singapore in 2005 [8] . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] . This pathogen was fully characterized by whole-genome sequencing [10] . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the", "serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes (HAdV-3, HAdV-7 and HAdV-14) [6] .", "Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU.", "Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation (NPPV) in ARDS patients was carried out in our center (ClinicalTrials.gov ID: NCT01585922). From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: (1) developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; (2) bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; (3) respiratory failure not fully explained by cardiac failure or fluid overload; (4) partial oxygen pressure/ fraction of inspired oxygen (PaO 2 /FiO 2 ) ≤100 mmHg with positive end-expiratory pressure (PEEP) ≥5 cmH 2 O; and (5) a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive", "mechanical ventilation (IMV) were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital (LLKYPJ2012031). Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication.", "Clinical information collected by investigators with a standardized data form included the following: demographic characteristics (age and sex), comorbidities, clinical symptoms (fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache), signs (body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs), laboratory tests (whole-blood cell count and blood chemistry) and microbiological findings and images of the lung (chest X-ray (CXR) and computed tomography). Concomitant medications, respiratory support, complications and outcomes were also recorded.", "Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay.\n\nViral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods.", "Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range).\n\nDuring the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs.", "All five patients were immunocompetent young men with a median age of 32 years (range, 28 to 40 years). All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay (Oxford Immunotec, Marlborough, MA, USA) and antibody of Mycobacterium tuberculosis, were negative.", "Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C (range, 39.0°C to 40.0°C), which persisted for 8 days (range, 6 to 11 days). Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days (range, 1 to 10 days). After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days (range, 8 to 11 days) ( Table 1) . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52). Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 (range = 49 to 62.5). White blood cell counts were low or in the normal range. All patients had elevated serum", "aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and hydroxybutyrate dehydrogenase (HBDH) ( Table 1) . At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range.", "Four patients had lower than normal T-cell subset counts (Table 2) .\n\nCXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ). Three patients were examined by highresolution computed tomography (HRCT). Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ). The mean duration from onset to a single-lobe consolidation on CXRs was 2 days (range = 1 to 5 days). The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days).", "All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days (Table 3) . Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy (Figure 3 ).", "Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours (range = 22 to 48 hours), and the mean time until IMV failure was 6.2 days (range 2 = to 13 days) ( Table 1) . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support.", "All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip). Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient (Table 3) . Four (80%) of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively.", "To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. (1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. (2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. (3) Viral load monitoring may help predict disease severity and patient outcome. (4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. (5) HAdV-55-induced severe ARDS", "has a very high mortality rate (80%) despite appropriate respiratory support.", "Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] . HAdV-55 was first completely characterized in Shaanxi, China [7] and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease [9] . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS.", "The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region.\n\nOur study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports [6, 9] .", "Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the", "reaction to therapy and patient outcome.", "The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation.\n\nOur study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response." ]

[ 2 ]

[ "This pathogen was fully characterized by whole-genome sequencing" ]

[ "Emergent severe acute respiratory distress syndrome caused by adenovirus type 55 in immunocompetent adults in 2013: a prospective observational study\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4243941/\n\nSHA: f5b706d0529bfcf7e2d1dfc037df5b6f95fc5ec0\n\nAuthors: Sun, Bing; He, Hangyong; Wang, Zheng; Qu, Jiuxin; Li, Xuyan; Ban, Chengjun; Wan, Jun; Cao, Bin; Tong, Zhaohui; Wang, Chen\nDate: 2014-08-12\nDOI: 10.1186/s13054-014-0456-6\nLicense: cc-by", "Abstract: INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 (HAdV-55) in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome (ARDS) caused by HAdV-55 requiring invasive mechanical ventilation (IMV) and/or extracorporeal membrane oxygenation (ECMO) are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was", "5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10(8) copies in three patients and was 1 × 10(4) in one patient. It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation (NPPV) failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four (80%) of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most", "frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012", "Text: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients [1] [2] [3] [4] [5] . Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 [7] and in Singapore in 2005 [8] . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] . This pathogen was fully characterized by whole-genome sequencing [10] . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the", "serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes (HAdV-3, HAdV-7 and HAdV-14) [6] .", "Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU.", "Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation (NPPV) in ARDS patients was carried out in our center (ClinicalTrials.gov ID: NCT01585922). From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: (1) developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; (2) bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; (3) respiratory failure not fully explained by cardiac failure or fluid overload; (4) partial oxygen pressure/ fraction of inspired oxygen (PaO 2 /FiO 2 ) ≤100 mmHg with positive end-expiratory pressure (PEEP) ≥5 cmH 2 O; and (5) a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive", "mechanical ventilation (IMV) were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital (LLKYPJ2012031). Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication.", "Clinical information collected by investigators with a standardized data form included the following: demographic characteristics (age and sex), comorbidities, clinical symptoms (fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache), signs (body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs), laboratory tests (whole-blood cell count and blood chemistry) and microbiological findings and images of the lung (chest X-ray (CXR) and computed tomography). Concomitant medications, respiratory support, complications and outcomes were also recorded.", "Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay.\n\nViral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods.", "Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range).\n\nDuring the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs.", "All five patients were immunocompetent young men with a median age of 32 years (range, 28 to 40 years). All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay (Oxford Immunotec, Marlborough, MA, USA) and antibody of Mycobacterium tuberculosis, were negative.", "Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C (range, 39.0°C to 40.0°C), which persisted for 8 days (range, 6 to 11 days). Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days (range, 1 to 10 days). After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days (range, 8 to 11 days) ( Table 1) . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52). Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 (range = 49 to 62.5). White blood cell counts were low or in the normal range. All patients had elevated serum", "aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and hydroxybutyrate dehydrogenase (HBDH) ( Table 1) . At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range.", "Four patients had lower than normal T-cell subset counts (Table 2) .\n\nCXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ). Three patients were examined by highresolution computed tomography (HRCT). Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ). The mean duration from onset to a single-lobe consolidation on CXRs was 2 days (range = 1 to 5 days). The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days).", "All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days (Table 3) . Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy (Figure 3 ).", "Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours (range = 22 to 48 hours), and the mean time until IMV failure was 6.2 days (range 2 = to 13 days) ( Table 1) . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support.", "All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip). Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient (Table 3) . Four (80%) of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively.", "To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. (1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. (2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. (3) Viral load monitoring may help predict disease severity and patient outcome. (4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. (5) HAdV-55-induced severe ARDS", "has a very high mortality rate (80%) despite appropriate respiratory support.", "Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] . HAdV-55 was first completely characterized in Shaanxi, China [7] and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease [9] . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS.", "The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region.\n\nOur study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports [6, 9] .", "Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the", "reaction to therapy and patient outcome.", "The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation.\n\nOur study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response." ]

[ 4 ]

[ "Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness" ]

[ "Emergent severe acute respiratory distress syndrome caused by adenovirus type 55 in immunocompetent adults in 2013: a prospective observational study\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4243941/\n\nSHA: f5b706d0529bfcf7e2d1dfc037df5b6f95fc5ec0\n\nAuthors: Sun, Bing; He, Hangyong; Wang, Zheng; Qu, Jiuxin; Li, Xuyan; Ban, Chengjun; Wan, Jun; Cao, Bin; Tong, Zhaohui; Wang, Chen\nDate: 2014-08-12\nDOI: 10.1186/s13054-014-0456-6\nLicense: cc-by", "Abstract: INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 (HAdV-55) in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome (ARDS) caused by HAdV-55 requiring invasive mechanical ventilation (IMV) and/or extracorporeal membrane oxygenation (ECMO) are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was", "5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10(8) copies in three patients and was 1 × 10(4) in one patient. It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation (NPPV) failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four (80%) of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most", "frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012", "Text: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients [1] [2] [3] [4] [5] . Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 [7] and in Singapore in 2005 [8] . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] . This pathogen was fully characterized by whole-genome sequencing [10] . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the", "serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes (HAdV-3, HAdV-7 and HAdV-14) [6] .", "Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU.", "Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation (NPPV) in ARDS patients was carried out in our center (ClinicalTrials.gov ID: NCT01585922). From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: (1) developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; (2) bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; (3) respiratory failure not fully explained by cardiac failure or fluid overload; (4) partial oxygen pressure/ fraction of inspired oxygen (PaO 2 /FiO 2 ) ≤100 mmHg with positive end-expiratory pressure (PEEP) ≥5 cmH 2 O; and (5) a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive", "mechanical ventilation (IMV) were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital (LLKYPJ2012031). Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication.", "Clinical information collected by investigators with a standardized data form included the following: demographic characteristics (age and sex), comorbidities, clinical symptoms (fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache), signs (body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs), laboratory tests (whole-blood cell count and blood chemistry) and microbiological findings and images of the lung (chest X-ray (CXR) and computed tomography). Concomitant medications, respiratory support, complications and outcomes were also recorded.", "Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay.\n\nViral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods.", "Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range).\n\nDuring the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs.", "All five patients were immunocompetent young men with a median age of 32 years (range, 28 to 40 years). All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay (Oxford Immunotec, Marlborough, MA, USA) and antibody of Mycobacterium tuberculosis, were negative.", "Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C (range, 39.0°C to 40.0°C), which persisted for 8 days (range, 6 to 11 days). Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days (range, 1 to 10 days). After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days (range, 8 to 11 days) ( Table 1) . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52). Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 (range = 49 to 62.5). White blood cell counts were low or in the normal range. All patients had elevated serum", "aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and hydroxybutyrate dehydrogenase (HBDH) ( Table 1) . At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range.", "Four patients had lower than normal T-cell subset counts (Table 2) .\n\nCXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ). Three patients were examined by highresolution computed tomography (HRCT). Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ). The mean duration from onset to a single-lobe consolidation on CXRs was 2 days (range = 1 to 5 days). The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days).", "All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days (Table 3) . Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy (Figure 3 ).", "Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours (range = 22 to 48 hours), and the mean time until IMV failure was 6.2 days (range 2 = to 13 days) ( Table 1) . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support.", "All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip). Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient (Table 3) . Four (80%) of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively.", "To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. (1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. (2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. (3) Viral load monitoring may help predict disease severity and patient outcome. (4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. (5) HAdV-55-induced severe ARDS", "has a very high mortality rate (80%) despite appropriate respiratory support.", "Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] . HAdV-55 was first completely characterized in Shaanxi, China [7] and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease [9] . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS.", "The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region.\n\nOur study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports [6, 9] .", "Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the", "reaction to therapy and patient outcome.", "The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation.\n\nOur study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response." ]

[ 13 ]

[ "5 days" ]

[ "Emergent severe acute respiratory distress syndrome caused by adenovirus type 55 in immunocompetent adults in 2013: a prospective observational study\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4243941/\n\nSHA: f5b706d0529bfcf7e2d1dfc037df5b6f95fc5ec0\n\nAuthors: Sun, Bing; He, Hangyong; Wang, Zheng; Qu, Jiuxin; Li, Xuyan; Ban, Chengjun; Wan, Jun; Cao, Bin; Tong, Zhaohui; Wang, Chen\nDate: 2014-08-12\nDOI: 10.1186/s13054-014-0456-6\nLicense: cc-by", "Abstract: INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 (HAdV-55) in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome (ARDS) caused by HAdV-55 requiring invasive mechanical ventilation (IMV) and/or extracorporeal membrane oxygenation (ECMO) are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was", "5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10(8) copies in three patients and was 1 × 10(4) in one patient. It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation (NPPV) failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four (80%) of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most", "frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012", "Text: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients [1] [2] [3] [4] [5] . Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 [7] and in Singapore in 2005 [8] . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] . This pathogen was fully characterized by whole-genome sequencing [10] . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the", "serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes (HAdV-3, HAdV-7 and HAdV-14) [6] .", "Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU.", "Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation (NPPV) in ARDS patients was carried out in our center (ClinicalTrials.gov ID: NCT01585922). From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: (1) developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; (2) bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; (3) respiratory failure not fully explained by cardiac failure or fluid overload; (4) partial oxygen pressure/ fraction of inspired oxygen (PaO 2 /FiO 2 ) ≤100 mmHg with positive end-expiratory pressure (PEEP) ≥5 cmH 2 O; and (5) a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive", "mechanical ventilation (IMV) were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital (LLKYPJ2012031). Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication.", "Clinical information collected by investigators with a standardized data form included the following: demographic characteristics (age and sex), comorbidities, clinical symptoms (fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache), signs (body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs), laboratory tests (whole-blood cell count and blood chemistry) and microbiological findings and images of the lung (chest X-ray (CXR) and computed tomography). Concomitant medications, respiratory support, complications and outcomes were also recorded.", "Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay.\n\nViral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods.", "Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range).\n\nDuring the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs.", "All five patients were immunocompetent young men with a median age of 32 years (range, 28 to 40 years). All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay (Oxford Immunotec, Marlborough, MA, USA) and antibody of Mycobacterium tuberculosis, were negative.", "Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C (range, 39.0°C to 40.0°C), which persisted for 8 days (range, 6 to 11 days). Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days (range, 1 to 10 days). After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days (range, 8 to 11 days) ( Table 1) . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52). Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 (range = 49 to 62.5). White blood cell counts were low or in the normal range. All patients had elevated serum", "aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and hydroxybutyrate dehydrogenase (HBDH) ( Table 1) . At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range.", "Four patients had lower than normal T-cell subset counts (Table 2) .\n\nCXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ). Three patients were examined by highresolution computed tomography (HRCT). Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ). The mean duration from onset to a single-lobe consolidation on CXRs was 2 days (range = 1 to 5 days). The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days).", "All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days (Table 3) . Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy (Figure 3 ).", "Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours (range = 22 to 48 hours), and the mean time until IMV failure was 6.2 days (range 2 = to 13 days) ( Table 1) . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support.", "All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip). Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient (Table 3) . Four (80%) of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively.", "To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. (1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. (2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. (3) Viral load monitoring may help predict disease severity and patient outcome. (4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. (5) HAdV-55-induced severe ARDS", "has a very high mortality rate (80%) despite appropriate respiratory support.", "Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] . HAdV-55 was first completely characterized in Shaanxi, China [7] and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease [9] . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS.", "The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region.\n\nOur study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports [6, 9] .", "Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the", "reaction to therapy and patient outcome.", "The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation.\n\nOur study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response." ]

[ 2 ]

[ "9.6 days" ]

[ "Emergent severe acute respiratory distress syndrome caused by adenovirus type 55 in immunocompetent adults in 2013: a prospective observational study\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4243941/\n\nSHA: f5b706d0529bfcf7e2d1dfc037df5b6f95fc5ec0\n\nAuthors: Sun, Bing; He, Hangyong; Wang, Zheng; Qu, Jiuxin; Li, Xuyan; Ban, Chengjun; Wan, Jun; Cao, Bin; Tong, Zhaohui; Wang, Chen\nDate: 2014-08-12\nDOI: 10.1186/s13054-014-0456-6\nLicense: cc-by", "Abstract: INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 (HAdV-55) in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome (ARDS) caused by HAdV-55 requiring invasive mechanical ventilation (IMV) and/or extracorporeal membrane oxygenation (ECMO) are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was", "5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10(8) copies in three patients and was 1 × 10(4) in one patient. It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation (NPPV) failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four (80%) of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most", "frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012", "Text: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients [1] [2] [3] [4] [5] . Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 [7] and in Singapore in 2005 [8] . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] . This pathogen was fully characterized by whole-genome sequencing [10] . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the", "serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes (HAdV-3, HAdV-7 and HAdV-14) [6] .", "Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU.", "Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation (NPPV) in ARDS patients was carried out in our center (ClinicalTrials.gov ID: NCT01585922). From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: (1) developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; (2) bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; (3) respiratory failure not fully explained by cardiac failure or fluid overload; (4) partial oxygen pressure/ fraction of inspired oxygen (PaO 2 /FiO 2 ) ≤100 mmHg with positive end-expiratory pressure (PEEP) ≥5 cmH 2 O; and (5) a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive", "mechanical ventilation (IMV) were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital (LLKYPJ2012031). Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication.", "Clinical information collected by investigators with a standardized data form included the following: demographic characteristics (age and sex), comorbidities, clinical symptoms (fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache), signs (body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs), laboratory tests (whole-blood cell count and blood chemistry) and microbiological findings and images of the lung (chest X-ray (CXR) and computed tomography). Concomitant medications, respiratory support, complications and outcomes were also recorded.", "Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay.\n\nViral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods.", "Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range).\n\nDuring the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs.", "All five patients were immunocompetent young men with a median age of 32 years (range, 28 to 40 years). All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay (Oxford Immunotec, Marlborough, MA, USA) and antibody of Mycobacterium tuberculosis, were negative.", "Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C (range, 39.0°C to 40.0°C), which persisted for 8 days (range, 6 to 11 days). Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days (range, 1 to 10 days). After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days (range, 8 to 11 days) ( Table 1) . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52). Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 (range = 49 to 62.5). White blood cell counts were low or in the normal range. All patients had elevated serum", "aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and hydroxybutyrate dehydrogenase (HBDH) ( Table 1) . At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range.", "Four patients had lower than normal T-cell subset counts (Table 2) .\n\nCXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ). Three patients were examined by highresolution computed tomography (HRCT). Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ). The mean duration from onset to a single-lobe consolidation on CXRs was 2 days (range = 1 to 5 days). The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days).", "All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days (Table 3) . Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy (Figure 3 ).", "Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours (range = 22 to 48 hours), and the mean time until IMV failure was 6.2 days (range 2 = to 13 days) ( Table 1) . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support.", "All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip). Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient (Table 3) . Four (80%) of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively.", "To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. (1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. (2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. (3) Viral load monitoring may help predict disease severity and patient outcome. (4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. (5) HAdV-55-induced severe ARDS", "has a very high mortality rate (80%) despite appropriate respiratory support.", "Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] . HAdV-55 was first completely characterized in Shaanxi, China [7] and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease [9] . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS.", "The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region.\n\nOur study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports [6, 9] .", "Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the", "reaction to therapy and patient outcome.", "The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation.\n\nOur study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response." ]

[ 13 ]

[ "43 breaths per minute" ]

[ "Emergent severe acute respiratory distress syndrome caused by adenovirus type 55 in immunocompetent adults in 2013: a prospective observational study\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4243941/\n\nSHA: f5b706d0529bfcf7e2d1dfc037df5b6f95fc5ec0\n\nAuthors: Sun, Bing; He, Hangyong; Wang, Zheng; Qu, Jiuxin; Li, Xuyan; Ban, Chengjun; Wan, Jun; Cao, Bin; Tong, Zhaohui; Wang, Chen\nDate: 2014-08-12\nDOI: 10.1186/s13054-014-0456-6\nLicense: cc-by", "Abstract: INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 (HAdV-55) in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome (ARDS) caused by HAdV-55 requiring invasive mechanical ventilation (IMV) and/or extracorporeal membrane oxygenation (ECMO) are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was", "5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10(8) copies in three patients and was 1 × 10(4) in one patient. It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation (NPPV) failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four (80%) of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most", "frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012", "Text: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients [1] [2] [3] [4] [5] . Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 [7] and in Singapore in 2005 [8] . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] . This pathogen was fully characterized by whole-genome sequencing [10] . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the", "serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes (HAdV-3, HAdV-7 and HAdV-14) [6] .", "Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU.", "Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation (NPPV) in ARDS patients was carried out in our center (ClinicalTrials.gov ID: NCT01585922). From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: (1) developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; (2) bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; (3) respiratory failure not fully explained by cardiac failure or fluid overload; (4) partial oxygen pressure/ fraction of inspired oxygen (PaO 2 /FiO 2 ) ≤100 mmHg with positive end-expiratory pressure (PEEP) ≥5 cmH 2 O; and (5) a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive", "mechanical ventilation (IMV) were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital (LLKYPJ2012031). Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication.", "Clinical information collected by investigators with a standardized data form included the following: demographic characteristics (age and sex), comorbidities, clinical symptoms (fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache), signs (body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs), laboratory tests (whole-blood cell count and blood chemistry) and microbiological findings and images of the lung (chest X-ray (CXR) and computed tomography). Concomitant medications, respiratory support, complications and outcomes were also recorded.", "Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay.\n\nViral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods.", "Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range).\n\nDuring the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs.", "All five patients were immunocompetent young men with a median age of 32 years (range, 28 to 40 years). All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay (Oxford Immunotec, Marlborough, MA, USA) and antibody of Mycobacterium tuberculosis, were negative.", "Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C (range, 39.0°C to 40.0°C), which persisted for 8 days (range, 6 to 11 days). Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days (range, 1 to 10 days). After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days (range, 8 to 11 days) ( Table 1) . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52). Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 (range = 49 to 62.5). White blood cell counts were low or in the normal range. All patients had elevated serum", "aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and hydroxybutyrate dehydrogenase (HBDH) ( Table 1) . At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range.", "Four patients had lower than normal T-cell subset counts (Table 2) .\n\nCXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ). Three patients were examined by highresolution computed tomography (HRCT). Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ). The mean duration from onset to a single-lobe consolidation on CXRs was 2 days (range = 1 to 5 days). The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days).", "All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days (Table 3) . Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy (Figure 3 ).", "Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours (range = 22 to 48 hours), and the mean time until IMV failure was 6.2 days (range 2 = to 13 days) ( Table 1) . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support.", "All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip). Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient (Table 3) . Four (80%) of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively.", "To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. (1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. (2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. (3) Viral load monitoring may help predict disease severity and patient outcome. (4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. (5) HAdV-55-induced severe ARDS", "has a very high mortality rate (80%) despite appropriate respiratory support.", "Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] . HAdV-55 was first completely characterized in Shaanxi, China [7] and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease [9] . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS.", "The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region.\n\nOur study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports [6, 9] .", "Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the", "reaction to therapy and patient outcome.", "The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation.\n\nOur study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response." ]

[ 13 ]

[ "low or in the normal range" ]

[ "Emergent severe acute respiratory distress syndrome caused by adenovirus type 55 in immunocompetent adults in 2013: a prospective observational study\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4243941/\n\nSHA: f5b706d0529bfcf7e2d1dfc037df5b6f95fc5ec0\n\nAuthors: Sun, Bing; He, Hangyong; Wang, Zheng; Qu, Jiuxin; Li, Xuyan; Ban, Chengjun; Wan, Jun; Cao, Bin; Tong, Zhaohui; Wang, Chen\nDate: 2014-08-12\nDOI: 10.1186/s13054-014-0456-6\nLicense: cc-by", "Abstract: INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 (HAdV-55) in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome (ARDS) caused by HAdV-55 requiring invasive mechanical ventilation (IMV) and/or extracorporeal membrane oxygenation (ECMO) are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was", "5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10(8) copies in three patients and was 1 × 10(4) in one patient. It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation (NPPV) failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four (80%) of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most", "frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012", "Text: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients [1] [2] [3] [4] [5] . Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 [7] and in Singapore in 2005 [8] . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] . This pathogen was fully characterized by whole-genome sequencing [10] . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the", "serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes (HAdV-3, HAdV-7 and HAdV-14) [6] .", "Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU.", "Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation (NPPV) in ARDS patients was carried out in our center (ClinicalTrials.gov ID: NCT01585922). From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: (1) developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; (2) bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; (3) respiratory failure not fully explained by cardiac failure or fluid overload; (4) partial oxygen pressure/ fraction of inspired oxygen (PaO 2 /FiO 2 ) ≤100 mmHg with positive end-expiratory pressure (PEEP) ≥5 cmH 2 O; and (5) a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive", "mechanical ventilation (IMV) were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital (LLKYPJ2012031). Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication.", "Clinical information collected by investigators with a standardized data form included the following: demographic characteristics (age and sex), comorbidities, clinical symptoms (fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache), signs (body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs), laboratory tests (whole-blood cell count and blood chemistry) and microbiological findings and images of the lung (chest X-ray (CXR) and computed tomography). Concomitant medications, respiratory support, complications and outcomes were also recorded.", "Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay.\n\nViral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods.", "Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range).\n\nDuring the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs.", "All five patients were immunocompetent young men with a median age of 32 years (range, 28 to 40 years). All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay (Oxford Immunotec, Marlborough, MA, USA) and antibody of Mycobacterium tuberculosis, were negative.", "Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C (range, 39.0°C to 40.0°C), which persisted for 8 days (range, 6 to 11 days). Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days (range, 1 to 10 days). After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days (range, 8 to 11 days) ( Table 1) . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52). Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 (range = 49 to 62.5). White blood cell counts were low or in the normal range. All patients had elevated serum", "aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and hydroxybutyrate dehydrogenase (HBDH) ( Table 1) . At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range.", "Four patients had lower than normal T-cell subset counts (Table 2) .\n\nCXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ). Three patients were examined by highresolution computed tomography (HRCT). Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ). The mean duration from onset to a single-lobe consolidation on CXRs was 2 days (range = 1 to 5 days). The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days).", "All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days (Table 3) . Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy (Figure 3 ).", "Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours (range = 22 to 48 hours), and the mean time until IMV failure was 6.2 days (range 2 = to 13 days) ( Table 1) . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support.", "All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip). Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient (Table 3) . Four (80%) of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively.", "To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. (1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. (2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. (3) Viral load monitoring may help predict disease severity and patient outcome. (4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. (5) HAdV-55-induced severe ARDS", "has a very high mortality rate (80%) despite appropriate respiratory support.", "Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] . HAdV-55 was first completely characterized in Shaanxi, China [7] and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease [9] . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS.", "The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region.\n\nOur study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports [6, 9] .", "Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the", "reaction to therapy and patient outcome.", "The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation.\n\nOur study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response." ]

[ 13 ]

[ "CXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission" ]

[ "Emergent severe acute respiratory distress syndrome caused by adenovirus type 55 in immunocompetent adults in 2013: a prospective observational study\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4243941/\n\nSHA: f5b706d0529bfcf7e2d1dfc037df5b6f95fc5ec0\n\nAuthors: Sun, Bing; He, Hangyong; Wang, Zheng; Qu, Jiuxin; Li, Xuyan; Ban, Chengjun; Wan, Jun; Cao, Bin; Tong, Zhaohui; Wang, Chen\nDate: 2014-08-12\nDOI: 10.1186/s13054-014-0456-6\nLicense: cc-by", "Abstract: INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 (HAdV-55) in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome (ARDS) caused by HAdV-55 requiring invasive mechanical ventilation (IMV) and/or extracorporeal membrane oxygenation (ECMO) are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was", "5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10(8) copies in three patients and was 1 × 10(4) in one patient. It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation (NPPV) failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four (80%) of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most", "frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012", "Text: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients [1] [2] [3] [4] [5] . Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 [7] and in Singapore in 2005 [8] . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] . This pathogen was fully characterized by whole-genome sequencing [10] . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the", "serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes (HAdV-3, HAdV-7 and HAdV-14) [6] .", "Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU.", "Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation (NPPV) in ARDS patients was carried out in our center (ClinicalTrials.gov ID: NCT01585922). From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: (1) developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; (2) bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; (3) respiratory failure not fully explained by cardiac failure or fluid overload; (4) partial oxygen pressure/ fraction of inspired oxygen (PaO 2 /FiO 2 ) ≤100 mmHg with positive end-expiratory pressure (PEEP) ≥5 cmH 2 O; and (5) a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive", "mechanical ventilation (IMV) were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital (LLKYPJ2012031). Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication.", "Clinical information collected by investigators with a standardized data form included the following: demographic characteristics (age and sex), comorbidities, clinical symptoms (fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache), signs (body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs), laboratory tests (whole-blood cell count and blood chemistry) and microbiological findings and images of the lung (chest X-ray (CXR) and computed tomography). Concomitant medications, respiratory support, complications and outcomes were also recorded.", "Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay.\n\nViral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods.", "Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range).\n\nDuring the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs.", "All five patients were immunocompetent young men with a median age of 32 years (range, 28 to 40 years). All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay (Oxford Immunotec, Marlborough, MA, USA) and antibody of Mycobacterium tuberculosis, were negative.", "Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C (range, 39.0°C to 40.0°C), which persisted for 8 days (range, 6 to 11 days). Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days (range, 1 to 10 days). After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days (range, 8 to 11 days) ( Table 1) . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52). Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 (range = 49 to 62.5). White blood cell counts were low or in the normal range. All patients had elevated serum", "aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and hydroxybutyrate dehydrogenase (HBDH) ( Table 1) . At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range.", "Four patients had lower than normal T-cell subset counts (Table 2) .\n\nCXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ). Three patients were examined by highresolution computed tomography (HRCT). Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ). The mean duration from onset to a single-lobe consolidation on CXRs was 2 days (range = 1 to 5 days). The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days).", "All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days (Table 3) . Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy (Figure 3 ).", "Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours (range = 22 to 48 hours), and the mean time until IMV failure was 6.2 days (range 2 = to 13 days) ( Table 1) . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support.", "All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip). Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient (Table 3) . Four (80%) of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively.", "To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. (1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. (2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. (3) Viral load monitoring may help predict disease severity and patient outcome. (4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. (5) HAdV-55-induced severe ARDS", "has a very high mortality rate (80%) despite appropriate respiratory support.", "Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] . HAdV-55 was first completely characterized in Shaanxi, China [7] and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease [9] . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS.", "The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region.\n\nOur study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports [6, 9] .", "Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the", "reaction to therapy and patient outcome.", "The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation.\n\nOur study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response." ]

[ 15 ]

[ "Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT" ]

[ "Emergent severe acute respiratory distress syndrome caused by adenovirus type 55 in immunocompetent adults in 2013: a prospective observational study\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4243941/\n\nSHA: f5b706d0529bfcf7e2d1dfc037df5b6f95fc5ec0\n\nAuthors: Sun, Bing; He, Hangyong; Wang, Zheng; Qu, Jiuxin; Li, Xuyan; Ban, Chengjun; Wan, Jun; Cao, Bin; Tong, Zhaohui; Wang, Chen\nDate: 2014-08-12\nDOI: 10.1186/s13054-014-0456-6\nLicense: cc-by", "Abstract: INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 (HAdV-55) in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome (ARDS) caused by HAdV-55 requiring invasive mechanical ventilation (IMV) and/or extracorporeal membrane oxygenation (ECMO) are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was", "5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10(8) copies in three patients and was 1 × 10(4) in one patient. It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation (NPPV) failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four (80%) of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most", "frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012", "Text: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients [1] [2] [3] [4] [5] . Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 [7] and in Singapore in 2005 [8] . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] . This pathogen was fully characterized by whole-genome sequencing [10] . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the", "serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes (HAdV-3, HAdV-7 and HAdV-14) [6] .", "Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU.", "Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation (NPPV) in ARDS patients was carried out in our center (ClinicalTrials.gov ID: NCT01585922). From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: (1) developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; (2) bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; (3) respiratory failure not fully explained by cardiac failure or fluid overload; (4) partial oxygen pressure/ fraction of inspired oxygen (PaO 2 /FiO 2 ) ≤100 mmHg with positive end-expiratory pressure (PEEP) ≥5 cmH 2 O; and (5) a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive", "mechanical ventilation (IMV) were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital (LLKYPJ2012031). Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication.", "Clinical information collected by investigators with a standardized data form included the following: demographic characteristics (age and sex), comorbidities, clinical symptoms (fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache), signs (body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs), laboratory tests (whole-blood cell count and blood chemistry) and microbiological findings and images of the lung (chest X-ray (CXR) and computed tomography). Concomitant medications, respiratory support, complications and outcomes were also recorded.", "Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay.\n\nViral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods.", "Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range).\n\nDuring the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs.", "All five patients were immunocompetent young men with a median age of 32 years (range, 28 to 40 years). All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay (Oxford Immunotec, Marlborough, MA, USA) and antibody of Mycobacterium tuberculosis, were negative.", "Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C (range, 39.0°C to 40.0°C), which persisted for 8 days (range, 6 to 11 days). Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days (range, 1 to 10 days). After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days (range, 8 to 11 days) ( Table 1) . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52). Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 (range = 49 to 62.5). White blood cell counts were low or in the normal range. All patients had elevated serum", "aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and hydroxybutyrate dehydrogenase (HBDH) ( Table 1) . At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range.", "Four patients had lower than normal T-cell subset counts (Table 2) .\n\nCXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ). Three patients were examined by highresolution computed tomography (HRCT). Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ). The mean duration from onset to a single-lobe consolidation on CXRs was 2 days (range = 1 to 5 days). The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days).", "All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days (Table 3) . Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy (Figure 3 ).", "Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours (range = 22 to 48 hours), and the mean time until IMV failure was 6.2 days (range 2 = to 13 days) ( Table 1) . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support.", "All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip). Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient (Table 3) . Four (80%) of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively.", "To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. (1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. (2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. (3) Viral load monitoring may help predict disease severity and patient outcome. (4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. (5) HAdV-55-induced severe ARDS", "has a very high mortality rate (80%) despite appropriate respiratory support.", "Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] . HAdV-55 was first completely characterized in Shaanxi, China [7] and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease [9] . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS.", "The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region.\n\nOur study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports [6, 9] .", "Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the", "reaction to therapy and patient outcome.", "The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation.\n\nOur study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response." ]

[ 15 ]

[ "All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples" ]

[ "Emergent severe acute respiratory distress syndrome caused by adenovirus type 55 in immunocompetent adults in 2013: a prospective observational study\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4243941/\n\nSHA: f5b706d0529bfcf7e2d1dfc037df5b6f95fc5ec0\n\nAuthors: Sun, Bing; He, Hangyong; Wang, Zheng; Qu, Jiuxin; Li, Xuyan; Ban, Chengjun; Wan, Jun; Cao, Bin; Tong, Zhaohui; Wang, Chen\nDate: 2014-08-12\nDOI: 10.1186/s13054-014-0456-6\nLicense: cc-by", "Abstract: INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 (HAdV-55) in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome (ARDS) caused by HAdV-55 requiring invasive mechanical ventilation (IMV) and/or extracorporeal membrane oxygenation (ECMO) are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was", "5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10(8) copies in three patients and was 1 × 10(4) in one patient. It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation (NPPV) failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four (80%) of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most", "frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012", "Text: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients [1] [2] [3] [4] [5] . Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 [7] and in Singapore in 2005 [8] . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] . This pathogen was fully characterized by whole-genome sequencing [10] . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the", "serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes (HAdV-3, HAdV-7 and HAdV-14) [6] .", "Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU.", "Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation (NPPV) in ARDS patients was carried out in our center (ClinicalTrials.gov ID: NCT01585922). From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: (1) developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; (2) bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; (3) respiratory failure not fully explained by cardiac failure or fluid overload; (4) partial oxygen pressure/ fraction of inspired oxygen (PaO 2 /FiO 2 ) ≤100 mmHg with positive end-expiratory pressure (PEEP) ≥5 cmH 2 O; and (5) a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive", "mechanical ventilation (IMV) were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital (LLKYPJ2012031). Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication.", "Clinical information collected by investigators with a standardized data form included the following: demographic characteristics (age and sex), comorbidities, clinical symptoms (fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache), signs (body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs), laboratory tests (whole-blood cell count and blood chemistry) and microbiological findings and images of the lung (chest X-ray (CXR) and computed tomography). Concomitant medications, respiratory support, complications and outcomes were also recorded.", "Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay.\n\nViral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods.", "Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range).\n\nDuring the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs.", "All five patients were immunocompetent young men with a median age of 32 years (range, 28 to 40 years). All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay (Oxford Immunotec, Marlborough, MA, USA) and antibody of Mycobacterium tuberculosis, were negative.", "Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C (range, 39.0°C to 40.0°C), which persisted for 8 days (range, 6 to 11 days). Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days (range, 1 to 10 days). After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days (range, 8 to 11 days) ( Table 1) . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52). Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 (range = 49 to 62.5). White blood cell counts were low or in the normal range. All patients had elevated serum", "aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and hydroxybutyrate dehydrogenase (HBDH) ( Table 1) . At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range.", "Four patients had lower than normal T-cell subset counts (Table 2) .\n\nCXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ). Three patients were examined by highresolution computed tomography (HRCT). Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ). The mean duration from onset to a single-lobe consolidation on CXRs was 2 days (range = 1 to 5 days). The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days).", "All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days (Table 3) . Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy (Figure 3 ).", "Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours (range = 22 to 48 hours), and the mean time until IMV failure was 6.2 days (range 2 = to 13 days) ( Table 1) . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support.", "All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip). Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient (Table 3) . Four (80%) of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively.", "To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. (1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. (2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. (3) Viral load monitoring may help predict disease severity and patient outcome. (4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. (5) HAdV-55-induced severe ARDS", "has a very high mortality rate (80%) despite appropriate respiratory support.", "Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] . HAdV-55 was first completely characterized in Shaanxi, China [7] and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease [9] . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS.", "The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region.\n\nOur study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports [6, 9] .", "Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the", "reaction to therapy and patient outcome.", "The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation.\n\nOur study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response." ]

[ 16 ]

[ "1 to 10 days" ]

[ "Emergent severe acute respiratory distress syndrome caused by adenovirus type 55 in immunocompetent adults in 2013: a prospective observational study\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4243941/\n\nSHA: f5b706d0529bfcf7e2d1dfc037df5b6f95fc5ec0\n\nAuthors: Sun, Bing; He, Hangyong; Wang, Zheng; Qu, Jiuxin; Li, Xuyan; Ban, Chengjun; Wan, Jun; Cao, Bin; Tong, Zhaohui; Wang, Chen\nDate: 2014-08-12\nDOI: 10.1186/s13054-014-0456-6\nLicense: cc-by", "Abstract: INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 (HAdV-55) in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome (ARDS) caused by HAdV-55 requiring invasive mechanical ventilation (IMV) and/or extracorporeal membrane oxygenation (ECMO) are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was", "5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10(8) copies in three patients and was 1 × 10(4) in one patient. It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation (NPPV) failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four (80%) of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most", "frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012", "Text: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients [1] [2] [3] [4] [5] . Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 [7] and in Singapore in 2005 [8] . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] . This pathogen was fully characterized by whole-genome sequencing [10] . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the", "serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes (HAdV-3, HAdV-7 and HAdV-14) [6] .", "Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU.", "Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation (NPPV) in ARDS patients was carried out in our center (ClinicalTrials.gov ID: NCT01585922). From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: (1) developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; (2) bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; (3) respiratory failure not fully explained by cardiac failure or fluid overload; (4) partial oxygen pressure/ fraction of inspired oxygen (PaO 2 /FiO 2 ) ≤100 mmHg with positive end-expiratory pressure (PEEP) ≥5 cmH 2 O; and (5) a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive", "mechanical ventilation (IMV) were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital (LLKYPJ2012031). Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication.", "Clinical information collected by investigators with a standardized data form included the following: demographic characteristics (age and sex), comorbidities, clinical symptoms (fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache), signs (body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs), laboratory tests (whole-blood cell count and blood chemistry) and microbiological findings and images of the lung (chest X-ray (CXR) and computed tomography). Concomitant medications, respiratory support, complications and outcomes were also recorded.", "Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay.\n\nViral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods.", "Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range).\n\nDuring the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs.", "All five patients were immunocompetent young men with a median age of 32 years (range, 28 to 40 years). All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay (Oxford Immunotec, Marlborough, MA, USA) and antibody of Mycobacterium tuberculosis, were negative.", "Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C (range, 39.0°C to 40.0°C), which persisted for 8 days (range, 6 to 11 days). Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days (range, 1 to 10 days). After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days (range, 8 to 11 days) ( Table 1) . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52). Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 (range = 49 to 62.5). White blood cell counts were low or in the normal range. All patients had elevated serum", "aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and hydroxybutyrate dehydrogenase (HBDH) ( Table 1) . At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range.", "Four patients had lower than normal T-cell subset counts (Table 2) .\n\nCXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ). Three patients were examined by highresolution computed tomography (HRCT). Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ). The mean duration from onset to a single-lobe consolidation on CXRs was 2 days (range = 1 to 5 days). The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days).", "All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days (Table 3) . Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy (Figure 3 ).", "Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours (range = 22 to 48 hours), and the mean time until IMV failure was 6.2 days (range 2 = to 13 days) ( Table 1) . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support.", "All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip). Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient (Table 3) . Four (80%) of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively.", "To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. (1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. (2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. (3) Viral load monitoring may help predict disease severity and patient outcome. (4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. (5) HAdV-55-induced severe ARDS", "has a very high mortality rate (80%) despite appropriate respiratory support.", "Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] . HAdV-55 was first completely characterized in Shaanxi, China [7] and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease [9] . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS.", "The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region.\n\nOur study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports [6, 9] .", "Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the", "reaction to therapy and patient outcome.", "The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation.\n\nOur study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response." ]

[ 13 ]

[ "HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B" ]

[ "Emergent severe acute respiratory distress syndrome caused by adenovirus type 55 in immunocompetent adults in 2013: a prospective observational study\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4243941/\n\nSHA: f5b706d0529bfcf7e2d1dfc037df5b6f95fc5ec0\n\nAuthors: Sun, Bing; He, Hangyong; Wang, Zheng; Qu, Jiuxin; Li, Xuyan; Ban, Chengjun; Wan, Jun; Cao, Bin; Tong, Zhaohui; Wang, Chen\nDate: 2014-08-12\nDOI: 10.1186/s13054-014-0456-6\nLicense: cc-by", "Abstract: INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 (HAdV-55) in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome (ARDS) caused by HAdV-55 requiring invasive mechanical ventilation (IMV) and/or extracorporeal membrane oxygenation (ECMO) are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was", "5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10(8) copies in three patients and was 1 × 10(4) in one patient. It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation (NPPV) failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four (80%) of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most", "frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012", "Text: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients [1] [2] [3] [4] [5] . Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 [7] and in Singapore in 2005 [8] . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] . This pathogen was fully characterized by whole-genome sequencing [10] . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the", "serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes (HAdV-3, HAdV-7 and HAdV-14) [6] .", "Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU.", "Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation (NPPV) in ARDS patients was carried out in our center (ClinicalTrials.gov ID: NCT01585922). From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: (1) developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; (2) bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; (3) respiratory failure not fully explained by cardiac failure or fluid overload; (4) partial oxygen pressure/ fraction of inspired oxygen (PaO 2 /FiO 2 ) ≤100 mmHg with positive end-expiratory pressure (PEEP) ≥5 cmH 2 O; and (5) a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive", "mechanical ventilation (IMV) were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital (LLKYPJ2012031). Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication.", "Clinical information collected by investigators with a standardized data form included the following: demographic characteristics (age and sex), comorbidities, clinical symptoms (fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache), signs (body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs), laboratory tests (whole-blood cell count and blood chemistry) and microbiological findings and images of the lung (chest X-ray (CXR) and computed tomography). Concomitant medications, respiratory support, complications and outcomes were also recorded.", "Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay.\n\nViral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods.", "Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range).\n\nDuring the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs.", "All five patients were immunocompetent young men with a median age of 32 years (range, 28 to 40 years). All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay (Oxford Immunotec, Marlborough, MA, USA) and antibody of Mycobacterium tuberculosis, were negative.", "Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C (range, 39.0°C to 40.0°C), which persisted for 8 days (range, 6 to 11 days). Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days (range, 1 to 10 days). After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days (range, 8 to 11 days) ( Table 1) . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52). Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 (range = 49 to 62.5). White blood cell counts were low or in the normal range. All patients had elevated serum", "aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and hydroxybutyrate dehydrogenase (HBDH) ( Table 1) . At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range.", "Four patients had lower than normal T-cell subset counts (Table 2) .\n\nCXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ). Three patients were examined by highresolution computed tomography (HRCT). Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ). The mean duration from onset to a single-lobe consolidation on CXRs was 2 days (range = 1 to 5 days). The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days).", "All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days (Table 3) . Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy (Figure 3 ).", "Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours (range = 22 to 48 hours), and the mean time until IMV failure was 6.2 days (range 2 = to 13 days) ( Table 1) . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support.", "All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip). Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient (Table 3) . Four (80%) of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively.", "To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. (1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. (2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. (3) Viral load monitoring may help predict disease severity and patient outcome. (4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. (5) HAdV-55-induced severe ARDS", "has a very high mortality rate (80%) despite appropriate respiratory support.", "Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] . HAdV-55 was first completely characterized in Shaanxi, China [7] and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease [9] . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS.", "The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region.\n\nOur study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports [6, 9] .", "Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the", "reaction to therapy and patient outcome.", "The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation.\n\nOur study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response." ]

[ 19 ]

[ "Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations" ]

[ "Emergent severe acute respiratory distress syndrome caused by adenovirus type 55 in immunocompetent adults in 2013: a prospective observational study\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4243941/\n\nSHA: f5b706d0529bfcf7e2d1dfc037df5b6f95fc5ec0\n\nAuthors: Sun, Bing; He, Hangyong; Wang, Zheng; Qu, Jiuxin; Li, Xuyan; Ban, Chengjun; Wan, Jun; Cao, Bin; Tong, Zhaohui; Wang, Chen\nDate: 2014-08-12\nDOI: 10.1186/s13054-014-0456-6\nLicense: cc-by", "Abstract: INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 (HAdV-55) in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome (ARDS) caused by HAdV-55 requiring invasive mechanical ventilation (IMV) and/or extracorporeal membrane oxygenation (ECMO) are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was", "5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10(8) copies in three patients and was 1 × 10(4) in one patient. It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation (NPPV) failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four (80%) of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most", "frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012", "Text: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients [1] [2] [3] [4] [5] . Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 [7] and in Singapore in 2005 [8] . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] . This pathogen was fully characterized by whole-genome sequencing [10] . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the", "serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes (HAdV-3, HAdV-7 and HAdV-14) [6] .", "Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU.", "Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation (NPPV) in ARDS patients was carried out in our center (ClinicalTrials.gov ID: NCT01585922). From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: (1) developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; (2) bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; (3) respiratory failure not fully explained by cardiac failure or fluid overload; (4) partial oxygen pressure/ fraction of inspired oxygen (PaO 2 /FiO 2 ) ≤100 mmHg with positive end-expiratory pressure (PEEP) ≥5 cmH 2 O; and (5) a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive", "mechanical ventilation (IMV) were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital (LLKYPJ2012031). Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication.", "Clinical information collected by investigators with a standardized data form included the following: demographic characteristics (age and sex), comorbidities, clinical symptoms (fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache), signs (body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs), laboratory tests (whole-blood cell count and blood chemistry) and microbiological findings and images of the lung (chest X-ray (CXR) and computed tomography). Concomitant medications, respiratory support, complications and outcomes were also recorded.", "Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay.\n\nViral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods.", "Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range).\n\nDuring the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs.", "All five patients were immunocompetent young men with a median age of 32 years (range, 28 to 40 years). All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay (Oxford Immunotec, Marlborough, MA, USA) and antibody of Mycobacterium tuberculosis, were negative.", "Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C (range, 39.0°C to 40.0°C), which persisted for 8 days (range, 6 to 11 days). Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days (range, 1 to 10 days). After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days (range, 8 to 11 days) ( Table 1) . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52). Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 (range = 49 to 62.5). White blood cell counts were low or in the normal range. All patients had elevated serum", "aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and hydroxybutyrate dehydrogenase (HBDH) ( Table 1) . At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range.", "Four patients had lower than normal T-cell subset counts (Table 2) .\n\nCXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ). Three patients were examined by highresolution computed tomography (HRCT). Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ). The mean duration from onset to a single-lobe consolidation on CXRs was 2 days (range = 1 to 5 days). The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days).", "All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days (Table 3) . Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy (Figure 3 ).", "Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours (range = 22 to 48 hours), and the mean time until IMV failure was 6.2 days (range 2 = to 13 days) ( Table 1) . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support.", "All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip). Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient (Table 3) . Four (80%) of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively.", "To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. (1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. (2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. (3) Viral load monitoring may help predict disease severity and patient outcome. (4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. (5) HAdV-55-induced severe ARDS", "has a very high mortality rate (80%) despite appropriate respiratory support.", "Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] . HAdV-55 was first completely characterized in Shaanxi, China [7] and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease [9] . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS.", "The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region.\n\nOur study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports [6, 9] .", "Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the", "reaction to therapy and patient outcome.", "The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation.\n\nOur study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response." ]

[ 19 ]

[ "HAdV-55-induced severe ARDS has a very high mortality rate (80%) despite appropriate respiratory support." ]

[ "Emergent severe acute respiratory distress syndrome caused by adenovirus type 55 in immunocompetent adults in 2013: a prospective observational study\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4243941/\n\nSHA: f5b706d0529bfcf7e2d1dfc037df5b6f95fc5ec0\n\nAuthors: Sun, Bing; He, Hangyong; Wang, Zheng; Qu, Jiuxin; Li, Xuyan; Ban, Chengjun; Wan, Jun; Cao, Bin; Tong, Zhaohui; Wang, Chen\nDate: 2014-08-12\nDOI: 10.1186/s13054-014-0456-6\nLicense: cc-by", "Abstract: INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 (HAdV-55) in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome (ARDS) caused by HAdV-55 requiring invasive mechanical ventilation (IMV) and/or extracorporeal membrane oxygenation (ECMO) are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was", "5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10(8) copies in three patients and was 1 × 10(4) in one patient. It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation (NPPV) failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four (80%) of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most", "frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012", "Text: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients [1] [2] [3] [4] [5] . Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 [7] and in Singapore in 2005 [8] . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] . This pathogen was fully characterized by whole-genome sequencing [10] . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the", "serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes (HAdV-3, HAdV-7 and HAdV-14) [6] .", "Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU.", "Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation (NPPV) in ARDS patients was carried out in our center (ClinicalTrials.gov ID: NCT01585922). From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: (1) developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; (2) bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; (3) respiratory failure not fully explained by cardiac failure or fluid overload; (4) partial oxygen pressure/ fraction of inspired oxygen (PaO 2 /FiO 2 ) ≤100 mmHg with positive end-expiratory pressure (PEEP) ≥5 cmH 2 O; and (5) a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive", "mechanical ventilation (IMV) were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital (LLKYPJ2012031). Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication.", "Clinical information collected by investigators with a standardized data form included the following: demographic characteristics (age and sex), comorbidities, clinical symptoms (fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache), signs (body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs), laboratory tests (whole-blood cell count and blood chemistry) and microbiological findings and images of the lung (chest X-ray (CXR) and computed tomography). Concomitant medications, respiratory support, complications and outcomes were also recorded.", "Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay.\n\nViral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods.", "Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range).\n\nDuring the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs.", "All five patients were immunocompetent young men with a median age of 32 years (range, 28 to 40 years). All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay (Oxford Immunotec, Marlborough, MA, USA) and antibody of Mycobacterium tuberculosis, were negative.", "Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C (range, 39.0°C to 40.0°C), which persisted for 8 days (range, 6 to 11 days). Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days (range, 1 to 10 days). After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days (range, 8 to 11 days) ( Table 1) . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52). Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 (range = 49 to 62.5). White blood cell counts were low or in the normal range. All patients had elevated serum", "aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and hydroxybutyrate dehydrogenase (HBDH) ( Table 1) . At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range.", "Four patients had lower than normal T-cell subset counts (Table 2) .\n\nCXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ). Three patients were examined by highresolution computed tomography (HRCT). Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ). The mean duration from onset to a single-lobe consolidation on CXRs was 2 days (range = 1 to 5 days). The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days).", "All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days (Table 3) . Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy (Figure 3 ).", "Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours (range = 22 to 48 hours), and the mean time until IMV failure was 6.2 days (range 2 = to 13 days) ( Table 1) . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support.", "All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip). Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient (Table 3) . Four (80%) of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively.", "To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. (1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. (2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. (3) Viral load monitoring may help predict disease severity and patient outcome. (4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. (5) HAdV-55-induced severe ARDS", "has a very high mortality rate (80%) despite appropriate respiratory support.", "Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] . HAdV-55 was first completely characterized in Shaanxi, China [7] and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease [9] . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS.", "The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region.\n\nOur study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports [6, 9] .", "Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the", "reaction to therapy and patient outcome.", "The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation.\n\nOur study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response." ]

[]

[ "a lower T-cell count may be a risk factor for HAdV-55 infection in young adults" ]

[ "Emergent severe acute respiratory distress syndrome caused by adenovirus type 55 in immunocompetent adults in 2013: a prospective observational study\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4243941/\n\nSHA: f5b706d0529bfcf7e2d1dfc037df5b6f95fc5ec0\n\nAuthors: Sun, Bing; He, Hangyong; Wang, Zheng; Qu, Jiuxin; Li, Xuyan; Ban, Chengjun; Wan, Jun; Cao, Bin; Tong, Zhaohui; Wang, Chen\nDate: 2014-08-12\nDOI: 10.1186/s13054-014-0456-6\nLicense: cc-by", "Abstract: INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 (HAdV-55) in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome (ARDS) caused by HAdV-55 requiring invasive mechanical ventilation (IMV) and/or extracorporeal membrane oxygenation (ECMO) are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was", "5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10(8) copies in three patients and was 1 × 10(4) in one patient. It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation (NPPV) failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four (80%) of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most", "frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012", "Text: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients [1] [2] [3] [4] [5] . Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 [7] and in Singapore in 2005 [8] . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] . This pathogen was fully characterized by whole-genome sequencing [10] . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the", "serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes (HAdV-3, HAdV-7 and HAdV-14) [6] .", "Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU.", "Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation (NPPV) in ARDS patients was carried out in our center (ClinicalTrials.gov ID: NCT01585922). From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: (1) developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; (2) bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; (3) respiratory failure not fully explained by cardiac failure or fluid overload; (4) partial oxygen pressure/ fraction of inspired oxygen (PaO 2 /FiO 2 ) ≤100 mmHg with positive end-expiratory pressure (PEEP) ≥5 cmH 2 O; and (5) a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive", "mechanical ventilation (IMV) were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital (LLKYPJ2012031). Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication.", "Clinical information collected by investigators with a standardized data form included the following: demographic characteristics (age and sex), comorbidities, clinical symptoms (fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache), signs (body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs), laboratory tests (whole-blood cell count and blood chemistry) and microbiological findings and images of the lung (chest X-ray (CXR) and computed tomography). Concomitant medications, respiratory support, complications and outcomes were also recorded.", "Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay.\n\nViral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods.", "Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range).\n\nDuring the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs.", "All five patients were immunocompetent young men with a median age of 32 years (range, 28 to 40 years). All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay (Oxford Immunotec, Marlborough, MA, USA) and antibody of Mycobacterium tuberculosis, were negative.", "Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C (range, 39.0°C to 40.0°C), which persisted for 8 days (range, 6 to 11 days). Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days (range, 1 to 10 days). After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days (range, 8 to 11 days) ( Table 1) . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52). Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 (range = 49 to 62.5). White blood cell counts were low or in the normal range. All patients had elevated serum", "aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and hydroxybutyrate dehydrogenase (HBDH) ( Table 1) . At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range.", "Four patients had lower than normal T-cell subset counts (Table 2) .\n\nCXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ). Three patients were examined by highresolution computed tomography (HRCT). Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ). The mean duration from onset to a single-lobe consolidation on CXRs was 2 days (range = 1 to 5 days). The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days).", "All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days (Table 3) . Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy (Figure 3 ).", "Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours (range = 22 to 48 hours), and the mean time until IMV failure was 6.2 days (range 2 = to 13 days) ( Table 1) . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support.", "All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip). Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient (Table 3) . Four (80%) of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively.", "To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. (1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. (2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. (3) Viral load monitoring may help predict disease severity and patient outcome. (4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. (5) HAdV-55-induced severe ARDS", "has a very high mortality rate (80%) despite appropriate respiratory support.", "Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] . HAdV-55 was first completely characterized in Shaanxi, China [7] and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease [9] . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS.", "The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region.\n\nOur study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports [6, 9] .", "Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the", "reaction to therapy and patient outcome.", "The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation.\n\nOur study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response." ]

[ 23 ]

[ "we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV" ]

[ "Emergent severe acute respiratory distress syndrome caused by adenovirus type 55 in immunocompetent adults in 2013: a prospective observational study\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4243941/\n\nSHA: f5b706d0529bfcf7e2d1dfc037df5b6f95fc5ec0\n\nAuthors: Sun, Bing; He, Hangyong; Wang, Zheng; Qu, Jiuxin; Li, Xuyan; Ban, Chengjun; Wan, Jun; Cao, Bin; Tong, Zhaohui; Wang, Chen\nDate: 2014-08-12\nDOI: 10.1186/s13054-014-0456-6\nLicense: cc-by", "Abstract: INTRODUCTION: Since 2008, severe cases of emerging human adenovirus type 55 (HAdV-55) in immunocompetent adults have been reported sporadically in China. The clinical features and outcomes of the most critically ill patients with severe acute respiratory distress syndrome (ARDS) caused by HAdV-55 requiring invasive mechanical ventilation (IMV) and/or extracorporeal membrane oxygenation (ECMO) are lacking. METHODS: We conducted a prospective, single-center observational study of pneumonia with ARDS in immunocompetent adults admitted to our respiratory ICU. We prospectively collected and analyzed clinical, laboratory, radiological characteristics, sequential tests of viral load in respiratory tract and blood, treatments and outcomes. RESULTS: The results for a total of five consecutive patients with severe ARDS with confirmed HAdV-55 infection were included. All five patients were immunocompetent young men with a median age of 32 years. The mean time from onset to dyspnea was", "5 days. Arterial blood gas analysis at ICU admission revealed profound hypoxia. Mean partial oxygen pressure/fraction of inspired oxygen was 58.1. Mean durations from onset to a single-lobe consolidation shown on chest X-rays (CXRs) and, from the first positive CXR to bilateral multilobar lung infiltrates, were 2 days and 4.8 days, respectively. The viral load was higher than 1 × 10(8) copies in three patients and was 1 × 10(4) in one patient. It was negative in the only patient who survived. The mean duration for noninvasive positive pressure ventilation (NPPV) failure and IMV failure were 30.8 hours and 6.2 days, respectively. Four patients received venovenous ECMO. Four (80%) of the five patients died despite receiving appropriate respiratory support. CONCLUSIONS: HAdV-55 may cause severe ARDS in immunocompetent young men. Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates, are the most", "frequent clinical manifestations of HAdV-55-induced severe ARDS. Viral load monitoring may help predict disease severity and outcome. The NPPV and IMV failure rates were very high, but ECMO may still be the respiratory support therapy of choice. TRIAL REGISTRATION: Clinicaltrials.gov NCT01585922. Registered 20 April 2012", "Text: Human adenoviruses (HAdVs) are notorious pathogens in people with compromised immune function and a frequent cause of outbreaks of acute respiratory disease among young children. Life-threatening adenoviral pneumonia has previously been documented among military trainees, patients with AIDS and transplant recipients [1] [2] [3] [4] [5] . Human adenovirus type 55 (HAdV-55), which is emerging as a highly virulent pathogen for acute fatal adenoviral pneumonia among immunocompetent adults in China, has gained increasing attention [6] . HAdV-55 is a newly identified, emergent acute respiratory disease pathogen causing two recent outbreaks in China in 2006 [7] and in Singapore in 2005 [8] . In 2011, this pathogen apparently re-emerged in Beijing, China, causing several cases of severe community-acquired pneumonia [9] . This pathogen was fully characterized by whole-genome sequencing [10] . Comparative studies showed that the ability of HAdV to cause severe disease may relate to the", "serotypes of HAdVs. Severe adenoviral pneumonia induced by HAdV-55 has been reported to be more closely related to severe cases compared to other serotypes (HAdV-3, HAdV-7 and HAdV-14) [6] .", "Current knowledge of HAdV-55-induced severe acute respiratory distress syndrome (ARDS) requiring invasive mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO) support in immunocompetent adults is derived from single case reports or relatively small, single-center series. As a result, little information is available on HAdV-55 pneumonia complicated with severe ARDS, the frequency of which is expected to increase in the coming years. Here we describe the clinical features and outcomes of five prospective cases of HAdV-55 pneumonia complicated with severe ARDS in immunocompetent adults in our ICU.", "Beginning in May 2012, a randomized trial of noninvasive positive pressure ventilation (NPPV) in ARDS patients was carried out in our center (ClinicalTrials.gov ID: NCT01585922). From May 2012 to April 2014, all adult patients with ARDS caused by pneumonia who were admitted to the respiratory ICU of Beijing Chao-Yang Hospital were prospectively enrolled. Severe ARDS was diagnosed according to the Berlin definition: (1) developing within 1 week of a known clinical insult or new or worsening respiratory symptoms; (2) bilateral opacities not fully explained by effusions, lobar and/or lung collapse, or nodules; (3) respiratory failure not fully explained by cardiac failure or fluid overload; (4) partial oxygen pressure/ fraction of inspired oxygen (PaO 2 /FiO 2 ) ≤100 mmHg with positive end-expiratory pressure (PEEP) ≥5 cmH 2 O; and (5) a chest radiograph with three or four quadrants with opacities. Patients with HAdV-55 infection and severe ARDS who failed conventional NPPV and invasive", "mechanical ventilation (IMV) were included in the analysis. This study was approved by the Institutional Review Board of Beijing Chao-Yang Hospital (LLKYPJ2012031). Data were analyzed anonymously. Each patient gave written informed consent for their data to be used for research and publication.", "Clinical information collected by investigators with a standardized data form included the following: demographic characteristics (age and sex), comorbidities, clinical symptoms (fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache), signs (body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs), laboratory tests (whole-blood cell count and blood chemistry) and microbiological findings and images of the lung (chest X-ray (CXR) and computed tomography). Concomitant medications, respiratory support, complications and outcomes were also recorded.", "Patients' specimens, including sputum, whole blood and serum samples, were collected upon admission and during hospitalization. Microbiological tests were performed at the Department of Infectious Disease and Clinical Microbiology in our center, and the detection methods used were described in our previous report [6] . Common viruses causing respiratory illness were screened using a kit with 15 different viral assays. Serum samples were used for Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila antibodies. All patients had their HAdV-55 infection confirmed by RT-PCR assay. Partial sequences of the hexon gene were analyzed to type the phylogeny of HAdV-55 strains. The adenoviral load was also performed on both respiratory specimens and blood by multiplex RT-PCR assay.\n\nViral pneumonia was diagnosed based on the presence of HAdV detected in sputum or throat swab samples by molecular methods.", "Continuous variables were summarized as mean ± standard deviation (SD) or median (interquartile range).\n\nDuring the study period, a total of eight patients diagnosed with HAdV infection and respiratory failure were admitted to our ICU, and seven of them received a diagnosis of ARDS. Five consecutive patients with severe ARDS with confirmed HAdV-55 infection were admitted to our ICU between April and July 2013. They were included in the analysis. The other two patients had mild ARDS and were infected with other types of HAdVs.", "All five patients were immunocompetent young men with a median age of 32 years (range, 28 to 40 years). All of the patients shared a B blood type and came from the same city: Baoding city, Hebei province, northern China. All patients had no exposure to farm animals, corn or hay. Patient 3 had tuberculosis pleuritis and received antituberculosis therapy at ICU admission. His blood tests, including the T-SPOT tuberculosis assay (Oxford Immunotec, Marlborough, MA, USA) and antibody of Mycobacterium tuberculosis, were negative.", "Flulike symptoms, such as fever, cough and little sputum, were commonly observed at the onset of illness. All patients presented with a high fever, with a mean body temperature of 39.5°C (range, 39.0°C to 40.0°C), which persisted for 8 days (range, 6 to 11 days). Productive cough was observed in two patients. Dull substernal chest pain and rash were also observed in two patients. All patients had dyspnea. The mean time from onset to dyspnea was 5 days (range, 1 to 10 days). After the onset of dyspnea, patients usually progressed to respiratory failure or hypoxemia. The mean time from onset to ICU admission was 9.6 days (range, 8 to 11 days) ( Table 1) . All patients had tachypnea when admitted to the ICU, with a mean rate of 43 breaths per minute (range = 38 to 52). Arterial blood gas analysis at ICU admission revealed profound hypoxia, with a mean PaO 2 /FiO 2 of 58.1 (range = 49 to 62.5). White blood cell counts were low or in the normal range. All patients had elevated serum", "aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and hydroxybutyrate dehydrogenase (HBDH) ( Table 1) . At admission, all patients' levels of immunoglobulin (serum immunoglobulins G and M) and components C3 and C4 were in the normal range.", "Four patients had lower than normal T-cell subset counts (Table 2) .\n\nCXRs revealed multiple bilateral lobar or segment consolidation in the lungs of all five patients, and radiographic lesions progressed rapidly after ICU admission ( Figure 1 ). Three patients were examined by highresolution computed tomography (HRCT). Unilateral or bilateral consolidations and infiltrates were found on HRCT scans of all three of these patients. Consolidations within a single lobe or several lobes with a clear border and air bronchogram were the most common findings on HRCT scans. Nodules, patches, pleural effusion, abscess and a cavity were also seen visualized by HRCT (Figure 2 ). The mean duration from onset to a single-lobe consolidation on CXRs was 2 days (range = 1 to 5 days). The mean duration from the first positive CXR to bilaterally multilobar lung infiltrates was 4.8 days (range = 4 to 7 days).", "All patients had HAdV-55 viremia. In four of the five patients, it was first detected in endotracheal aspirate (ETA) samples. The time between initial ETA sample collection of adenoviruses and positive results for HAdV-55 nucleic acid in the blood was 1 to 10 days (Table 3) . Virus DNA copies in ETAs were determined for all patients during their ICU stay. The viral load was higher than 1 × 10 8 copies in three patients and 1 × 10 4 in one patient. The viral load became negative in the only patient who survived. In the four patients who did not survive, DNA copies did not decrease, even with antiviral therapy (Figure 3 ).", "Oxygenation was not maintained with conventional NPPV or IMV support in any of the patients. The mean duration until NPPV failure was 30.8 hours (range = 22 to 48 hours), and the mean time until IMV failure was 6.2 days (range 2 = to 13 days) ( Table 1) . Four patients received venovenous ECMO to maintain oxygen saturation, and one patient refused ECMO support and received high-frequency oscillatory ventilation instead. Table 4 gives the oxygenation data of patients before and after venovenous ECMO support.", "All patients received antiviral therapy, including acyclovir (10 mg/kg, every 8 hours, intravenous drip), ganciclovir (5 mg/kg, every 12 hours, intravenous drip) and ribavirin (250 mg, twice daily, intravenous drip). Considering that bacterial coinfection may combine with a severe viral infection, broad-spectrum intravenous antibiotics were given to all patients. Tests for bacterial pathogens were negative for only one patient (Table 3) . Four (80%) of the five patients died. Among the four patients receiving venovenous ECMO, only one patient survived. The other four patients died due to ARDS, Aspergillus fumigatus coinfection, septic shock and catheter-related bloodstream infection due to Acinetobacter baumannii, respectively.", "To the best of our knowledge, this is the first cohort observational study on the clinical characteristics of patients with severe ARDS caused by emergent HAdV-55 infection and also the first on the evaluation of a viral load test for monitoring the reaction to therapy and for prediction of patient outcome. The following are the main findings of this study. (1) HAdV-55 may cause severe ARDS in immunocompetent young men with blood type B. All of our patients were from the same city of Hebei province, northern China. (2) Persistent high fever, dyspnea and rapid progression to respiratory failure within 2 weeks, together with bilateral consolidations and infiltrates at the same time, are the most frequent clinical manifestations of severe HAdV-55induced ARDS. (3) Viral load monitoring may help predict disease severity and patient outcome. (4) The NPPV and IMV failure rates were very high, and ECMO may be the last support method for this group of patients. (5) HAdV-55-induced severe ARDS", "has a very high mortality rate (80%) despite appropriate respiratory support.", "Sporadic severe adenoviral infection in healthy adults has historically been described for serotype 4 [11] , serotype 7 [4, 12] and, more recently, serotype 14 in the general population and in military trainees [13, 14] . HAdV-55 was first completely characterized in Shaanxi, China [7] and then reemerged in Hebei, a province close to Beijing, where it caused several cases of acute respiratory disease [9] . It was presumed that HAdV-55 was a recombinant form of the B2 species of HAdV-14 and HAdV-11 [7, 15] due to its sharing a hexon gene with the HAdV-11 and HAdV-14 chassis [16] . The results of our study show that HAdV-55, as an emerging pathogen among immunocompetent adults, may cause severe ARDS.", "The prevalence of severe fatal adenoviral pneumonia induced by HAdV-55 in our study is somewhat similar to that described by Cao and colleagues [6] . All cases of reported HAdV-55 in our study were from the same city: Baoding, Hebei province, northern China. They occurred between April and July 2013, just partly overlapping or following the influenza epidemic. The patients with severe disease also came from the same region and were treated during a similar time period, which suggests that HAdV-55 may be an important viral pathogen derived from this region.\n\nOur study results suggest that the following may be clinical features of ARDS caused by HAdV-55: persistent high fever, rapid progression of dyspnea, need for mechanical ventilation support, elevated AST level and rapid progression from unilateral infiltrates to bilateral consolidations. These clinical features are highly similar to those of ARDS caused by other types of HAdV described in previous reports [6, 9] .", "Recent studies have shown that the immune system plays a crucial role in the clearance of HAdV viremia and survival of the host [17] . Chen et al. reported that, in the acute phase of HAdV-55 infection, patients with severe disease may have high levels of dendritic cells and Th17 cells [18] . In our study, the only patient who recovered from severe infection had higher T-cell counts. Three of the five patients had relatively low T-cell counts when admitted. Our results suggest that these three patients may have been relatively immunocompromised and that a lower T-cell count may be a risk factor for HAdV-55 infection in young adults. HAdV-55 DNA was previously reported in 41.2% of patients with severe infection [18] . In our study, HAdV-55 DNA was detected and monitored in all patients with severe ARDS. The initial, and trend of, viral load that presented as HAdV-55 DNA copies in the respiratory tract samples and blood may suggest the severity of infection and may predict both the", "reaction to therapy and patient outcome.", "The use of mechanical ventilation and ECMO in patients with ARDS caused by HAdV-55 has not been detailed in previous studies. In our cohort, we found that severe HAdV-55 infection could cause a rapid progression of respiratory failure, with a very high failure rate for NPPV and IMV. This failure rate may be a result of the large area of consolidation that induced a severe shunt in the lung, which may lead to lack of response to positive pressure ventilation. For patients with severe ARDS, ECMO should be considered a better choice for oxygenation.\n\nOur study has limitations. It is an observational study with no comparison group, so the difference between the severe and modest infections could not be clarified in terms of immune status, clinical features, radiological findings, viral load and treatment effects on respiratory support and antiviral therapy. Sequential dynamic analysis is needed to determine the relationship between HAdV-55 viremia and treatment response." ]

[ 25 ]

[ "Two hundred" ]

[ "Diagnostic accuracy of C-reactive protein and procalcitonin in suspected community-acquired pneumonia adults visiting emergency department and having a systematic thoracic CT scan\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4608327/\n\nSHA: f3d150545162ff3cc253c235011a02a91ee676cb\n\nAuthors: Le Bel, Josselin; Hausfater, Pierre; Chenevier-Gobeaux, Camille; Blanc, François-Xavier; Benjoar, Mikhael; Ficko, Cécile; Ray, Patrick; Choquet, Christophe; Duval, Xavier; Claessens, Yann-Erick\nDate: 2015-10-16\nDOI: 10.1186/s13054-015-1083-6\nLicense: cc-by", "Abstract: INTRODUCTION: Community-acquired pneumonia (CAP) requires prompt treatment, but its diagnosis is complex. Improvement of bacterial CAP diagnosis by biomarkers has been evaluated using chest X-ray infiltrate as the CAP gold standard, producing conflicting results. We analyzed the diagnostic accuracy of biomarkers in suspected CAP adults visiting emergency departments for whom CAP diagnosis was established by an adjudication committee which founded its judgment on a systematic multidetector thoracic CT scan. METHODS: In an ancillary study of a multi-center prospective study evaluating the impact of systematic thoracic CT scan on CAP diagnosis, sensitivity and specificity of C-reactive protein (CRP) and procalcitonin (PCT) were evaluated. Systematic nasopharyngeal multiplex respiratory virus PCR was performed at inclusion. An adjudication committee classified CAP diagnostic probability on a 4-level Likert scale, based on all available data. RESULTS: Two hundred patients with", "suspected CAP were analyzed. The adjudication committee classified 98 patients (49.0 %) as definite CAP, 8 (4.0 %) as probable, 23 (11.5 %) as possible and excluded in 71 (35.5 %, including 29 patients with pulmonary infiltrates on chest X-ray). Among patients with radiological pulmonary infiltrate, 23 % were finally classified as excluded. Viruses were identified by PCR in 29 % of patients classified as definite. Area under the curve was 0.787 [95 % confidence interval (95 % CI), 0.717 to 0.857] for CRP and 0.655 (95 % CI, 0.570 to 0.739) for PCT to detect definite CAP. CRP threshold at 50 mg/L resulted in a positive predictive value of 0.76 and a negative predictive value of 0.75. No PCT cut-off resulted in satisfactory positive or negative predictive values. CRP and PCT accuracy was not improved by exclusion of the 25 (25.5 %) definite viral CAP cases. CONCLUSIONS: For patients with suspected CAP visiting emergency departments, diagnostic accuracy of CRP and PCT are insufficient to", "confirm the CAP diagnosis established using a gold standard that includes thoracic CT scan. Diagnostic accuracy of these biomarkers is also insufficient to distinguish bacterial CAP from viral CAP. TRIAL REGISTRATION: ClinicalTrials.gov registry NCT01574066 (February 7, 2012) ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13054-015-1083-6) contains supplementary material, which is available to authorized users.", "Text: Community-acquired pneumonia (CAP) is a frequently seen disease, with high morbidity and mortality, accounting for 600,000 hospitalizations each year. It represents the seventh leading cause of death in the USA [1] . CAP prognosis depends on the rapidity of specific treatment, which should ideally be initiated within four hours and no later than eight hours after diagnosis [2, 3] . CAP diagnosis is based on the clustering of non-specific pulmonary and general symptoms [4, 5] , an increase in biomarkers reflecting systemic inflammatory response syndrome (SIRS), and the presence of new parenchymal infiltrates on chest X-ray. However, CAP diagnosis remains uncertain in many cases with alternative diagnoses, such as cardiac failure, acute bronchitis, chronic obstructive pulmonary disease (COPD) exacerbations, pulmonary embolism, neoplasia, and sepsis [6, 7] .", "Part of the uncertainty of CAP diagnosis may be due to the high rate of chest X-ray misdiagnosis [8, 9] ; over diagnosis of CAP is frequent when infiltrates of noninfectious origin coexist with pulmonary or general symptoms, and the diagnosis of CAP is often ignored when the lung infiltrates are at the limit of visibility or are hidden due to superposition [10] . We recently published a study in which thoracic CT scan was systematically performed in a population of clinically suspected CAP patients visiting the emergency department for CAP (the ESCAPED study) [11] . We showed that CAP diagnosis based on chest X-ray led to a false CAP diagnosis in many patients: among CAP suspected patients with radiological pulmonary infiltrate, CAP diagnosis was excluded in around 30 % of patients based on CT scan results; on the contrary, among patients without radiological pulmonary infiltrate, one-third had a pulmonary infiltrate on thoracic CT-scan. We also reported the isolation of viruses in", "one-third of patients [11, 12] .", "Several attempts have been made to improve CAP diagnosis based on biomarkers, such as C-reactive protein (CRP) and procalcitonin (PCT); however, there are conflicting data on their reliability [13] [14] [15] [16] [17] . This could be due to the consideration of CAP diagnosis based on chest X-ray as establishing pulmonary infection. In the present study, we aimed to analyze CRP and PCT values in the population of the ESCAPED study reported above for whom CAP diagnosis was established by an adjudication committee which founded its judgment on all usual available data, systematic multidetector thoracic CT scan performed at inclusion, and results from a day-28 follow-up. We also analyzed whether the viral etiology of definite CAP based on polymerase chain reaction (PCR) multiplex naso-pharyngeal swab interfered with the accuracy of the biomarkers.", "Setting ESCAPED was a multicenter, prospective, interventional study, entitled \"Early Thoracic CT-Scan for Community-Acquired Pneumonia at the Emergency Department (ESCAPED)\" [11] , conducted from November 2011 to January 2013, in four emergency departments (EDs) of four tertiary teaching hospitals in Paris, France, designed to measure the impact of thoracic CT scan on clinical decision. The study was sponsored and monitored by the Paris public health hospitals, and funded by the French Ministry of Health. The French health authorities (Agence nationale de sécurité des medicaments et produits de santé, ANSM) and the institutional review board for the protection of human subjects approved the study protocol and patient informed consent procedures. All enrolled patients provided written informed consent for inclusion. The protocol was registered in the clinicaltrial.gov website under the PACSCAN acronym, the French translation of the English ESCAPED acronym (NCT01574066). The Ethics", "Committee of Ile de France (Comité de Protection des Personnes. Paris N°2 011-oct-12749) approved the study protocol.", "The primary objective was to compare CRP and PCT values in the four different categories of CAP level of certainty using the day-28 adjudication committee classification. The four categories were: 1) absence of CAP hereafter referred to as excluded CAP diagnosis; 2) possible CAP; 3) probable CAP; and 4) definite CAP. The secondary objectives were to assess whether CRP and PCT were associated with CAP diagnosis using sensitivity analyses in three successive subgroups chosen a priori; 1) when specifically considering patients classified as having excluded CAP diagnosis and definite CAP (i.e., the patients for whom the level of certainty was the highest); 2) when patients with excluded CAP diagnosis and diagnosed extra-pulmonary infectious disease (which may increase biomarker values) were not taken into account, in the excluded CAP group; and 3) when patients classified as viral CAP were not taken into account in the definite CAP group, as PCT has been reported to be lower in viral", "infections as compared to bacterial infections [18] .", "Consecutive adults ( [19] .\n\nMultidetector thoracic CT-scan was performed after chest X-ray, ideally within the four hours following inclusion. Chest X-ray and thoracic CT-scan were performed using a standardized protocol. The four levels of CAP probability according to CT scan were defined as definite (systematic alveolar condensation, alveolar condensation with peripheral and localized ground glass opacities, bronchiolar focal or multifocal micronodules), probable (peripheral alveolar condensation, retractile systematic alveolar condensation, or diffuse ground glass opacities), possible (pulmonary infarct), or excluded (pulmonary mass, other abnormalities, or normal images). Scan views were recorded on a DVD.", "Based on data collected from baseline standardized case report forms, DVD recorded pictures of X-ray and CTscan, and blinded to local interpretations, an adjudication committee consisting of three independent senior experts in infectious diseases, pneumology and radiology retrospectively assigned the probability of CAP diagnosis using the same 4-level Likert scale, with all available data including patients' discharge summary, and follow-up data obtained by assistant investigators who contacted by phone either the patient, relatives or general practitioners at day 28. For this study, the gold standard of CAP was the diagnosis assessed by this adjudication committee. Alternative diagnoses were established for excluded CAP and classified as non-CAP pulmonary diseases and extra-pulmonary infectious diseases and others.", "Blood samples were collected at inclusion in sodium heparin-treated tubes, centrifuged, and stored at −40°C until completion of the study. CRP and PCT concentrations were measured a posteriori on plasma collection (see Additional file 1 for methodology), except for patients in whom marker dosage was performed by the emergency practitioner on his own initiative.", "Naso-pharyngeal swabs were collected at enrollment and placed in a Middle Virocult MWE (Sigma®) transport medium. Samples were kept at room temperature and sent to the virology laboratory of Bichat -Claude Bernard Hospital (Paris) as soon as possible after collection. The samples were not frozen and thawed. Multiplex PCR (RespiFinder-19 assay (Pathofinder®, Maastricht, Netherlands)) was performed on naso-pharyngeal swabs to detect 15 respiratory viruses -coronavirus 229E, NL63, OC43, human metapneumovirus (hMPV), influenza A, A (H1N1) pdm2009 and B viruses, parainfluenza viruses 1, 2, 3, and 4, respiratory syncytial virus (RSV) A and B, rhinovirus, adenovirus, and 4 intracellular bacteria -Bordetella pertussis, Chlamydophila pneumoniae, Legionella pneumophila, Mycoplasma pneumoniae, in one reaction. The multiplex PCR results were not available to the adjudication committee. Routine microbiological examinations were also performed at the discretion of the emergency physicians and", "included blood culture, sputum culture, and antigenuria (see Additional file 1 for methodology). CAP, classified as definite, was considered as being of viral origin when multiplex PCR was positive for at least one of the 15 respiratory viruses and no bacteria were found using PCR and routine bacterial microbiological samples (sputum, blood culture, antigenuria) when performed.", "Baseline and follow-up characteristics were described by means and standard deviations (SD) or by median and interquartile range (IQR) for continuous variables normally distributed or with skewed distribution, respectively, and by percentages for categorical variables, for the total study population and for the study groups. We performed chi-square or Fisher exact tests when appropriate for qualitative variables, and the Student or Mann-Whitney tests for continuous variables with skewed distributions to compare baseline patient characteristics and study outcomes between study groups.", "The distribution values of the biomarkers were determined in the different populations of patients using boxplots. The performances of CRP and PCT in predicting definite CAP were evaluated by sensitivity analysis (definite CAP vs excluded CAP). CRP was evaluated at several cut-off points of 20 mg/L, 30 mg/L, 50 mg/L, 70 mg/L, and 100 mg/L, values used in previous studies [15, 20, 21] . Several cut-off points for PCT were chosen at the level of 0.10 μg/L [18] , and at the two levels for suspected bacterial infection as stated by the manufacturer, i.e., 0.25 μg/L and 0.50 μg/L. Sensitivities, specificities, positive predictive values (PPVs), negative predictive values (NPVs), and likelihood ratio were calculated. Receiver operating characteristic (ROC) curves were drawn, area under the curve AUC was computed and optimal cut-off was identified by the maximization of the Youden's index, comparing biomarker values in patients with excluded CAP and definite CAP. From these optimal cut-offs", "for CRP and PCT, sensitivity analyses were performed combining the CRP and PCT cut-offs.", "A multivariate logistic regression model was built to identify factors associated with having definite CAP as compared to having an excluded CAP diagnosis. We excluded from the excluded CAP diagnosis group, patients with an extra-pulmonary infectious disease. All variables with a p value of < 0.25 in the bivariate analysis were entered into a multivariate logistic regression with a backward stepwise approach; the discrimination was evaluated by the C-index and its 95 % confidence interval (95 % CI) and the calibration was evaluated by the Hosmer Lemeshow goodness-of-fit test.\n\nAll tests were two-sided, and p-values below 0.05 were considered to denote statistical significance. All statistical analyses were performed using SPSS statistical software version 21.0 (SPSS Inc., Chicago, IL, USA).", "Two hundred patients with suspected CAP out of the 319 in the ESCAPED study were included in the present study, for which CRP and PCT assays and nasopharyngeal swab for multiplex PCR were available (Fig. 1) . Characteristics of the 200 patients (age, age more than 65, gender, probability of CAP diagnosis by adjudication committee) were not significantly different from those of the 119 other patients of the ESCAPED study and are summarized in Table 1 . CRP and PCT assays were performed based on the emergency practitioner's own initiative in 70 patients for CRP and 131 for PCT, or performed a posteriori on plasma samples of the remaining patients. Sex ratio was approximately 1. More than half of the patients (54 %) were 65 years of age or older. The", "Pulmonary infiltrates were seen on chest X-ray in 127 (63.5 %) patients. Thoracic CT-scan excluded a CAP diagnosis in 16.5 % of these 127 patients; on the contrary, thoracic CT-scan revealed a parenchymal infiltrate in 27 % of the 73 patients without infiltrate on chest X-ray.\n\nBased on all available data including multidetector CT scan results (but excluding PCR results), the adjudication", "The CRP and PCT distributions in the 200 patients are presented in Fig. 2 A statistically significant difference between the two groups (excluded CAP vs definite CAP) was demonstrated for several cut-off points for CRP and PCT ( Table 2 ). For CRP, the value of 50 mg/L resulted in a PPV of 0.76 and a NPV of 0.75. For PCT, no value resulted in a satisfactory PPV or NPV. For these two biochemical markers, the ability to predict CAP was evaluated by a ROC curve. The AUC was 0.787 (95 % CI 0.717-0.857), optimal cut-off = 45.9 mg/L for CRP (Fig. 3 ) and 0.655 (95 % CI 0.570-0.739), optimal cut-off = 0.13 μg/ L for PCT (Fig. 4) .\n\nSensitivity analyses for the combination of CRP and PCT, using these optimal cut-offs, resulted in a PPV of 0.74 and a NPV of 0.58. Use of the other PCT cut-offs did not result in better PPV or NPV ( Table 2) .", "The present study is novel as patients prospectively benefited from extensive investigation to determine the diagnosis of CAP in the ED, including both early multidetector thoracic CT-scan and day-28 adjudication committee. This led to the correction of CAP diagnosis previously based on chest X-ray in a high number of patients. In these extensively characterized patients, both CRP and PCT lacked operational precision to allow the decisionmaking process to rule out or confirm diagnosis of CAP even in selected subgroups.", "The clinical characteristics of the patients included in this sub-study are consistent with those in the current literature. As previously reported, patients frequently had a history of respiratory disorders, cancer and congestive heart failure [21, 22] . The design of the ESCAPED study required exclusion of patients within the highest CRB 65 categories, which limited the inclusion of patients older than 65. This may explain why the mean age of our patients (64 years) falls within the lower values of those reported elsewhere [19] . Data to identify the microbial agent responsible for the disease were collected by the usual techniques and multiplex PCR. Viral identification using naso-pharyngeal PCR that revealed viral respiratory infection in approximately one-third of cases was concordant with values reported in the literature [23] . Therefore, we believe that our results can be extrapolated to most emergency patients suffering from CAP.", "In the present study, patients were recruited on the basis of initial clinical assessment for the diagnosis of CAP. Therefore, we believe that the characteristics of the patients closely correspond to those that lead practitioners to consider a possible diagnosis of CAP. In these patients, the design of our study allowed us to confirm or refute CAP diagnosis with a high level of certainty. Results confirmed the poor predictive value of clinical symptoms (new onset of systemic features and symptoms of an acute lower respiratory tract illness) in identifying CAP patients [21] . Indeed, clinical presentation of excluded CAP patients was similar to that of definite CAP patients except for fever and cough that were more frequent in definite CAP patients. Furthermore, the design also revealed that the combination of clinical symptoms and chest X-ray results led to CAP misdiagnosis in a high number of patients, including the 98 whose CAP diagnosis was excluded by the adjudication committee", "and who would have been considered as possible, probable or definite CAP without the use of the CT scan. This low specificity of clinical-standard radiological evaluation led to the consideration of either non-infectious pulmonary diseases (such as, cardiac failure, pulmonary embolism, pulmonary neoplasia or bronchitis) or extra-pulmonary infectious diseases as CAP. Of note, some of these diseases are also associated with increased biomarker values. This raises concerns about previous evaluations of biomarkers in CAP-suspected patients, which used clinical and standard radiological (chest X-ray) evaluations as the gold standard for CAP diagnosis [15] . The use of biomarkers has been advocated to improve diagnosis and management of patients with lower respiratory tract infections [14] . However, this issue is still unresolved [24] , with conflicting positions [14, 15, 25, 26] . In our study, while median values of both biomarkers did increase with level of certainty for CAP diagnosis,", "we were unable to establish discriminating values for PCT. Recent data suggested that CRP could be of more help in assisting in the diagnosis of lower respiratory tract infections (LRTI) [15, 27, 28] . In our study, although CRP seems more discriminating than PCT, neither the experimental exclusion of extra-pulmonary bacterial infections from the excluded CAP group, nor the exclusion of viral CAP from the definite CAP patients group, made possible the determination of a discriminant cutoff. The combination of CRP and PCT was not more discriminating than each biomarker separately. An operational algorithm has been released to assist physicians in prescribing antimicrobial therapy [14, 26, 29] . According to this strategy, a PCT concentration higher than 0.25 μg/L should prompt administration of antibiotics to patients with suspected LRTI. In our study, this value was associated with poor performance. Additionally, mean PCT levels remained above this threshold both in excluded CAP", "patients without infectious disorders and in definite CAP presumably related to virus. Therefore, the gold standard for the diagnosis of CAP may influence the performance and utility of PCT in this setting.", "This study has some limitations. First, the adjudication committee was not blinded to the value of biomarkers measured at bedside in some patients (70 for CRP and 131 for PCT) and its CAP classification could thus have been influenced by these results. However, the lack of statistically significant differences in the mean CRP and PCT values in the definite CAP cases, whether or not these biomarkers were available for the adjudication committee, argues against a major impact of these results on adjudication committee classification. Second, another critical point is the prescription of antibiotic therapy (34 %) previous to inclusion. We cannot exclude that these previously-treated CAP patients may have altered biomarker performance and reduced the yield of bacterial cultures, although such a population reflects the usual emergency department practice. Third, multiplex PCR was performed on naso-pharyngeal sampling and not on lower respiratory tract samples, which does not allow definite", "confirmation of the viral origin of CAP. However, a recent large study on CAP patients which reported a viral etiology of CAP at a comparable rate, did not find upper respiratory tract shedding in a control population without CAP explored during the same year and season [30] . Finally, even if multidetector thoracic CT scan is a better imaging examination than X-ray to explore the chest, only invasive local microbiological samples would have provided a diagnosis with certainty.", "Given the diversity of the clinical and radiological CAP presentations, CAP diagnosis is often uncertain. In our population of patients treated in the emergency room with clinical symptoms evoking CAP, neither CRP nor PCT cut-off values carried sufficient weight to confirm or refute CAP diagnosis at bedside; this underlines that these biomarkers are telltales of the host inflammatory response to the intrusion of microorganisms independent of the site of infection. These results, based on a systematic thoracic CT scan evaluation of CAP-suspected patients, do not argue for the use of CRP and PCT in routine care to diagnose CAP with certainty in patients visiting the ED for suspected CAP." ]

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[ "Diagnostic accuracy of C-reactive protein and procalcitonin in suspected community-acquired pneumonia adults visiting emergency department and having a systematic thoracic CT scan\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4608327/\n\nSHA: f3d150545162ff3cc253c235011a02a91ee676cb\n\nAuthors: Le Bel, Josselin; Hausfater, Pierre; Chenevier-Gobeaux, Camille; Blanc, François-Xavier; Benjoar, Mikhael; Ficko, Cécile; Ray, Patrick; Choquet, Christophe; Duval, Xavier; Claessens, Yann-Erick\nDate: 2015-10-16\nDOI: 10.1186/s13054-015-1083-6\nLicense: cc-by", "Abstract: INTRODUCTION: Community-acquired pneumonia (CAP) requires prompt treatment, but its diagnosis is complex. Improvement of bacterial CAP diagnosis by biomarkers has been evaluated using chest X-ray infiltrate as the CAP gold standard, producing conflicting results. We analyzed the diagnostic accuracy of biomarkers in suspected CAP adults visiting emergency departments for whom CAP diagnosis was established by an adjudication committee which founded its judgment on a systematic multidetector thoracic CT scan. METHODS: In an ancillary study of a multi-center prospective study evaluating the impact of systematic thoracic CT scan on CAP diagnosis, sensitivity and specificity of C-reactive protein (CRP) and procalcitonin (PCT) were evaluated. Systematic nasopharyngeal multiplex respiratory virus PCR was performed at inclusion. An adjudication committee classified CAP diagnostic probability on a 4-level Likert scale, based on all available data. RESULTS: Two hundred patients with", "suspected CAP were analyzed. The adjudication committee classified 98 patients (49.0 %) as definite CAP, 8 (4.0 %) as probable, 23 (11.5 %) as possible and excluded in 71 (35.5 %, including 29 patients with pulmonary infiltrates on chest X-ray). Among patients with radiological pulmonary infiltrate, 23 % were finally classified as excluded. Viruses were identified by PCR in 29 % of patients classified as definite. Area under the curve was 0.787 [95 % confidence interval (95 % CI), 0.717 to 0.857] for CRP and 0.655 (95 % CI, 0.570 to 0.739) for PCT to detect definite CAP. CRP threshold at 50 mg/L resulted in a positive predictive value of 0.76 and a negative predictive value of 0.75. No PCT cut-off resulted in satisfactory positive or negative predictive values. CRP and PCT accuracy was not improved by exclusion of the 25 (25.5 %) definite viral CAP cases. CONCLUSIONS: For patients with suspected CAP visiting emergency departments, diagnostic accuracy of CRP and PCT are insufficient to", "confirm the CAP diagnosis established using a gold standard that includes thoracic CT scan. Diagnostic accuracy of these biomarkers is also insufficient to distinguish bacterial CAP from viral CAP. TRIAL REGISTRATION: ClinicalTrials.gov registry NCT01574066 (February 7, 2012) ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13054-015-1083-6) contains supplementary material, which is available to authorized users.", "Text: Community-acquired pneumonia (CAP) is a frequently seen disease, with high morbidity and mortality, accounting for 600,000 hospitalizations each year. It represents the seventh leading cause of death in the USA [1] . CAP prognosis depends on the rapidity of specific treatment, which should ideally be initiated within four hours and no later than eight hours after diagnosis [2, 3] . CAP diagnosis is based on the clustering of non-specific pulmonary and general symptoms [4, 5] , an increase in biomarkers reflecting systemic inflammatory response syndrome (SIRS), and the presence of new parenchymal infiltrates on chest X-ray. However, CAP diagnosis remains uncertain in many cases with alternative diagnoses, such as cardiac failure, acute bronchitis, chronic obstructive pulmonary disease (COPD) exacerbations, pulmonary embolism, neoplasia, and sepsis [6, 7] .", "Part of the uncertainty of CAP diagnosis may be due to the high rate of chest X-ray misdiagnosis [8, 9] ; over diagnosis of CAP is frequent when infiltrates of noninfectious origin coexist with pulmonary or general symptoms, and the diagnosis of CAP is often ignored when the lung infiltrates are at the limit of visibility or are hidden due to superposition [10] . We recently published a study in which thoracic CT scan was systematically performed in a population of clinically suspected CAP patients visiting the emergency department for CAP (the ESCAPED study) [11] . We showed that CAP diagnosis based on chest X-ray led to a false CAP diagnosis in many patients: among CAP suspected patients with radiological pulmonary infiltrate, CAP diagnosis was excluded in around 30 % of patients based on CT scan results; on the contrary, among patients without radiological pulmonary infiltrate, one-third had a pulmonary infiltrate on thoracic CT-scan. We also reported the isolation of viruses in", "one-third of patients [11, 12] .", "Several attempts have been made to improve CAP diagnosis based on biomarkers, such as C-reactive protein (CRP) and procalcitonin (PCT); however, there are conflicting data on their reliability [13] [14] [15] [16] [17] . This could be due to the consideration of CAP diagnosis based on chest X-ray as establishing pulmonary infection. In the present study, we aimed to analyze CRP and PCT values in the population of the ESCAPED study reported above for whom CAP diagnosis was established by an adjudication committee which founded its judgment on all usual available data, systematic multidetector thoracic CT scan performed at inclusion, and results from a day-28 follow-up. We also analyzed whether the viral etiology of definite CAP based on polymerase chain reaction (PCR) multiplex naso-pharyngeal swab interfered with the accuracy of the biomarkers.", "Setting ESCAPED was a multicenter, prospective, interventional study, entitled \"Early Thoracic CT-Scan for Community-Acquired Pneumonia at the Emergency Department (ESCAPED)\" [11] , conducted from November 2011 to January 2013, in four emergency departments (EDs) of four tertiary teaching hospitals in Paris, France, designed to measure the impact of thoracic CT scan on clinical decision. The study was sponsored and monitored by the Paris public health hospitals, and funded by the French Ministry of Health. The French health authorities (Agence nationale de sécurité des medicaments et produits de santé, ANSM) and the institutional review board for the protection of human subjects approved the study protocol and patient informed consent procedures. All enrolled patients provided written informed consent for inclusion. The protocol was registered in the clinicaltrial.gov website under the PACSCAN acronym, the French translation of the English ESCAPED acronym (NCT01574066). The Ethics", "Committee of Ile de France (Comité de Protection des Personnes. Paris N°2 011-oct-12749) approved the study protocol.", "The primary objective was to compare CRP and PCT values in the four different categories of CAP level of certainty using the day-28 adjudication committee classification. The four categories were: 1) absence of CAP hereafter referred to as excluded CAP diagnosis; 2) possible CAP; 3) probable CAP; and 4) definite CAP. The secondary objectives were to assess whether CRP and PCT were associated with CAP diagnosis using sensitivity analyses in three successive subgroups chosen a priori; 1) when specifically considering patients classified as having excluded CAP diagnosis and definite CAP (i.e., the patients for whom the level of certainty was the highest); 2) when patients with excluded CAP diagnosis and diagnosed extra-pulmonary infectious disease (which may increase biomarker values) were not taken into account, in the excluded CAP group; and 3) when patients classified as viral CAP were not taken into account in the definite CAP group, as PCT has been reported to be lower in viral", "infections as compared to bacterial infections [18] .", "Consecutive adults ( [19] .\n\nMultidetector thoracic CT-scan was performed after chest X-ray, ideally within the four hours following inclusion. Chest X-ray and thoracic CT-scan were performed using a standardized protocol. The four levels of CAP probability according to CT scan were defined as definite (systematic alveolar condensation, alveolar condensation with peripheral and localized ground glass opacities, bronchiolar focal or multifocal micronodules), probable (peripheral alveolar condensation, retractile systematic alveolar condensation, or diffuse ground glass opacities), possible (pulmonary infarct), or excluded (pulmonary mass, other abnormalities, or normal images). Scan views were recorded on a DVD.", "Based on data collected from baseline standardized case report forms, DVD recorded pictures of X-ray and CTscan, and blinded to local interpretations, an adjudication committee consisting of three independent senior experts in infectious diseases, pneumology and radiology retrospectively assigned the probability of CAP diagnosis using the same 4-level Likert scale, with all available data including patients' discharge summary, and follow-up data obtained by assistant investigators who contacted by phone either the patient, relatives or general practitioners at day 28. For this study, the gold standard of CAP was the diagnosis assessed by this adjudication committee. Alternative diagnoses were established for excluded CAP and classified as non-CAP pulmonary diseases and extra-pulmonary infectious diseases and others.", "Blood samples were collected at inclusion in sodium heparin-treated tubes, centrifuged, and stored at −40°C until completion of the study. CRP and PCT concentrations were measured a posteriori on plasma collection (see Additional file 1 for methodology), except for patients in whom marker dosage was performed by the emergency practitioner on his own initiative.", "Naso-pharyngeal swabs were collected at enrollment and placed in a Middle Virocult MWE (Sigma®) transport medium. Samples were kept at room temperature and sent to the virology laboratory of Bichat -Claude Bernard Hospital (Paris) as soon as possible after collection. The samples were not frozen and thawed. Multiplex PCR (RespiFinder-19 assay (Pathofinder®, Maastricht, Netherlands)) was performed on naso-pharyngeal swabs to detect 15 respiratory viruses -coronavirus 229E, NL63, OC43, human metapneumovirus (hMPV), influenza A, A (H1N1) pdm2009 and B viruses, parainfluenza viruses 1, 2, 3, and 4, respiratory syncytial virus (RSV) A and B, rhinovirus, adenovirus, and 4 intracellular bacteria -Bordetella pertussis, Chlamydophila pneumoniae, Legionella pneumophila, Mycoplasma pneumoniae, in one reaction. The multiplex PCR results were not available to the adjudication committee. Routine microbiological examinations were also performed at the discretion of the emergency physicians and", "included blood culture, sputum culture, and antigenuria (see Additional file 1 for methodology). CAP, classified as definite, was considered as being of viral origin when multiplex PCR was positive for at least one of the 15 respiratory viruses and no bacteria were found using PCR and routine bacterial microbiological samples (sputum, blood culture, antigenuria) when performed.", "Baseline and follow-up characteristics were described by means and standard deviations (SD) or by median and interquartile range (IQR) for continuous variables normally distributed or with skewed distribution, respectively, and by percentages for categorical variables, for the total study population and for the study groups. We performed chi-square or Fisher exact tests when appropriate for qualitative variables, and the Student or Mann-Whitney tests for continuous variables with skewed distributions to compare baseline patient characteristics and study outcomes between study groups.", "The distribution values of the biomarkers were determined in the different populations of patients using boxplots. The performances of CRP and PCT in predicting definite CAP were evaluated by sensitivity analysis (definite CAP vs excluded CAP). CRP was evaluated at several cut-off points of 20 mg/L, 30 mg/L, 50 mg/L, 70 mg/L, and 100 mg/L, values used in previous studies [15, 20, 21] . Several cut-off points for PCT were chosen at the level of 0.10 μg/L [18] , and at the two levels for suspected bacterial infection as stated by the manufacturer, i.e., 0.25 μg/L and 0.50 μg/L. Sensitivities, specificities, positive predictive values (PPVs), negative predictive values (NPVs), and likelihood ratio were calculated. Receiver operating characteristic (ROC) curves were drawn, area under the curve AUC was computed and optimal cut-off was identified by the maximization of the Youden's index, comparing biomarker values in patients with excluded CAP and definite CAP. From these optimal cut-offs", "for CRP and PCT, sensitivity analyses were performed combining the CRP and PCT cut-offs.", "A multivariate logistic regression model was built to identify factors associated with having definite CAP as compared to having an excluded CAP diagnosis. We excluded from the excluded CAP diagnosis group, patients with an extra-pulmonary infectious disease. All variables with a p value of < 0.25 in the bivariate analysis were entered into a multivariate logistic regression with a backward stepwise approach; the discrimination was evaluated by the C-index and its 95 % confidence interval (95 % CI) and the calibration was evaluated by the Hosmer Lemeshow goodness-of-fit test.\n\nAll tests were two-sided, and p-values below 0.05 were considered to denote statistical significance. All statistical analyses were performed using SPSS statistical software version 21.0 (SPSS Inc., Chicago, IL, USA).", "Two hundred patients with suspected CAP out of the 319 in the ESCAPED study were included in the present study, for which CRP and PCT assays and nasopharyngeal swab for multiplex PCR were available (Fig. 1) . Characteristics of the 200 patients (age, age more than 65, gender, probability of CAP diagnosis by adjudication committee) were not significantly different from those of the 119 other patients of the ESCAPED study and are summarized in Table 1 . CRP and PCT assays were performed based on the emergency practitioner's own initiative in 70 patients for CRP and 131 for PCT, or performed a posteriori on plasma samples of the remaining patients. Sex ratio was approximately 1. More than half of the patients (54 %) were 65 years of age or older. The", "Pulmonary infiltrates were seen on chest X-ray in 127 (63.5 %) patients. Thoracic CT-scan excluded a CAP diagnosis in 16.5 % of these 127 patients; on the contrary, thoracic CT-scan revealed a parenchymal infiltrate in 27 % of the 73 patients without infiltrate on chest X-ray.\n\nBased on all available data including multidetector CT scan results (but excluding PCR results), the adjudication", "The CRP and PCT distributions in the 200 patients are presented in Fig. 2 A statistically significant difference between the two groups (excluded CAP vs definite CAP) was demonstrated for several cut-off points for CRP and PCT ( Table 2 ). For CRP, the value of 50 mg/L resulted in a PPV of 0.76 and a NPV of 0.75. For PCT, no value resulted in a satisfactory PPV or NPV. For these two biochemical markers, the ability to predict CAP was evaluated by a ROC curve. The AUC was 0.787 (95 % CI 0.717-0.857), optimal cut-off = 45.9 mg/L for CRP (Fig. 3 ) and 0.655 (95 % CI 0.570-0.739), optimal cut-off = 0.13 μg/ L for PCT (Fig. 4) .\n\nSensitivity analyses for the combination of CRP and PCT, using these optimal cut-offs, resulted in a PPV of 0.74 and a NPV of 0.58. Use of the other PCT cut-offs did not result in better PPV or NPV ( Table 2) .", "The present study is novel as patients prospectively benefited from extensive investigation to determine the diagnosis of CAP in the ED, including both early multidetector thoracic CT-scan and day-28 adjudication committee. This led to the correction of CAP diagnosis previously based on chest X-ray in a high number of patients. In these extensively characterized patients, both CRP and PCT lacked operational precision to allow the decisionmaking process to rule out or confirm diagnosis of CAP even in selected subgroups.", "The clinical characteristics of the patients included in this sub-study are consistent with those in the current literature. As previously reported, patients frequently had a history of respiratory disorders, cancer and congestive heart failure [21, 22] . The design of the ESCAPED study required exclusion of patients within the highest CRB 65 categories, which limited the inclusion of patients older than 65. This may explain why the mean age of our patients (64 years) falls within the lower values of those reported elsewhere [19] . Data to identify the microbial agent responsible for the disease were collected by the usual techniques and multiplex PCR. Viral identification using naso-pharyngeal PCR that revealed viral respiratory infection in approximately one-third of cases was concordant with values reported in the literature [23] . Therefore, we believe that our results can be extrapolated to most emergency patients suffering from CAP.", "In the present study, patients were recruited on the basis of initial clinical assessment for the diagnosis of CAP. Therefore, we believe that the characteristics of the patients closely correspond to those that lead practitioners to consider a possible diagnosis of CAP. In these patients, the design of our study allowed us to confirm or refute CAP diagnosis with a high level of certainty. Results confirmed the poor predictive value of clinical symptoms (new onset of systemic features and symptoms of an acute lower respiratory tract illness) in identifying CAP patients [21] . Indeed, clinical presentation of excluded CAP patients was similar to that of definite CAP patients except for fever and cough that were more frequent in definite CAP patients. Furthermore, the design also revealed that the combination of clinical symptoms and chest X-ray results led to CAP misdiagnosis in a high number of patients, including the 98 whose CAP diagnosis was excluded by the adjudication committee", "and who would have been considered as possible, probable or definite CAP without the use of the CT scan. This low specificity of clinical-standard radiological evaluation led to the consideration of either non-infectious pulmonary diseases (such as, cardiac failure, pulmonary embolism, pulmonary neoplasia or bronchitis) or extra-pulmonary infectious diseases as CAP. Of note, some of these diseases are also associated with increased biomarker values. This raises concerns about previous evaluations of biomarkers in CAP-suspected patients, which used clinical and standard radiological (chest X-ray) evaluations as the gold standard for CAP diagnosis [15] . The use of biomarkers has been advocated to improve diagnosis and management of patients with lower respiratory tract infections [14] . However, this issue is still unresolved [24] , with conflicting positions [14, 15, 25, 26] . In our study, while median values of both biomarkers did increase with level of certainty for CAP diagnosis,", "we were unable to establish discriminating values for PCT. Recent data suggested that CRP could be of more help in assisting in the diagnosis of lower respiratory tract infections (LRTI) [15, 27, 28] . In our study, although CRP seems more discriminating than PCT, neither the experimental exclusion of extra-pulmonary bacterial infections from the excluded CAP group, nor the exclusion of viral CAP from the definite CAP patients group, made possible the determination of a discriminant cutoff. The combination of CRP and PCT was not more discriminating than each biomarker separately. An operational algorithm has been released to assist physicians in prescribing antimicrobial therapy [14, 26, 29] . According to this strategy, a PCT concentration higher than 0.25 μg/L should prompt administration of antibiotics to patients with suspected LRTI. In our study, this value was associated with poor performance. Additionally, mean PCT levels remained above this threshold both in excluded CAP", "patients without infectious disorders and in definite CAP presumably related to virus. Therefore, the gold standard for the diagnosis of CAP may influence the performance and utility of PCT in this setting.", "This study has some limitations. First, the adjudication committee was not blinded to the value of biomarkers measured at bedside in some patients (70 for CRP and 131 for PCT) and its CAP classification could thus have been influenced by these results. However, the lack of statistically significant differences in the mean CRP and PCT values in the definite CAP cases, whether or not these biomarkers were available for the adjudication committee, argues against a major impact of these results on adjudication committee classification. Second, another critical point is the prescription of antibiotic therapy (34 %) previous to inclusion. We cannot exclude that these previously-treated CAP patients may have altered biomarker performance and reduced the yield of bacterial cultures, although such a population reflects the usual emergency department practice. Third, multiplex PCR was performed on naso-pharyngeal sampling and not on lower respiratory tract samples, which does not allow definite", "confirmation of the viral origin of CAP. However, a recent large study on CAP patients which reported a viral etiology of CAP at a comparable rate, did not find upper respiratory tract shedding in a control population without CAP explored during the same year and season [30] . Finally, even if multidetector thoracic CT scan is a better imaging examination than X-ray to explore the chest, only invasive local microbiological samples would have provided a diagnosis with certainty.", "Given the diversity of the clinical and radiological CAP presentations, CAP diagnosis is often uncertain. In our population of patients treated in the emergency room with clinical symptoms evoking CAP, neither CRP nor PCT cut-off values carried sufficient weight to confirm or refute CAP diagnosis at bedside; this underlines that these biomarkers are telltales of the host inflammatory response to the intrusion of microorganisms independent of the site of infection. These results, based on a systematic thoracic CT scan evaluation of CAP-suspected patients, do not argue for the use of CRP and PCT in routine care to diagnose CAP with certainty in patients visiting the ED for suspected CAP." ]

[ 4 ]

[ "the presence of new parenchymal infiltrates" ]

[ "Diagnostic accuracy of C-reactive protein and procalcitonin in suspected community-acquired pneumonia adults visiting emergency department and having a systematic thoracic CT scan\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4608327/\n\nSHA: f3d150545162ff3cc253c235011a02a91ee676cb\n\nAuthors: Le Bel, Josselin; Hausfater, Pierre; Chenevier-Gobeaux, Camille; Blanc, François-Xavier; Benjoar, Mikhael; Ficko, Cécile; Ray, Patrick; Choquet, Christophe; Duval, Xavier; Claessens, Yann-Erick\nDate: 2015-10-16\nDOI: 10.1186/s13054-015-1083-6\nLicense: cc-by", "Abstract: INTRODUCTION: Community-acquired pneumonia (CAP) requires prompt treatment, but its diagnosis is complex. Improvement of bacterial CAP diagnosis by biomarkers has been evaluated using chest X-ray infiltrate as the CAP gold standard, producing conflicting results. We analyzed the diagnostic accuracy of biomarkers in suspected CAP adults visiting emergency departments for whom CAP diagnosis was established by an adjudication committee which founded its judgment on a systematic multidetector thoracic CT scan. METHODS: In an ancillary study of a multi-center prospective study evaluating the impact of systematic thoracic CT scan on CAP diagnosis, sensitivity and specificity of C-reactive protein (CRP) and procalcitonin (PCT) were evaluated. Systematic nasopharyngeal multiplex respiratory virus PCR was performed at inclusion. An adjudication committee classified CAP diagnostic probability on a 4-level Likert scale, based on all available data. RESULTS: Two hundred patients with", "suspected CAP were analyzed. The adjudication committee classified 98 patients (49.0 %) as definite CAP, 8 (4.0 %) as probable, 23 (11.5 %) as possible and excluded in 71 (35.5 %, including 29 patients with pulmonary infiltrates on chest X-ray). Among patients with radiological pulmonary infiltrate, 23 % were finally classified as excluded. Viruses were identified by PCR in 29 % of patients classified as definite. Area under the curve was 0.787 [95 % confidence interval (95 % CI), 0.717 to 0.857] for CRP and 0.655 (95 % CI, 0.570 to 0.739) for PCT to detect definite CAP. CRP threshold at 50 mg/L resulted in a positive predictive value of 0.76 and a negative predictive value of 0.75. No PCT cut-off resulted in satisfactory positive or negative predictive values. CRP and PCT accuracy was not improved by exclusion of the 25 (25.5 %) definite viral CAP cases. CONCLUSIONS: For patients with suspected CAP visiting emergency departments, diagnostic accuracy of CRP and PCT are insufficient to", "confirm the CAP diagnosis established using a gold standard that includes thoracic CT scan. Diagnostic accuracy of these biomarkers is also insufficient to distinguish bacterial CAP from viral CAP. TRIAL REGISTRATION: ClinicalTrials.gov registry NCT01574066 (February 7, 2012) ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13054-015-1083-6) contains supplementary material, which is available to authorized users.", "Text: Community-acquired pneumonia (CAP) is a frequently seen disease, with high morbidity and mortality, accounting for 600,000 hospitalizations each year. It represents the seventh leading cause of death in the USA [1] . CAP prognosis depends on the rapidity of specific treatment, which should ideally be initiated within four hours and no later than eight hours after diagnosis [2, 3] . CAP diagnosis is based on the clustering of non-specific pulmonary and general symptoms [4, 5] , an increase in biomarkers reflecting systemic inflammatory response syndrome (SIRS), and the presence of new parenchymal infiltrates on chest X-ray. However, CAP diagnosis remains uncertain in many cases with alternative diagnoses, such as cardiac failure, acute bronchitis, chronic obstructive pulmonary disease (COPD) exacerbations, pulmonary embolism, neoplasia, and sepsis [6, 7] .", "Part of the uncertainty of CAP diagnosis may be due to the high rate of chest X-ray misdiagnosis [8, 9] ; over diagnosis of CAP is frequent when infiltrates of noninfectious origin coexist with pulmonary or general symptoms, and the diagnosis of CAP is often ignored when the lung infiltrates are at the limit of visibility or are hidden due to superposition [10] . We recently published a study in which thoracic CT scan was systematically performed in a population of clinically suspected CAP patients visiting the emergency department for CAP (the ESCAPED study) [11] . We showed that CAP diagnosis based on chest X-ray led to a false CAP diagnosis in many patients: among CAP suspected patients with radiological pulmonary infiltrate, CAP diagnosis was excluded in around 30 % of patients based on CT scan results; on the contrary, among patients without radiological pulmonary infiltrate, one-third had a pulmonary infiltrate on thoracic CT-scan. We also reported the isolation of viruses in", "one-third of patients [11, 12] .", "Several attempts have been made to improve CAP diagnosis based on biomarkers, such as C-reactive protein (CRP) and procalcitonin (PCT); however, there are conflicting data on their reliability [13] [14] [15] [16] [17] . This could be due to the consideration of CAP diagnosis based on chest X-ray as establishing pulmonary infection. In the present study, we aimed to analyze CRP and PCT values in the population of the ESCAPED study reported above for whom CAP diagnosis was established by an adjudication committee which founded its judgment on all usual available data, systematic multidetector thoracic CT scan performed at inclusion, and results from a day-28 follow-up. We also analyzed whether the viral etiology of definite CAP based on polymerase chain reaction (PCR) multiplex naso-pharyngeal swab interfered with the accuracy of the biomarkers.", "Setting ESCAPED was a multicenter, prospective, interventional study, entitled \"Early Thoracic CT-Scan for Community-Acquired Pneumonia at the Emergency Department (ESCAPED)\" [11] , conducted from November 2011 to January 2013, in four emergency departments (EDs) of four tertiary teaching hospitals in Paris, France, designed to measure the impact of thoracic CT scan on clinical decision. The study was sponsored and monitored by the Paris public health hospitals, and funded by the French Ministry of Health. The French health authorities (Agence nationale de sécurité des medicaments et produits de santé, ANSM) and the institutional review board for the protection of human subjects approved the study protocol and patient informed consent procedures. All enrolled patients provided written informed consent for inclusion. The protocol was registered in the clinicaltrial.gov website under the PACSCAN acronym, the French translation of the English ESCAPED acronym (NCT01574066). The Ethics", "Committee of Ile de France (Comité de Protection des Personnes. Paris N°2 011-oct-12749) approved the study protocol.", "The primary objective was to compare CRP and PCT values in the four different categories of CAP level of certainty using the day-28 adjudication committee classification. The four categories were: 1) absence of CAP hereafter referred to as excluded CAP diagnosis; 2) possible CAP; 3) probable CAP; and 4) definite CAP. The secondary objectives were to assess whether CRP and PCT were associated with CAP diagnosis using sensitivity analyses in three successive subgroups chosen a priori; 1) when specifically considering patients classified as having excluded CAP diagnosis and definite CAP (i.e., the patients for whom the level of certainty was the highest); 2) when patients with excluded CAP diagnosis and diagnosed extra-pulmonary infectious disease (which may increase biomarker values) were not taken into account, in the excluded CAP group; and 3) when patients classified as viral CAP were not taken into account in the definite CAP group, as PCT has been reported to be lower in viral", "infections as compared to bacterial infections [18] .", "Consecutive adults ( [19] .\n\nMultidetector thoracic CT-scan was performed after chest X-ray, ideally within the four hours following inclusion. Chest X-ray and thoracic CT-scan were performed using a standardized protocol. The four levels of CAP probability according to CT scan were defined as definite (systematic alveolar condensation, alveolar condensation with peripheral and localized ground glass opacities, bronchiolar focal or multifocal micronodules), probable (peripheral alveolar condensation, retractile systematic alveolar condensation, or diffuse ground glass opacities), possible (pulmonary infarct), or excluded (pulmonary mass, other abnormalities, or normal images). Scan views were recorded on a DVD.", "Based on data collected from baseline standardized case report forms, DVD recorded pictures of X-ray and CTscan, and blinded to local interpretations, an adjudication committee consisting of three independent senior experts in infectious diseases, pneumology and radiology retrospectively assigned the probability of CAP diagnosis using the same 4-level Likert scale, with all available data including patients' discharge summary, and follow-up data obtained by assistant investigators who contacted by phone either the patient, relatives or general practitioners at day 28. For this study, the gold standard of CAP was the diagnosis assessed by this adjudication committee. Alternative diagnoses were established for excluded CAP and classified as non-CAP pulmonary diseases and extra-pulmonary infectious diseases and others.", "Blood samples were collected at inclusion in sodium heparin-treated tubes, centrifuged, and stored at −40°C until completion of the study. CRP and PCT concentrations were measured a posteriori on plasma collection (see Additional file 1 for methodology), except for patients in whom marker dosage was performed by the emergency practitioner on his own initiative.", "Naso-pharyngeal swabs were collected at enrollment and placed in a Middle Virocult MWE (Sigma®) transport medium. Samples were kept at room temperature and sent to the virology laboratory of Bichat -Claude Bernard Hospital (Paris) as soon as possible after collection. The samples were not frozen and thawed. Multiplex PCR (RespiFinder-19 assay (Pathofinder®, Maastricht, Netherlands)) was performed on naso-pharyngeal swabs to detect 15 respiratory viruses -coronavirus 229E, NL63, OC43, human metapneumovirus (hMPV), influenza A, A (H1N1) pdm2009 and B viruses, parainfluenza viruses 1, 2, 3, and 4, respiratory syncytial virus (RSV) A and B, rhinovirus, adenovirus, and 4 intracellular bacteria -Bordetella pertussis, Chlamydophila pneumoniae, Legionella pneumophila, Mycoplasma pneumoniae, in one reaction. The multiplex PCR results were not available to the adjudication committee. Routine microbiological examinations were also performed at the discretion of the emergency physicians and", "included blood culture, sputum culture, and antigenuria (see Additional file 1 for methodology). CAP, classified as definite, was considered as being of viral origin when multiplex PCR was positive for at least one of the 15 respiratory viruses and no bacteria were found using PCR and routine bacterial microbiological samples (sputum, blood culture, antigenuria) when performed.", "Baseline and follow-up characteristics were described by means and standard deviations (SD) or by median and interquartile range (IQR) for continuous variables normally distributed or with skewed distribution, respectively, and by percentages for categorical variables, for the total study population and for the study groups. We performed chi-square or Fisher exact tests when appropriate for qualitative variables, and the Student or Mann-Whitney tests for continuous variables with skewed distributions to compare baseline patient characteristics and study outcomes between study groups.", "The distribution values of the biomarkers were determined in the different populations of patients using boxplots. The performances of CRP and PCT in predicting definite CAP were evaluated by sensitivity analysis (definite CAP vs excluded CAP). CRP was evaluated at several cut-off points of 20 mg/L, 30 mg/L, 50 mg/L, 70 mg/L, and 100 mg/L, values used in previous studies [15, 20, 21] . Several cut-off points for PCT were chosen at the level of 0.10 μg/L [18] , and at the two levels for suspected bacterial infection as stated by the manufacturer, i.e., 0.25 μg/L and 0.50 μg/L. Sensitivities, specificities, positive predictive values (PPVs), negative predictive values (NPVs), and likelihood ratio were calculated. Receiver operating characteristic (ROC) curves were drawn, area under the curve AUC was computed and optimal cut-off was identified by the maximization of the Youden's index, comparing biomarker values in patients with excluded CAP and definite CAP. From these optimal cut-offs", "for CRP and PCT, sensitivity analyses were performed combining the CRP and PCT cut-offs.", "A multivariate logistic regression model was built to identify factors associated with having definite CAP as compared to having an excluded CAP diagnosis. We excluded from the excluded CAP diagnosis group, patients with an extra-pulmonary infectious disease. All variables with a p value of < 0.25 in the bivariate analysis were entered into a multivariate logistic regression with a backward stepwise approach; the discrimination was evaluated by the C-index and its 95 % confidence interval (95 % CI) and the calibration was evaluated by the Hosmer Lemeshow goodness-of-fit test.\n\nAll tests were two-sided, and p-values below 0.05 were considered to denote statistical significance. All statistical analyses were performed using SPSS statistical software version 21.0 (SPSS Inc., Chicago, IL, USA).", "Two hundred patients with suspected CAP out of the 319 in the ESCAPED study were included in the present study, for which CRP and PCT assays and nasopharyngeal swab for multiplex PCR were available (Fig. 1) . Characteristics of the 200 patients (age, age more than 65, gender, probability of CAP diagnosis by adjudication committee) were not significantly different from those of the 119 other patients of the ESCAPED study and are summarized in Table 1 . CRP and PCT assays were performed based on the emergency practitioner's own initiative in 70 patients for CRP and 131 for PCT, or performed a posteriori on plasma samples of the remaining patients. Sex ratio was approximately 1. More than half of the patients (54 %) were 65 years of age or older. The", "Pulmonary infiltrates were seen on chest X-ray in 127 (63.5 %) patients. Thoracic CT-scan excluded a CAP diagnosis in 16.5 % of these 127 patients; on the contrary, thoracic CT-scan revealed a parenchymal infiltrate in 27 % of the 73 patients without infiltrate on chest X-ray.\n\nBased on all available data including multidetector CT scan results (but excluding PCR results), the adjudication", "The CRP and PCT distributions in the 200 patients are presented in Fig. 2 A statistically significant difference between the two groups (excluded CAP vs definite CAP) was demonstrated for several cut-off points for CRP and PCT ( Table 2 ). For CRP, the value of 50 mg/L resulted in a PPV of 0.76 and a NPV of 0.75. For PCT, no value resulted in a satisfactory PPV or NPV. For these two biochemical markers, the ability to predict CAP was evaluated by a ROC curve. The AUC was 0.787 (95 % CI 0.717-0.857), optimal cut-off = 45.9 mg/L for CRP (Fig. 3 ) and 0.655 (95 % CI 0.570-0.739), optimal cut-off = 0.13 μg/ L for PCT (Fig. 4) .\n\nSensitivity analyses for the combination of CRP and PCT, using these optimal cut-offs, resulted in a PPV of 0.74 and a NPV of 0.58. Use of the other PCT cut-offs did not result in better PPV or NPV ( Table 2) .", "The present study is novel as patients prospectively benefited from extensive investigation to determine the diagnosis of CAP in the ED, including both early multidetector thoracic CT-scan and day-28 adjudication committee. This led to the correction of CAP diagnosis previously based on chest X-ray in a high number of patients. In these extensively characterized patients, both CRP and PCT lacked operational precision to allow the decisionmaking process to rule out or confirm diagnosis of CAP even in selected subgroups.", "The clinical characteristics of the patients included in this sub-study are consistent with those in the current literature. As previously reported, patients frequently had a history of respiratory disorders, cancer and congestive heart failure [21, 22] . The design of the ESCAPED study required exclusion of patients within the highest CRB 65 categories, which limited the inclusion of patients older than 65. This may explain why the mean age of our patients (64 years) falls within the lower values of those reported elsewhere [19] . Data to identify the microbial agent responsible for the disease were collected by the usual techniques and multiplex PCR. Viral identification using naso-pharyngeal PCR that revealed viral respiratory infection in approximately one-third of cases was concordant with values reported in the literature [23] . Therefore, we believe that our results can be extrapolated to most emergency patients suffering from CAP.", "In the present study, patients were recruited on the basis of initial clinical assessment for the diagnosis of CAP. Therefore, we believe that the characteristics of the patients closely correspond to those that lead practitioners to consider a possible diagnosis of CAP. In these patients, the design of our study allowed us to confirm or refute CAP diagnosis with a high level of certainty. Results confirmed the poor predictive value of clinical symptoms (new onset of systemic features and symptoms of an acute lower respiratory tract illness) in identifying CAP patients [21] . Indeed, clinical presentation of excluded CAP patients was similar to that of definite CAP patients except for fever and cough that were more frequent in definite CAP patients. Furthermore, the design also revealed that the combination of clinical symptoms and chest X-ray results led to CAP misdiagnosis in a high number of patients, including the 98 whose CAP diagnosis was excluded by the adjudication committee", "and who would have been considered as possible, probable or definite CAP without the use of the CT scan. This low specificity of clinical-standard radiological evaluation led to the consideration of either non-infectious pulmonary diseases (such as, cardiac failure, pulmonary embolism, pulmonary neoplasia or bronchitis) or extra-pulmonary infectious diseases as CAP. Of note, some of these diseases are also associated with increased biomarker values. This raises concerns about previous evaluations of biomarkers in CAP-suspected patients, which used clinical and standard radiological (chest X-ray) evaluations as the gold standard for CAP diagnosis [15] . The use of biomarkers has been advocated to improve diagnosis and management of patients with lower respiratory tract infections [14] . However, this issue is still unresolved [24] , with conflicting positions [14, 15, 25, 26] . In our study, while median values of both biomarkers did increase with level of certainty for CAP diagnosis,", "we were unable to establish discriminating values for PCT. Recent data suggested that CRP could be of more help in assisting in the diagnosis of lower respiratory tract infections (LRTI) [15, 27, 28] . In our study, although CRP seems more discriminating than PCT, neither the experimental exclusion of extra-pulmonary bacterial infections from the excluded CAP group, nor the exclusion of viral CAP from the definite CAP patients group, made possible the determination of a discriminant cutoff. The combination of CRP and PCT was not more discriminating than each biomarker separately. An operational algorithm has been released to assist physicians in prescribing antimicrobial therapy [14, 26, 29] . According to this strategy, a PCT concentration higher than 0.25 μg/L should prompt administration of antibiotics to patients with suspected LRTI. In our study, this value was associated with poor performance. Additionally, mean PCT levels remained above this threshold both in excluded CAP", "patients without infectious disorders and in definite CAP presumably related to virus. Therefore, the gold standard for the diagnosis of CAP may influence the performance and utility of PCT in this setting.", "This study has some limitations. First, the adjudication committee was not blinded to the value of biomarkers measured at bedside in some patients (70 for CRP and 131 for PCT) and its CAP classification could thus have been influenced by these results. However, the lack of statistically significant differences in the mean CRP and PCT values in the definite CAP cases, whether or not these biomarkers were available for the adjudication committee, argues against a major impact of these results on adjudication committee classification. Second, another critical point is the prescription of antibiotic therapy (34 %) previous to inclusion. We cannot exclude that these previously-treated CAP patients may have altered biomarker performance and reduced the yield of bacterial cultures, although such a population reflects the usual emergency department practice. Third, multiplex PCR was performed on naso-pharyngeal sampling and not on lower respiratory tract samples, which does not allow definite", "confirmation of the viral origin of CAP. However, a recent large study on CAP patients which reported a viral etiology of CAP at a comparable rate, did not find upper respiratory tract shedding in a control population without CAP explored during the same year and season [30] . Finally, even if multidetector thoracic CT scan is a better imaging examination than X-ray to explore the chest, only invasive local microbiological samples would have provided a diagnosis with certainty.", "Given the diversity of the clinical and radiological CAP presentations, CAP diagnosis is often uncertain. In our population of patients treated in the emergency room with clinical symptoms evoking CAP, neither CRP nor PCT cut-off values carried sufficient weight to confirm or refute CAP diagnosis at bedside; this underlines that these biomarkers are telltales of the host inflammatory response to the intrusion of microorganisms independent of the site of infection. These results, based on a systematic thoracic CT scan evaluation of CAP-suspected patients, do not argue for the use of CRP and PCT in routine care to diagnose CAP with certainty in patients visiting the ED for suspected CAP." ]

[ 4 ]

[ "11 June 2009" ]

[ "Pandemic Influenza Due to pH1N1/2009 Virus: Estimation of Infection Burden in Reunion Island through a Prospective Serosurvey, Austral Winter 2009\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3183080/\n\nSHA: ee6d70a53e3262cea6f85bd8b226f6b4c8b5f64b\n\nAuthors: Dellagi, Koussay; Rollot, Olivier; Temmam, Sarah; Salez, Nicolas; Guernier, Vanina; Pascalis, Hervé; Gérardin, Patrick; Fianu, Adrian; Lapidus, Nathanael; Naty, Nadège; Tortosa, Pablo; Boussaïd, Karim; Jaffar-Banjee, Marie-Christine; Filleul, Laurent; Flahault, Antoine; Carrat, Fabrice; Favier, Francois; de Lamballerie, Xavier\nDate: 2011-09-29\nDOI: 10.1371/journal.pone.0025738\nLicense: cc-by", "Abstract: BACKGROUND: To date, there is little information that reflects the true extent of spread of the pH1N1/2009v influenza pandemic at the community level as infection often results in mild or no clinical symptoms. This study aimed at assessing through a prospective study, the attack rate of pH1N1/2009 virus in Reunion Island and risk factors of infection, during the 2009 season. METHODOLOGY/PRINCIPAL FINDINGS: A serosurvey was conducted during the 2009 austral winter, in the frame of a prospective population study. Pairs of sera were collected from 1687 individuals belonging to 772 households, during and after passage of the pandemic wave. Antibodies to pH1N1/2009v were titered using the hemagglutination inhibition assay (HIA) with titers ≥1/40 being considered positive. Seroprevalence during the first two weeks of detection of pH1N1/2009v in Reunion Island was 29.8% in people under 20 years of age, 35.6% in adults (20–59 years) and 73.3% in the elderly (≥60 years) (P<0.0001).", "Baseline corrected cumulative incidence rates, were 42.9%, 13.9% and 0% in these age groups respectively (P<0.0001). A significant decline in antibody titers occurred soon after the passage of the epidemic wave. Seroconversion rates to pH1N1/2009 correlated negatively with age: 63.2%, 39.4% and 16.7%, in each age group respectively (P<0.0001). Seroconversion occurred in 65.2% of individuals who were seronegative at inclusion compared to 6.8% in those who were initially seropositive. CONCLUSIONS: Seroincidence of pH1N1/2009v infection was three times that estimated from clinical surveillance, indicating that almost two thirds of infections occurring at the community level have escaped medical detection. People under 20 years of age were the most affected group. Pre-epidemic titers ≥1/40 prevented seroconversion and are likely protective against infection. A concern was raised about the long term stability of the antibody responses.", "Text: In April 2009, the first cases of acute respiratory infections caused by a novel triple-reassortant influenza virus, pH1N1/ 2009v, occurred in Mexico and the United States [1] . The rapid spread of infection to other continents led the World Health Organization (WHO) to declare on 11 June 2009 that a pandemic of pH1N1/2009v influenza was under way, which raised major international concern about the risk of high morbidity and lethality and the potential for severe socio-economic impact. Actually, the potential impact of this first third-millenium influenza pandemic has been revisited downwards as morbidity and case-fatality rates were less severe than initially anticipated [2] . Illness surveillance data do not allow to an accurate estimate of the true influenza infection rate, as a substantial proportion of infections are asymptomatic or mild [3] . Serological surveys can overcome this limitation, but must take into account that a significant proportion of the population that", "exhibited crossprotective antibody titers before circulation of the pH1N1/2009v [4] . This so-called ''baseline immunity'' has to be subtracted from the seroprevalence observed after the pandemic wave, to determine seroincidence in serosurveys [5] [6] [7] [8] . However, except for few studies [9] [10] [11] , most of these serosurveys did not use serial measurements in the same person, which allows for a better understanding of antibody kinetics and the dynamics of infection within individuals and communities.", "Reunion Island (805,500 inhabitants) is a French overseas department located in the southwestern Indian Ocean, 700 km east of Madagascar and 200 km southwest of Mauritius. The first imported case of pH1N1/2009v was identified on 5 th July 2009 (week 29) in a traveller returning from Australia. The first case indicating community transmission was detected on 21 st July (week 30). pH1N1/2009v became the predominant circulating influenza virus within four weeks of its first detection, its activity peaked during week 35 (24) (25) (26) (27) (28) (29) (30) and ended at week 38 [12] . Contrary to initial fears, the health care system was not overwhelmed, as morbidity and mortality rates were lower than predicted [12] [13] [14] .", "In order to assess at the community level, the actual magnitude of the pH1N1/2009v pandemic and the extent of the herd immunity acquired after passage of the epidemic wave, a prospective population serosurvey was conducted in Reunion Island during the passage of the epidemic wave in the 2009 austral winter season (July-December 2009): prevalence of infection was assessed on a weekly basis and seroconversion rates were measured using paired sera.", "The CoPanFLu-RUN was part of the CoPanFLu international project, a consortium between the French National Institute of Health and Medical Research (INSERM), the Institute of Research for Development (IRD) and the Mérieux Fondation under the promotion of the School of Advanced Studies in Public Health (EHESP). To enable the rapid implementation of the study in anticipation of the imminent spread of the pandemic wave, we used a pre-existing sample of 2442 households established in October 2006 for the investigation of the Chikungunya outbreak (SEROCHIK) and updated in May 2008 throughout a follow-up telephone survey (TELECHIK) on a basis of 1148 households [15, 16] . We took special attention to select households representing a wide range of geographic locations in order to minimize the repartition bias.", "The inclusion phase started on July 21 st (week 30) and was continued up to week 44, throughout the epidemic wave and beyond. A first serum sample (sample 1) was obtained from each household member. An active telephonic inquiry was then conducted twice a week to record symptoms compatible with influenza-like illness (ILI) occurring in households. Report of ILI (fever $37.8uC associated with any respiratory or systemic symptom) led to three consecutive visits of a nurse to the incident case-dwelling (on day 0, +3 and +8 post-report) to record symptoms and collect nasal swabs from all family members (for qRT-PCR detection of pH1N1/2009v. At week 45, the active inquiry was discontinued and a second (post-epidemic) serum sample (sample 2) was obtained (weeks 45-52) to determine seroconversion rates. Sera were aliquoted and stored at 280uC.", "The protocol was conducted in accordance with the Declaration of Helsinki and French law for biomedical research (Nu ID RCB AFSSAPS: 2009-A00689-48) and was approved by the local Ethics Committee (Comité de Protection des Personnes of Bordeaux 2 University). Every eligible person for participation was asked for giving their written informed consent.\n\nViral genome detection by RT-PCR. Viral RNA was extracted from 140 mL of nasal swab eluate using the QIAamp Viral RNA kit (Qiagen) and processed for detection by TaqMan qRT-PCR targeting the heamagglutinin HA gene (SuperScript III Platinum one-step qRT-PCR system, Invitrogen) according to the recommendations of the Pasteur Institute (Van der Werf S. & Enouf V., SOP/FluA/130509). Confirmed pH1N1/2009v infection was defined as a positive qRT-PCR detection of the HA gene in at least one nasal swab.", "Hemagglutination inhibition assay (HIA). A standard hemagglutination inhibition technique was adapted to detect and quantify pH1N1/2009v antibodies [17] . The antigen was prepared by diluting a non-inactivated cell culture supernatant producing a pdm H1N1v strain (strain OPYFLU-1 isolated from a young patient returning from Mexico in early May 2009) [18] . Briefly, the virus was propagated onto MDCK cells under standard conditions. The last passage (used for antigen preparation) was performed in the absence of trypsin and ht-FBS. The supernatant was collected at day seven p.i. clarified by centrifugation at 8006 g for 10 min at room temperature, aliquoted and conserved at 280uC. The hemagglutinating titer of the non inactivated viral antigen was immediately determined under the HIA format described below. The dilution providing 5.33 hemagglutinating units in a volume of 25 mL was used for subsequent HIA. Sera were heat-inactivated at 56uC for 30 min prior to use. Sequential twofold", "dilutions in PBS (1/10 to 1/1280) in volumes of 25 mL were performed and distributed in V-bottom 96 well microplates. Human red blood cells (RBC) were used for hemagglutination experiments. Detection and quantification of antibody to pH1N1/2009v was performed as follows: 25 mL of virus suspension was added to the serum dilution (25 mL) and incubated for 1 hour at room temperature. Each well was then filled with 25 mL of a 1% RBC suspension in PBS (v/v: 0.33%), followed by another 30 min incubation at room temperature. The HIA titer was determined as the last dilution providing clear inhibition of hemagglutination. All experiments were performed in the presence of the same negative and positive controls, the latter including sera with 1/40, 1/80, 1/160 and 1/320 antibody titers.", "The results reported in this study were based only on serological analysis of paired sera. For the sake of analysis, four successive phases were identified throughout the pandemic wave: phase A (weeks 30-31) corresponded to early epidemic time, phase B (W32-39) to the epidemic unfolding, phase C (W40-44) to the immediate post-epidemic stage and phase D (W45-52) to the late post-epidemic stage. Seropositivity was defined as a HIA titer of 1/ 40 or more. The baseline-proxy seroprevalence rate was estimated on serum samples collected in phase A. The cumulative incidence rate of infection measured the raise between the raw seroprevalence rate at any given time during the epidemic phases (S2pi) and the age-specific baseline-proxy seroprevalence rate (S1pA) (s2 pi -s1 pA ). Seroconversion was defined as a shift from seronegative at inclusion (sample 1: HIA ,1/40) to seropositive on follow-up (sample 2: HIA $1/40), or for sera tested seropositive on inclusion as a four-fold increase of HIA", "titers between sample 1 and sample 2 paired sera. We also calculated the proportion of sera that tested seropositive in sample 1 for which the HIA titer decreased fourfold and passed under the cut-off value of 1/40 in sample 2. We considered this proportion as a ''seronegation'' rate.", "The sample size was calculated for identifying risk factors in the prospective cohort study. Considering on average three individuals per household, an intra-household correlation of 0.3, a power greater than 80% could be obtained with a sample size of 840 comprising 2500 individuals, assuming exposure levels ranging from 10% to 90% and a relative risk greater than 1.3. With 2,500 subjects, the study allowed 1-2% absolute precision around the estimated values for seroconversion rates. Data entry used EpiData version 3.1 (The Epidata Association, Odense, Denmark). SAS version 9.1 (SAS Inc., Cary, NC, USA) was used for statistical analysis. The characteristics of the study cohort were compared to those of the population of Reunion Island and a Chi2 test (or Fisher's exact test when non applicable) was used to analyse differences in age, sex and geographic location. Cumulative incidence rates of infection (i.e. seroincidence) and seroconversion rates were standardized according to the", "age structure of the community (French National Institute for Statistics and Economical Studies (INSEE) source).", "Baseline-proxy seroprevalence, cumulative incidence rates of infection, as well as seroconversion and seronegation rates, were expressed as percentages. Cumulative reverse distribution curves were used to show the distribution of antibody titers. In all tests, a P value,0.05 was considered significant.\n\nWe estimated 95% confidence intervals (CIs) of proportions by using a cluster bootstrap technique with 1000 re-samples [19] . After bootstraping, we used an ANOVA model to compare mean cumulative incidence proportions between pandemic phases, within each age group. We used an alternating logistic regression model (ALR) with an exchangeable log Odds Ratio (OR) to test the intra-household correlation-adjusted association between factors and the seroconversion outcome.", "Data were analysed with respect to subject age. Initially, four age groups were considered: the children and adolescents (,20 yrs), young adults (20-39 yrs), middle-age adults (40-59 yrs), and elderly adults ($60 yrs). As the cumulative incidence of infection of the second and third groups were very close, both groups were merged into one adults group (20-59 yrs). Therefore we refer further in our study to three age groups: children and adolescents (,20 yrs), adults (20-59 yrs), elderly ($60 yrs).", "A total of 2,164 individuals from 772 households were enrolled between weeks 30 and 44 in the CoPanFlu-RUN cohort, allowing the collection of 1,932 sera at inclusion (sample 1). During this period, 136 households (17.7% of households) containing 464 individuals (21.4% of individuals) reported at least one case of ILI. Sixty subjects among the 464 individuals (12.9%, belonging to 33 households [24.3%]) were qRT-PCR positive, which documented the pH1N1/2009v infection. No positive qRT-PCR could be detected after week 37 and no ILI was reported after week 40, the end of the epidemic wave. The second follow up serum sample (sample 2) was obtained for 1,759 subjects at least five weeks after the end of the epidemic wave (weeks 45-52) which allowed the constitution of a serobank of 1,687 paired-sera. The profile of the cohort and the major outcomes are displayed in Figure 1 . Details on inclusions and serum sample timing with respect to the circulation of pH1N1/2009v over the island are", "provided in figure 2 . The socio-demographic and space-time characteristics of the cohort are detailed in Table 1 . Compared to the community of Reunion Island, the sample of 1,687 individuals for whom pairedsera were available, was older (,20 yrs: 27% vs 35%, and $60 yrs: 17,9% vs 11,3%) and composed of a slight excess of females (54.1% vs 51.5%). The imbalance was due to a deficit in subjects aged under 40 years, reflecting men at work and the fact that parents declined the second serum for children younger than five.", "Baseline-proxy (,pre-epidemic) HIA titers to the pH1N1/ 2009v were measured on sample 1 ( Table 2) , obtained from 249 subjects (103 households) recruited at the very beginning of the investigation during weeks 30 and 31 (phase A, Figure 2 ), when the epidemic activity in the cohort was still very low. Age distribution in this group was similar to that of the whole cohort (data not shown). The overall, the baseline-proxy seroprevalence rate (HIA $1/40), over all ages, was 43.4% (95%CI: 37.4%-49.6%). However the majority of positive sera had low antibody titers, at the cut off value for confirmation (i.e. = 1/40). The proportions of sera with HIA titer .1/40 were 0%, 3.0% and 24.6% in the young, middle-aged and older age groups respectively. These results indicate that pre-epidemic baseline antibody cross reactivity was stronger in the elderly ($60 yrs) and weaker in children and adolescents (,20 yrs) and adults (20-59 yrs), with highly significant differences between age groups", "(P,0.0001).", "The reverse cumulative distribution curves of HIA titers are displayed for each age group and for the whole cohort on Figure 3 . The proportion of seropositive sera (HI $1/40) steadily increased during the epidemic unfolding (phase B, W32-39) and in immediate post epidemic period (phase C, W40-44) when it reached its maximum level, then declined in the late post epidemic period (phase D, W45-52). This decline was significant enough to return the reverse cumulative distribution curve to baseline levels in the elderly. The cumulative incidence rates, obtained after subtraction of the age-specific baseline-proxy seroprevalence from the raw seroprevalence at each phase of the epidemic are shown in Table 2 (note that the cumulative incidence rates of infection represented for the group ''all ages'' were standardized according to age structure of the community). The cumulative incidence rates were much higher in children and adolescents (,20 yrs), indicating very active transmission of", "infection within this age group. As mentioned earlier, cumulative incidence rates peaked in phase C (W40-44), and then declined indicating some lability of the humoral immune response against the pH1N1/2009v. The age-related difference observed in the incidence rates was highly statistically significant (P,0.0001).", "To estimate more appropriately the decline of antibody titers occurring after the peak of the humoral response to the pH1N1/ 2009v, we considered paired-sera from the group of 264 subjects for whom the first serum sample (sample 1) was obtained just after the epidemic wave (phase C, W40-44), and the corresponding second sample was collected at the end of the survey (phase D, W45-52). Seronegation rates were 27.0% (61/226) for all age groups, 17.4% (12/69) in children and adolescents (,20 yrs), 32.3% (41/127) in adults (20-59 yrs) and 26.7% (8/30) in the elderly ($60 yrs). Differences between the seronegation rates according to age were statistically weakly significant (P = 0.0671).", "We then considered the 1687 individuals for whom paired sera were available and we measured the seroconversion rates according to age and to the time of first serum sample collection (phase A, B or C). Criteria of seroconversion were defined in the method section. As shown in table 3, there was a sharp decline in seroconversion rates across all the age groups, depending on whether participants were enrolled during phase A, phase B, or phase C (P,0.0001). To interpret these data, one should remember that antibodies at seroprotective levels (HIA $1/40), in serum samples 1 collected during the per epidemic phase B or early post epidemic phase C could represent either base line cross reactive antibodies or rising pH1N1/2009 specific antibodies due to a recent or ongoing infection. This ambiguity could lead to underestimation of the seroconversion rate for subjects enrolled in phases B and C. In order to solve this ambiguity, we specifically considered the group of 249 subjects in whom", "cross reactive antibodies were detected at the time of phase A (W30-31). The seroconversion rate of this group is the most indicative of the exposure of individuals to the whole epidemic wave. It was the highest (63,2%, P,0.0001) in children and adolescents (,20 yrs), and still significantly high in adults (39.4%, P,0.0001).", "We then tested in this particular group, the impact of (baseline) pre-epidemic cross reactive antibodies on the rate of seroconversion to pH1N1/2009 (Table 4) . No subject with HIA titer superior to 1/40 had evidence of seroconversion to pH1N1/2009. The seroconversion rate in individuals with a HIA titer equal to 1/40 was linked with age, being more important in children and adolescents (,20 yrs). The highest seroconversion rate (.56%) was registered in subjects with HIA titers inferior to 1/40, particularly for the under 20 years where it reached 85%. Hence, the risk of seroconversion decreased when pre-epidemic HIA titer was high after controlling for age (P,0.0001) (Figure 4) . The multivariate adjusted odds ratio for seroconversion were 0.15 (95%CI: 0.06-0.37, P,0.0001) per two-fold increase in baseline titer, 1.79 (95%CI: 1.23-2.59, P,0.003) per other household members who seroconverted, 5.33 (95%CI: 1.56-19.27, P,0.008) Figure 1 . The cohort profile and major outcomes. Figure 1", "details the three phases of the protocol: i) inclusion (weeks 30-44) and serum samples S1 collection; ii) follow up for detection of ILI in households, qRT-PCR on nasal swabs and estimation of cumulative seroincidence rates; iii) end of the study (weeks 45-52) and samples S2 collection. HIA on paired sera (S1+S2) allowed estimating seroconversion rates. doi:10.1371/journal.pone.0025738.g001 Bp (baseline-proxy) seroprevalence rates were estimated on weeks 30-31 in each age group. b Cumulative incidence rates measured the raise between raw seroprevalence rates and age-specific baseline-proxy seroprevalence rate. In the group ''All ages'', cumulative incidence rates were standardized according to age structure of the community. doi:10.1371/journal.pone.0025738.t002 Data are numbers, percentages (95% confidence intervals) and ALR parameter test P value for comparison of seroconversion proportions according to time of first sample (S1) collection at inclusion, in each age group, after", "controlling for household selection. In the group ''All ages'', rates of seroconversion were standardized according to age structure of the community. NA: not assessed. Seroconversion was defined as a shift from seronegative at inclusion (i.e. HIA titer ,1/40) to seropositive on follow-up sample, or as a 4-fold increase of reciprocal HIA titer between first and second paired samples for sera tested seropositive on inclusion (i.e. HIA titer $1/40). for age ,20 years (vs age $60 years) and 11.35 (95%CI: 0.41-4.47, P = 0.62) for age 20-60 years (vs age $60 years). The observed and predicted seroconversion rates according to age and baseline HIA titer are displayed Figure 4 .", "Finally, we considered the 46 subjects who had been infected by the pandemic virus over the course of the study, verified by a positive qRT-PCR nasal swab, and for whom paired sera were available. Initial HIA antibody titers in this group were ,1/40,", "The CoPanFlu-RUN cohort was set up to conduct a prospective population-based study investigating the herd immunity induced by the 2009 pandemic influenza virus and identifying risk factors for pH1N1/2009v infection from paired sera collected in an entire community. Most works published to date have used either extensive cross-sectional serosurveys on pre-and post-epidemic independent serum samples, the baseline immunity being assessed from stored frozen samples [5, 7, 8] , or non representative adult cohorts (military, health care workers, long-stay patients). Antibody titers were measured by HIA using a cut-off value set at 1/40 as classically recommended. This HIA titer at 1/40 is considered protective, i.e. conferring 50% protection against a viral challenge [20] . Our assay has introduced some changes in the experimental protocol compared to the classic one. The use of a non-inactivated viral antigen, i.e. a native virus, with nondenatured epitopes probably allows detection of", "antibodies to epitopes of the hemagglutinin not detected in the classic HIA test. This can induce slight differences in the sensitivity of detection of cross-reacting antibodies, but this does not modify the kinetics of Ab and the epidemiological evolution of seroprevalence and does not jeopardize the global comparability of serological results. This is confirmed by the fact that our HI assay detected seroprotective antibody titers in 93.5% and gave evidence seroconversion in 73.9% of qRT-PCR confirmed pH1N1/2009 influenza, all figures close to those reported in the literature [5, 21] .", "We considered that titers of .1/40, in sera collected from individuals enrolled during weeks 30 and 31 were cross reactive antibodies and not de novo antibodies triggered by the pandemic virus and hence used them as a proxy for baseline pre epidemic immunity. Several arguments support this assumption: i) the first case indicating autochthonous transmission in Reunion Island was reported by the epidemiological surveillance department of La Réunion on 21st July (week 30), i.e. the same day when inclusion started in our study cohort; ii) 7 to 15 days are required to develop an antibody response after viral infection; iii) On weeks 30 and 31, the epidemic activity due to the pandemic virus was very low in our study cohort and it became significant only after week 32. Hence, during weeks 30-31, 103 households were recruited and only 2 households reported ILI cases. Nasal swabs collected from these 2 individuals were tested qRT-PCR negative to the pandemic virus whereas one had evidence of", "coronavirus and rhinovirus using a multiplex RT-PCR to respiratory viruses (H. Pascalis, manuscript in preparation). In contrast, during weeks 32 to 39, 199 individuals belonging to 99 households reported ILI, among whom 60 individuals had documented infection by the pandemic virus.", "Our study shows that a substantial proportion of Reunion Island's population had pre-existing immunity to 2009 pandemic influenza virus with the highest baseline-proxy seroprevalence rate observed among adults aged of 60 years or more. Other studies from all continents had also reported high pre-epidemic seropositivity rates among the elderly [5, 6, 8, [22] [23] [24] [25] [26] , though large variations do exist between countries [10, 11, 23, 27, 28] . These cross reactive antibodies have been interpreted as being the residual signature of the remote exposure of these individuals to H1N1 viruses circulating before 1957 [24, 25, 29, 30] . Baseline seropositivity rates that we report in children and in younger adults (i.e. 30%-35%) were notably higher than those reported from other parts of the world [6, 8, 22, 23, [31] [32] [33] . However one should note that these baseline antibodies were of low titer, just at the level of the HIA threshold (i.e. 1/40). Several factors could have", "contributed to this comparatively high baseline rates found in our study: i) It may reflect the fact that the HI test used in our study was marginally more sensitive than the classic one [17] ; ii) Some individuals may have already been infected with pH1N1/ 2009 virus at weeks 30 and 31 and may have triggered an antibody response to the virus. This hypothesis seems unlikely in view of the arguments presented above and of a similar high proportion of sera titering HIA = 1/40 among 122 sera from adult patients sent for diagnostic purposes to the Regional Hospital microbiology laboratory, during the first half of 2009 (i.e. before the 2009 pandemic) (data not shown). However we cannot formally exclude this hypothesis in view of a recently reported study from Taiwan [11] that showed evidence of subclinical community transmission with proved seroconversion several weeks before report of the first documented case in the island. A similar conclusion was also drawn from Australia [34] ; iii)", "our serological test might detect cross-reactive antibodies triggered by recent vaccination with trivalent seasonal influenza vaccine as reported [4, [35] [36] [37] [38] [39] . However, seasonal influenza vaccines were of rather limited use in Reunion Island, especially in children and young adults; iv) Finally the high baseline titers may reflect the infectious history of the individuals to seasonal influenza viruses cross antigenic with pH1N1/2009 virus as recently suggested for seasonal 2007 H1N1 infection [40] . This serosurvey indicates that a large fraction of the Reunion Island population was infected with the pandemic virus. Younger people, have paid the main tribute to the epidemic as almost two thirds show evidence of seroconversion, confirming earlier clinical reports from the island [12] and accumulating reports from other countries [17, 32, 41, 42] and suggesting that school children have likely played the central role in the epidemic diffusion of the pandemic virus.", "Lower infection rates were found in adults and the lowest rates were recorded in the elderly.", "Based on clinical cases reported to the epidemiological surveillance services [12] , it was estimated that 66,915 persons in Reunion Island who consulted a physician were infected by the pH1N1/2009 virus during the 9 weeks of the epidemic, giving a cumulative attack rate of 8.26%. Taking into account those who did not consult a physician, the number of symptomatic infected persons was estimated to 104,067 (attack rate: 12.85%). In fact, the attack rate of pH1N1/2009 infection in our serosurvey was about 42%-44% at the peak of the antibody response (i.e., weeks 40-44), a figure which is at least 3 to 4 times higher than rates of infection based on clinical cases The wide gap between the two estimates indicates that a large fraction (almost two thirds) of those who got infected by pH1N1/2009 virus escaped medical detection, probably because they developed mild disease or asymptomatic infection, a further indication of the benign nature of the virus, at least at the community level. In", "England, Baguelin et al. [43] estimated that the cumulative incidence rates of infection by the pandemic virus in children were 20 to 40 times higher than that estimated from clinical surveillance.", "Our study, as others [6] , indicates that pre-existing cross reactive antibodies to pH1N1/2009 at titers $1/40 prevented from seroconversion in response to the pandemic virus. This level of pre-existing cross reactive immunity likely confers true protection against infection as about two thirds and one third of documented infection (qRT-PCR positive) in our series have occurred in individuals with baseline HIA titers ,1/40 and = 1/ 40 respectively and less than 5% of documented infections occurred in individuals with base line titers .1/40. The protection was effective not only in older adults but also in younger persons. This indicates that protection was conferred not only by baseline cross reactive antibodies triggered by close pH1N1/2009 viruses that circulated before 1957 (as in the elderly), but also by antibodies likely resulting from recent exposure to seasonal influenza epidemics (as shown in younger persons) [40] . The observed seroconversion rates depend on age, after", "adjusting for baseline pH1N1/2009 titers. The protective role of increasing age might be explained by a stronger cross-immunity in adults and elderly or by a higher exposure of young subjects to the virus during the 2009 epidemic (due to social contacts and mixing patterns). It may also indicate that immune mechanisms other than cross reactive antibodies detected by HIA (i.e. immunity to neuraminidase and conserved T cells epitopes [44] might develop throughout life, providing additional protection from infection or severe disease, especially in the elderly. Interestingly, evidence is seen for a decline in antibody titers, which occurred soon after the passage of the epidemic wave. In paired sera, this decline was significant enough to bring, within a few weeks, almost 27% of sera that tested positive (i.e. HI titers $1/40) in the immediate post epidemic phase to levels under the cut-off value in the second serum sample. This decay accounts for the observation that older adults ($60", "yrs) in the study cohort were apparently almost completely spared by the epidemic if one only considers cumulative incidence rates derived from IHA titration on samples 2 (weeks 45-52). In fact, the cumulative incidence rate in older adults measured just after the epidemic peak (i.e. weeks 40-44) was 20.4%. Similar results of early antibody decay were recently reported [10, 45] . More generally, these data show that serosurveys conducted months after passage of the epidemic, likely underestimate the real extent of pH1N1/2009 infection, compared to antibody titration performed earlier, when humoral responses are at their highest level. Whether the decline in antibody titers has functional immunologic consequence to individuals or within the communities warrants further investigation. However, one should note that there was no second epidemic wave in Reunion Island during the subsequent austral winter seasons in 2010 and 2011.", "Influenza during the 2010 winter was at a level not higher than the usual passages of seasonal flu, though almost two thirds of documented cases in 2010 were also due to pH1N1/2009v [46] . In addition many fewer pandemic virus isolates were noted during the ongoing 2011 austral winter, strongly suggesting that the first epidemic wave had conferred a solid herd immunity, at the community level.", "Our study has some limitations. The fact that the epidemic progression coincided with the implementation of the prospective study, we were not able to collect, strictly speaking, pre-epidemic sera from the cohort members. Therefore we used as proxy base line seroprevalence data from individuals recruited at the very beginning of the investigation when the epidemic activity in the cohort was very low. This may overestimate the base line immunity if subclinical community transmission had occurred before the first cases of pH1N1/2009 influenza were reported. Antibodies to the pandemic virus were detected by HIA, a test that has a good specificity but a rather low sensitivity [46] . Hence, the threshold of 1/40 may underestimate the number of infected individuals. However, rates of seroconversion, the serologic gold standard test based on paired sera, likely gave the most accurate picture of the pandemic in at the community level in Reunion Island." ]

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[ "Pandemic Influenza Due to pH1N1/2009 Virus: Estimation of Infection Burden in Reunion Island through a Prospective Serosurvey, Austral Winter 2009\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3183080/\n\nSHA: ee6d70a53e3262cea6f85bd8b226f6b4c8b5f64b\n\nAuthors: Dellagi, Koussay; Rollot, Olivier; Temmam, Sarah; Salez, Nicolas; Guernier, Vanina; Pascalis, Hervé; Gérardin, Patrick; Fianu, Adrian; Lapidus, Nathanael; Naty, Nadège; Tortosa, Pablo; Boussaïd, Karim; Jaffar-Banjee, Marie-Christine; Filleul, Laurent; Flahault, Antoine; Carrat, Fabrice; Favier, Francois; de Lamballerie, Xavier\nDate: 2011-09-29\nDOI: 10.1371/journal.pone.0025738\nLicense: cc-by", "Abstract: BACKGROUND: To date, there is little information that reflects the true extent of spread of the pH1N1/2009v influenza pandemic at the community level as infection often results in mild or no clinical symptoms. This study aimed at assessing through a prospective study, the attack rate of pH1N1/2009 virus in Reunion Island and risk factors of infection, during the 2009 season. METHODOLOGY/PRINCIPAL FINDINGS: A serosurvey was conducted during the 2009 austral winter, in the frame of a prospective population study. Pairs of sera were collected from 1687 individuals belonging to 772 households, during and after passage of the pandemic wave. Antibodies to pH1N1/2009v were titered using the hemagglutination inhibition assay (HIA) with titers ≥1/40 being considered positive. Seroprevalence during the first two weeks of detection of pH1N1/2009v in Reunion Island was 29.8% in people under 20 years of age, 35.6% in adults (20–59 years) and 73.3% in the elderly (≥60 years) (P<0.0001).", "Baseline corrected cumulative incidence rates, were 42.9%, 13.9% and 0% in these age groups respectively (P<0.0001). A significant decline in antibody titers occurred soon after the passage of the epidemic wave. Seroconversion rates to pH1N1/2009 correlated negatively with age: 63.2%, 39.4% and 16.7%, in each age group respectively (P<0.0001). Seroconversion occurred in 65.2% of individuals who were seronegative at inclusion compared to 6.8% in those who were initially seropositive. CONCLUSIONS: Seroincidence of pH1N1/2009v infection was three times that estimated from clinical surveillance, indicating that almost two thirds of infections occurring at the community level have escaped medical detection. People under 20 years of age were the most affected group. Pre-epidemic titers ≥1/40 prevented seroconversion and are likely protective against infection. A concern was raised about the long term stability of the antibody responses.", "Text: In April 2009, the first cases of acute respiratory infections caused by a novel triple-reassortant influenza virus, pH1N1/ 2009v, occurred in Mexico and the United States [1] . The rapid spread of infection to other continents led the World Health Organization (WHO) to declare on 11 June 2009 that a pandemic of pH1N1/2009v influenza was under way, which raised major international concern about the risk of high morbidity and lethality and the potential for severe socio-economic impact. Actually, the potential impact of this first third-millenium influenza pandemic has been revisited downwards as morbidity and case-fatality rates were less severe than initially anticipated [2] . Illness surveillance data do not allow to an accurate estimate of the true influenza infection rate, as a substantial proportion of infections are asymptomatic or mild [3] . Serological surveys can overcome this limitation, but must take into account that a significant proportion of the population that", "exhibited crossprotective antibody titers before circulation of the pH1N1/2009v [4] . This so-called ''baseline immunity'' has to be subtracted from the seroprevalence observed after the pandemic wave, to determine seroincidence in serosurveys [5] [6] [7] [8] . However, except for few studies [9] [10] [11] , most of these serosurveys did not use serial measurements in the same person, which allows for a better understanding of antibody kinetics and the dynamics of infection within individuals and communities.", "Reunion Island (805,500 inhabitants) is a French overseas department located in the southwestern Indian Ocean, 700 km east of Madagascar and 200 km southwest of Mauritius. The first imported case of pH1N1/2009v was identified on 5 th July 2009 (week 29) in a traveller returning from Australia. The first case indicating community transmission was detected on 21 st July (week 30). pH1N1/2009v became the predominant circulating influenza virus within four weeks of its first detection, its activity peaked during week 35 (24) (25) (26) (27) (28) (29) (30) and ended at week 38 [12] . Contrary to initial fears, the health care system was not overwhelmed, as morbidity and mortality rates were lower than predicted [12] [13] [14] .", "In order to assess at the community level, the actual magnitude of the pH1N1/2009v pandemic and the extent of the herd immunity acquired after passage of the epidemic wave, a prospective population serosurvey was conducted in Reunion Island during the passage of the epidemic wave in the 2009 austral winter season (July-December 2009): prevalence of infection was assessed on a weekly basis and seroconversion rates were measured using paired sera.", "The CoPanFLu-RUN was part of the CoPanFLu international project, a consortium between the French National Institute of Health and Medical Research (INSERM), the Institute of Research for Development (IRD) and the Mérieux Fondation under the promotion of the School of Advanced Studies in Public Health (EHESP). To enable the rapid implementation of the study in anticipation of the imminent spread of the pandemic wave, we used a pre-existing sample of 2442 households established in October 2006 for the investigation of the Chikungunya outbreak (SEROCHIK) and updated in May 2008 throughout a follow-up telephone survey (TELECHIK) on a basis of 1148 households [15, 16] . We took special attention to select households representing a wide range of geographic locations in order to minimize the repartition bias.", "The inclusion phase started on July 21 st (week 30) and was continued up to week 44, throughout the epidemic wave and beyond. A first serum sample (sample 1) was obtained from each household member. An active telephonic inquiry was then conducted twice a week to record symptoms compatible with influenza-like illness (ILI) occurring in households. Report of ILI (fever $37.8uC associated with any respiratory or systemic symptom) led to three consecutive visits of a nurse to the incident case-dwelling (on day 0, +3 and +8 post-report) to record symptoms and collect nasal swabs from all family members (for qRT-PCR detection of pH1N1/2009v. At week 45, the active inquiry was discontinued and a second (post-epidemic) serum sample (sample 2) was obtained (weeks 45-52) to determine seroconversion rates. Sera were aliquoted and stored at 280uC.", "The protocol was conducted in accordance with the Declaration of Helsinki and French law for biomedical research (Nu ID RCB AFSSAPS: 2009-A00689-48) and was approved by the local Ethics Committee (Comité de Protection des Personnes of Bordeaux 2 University). Every eligible person for participation was asked for giving their written informed consent.\n\nViral genome detection by RT-PCR. Viral RNA was extracted from 140 mL of nasal swab eluate using the QIAamp Viral RNA kit (Qiagen) and processed for detection by TaqMan qRT-PCR targeting the heamagglutinin HA gene (SuperScript III Platinum one-step qRT-PCR system, Invitrogen) according to the recommendations of the Pasteur Institute (Van der Werf S. & Enouf V., SOP/FluA/130509). Confirmed pH1N1/2009v infection was defined as a positive qRT-PCR detection of the HA gene in at least one nasal swab.", "Hemagglutination inhibition assay (HIA). A standard hemagglutination inhibition technique was adapted to detect and quantify pH1N1/2009v antibodies [17] . The antigen was prepared by diluting a non-inactivated cell culture supernatant producing a pdm H1N1v strain (strain OPYFLU-1 isolated from a young patient returning from Mexico in early May 2009) [18] . Briefly, the virus was propagated onto MDCK cells under standard conditions. The last passage (used for antigen preparation) was performed in the absence of trypsin and ht-FBS. The supernatant was collected at day seven p.i. clarified by centrifugation at 8006 g for 10 min at room temperature, aliquoted and conserved at 280uC. The hemagglutinating titer of the non inactivated viral antigen was immediately determined under the HIA format described below. The dilution providing 5.33 hemagglutinating units in a volume of 25 mL was used for subsequent HIA. Sera were heat-inactivated at 56uC for 30 min prior to use. Sequential twofold", "dilutions in PBS (1/10 to 1/1280) in volumes of 25 mL were performed and distributed in V-bottom 96 well microplates. Human red blood cells (RBC) were used for hemagglutination experiments. Detection and quantification of antibody to pH1N1/2009v was performed as follows: 25 mL of virus suspension was added to the serum dilution (25 mL) and incubated for 1 hour at room temperature. Each well was then filled with 25 mL of a 1% RBC suspension in PBS (v/v: 0.33%), followed by another 30 min incubation at room temperature. The HIA titer was determined as the last dilution providing clear inhibition of hemagglutination. All experiments were performed in the presence of the same negative and positive controls, the latter including sera with 1/40, 1/80, 1/160 and 1/320 antibody titers.", "The results reported in this study were based only on serological analysis of paired sera. For the sake of analysis, four successive phases were identified throughout the pandemic wave: phase A (weeks 30-31) corresponded to early epidemic time, phase B (W32-39) to the epidemic unfolding, phase C (W40-44) to the immediate post-epidemic stage and phase D (W45-52) to the late post-epidemic stage. Seropositivity was defined as a HIA titer of 1/ 40 or more. The baseline-proxy seroprevalence rate was estimated on serum samples collected in phase A. The cumulative incidence rate of infection measured the raise between the raw seroprevalence rate at any given time during the epidemic phases (S2pi) and the age-specific baseline-proxy seroprevalence rate (S1pA) (s2 pi -s1 pA ). Seroconversion was defined as a shift from seronegative at inclusion (sample 1: HIA ,1/40) to seropositive on follow-up (sample 2: HIA $1/40), or for sera tested seropositive on inclusion as a four-fold increase of HIA", "titers between sample 1 and sample 2 paired sera. We also calculated the proportion of sera that tested seropositive in sample 1 for which the HIA titer decreased fourfold and passed under the cut-off value of 1/40 in sample 2. We considered this proportion as a ''seronegation'' rate.", "The sample size was calculated for identifying risk factors in the prospective cohort study. Considering on average three individuals per household, an intra-household correlation of 0.3, a power greater than 80% could be obtained with a sample size of 840 comprising 2500 individuals, assuming exposure levels ranging from 10% to 90% and a relative risk greater than 1.3. With 2,500 subjects, the study allowed 1-2% absolute precision around the estimated values for seroconversion rates. Data entry used EpiData version 3.1 (The Epidata Association, Odense, Denmark). SAS version 9.1 (SAS Inc., Cary, NC, USA) was used for statistical analysis. The characteristics of the study cohort were compared to those of the population of Reunion Island and a Chi2 test (or Fisher's exact test when non applicable) was used to analyse differences in age, sex and geographic location. Cumulative incidence rates of infection (i.e. seroincidence) and seroconversion rates were standardized according to the", "age structure of the community (French National Institute for Statistics and Economical Studies (INSEE) source).", "Baseline-proxy seroprevalence, cumulative incidence rates of infection, as well as seroconversion and seronegation rates, were expressed as percentages. Cumulative reverse distribution curves were used to show the distribution of antibody titers. In all tests, a P value,0.05 was considered significant.\n\nWe estimated 95% confidence intervals (CIs) of proportions by using a cluster bootstrap technique with 1000 re-samples [19] . After bootstraping, we used an ANOVA model to compare mean cumulative incidence proportions between pandemic phases, within each age group. We used an alternating logistic regression model (ALR) with an exchangeable log Odds Ratio (OR) to test the intra-household correlation-adjusted association between factors and the seroconversion outcome.", "Data were analysed with respect to subject age. Initially, four age groups were considered: the children and adolescents (,20 yrs), young adults (20-39 yrs), middle-age adults (40-59 yrs), and elderly adults ($60 yrs). As the cumulative incidence of infection of the second and third groups were very close, both groups were merged into one adults group (20-59 yrs). Therefore we refer further in our study to three age groups: children and adolescents (,20 yrs), adults (20-59 yrs), elderly ($60 yrs).", "A total of 2,164 individuals from 772 households were enrolled between weeks 30 and 44 in the CoPanFlu-RUN cohort, allowing the collection of 1,932 sera at inclusion (sample 1). During this period, 136 households (17.7% of households) containing 464 individuals (21.4% of individuals) reported at least one case of ILI. Sixty subjects among the 464 individuals (12.9%, belonging to 33 households [24.3%]) were qRT-PCR positive, which documented the pH1N1/2009v infection. No positive qRT-PCR could be detected after week 37 and no ILI was reported after week 40, the end of the epidemic wave. The second follow up serum sample (sample 2) was obtained for 1,759 subjects at least five weeks after the end of the epidemic wave (weeks 45-52) which allowed the constitution of a serobank of 1,687 paired-sera. The profile of the cohort and the major outcomes are displayed in Figure 1 . Details on inclusions and serum sample timing with respect to the circulation of pH1N1/2009v over the island are", "provided in figure 2 . The socio-demographic and space-time characteristics of the cohort are detailed in Table 1 . Compared to the community of Reunion Island, the sample of 1,687 individuals for whom pairedsera were available, was older (,20 yrs: 27% vs 35%, and $60 yrs: 17,9% vs 11,3%) and composed of a slight excess of females (54.1% vs 51.5%). The imbalance was due to a deficit in subjects aged under 40 years, reflecting men at work and the fact that parents declined the second serum for children younger than five.", "Baseline-proxy (,pre-epidemic) HIA titers to the pH1N1/ 2009v were measured on sample 1 ( Table 2) , obtained from 249 subjects (103 households) recruited at the very beginning of the investigation during weeks 30 and 31 (phase A, Figure 2 ), when the epidemic activity in the cohort was still very low. Age distribution in this group was similar to that of the whole cohort (data not shown). The overall, the baseline-proxy seroprevalence rate (HIA $1/40), over all ages, was 43.4% (95%CI: 37.4%-49.6%). However the majority of positive sera had low antibody titers, at the cut off value for confirmation (i.e. = 1/40). The proportions of sera with HIA titer .1/40 were 0%, 3.0% and 24.6% in the young, middle-aged and older age groups respectively. These results indicate that pre-epidemic baseline antibody cross reactivity was stronger in the elderly ($60 yrs) and weaker in children and adolescents (,20 yrs) and adults (20-59 yrs), with highly significant differences between age groups", "(P,0.0001).", "The reverse cumulative distribution curves of HIA titers are displayed for each age group and for the whole cohort on Figure 3 . The proportion of seropositive sera (HI $1/40) steadily increased during the epidemic unfolding (phase B, W32-39) and in immediate post epidemic period (phase C, W40-44) when it reached its maximum level, then declined in the late post epidemic period (phase D, W45-52). This decline was significant enough to return the reverse cumulative distribution curve to baseline levels in the elderly. The cumulative incidence rates, obtained after subtraction of the age-specific baseline-proxy seroprevalence from the raw seroprevalence at each phase of the epidemic are shown in Table 2 (note that the cumulative incidence rates of infection represented for the group ''all ages'' were standardized according to age structure of the community). The cumulative incidence rates were much higher in children and adolescents (,20 yrs), indicating very active transmission of", "infection within this age group. As mentioned earlier, cumulative incidence rates peaked in phase C (W40-44), and then declined indicating some lability of the humoral immune response against the pH1N1/2009v. The age-related difference observed in the incidence rates was highly statistically significant (P,0.0001).", "To estimate more appropriately the decline of antibody titers occurring after the peak of the humoral response to the pH1N1/ 2009v, we considered paired-sera from the group of 264 subjects for whom the first serum sample (sample 1) was obtained just after the epidemic wave (phase C, W40-44), and the corresponding second sample was collected at the end of the survey (phase D, W45-52). Seronegation rates were 27.0% (61/226) for all age groups, 17.4% (12/69) in children and adolescents (,20 yrs), 32.3% (41/127) in adults (20-59 yrs) and 26.7% (8/30) in the elderly ($60 yrs). Differences between the seronegation rates according to age were statistically weakly significant (P = 0.0671).", "We then considered the 1687 individuals for whom paired sera were available and we measured the seroconversion rates according to age and to the time of first serum sample collection (phase A, B or C). Criteria of seroconversion were defined in the method section. As shown in table 3, there was a sharp decline in seroconversion rates across all the age groups, depending on whether participants were enrolled during phase A, phase B, or phase C (P,0.0001). To interpret these data, one should remember that antibodies at seroprotective levels (HIA $1/40), in serum samples 1 collected during the per epidemic phase B or early post epidemic phase C could represent either base line cross reactive antibodies or rising pH1N1/2009 specific antibodies due to a recent or ongoing infection. This ambiguity could lead to underestimation of the seroconversion rate for subjects enrolled in phases B and C. In order to solve this ambiguity, we specifically considered the group of 249 subjects in whom", "cross reactive antibodies were detected at the time of phase A (W30-31). The seroconversion rate of this group is the most indicative of the exposure of individuals to the whole epidemic wave. It was the highest (63,2%, P,0.0001) in children and adolescents (,20 yrs), and still significantly high in adults (39.4%, P,0.0001).", "We then tested in this particular group, the impact of (baseline) pre-epidemic cross reactive antibodies on the rate of seroconversion to pH1N1/2009 (Table 4) . No subject with HIA titer superior to 1/40 had evidence of seroconversion to pH1N1/2009. The seroconversion rate in individuals with a HIA titer equal to 1/40 was linked with age, being more important in children and adolescents (,20 yrs). The highest seroconversion rate (.56%) was registered in subjects with HIA titers inferior to 1/40, particularly for the under 20 years where it reached 85%. Hence, the risk of seroconversion decreased when pre-epidemic HIA titer was high after controlling for age (P,0.0001) (Figure 4) . The multivariate adjusted odds ratio for seroconversion were 0.15 (95%CI: 0.06-0.37, P,0.0001) per two-fold increase in baseline titer, 1.79 (95%CI: 1.23-2.59, P,0.003) per other household members who seroconverted, 5.33 (95%CI: 1.56-19.27, P,0.008) Figure 1 . The cohort profile and major outcomes. Figure 1", "details the three phases of the protocol: i) inclusion (weeks 30-44) and serum samples S1 collection; ii) follow up for detection of ILI in households, qRT-PCR on nasal swabs and estimation of cumulative seroincidence rates; iii) end of the study (weeks 45-52) and samples S2 collection. HIA on paired sera (S1+S2) allowed estimating seroconversion rates. doi:10.1371/journal.pone.0025738.g001 Bp (baseline-proxy) seroprevalence rates were estimated on weeks 30-31 in each age group. b Cumulative incidence rates measured the raise between raw seroprevalence rates and age-specific baseline-proxy seroprevalence rate. In the group ''All ages'', cumulative incidence rates were standardized according to age structure of the community. doi:10.1371/journal.pone.0025738.t002 Data are numbers, percentages (95% confidence intervals) and ALR parameter test P value for comparison of seroconversion proportions according to time of first sample (S1) collection at inclusion, in each age group, after", "controlling for household selection. In the group ''All ages'', rates of seroconversion were standardized according to age structure of the community. NA: not assessed. Seroconversion was defined as a shift from seronegative at inclusion (i.e. HIA titer ,1/40) to seropositive on follow-up sample, or as a 4-fold increase of reciprocal HIA titer between first and second paired samples for sera tested seropositive on inclusion (i.e. HIA titer $1/40). for age ,20 years (vs age $60 years) and 11.35 (95%CI: 0.41-4.47, P = 0.62) for age 20-60 years (vs age $60 years). The observed and predicted seroconversion rates according to age and baseline HIA titer are displayed Figure 4 .", "Finally, we considered the 46 subjects who had been infected by the pandemic virus over the course of the study, verified by a positive qRT-PCR nasal swab, and for whom paired sera were available. Initial HIA antibody titers in this group were ,1/40,", "The CoPanFlu-RUN cohort was set up to conduct a prospective population-based study investigating the herd immunity induced by the 2009 pandemic influenza virus and identifying risk factors for pH1N1/2009v infection from paired sera collected in an entire community. Most works published to date have used either extensive cross-sectional serosurveys on pre-and post-epidemic independent serum samples, the baseline immunity being assessed from stored frozen samples [5, 7, 8] , or non representative adult cohorts (military, health care workers, long-stay patients). Antibody titers were measured by HIA using a cut-off value set at 1/40 as classically recommended. This HIA titer at 1/40 is considered protective, i.e. conferring 50% protection against a viral challenge [20] . Our assay has introduced some changes in the experimental protocol compared to the classic one. The use of a non-inactivated viral antigen, i.e. a native virus, with nondenatured epitopes probably allows detection of", "antibodies to epitopes of the hemagglutinin not detected in the classic HIA test. This can induce slight differences in the sensitivity of detection of cross-reacting antibodies, but this does not modify the kinetics of Ab and the epidemiological evolution of seroprevalence and does not jeopardize the global comparability of serological results. This is confirmed by the fact that our HI assay detected seroprotective antibody titers in 93.5% and gave evidence seroconversion in 73.9% of qRT-PCR confirmed pH1N1/2009 influenza, all figures close to those reported in the literature [5, 21] .", "We considered that titers of .1/40, in sera collected from individuals enrolled during weeks 30 and 31 were cross reactive antibodies and not de novo antibodies triggered by the pandemic virus and hence used them as a proxy for baseline pre epidemic immunity. Several arguments support this assumption: i) the first case indicating autochthonous transmission in Reunion Island was reported by the epidemiological surveillance department of La Réunion on 21st July (week 30), i.e. the same day when inclusion started in our study cohort; ii) 7 to 15 days are required to develop an antibody response after viral infection; iii) On weeks 30 and 31, the epidemic activity due to the pandemic virus was very low in our study cohort and it became significant only after week 32. Hence, during weeks 30-31, 103 households were recruited and only 2 households reported ILI cases. Nasal swabs collected from these 2 individuals were tested qRT-PCR negative to the pandemic virus whereas one had evidence of", "coronavirus and rhinovirus using a multiplex RT-PCR to respiratory viruses (H. Pascalis, manuscript in preparation). In contrast, during weeks 32 to 39, 199 individuals belonging to 99 households reported ILI, among whom 60 individuals had documented infection by the pandemic virus.", "Our study shows that a substantial proportion of Reunion Island's population had pre-existing immunity to 2009 pandemic influenza virus with the highest baseline-proxy seroprevalence rate observed among adults aged of 60 years or more. Other studies from all continents had also reported high pre-epidemic seropositivity rates among the elderly [5, 6, 8, [22] [23] [24] [25] [26] , though large variations do exist between countries [10, 11, 23, 27, 28] . These cross reactive antibodies have been interpreted as being the residual signature of the remote exposure of these individuals to H1N1 viruses circulating before 1957 [24, 25, 29, 30] . Baseline seropositivity rates that we report in children and in younger adults (i.e. 30%-35%) were notably higher than those reported from other parts of the world [6, 8, 22, 23, [31] [32] [33] . However one should note that these baseline antibodies were of low titer, just at the level of the HIA threshold (i.e. 1/40). Several factors could have", "contributed to this comparatively high baseline rates found in our study: i) It may reflect the fact that the HI test used in our study was marginally more sensitive than the classic one [17] ; ii) Some individuals may have already been infected with pH1N1/ 2009 virus at weeks 30 and 31 and may have triggered an antibody response to the virus. This hypothesis seems unlikely in view of the arguments presented above and of a similar high proportion of sera titering HIA = 1/40 among 122 sera from adult patients sent for diagnostic purposes to the Regional Hospital microbiology laboratory, during the first half of 2009 (i.e. before the 2009 pandemic) (data not shown). However we cannot formally exclude this hypothesis in view of a recently reported study from Taiwan [11] that showed evidence of subclinical community transmission with proved seroconversion several weeks before report of the first documented case in the island. A similar conclusion was also drawn from Australia [34] ; iii)", "our serological test might detect cross-reactive antibodies triggered by recent vaccination with trivalent seasonal influenza vaccine as reported [4, [35] [36] [37] [38] [39] . However, seasonal influenza vaccines were of rather limited use in Reunion Island, especially in children and young adults; iv) Finally the high baseline titers may reflect the infectious history of the individuals to seasonal influenza viruses cross antigenic with pH1N1/2009 virus as recently suggested for seasonal 2007 H1N1 infection [40] . This serosurvey indicates that a large fraction of the Reunion Island population was infected with the pandemic virus. Younger people, have paid the main tribute to the epidemic as almost two thirds show evidence of seroconversion, confirming earlier clinical reports from the island [12] and accumulating reports from other countries [17, 32, 41, 42] and suggesting that school children have likely played the central role in the epidemic diffusion of the pandemic virus.", "Lower infection rates were found in adults and the lowest rates were recorded in the elderly.", "Based on clinical cases reported to the epidemiological surveillance services [12] , it was estimated that 66,915 persons in Reunion Island who consulted a physician were infected by the pH1N1/2009 virus during the 9 weeks of the epidemic, giving a cumulative attack rate of 8.26%. Taking into account those who did not consult a physician, the number of symptomatic infected persons was estimated to 104,067 (attack rate: 12.85%). In fact, the attack rate of pH1N1/2009 infection in our serosurvey was about 42%-44% at the peak of the antibody response (i.e., weeks 40-44), a figure which is at least 3 to 4 times higher than rates of infection based on clinical cases The wide gap between the two estimates indicates that a large fraction (almost two thirds) of those who got infected by pH1N1/2009 virus escaped medical detection, probably because they developed mild disease or asymptomatic infection, a further indication of the benign nature of the virus, at least at the community level. In", "England, Baguelin et al. [43] estimated that the cumulative incidence rates of infection by the pandemic virus in children were 20 to 40 times higher than that estimated from clinical surveillance.", "Our study, as others [6] , indicates that pre-existing cross reactive antibodies to pH1N1/2009 at titers $1/40 prevented from seroconversion in response to the pandemic virus. This level of pre-existing cross reactive immunity likely confers true protection against infection as about two thirds and one third of documented infection (qRT-PCR positive) in our series have occurred in individuals with baseline HIA titers ,1/40 and = 1/ 40 respectively and less than 5% of documented infections occurred in individuals with base line titers .1/40. The protection was effective not only in older adults but also in younger persons. This indicates that protection was conferred not only by baseline cross reactive antibodies triggered by close pH1N1/2009 viruses that circulated before 1957 (as in the elderly), but also by antibodies likely resulting from recent exposure to seasonal influenza epidemics (as shown in younger persons) [40] . The observed seroconversion rates depend on age, after", "adjusting for baseline pH1N1/2009 titers. The protective role of increasing age might be explained by a stronger cross-immunity in adults and elderly or by a higher exposure of young subjects to the virus during the 2009 epidemic (due to social contacts and mixing patterns). It may also indicate that immune mechanisms other than cross reactive antibodies detected by HIA (i.e. immunity to neuraminidase and conserved T cells epitopes [44] might develop throughout life, providing additional protection from infection or severe disease, especially in the elderly. Interestingly, evidence is seen for a decline in antibody titers, which occurred soon after the passage of the epidemic wave. In paired sera, this decline was significant enough to bring, within a few weeks, almost 27% of sera that tested positive (i.e. HI titers $1/40) in the immediate post epidemic phase to levels under the cut-off value in the second serum sample. This decay accounts for the observation that older adults ($60", "yrs) in the study cohort were apparently almost completely spared by the epidemic if one only considers cumulative incidence rates derived from IHA titration on samples 2 (weeks 45-52). In fact, the cumulative incidence rate in older adults measured just after the epidemic peak (i.e. weeks 40-44) was 20.4%. Similar results of early antibody decay were recently reported [10, 45] . More generally, these data show that serosurveys conducted months after passage of the epidemic, likely underestimate the real extent of pH1N1/2009 infection, compared to antibody titration performed earlier, when humoral responses are at their highest level. Whether the decline in antibody titers has functional immunologic consequence to individuals or within the communities warrants further investigation. However, one should note that there was no second epidemic wave in Reunion Island during the subsequent austral winter seasons in 2010 and 2011.", "Influenza during the 2010 winter was at a level not higher than the usual passages of seasonal flu, though almost two thirds of documented cases in 2010 were also due to pH1N1/2009v [46] . In addition many fewer pandemic virus isolates were noted during the ongoing 2011 austral winter, strongly suggesting that the first epidemic wave had conferred a solid herd immunity, at the community level.", "Our study has some limitations. The fact that the epidemic progression coincided with the implementation of the prospective study, we were not able to collect, strictly speaking, pre-epidemic sera from the cohort members. Therefore we used as proxy base line seroprevalence data from individuals recruited at the very beginning of the investigation when the epidemic activity in the cohort was very low. This may overestimate the base line immunity if subclinical community transmission had occurred before the first cases of pH1N1/2009 influenza were reported. Antibodies to the pandemic virus were detected by HIA, a test that has a good specificity but a rather low sensitivity [46] . Hence, the threshold of 1/40 may underestimate the number of infected individuals. However, rates of seroconversion, the serologic gold standard test based on paired sera, likely gave the most accurate picture of the pandemic in at the community level in Reunion Island." ]

[ 1 ]

[ "conferring 50% protection against a viral challenge" ]

[ "Pandemic Influenza Due to pH1N1/2009 Virus: Estimation of Infection Burden in Reunion Island through a Prospective Serosurvey, Austral Winter 2009\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3183080/\n\nSHA: ee6d70a53e3262cea6f85bd8b226f6b4c8b5f64b\n\nAuthors: Dellagi, Koussay; Rollot, Olivier; Temmam, Sarah; Salez, Nicolas; Guernier, Vanina; Pascalis, Hervé; Gérardin, Patrick; Fianu, Adrian; Lapidus, Nathanael; Naty, Nadège; Tortosa, Pablo; Boussaïd, Karim; Jaffar-Banjee, Marie-Christine; Filleul, Laurent; Flahault, Antoine; Carrat, Fabrice; Favier, Francois; de Lamballerie, Xavier\nDate: 2011-09-29\nDOI: 10.1371/journal.pone.0025738\nLicense: cc-by", "Abstract: BACKGROUND: To date, there is little information that reflects the true extent of spread of the pH1N1/2009v influenza pandemic at the community level as infection often results in mild or no clinical symptoms. This study aimed at assessing through a prospective study, the attack rate of pH1N1/2009 virus in Reunion Island and risk factors of infection, during the 2009 season. METHODOLOGY/PRINCIPAL FINDINGS: A serosurvey was conducted during the 2009 austral winter, in the frame of a prospective population study. Pairs of sera were collected from 1687 individuals belonging to 772 households, during and after passage of the pandemic wave. Antibodies to pH1N1/2009v were titered using the hemagglutination inhibition assay (HIA) with titers ≥1/40 being considered positive. Seroprevalence during the first two weeks of detection of pH1N1/2009v in Reunion Island was 29.8% in people under 20 years of age, 35.6% in adults (20–59 years) and 73.3% in the elderly (≥60 years) (P<0.0001).", "Baseline corrected cumulative incidence rates, were 42.9%, 13.9% and 0% in these age groups respectively (P<0.0001). A significant decline in antibody titers occurred soon after the passage of the epidemic wave. Seroconversion rates to pH1N1/2009 correlated negatively with age: 63.2%, 39.4% and 16.7%, in each age group respectively (P<0.0001). Seroconversion occurred in 65.2% of individuals who were seronegative at inclusion compared to 6.8% in those who were initially seropositive. CONCLUSIONS: Seroincidence of pH1N1/2009v infection was three times that estimated from clinical surveillance, indicating that almost two thirds of infections occurring at the community level have escaped medical detection. People under 20 years of age were the most affected group. Pre-epidemic titers ≥1/40 prevented seroconversion and are likely protective against infection. A concern was raised about the long term stability of the antibody responses.", "Text: In April 2009, the first cases of acute respiratory infections caused by a novel triple-reassortant influenza virus, pH1N1/ 2009v, occurred in Mexico and the United States [1] . The rapid spread of infection to other continents led the World Health Organization (WHO) to declare on 11 June 2009 that a pandemic of pH1N1/2009v influenza was under way, which raised major international concern about the risk of high morbidity and lethality and the potential for severe socio-economic impact. Actually, the potential impact of this first third-millenium influenza pandemic has been revisited downwards as morbidity and case-fatality rates were less severe than initially anticipated [2] . Illness surveillance data do not allow to an accurate estimate of the true influenza infection rate, as a substantial proportion of infections are asymptomatic or mild [3] . Serological surveys can overcome this limitation, but must take into account that a significant proportion of the population that", "exhibited crossprotective antibody titers before circulation of the pH1N1/2009v [4] . This so-called ''baseline immunity'' has to be subtracted from the seroprevalence observed after the pandemic wave, to determine seroincidence in serosurveys [5] [6] [7] [8] . However, except for few studies [9] [10] [11] , most of these serosurveys did not use serial measurements in the same person, which allows for a better understanding of antibody kinetics and the dynamics of infection within individuals and communities.", "Reunion Island (805,500 inhabitants) is a French overseas department located in the southwestern Indian Ocean, 700 km east of Madagascar and 200 km southwest of Mauritius. The first imported case of pH1N1/2009v was identified on 5 th July 2009 (week 29) in a traveller returning from Australia. The first case indicating community transmission was detected on 21 st July (week 30). pH1N1/2009v became the predominant circulating influenza virus within four weeks of its first detection, its activity peaked during week 35 (24) (25) (26) (27) (28) (29) (30) and ended at week 38 [12] . Contrary to initial fears, the health care system was not overwhelmed, as morbidity and mortality rates were lower than predicted [12] [13] [14] .", "In order to assess at the community level, the actual magnitude of the pH1N1/2009v pandemic and the extent of the herd immunity acquired after passage of the epidemic wave, a prospective population serosurvey was conducted in Reunion Island during the passage of the epidemic wave in the 2009 austral winter season (July-December 2009): prevalence of infection was assessed on a weekly basis and seroconversion rates were measured using paired sera.", "The CoPanFLu-RUN was part of the CoPanFLu international project, a consortium between the French National Institute of Health and Medical Research (INSERM), the Institute of Research for Development (IRD) and the Mérieux Fondation under the promotion of the School of Advanced Studies in Public Health (EHESP). To enable the rapid implementation of the study in anticipation of the imminent spread of the pandemic wave, we used a pre-existing sample of 2442 households established in October 2006 for the investigation of the Chikungunya outbreak (SEROCHIK) and updated in May 2008 throughout a follow-up telephone survey (TELECHIK) on a basis of 1148 households [15, 16] . We took special attention to select households representing a wide range of geographic locations in order to minimize the repartition bias.", "The inclusion phase started on July 21 st (week 30) and was continued up to week 44, throughout the epidemic wave and beyond. A first serum sample (sample 1) was obtained from each household member. An active telephonic inquiry was then conducted twice a week to record symptoms compatible with influenza-like illness (ILI) occurring in households. Report of ILI (fever $37.8uC associated with any respiratory or systemic symptom) led to three consecutive visits of a nurse to the incident case-dwelling (on day 0, +3 and +8 post-report) to record symptoms and collect nasal swabs from all family members (for qRT-PCR detection of pH1N1/2009v. At week 45, the active inquiry was discontinued and a second (post-epidemic) serum sample (sample 2) was obtained (weeks 45-52) to determine seroconversion rates. Sera were aliquoted and stored at 280uC.", "The protocol was conducted in accordance with the Declaration of Helsinki and French law for biomedical research (Nu ID RCB AFSSAPS: 2009-A00689-48) and was approved by the local Ethics Committee (Comité de Protection des Personnes of Bordeaux 2 University). Every eligible person for participation was asked for giving their written informed consent.\n\nViral genome detection by RT-PCR. Viral RNA was extracted from 140 mL of nasal swab eluate using the QIAamp Viral RNA kit (Qiagen) and processed for detection by TaqMan qRT-PCR targeting the heamagglutinin HA gene (SuperScript III Platinum one-step qRT-PCR system, Invitrogen) according to the recommendations of the Pasteur Institute (Van der Werf S. & Enouf V., SOP/FluA/130509). Confirmed pH1N1/2009v infection was defined as a positive qRT-PCR detection of the HA gene in at least one nasal swab.", "Hemagglutination inhibition assay (HIA). A standard hemagglutination inhibition technique was adapted to detect and quantify pH1N1/2009v antibodies [17] . The antigen was prepared by diluting a non-inactivated cell culture supernatant producing a pdm H1N1v strain (strain OPYFLU-1 isolated from a young patient returning from Mexico in early May 2009) [18] . Briefly, the virus was propagated onto MDCK cells under standard conditions. The last passage (used for antigen preparation) was performed in the absence of trypsin and ht-FBS. The supernatant was collected at day seven p.i. clarified by centrifugation at 8006 g for 10 min at room temperature, aliquoted and conserved at 280uC. The hemagglutinating titer of the non inactivated viral antigen was immediately determined under the HIA format described below. The dilution providing 5.33 hemagglutinating units in a volume of 25 mL was used for subsequent HIA. Sera were heat-inactivated at 56uC for 30 min prior to use. Sequential twofold", "dilutions in PBS (1/10 to 1/1280) in volumes of 25 mL were performed and distributed in V-bottom 96 well microplates. Human red blood cells (RBC) were used for hemagglutination experiments. Detection and quantification of antibody to pH1N1/2009v was performed as follows: 25 mL of virus suspension was added to the serum dilution (25 mL) and incubated for 1 hour at room temperature. Each well was then filled with 25 mL of a 1% RBC suspension in PBS (v/v: 0.33%), followed by another 30 min incubation at room temperature. The HIA titer was determined as the last dilution providing clear inhibition of hemagglutination. All experiments were performed in the presence of the same negative and positive controls, the latter including sera with 1/40, 1/80, 1/160 and 1/320 antibody titers.", "The results reported in this study were based only on serological analysis of paired sera. For the sake of analysis, four successive phases were identified throughout the pandemic wave: phase A (weeks 30-31) corresponded to early epidemic time, phase B (W32-39) to the epidemic unfolding, phase C (W40-44) to the immediate post-epidemic stage and phase D (W45-52) to the late post-epidemic stage. Seropositivity was defined as a HIA titer of 1/ 40 or more. The baseline-proxy seroprevalence rate was estimated on serum samples collected in phase A. The cumulative incidence rate of infection measured the raise between the raw seroprevalence rate at any given time during the epidemic phases (S2pi) and the age-specific baseline-proxy seroprevalence rate (S1pA) (s2 pi -s1 pA ). Seroconversion was defined as a shift from seronegative at inclusion (sample 1: HIA ,1/40) to seropositive on follow-up (sample 2: HIA $1/40), or for sera tested seropositive on inclusion as a four-fold increase of HIA", "titers between sample 1 and sample 2 paired sera. We also calculated the proportion of sera that tested seropositive in sample 1 for which the HIA titer decreased fourfold and passed under the cut-off value of 1/40 in sample 2. We considered this proportion as a ''seronegation'' rate.", "The sample size was calculated for identifying risk factors in the prospective cohort study. Considering on average three individuals per household, an intra-household correlation of 0.3, a power greater than 80% could be obtained with a sample size of 840 comprising 2500 individuals, assuming exposure levels ranging from 10% to 90% and a relative risk greater than 1.3. With 2,500 subjects, the study allowed 1-2% absolute precision around the estimated values for seroconversion rates. Data entry used EpiData version 3.1 (The Epidata Association, Odense, Denmark). SAS version 9.1 (SAS Inc., Cary, NC, USA) was used for statistical analysis. The characteristics of the study cohort were compared to those of the population of Reunion Island and a Chi2 test (or Fisher's exact test when non applicable) was used to analyse differences in age, sex and geographic location. Cumulative incidence rates of infection (i.e. seroincidence) and seroconversion rates were standardized according to the", "age structure of the community (French National Institute for Statistics and Economical Studies (INSEE) source).", "Baseline-proxy seroprevalence, cumulative incidence rates of infection, as well as seroconversion and seronegation rates, were expressed as percentages. Cumulative reverse distribution curves were used to show the distribution of antibody titers. In all tests, a P value,0.05 was considered significant.\n\nWe estimated 95% confidence intervals (CIs) of proportions by using a cluster bootstrap technique with 1000 re-samples [19] . After bootstraping, we used an ANOVA model to compare mean cumulative incidence proportions between pandemic phases, within each age group. We used an alternating logistic regression model (ALR) with an exchangeable log Odds Ratio (OR) to test the intra-household correlation-adjusted association between factors and the seroconversion outcome.", "Data were analysed with respect to subject age. Initially, four age groups were considered: the children and adolescents (,20 yrs), young adults (20-39 yrs), middle-age adults (40-59 yrs), and elderly adults ($60 yrs). As the cumulative incidence of infection of the second and third groups were very close, both groups were merged into one adults group (20-59 yrs). Therefore we refer further in our study to three age groups: children and adolescents (,20 yrs), adults (20-59 yrs), elderly ($60 yrs).", "A total of 2,164 individuals from 772 households were enrolled between weeks 30 and 44 in the CoPanFlu-RUN cohort, allowing the collection of 1,932 sera at inclusion (sample 1). During this period, 136 households (17.7% of households) containing 464 individuals (21.4% of individuals) reported at least one case of ILI. Sixty subjects among the 464 individuals (12.9%, belonging to 33 households [24.3%]) were qRT-PCR positive, which documented the pH1N1/2009v infection. No positive qRT-PCR could be detected after week 37 and no ILI was reported after week 40, the end of the epidemic wave. The second follow up serum sample (sample 2) was obtained for 1,759 subjects at least five weeks after the end of the epidemic wave (weeks 45-52) which allowed the constitution of a serobank of 1,687 paired-sera. The profile of the cohort and the major outcomes are displayed in Figure 1 . Details on inclusions and serum sample timing with respect to the circulation of pH1N1/2009v over the island are", "provided in figure 2 . The socio-demographic and space-time characteristics of the cohort are detailed in Table 1 . Compared to the community of Reunion Island, the sample of 1,687 individuals for whom pairedsera were available, was older (,20 yrs: 27% vs 35%, and $60 yrs: 17,9% vs 11,3%) and composed of a slight excess of females (54.1% vs 51.5%). The imbalance was due to a deficit in subjects aged under 40 years, reflecting men at work and the fact that parents declined the second serum for children younger than five.", "Baseline-proxy (,pre-epidemic) HIA titers to the pH1N1/ 2009v were measured on sample 1 ( Table 2) , obtained from 249 subjects (103 households) recruited at the very beginning of the investigation during weeks 30 and 31 (phase A, Figure 2 ), when the epidemic activity in the cohort was still very low. Age distribution in this group was similar to that of the whole cohort (data not shown). The overall, the baseline-proxy seroprevalence rate (HIA $1/40), over all ages, was 43.4% (95%CI: 37.4%-49.6%). However the majority of positive sera had low antibody titers, at the cut off value for confirmation (i.e. = 1/40). The proportions of sera with HIA titer .1/40 were 0%, 3.0% and 24.6% in the young, middle-aged and older age groups respectively. These results indicate that pre-epidemic baseline antibody cross reactivity was stronger in the elderly ($60 yrs) and weaker in children and adolescents (,20 yrs) and adults (20-59 yrs), with highly significant differences between age groups", "(P,0.0001).", "The reverse cumulative distribution curves of HIA titers are displayed for each age group and for the whole cohort on Figure 3 . The proportion of seropositive sera (HI $1/40) steadily increased during the epidemic unfolding (phase B, W32-39) and in immediate post epidemic period (phase C, W40-44) when it reached its maximum level, then declined in the late post epidemic period (phase D, W45-52). This decline was significant enough to return the reverse cumulative distribution curve to baseline levels in the elderly. The cumulative incidence rates, obtained after subtraction of the age-specific baseline-proxy seroprevalence from the raw seroprevalence at each phase of the epidemic are shown in Table 2 (note that the cumulative incidence rates of infection represented for the group ''all ages'' were standardized according to age structure of the community). The cumulative incidence rates were much higher in children and adolescents (,20 yrs), indicating very active transmission of", "infection within this age group. As mentioned earlier, cumulative incidence rates peaked in phase C (W40-44), and then declined indicating some lability of the humoral immune response against the pH1N1/2009v. The age-related difference observed in the incidence rates was highly statistically significant (P,0.0001).", "To estimate more appropriately the decline of antibody titers occurring after the peak of the humoral response to the pH1N1/ 2009v, we considered paired-sera from the group of 264 subjects for whom the first serum sample (sample 1) was obtained just after the epidemic wave (phase C, W40-44), and the corresponding second sample was collected at the end of the survey (phase D, W45-52). Seronegation rates were 27.0% (61/226) for all age groups, 17.4% (12/69) in children and adolescents (,20 yrs), 32.3% (41/127) in adults (20-59 yrs) and 26.7% (8/30) in the elderly ($60 yrs). Differences between the seronegation rates according to age were statistically weakly significant (P = 0.0671).", "We then considered the 1687 individuals for whom paired sera were available and we measured the seroconversion rates according to age and to the time of first serum sample collection (phase A, B or C). Criteria of seroconversion were defined in the method section. As shown in table 3, there was a sharp decline in seroconversion rates across all the age groups, depending on whether participants were enrolled during phase A, phase B, or phase C (P,0.0001). To interpret these data, one should remember that antibodies at seroprotective levels (HIA $1/40), in serum samples 1 collected during the per epidemic phase B or early post epidemic phase C could represent either base line cross reactive antibodies or rising pH1N1/2009 specific antibodies due to a recent or ongoing infection. This ambiguity could lead to underestimation of the seroconversion rate for subjects enrolled in phases B and C. In order to solve this ambiguity, we specifically considered the group of 249 subjects in whom", "cross reactive antibodies were detected at the time of phase A (W30-31). The seroconversion rate of this group is the most indicative of the exposure of individuals to the whole epidemic wave. It was the highest (63,2%, P,0.0001) in children and adolescents (,20 yrs), and still significantly high in adults (39.4%, P,0.0001).", "We then tested in this particular group, the impact of (baseline) pre-epidemic cross reactive antibodies on the rate of seroconversion to pH1N1/2009 (Table 4) . No subject with HIA titer superior to 1/40 had evidence of seroconversion to pH1N1/2009. The seroconversion rate in individuals with a HIA titer equal to 1/40 was linked with age, being more important in children and adolescents (,20 yrs). The highest seroconversion rate (.56%) was registered in subjects with HIA titers inferior to 1/40, particularly for the under 20 years where it reached 85%. Hence, the risk of seroconversion decreased when pre-epidemic HIA titer was high after controlling for age (P,0.0001) (Figure 4) . The multivariate adjusted odds ratio for seroconversion were 0.15 (95%CI: 0.06-0.37, P,0.0001) per two-fold increase in baseline titer, 1.79 (95%CI: 1.23-2.59, P,0.003) per other household members who seroconverted, 5.33 (95%CI: 1.56-19.27, P,0.008) Figure 1 . The cohort profile and major outcomes. Figure 1", "details the three phases of the protocol: i) inclusion (weeks 30-44) and serum samples S1 collection; ii) follow up for detection of ILI in households, qRT-PCR on nasal swabs and estimation of cumulative seroincidence rates; iii) end of the study (weeks 45-52) and samples S2 collection. HIA on paired sera (S1+S2) allowed estimating seroconversion rates. doi:10.1371/journal.pone.0025738.g001 Bp (baseline-proxy) seroprevalence rates were estimated on weeks 30-31 in each age group. b Cumulative incidence rates measured the raise between raw seroprevalence rates and age-specific baseline-proxy seroprevalence rate. In the group ''All ages'', cumulative incidence rates were standardized according to age structure of the community. doi:10.1371/journal.pone.0025738.t002 Data are numbers, percentages (95% confidence intervals) and ALR parameter test P value for comparison of seroconversion proportions according to time of first sample (S1) collection at inclusion, in each age group, after", "controlling for household selection. In the group ''All ages'', rates of seroconversion were standardized according to age structure of the community. NA: not assessed. Seroconversion was defined as a shift from seronegative at inclusion (i.e. HIA titer ,1/40) to seropositive on follow-up sample, or as a 4-fold increase of reciprocal HIA titer between first and second paired samples for sera tested seropositive on inclusion (i.e. HIA titer $1/40). for age ,20 years (vs age $60 years) and 11.35 (95%CI: 0.41-4.47, P = 0.62) for age 20-60 years (vs age $60 years). The observed and predicted seroconversion rates according to age and baseline HIA titer are displayed Figure 4 .", "Finally, we considered the 46 subjects who had been infected by the pandemic virus over the course of the study, verified by a positive qRT-PCR nasal swab, and for whom paired sera were available. Initial HIA antibody titers in this group were ,1/40,", "The CoPanFlu-RUN cohort was set up to conduct a prospective population-based study investigating the herd immunity induced by the 2009 pandemic influenza virus and identifying risk factors for pH1N1/2009v infection from paired sera collected in an entire community. Most works published to date have used either extensive cross-sectional serosurveys on pre-and post-epidemic independent serum samples, the baseline immunity being assessed from stored frozen samples [5, 7, 8] , or non representative adult cohorts (military, health care workers, long-stay patients). Antibody titers were measured by HIA using a cut-off value set at 1/40 as classically recommended. This HIA titer at 1/40 is considered protective, i.e. conferring 50% protection against a viral challenge [20] . Our assay has introduced some changes in the experimental protocol compared to the classic one. The use of a non-inactivated viral antigen, i.e. a native virus, with nondenatured epitopes probably allows detection of", "antibodies to epitopes of the hemagglutinin not detected in the classic HIA test. This can induce slight differences in the sensitivity of detection of cross-reacting antibodies, but this does not modify the kinetics of Ab and the epidemiological evolution of seroprevalence and does not jeopardize the global comparability of serological results. This is confirmed by the fact that our HI assay detected seroprotective antibody titers in 93.5% and gave evidence seroconversion in 73.9% of qRT-PCR confirmed pH1N1/2009 influenza, all figures close to those reported in the literature [5, 21] .", "We considered that titers of .1/40, in sera collected from individuals enrolled during weeks 30 and 31 were cross reactive antibodies and not de novo antibodies triggered by the pandemic virus and hence used them as a proxy for baseline pre epidemic immunity. Several arguments support this assumption: i) the first case indicating autochthonous transmission in Reunion Island was reported by the epidemiological surveillance department of La Réunion on 21st July (week 30), i.e. the same day when inclusion started in our study cohort; ii) 7 to 15 days are required to develop an antibody response after viral infection; iii) On weeks 30 and 31, the epidemic activity due to the pandemic virus was very low in our study cohort and it became significant only after week 32. Hence, during weeks 30-31, 103 households were recruited and only 2 households reported ILI cases. Nasal swabs collected from these 2 individuals were tested qRT-PCR negative to the pandemic virus whereas one had evidence of", "coronavirus and rhinovirus using a multiplex RT-PCR to respiratory viruses (H. Pascalis, manuscript in preparation). In contrast, during weeks 32 to 39, 199 individuals belonging to 99 households reported ILI, among whom 60 individuals had documented infection by the pandemic virus.", "Our study shows that a substantial proportion of Reunion Island's population had pre-existing immunity to 2009 pandemic influenza virus with the highest baseline-proxy seroprevalence rate observed among adults aged of 60 years or more. Other studies from all continents had also reported high pre-epidemic seropositivity rates among the elderly [5, 6, 8, [22] [23] [24] [25] [26] , though large variations do exist between countries [10, 11, 23, 27, 28] . These cross reactive antibodies have been interpreted as being the residual signature of the remote exposure of these individuals to H1N1 viruses circulating before 1957 [24, 25, 29, 30] . Baseline seropositivity rates that we report in children and in younger adults (i.e. 30%-35%) were notably higher than those reported from other parts of the world [6, 8, 22, 23, [31] [32] [33] . However one should note that these baseline antibodies were of low titer, just at the level of the HIA threshold (i.e. 1/40). Several factors could have", "contributed to this comparatively high baseline rates found in our study: i) It may reflect the fact that the HI test used in our study was marginally more sensitive than the classic one [17] ; ii) Some individuals may have already been infected with pH1N1/ 2009 virus at weeks 30 and 31 and may have triggered an antibody response to the virus. This hypothesis seems unlikely in view of the arguments presented above and of a similar high proportion of sera titering HIA = 1/40 among 122 sera from adult patients sent for diagnostic purposes to the Regional Hospital microbiology laboratory, during the first half of 2009 (i.e. before the 2009 pandemic) (data not shown). However we cannot formally exclude this hypothesis in view of a recently reported study from Taiwan [11] that showed evidence of subclinical community transmission with proved seroconversion several weeks before report of the first documented case in the island. A similar conclusion was also drawn from Australia [34] ; iii)", "our serological test might detect cross-reactive antibodies triggered by recent vaccination with trivalent seasonal influenza vaccine as reported [4, [35] [36] [37] [38] [39] . However, seasonal influenza vaccines were of rather limited use in Reunion Island, especially in children and young adults; iv) Finally the high baseline titers may reflect the infectious history of the individuals to seasonal influenza viruses cross antigenic with pH1N1/2009 virus as recently suggested for seasonal 2007 H1N1 infection [40] . This serosurvey indicates that a large fraction of the Reunion Island population was infected with the pandemic virus. Younger people, have paid the main tribute to the epidemic as almost two thirds show evidence of seroconversion, confirming earlier clinical reports from the island [12] and accumulating reports from other countries [17, 32, 41, 42] and suggesting that school children have likely played the central role in the epidemic diffusion of the pandemic virus.", "Lower infection rates were found in adults and the lowest rates were recorded in the elderly.", "Based on clinical cases reported to the epidemiological surveillance services [12] , it was estimated that 66,915 persons in Reunion Island who consulted a physician were infected by the pH1N1/2009 virus during the 9 weeks of the epidemic, giving a cumulative attack rate of 8.26%. Taking into account those who did not consult a physician, the number of symptomatic infected persons was estimated to 104,067 (attack rate: 12.85%). In fact, the attack rate of pH1N1/2009 infection in our serosurvey was about 42%-44% at the peak of the antibody response (i.e., weeks 40-44), a figure which is at least 3 to 4 times higher than rates of infection based on clinical cases The wide gap between the two estimates indicates that a large fraction (almost two thirds) of those who got infected by pH1N1/2009 virus escaped medical detection, probably because they developed mild disease or asymptomatic infection, a further indication of the benign nature of the virus, at least at the community level. In", "England, Baguelin et al. [43] estimated that the cumulative incidence rates of infection by the pandemic virus in children were 20 to 40 times higher than that estimated from clinical surveillance.", "Our study, as others [6] , indicates that pre-existing cross reactive antibodies to pH1N1/2009 at titers $1/40 prevented from seroconversion in response to the pandemic virus. This level of pre-existing cross reactive immunity likely confers true protection against infection as about two thirds and one third of documented infection (qRT-PCR positive) in our series have occurred in individuals with baseline HIA titers ,1/40 and = 1/ 40 respectively and less than 5% of documented infections occurred in individuals with base line titers .1/40. The protection was effective not only in older adults but also in younger persons. This indicates that protection was conferred not only by baseline cross reactive antibodies triggered by close pH1N1/2009 viruses that circulated before 1957 (as in the elderly), but also by antibodies likely resulting from recent exposure to seasonal influenza epidemics (as shown in younger persons) [40] . The observed seroconversion rates depend on age, after", "adjusting for baseline pH1N1/2009 titers. The protective role of increasing age might be explained by a stronger cross-immunity in adults and elderly or by a higher exposure of young subjects to the virus during the 2009 epidemic (due to social contacts and mixing patterns). It may also indicate that immune mechanisms other than cross reactive antibodies detected by HIA (i.e. immunity to neuraminidase and conserved T cells epitopes [44] might develop throughout life, providing additional protection from infection or severe disease, especially in the elderly. Interestingly, evidence is seen for a decline in antibody titers, which occurred soon after the passage of the epidemic wave. In paired sera, this decline was significant enough to bring, within a few weeks, almost 27% of sera that tested positive (i.e. HI titers $1/40) in the immediate post epidemic phase to levels under the cut-off value in the second serum sample. This decay accounts for the observation that older adults ($60", "yrs) in the study cohort were apparently almost completely spared by the epidemic if one only considers cumulative incidence rates derived from IHA titration on samples 2 (weeks 45-52). In fact, the cumulative incidence rate in older adults measured just after the epidemic peak (i.e. weeks 40-44) was 20.4%. Similar results of early antibody decay were recently reported [10, 45] . More generally, these data show that serosurveys conducted months after passage of the epidemic, likely underestimate the real extent of pH1N1/2009 infection, compared to antibody titration performed earlier, when humoral responses are at their highest level. Whether the decline in antibody titers has functional immunologic consequence to individuals or within the communities warrants further investigation. However, one should note that there was no second epidemic wave in Reunion Island during the subsequent austral winter seasons in 2010 and 2011.", "Influenza during the 2010 winter was at a level not higher than the usual passages of seasonal flu, though almost two thirds of documented cases in 2010 were also due to pH1N1/2009v [46] . In addition many fewer pandemic virus isolates were noted during the ongoing 2011 austral winter, strongly suggesting that the first epidemic wave had conferred a solid herd immunity, at the community level.", "Our study has some limitations. The fact that the epidemic progression coincided with the implementation of the prospective study, we were not able to collect, strictly speaking, pre-epidemic sera from the cohort members. Therefore we used as proxy base line seroprevalence data from individuals recruited at the very beginning of the investigation when the epidemic activity in the cohort was very low. This may overestimate the base line immunity if subclinical community transmission had occurred before the first cases of pH1N1/2009 influenza were reported. Antibodies to the pandemic virus were detected by HIA, a test that has a good specificity but a rather low sensitivity [46] . Hence, the threshold of 1/40 may underestimate the number of infected individuals. However, rates of seroconversion, the serologic gold standard test based on paired sera, likely gave the most accurate picture of the pandemic in at the community level in Reunion Island." ]

[ 31 ]

[ "a substantial proportion of Reunion Island's population had pre-existing immunity to 2009 pandemic influenza virus" ]

[ "Pandemic Influenza Due to pH1N1/2009 Virus: Estimation of Infection Burden in Reunion Island through a Prospective Serosurvey, Austral Winter 2009\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3183080/\n\nSHA: ee6d70a53e3262cea6f85bd8b226f6b4c8b5f64b\n\nAuthors: Dellagi, Koussay; Rollot, Olivier; Temmam, Sarah; Salez, Nicolas; Guernier, Vanina; Pascalis, Hervé; Gérardin, Patrick; Fianu, Adrian; Lapidus, Nathanael; Naty, Nadège; Tortosa, Pablo; Boussaïd, Karim; Jaffar-Banjee, Marie-Christine; Filleul, Laurent; Flahault, Antoine; Carrat, Fabrice; Favier, Francois; de Lamballerie, Xavier\nDate: 2011-09-29\nDOI: 10.1371/journal.pone.0025738\nLicense: cc-by", "Abstract: BACKGROUND: To date, there is little information that reflects the true extent of spread of the pH1N1/2009v influenza pandemic at the community level as infection often results in mild or no clinical symptoms. This study aimed at assessing through a prospective study, the attack rate of pH1N1/2009 virus in Reunion Island and risk factors of infection, during the 2009 season. METHODOLOGY/PRINCIPAL FINDINGS: A serosurvey was conducted during the 2009 austral winter, in the frame of a prospective population study. Pairs of sera were collected from 1687 individuals belonging to 772 households, during and after passage of the pandemic wave. Antibodies to pH1N1/2009v were titered using the hemagglutination inhibition assay (HIA) with titers ≥1/40 being considered positive. Seroprevalence during the first two weeks of detection of pH1N1/2009v in Reunion Island was 29.8% in people under 20 years of age, 35.6% in adults (20–59 years) and 73.3% in the elderly (≥60 years) (P<0.0001).", "Baseline corrected cumulative incidence rates, were 42.9%, 13.9% and 0% in these age groups respectively (P<0.0001). A significant decline in antibody titers occurred soon after the passage of the epidemic wave. Seroconversion rates to pH1N1/2009 correlated negatively with age: 63.2%, 39.4% and 16.7%, in each age group respectively (P<0.0001). Seroconversion occurred in 65.2% of individuals who were seronegative at inclusion compared to 6.8% in those who were initially seropositive. CONCLUSIONS: Seroincidence of pH1N1/2009v infection was three times that estimated from clinical surveillance, indicating that almost two thirds of infections occurring at the community level have escaped medical detection. People under 20 years of age were the most affected group. Pre-epidemic titers ≥1/40 prevented seroconversion and are likely protective against infection. A concern was raised about the long term stability of the antibody responses.", "Text: In April 2009, the first cases of acute respiratory infections caused by a novel triple-reassortant influenza virus, pH1N1/ 2009v, occurred in Mexico and the United States [1] . The rapid spread of infection to other continents led the World Health Organization (WHO) to declare on 11 June 2009 that a pandemic of pH1N1/2009v influenza was under way, which raised major international concern about the risk of high morbidity and lethality and the potential for severe socio-economic impact. Actually, the potential impact of this first third-millenium influenza pandemic has been revisited downwards as morbidity and case-fatality rates were less severe than initially anticipated [2] . Illness surveillance data do not allow to an accurate estimate of the true influenza infection rate, as a substantial proportion of infections are asymptomatic or mild [3] . Serological surveys can overcome this limitation, but must take into account that a significant proportion of the population that", "exhibited crossprotective antibody titers before circulation of the pH1N1/2009v [4] . This so-called ''baseline immunity'' has to be subtracted from the seroprevalence observed after the pandemic wave, to determine seroincidence in serosurveys [5] [6] [7] [8] . However, except for few studies [9] [10] [11] , most of these serosurveys did not use serial measurements in the same person, which allows for a better understanding of antibody kinetics and the dynamics of infection within individuals and communities.", "Reunion Island (805,500 inhabitants) is a French overseas department located in the southwestern Indian Ocean, 700 km east of Madagascar and 200 km southwest of Mauritius. The first imported case of pH1N1/2009v was identified on 5 th July 2009 (week 29) in a traveller returning from Australia. The first case indicating community transmission was detected on 21 st July (week 30). pH1N1/2009v became the predominant circulating influenza virus within four weeks of its first detection, its activity peaked during week 35 (24) (25) (26) (27) (28) (29) (30) and ended at week 38 [12] . Contrary to initial fears, the health care system was not overwhelmed, as morbidity and mortality rates were lower than predicted [12] [13] [14] .", "In order to assess at the community level, the actual magnitude of the pH1N1/2009v pandemic and the extent of the herd immunity acquired after passage of the epidemic wave, a prospective population serosurvey was conducted in Reunion Island during the passage of the epidemic wave in the 2009 austral winter season (July-December 2009): prevalence of infection was assessed on a weekly basis and seroconversion rates were measured using paired sera.", "The CoPanFLu-RUN was part of the CoPanFLu international project, a consortium between the French National Institute of Health and Medical Research (INSERM), the Institute of Research for Development (IRD) and the Mérieux Fondation under the promotion of the School of Advanced Studies in Public Health (EHESP). To enable the rapid implementation of the study in anticipation of the imminent spread of the pandemic wave, we used a pre-existing sample of 2442 households established in October 2006 for the investigation of the Chikungunya outbreak (SEROCHIK) and updated in May 2008 throughout a follow-up telephone survey (TELECHIK) on a basis of 1148 households [15, 16] . We took special attention to select households representing a wide range of geographic locations in order to minimize the repartition bias.", "The inclusion phase started on July 21 st (week 30) and was continued up to week 44, throughout the epidemic wave and beyond. A first serum sample (sample 1) was obtained from each household member. An active telephonic inquiry was then conducted twice a week to record symptoms compatible with influenza-like illness (ILI) occurring in households. Report of ILI (fever $37.8uC associated with any respiratory or systemic symptom) led to three consecutive visits of a nurse to the incident case-dwelling (on day 0, +3 and +8 post-report) to record symptoms and collect nasal swabs from all family members (for qRT-PCR detection of pH1N1/2009v. At week 45, the active inquiry was discontinued and a second (post-epidemic) serum sample (sample 2) was obtained (weeks 45-52) to determine seroconversion rates. Sera were aliquoted and stored at 280uC.", "The protocol was conducted in accordance with the Declaration of Helsinki and French law for biomedical research (Nu ID RCB AFSSAPS: 2009-A00689-48) and was approved by the local Ethics Committee (Comité de Protection des Personnes of Bordeaux 2 University). Every eligible person for participation was asked for giving their written informed consent.\n\nViral genome detection by RT-PCR. Viral RNA was extracted from 140 mL of nasal swab eluate using the QIAamp Viral RNA kit (Qiagen) and processed for detection by TaqMan qRT-PCR targeting the heamagglutinin HA gene (SuperScript III Platinum one-step qRT-PCR system, Invitrogen) according to the recommendations of the Pasteur Institute (Van der Werf S. & Enouf V., SOP/FluA/130509). Confirmed pH1N1/2009v infection was defined as a positive qRT-PCR detection of the HA gene in at least one nasal swab.", "Hemagglutination inhibition assay (HIA). A standard hemagglutination inhibition technique was adapted to detect and quantify pH1N1/2009v antibodies [17] . The antigen was prepared by diluting a non-inactivated cell culture supernatant producing a pdm H1N1v strain (strain OPYFLU-1 isolated from a young patient returning from Mexico in early May 2009) [18] . Briefly, the virus was propagated onto MDCK cells under standard conditions. The last passage (used for antigen preparation) was performed in the absence of trypsin and ht-FBS. The supernatant was collected at day seven p.i. clarified by centrifugation at 8006 g for 10 min at room temperature, aliquoted and conserved at 280uC. The hemagglutinating titer of the non inactivated viral antigen was immediately determined under the HIA format described below. The dilution providing 5.33 hemagglutinating units in a volume of 25 mL was used for subsequent HIA. Sera were heat-inactivated at 56uC for 30 min prior to use. Sequential twofold", "dilutions in PBS (1/10 to 1/1280) in volumes of 25 mL were performed and distributed in V-bottom 96 well microplates. Human red blood cells (RBC) were used for hemagglutination experiments. Detection and quantification of antibody to pH1N1/2009v was performed as follows: 25 mL of virus suspension was added to the serum dilution (25 mL) and incubated for 1 hour at room temperature. Each well was then filled with 25 mL of a 1% RBC suspension in PBS (v/v: 0.33%), followed by another 30 min incubation at room temperature. The HIA titer was determined as the last dilution providing clear inhibition of hemagglutination. All experiments were performed in the presence of the same negative and positive controls, the latter including sera with 1/40, 1/80, 1/160 and 1/320 antibody titers.", "The results reported in this study were based only on serological analysis of paired sera. For the sake of analysis, four successive phases were identified throughout the pandemic wave: phase A (weeks 30-31) corresponded to early epidemic time, phase B (W32-39) to the epidemic unfolding, phase C (W40-44) to the immediate post-epidemic stage and phase D (W45-52) to the late post-epidemic stage. Seropositivity was defined as a HIA titer of 1/ 40 or more. The baseline-proxy seroprevalence rate was estimated on serum samples collected in phase A. The cumulative incidence rate of infection measured the raise between the raw seroprevalence rate at any given time during the epidemic phases (S2pi) and the age-specific baseline-proxy seroprevalence rate (S1pA) (s2 pi -s1 pA ). Seroconversion was defined as a shift from seronegative at inclusion (sample 1: HIA ,1/40) to seropositive on follow-up (sample 2: HIA $1/40), or for sera tested seropositive on inclusion as a four-fold increase of HIA", "titers between sample 1 and sample 2 paired sera. We also calculated the proportion of sera that tested seropositive in sample 1 for which the HIA titer decreased fourfold and passed under the cut-off value of 1/40 in sample 2. We considered this proportion as a ''seronegation'' rate.", "The sample size was calculated for identifying risk factors in the prospective cohort study. Considering on average three individuals per household, an intra-household correlation of 0.3, a power greater than 80% could be obtained with a sample size of 840 comprising 2500 individuals, assuming exposure levels ranging from 10% to 90% and a relative risk greater than 1.3. With 2,500 subjects, the study allowed 1-2% absolute precision around the estimated values for seroconversion rates. Data entry used EpiData version 3.1 (The Epidata Association, Odense, Denmark). SAS version 9.1 (SAS Inc., Cary, NC, USA) was used for statistical analysis. The characteristics of the study cohort were compared to those of the population of Reunion Island and a Chi2 test (or Fisher's exact test when non applicable) was used to analyse differences in age, sex and geographic location. Cumulative incidence rates of infection (i.e. seroincidence) and seroconversion rates were standardized according to the", "age structure of the community (French National Institute for Statistics and Economical Studies (INSEE) source).", "Baseline-proxy seroprevalence, cumulative incidence rates of infection, as well as seroconversion and seronegation rates, were expressed as percentages. Cumulative reverse distribution curves were used to show the distribution of antibody titers. In all tests, a P value,0.05 was considered significant.\n\nWe estimated 95% confidence intervals (CIs) of proportions by using a cluster bootstrap technique with 1000 re-samples [19] . After bootstraping, we used an ANOVA model to compare mean cumulative incidence proportions between pandemic phases, within each age group. We used an alternating logistic regression model (ALR) with an exchangeable log Odds Ratio (OR) to test the intra-household correlation-adjusted association between factors and the seroconversion outcome.", "Data were analysed with respect to subject age. Initially, four age groups were considered: the children and adolescents (,20 yrs), young adults (20-39 yrs), middle-age adults (40-59 yrs), and elderly adults ($60 yrs). As the cumulative incidence of infection of the second and third groups were very close, both groups were merged into one adults group (20-59 yrs). Therefore we refer further in our study to three age groups: children and adolescents (,20 yrs), adults (20-59 yrs), elderly ($60 yrs).", "A total of 2,164 individuals from 772 households were enrolled between weeks 30 and 44 in the CoPanFlu-RUN cohort, allowing the collection of 1,932 sera at inclusion (sample 1). During this period, 136 households (17.7% of households) containing 464 individuals (21.4% of individuals) reported at least one case of ILI. Sixty subjects among the 464 individuals (12.9%, belonging to 33 households [24.3%]) were qRT-PCR positive, which documented the pH1N1/2009v infection. No positive qRT-PCR could be detected after week 37 and no ILI was reported after week 40, the end of the epidemic wave. The second follow up serum sample (sample 2) was obtained for 1,759 subjects at least five weeks after the end of the epidemic wave (weeks 45-52) which allowed the constitution of a serobank of 1,687 paired-sera. The profile of the cohort and the major outcomes are displayed in Figure 1 . Details on inclusions and serum sample timing with respect to the circulation of pH1N1/2009v over the island are", "provided in figure 2 . The socio-demographic and space-time characteristics of the cohort are detailed in Table 1 . Compared to the community of Reunion Island, the sample of 1,687 individuals for whom pairedsera were available, was older (,20 yrs: 27% vs 35%, and $60 yrs: 17,9% vs 11,3%) and composed of a slight excess of females (54.1% vs 51.5%). The imbalance was due to a deficit in subjects aged under 40 years, reflecting men at work and the fact that parents declined the second serum for children younger than five.", "Baseline-proxy (,pre-epidemic) HIA titers to the pH1N1/ 2009v were measured on sample 1 ( Table 2) , obtained from 249 subjects (103 households) recruited at the very beginning of the investigation during weeks 30 and 31 (phase A, Figure 2 ), when the epidemic activity in the cohort was still very low. Age distribution in this group was similar to that of the whole cohort (data not shown). The overall, the baseline-proxy seroprevalence rate (HIA $1/40), over all ages, was 43.4% (95%CI: 37.4%-49.6%). However the majority of positive sera had low antibody titers, at the cut off value for confirmation (i.e. = 1/40). The proportions of sera with HIA titer .1/40 were 0%, 3.0% and 24.6% in the young, middle-aged and older age groups respectively. These results indicate that pre-epidemic baseline antibody cross reactivity was stronger in the elderly ($60 yrs) and weaker in children and adolescents (,20 yrs) and adults (20-59 yrs), with highly significant differences between age groups", "(P,0.0001).", "The reverse cumulative distribution curves of HIA titers are displayed for each age group and for the whole cohort on Figure 3 . The proportion of seropositive sera (HI $1/40) steadily increased during the epidemic unfolding (phase B, W32-39) and in immediate post epidemic period (phase C, W40-44) when it reached its maximum level, then declined in the late post epidemic period (phase D, W45-52). This decline was significant enough to return the reverse cumulative distribution curve to baseline levels in the elderly. The cumulative incidence rates, obtained after subtraction of the age-specific baseline-proxy seroprevalence from the raw seroprevalence at each phase of the epidemic are shown in Table 2 (note that the cumulative incidence rates of infection represented for the group ''all ages'' were standardized according to age structure of the community). The cumulative incidence rates were much higher in children and adolescents (,20 yrs), indicating very active transmission of", "infection within this age group. As mentioned earlier, cumulative incidence rates peaked in phase C (W40-44), and then declined indicating some lability of the humoral immune response against the pH1N1/2009v. The age-related difference observed in the incidence rates was highly statistically significant (P,0.0001).", "To estimate more appropriately the decline of antibody titers occurring after the peak of the humoral response to the pH1N1/ 2009v, we considered paired-sera from the group of 264 subjects for whom the first serum sample (sample 1) was obtained just after the epidemic wave (phase C, W40-44), and the corresponding second sample was collected at the end of the survey (phase D, W45-52). Seronegation rates were 27.0% (61/226) for all age groups, 17.4% (12/69) in children and adolescents (,20 yrs), 32.3% (41/127) in adults (20-59 yrs) and 26.7% (8/30) in the elderly ($60 yrs). Differences between the seronegation rates according to age were statistically weakly significant (P = 0.0671).", "We then considered the 1687 individuals for whom paired sera were available and we measured the seroconversion rates according to age and to the time of first serum sample collection (phase A, B or C). Criteria of seroconversion were defined in the method section. As shown in table 3, there was a sharp decline in seroconversion rates across all the age groups, depending on whether participants were enrolled during phase A, phase B, or phase C (P,0.0001). To interpret these data, one should remember that antibodies at seroprotective levels (HIA $1/40), in serum samples 1 collected during the per epidemic phase B or early post epidemic phase C could represent either base line cross reactive antibodies or rising pH1N1/2009 specific antibodies due to a recent or ongoing infection. This ambiguity could lead to underestimation of the seroconversion rate for subjects enrolled in phases B and C. In order to solve this ambiguity, we specifically considered the group of 249 subjects in whom", "cross reactive antibodies were detected at the time of phase A (W30-31). The seroconversion rate of this group is the most indicative of the exposure of individuals to the whole epidemic wave. It was the highest (63,2%, P,0.0001) in children and adolescents (,20 yrs), and still significantly high in adults (39.4%, P,0.0001).", "We then tested in this particular group, the impact of (baseline) pre-epidemic cross reactive antibodies on the rate of seroconversion to pH1N1/2009 (Table 4) . No subject with HIA titer superior to 1/40 had evidence of seroconversion to pH1N1/2009. The seroconversion rate in individuals with a HIA titer equal to 1/40 was linked with age, being more important in children and adolescents (,20 yrs). The highest seroconversion rate (.56%) was registered in subjects with HIA titers inferior to 1/40, particularly for the under 20 years where it reached 85%. Hence, the risk of seroconversion decreased when pre-epidemic HIA titer was high after controlling for age (P,0.0001) (Figure 4) . The multivariate adjusted odds ratio for seroconversion were 0.15 (95%CI: 0.06-0.37, P,0.0001) per two-fold increase in baseline titer, 1.79 (95%CI: 1.23-2.59, P,0.003) per other household members who seroconverted, 5.33 (95%CI: 1.56-19.27, P,0.008) Figure 1 . The cohort profile and major outcomes. Figure 1", "details the three phases of the protocol: i) inclusion (weeks 30-44) and serum samples S1 collection; ii) follow up for detection of ILI in households, qRT-PCR on nasal swabs and estimation of cumulative seroincidence rates; iii) end of the study (weeks 45-52) and samples S2 collection. HIA on paired sera (S1+S2) allowed estimating seroconversion rates. doi:10.1371/journal.pone.0025738.g001 Bp (baseline-proxy) seroprevalence rates were estimated on weeks 30-31 in each age group. b Cumulative incidence rates measured the raise between raw seroprevalence rates and age-specific baseline-proxy seroprevalence rate. In the group ''All ages'', cumulative incidence rates were standardized according to age structure of the community. doi:10.1371/journal.pone.0025738.t002 Data are numbers, percentages (95% confidence intervals) and ALR parameter test P value for comparison of seroconversion proportions according to time of first sample (S1) collection at inclusion, in each age group, after", "controlling for household selection. In the group ''All ages'', rates of seroconversion were standardized according to age structure of the community. NA: not assessed. Seroconversion was defined as a shift from seronegative at inclusion (i.e. HIA titer ,1/40) to seropositive on follow-up sample, or as a 4-fold increase of reciprocal HIA titer between first and second paired samples for sera tested seropositive on inclusion (i.e. HIA titer $1/40). for age ,20 years (vs age $60 years) and 11.35 (95%CI: 0.41-4.47, P = 0.62) for age 20-60 years (vs age $60 years). The observed and predicted seroconversion rates according to age and baseline HIA titer are displayed Figure 4 .", "Finally, we considered the 46 subjects who had been infected by the pandemic virus over the course of the study, verified by a positive qRT-PCR nasal swab, and for whom paired sera were available. Initial HIA antibody titers in this group were ,1/40,", "The CoPanFlu-RUN cohort was set up to conduct a prospective population-based study investigating the herd immunity induced by the 2009 pandemic influenza virus and identifying risk factors for pH1N1/2009v infection from paired sera collected in an entire community. Most works published to date have used either extensive cross-sectional serosurveys on pre-and post-epidemic independent serum samples, the baseline immunity being assessed from stored frozen samples [5, 7, 8] , or non representative adult cohorts (military, health care workers, long-stay patients). Antibody titers were measured by HIA using a cut-off value set at 1/40 as classically recommended. This HIA titer at 1/40 is considered protective, i.e. conferring 50% protection against a viral challenge [20] . Our assay has introduced some changes in the experimental protocol compared to the classic one. The use of a non-inactivated viral antigen, i.e. a native virus, with nondenatured epitopes probably allows detection of", "antibodies to epitopes of the hemagglutinin not detected in the classic HIA test. This can induce slight differences in the sensitivity of detection of cross-reacting antibodies, but this does not modify the kinetics of Ab and the epidemiological evolution of seroprevalence and does not jeopardize the global comparability of serological results. This is confirmed by the fact that our HI assay detected seroprotective antibody titers in 93.5% and gave evidence seroconversion in 73.9% of qRT-PCR confirmed pH1N1/2009 influenza, all figures close to those reported in the literature [5, 21] .", "We considered that titers of .1/40, in sera collected from individuals enrolled during weeks 30 and 31 were cross reactive antibodies and not de novo antibodies triggered by the pandemic virus and hence used them as a proxy for baseline pre epidemic immunity. Several arguments support this assumption: i) the first case indicating autochthonous transmission in Reunion Island was reported by the epidemiological surveillance department of La Réunion on 21st July (week 30), i.e. the same day when inclusion started in our study cohort; ii) 7 to 15 days are required to develop an antibody response after viral infection; iii) On weeks 30 and 31, the epidemic activity due to the pandemic virus was very low in our study cohort and it became significant only after week 32. Hence, during weeks 30-31, 103 households were recruited and only 2 households reported ILI cases. Nasal swabs collected from these 2 individuals were tested qRT-PCR negative to the pandemic virus whereas one had evidence of", "coronavirus and rhinovirus using a multiplex RT-PCR to respiratory viruses (H. Pascalis, manuscript in preparation). In contrast, during weeks 32 to 39, 199 individuals belonging to 99 households reported ILI, among whom 60 individuals had documented infection by the pandemic virus.", "Our study shows that a substantial proportion of Reunion Island's population had pre-existing immunity to 2009 pandemic influenza virus with the highest baseline-proxy seroprevalence rate observed among adults aged of 60 years or more. Other studies from all continents had also reported high pre-epidemic seropositivity rates among the elderly [5, 6, 8, [22] [23] [24] [25] [26] , though large variations do exist between countries [10, 11, 23, 27, 28] . These cross reactive antibodies have been interpreted as being the residual signature of the remote exposure of these individuals to H1N1 viruses circulating before 1957 [24, 25, 29, 30] . Baseline seropositivity rates that we report in children and in younger adults (i.e. 30%-35%) were notably higher than those reported from other parts of the world [6, 8, 22, 23, [31] [32] [33] . However one should note that these baseline antibodies were of low titer, just at the level of the HIA threshold (i.e. 1/40). Several factors could have", "contributed to this comparatively high baseline rates found in our study: i) It may reflect the fact that the HI test used in our study was marginally more sensitive than the classic one [17] ; ii) Some individuals may have already been infected with pH1N1/ 2009 virus at weeks 30 and 31 and may have triggered an antibody response to the virus. This hypothesis seems unlikely in view of the arguments presented above and of a similar high proportion of sera titering HIA = 1/40 among 122 sera from adult patients sent for diagnostic purposes to the Regional Hospital microbiology laboratory, during the first half of 2009 (i.e. before the 2009 pandemic) (data not shown). However we cannot formally exclude this hypothesis in view of a recently reported study from Taiwan [11] that showed evidence of subclinical community transmission with proved seroconversion several weeks before report of the first documented case in the island. A similar conclusion was also drawn from Australia [34] ; iii)", "our serological test might detect cross-reactive antibodies triggered by recent vaccination with trivalent seasonal influenza vaccine as reported [4, [35] [36] [37] [38] [39] . However, seasonal influenza vaccines were of rather limited use in Reunion Island, especially in children and young adults; iv) Finally the high baseline titers may reflect the infectious history of the individuals to seasonal influenza viruses cross antigenic with pH1N1/2009 virus as recently suggested for seasonal 2007 H1N1 infection [40] . This serosurvey indicates that a large fraction of the Reunion Island population was infected with the pandemic virus. Younger people, have paid the main tribute to the epidemic as almost two thirds show evidence of seroconversion, confirming earlier clinical reports from the island [12] and accumulating reports from other countries [17, 32, 41, 42] and suggesting that school children have likely played the central role in the epidemic diffusion of the pandemic virus.", "Lower infection rates were found in adults and the lowest rates were recorded in the elderly.", "Based on clinical cases reported to the epidemiological surveillance services [12] , it was estimated that 66,915 persons in Reunion Island who consulted a physician were infected by the pH1N1/2009 virus during the 9 weeks of the epidemic, giving a cumulative attack rate of 8.26%. Taking into account those who did not consult a physician, the number of symptomatic infected persons was estimated to 104,067 (attack rate: 12.85%). In fact, the attack rate of pH1N1/2009 infection in our serosurvey was about 42%-44% at the peak of the antibody response (i.e., weeks 40-44), a figure which is at least 3 to 4 times higher than rates of infection based on clinical cases The wide gap between the two estimates indicates that a large fraction (almost two thirds) of those who got infected by pH1N1/2009 virus escaped medical detection, probably because they developed mild disease or asymptomatic infection, a further indication of the benign nature of the virus, at least at the community level. In", "England, Baguelin et al. [43] estimated that the cumulative incidence rates of infection by the pandemic virus in children were 20 to 40 times higher than that estimated from clinical surveillance.", "Our study, as others [6] , indicates that pre-existing cross reactive antibodies to pH1N1/2009 at titers $1/40 prevented from seroconversion in response to the pandemic virus. This level of pre-existing cross reactive immunity likely confers true protection against infection as about two thirds and one third of documented infection (qRT-PCR positive) in our series have occurred in individuals with baseline HIA titers ,1/40 and = 1/ 40 respectively and less than 5% of documented infections occurred in individuals with base line titers .1/40. The protection was effective not only in older adults but also in younger persons. This indicates that protection was conferred not only by baseline cross reactive antibodies triggered by close pH1N1/2009 viruses that circulated before 1957 (as in the elderly), but also by antibodies likely resulting from recent exposure to seasonal influenza epidemics (as shown in younger persons) [40] . The observed seroconversion rates depend on age, after", "adjusting for baseline pH1N1/2009 titers. The protective role of increasing age might be explained by a stronger cross-immunity in adults and elderly or by a higher exposure of young subjects to the virus during the 2009 epidemic (due to social contacts and mixing patterns). It may also indicate that immune mechanisms other than cross reactive antibodies detected by HIA (i.e. immunity to neuraminidase and conserved T cells epitopes [44] might develop throughout life, providing additional protection from infection or severe disease, especially in the elderly. Interestingly, evidence is seen for a decline in antibody titers, which occurred soon after the passage of the epidemic wave. In paired sera, this decline was significant enough to bring, within a few weeks, almost 27% of sera that tested positive (i.e. HI titers $1/40) in the immediate post epidemic phase to levels under the cut-off value in the second serum sample. This decay accounts for the observation that older adults ($60", "yrs) in the study cohort were apparently almost completely spared by the epidemic if one only considers cumulative incidence rates derived from IHA titration on samples 2 (weeks 45-52). In fact, the cumulative incidence rate in older adults measured just after the epidemic peak (i.e. weeks 40-44) was 20.4%. Similar results of early antibody decay were recently reported [10, 45] . More generally, these data show that serosurveys conducted months after passage of the epidemic, likely underestimate the real extent of pH1N1/2009 infection, compared to antibody titration performed earlier, when humoral responses are at their highest level. Whether the decline in antibody titers has functional immunologic consequence to individuals or within the communities warrants further investigation. However, one should note that there was no second epidemic wave in Reunion Island during the subsequent austral winter seasons in 2010 and 2011.", "Influenza during the 2010 winter was at a level not higher than the usual passages of seasonal flu, though almost two thirds of documented cases in 2010 were also due to pH1N1/2009v [46] . In addition many fewer pandemic virus isolates were noted during the ongoing 2011 austral winter, strongly suggesting that the first epidemic wave had conferred a solid herd immunity, at the community level.", "Our study has some limitations. The fact that the epidemic progression coincided with the implementation of the prospective study, we were not able to collect, strictly speaking, pre-epidemic sera from the cohort members. Therefore we used as proxy base line seroprevalence data from individuals recruited at the very beginning of the investigation when the epidemic activity in the cohort was very low. This may overestimate the base line immunity if subclinical community transmission had occurred before the first cases of pH1N1/2009 influenza were reported. Antibodies to the pandemic virus were detected by HIA, a test that has a good specificity but a rather low sensitivity [46] . Hence, the threshold of 1/40 may underestimate the number of infected individuals. However, rates of seroconversion, the serologic gold standard test based on paired sera, likely gave the most accurate picture of the pandemic in at the community level in Reunion Island." ]

[ 35 ]

[ "the remote exposure of these individuals to H1N1 viruses circulating before 1957" ]

[ "Pandemic Influenza Due to pH1N1/2009 Virus: Estimation of Infection Burden in Reunion Island through a Prospective Serosurvey, Austral Winter 2009\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3183080/\n\nSHA: ee6d70a53e3262cea6f85bd8b226f6b4c8b5f64b\n\nAuthors: Dellagi, Koussay; Rollot, Olivier; Temmam, Sarah; Salez, Nicolas; Guernier, Vanina; Pascalis, Hervé; Gérardin, Patrick; Fianu, Adrian; Lapidus, Nathanael; Naty, Nadège; Tortosa, Pablo; Boussaïd, Karim; Jaffar-Banjee, Marie-Christine; Filleul, Laurent; Flahault, Antoine; Carrat, Fabrice; Favier, Francois; de Lamballerie, Xavier\nDate: 2011-09-29\nDOI: 10.1371/journal.pone.0025738\nLicense: cc-by", "Abstract: BACKGROUND: To date, there is little information that reflects the true extent of spread of the pH1N1/2009v influenza pandemic at the community level as infection often results in mild or no clinical symptoms. This study aimed at assessing through a prospective study, the attack rate of pH1N1/2009 virus in Reunion Island and risk factors of infection, during the 2009 season. METHODOLOGY/PRINCIPAL FINDINGS: A serosurvey was conducted during the 2009 austral winter, in the frame of a prospective population study. Pairs of sera were collected from 1687 individuals belonging to 772 households, during and after passage of the pandemic wave. Antibodies to pH1N1/2009v were titered using the hemagglutination inhibition assay (HIA) with titers ≥1/40 being considered positive. Seroprevalence during the first two weeks of detection of pH1N1/2009v in Reunion Island was 29.8% in people under 20 years of age, 35.6% in adults (20–59 years) and 73.3% in the elderly (≥60 years) (P<0.0001).", "Baseline corrected cumulative incidence rates, were 42.9%, 13.9% and 0% in these age groups respectively (P<0.0001). A significant decline in antibody titers occurred soon after the passage of the epidemic wave. Seroconversion rates to pH1N1/2009 correlated negatively with age: 63.2%, 39.4% and 16.7%, in each age group respectively (P<0.0001). Seroconversion occurred in 65.2% of individuals who were seronegative at inclusion compared to 6.8% in those who were initially seropositive. CONCLUSIONS: Seroincidence of pH1N1/2009v infection was three times that estimated from clinical surveillance, indicating that almost two thirds of infections occurring at the community level have escaped medical detection. People under 20 years of age were the most affected group. Pre-epidemic titers ≥1/40 prevented seroconversion and are likely protective against infection. A concern was raised about the long term stability of the antibody responses.", "Text: In April 2009, the first cases of acute respiratory infections caused by a novel triple-reassortant influenza virus, pH1N1/ 2009v, occurred in Mexico and the United States [1] . The rapid spread of infection to other continents led the World Health Organization (WHO) to declare on 11 June 2009 that a pandemic of pH1N1/2009v influenza was under way, which raised major international concern about the risk of high morbidity and lethality and the potential for severe socio-economic impact. Actually, the potential impact of this first third-millenium influenza pandemic has been revisited downwards as morbidity and case-fatality rates were less severe than initially anticipated [2] . Illness surveillance data do not allow to an accurate estimate of the true influenza infection rate, as a substantial proportion of infections are asymptomatic or mild [3] . Serological surveys can overcome this limitation, but must take into account that a significant proportion of the population that", "exhibited crossprotective antibody titers before circulation of the pH1N1/2009v [4] . This so-called ''baseline immunity'' has to be subtracted from the seroprevalence observed after the pandemic wave, to determine seroincidence in serosurveys [5] [6] [7] [8] . However, except for few studies [9] [10] [11] , most of these serosurveys did not use serial measurements in the same person, which allows for a better understanding of antibody kinetics and the dynamics of infection within individuals and communities.", "Reunion Island (805,500 inhabitants) is a French overseas department located in the southwestern Indian Ocean, 700 km east of Madagascar and 200 km southwest of Mauritius. The first imported case of pH1N1/2009v was identified on 5 th July 2009 (week 29) in a traveller returning from Australia. The first case indicating community transmission was detected on 21 st July (week 30). pH1N1/2009v became the predominant circulating influenza virus within four weeks of its first detection, its activity peaked during week 35 (24) (25) (26) (27) (28) (29) (30) and ended at week 38 [12] . Contrary to initial fears, the health care system was not overwhelmed, as morbidity and mortality rates were lower than predicted [12] [13] [14] .", "In order to assess at the community level, the actual magnitude of the pH1N1/2009v pandemic and the extent of the herd immunity acquired after passage of the epidemic wave, a prospective population serosurvey was conducted in Reunion Island during the passage of the epidemic wave in the 2009 austral winter season (July-December 2009): prevalence of infection was assessed on a weekly basis and seroconversion rates were measured using paired sera.", "The CoPanFLu-RUN was part of the CoPanFLu international project, a consortium between the French National Institute of Health and Medical Research (INSERM), the Institute of Research for Development (IRD) and the Mérieux Fondation under the promotion of the School of Advanced Studies in Public Health (EHESP). To enable the rapid implementation of the study in anticipation of the imminent spread of the pandemic wave, we used a pre-existing sample of 2442 households established in October 2006 for the investigation of the Chikungunya outbreak (SEROCHIK) and updated in May 2008 throughout a follow-up telephone survey (TELECHIK) on a basis of 1148 households [15, 16] . We took special attention to select households representing a wide range of geographic locations in order to minimize the repartition bias.", "The inclusion phase started on July 21 st (week 30) and was continued up to week 44, throughout the epidemic wave and beyond. A first serum sample (sample 1) was obtained from each household member. An active telephonic inquiry was then conducted twice a week to record symptoms compatible with influenza-like illness (ILI) occurring in households. Report of ILI (fever $37.8uC associated with any respiratory or systemic symptom) led to three consecutive visits of a nurse to the incident case-dwelling (on day 0, +3 and +8 post-report) to record symptoms and collect nasal swabs from all family members (for qRT-PCR detection of pH1N1/2009v. At week 45, the active inquiry was discontinued and a second (post-epidemic) serum sample (sample 2) was obtained (weeks 45-52) to determine seroconversion rates. Sera were aliquoted and stored at 280uC.", "The protocol was conducted in accordance with the Declaration of Helsinki and French law for biomedical research (Nu ID RCB AFSSAPS: 2009-A00689-48) and was approved by the local Ethics Committee (Comité de Protection des Personnes of Bordeaux 2 University). Every eligible person for participation was asked for giving their written informed consent.\n\nViral genome detection by RT-PCR. Viral RNA was extracted from 140 mL of nasal swab eluate using the QIAamp Viral RNA kit (Qiagen) and processed for detection by TaqMan qRT-PCR targeting the heamagglutinin HA gene (SuperScript III Platinum one-step qRT-PCR system, Invitrogen) according to the recommendations of the Pasteur Institute (Van der Werf S. & Enouf V., SOP/FluA/130509). Confirmed pH1N1/2009v infection was defined as a positive qRT-PCR detection of the HA gene in at least one nasal swab.", "Hemagglutination inhibition assay (HIA). A standard hemagglutination inhibition technique was adapted to detect and quantify pH1N1/2009v antibodies [17] . The antigen was prepared by diluting a non-inactivated cell culture supernatant producing a pdm H1N1v strain (strain OPYFLU-1 isolated from a young patient returning from Mexico in early May 2009) [18] . Briefly, the virus was propagated onto MDCK cells under standard conditions. The last passage (used for antigen preparation) was performed in the absence of trypsin and ht-FBS. The supernatant was collected at day seven p.i. clarified by centrifugation at 8006 g for 10 min at room temperature, aliquoted and conserved at 280uC. The hemagglutinating titer of the non inactivated viral antigen was immediately determined under the HIA format described below. The dilution providing 5.33 hemagglutinating units in a volume of 25 mL was used for subsequent HIA. Sera were heat-inactivated at 56uC for 30 min prior to use. Sequential twofold", "dilutions in PBS (1/10 to 1/1280) in volumes of 25 mL were performed and distributed in V-bottom 96 well microplates. Human red blood cells (RBC) were used for hemagglutination experiments. Detection and quantification of antibody to pH1N1/2009v was performed as follows: 25 mL of virus suspension was added to the serum dilution (25 mL) and incubated for 1 hour at room temperature. Each well was then filled with 25 mL of a 1% RBC suspension in PBS (v/v: 0.33%), followed by another 30 min incubation at room temperature. The HIA titer was determined as the last dilution providing clear inhibition of hemagglutination. All experiments were performed in the presence of the same negative and positive controls, the latter including sera with 1/40, 1/80, 1/160 and 1/320 antibody titers.", "The results reported in this study were based only on serological analysis of paired sera. For the sake of analysis, four successive phases were identified throughout the pandemic wave: phase A (weeks 30-31) corresponded to early epidemic time, phase B (W32-39) to the epidemic unfolding, phase C (W40-44) to the immediate post-epidemic stage and phase D (W45-52) to the late post-epidemic stage. Seropositivity was defined as a HIA titer of 1/ 40 or more. The baseline-proxy seroprevalence rate was estimated on serum samples collected in phase A. The cumulative incidence rate of infection measured the raise between the raw seroprevalence rate at any given time during the epidemic phases (S2pi) and the age-specific baseline-proxy seroprevalence rate (S1pA) (s2 pi -s1 pA ). Seroconversion was defined as a shift from seronegative at inclusion (sample 1: HIA ,1/40) to seropositive on follow-up (sample 2: HIA $1/40), or for sera tested seropositive on inclusion as a four-fold increase of HIA", "titers between sample 1 and sample 2 paired sera. We also calculated the proportion of sera that tested seropositive in sample 1 for which the HIA titer decreased fourfold and passed under the cut-off value of 1/40 in sample 2. We considered this proportion as a ''seronegation'' rate.", "The sample size was calculated for identifying risk factors in the prospective cohort study. Considering on average three individuals per household, an intra-household correlation of 0.3, a power greater than 80% could be obtained with a sample size of 840 comprising 2500 individuals, assuming exposure levels ranging from 10% to 90% and a relative risk greater than 1.3. With 2,500 subjects, the study allowed 1-2% absolute precision around the estimated values for seroconversion rates. Data entry used EpiData version 3.1 (The Epidata Association, Odense, Denmark). SAS version 9.1 (SAS Inc., Cary, NC, USA) was used for statistical analysis. The characteristics of the study cohort were compared to those of the population of Reunion Island and a Chi2 test (or Fisher's exact test when non applicable) was used to analyse differences in age, sex and geographic location. Cumulative incidence rates of infection (i.e. seroincidence) and seroconversion rates were standardized according to the", "age structure of the community (French National Institute for Statistics and Economical Studies (INSEE) source).", "Baseline-proxy seroprevalence, cumulative incidence rates of infection, as well as seroconversion and seronegation rates, were expressed as percentages. Cumulative reverse distribution curves were used to show the distribution of antibody titers. In all tests, a P value,0.05 was considered significant.\n\nWe estimated 95% confidence intervals (CIs) of proportions by using a cluster bootstrap technique with 1000 re-samples [19] . After bootstraping, we used an ANOVA model to compare mean cumulative incidence proportions between pandemic phases, within each age group. We used an alternating logistic regression model (ALR) with an exchangeable log Odds Ratio (OR) to test the intra-household correlation-adjusted association between factors and the seroconversion outcome.", "Data were analysed with respect to subject age. Initially, four age groups were considered: the children and adolescents (,20 yrs), young adults (20-39 yrs), middle-age adults (40-59 yrs), and elderly adults ($60 yrs). As the cumulative incidence of infection of the second and third groups were very close, both groups were merged into one adults group (20-59 yrs). Therefore we refer further in our study to three age groups: children and adolescents (,20 yrs), adults (20-59 yrs), elderly ($60 yrs).", "A total of 2,164 individuals from 772 households were enrolled between weeks 30 and 44 in the CoPanFlu-RUN cohort, allowing the collection of 1,932 sera at inclusion (sample 1). During this period, 136 households (17.7% of households) containing 464 individuals (21.4% of individuals) reported at least one case of ILI. Sixty subjects among the 464 individuals (12.9%, belonging to 33 households [24.3%]) were qRT-PCR positive, which documented the pH1N1/2009v infection. No positive qRT-PCR could be detected after week 37 and no ILI was reported after week 40, the end of the epidemic wave. The second follow up serum sample (sample 2) was obtained for 1,759 subjects at least five weeks after the end of the epidemic wave (weeks 45-52) which allowed the constitution of a serobank of 1,687 paired-sera. The profile of the cohort and the major outcomes are displayed in Figure 1 . Details on inclusions and serum sample timing with respect to the circulation of pH1N1/2009v over the island are", "provided in figure 2 . The socio-demographic and space-time characteristics of the cohort are detailed in Table 1 . Compared to the community of Reunion Island, the sample of 1,687 individuals for whom pairedsera were available, was older (,20 yrs: 27% vs 35%, and $60 yrs: 17,9% vs 11,3%) and composed of a slight excess of females (54.1% vs 51.5%). The imbalance was due to a deficit in subjects aged under 40 years, reflecting men at work and the fact that parents declined the second serum for children younger than five.", "Baseline-proxy (,pre-epidemic) HIA titers to the pH1N1/ 2009v were measured on sample 1 ( Table 2) , obtained from 249 subjects (103 households) recruited at the very beginning of the investigation during weeks 30 and 31 (phase A, Figure 2 ), when the epidemic activity in the cohort was still very low. Age distribution in this group was similar to that of the whole cohort (data not shown). The overall, the baseline-proxy seroprevalence rate (HIA $1/40), over all ages, was 43.4% (95%CI: 37.4%-49.6%). However the majority of positive sera had low antibody titers, at the cut off value for confirmation (i.e. = 1/40). The proportions of sera with HIA titer .1/40 were 0%, 3.0% and 24.6% in the young, middle-aged and older age groups respectively. These results indicate that pre-epidemic baseline antibody cross reactivity was stronger in the elderly ($60 yrs) and weaker in children and adolescents (,20 yrs) and adults (20-59 yrs), with highly significant differences between age groups", "(P,0.0001).", "The reverse cumulative distribution curves of HIA titers are displayed for each age group and for the whole cohort on Figure 3 . The proportion of seropositive sera (HI $1/40) steadily increased during the epidemic unfolding (phase B, W32-39) and in immediate post epidemic period (phase C, W40-44) when it reached its maximum level, then declined in the late post epidemic period (phase D, W45-52). This decline was significant enough to return the reverse cumulative distribution curve to baseline levels in the elderly. The cumulative incidence rates, obtained after subtraction of the age-specific baseline-proxy seroprevalence from the raw seroprevalence at each phase of the epidemic are shown in Table 2 (note that the cumulative incidence rates of infection represented for the group ''all ages'' were standardized according to age structure of the community). The cumulative incidence rates were much higher in children and adolescents (,20 yrs), indicating very active transmission of", "infection within this age group. As mentioned earlier, cumulative incidence rates peaked in phase C (W40-44), and then declined indicating some lability of the humoral immune response against the pH1N1/2009v. The age-related difference observed in the incidence rates was highly statistically significant (P,0.0001).", "To estimate more appropriately the decline of antibody titers occurring after the peak of the humoral response to the pH1N1/ 2009v, we considered paired-sera from the group of 264 subjects for whom the first serum sample (sample 1) was obtained just after the epidemic wave (phase C, W40-44), and the corresponding second sample was collected at the end of the survey (phase D, W45-52). Seronegation rates were 27.0% (61/226) for all age groups, 17.4% (12/69) in children and adolescents (,20 yrs), 32.3% (41/127) in adults (20-59 yrs) and 26.7% (8/30) in the elderly ($60 yrs). Differences between the seronegation rates according to age were statistically weakly significant (P = 0.0671).", "We then considered the 1687 individuals for whom paired sera were available and we measured the seroconversion rates according to age and to the time of first serum sample collection (phase A, B or C). Criteria of seroconversion were defined in the method section. As shown in table 3, there was a sharp decline in seroconversion rates across all the age groups, depending on whether participants were enrolled during phase A, phase B, or phase C (P,0.0001). To interpret these data, one should remember that antibodies at seroprotective levels (HIA $1/40), in serum samples 1 collected during the per epidemic phase B or early post epidemic phase C could represent either base line cross reactive antibodies or rising pH1N1/2009 specific antibodies due to a recent or ongoing infection. This ambiguity could lead to underestimation of the seroconversion rate for subjects enrolled in phases B and C. In order to solve this ambiguity, we specifically considered the group of 249 subjects in whom", "cross reactive antibodies were detected at the time of phase A (W30-31). The seroconversion rate of this group is the most indicative of the exposure of individuals to the whole epidemic wave. It was the highest (63,2%, P,0.0001) in children and adolescents (,20 yrs), and still significantly high in adults (39.4%, P,0.0001).", "We then tested in this particular group, the impact of (baseline) pre-epidemic cross reactive antibodies on the rate of seroconversion to pH1N1/2009 (Table 4) . No subject with HIA titer superior to 1/40 had evidence of seroconversion to pH1N1/2009. The seroconversion rate in individuals with a HIA titer equal to 1/40 was linked with age, being more important in children and adolescents (,20 yrs). The highest seroconversion rate (.56%) was registered in subjects with HIA titers inferior to 1/40, particularly for the under 20 years where it reached 85%. Hence, the risk of seroconversion decreased when pre-epidemic HIA titer was high after controlling for age (P,0.0001) (Figure 4) . The multivariate adjusted odds ratio for seroconversion were 0.15 (95%CI: 0.06-0.37, P,0.0001) per two-fold increase in baseline titer, 1.79 (95%CI: 1.23-2.59, P,0.003) per other household members who seroconverted, 5.33 (95%CI: 1.56-19.27, P,0.008) Figure 1 . The cohort profile and major outcomes. Figure 1", "details the three phases of the protocol: i) inclusion (weeks 30-44) and serum samples S1 collection; ii) follow up for detection of ILI in households, qRT-PCR on nasal swabs and estimation of cumulative seroincidence rates; iii) end of the study (weeks 45-52) and samples S2 collection. HIA on paired sera (S1+S2) allowed estimating seroconversion rates. doi:10.1371/journal.pone.0025738.g001 Bp (baseline-proxy) seroprevalence rates were estimated on weeks 30-31 in each age group. b Cumulative incidence rates measured the raise between raw seroprevalence rates and age-specific baseline-proxy seroprevalence rate. In the group ''All ages'', cumulative incidence rates were standardized according to age structure of the community. doi:10.1371/journal.pone.0025738.t002 Data are numbers, percentages (95% confidence intervals) and ALR parameter test P value for comparison of seroconversion proportions according to time of first sample (S1) collection at inclusion, in each age group, after", "controlling for household selection. In the group ''All ages'', rates of seroconversion were standardized according to age structure of the community. NA: not assessed. Seroconversion was defined as a shift from seronegative at inclusion (i.e. HIA titer ,1/40) to seropositive on follow-up sample, or as a 4-fold increase of reciprocal HIA titer between first and second paired samples for sera tested seropositive on inclusion (i.e. HIA titer $1/40). for age ,20 years (vs age $60 years) and 11.35 (95%CI: 0.41-4.47, P = 0.62) for age 20-60 years (vs age $60 years). The observed and predicted seroconversion rates according to age and baseline HIA titer are displayed Figure 4 .", "Finally, we considered the 46 subjects who had been infected by the pandemic virus over the course of the study, verified by a positive qRT-PCR nasal swab, and for whom paired sera were available. Initial HIA antibody titers in this group were ,1/40,", "The CoPanFlu-RUN cohort was set up to conduct a prospective population-based study investigating the herd immunity induced by the 2009 pandemic influenza virus and identifying risk factors for pH1N1/2009v infection from paired sera collected in an entire community. Most works published to date have used either extensive cross-sectional serosurveys on pre-and post-epidemic independent serum samples, the baseline immunity being assessed from stored frozen samples [5, 7, 8] , or non representative adult cohorts (military, health care workers, long-stay patients). Antibody titers were measured by HIA using a cut-off value set at 1/40 as classically recommended. This HIA titer at 1/40 is considered protective, i.e. conferring 50% protection against a viral challenge [20] . Our assay has introduced some changes in the experimental protocol compared to the classic one. The use of a non-inactivated viral antigen, i.e. a native virus, with nondenatured epitopes probably allows detection of", "antibodies to epitopes of the hemagglutinin not detected in the classic HIA test. This can induce slight differences in the sensitivity of detection of cross-reacting antibodies, but this does not modify the kinetics of Ab and the epidemiological evolution of seroprevalence and does not jeopardize the global comparability of serological results. This is confirmed by the fact that our HI assay detected seroprotective antibody titers in 93.5% and gave evidence seroconversion in 73.9% of qRT-PCR confirmed pH1N1/2009 influenza, all figures close to those reported in the literature [5, 21] .", "We considered that titers of .1/40, in sera collected from individuals enrolled during weeks 30 and 31 were cross reactive antibodies and not de novo antibodies triggered by the pandemic virus and hence used them as a proxy for baseline pre epidemic immunity. Several arguments support this assumption: i) the first case indicating autochthonous transmission in Reunion Island was reported by the epidemiological surveillance department of La Réunion on 21st July (week 30), i.e. the same day when inclusion started in our study cohort; ii) 7 to 15 days are required to develop an antibody response after viral infection; iii) On weeks 30 and 31, the epidemic activity due to the pandemic virus was very low in our study cohort and it became significant only after week 32. Hence, during weeks 30-31, 103 households were recruited and only 2 households reported ILI cases. Nasal swabs collected from these 2 individuals were tested qRT-PCR negative to the pandemic virus whereas one had evidence of", "coronavirus and rhinovirus using a multiplex RT-PCR to respiratory viruses (H. Pascalis, manuscript in preparation). In contrast, during weeks 32 to 39, 199 individuals belonging to 99 households reported ILI, among whom 60 individuals had documented infection by the pandemic virus.", "Our study shows that a substantial proportion of Reunion Island's population had pre-existing immunity to 2009 pandemic influenza virus with the highest baseline-proxy seroprevalence rate observed among adults aged of 60 years or more. Other studies from all continents had also reported high pre-epidemic seropositivity rates among the elderly [5, 6, 8, [22] [23] [24] [25] [26] , though large variations do exist between countries [10, 11, 23, 27, 28] . These cross reactive antibodies have been interpreted as being the residual signature of the remote exposure of these individuals to H1N1 viruses circulating before 1957 [24, 25, 29, 30] . Baseline seropositivity rates that we report in children and in younger adults (i.e. 30%-35%) were notably higher than those reported from other parts of the world [6, 8, 22, 23, [31] [32] [33] . However one should note that these baseline antibodies were of low titer, just at the level of the HIA threshold (i.e. 1/40). Several factors could have", "contributed to this comparatively high baseline rates found in our study: i) It may reflect the fact that the HI test used in our study was marginally more sensitive than the classic one [17] ; ii) Some individuals may have already been infected with pH1N1/ 2009 virus at weeks 30 and 31 and may have triggered an antibody response to the virus. This hypothesis seems unlikely in view of the arguments presented above and of a similar high proportion of sera titering HIA = 1/40 among 122 sera from adult patients sent for diagnostic purposes to the Regional Hospital microbiology laboratory, during the first half of 2009 (i.e. before the 2009 pandemic) (data not shown). However we cannot formally exclude this hypothesis in view of a recently reported study from Taiwan [11] that showed evidence of subclinical community transmission with proved seroconversion several weeks before report of the first documented case in the island. A similar conclusion was also drawn from Australia [34] ; iii)", "our serological test might detect cross-reactive antibodies triggered by recent vaccination with trivalent seasonal influenza vaccine as reported [4, [35] [36] [37] [38] [39] . However, seasonal influenza vaccines were of rather limited use in Reunion Island, especially in children and young adults; iv) Finally the high baseline titers may reflect the infectious history of the individuals to seasonal influenza viruses cross antigenic with pH1N1/2009 virus as recently suggested for seasonal 2007 H1N1 infection [40] . This serosurvey indicates that a large fraction of the Reunion Island population was infected with the pandemic virus. Younger people, have paid the main tribute to the epidemic as almost two thirds show evidence of seroconversion, confirming earlier clinical reports from the island [12] and accumulating reports from other countries [17, 32, 41, 42] and suggesting that school children have likely played the central role in the epidemic diffusion of the pandemic virus.", "Lower infection rates were found in adults and the lowest rates were recorded in the elderly.", "Based on clinical cases reported to the epidemiological surveillance services [12] , it was estimated that 66,915 persons in Reunion Island who consulted a physician were infected by the pH1N1/2009 virus during the 9 weeks of the epidemic, giving a cumulative attack rate of 8.26%. Taking into account those who did not consult a physician, the number of symptomatic infected persons was estimated to 104,067 (attack rate: 12.85%). In fact, the attack rate of pH1N1/2009 infection in our serosurvey was about 42%-44% at the peak of the antibody response (i.e., weeks 40-44), a figure which is at least 3 to 4 times higher than rates of infection based on clinical cases The wide gap between the two estimates indicates that a large fraction (almost two thirds) of those who got infected by pH1N1/2009 virus escaped medical detection, probably because they developed mild disease or asymptomatic infection, a further indication of the benign nature of the virus, at least at the community level. In", "England, Baguelin et al. [43] estimated that the cumulative incidence rates of infection by the pandemic virus in children were 20 to 40 times higher than that estimated from clinical surveillance.", "Our study, as others [6] , indicates that pre-existing cross reactive antibodies to pH1N1/2009 at titers $1/40 prevented from seroconversion in response to the pandemic virus. This level of pre-existing cross reactive immunity likely confers true protection against infection as about two thirds and one third of documented infection (qRT-PCR positive) in our series have occurred in individuals with baseline HIA titers ,1/40 and = 1/ 40 respectively and less than 5% of documented infections occurred in individuals with base line titers .1/40. The protection was effective not only in older adults but also in younger persons. This indicates that protection was conferred not only by baseline cross reactive antibodies triggered by close pH1N1/2009 viruses that circulated before 1957 (as in the elderly), but also by antibodies likely resulting from recent exposure to seasonal influenza epidemics (as shown in younger persons) [40] . The observed seroconversion rates depend on age, after", "adjusting for baseline pH1N1/2009 titers. The protective role of increasing age might be explained by a stronger cross-immunity in adults and elderly or by a higher exposure of young subjects to the virus during the 2009 epidemic (due to social contacts and mixing patterns). It may also indicate that immune mechanisms other than cross reactive antibodies detected by HIA (i.e. immunity to neuraminidase and conserved T cells epitopes [44] might develop throughout life, providing additional protection from infection or severe disease, especially in the elderly. Interestingly, evidence is seen for a decline in antibody titers, which occurred soon after the passage of the epidemic wave. In paired sera, this decline was significant enough to bring, within a few weeks, almost 27% of sera that tested positive (i.e. HI titers $1/40) in the immediate post epidemic phase to levels under the cut-off value in the second serum sample. This decay accounts for the observation that older adults ($60", "yrs) in the study cohort were apparently almost completely spared by the epidemic if one only considers cumulative incidence rates derived from IHA titration on samples 2 (weeks 45-52). In fact, the cumulative incidence rate in older adults measured just after the epidemic peak (i.e. weeks 40-44) was 20.4%. Similar results of early antibody decay were recently reported [10, 45] . More generally, these data show that serosurveys conducted months after passage of the epidemic, likely underestimate the real extent of pH1N1/2009 infection, compared to antibody titration performed earlier, when humoral responses are at their highest level. Whether the decline in antibody titers has functional immunologic consequence to individuals or within the communities warrants further investigation. However, one should note that there was no second epidemic wave in Reunion Island during the subsequent austral winter seasons in 2010 and 2011.", "Influenza during the 2010 winter was at a level not higher than the usual passages of seasonal flu, though almost two thirds of documented cases in 2010 were also due to pH1N1/2009v [46] . In addition many fewer pandemic virus isolates were noted during the ongoing 2011 austral winter, strongly suggesting that the first epidemic wave had conferred a solid herd immunity, at the community level.", "Our study has some limitations. The fact that the epidemic progression coincided with the implementation of the prospective study, we were not able to collect, strictly speaking, pre-epidemic sera from the cohort members. Therefore we used as proxy base line seroprevalence data from individuals recruited at the very beginning of the investigation when the epidemic activity in the cohort was very low. This may overestimate the base line immunity if subclinical community transmission had occurred before the first cases of pH1N1/2009 influenza were reported. Antibodies to the pandemic virus were detected by HIA, a test that has a good specificity but a rather low sensitivity [46] . Hence, the threshold of 1/40 may underestimate the number of infected individuals. However, rates of seroconversion, the serologic gold standard test based on paired sera, likely gave the most accurate picture of the pandemic in at the community level in Reunion Island." ]

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[ "Pandemic Influenza Due to pH1N1/2009 Virus: Estimation of Infection Burden in Reunion Island through a Prospective Serosurvey, Austral Winter 2009\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3183080/\n\nSHA: ee6d70a53e3262cea6f85bd8b226f6b4c8b5f64b\n\nAuthors: Dellagi, Koussay; Rollot, Olivier; Temmam, Sarah; Salez, Nicolas; Guernier, Vanina; Pascalis, Hervé; Gérardin, Patrick; Fianu, Adrian; Lapidus, Nathanael; Naty, Nadège; Tortosa, Pablo; Boussaïd, Karim; Jaffar-Banjee, Marie-Christine; Filleul, Laurent; Flahault, Antoine; Carrat, Fabrice; Favier, Francois; de Lamballerie, Xavier\nDate: 2011-09-29\nDOI: 10.1371/journal.pone.0025738\nLicense: cc-by", "Abstract: BACKGROUND: To date, there is little information that reflects the true extent of spread of the pH1N1/2009v influenza pandemic at the community level as infection often results in mild or no clinical symptoms. This study aimed at assessing through a prospective study, the attack rate of pH1N1/2009 virus in Reunion Island and risk factors of infection, during the 2009 season. METHODOLOGY/PRINCIPAL FINDINGS: A serosurvey was conducted during the 2009 austral winter, in the frame of a prospective population study. Pairs of sera were collected from 1687 individuals belonging to 772 households, during and after passage of the pandemic wave. Antibodies to pH1N1/2009v were titered using the hemagglutination inhibition assay (HIA) with titers ≥1/40 being considered positive. Seroprevalence during the first two weeks of detection of pH1N1/2009v in Reunion Island was 29.8% in people under 20 years of age, 35.6% in adults (20–59 years) and 73.3% in the elderly (≥60 years) (P<0.0001).", "Baseline corrected cumulative incidence rates, were 42.9%, 13.9% and 0% in these age groups respectively (P<0.0001). A significant decline in antibody titers occurred soon after the passage of the epidemic wave. Seroconversion rates to pH1N1/2009 correlated negatively with age: 63.2%, 39.4% and 16.7%, in each age group respectively (P<0.0001). Seroconversion occurred in 65.2% of individuals who were seronegative at inclusion compared to 6.8% in those who were initially seropositive. CONCLUSIONS: Seroincidence of pH1N1/2009v infection was three times that estimated from clinical surveillance, indicating that almost two thirds of infections occurring at the community level have escaped medical detection. People under 20 years of age were the most affected group. Pre-epidemic titers ≥1/40 prevented seroconversion and are likely protective against infection. A concern was raised about the long term stability of the antibody responses.", "Text: In April 2009, the first cases of acute respiratory infections caused by a novel triple-reassortant influenza virus, pH1N1/ 2009v, occurred in Mexico and the United States [1] . The rapid spread of infection to other continents led the World Health Organization (WHO) to declare on 11 June 2009 that a pandemic of pH1N1/2009v influenza was under way, which raised major international concern about the risk of high morbidity and lethality and the potential for severe socio-economic impact. Actually, the potential impact of this first third-millenium influenza pandemic has been revisited downwards as morbidity and case-fatality rates were less severe than initially anticipated [2] . Illness surveillance data do not allow to an accurate estimate of the true influenza infection rate, as a substantial proportion of infections are asymptomatic or mild [3] . Serological surveys can overcome this limitation, but must take into account that a significant proportion of the population that", "exhibited crossprotective antibody titers before circulation of the pH1N1/2009v [4] . This so-called ''baseline immunity'' has to be subtracted from the seroprevalence observed after the pandemic wave, to determine seroincidence in serosurveys [5] [6] [7] [8] . However, except for few studies [9] [10] [11] , most of these serosurveys did not use serial measurements in the same person, which allows for a better understanding of antibody kinetics and the dynamics of infection within individuals and communities.", "Reunion Island (805,500 inhabitants) is a French overseas department located in the southwestern Indian Ocean, 700 km east of Madagascar and 200 km southwest of Mauritius. The first imported case of pH1N1/2009v was identified on 5 th July 2009 (week 29) in a traveller returning from Australia. The first case indicating community transmission was detected on 21 st July (week 30). pH1N1/2009v became the predominant circulating influenza virus within four weeks of its first detection, its activity peaked during week 35 (24) (25) (26) (27) (28) (29) (30) and ended at week 38 [12] . Contrary to initial fears, the health care system was not overwhelmed, as morbidity and mortality rates were lower than predicted [12] [13] [14] .", "In order to assess at the community level, the actual magnitude of the pH1N1/2009v pandemic and the extent of the herd immunity acquired after passage of the epidemic wave, a prospective population serosurvey was conducted in Reunion Island during the passage of the epidemic wave in the 2009 austral winter season (July-December 2009): prevalence of infection was assessed on a weekly basis and seroconversion rates were measured using paired sera.", "The CoPanFLu-RUN was part of the CoPanFLu international project, a consortium between the French National Institute of Health and Medical Research (INSERM), the Institute of Research for Development (IRD) and the Mérieux Fondation under the promotion of the School of Advanced Studies in Public Health (EHESP). To enable the rapid implementation of the study in anticipation of the imminent spread of the pandemic wave, we used a pre-existing sample of 2442 households established in October 2006 for the investigation of the Chikungunya outbreak (SEROCHIK) and updated in May 2008 throughout a follow-up telephone survey (TELECHIK) on a basis of 1148 households [15, 16] . We took special attention to select households representing a wide range of geographic locations in order to minimize the repartition bias.", "The inclusion phase started on July 21 st (week 30) and was continued up to week 44, throughout the epidemic wave and beyond. A first serum sample (sample 1) was obtained from each household member. An active telephonic inquiry was then conducted twice a week to record symptoms compatible with influenza-like illness (ILI) occurring in households. Report of ILI (fever $37.8uC associated with any respiratory or systemic symptom) led to three consecutive visits of a nurse to the incident case-dwelling (on day 0, +3 and +8 post-report) to record symptoms and collect nasal swabs from all family members (for qRT-PCR detection of pH1N1/2009v. At week 45, the active inquiry was discontinued and a second (post-epidemic) serum sample (sample 2) was obtained (weeks 45-52) to determine seroconversion rates. Sera were aliquoted and stored at 280uC.", "The protocol was conducted in accordance with the Declaration of Helsinki and French law for biomedical research (Nu ID RCB AFSSAPS: 2009-A00689-48) and was approved by the local Ethics Committee (Comité de Protection des Personnes of Bordeaux 2 University). Every eligible person for participation was asked for giving their written informed consent.\n\nViral genome detection by RT-PCR. Viral RNA was extracted from 140 mL of nasal swab eluate using the QIAamp Viral RNA kit (Qiagen) and processed for detection by TaqMan qRT-PCR targeting the heamagglutinin HA gene (SuperScript III Platinum one-step qRT-PCR system, Invitrogen) according to the recommendations of the Pasteur Institute (Van der Werf S. & Enouf V., SOP/FluA/130509). Confirmed pH1N1/2009v infection was defined as a positive qRT-PCR detection of the HA gene in at least one nasal swab.", "Hemagglutination inhibition assay (HIA). A standard hemagglutination inhibition technique was adapted to detect and quantify pH1N1/2009v antibodies [17] . The antigen was prepared by diluting a non-inactivated cell culture supernatant producing a pdm H1N1v strain (strain OPYFLU-1 isolated from a young patient returning from Mexico in early May 2009) [18] . Briefly, the virus was propagated onto MDCK cells under standard conditions. The last passage (used for antigen preparation) was performed in the absence of trypsin and ht-FBS. The supernatant was collected at day seven p.i. clarified by centrifugation at 8006 g for 10 min at room temperature, aliquoted and conserved at 280uC. The hemagglutinating titer of the non inactivated viral antigen was immediately determined under the HIA format described below. The dilution providing 5.33 hemagglutinating units in a volume of 25 mL was used for subsequent HIA. Sera were heat-inactivated at 56uC for 30 min prior to use. Sequential twofold", "dilutions in PBS (1/10 to 1/1280) in volumes of 25 mL were performed and distributed in V-bottom 96 well microplates. Human red blood cells (RBC) were used for hemagglutination experiments. Detection and quantification of antibody to pH1N1/2009v was performed as follows: 25 mL of virus suspension was added to the serum dilution (25 mL) and incubated for 1 hour at room temperature. Each well was then filled with 25 mL of a 1% RBC suspension in PBS (v/v: 0.33%), followed by another 30 min incubation at room temperature. The HIA titer was determined as the last dilution providing clear inhibition of hemagglutination. All experiments were performed in the presence of the same negative and positive controls, the latter including sera with 1/40, 1/80, 1/160 and 1/320 antibody titers.", "The results reported in this study were based only on serological analysis of paired sera. For the sake of analysis, four successive phases were identified throughout the pandemic wave: phase A (weeks 30-31) corresponded to early epidemic time, phase B (W32-39) to the epidemic unfolding, phase C (W40-44) to the immediate post-epidemic stage and phase D (W45-52) to the late post-epidemic stage. Seropositivity was defined as a HIA titer of 1/ 40 or more. The baseline-proxy seroprevalence rate was estimated on serum samples collected in phase A. The cumulative incidence rate of infection measured the raise between the raw seroprevalence rate at any given time during the epidemic phases (S2pi) and the age-specific baseline-proxy seroprevalence rate (S1pA) (s2 pi -s1 pA ). Seroconversion was defined as a shift from seronegative at inclusion (sample 1: HIA ,1/40) to seropositive on follow-up (sample 2: HIA $1/40), or for sera tested seropositive on inclusion as a four-fold increase of HIA", "titers between sample 1 and sample 2 paired sera. We also calculated the proportion of sera that tested seropositive in sample 1 for which the HIA titer decreased fourfold and passed under the cut-off value of 1/40 in sample 2. We considered this proportion as a ''seronegation'' rate.", "The sample size was calculated for identifying risk factors in the prospective cohort study. Considering on average three individuals per household, an intra-household correlation of 0.3, a power greater than 80% could be obtained with a sample size of 840 comprising 2500 individuals, assuming exposure levels ranging from 10% to 90% and a relative risk greater than 1.3. With 2,500 subjects, the study allowed 1-2% absolute precision around the estimated values for seroconversion rates. Data entry used EpiData version 3.1 (The Epidata Association, Odense, Denmark). SAS version 9.1 (SAS Inc., Cary, NC, USA) was used for statistical analysis. The characteristics of the study cohort were compared to those of the population of Reunion Island and a Chi2 test (or Fisher's exact test when non applicable) was used to analyse differences in age, sex and geographic location. Cumulative incidence rates of infection (i.e. seroincidence) and seroconversion rates were standardized according to the", "age structure of the community (French National Institute for Statistics and Economical Studies (INSEE) source).", "Baseline-proxy seroprevalence, cumulative incidence rates of infection, as well as seroconversion and seronegation rates, were expressed as percentages. Cumulative reverse distribution curves were used to show the distribution of antibody titers. In all tests, a P value,0.05 was considered significant.\n\nWe estimated 95% confidence intervals (CIs) of proportions by using a cluster bootstrap technique with 1000 re-samples [19] . After bootstraping, we used an ANOVA model to compare mean cumulative incidence proportions between pandemic phases, within each age group. We used an alternating logistic regression model (ALR) with an exchangeable log Odds Ratio (OR) to test the intra-household correlation-adjusted association between factors and the seroconversion outcome.", "Data were analysed with respect to subject age. Initially, four age groups were considered: the children and adolescents (,20 yrs), young adults (20-39 yrs), middle-age adults (40-59 yrs), and elderly adults ($60 yrs). As the cumulative incidence of infection of the second and third groups were very close, both groups were merged into one adults group (20-59 yrs). Therefore we refer further in our study to three age groups: children and adolescents (,20 yrs), adults (20-59 yrs), elderly ($60 yrs).", "A total of 2,164 individuals from 772 households were enrolled between weeks 30 and 44 in the CoPanFlu-RUN cohort, allowing the collection of 1,932 sera at inclusion (sample 1). During this period, 136 households (17.7% of households) containing 464 individuals (21.4% of individuals) reported at least one case of ILI. Sixty subjects among the 464 individuals (12.9%, belonging to 33 households [24.3%]) were qRT-PCR positive, which documented the pH1N1/2009v infection. No positive qRT-PCR could be detected after week 37 and no ILI was reported after week 40, the end of the epidemic wave. The second follow up serum sample (sample 2) was obtained for 1,759 subjects at least five weeks after the end of the epidemic wave (weeks 45-52) which allowed the constitution of a serobank of 1,687 paired-sera. The profile of the cohort and the major outcomes are displayed in Figure 1 . Details on inclusions and serum sample timing with respect to the circulation of pH1N1/2009v over the island are", "provided in figure 2 . The socio-demographic and space-time characteristics of the cohort are detailed in Table 1 . Compared to the community of Reunion Island, the sample of 1,687 individuals for whom pairedsera were available, was older (,20 yrs: 27% vs 35%, and $60 yrs: 17,9% vs 11,3%) and composed of a slight excess of females (54.1% vs 51.5%). The imbalance was due to a deficit in subjects aged under 40 years, reflecting men at work and the fact that parents declined the second serum for children younger than five.", "Baseline-proxy (,pre-epidemic) HIA titers to the pH1N1/ 2009v were measured on sample 1 ( Table 2) , obtained from 249 subjects (103 households) recruited at the very beginning of the investigation during weeks 30 and 31 (phase A, Figure 2 ), when the epidemic activity in the cohort was still very low. Age distribution in this group was similar to that of the whole cohort (data not shown). The overall, the baseline-proxy seroprevalence rate (HIA $1/40), over all ages, was 43.4% (95%CI: 37.4%-49.6%). However the majority of positive sera had low antibody titers, at the cut off value for confirmation (i.e. = 1/40). The proportions of sera with HIA titer .1/40 were 0%, 3.0% and 24.6% in the young, middle-aged and older age groups respectively. These results indicate that pre-epidemic baseline antibody cross reactivity was stronger in the elderly ($60 yrs) and weaker in children and adolescents (,20 yrs) and adults (20-59 yrs), with highly significant differences between age groups", "(P,0.0001).", "The reverse cumulative distribution curves of HIA titers are displayed for each age group and for the whole cohort on Figure 3 . The proportion of seropositive sera (HI $1/40) steadily increased during the epidemic unfolding (phase B, W32-39) and in immediate post epidemic period (phase C, W40-44) when it reached its maximum level, then declined in the late post epidemic period (phase D, W45-52). This decline was significant enough to return the reverse cumulative distribution curve to baseline levels in the elderly. The cumulative incidence rates, obtained after subtraction of the age-specific baseline-proxy seroprevalence from the raw seroprevalence at each phase of the epidemic are shown in Table 2 (note that the cumulative incidence rates of infection represented for the group ''all ages'' were standardized according to age structure of the community). The cumulative incidence rates were much higher in children and adolescents (,20 yrs), indicating very active transmission of", "infection within this age group. As mentioned earlier, cumulative incidence rates peaked in phase C (W40-44), and then declined indicating some lability of the humoral immune response against the pH1N1/2009v. The age-related difference observed in the incidence rates was highly statistically significant (P,0.0001).", "To estimate more appropriately the decline of antibody titers occurring after the peak of the humoral response to the pH1N1/ 2009v, we considered paired-sera from the group of 264 subjects for whom the first serum sample (sample 1) was obtained just after the epidemic wave (phase C, W40-44), and the corresponding second sample was collected at the end of the survey (phase D, W45-52). Seronegation rates were 27.0% (61/226) for all age groups, 17.4% (12/69) in children and adolescents (,20 yrs), 32.3% (41/127) in adults (20-59 yrs) and 26.7% (8/30) in the elderly ($60 yrs). Differences between the seronegation rates according to age were statistically weakly significant (P = 0.0671).", "We then considered the 1687 individuals for whom paired sera were available and we measured the seroconversion rates according to age and to the time of first serum sample collection (phase A, B or C). Criteria of seroconversion were defined in the method section. As shown in table 3, there was a sharp decline in seroconversion rates across all the age groups, depending on whether participants were enrolled during phase A, phase B, or phase C (P,0.0001). To interpret these data, one should remember that antibodies at seroprotective levels (HIA $1/40), in serum samples 1 collected during the per epidemic phase B or early post epidemic phase C could represent either base line cross reactive antibodies or rising pH1N1/2009 specific antibodies due to a recent or ongoing infection. This ambiguity could lead to underestimation of the seroconversion rate for subjects enrolled in phases B and C. In order to solve this ambiguity, we specifically considered the group of 249 subjects in whom", "cross reactive antibodies were detected at the time of phase A (W30-31). The seroconversion rate of this group is the most indicative of the exposure of individuals to the whole epidemic wave. It was the highest (63,2%, P,0.0001) in children and adolescents (,20 yrs), and still significantly high in adults (39.4%, P,0.0001).", "We then tested in this particular group, the impact of (baseline) pre-epidemic cross reactive antibodies on the rate of seroconversion to pH1N1/2009 (Table 4) . No subject with HIA titer superior to 1/40 had evidence of seroconversion to pH1N1/2009. The seroconversion rate in individuals with a HIA titer equal to 1/40 was linked with age, being more important in children and adolescents (,20 yrs). The highest seroconversion rate (.56%) was registered in subjects with HIA titers inferior to 1/40, particularly for the under 20 years where it reached 85%. Hence, the risk of seroconversion decreased when pre-epidemic HIA titer was high after controlling for age (P,0.0001) (Figure 4) . The multivariate adjusted odds ratio for seroconversion were 0.15 (95%CI: 0.06-0.37, P,0.0001) per two-fold increase in baseline titer, 1.79 (95%CI: 1.23-2.59, P,0.003) per other household members who seroconverted, 5.33 (95%CI: 1.56-19.27, P,0.008) Figure 1 . The cohort profile and major outcomes. Figure 1", "details the three phases of the protocol: i) inclusion (weeks 30-44) and serum samples S1 collection; ii) follow up for detection of ILI in households, qRT-PCR on nasal swabs and estimation of cumulative seroincidence rates; iii) end of the study (weeks 45-52) and samples S2 collection. HIA on paired sera (S1+S2) allowed estimating seroconversion rates. doi:10.1371/journal.pone.0025738.g001 Bp (baseline-proxy) seroprevalence rates were estimated on weeks 30-31 in each age group. b Cumulative incidence rates measured the raise between raw seroprevalence rates and age-specific baseline-proxy seroprevalence rate. In the group ''All ages'', cumulative incidence rates were standardized according to age structure of the community. doi:10.1371/journal.pone.0025738.t002 Data are numbers, percentages (95% confidence intervals) and ALR parameter test P value for comparison of seroconversion proportions according to time of first sample (S1) collection at inclusion, in each age group, after", "controlling for household selection. In the group ''All ages'', rates of seroconversion were standardized according to age structure of the community. NA: not assessed. Seroconversion was defined as a shift from seronegative at inclusion (i.e. HIA titer ,1/40) to seropositive on follow-up sample, or as a 4-fold increase of reciprocal HIA titer between first and second paired samples for sera tested seropositive on inclusion (i.e. HIA titer $1/40). for age ,20 years (vs age $60 years) and 11.35 (95%CI: 0.41-4.47, P = 0.62) for age 20-60 years (vs age $60 years). The observed and predicted seroconversion rates according to age and baseline HIA titer are displayed Figure 4 .", "Finally, we considered the 46 subjects who had been infected by the pandemic virus over the course of the study, verified by a positive qRT-PCR nasal swab, and for whom paired sera were available. Initial HIA antibody titers in this group were ,1/40,", "The CoPanFlu-RUN cohort was set up to conduct a prospective population-based study investigating the herd immunity induced by the 2009 pandemic influenza virus and identifying risk factors for pH1N1/2009v infection from paired sera collected in an entire community. Most works published to date have used either extensive cross-sectional serosurveys on pre-and post-epidemic independent serum samples, the baseline immunity being assessed from stored frozen samples [5, 7, 8] , or non representative adult cohorts (military, health care workers, long-stay patients). Antibody titers were measured by HIA using a cut-off value set at 1/40 as classically recommended. This HIA titer at 1/40 is considered protective, i.e. conferring 50% protection against a viral challenge [20] . Our assay has introduced some changes in the experimental protocol compared to the classic one. The use of a non-inactivated viral antigen, i.e. a native virus, with nondenatured epitopes probably allows detection of", "antibodies to epitopes of the hemagglutinin not detected in the classic HIA test. This can induce slight differences in the sensitivity of detection of cross-reacting antibodies, but this does not modify the kinetics of Ab and the epidemiological evolution of seroprevalence and does not jeopardize the global comparability of serological results. This is confirmed by the fact that our HI assay detected seroprotective antibody titers in 93.5% and gave evidence seroconversion in 73.9% of qRT-PCR confirmed pH1N1/2009 influenza, all figures close to those reported in the literature [5, 21] .", "We considered that titers of .1/40, in sera collected from individuals enrolled during weeks 30 and 31 were cross reactive antibodies and not de novo antibodies triggered by the pandemic virus and hence used them as a proxy for baseline pre epidemic immunity. Several arguments support this assumption: i) the first case indicating autochthonous transmission in Reunion Island was reported by the epidemiological surveillance department of La Réunion on 21st July (week 30), i.e. the same day when inclusion started in our study cohort; ii) 7 to 15 days are required to develop an antibody response after viral infection; iii) On weeks 30 and 31, the epidemic activity due to the pandemic virus was very low in our study cohort and it became significant only after week 32. Hence, during weeks 30-31, 103 households were recruited and only 2 households reported ILI cases. Nasal swabs collected from these 2 individuals were tested qRT-PCR negative to the pandemic virus whereas one had evidence of", "coronavirus and rhinovirus using a multiplex RT-PCR to respiratory viruses (H. Pascalis, manuscript in preparation). In contrast, during weeks 32 to 39, 199 individuals belonging to 99 households reported ILI, among whom 60 individuals had documented infection by the pandemic virus.", "Our study shows that a substantial proportion of Reunion Island's population had pre-existing immunity to 2009 pandemic influenza virus with the highest baseline-proxy seroprevalence rate observed among adults aged of 60 years or more. Other studies from all continents had also reported high pre-epidemic seropositivity rates among the elderly [5, 6, 8, [22] [23] [24] [25] [26] , though large variations do exist between countries [10, 11, 23, 27, 28] . These cross reactive antibodies have been interpreted as being the residual signature of the remote exposure of these individuals to H1N1 viruses circulating before 1957 [24, 25, 29, 30] . Baseline seropositivity rates that we report in children and in younger adults (i.e. 30%-35%) were notably higher than those reported from other parts of the world [6, 8, 22, 23, [31] [32] [33] . However one should note that these baseline antibodies were of low titer, just at the level of the HIA threshold (i.e. 1/40). Several factors could have", "contributed to this comparatively high baseline rates found in our study: i) It may reflect the fact that the HI test used in our study was marginally more sensitive than the classic one [17] ; ii) Some individuals may have already been infected with pH1N1/ 2009 virus at weeks 30 and 31 and may have triggered an antibody response to the virus. This hypothesis seems unlikely in view of the arguments presented above and of a similar high proportion of sera titering HIA = 1/40 among 122 sera from adult patients sent for diagnostic purposes to the Regional Hospital microbiology laboratory, during the first half of 2009 (i.e. before the 2009 pandemic) (data not shown). However we cannot formally exclude this hypothesis in view of a recently reported study from Taiwan [11] that showed evidence of subclinical community transmission with proved seroconversion several weeks before report of the first documented case in the island. A similar conclusion was also drawn from Australia [34] ; iii)", "our serological test might detect cross-reactive antibodies triggered by recent vaccination with trivalent seasonal influenza vaccine as reported [4, [35] [36] [37] [38] [39] . However, seasonal influenza vaccines were of rather limited use in Reunion Island, especially in children and young adults; iv) Finally the high baseline titers may reflect the infectious history of the individuals to seasonal influenza viruses cross antigenic with pH1N1/2009 virus as recently suggested for seasonal 2007 H1N1 infection [40] . This serosurvey indicates that a large fraction of the Reunion Island population was infected with the pandemic virus. Younger people, have paid the main tribute to the epidemic as almost two thirds show evidence of seroconversion, confirming earlier clinical reports from the island [12] and accumulating reports from other countries [17, 32, 41, 42] and suggesting that school children have likely played the central role in the epidemic diffusion of the pandemic virus.", "Lower infection rates were found in adults and the lowest rates were recorded in the elderly.", "Based on clinical cases reported to the epidemiological surveillance services [12] , it was estimated that 66,915 persons in Reunion Island who consulted a physician were infected by the pH1N1/2009 virus during the 9 weeks of the epidemic, giving a cumulative attack rate of 8.26%. Taking into account those who did not consult a physician, the number of symptomatic infected persons was estimated to 104,067 (attack rate: 12.85%). In fact, the attack rate of pH1N1/2009 infection in our serosurvey was about 42%-44% at the peak of the antibody response (i.e., weeks 40-44), a figure which is at least 3 to 4 times higher than rates of infection based on clinical cases The wide gap between the two estimates indicates that a large fraction (almost two thirds) of those who got infected by pH1N1/2009 virus escaped medical detection, probably because they developed mild disease or asymptomatic infection, a further indication of the benign nature of the virus, at least at the community level. In", "England, Baguelin et al. [43] estimated that the cumulative incidence rates of infection by the pandemic virus in children were 20 to 40 times higher than that estimated from clinical surveillance.", "Our study, as others [6] , indicates that pre-existing cross reactive antibodies to pH1N1/2009 at titers $1/40 prevented from seroconversion in response to the pandemic virus. This level of pre-existing cross reactive immunity likely confers true protection against infection as about two thirds and one third of documented infection (qRT-PCR positive) in our series have occurred in individuals with baseline HIA titers ,1/40 and = 1/ 40 respectively and less than 5% of documented infections occurred in individuals with base line titers .1/40. The protection was effective not only in older adults but also in younger persons. This indicates that protection was conferred not only by baseline cross reactive antibodies triggered by close pH1N1/2009 viruses that circulated before 1957 (as in the elderly), but also by antibodies likely resulting from recent exposure to seasonal influenza epidemics (as shown in younger persons) [40] . The observed seroconversion rates depend on age, after", "adjusting for baseline pH1N1/2009 titers. The protective role of increasing age might be explained by a stronger cross-immunity in adults and elderly or by a higher exposure of young subjects to the virus during the 2009 epidemic (due to social contacts and mixing patterns). It may also indicate that immune mechanisms other than cross reactive antibodies detected by HIA (i.e. immunity to neuraminidase and conserved T cells epitopes [44] might develop throughout life, providing additional protection from infection or severe disease, especially in the elderly. Interestingly, evidence is seen for a decline in antibody titers, which occurred soon after the passage of the epidemic wave. In paired sera, this decline was significant enough to bring, within a few weeks, almost 27% of sera that tested positive (i.e. HI titers $1/40) in the immediate post epidemic phase to levels under the cut-off value in the second serum sample. This decay accounts for the observation that older adults ($60", "yrs) in the study cohort were apparently almost completely spared by the epidemic if one only considers cumulative incidence rates derived from IHA titration on samples 2 (weeks 45-52). In fact, the cumulative incidence rate in older adults measured just after the epidemic peak (i.e. weeks 40-44) was 20.4%. Similar results of early antibody decay were recently reported [10, 45] . More generally, these data show that serosurveys conducted months after passage of the epidemic, likely underestimate the real extent of pH1N1/2009 infection, compared to antibody titration performed earlier, when humoral responses are at their highest level. Whether the decline in antibody titers has functional immunologic consequence to individuals or within the communities warrants further investigation. However, one should note that there was no second epidemic wave in Reunion Island during the subsequent austral winter seasons in 2010 and 2011.", "Influenza during the 2010 winter was at a level not higher than the usual passages of seasonal flu, though almost two thirds of documented cases in 2010 were also due to pH1N1/2009v [46] . In addition many fewer pandemic virus isolates were noted during the ongoing 2011 austral winter, strongly suggesting that the first epidemic wave had conferred a solid herd immunity, at the community level.", "Our study has some limitations. The fact that the epidemic progression coincided with the implementation of the prospective study, we were not able to collect, strictly speaking, pre-epidemic sera from the cohort members. Therefore we used as proxy base line seroprevalence data from individuals recruited at the very beginning of the investigation when the epidemic activity in the cohort was very low. This may overestimate the base line immunity if subclinical community transmission had occurred before the first cases of pH1N1/2009 influenza were reported. Antibodies to the pandemic virus were detected by HIA, a test that has a good specificity but a rather low sensitivity [46] . Hence, the threshold of 1/40 may underestimate the number of infected individuals. However, rates of seroconversion, the serologic gold standard test based on paired sera, likely gave the most accurate picture of the pandemic in at the community level in Reunion Island." ]

[ 39 ]

[ "to investigate the different pathogens involved in ILI and describe the associated symptoms" ]

[ "High Burden of Non-Influenza Viruses in Influenza-Like Illness in the Early Weeks of H1N1v Epidemic in France\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3157400/\n\nSHA: f4c1afe385e9e31eb5678e15a3c280ba97326554\n\nAuthors: Schnepf, Nathalie; Resche-Rigon, Matthieu; Chaillon, Antoine; Scemla, Anne; Gras, Guillaume; Semoun, Oren; Taboulet, Pierre; Molina, Jean-Michel; Simon, François; Goudeau, Alain; LeGoff, Jérôme\nDate: 2011-08-17\nDOI: 10.1371/journal.pone.0023514\nLicense: cc-by", "Abstract: BACKGROUND: Influenza-like illness (ILI) may be caused by a variety of pathogens. Clinical observations are of little help to recognise myxovirus infection and implement appropriate prevention measures. The limited use of molecular tools underestimates the role of other common pathogens. OBJECTIVES: During the early weeks of the 2009–2010 flu pandemic, a clinical and virological survey was conducted in adult and paediatric patients with ILI referred to two French University hospitals in Paris and Tours. Aims were to investigate the different pathogens involved in ILI and describe the associated symptoms. METHODS: H1N1v pandemic influenza diagnosis was performed with real time RT-PCR assay. Other viral aetiologies were investigated by the molecular multiplex assay RespiFinder19®. Clinical data were collected prospectively by physicians using a standard questionnaire. RESULTS: From week 35 to 44, endonasal swabs were collected in 413 patients. Overall, 68 samples (16.5%) were", "positive for H1N1v. In 13 of them, other respiratory pathogens were also detected. Among H1N1v negative samples, 213 (61.9%) were positive for various respiratory agents, 190 in single infections and 23 in mixed infections. The most prevalent viruses in H1N1v negative single infections were rhinovirus (62.6%), followed by parainfluenza viruses (24.2%) and adenovirus (5.3%). 70.6% of H1N1v cases were identified in patients under 40 years and none after 65 years. There was no difference between clinical symptoms observed in patients infected with H1N1v or with other pathogens. CONCLUSION: Our results highlight the high frequency of non-influenza viruses involved in ILI during the pre-epidemic period of a flu alert and the lack of specific clinical signs associated with influenza infections. Rapid diagnostic screening of a large panel of respiratory pathogens may be critical to define and survey the epidemic situation and to provide critical information for patient management.", "Text: In order to monitor the spread of influenza and alert health handlers, several epidemiological tools have been developed. In France, a network of 1300 general practitioners, ''Réseau Sentinelles'', working throughout the country, provides real-time clinical data used to evaluate regional and national influenza spreading [1, 2] . The criteria used by this network to define clinical influenza-like illness (ILI) are the occurrence of a sudden fever above 39uC with myalgia and respiratory signs. In general no formal viral diagnosis is carried out. The Groupes Régionaux d'Observation de la Grippe (GROG) is a second French network that surveys the emergence and the spread of the influenza viruses [3, 4] . This network is based on clinical surveillance of acute respiratory infections and laboratory analysis of nasal specimens collected from adults and children by volunteer general practitioners and pediatricians.", "According to the sentinel network's criteria, French health authorities proclaimed that flu epidemic level was reached during the second week of September 2009 (week 37) [5, 6] . On the contrary, data provided by the GROG showed only sporadic H1N1v activity until the last week of October (week 44) [6, 7] . Thus, it became rapidly obvious that a variety of viruses were circulating in the community and that an overestimation of myxovirus infection was at stake [8, 9, 10, 11] .", "As a better knowledge of the epidemic status was a key feature for national healthcare organization, hospital preparedness, patient management and disease control, unambiguous viral diagnosis appeared critical. In France, data on viral aetiologies associated with ILI were at best sporadic and correlations with clinical symptoms were often lacking. Extensive molecular assays to screening for respiratory viruses were not available countrywide for routine diagnosis. Therefore the epidemiological pattern of respiratory pathogens with overlapping seasonality was poorly known.\n\nThe aim of the present study was to investigate respiratory pathogens involved in ILI during the early weeks of the 2009-2010 H1N1v diffusion in France (weeks 35 through 44) and describe the associated symptoms in paediatric and adult populations.", "This study was a non-interventional study with no addition to usual proceedures. Biological material and clinical data were obtained only for standard viral diagnostic following physicians' prescriptions (no specific sampling, no modification of the sampling protocol, no supplementary question in the national standardized questionnaire). Data analyses were carried out using an anonymized database. According to the French Health Public Law (CSP Art L 1121-1.1), such protocol does not require approval of an ethics committee and is exempted from informed consent application.", "In the two academic hospitals, Saint-Louis hospital (SLS) in Paris and Tours hospital (TRS), influenza-like illness (ILI) was defined as a patient suffering from at least one general symptom (fever above 38uC, asthenia, myalgia, shivers or headache) and one respiratory symptom (cough, dyspnoea, rhinitis or pharyngitis), in agreement with the guidelines from the French Institut de Veille Sanitaire (InVS), a governmental institution responsible for surveillance and alert in all domains of public health [12] . Criteria for severe clinical presentation were temperature below 35uC or above 39uC despite antipyretic, cardiac frequency above 120/min, respiratory frequency above 30/min, respiratory distress, systolic arterial pressure below 90 mmHg or altered consciousness. Predisposing factors of critical illness were children younger than one year old, pregnant women, diabetes, chronic pre-existing disease (such as respiratory, cardiovascular, neurologic, renal, hepatic or hematologic", "diseases) and immunosuppression (associated with HIV infection, organ or hematopoietic stem cells transplantation, receipt of chemotherapy or corticosteroids) [13, 14] . A cluster of suspected influenza infections was defined as at least three possible cases in a week in a closed community (household, school,…) [15] .", "In the two institutions, the prescription of H1N1v molecular testing was recommended for patients with ILI and with either a severe clinical presentation, an underlying risk factor of complications or a condition which was not improving under antiviral treatment. Investigation of grouped suspected cases was also recommended. From week 35 (last week of August) to 44 (last week of October), 413 endonasal swabs were collected in 3 ml of Universal Transport Medium (Copan Diagnostics Inc, Murrieta, CA) from adults and children seen in emergency rooms for suspected ILI (Table 1 ) and sent to SLS and TRS laboratories for H1N1v detection. The two microbiology laboratories participated in the reference laboratories network for the detection of pandemic influenza H1N1v.", "Clinical data were collected at the time of medical attention and reported by clinicians on a national standardized questionnaire provided by InVS [1, 12] . This questionnaire included the presence or absence of the main general and respiratory symptoms associated with ILI (fever, asthenia, myalgia, shivers, headache, cough, rhinitis, pharyngitis, sudden onset) [12] .\n\nTotal nucleic acid was extracted from 400 mL of Universal Transport Medium using the EasyMag System (Biomérieux, Marcy l'Etoile, France) in SLS or the EZ1 Advanced XL (Qiagen, Courtaboeuf, France) in TRS, according to the manufacturers' instructions (elution volume: 100 mL in SLS or 90 mL in TRS). Before extraction, 5 ml of an Internal Amplification Control (IAC) which contained an encephalomyocarditis virus (EMC) RNA transcript was added into the sample.", "Pandemic H1N1v infection was diagnosed by real-time reverse transcription-PCR (RT-PCR) assay on a 7500 Real Time PCR System (Applied Biosystems, Foster City, CA) according to the protocol of the Centers for Disease Control (CDC) [16] . Other respiratory infections were investigated by a multiplex molecular assay based on the Multiplex Ligation-dependent Probe-Amplification (MLPA) technology (RespiFinder19H, Pathofinder, Maastricht, The Netherlands) that allows the detection and differentiation of 14 respiratory viruses, including influenza virus A (InfA), influenza virus B (InfB), rhinovirus (RHV), parainfluenza viruses 1 to 4 (PIV-1 to PIV-4), human metapneumovirus (hMPV), adenovirus (ADV), respiratory syncytial virus A (RSVA), respiratory syncytial virus B (RSVB) and human coronaviruses 229E, OC43 and NL63 (Cor-229E, Cor-OC43, Cor-NL63) [17] . The test allows also the detection of H5N1 influenza A virus and of four bacteria: Chlamydophila pneumoniae (CP), Mycoplasma pneumoniae (MP),", "Legionella pneumophila (LP) and Bordetella pertussis (BP). The amplified MLPA products were analyzed on an ABI 3100 genetic analyzer (Applied Biosystems, Foster City, CA). Fragment sizing analysis was performed with the GeneMarker software (SoftGenetics, LLC, State College, PA).", "Further testing for H1N1v was carried out with Simplexa TM Influenza A H1N1 (2009) (Focus Diagnostics, Cypress, California) when the CDC real time RT-PCR assay was negative for H1N1 and the RespiFinder19H assay was positive for Influenza A. If this latter assay was negative, H3N2 typing was performed as previously described [18] .\n\nData from our study are summarized as frequencies and percentages for categorical variables. Quantitative variables are presented as medians, 25th and 75th percentiles. To compare those variables according to the viral infection status, Fisher tests", "By using CDC reference assay, H1N1v was detected in 66 samples out of 413 (16.6%), more frequently in SLS (38 samples) than in TRS (28 samples) (p,10 24 ). Overall, weekly percentage of H1N1v positive endonasal swabs remained under 10% until week 41 and increase significantly after (P Trend ,0.0001) ( Figure 1 ). Rate of H1N1v detection reached 30% in SLS at week 42 and in TRS at week 44. Overall, this rate was in agreement with results provided by the GROG network, showing an earlier start of H1N1v epidemic in Paris area [7, 19] .", "All 413 nucleic acid extracts were analyzed using the RespiFinder19H assay ( Figure 2 ). Sixty six patients tested H1N1v positive with CDC real time RT-PCR assay were confirmed with the multiplex assay. Thirteen were also co-infected by one or two other respiratory pathogens (multiple infections) ( Figure 2 ). Three of the 347 H1N1v negative samples could not be studied with the multiplex assay because they contained RT-PCR inhibitors (no amplification of the internal control). Two hundred and fifteen (62.5%) of the remaining 344 H1N1v negative samples were found positive for at least one respiratory pathogen ( Figure 2 ). Two hundred and twelve were positive for non influenza pathogens (189 single infections and 23 mixed infections with two, three or four viruses) and three additional single infections by influenza A were identified in SLS, including two by pandemic H1N1v and one by seasonal H3N2, as determined after molecular typing (data not shown).", "Overall, 68 patients (16.5%) were then positive for H1N1v, one for H3N2 and 212 for non influenza pathogens. There were 245 single infections (55 with H1N1v and 190 with other respiratory pathogens) and 36 mixed infections (13 with H1N1v and 23 without H1N1v) ( Figure 2 ).", "Among H1N1v negative single infections, the most prevalent viruses were rhinovirus (62.6%, 119 patients), followed by parainfluenza viruses 1 to 4 (24.2%, 46 patients), adenovirus (5.3%, 10 patients), human coronavirus 229E, OC43 and NL63 (3.2%, 6 patients) and respiratory syncytial virus A and B (2.6%, 5 patients) (Figure 2 ). In addition, RespiFinder19H assay identified three patients with bacterial infection, two with Mycoplasma pneumoniae (one 25 years old female in SLS and one 39 years old female in TRS) and one with Bordetella pertussis (one 60 years old male in SLS). No single infection by influenza B, hMPV, Chlamydophila pneumoniae or Legionella pneumophila was identified ( Figure 2 To analyze if viral co-infections occurred more frequently for some viruses, we carried out a two by two comparisons, that showed a higher proportion of co-infection only for ADV (p = 0.05).", "Non-influenza respiratory viruses presented a different epidemic profile compared to H1N1v. Overall, in both hospitals, weekly rate of non-H1N1v respiratory viruses whether alone or involved in co-infection increased between week 37 and 39 (from 51.4% to 81.3%) and then consistently decreased ( Figure 3 ). RHV infections that represented nearly half of non-H1N1v viral infections (141 out of 213, 66.2%) were a significant contributing factor. In both hospitals, emergence of H1N1v cases was associated with a rapid decline of RHV rate of infection from 50-60% down to less than 20% with a one to two weeks gap between SLS and TRS.", "Data on age ( In both institutions, 85.5% (106/124) children younger than 15 years of age were infected by at least one respiratory pathogen ( Table 2 ). H1N1v infected patients were not significantly younger than H1N1v non infected patients (27 years old vs. 25 years old, p = 0.80) (Figure 4) . However, 70.6% (48/68) of H1N1v cases were identified in patients under 40 years old (22 in SLS and 26 in TRS) and no case was observed in patients older than 65 years ( Table 2) . PIV infection occurred in very young patients (median (Figure 4) . Consequently, PIV and ADV were more frequently detected in the younger population of TRS versus SLS (p,10 24 and p,10 23 respectively). In contrast, although individuals with RHV infection were slightly younger than individuals without (median age = 24 vs. 29 for patients without RHV, p = 0.05) (Figure 4) , influenza-like illness associated with RHV was more frequent in SLS than in TRS (p = 0.012). Finally, patients with viral multiple infection were", "significantly younger than those with single infection (median, IDR: 4, 2-18.5 vs. 25, 6-43) and rates of mixed infection At the time of medical attention, 383 (92.7%) standardized clinical questionnaires were collected out of 413 patients. Four of them could not be exploited because they were too incomplete. A review of the 379 workable questionnaires showed that 90.8% (344/379) of the patients included in this study fulfilled the criteria of ILI as defined above, and 52.5% had either a severe clinical presentation or an underlying risk factor of complications (45.9%, 174/379), or were in a suspected cluster of grouped cases (6.6%, 25/379).", "Overall, most patients have fever (93.9%) and cough (86.1%) ( Table 3) . Other classical clinical signs associated with ILI such as asthenia, myalgia, shivers, headache, rhinitis or pharyngitis were less frequent. A sudden onset was also described in 59.2% of cases. Only 32.5% of the patients had a temperature above 39uC; the age of these patients ranged from zero to 86 years, with a median age of 32 years and a mean age of 34 years (data not shown).", "In H1N1v infected patients (including single and multiple infections), the main symptoms were also fever (98.2%) and cough (89.5%) ( We then compared clinical characteristics between patients positive for H1N1v, patients positive for other respiratory pathogens and negative for H1N1v and patients without any detection of respiratory pathogens (as detected with RespiFin-der19H) ( Table 3 ). There was no difference between the three groups except for fever, cough, pharyngitis. However for these latter symptoms, the comparison between patients positive for H1N1v and those positive for other respiratory pathogens or between patients positive for H1N1v and those without any detection of respiratory pathogens, showed no difference except for pharyngitis, which was less frequent in patients positive for H1N1v than in patients positive for other respiratory pathogens ( Table 3) .", "As RHV was the most frequent aetiology in ILI, we also compared clinical symptoms observed in patients with a single infection by RHV or by H1N1v (data not shown). There was no difference except that rhinitis and pharyngitis were significantly more frequent in RHV infection (62.7% vs. 34.1% [p = 0.006] and 39.0% vs. 10.0% [p = 0.001], respectively). Viral multiple infection (including samples with H1N1v) was not associated with a different clinical presentation. Fever and cough were observed in over 90% of the patients (90.6% and 90.3%, respectively), but only 33.3% of these patients had a temperature above 39uC, which was not different from patients with single viral infection (28.6%).", "Our results highlight the high frequency of non-influenza viruses involved in acute respiratory infections during the epidemic period of a flu alert as defined by the Réseau Sentinelles according to ILI definition (a sudden fever above 39uC accompanied by myalgia and respiratory signs). These data extent previous observations in Europe reporting high prevalence of RHV infections before seasonal influenza [4, 20] or in 2009, before H1N1v pandemic influenza [1, 8, 9, 11, 21] . We confirm that RHV represent the most frequent aetiology of acute respiratory Table 2 . Age of patients with respiratory samples positive for H1N1v, positive for other respiratory pathogens or negative. infections both in adult and paediatric populations and may represent more than 50% of cases. We show that other viral infections than influenza and RHV may represent up to 30% of aetiologies. We observed differences between the two hospitals, with a higher frequency of parainfluenza and ADV infections in Tours in", "contrast with a higher frequency of RHV in Paris, likely explained by the higher proportion of paediatric samples collected in Tours. However, despite the distance between the two institutions (about 250 km) and differences between the two populations, both presented similar patterns of high frequency of non-influenza viruses in acute respiratory infections before the flu epidemic wave and a decline when influenza reached epidemic levels.", "In the two cities, high frequencies of RHV were seen at the same level with a likely different evolution speed, with sudden increase and decrease in SLS and more progressive variation in TRS. In both institutions, there was a decrease in the proportion and number of RHV diagnoses roughly in parallel with the increase of influenza diagnoses. Indeed, H1N1v exceeds 20% of positive detection's rate only when RHV dropped under 40%. These data are thus consistent with negative interaction of the two epidemics at the population level. It was previously hypothesised that RHV epidemic could interfere with the spread of pandemic influenza [20, 21, 22] . Few in vitro data support this hypothesis. It has been reported that interferon and other cytokines production by RHV infected cells induced a refractory state to virus infection These data include the three patients whose respiratory samples could not be studied with the multiplex assay because of RT-PCR inhibitors. of neighbouring cells [23] .", "Further work is needed to confirm in vitro and in vivo such negative interactions and if viral interference are really translated to a population level. Analysis of rhinovirus and influenza epidemics in previous years should also help to determine if similar interferences were observed with seasonal influenza and to elaborate modelling and prediction of the spread of influenza according to respiratory viruses' circulation. Systematic extensive screening of respiratory viruses at a national level should be implemented for this purpose.", "Very few RSV infections were observed in contrast to usual epidemiology which was characterized the last four past years by a start of epidemics in weeks 44-45 [1] . It has been confirmed by other laboratories and the French InVS that the 2009-10 RSV epidemic was delayed and had a lower impact compared with the previous winter season [1, 24] . Delayed and reduced RSV spread may be due to viral interference between RSV and influenza. Another possible explanation is better prevention behaviour about respiratory infections as recommended by a national campaign including recommendations for hands washing after sneezing and the use of mask [1] .", "Influenza infections were mainly detected in patient under 40 years old and no case was found in patients older than 65. These results corroborate previous data suggesting that past seasonal H1N1 infections or vaccination may give partial crossed protection [10, 13, 25] . We have previously shown that the neutralizing titers against pandemic H1N1v virus correlate significantly with neutralizing titers against a seasonal H1N1 virus, and that the H1N1v pandemic influenza virus neutralizing titer was significantly higher in subjects who had recently been inoculated by a seasonal trivalent influenza vaccine [26] .\n\nViral co-infections were predominantly seen in paediatric patients, as previously described [4, 27, 28, 29] , both in influenza and non-influenza cases at a similar rate. No evidence of more pronounced respiratory impact was seen in these patients.", "Our results showed the lack of specific clinical signs associated with proven H1N1v infections. Clinical characteristics did not differ between influenza infections or other viral infections. In particular, the proportion of patients with fever above 39uC was not higher in H1N1v positive patients. In addition, the patients without any evidence of respiratory viral infections did not have different symptoms. These patients may have been infected with other virus not included in the multiplex assay (human Bocavirus, coronavirus HKU1) [9, 10, 11] or were seen too late at the time of viral shedding was cleared [30] . However, to determine how specific the symptoms are for influenza would require to assess also the distribution of respiratory pathogens (H1N1v and other respiratory viruses) and related symptoms in patients presented at the emergency departments in SLS and TRS with respiratory syndromes, but not tested for H1N1v. In addition, despite some underlying conditions that were", "associated with complications not previously observed in seasonal influenza, most illnesses caused by the H1N1v virus were acute and self-limited [13, 31] . The higher proportion of non influenza viruses reported in ILI in 2009 was thus most likely a consequence of more frequent visits to a doctor for respiratory tract infections than usually observed for fear of the flu pandemic. The general lack of difference in symptoms in the particular context of H1N1v pandemic has therefore to be considered with caution and does not rule out that more significant differences may arise in future influenza epidemics with other influenza viruses. Our data confirm that it may be virtually impossible to recognize symptoms heralding H1N1v infections and virological data should be helpful along with clinical reports to monitor influenza epidemic [10] .", "Molecular multiplex detection has recently emerged as a potent diagnostic tool to determine acute respiratory infections' aetiologies [11, 32, 33] . These data show that sensitive molecular multiplex detection of respiratory viruses is feasible and efficient for the detection of virus involved in acute respiratory infections and provides insights into their epidemic profile. Our results confirm the performance of RespiFinder19H assay to detecting respiratory viruses in the general population as recently shown in transplant patients with ILI [34] . RespiFinder19H confirmed all H1N1 infections detected by the CDC reference assay and was able to identify two additional H1N1 cases suggesting a high sensitivity of this multiplex assay to detect influenza A infections.", "In conclusion, our results highlight that successive and mixed outbreaks of respiratory viral infections may affect influenza epidemiology and can lead to misinterpret the early development of a flu epidemic. Rapid diagnostic screening of a large panel of respiratory pathogens may be critical to define and survey the epidemic situation and to provide critical information for patient management." ]

[ 1 ]

[ "Réseau Sentinelles" ]

[ "High Burden of Non-Influenza Viruses in Influenza-Like Illness in the Early Weeks of H1N1v Epidemic in France\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3157400/\n\nSHA: f4c1afe385e9e31eb5678e15a3c280ba97326554\n\nAuthors: Schnepf, Nathalie; Resche-Rigon, Matthieu; Chaillon, Antoine; Scemla, Anne; Gras, Guillaume; Semoun, Oren; Taboulet, Pierre; Molina, Jean-Michel; Simon, François; Goudeau, Alain; LeGoff, Jérôme\nDate: 2011-08-17\nDOI: 10.1371/journal.pone.0023514\nLicense: cc-by", "Abstract: BACKGROUND: Influenza-like illness (ILI) may be caused by a variety of pathogens. Clinical observations are of little help to recognise myxovirus infection and implement appropriate prevention measures. The limited use of molecular tools underestimates the role of other common pathogens. OBJECTIVES: During the early weeks of the 2009–2010 flu pandemic, a clinical and virological survey was conducted in adult and paediatric patients with ILI referred to two French University hospitals in Paris and Tours. Aims were to investigate the different pathogens involved in ILI and describe the associated symptoms. METHODS: H1N1v pandemic influenza diagnosis was performed with real time RT-PCR assay. Other viral aetiologies were investigated by the molecular multiplex assay RespiFinder19®. Clinical data were collected prospectively by physicians using a standard questionnaire. RESULTS: From week 35 to 44, endonasal swabs were collected in 413 patients. Overall, 68 samples (16.5%) were", "positive for H1N1v. In 13 of them, other respiratory pathogens were also detected. Among H1N1v negative samples, 213 (61.9%) were positive for various respiratory agents, 190 in single infections and 23 in mixed infections. The most prevalent viruses in H1N1v negative single infections were rhinovirus (62.6%), followed by parainfluenza viruses (24.2%) and adenovirus (5.3%). 70.6% of H1N1v cases were identified in patients under 40 years and none after 65 years. There was no difference between clinical symptoms observed in patients infected with H1N1v or with other pathogens. CONCLUSION: Our results highlight the high frequency of non-influenza viruses involved in ILI during the pre-epidemic period of a flu alert and the lack of specific clinical signs associated with influenza infections. Rapid diagnostic screening of a large panel of respiratory pathogens may be critical to define and survey the epidemic situation and to provide critical information for patient management.", "Text: In order to monitor the spread of influenza and alert health handlers, several epidemiological tools have been developed. In France, a network of 1300 general practitioners, ''Réseau Sentinelles'', working throughout the country, provides real-time clinical data used to evaluate regional and national influenza spreading [1, 2] . The criteria used by this network to define clinical influenza-like illness (ILI) are the occurrence of a sudden fever above 39uC with myalgia and respiratory signs. In general no formal viral diagnosis is carried out. The Groupes Régionaux d'Observation de la Grippe (GROG) is a second French network that surveys the emergence and the spread of the influenza viruses [3, 4] . This network is based on clinical surveillance of acute respiratory infections and laboratory analysis of nasal specimens collected from adults and children by volunteer general practitioners and pediatricians.", "According to the sentinel network's criteria, French health authorities proclaimed that flu epidemic level was reached during the second week of September 2009 (week 37) [5, 6] . On the contrary, data provided by the GROG showed only sporadic H1N1v activity until the last week of October (week 44) [6, 7] . Thus, it became rapidly obvious that a variety of viruses were circulating in the community and that an overestimation of myxovirus infection was at stake [8, 9, 10, 11] .", "As a better knowledge of the epidemic status was a key feature for national healthcare organization, hospital preparedness, patient management and disease control, unambiguous viral diagnosis appeared critical. In France, data on viral aetiologies associated with ILI were at best sporadic and correlations with clinical symptoms were often lacking. Extensive molecular assays to screening for respiratory viruses were not available countrywide for routine diagnosis. Therefore the epidemiological pattern of respiratory pathogens with overlapping seasonality was poorly known.\n\nThe aim of the present study was to investigate respiratory pathogens involved in ILI during the early weeks of the 2009-2010 H1N1v diffusion in France (weeks 35 through 44) and describe the associated symptoms in paediatric and adult populations.", "This study was a non-interventional study with no addition to usual proceedures. Biological material and clinical data were obtained only for standard viral diagnostic following physicians' prescriptions (no specific sampling, no modification of the sampling protocol, no supplementary question in the national standardized questionnaire). Data analyses were carried out using an anonymized database. According to the French Health Public Law (CSP Art L 1121-1.1), such protocol does not require approval of an ethics committee and is exempted from informed consent application.", "In the two academic hospitals, Saint-Louis hospital (SLS) in Paris and Tours hospital (TRS), influenza-like illness (ILI) was defined as a patient suffering from at least one general symptom (fever above 38uC, asthenia, myalgia, shivers or headache) and one respiratory symptom (cough, dyspnoea, rhinitis or pharyngitis), in agreement with the guidelines from the French Institut de Veille Sanitaire (InVS), a governmental institution responsible for surveillance and alert in all domains of public health [12] . Criteria for severe clinical presentation were temperature below 35uC or above 39uC despite antipyretic, cardiac frequency above 120/min, respiratory frequency above 30/min, respiratory distress, systolic arterial pressure below 90 mmHg or altered consciousness. Predisposing factors of critical illness were children younger than one year old, pregnant women, diabetes, chronic pre-existing disease (such as respiratory, cardiovascular, neurologic, renal, hepatic or hematologic", "diseases) and immunosuppression (associated with HIV infection, organ or hematopoietic stem cells transplantation, receipt of chemotherapy or corticosteroids) [13, 14] . A cluster of suspected influenza infections was defined as at least three possible cases in a week in a closed community (household, school,…) [15] .", "In the two institutions, the prescription of H1N1v molecular testing was recommended for patients with ILI and with either a severe clinical presentation, an underlying risk factor of complications or a condition which was not improving under antiviral treatment. Investigation of grouped suspected cases was also recommended. From week 35 (last week of August) to 44 (last week of October), 413 endonasal swabs were collected in 3 ml of Universal Transport Medium (Copan Diagnostics Inc, Murrieta, CA) from adults and children seen in emergency rooms for suspected ILI (Table 1 ) and sent to SLS and TRS laboratories for H1N1v detection. The two microbiology laboratories participated in the reference laboratories network for the detection of pandemic influenza H1N1v.", "Clinical data were collected at the time of medical attention and reported by clinicians on a national standardized questionnaire provided by InVS [1, 12] . This questionnaire included the presence or absence of the main general and respiratory symptoms associated with ILI (fever, asthenia, myalgia, shivers, headache, cough, rhinitis, pharyngitis, sudden onset) [12] .\n\nTotal nucleic acid was extracted from 400 mL of Universal Transport Medium using the EasyMag System (Biomérieux, Marcy l'Etoile, France) in SLS or the EZ1 Advanced XL (Qiagen, Courtaboeuf, France) in TRS, according to the manufacturers' instructions (elution volume: 100 mL in SLS or 90 mL in TRS). Before extraction, 5 ml of an Internal Amplification Control (IAC) which contained an encephalomyocarditis virus (EMC) RNA transcript was added into the sample.", "Pandemic H1N1v infection was diagnosed by real-time reverse transcription-PCR (RT-PCR) assay on a 7500 Real Time PCR System (Applied Biosystems, Foster City, CA) according to the protocol of the Centers for Disease Control (CDC) [16] . Other respiratory infections were investigated by a multiplex molecular assay based on the Multiplex Ligation-dependent Probe-Amplification (MLPA) technology (RespiFinder19H, Pathofinder, Maastricht, The Netherlands) that allows the detection and differentiation of 14 respiratory viruses, including influenza virus A (InfA), influenza virus B (InfB), rhinovirus (RHV), parainfluenza viruses 1 to 4 (PIV-1 to PIV-4), human metapneumovirus (hMPV), adenovirus (ADV), respiratory syncytial virus A (RSVA), respiratory syncytial virus B (RSVB) and human coronaviruses 229E, OC43 and NL63 (Cor-229E, Cor-OC43, Cor-NL63) [17] . The test allows also the detection of H5N1 influenza A virus and of four bacteria: Chlamydophila pneumoniae (CP), Mycoplasma pneumoniae (MP),", "Legionella pneumophila (LP) and Bordetella pertussis (BP). The amplified MLPA products were analyzed on an ABI 3100 genetic analyzer (Applied Biosystems, Foster City, CA). Fragment sizing analysis was performed with the GeneMarker software (SoftGenetics, LLC, State College, PA).", "Further testing for H1N1v was carried out with Simplexa TM Influenza A H1N1 (2009) (Focus Diagnostics, Cypress, California) when the CDC real time RT-PCR assay was negative for H1N1 and the RespiFinder19H assay was positive for Influenza A. If this latter assay was negative, H3N2 typing was performed as previously described [18] .\n\nData from our study are summarized as frequencies and percentages for categorical variables. Quantitative variables are presented as medians, 25th and 75th percentiles. To compare those variables according to the viral infection status, Fisher tests", "By using CDC reference assay, H1N1v was detected in 66 samples out of 413 (16.6%), more frequently in SLS (38 samples) than in TRS (28 samples) (p,10 24 ). Overall, weekly percentage of H1N1v positive endonasal swabs remained under 10% until week 41 and increase significantly after (P Trend ,0.0001) ( Figure 1 ). Rate of H1N1v detection reached 30% in SLS at week 42 and in TRS at week 44. Overall, this rate was in agreement with results provided by the GROG network, showing an earlier start of H1N1v epidemic in Paris area [7, 19] .", "All 413 nucleic acid extracts were analyzed using the RespiFinder19H assay ( Figure 2 ). Sixty six patients tested H1N1v positive with CDC real time RT-PCR assay were confirmed with the multiplex assay. Thirteen were also co-infected by one or two other respiratory pathogens (multiple infections) ( Figure 2 ). Three of the 347 H1N1v negative samples could not be studied with the multiplex assay because they contained RT-PCR inhibitors (no amplification of the internal control). Two hundred and fifteen (62.5%) of the remaining 344 H1N1v negative samples were found positive for at least one respiratory pathogen ( Figure 2 ). Two hundred and twelve were positive for non influenza pathogens (189 single infections and 23 mixed infections with two, three or four viruses) and three additional single infections by influenza A were identified in SLS, including two by pandemic H1N1v and one by seasonal H3N2, as determined after molecular typing (data not shown).", "Overall, 68 patients (16.5%) were then positive for H1N1v, one for H3N2 and 212 for non influenza pathogens. There were 245 single infections (55 with H1N1v and 190 with other respiratory pathogens) and 36 mixed infections (13 with H1N1v and 23 without H1N1v) ( Figure 2 ).", "Among H1N1v negative single infections, the most prevalent viruses were rhinovirus (62.6%, 119 patients), followed by parainfluenza viruses 1 to 4 (24.2%, 46 patients), adenovirus (5.3%, 10 patients), human coronavirus 229E, OC43 and NL63 (3.2%, 6 patients) and respiratory syncytial virus A and B (2.6%, 5 patients) (Figure 2 ). In addition, RespiFinder19H assay identified three patients with bacterial infection, two with Mycoplasma pneumoniae (one 25 years old female in SLS and one 39 years old female in TRS) and one with Bordetella pertussis (one 60 years old male in SLS). No single infection by influenza B, hMPV, Chlamydophila pneumoniae or Legionella pneumophila was identified ( Figure 2 To analyze if viral co-infections occurred more frequently for some viruses, we carried out a two by two comparisons, that showed a higher proportion of co-infection only for ADV (p = 0.05).", "Non-influenza respiratory viruses presented a different epidemic profile compared to H1N1v. Overall, in both hospitals, weekly rate of non-H1N1v respiratory viruses whether alone or involved in co-infection increased between week 37 and 39 (from 51.4% to 81.3%) and then consistently decreased ( Figure 3 ). RHV infections that represented nearly half of non-H1N1v viral infections (141 out of 213, 66.2%) were a significant contributing factor. In both hospitals, emergence of H1N1v cases was associated with a rapid decline of RHV rate of infection from 50-60% down to less than 20% with a one to two weeks gap between SLS and TRS.", "Data on age ( In both institutions, 85.5% (106/124) children younger than 15 years of age were infected by at least one respiratory pathogen ( Table 2 ). H1N1v infected patients were not significantly younger than H1N1v non infected patients (27 years old vs. 25 years old, p = 0.80) (Figure 4) . However, 70.6% (48/68) of H1N1v cases were identified in patients under 40 years old (22 in SLS and 26 in TRS) and no case was observed in patients older than 65 years ( Table 2) . PIV infection occurred in very young patients (median (Figure 4) . Consequently, PIV and ADV were more frequently detected in the younger population of TRS versus SLS (p,10 24 and p,10 23 respectively). In contrast, although individuals with RHV infection were slightly younger than individuals without (median age = 24 vs. 29 for patients without RHV, p = 0.05) (Figure 4) , influenza-like illness associated with RHV was more frequent in SLS than in TRS (p = 0.012). Finally, patients with viral multiple infection were", "significantly younger than those with single infection (median, IDR: 4, 2-18.5 vs. 25, 6-43) and rates of mixed infection At the time of medical attention, 383 (92.7%) standardized clinical questionnaires were collected out of 413 patients. Four of them could not be exploited because they were too incomplete. A review of the 379 workable questionnaires showed that 90.8% (344/379) of the patients included in this study fulfilled the criteria of ILI as defined above, and 52.5% had either a severe clinical presentation or an underlying risk factor of complications (45.9%, 174/379), or were in a suspected cluster of grouped cases (6.6%, 25/379).", "Overall, most patients have fever (93.9%) and cough (86.1%) ( Table 3) . Other classical clinical signs associated with ILI such as asthenia, myalgia, shivers, headache, rhinitis or pharyngitis were less frequent. A sudden onset was also described in 59.2% of cases. Only 32.5% of the patients had a temperature above 39uC; the age of these patients ranged from zero to 86 years, with a median age of 32 years and a mean age of 34 years (data not shown).", "In H1N1v infected patients (including single and multiple infections), the main symptoms were also fever (98.2%) and cough (89.5%) ( We then compared clinical characteristics between patients positive for H1N1v, patients positive for other respiratory pathogens and negative for H1N1v and patients without any detection of respiratory pathogens (as detected with RespiFin-der19H) ( Table 3 ). There was no difference between the three groups except for fever, cough, pharyngitis. However for these latter symptoms, the comparison between patients positive for H1N1v and those positive for other respiratory pathogens or between patients positive for H1N1v and those without any detection of respiratory pathogens, showed no difference except for pharyngitis, which was less frequent in patients positive for H1N1v than in patients positive for other respiratory pathogens ( Table 3) .", "As RHV was the most frequent aetiology in ILI, we also compared clinical symptoms observed in patients with a single infection by RHV or by H1N1v (data not shown). There was no difference except that rhinitis and pharyngitis were significantly more frequent in RHV infection (62.7% vs. 34.1% [p = 0.006] and 39.0% vs. 10.0% [p = 0.001], respectively). Viral multiple infection (including samples with H1N1v) was not associated with a different clinical presentation. Fever and cough were observed in over 90% of the patients (90.6% and 90.3%, respectively), but only 33.3% of these patients had a temperature above 39uC, which was not different from patients with single viral infection (28.6%).", "Our results highlight the high frequency of non-influenza viruses involved in acute respiratory infections during the epidemic period of a flu alert as defined by the Réseau Sentinelles according to ILI definition (a sudden fever above 39uC accompanied by myalgia and respiratory signs). These data extent previous observations in Europe reporting high prevalence of RHV infections before seasonal influenza [4, 20] or in 2009, before H1N1v pandemic influenza [1, 8, 9, 11, 21] . We confirm that RHV represent the most frequent aetiology of acute respiratory Table 2 . Age of patients with respiratory samples positive for H1N1v, positive for other respiratory pathogens or negative. infections both in adult and paediatric populations and may represent more than 50% of cases. We show that other viral infections than influenza and RHV may represent up to 30% of aetiologies. We observed differences between the two hospitals, with a higher frequency of parainfluenza and ADV infections in Tours in", "contrast with a higher frequency of RHV in Paris, likely explained by the higher proportion of paediatric samples collected in Tours. However, despite the distance between the two institutions (about 250 km) and differences between the two populations, both presented similar patterns of high frequency of non-influenza viruses in acute respiratory infections before the flu epidemic wave and a decline when influenza reached epidemic levels.", "In the two cities, high frequencies of RHV were seen at the same level with a likely different evolution speed, with sudden increase and decrease in SLS and more progressive variation in TRS. In both institutions, there was a decrease in the proportion and number of RHV diagnoses roughly in parallel with the increase of influenza diagnoses. Indeed, H1N1v exceeds 20% of positive detection's rate only when RHV dropped under 40%. These data are thus consistent with negative interaction of the two epidemics at the population level. It was previously hypothesised that RHV epidemic could interfere with the spread of pandemic influenza [20, 21, 22] . Few in vitro data support this hypothesis. It has been reported that interferon and other cytokines production by RHV infected cells induced a refractory state to virus infection These data include the three patients whose respiratory samples could not be studied with the multiplex assay because of RT-PCR inhibitors. of neighbouring cells [23] .", "Further work is needed to confirm in vitro and in vivo such negative interactions and if viral interference are really translated to a population level. Analysis of rhinovirus and influenza epidemics in previous years should also help to determine if similar interferences were observed with seasonal influenza and to elaborate modelling and prediction of the spread of influenza according to respiratory viruses' circulation. Systematic extensive screening of respiratory viruses at a national level should be implemented for this purpose.", "Very few RSV infections were observed in contrast to usual epidemiology which was characterized the last four past years by a start of epidemics in weeks 44-45 [1] . It has been confirmed by other laboratories and the French InVS that the 2009-10 RSV epidemic was delayed and had a lower impact compared with the previous winter season [1, 24] . Delayed and reduced RSV spread may be due to viral interference between RSV and influenza. Another possible explanation is better prevention behaviour about respiratory infections as recommended by a national campaign including recommendations for hands washing after sneezing and the use of mask [1] .", "Influenza infections were mainly detected in patient under 40 years old and no case was found in patients older than 65. These results corroborate previous data suggesting that past seasonal H1N1 infections or vaccination may give partial crossed protection [10, 13, 25] . We have previously shown that the neutralizing titers against pandemic H1N1v virus correlate significantly with neutralizing titers against a seasonal H1N1 virus, and that the H1N1v pandemic influenza virus neutralizing titer was significantly higher in subjects who had recently been inoculated by a seasonal trivalent influenza vaccine [26] .\n\nViral co-infections were predominantly seen in paediatric patients, as previously described [4, 27, 28, 29] , both in influenza and non-influenza cases at a similar rate. No evidence of more pronounced respiratory impact was seen in these patients.", "Our results showed the lack of specific clinical signs associated with proven H1N1v infections. Clinical characteristics did not differ between influenza infections or other viral infections. In particular, the proportion of patients with fever above 39uC was not higher in H1N1v positive patients. In addition, the patients without any evidence of respiratory viral infections did not have different symptoms. These patients may have been infected with other virus not included in the multiplex assay (human Bocavirus, coronavirus HKU1) [9, 10, 11] or were seen too late at the time of viral shedding was cleared [30] . However, to determine how specific the symptoms are for influenza would require to assess also the distribution of respiratory pathogens (H1N1v and other respiratory viruses) and related symptoms in patients presented at the emergency departments in SLS and TRS with respiratory syndromes, but not tested for H1N1v. In addition, despite some underlying conditions that were", "associated with complications not previously observed in seasonal influenza, most illnesses caused by the H1N1v virus were acute and self-limited [13, 31] . The higher proportion of non influenza viruses reported in ILI in 2009 was thus most likely a consequence of more frequent visits to a doctor for respiratory tract infections than usually observed for fear of the flu pandemic. The general lack of difference in symptoms in the particular context of H1N1v pandemic has therefore to be considered with caution and does not rule out that more significant differences may arise in future influenza epidemics with other influenza viruses. Our data confirm that it may be virtually impossible to recognize symptoms heralding H1N1v infections and virological data should be helpful along with clinical reports to monitor influenza epidemic [10] .", "Molecular multiplex detection has recently emerged as a potent diagnostic tool to determine acute respiratory infections' aetiologies [11, 32, 33] . These data show that sensitive molecular multiplex detection of respiratory viruses is feasible and efficient for the detection of virus involved in acute respiratory infections and provides insights into their epidemic profile. Our results confirm the performance of RespiFinder19H assay to detecting respiratory viruses in the general population as recently shown in transplant patients with ILI [34] . RespiFinder19H confirmed all H1N1 infections detected by the CDC reference assay and was able to identify two additional H1N1 cases suggesting a high sensitivity of this multiplex assay to detect influenza A infections.", "In conclusion, our results highlight that successive and mixed outbreaks of respiratory viral infections may affect influenza epidemiology and can lead to misinterpret the early development of a flu epidemic. Rapid diagnostic screening of a large panel of respiratory pathogens may be critical to define and survey the epidemic situation and to provide critical information for patient management." ]

[ 3 ]

[ "a sudden fever above 39uC with myalgia and respiratory signs" ]

[ "High Burden of Non-Influenza Viruses in Influenza-Like Illness in the Early Weeks of H1N1v Epidemic in France\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3157400/\n\nSHA: f4c1afe385e9e31eb5678e15a3c280ba97326554\n\nAuthors: Schnepf, Nathalie; Resche-Rigon, Matthieu; Chaillon, Antoine; Scemla, Anne; Gras, Guillaume; Semoun, Oren; Taboulet, Pierre; Molina, Jean-Michel; Simon, François; Goudeau, Alain; LeGoff, Jérôme\nDate: 2011-08-17\nDOI: 10.1371/journal.pone.0023514\nLicense: cc-by", "Abstract: BACKGROUND: Influenza-like illness (ILI) may be caused by a variety of pathogens. Clinical observations are of little help to recognise myxovirus infection and implement appropriate prevention measures. The limited use of molecular tools underestimates the role of other common pathogens. OBJECTIVES: During the early weeks of the 2009–2010 flu pandemic, a clinical and virological survey was conducted in adult and paediatric patients with ILI referred to two French University hospitals in Paris and Tours. Aims were to investigate the different pathogens involved in ILI and describe the associated symptoms. METHODS: H1N1v pandemic influenza diagnosis was performed with real time RT-PCR assay. Other viral aetiologies were investigated by the molecular multiplex assay RespiFinder19®. Clinical data were collected prospectively by physicians using a standard questionnaire. RESULTS: From week 35 to 44, endonasal swabs were collected in 413 patients. Overall, 68 samples (16.5%) were", "positive for H1N1v. In 13 of them, other respiratory pathogens were also detected. Among H1N1v negative samples, 213 (61.9%) were positive for various respiratory agents, 190 in single infections and 23 in mixed infections. The most prevalent viruses in H1N1v negative single infections were rhinovirus (62.6%), followed by parainfluenza viruses (24.2%) and adenovirus (5.3%). 70.6% of H1N1v cases were identified in patients under 40 years and none after 65 years. There was no difference between clinical symptoms observed in patients infected with H1N1v or with other pathogens. CONCLUSION: Our results highlight the high frequency of non-influenza viruses involved in ILI during the pre-epidemic period of a flu alert and the lack of specific clinical signs associated with influenza infections. Rapid diagnostic screening of a large panel of respiratory pathogens may be critical to define and survey the epidemic situation and to provide critical information for patient management.", "Text: In order to monitor the spread of influenza and alert health handlers, several epidemiological tools have been developed. In France, a network of 1300 general practitioners, ''Réseau Sentinelles'', working throughout the country, provides real-time clinical data used to evaluate regional and national influenza spreading [1, 2] . The criteria used by this network to define clinical influenza-like illness (ILI) are the occurrence of a sudden fever above 39uC with myalgia and respiratory signs. In general no formal viral diagnosis is carried out. The Groupes Régionaux d'Observation de la Grippe (GROG) is a second French network that surveys the emergence and the spread of the influenza viruses [3, 4] . This network is based on clinical surveillance of acute respiratory infections and laboratory analysis of nasal specimens collected from adults and children by volunteer general practitioners and pediatricians.", "According to the sentinel network's criteria, French health authorities proclaimed that flu epidemic level was reached during the second week of September 2009 (week 37) [5, 6] . On the contrary, data provided by the GROG showed only sporadic H1N1v activity until the last week of October (week 44) [6, 7] . Thus, it became rapidly obvious that a variety of viruses were circulating in the community and that an overestimation of myxovirus infection was at stake [8, 9, 10, 11] .", "As a better knowledge of the epidemic status was a key feature for national healthcare organization, hospital preparedness, patient management and disease control, unambiguous viral diagnosis appeared critical. In France, data on viral aetiologies associated with ILI were at best sporadic and correlations with clinical symptoms were often lacking. Extensive molecular assays to screening for respiratory viruses were not available countrywide for routine diagnosis. Therefore the epidemiological pattern of respiratory pathogens with overlapping seasonality was poorly known.\n\nThe aim of the present study was to investigate respiratory pathogens involved in ILI during the early weeks of the 2009-2010 H1N1v diffusion in France (weeks 35 through 44) and describe the associated symptoms in paediatric and adult populations.", "This study was a non-interventional study with no addition to usual proceedures. Biological material and clinical data were obtained only for standard viral diagnostic following physicians' prescriptions (no specific sampling, no modification of the sampling protocol, no supplementary question in the national standardized questionnaire). Data analyses were carried out using an anonymized database. According to the French Health Public Law (CSP Art L 1121-1.1), such protocol does not require approval of an ethics committee and is exempted from informed consent application.", "In the two academic hospitals, Saint-Louis hospital (SLS) in Paris and Tours hospital (TRS), influenza-like illness (ILI) was defined as a patient suffering from at least one general symptom (fever above 38uC, asthenia, myalgia, shivers or headache) and one respiratory symptom (cough, dyspnoea, rhinitis or pharyngitis), in agreement with the guidelines from the French Institut de Veille Sanitaire (InVS), a governmental institution responsible for surveillance and alert in all domains of public health [12] . Criteria for severe clinical presentation were temperature below 35uC or above 39uC despite antipyretic, cardiac frequency above 120/min, respiratory frequency above 30/min, respiratory distress, systolic arterial pressure below 90 mmHg or altered consciousness. Predisposing factors of critical illness were children younger than one year old, pregnant women, diabetes, chronic pre-existing disease (such as respiratory, cardiovascular, neurologic, renal, hepatic or hematologic", "diseases) and immunosuppression (associated with HIV infection, organ or hematopoietic stem cells transplantation, receipt of chemotherapy or corticosteroids) [13, 14] . A cluster of suspected influenza infections was defined as at least three possible cases in a week in a closed community (household, school,…) [15] .", "In the two institutions, the prescription of H1N1v molecular testing was recommended for patients with ILI and with either a severe clinical presentation, an underlying risk factor of complications or a condition which was not improving under antiviral treatment. Investigation of grouped suspected cases was also recommended. From week 35 (last week of August) to 44 (last week of October), 413 endonasal swabs were collected in 3 ml of Universal Transport Medium (Copan Diagnostics Inc, Murrieta, CA) from adults and children seen in emergency rooms for suspected ILI (Table 1 ) and sent to SLS and TRS laboratories for H1N1v detection. The two microbiology laboratories participated in the reference laboratories network for the detection of pandemic influenza H1N1v.", "Clinical data were collected at the time of medical attention and reported by clinicians on a national standardized questionnaire provided by InVS [1, 12] . This questionnaire included the presence or absence of the main general and respiratory symptoms associated with ILI (fever, asthenia, myalgia, shivers, headache, cough, rhinitis, pharyngitis, sudden onset) [12] .\n\nTotal nucleic acid was extracted from 400 mL of Universal Transport Medium using the EasyMag System (Biomérieux, Marcy l'Etoile, France) in SLS or the EZ1 Advanced XL (Qiagen, Courtaboeuf, France) in TRS, according to the manufacturers' instructions (elution volume: 100 mL in SLS or 90 mL in TRS). Before extraction, 5 ml of an Internal Amplification Control (IAC) which contained an encephalomyocarditis virus (EMC) RNA transcript was added into the sample.", "Pandemic H1N1v infection was diagnosed by real-time reverse transcription-PCR (RT-PCR) assay on a 7500 Real Time PCR System (Applied Biosystems, Foster City, CA) according to the protocol of the Centers for Disease Control (CDC) [16] . Other respiratory infections were investigated by a multiplex molecular assay based on the Multiplex Ligation-dependent Probe-Amplification (MLPA) technology (RespiFinder19H, Pathofinder, Maastricht, The Netherlands) that allows the detection and differentiation of 14 respiratory viruses, including influenza virus A (InfA), influenza virus B (InfB), rhinovirus (RHV), parainfluenza viruses 1 to 4 (PIV-1 to PIV-4), human metapneumovirus (hMPV), adenovirus (ADV), respiratory syncytial virus A (RSVA), respiratory syncytial virus B (RSVB) and human coronaviruses 229E, OC43 and NL63 (Cor-229E, Cor-OC43, Cor-NL63) [17] . The test allows also the detection of H5N1 influenza A virus and of four bacteria: Chlamydophila pneumoniae (CP), Mycoplasma pneumoniae (MP),", "Legionella pneumophila (LP) and Bordetella pertussis (BP). The amplified MLPA products were analyzed on an ABI 3100 genetic analyzer (Applied Biosystems, Foster City, CA). Fragment sizing analysis was performed with the GeneMarker software (SoftGenetics, LLC, State College, PA).", "Further testing for H1N1v was carried out with Simplexa TM Influenza A H1N1 (2009) (Focus Diagnostics, Cypress, California) when the CDC real time RT-PCR assay was negative for H1N1 and the RespiFinder19H assay was positive for Influenza A. If this latter assay was negative, H3N2 typing was performed as previously described [18] .\n\nData from our study are summarized as frequencies and percentages for categorical variables. Quantitative variables are presented as medians, 25th and 75th percentiles. To compare those variables according to the viral infection status, Fisher tests", "By using CDC reference assay, H1N1v was detected in 66 samples out of 413 (16.6%), more frequently in SLS (38 samples) than in TRS (28 samples) (p,10 24 ). Overall, weekly percentage of H1N1v positive endonasal swabs remained under 10% until week 41 and increase significantly after (P Trend ,0.0001) ( Figure 1 ). Rate of H1N1v detection reached 30% in SLS at week 42 and in TRS at week 44. Overall, this rate was in agreement with results provided by the GROG network, showing an earlier start of H1N1v epidemic in Paris area [7, 19] .", "All 413 nucleic acid extracts were analyzed using the RespiFinder19H assay ( Figure 2 ). Sixty six patients tested H1N1v positive with CDC real time RT-PCR assay were confirmed with the multiplex assay. Thirteen were also co-infected by one or two other respiratory pathogens (multiple infections) ( Figure 2 ). Three of the 347 H1N1v negative samples could not be studied with the multiplex assay because they contained RT-PCR inhibitors (no amplification of the internal control). Two hundred and fifteen (62.5%) of the remaining 344 H1N1v negative samples were found positive for at least one respiratory pathogen ( Figure 2 ). Two hundred and twelve were positive for non influenza pathogens (189 single infections and 23 mixed infections with two, three or four viruses) and three additional single infections by influenza A were identified in SLS, including two by pandemic H1N1v and one by seasonal H3N2, as determined after molecular typing (data not shown).", "Overall, 68 patients (16.5%) were then positive for H1N1v, one for H3N2 and 212 for non influenza pathogens. There were 245 single infections (55 with H1N1v and 190 with other respiratory pathogens) and 36 mixed infections (13 with H1N1v and 23 without H1N1v) ( Figure 2 ).", "Among H1N1v negative single infections, the most prevalent viruses were rhinovirus (62.6%, 119 patients), followed by parainfluenza viruses 1 to 4 (24.2%, 46 patients), adenovirus (5.3%, 10 patients), human coronavirus 229E, OC43 and NL63 (3.2%, 6 patients) and respiratory syncytial virus A and B (2.6%, 5 patients) (Figure 2 ). In addition, RespiFinder19H assay identified three patients with bacterial infection, two with Mycoplasma pneumoniae (one 25 years old female in SLS and one 39 years old female in TRS) and one with Bordetella pertussis (one 60 years old male in SLS). No single infection by influenza B, hMPV, Chlamydophila pneumoniae or Legionella pneumophila was identified ( Figure 2 To analyze if viral co-infections occurred more frequently for some viruses, we carried out a two by two comparisons, that showed a higher proportion of co-infection only for ADV (p = 0.05).", "Non-influenza respiratory viruses presented a different epidemic profile compared to H1N1v. Overall, in both hospitals, weekly rate of non-H1N1v respiratory viruses whether alone or involved in co-infection increased between week 37 and 39 (from 51.4% to 81.3%) and then consistently decreased ( Figure 3 ). RHV infections that represented nearly half of non-H1N1v viral infections (141 out of 213, 66.2%) were a significant contributing factor. In both hospitals, emergence of H1N1v cases was associated with a rapid decline of RHV rate of infection from 50-60% down to less than 20% with a one to two weeks gap between SLS and TRS.", "Data on age ( In both institutions, 85.5% (106/124) children younger than 15 years of age were infected by at least one respiratory pathogen ( Table 2 ). H1N1v infected patients were not significantly younger than H1N1v non infected patients (27 years old vs. 25 years old, p = 0.80) (Figure 4) . However, 70.6% (48/68) of H1N1v cases were identified in patients under 40 years old (22 in SLS and 26 in TRS) and no case was observed in patients older than 65 years ( Table 2) . PIV infection occurred in very young patients (median (Figure 4) . Consequently, PIV and ADV were more frequently detected in the younger population of TRS versus SLS (p,10 24 and p,10 23 respectively). In contrast, although individuals with RHV infection were slightly younger than individuals without (median age = 24 vs. 29 for patients without RHV, p = 0.05) (Figure 4) , influenza-like illness associated with RHV was more frequent in SLS than in TRS (p = 0.012). Finally, patients with viral multiple infection were", "significantly younger than those with single infection (median, IDR: 4, 2-18.5 vs. 25, 6-43) and rates of mixed infection At the time of medical attention, 383 (92.7%) standardized clinical questionnaires were collected out of 413 patients. Four of them could not be exploited because they were too incomplete. A review of the 379 workable questionnaires showed that 90.8% (344/379) of the patients included in this study fulfilled the criteria of ILI as defined above, and 52.5% had either a severe clinical presentation or an underlying risk factor of complications (45.9%, 174/379), or were in a suspected cluster of grouped cases (6.6%, 25/379).", "Overall, most patients have fever (93.9%) and cough (86.1%) ( Table 3) . Other classical clinical signs associated with ILI such as asthenia, myalgia, shivers, headache, rhinitis or pharyngitis were less frequent. A sudden onset was also described in 59.2% of cases. Only 32.5% of the patients had a temperature above 39uC; the age of these patients ranged from zero to 86 years, with a median age of 32 years and a mean age of 34 years (data not shown).", "In H1N1v infected patients (including single and multiple infections), the main symptoms were also fever (98.2%) and cough (89.5%) ( We then compared clinical characteristics between patients positive for H1N1v, patients positive for other respiratory pathogens and negative for H1N1v and patients without any detection of respiratory pathogens (as detected with RespiFin-der19H) ( Table 3 ). There was no difference between the three groups except for fever, cough, pharyngitis. However for these latter symptoms, the comparison between patients positive for H1N1v and those positive for other respiratory pathogens or between patients positive for H1N1v and those without any detection of respiratory pathogens, showed no difference except for pharyngitis, which was less frequent in patients positive for H1N1v than in patients positive for other respiratory pathogens ( Table 3) .", "As RHV was the most frequent aetiology in ILI, we also compared clinical symptoms observed in patients with a single infection by RHV or by H1N1v (data not shown). There was no difference except that rhinitis and pharyngitis were significantly more frequent in RHV infection (62.7% vs. 34.1% [p = 0.006] and 39.0% vs. 10.0% [p = 0.001], respectively). Viral multiple infection (including samples with H1N1v) was not associated with a different clinical presentation. Fever and cough were observed in over 90% of the patients (90.6% and 90.3%, respectively), but only 33.3% of these patients had a temperature above 39uC, which was not different from patients with single viral infection (28.6%).", "Our results highlight the high frequency of non-influenza viruses involved in acute respiratory infections during the epidemic period of a flu alert as defined by the Réseau Sentinelles according to ILI definition (a sudden fever above 39uC accompanied by myalgia and respiratory signs). These data extent previous observations in Europe reporting high prevalence of RHV infections before seasonal influenza [4, 20] or in 2009, before H1N1v pandemic influenza [1, 8, 9, 11, 21] . We confirm that RHV represent the most frequent aetiology of acute respiratory Table 2 . Age of patients with respiratory samples positive for H1N1v, positive for other respiratory pathogens or negative. infections both in adult and paediatric populations and may represent more than 50% of cases. We show that other viral infections than influenza and RHV may represent up to 30% of aetiologies. We observed differences between the two hospitals, with a higher frequency of parainfluenza and ADV infections in Tours in", "contrast with a higher frequency of RHV in Paris, likely explained by the higher proportion of paediatric samples collected in Tours. However, despite the distance between the two institutions (about 250 km) and differences between the two populations, both presented similar patterns of high frequency of non-influenza viruses in acute respiratory infections before the flu epidemic wave and a decline when influenza reached epidemic levels.", "In the two cities, high frequencies of RHV were seen at the same level with a likely different evolution speed, with sudden increase and decrease in SLS and more progressive variation in TRS. In both institutions, there was a decrease in the proportion and number of RHV diagnoses roughly in parallel with the increase of influenza diagnoses. Indeed, H1N1v exceeds 20% of positive detection's rate only when RHV dropped under 40%. These data are thus consistent with negative interaction of the two epidemics at the population level. It was previously hypothesised that RHV epidemic could interfere with the spread of pandemic influenza [20, 21, 22] . Few in vitro data support this hypothesis. It has been reported that interferon and other cytokines production by RHV infected cells induced a refractory state to virus infection These data include the three patients whose respiratory samples could not be studied with the multiplex assay because of RT-PCR inhibitors. of neighbouring cells [23] .", "Further work is needed to confirm in vitro and in vivo such negative interactions and if viral interference are really translated to a population level. Analysis of rhinovirus and influenza epidemics in previous years should also help to determine if similar interferences were observed with seasonal influenza and to elaborate modelling and prediction of the spread of influenza according to respiratory viruses' circulation. Systematic extensive screening of respiratory viruses at a national level should be implemented for this purpose.", "Very few RSV infections were observed in contrast to usual epidemiology which was characterized the last four past years by a start of epidemics in weeks 44-45 [1] . It has been confirmed by other laboratories and the French InVS that the 2009-10 RSV epidemic was delayed and had a lower impact compared with the previous winter season [1, 24] . Delayed and reduced RSV spread may be due to viral interference between RSV and influenza. Another possible explanation is better prevention behaviour about respiratory infections as recommended by a national campaign including recommendations for hands washing after sneezing and the use of mask [1] .", "Influenza infections were mainly detected in patient under 40 years old and no case was found in patients older than 65. These results corroborate previous data suggesting that past seasonal H1N1 infections or vaccination may give partial crossed protection [10, 13, 25] . We have previously shown that the neutralizing titers against pandemic H1N1v virus correlate significantly with neutralizing titers against a seasonal H1N1 virus, and that the H1N1v pandemic influenza virus neutralizing titer was significantly higher in subjects who had recently been inoculated by a seasonal trivalent influenza vaccine [26] .\n\nViral co-infections were predominantly seen in paediatric patients, as previously described [4, 27, 28, 29] , both in influenza and non-influenza cases at a similar rate. No evidence of more pronounced respiratory impact was seen in these patients.", "Our results showed the lack of specific clinical signs associated with proven H1N1v infections. Clinical characteristics did not differ between influenza infections or other viral infections. In particular, the proportion of patients with fever above 39uC was not higher in H1N1v positive patients. In addition, the patients without any evidence of respiratory viral infections did not have different symptoms. These patients may have been infected with other virus not included in the multiplex assay (human Bocavirus, coronavirus HKU1) [9, 10, 11] or were seen too late at the time of viral shedding was cleared [30] . However, to determine how specific the symptoms are for influenza would require to assess also the distribution of respiratory pathogens (H1N1v and other respiratory viruses) and related symptoms in patients presented at the emergency departments in SLS and TRS with respiratory syndromes, but not tested for H1N1v. In addition, despite some underlying conditions that were", "associated with complications not previously observed in seasonal influenza, most illnesses caused by the H1N1v virus were acute and self-limited [13, 31] . The higher proportion of non influenza viruses reported in ILI in 2009 was thus most likely a consequence of more frequent visits to a doctor for respiratory tract infections than usually observed for fear of the flu pandemic. The general lack of difference in symptoms in the particular context of H1N1v pandemic has therefore to be considered with caution and does not rule out that more significant differences may arise in future influenza epidemics with other influenza viruses. Our data confirm that it may be virtually impossible to recognize symptoms heralding H1N1v infections and virological data should be helpful along with clinical reports to monitor influenza epidemic [10] .", "Molecular multiplex detection has recently emerged as a potent diagnostic tool to determine acute respiratory infections' aetiologies [11, 32, 33] . These data show that sensitive molecular multiplex detection of respiratory viruses is feasible and efficient for the detection of virus involved in acute respiratory infections and provides insights into their epidemic profile. Our results confirm the performance of RespiFinder19H assay to detecting respiratory viruses in the general population as recently shown in transplant patients with ILI [34] . RespiFinder19H confirmed all H1N1 infections detected by the CDC reference assay and was able to identify two additional H1N1 cases suggesting a high sensitivity of this multiplex assay to detect influenza A infections.", "In conclusion, our results highlight that successive and mixed outbreaks of respiratory viral infections may affect influenza epidemiology and can lead to misinterpret the early development of a flu epidemic. Rapid diagnostic screening of a large panel of respiratory pathogens may be critical to define and survey the epidemic situation and to provide critical information for patient management." ]

[ 3 ]

[ "rhinovirus" ]

[ "High Burden of Non-Influenza Viruses in Influenza-Like Illness in the Early Weeks of H1N1v Epidemic in France\n\nhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3157400/\n\nSHA: f4c1afe385e9e31eb5678e15a3c280ba97326554\n\nAuthors: Schnepf, Nathalie; Resche-Rigon, Matthieu; Chaillon, Antoine; Scemla, Anne; Gras, Guillaume; Semoun, Oren; Taboulet, Pierre; Molina, Jean-Michel; Simon, François; Goudeau, Alain; LeGoff, Jérôme\nDate: 2011-08-17\nDOI: 10.1371/journal.pone.0023514\nLicense: cc-by", "Abstract: BACKGROUND: Influenza-like illness (ILI) may be caused by a variety of pathogens. Clinical observations are of little help to recognise myxovirus infection and implement appropriate prevention measures. The limited use of molecular tools underestimates the role of other common pathogens. OBJECTIVES: During the early weeks of the 2009–2010 flu pandemic, a clinical and virological survey was conducted in adult and paediatric patients with ILI referred to two French University hospitals in Paris and Tours. Aims were to investigate the different pathogens involved in ILI and describe the associated symptoms. METHODS: H1N1v pandemic influenza diagnosis was performed with real time RT-PCR assay. Other viral aetiologies were investigated by the molecular multiplex assay RespiFinder19®. Clinical data were collected prospectively by physicians using a standard questionnaire. RESULTS: From week 35 to 44, endonasal swabs were collected in 413 patients. Overall, 68 samples (16.5%) were", "positive for H1N1v. In 13 of them, other respiratory pathogens were also detected. Among H1N1v negative samples, 213 (61.9%) were positive for various respiratory agents, 190 in single infections and 23 in mixed infections. The most prevalent viruses in H1N1v negative single infections were rhinovirus (62.6%), followed by parainfluenza viruses (24.2%) and adenovirus (5.3%). 70.6% of H1N1v cases were identified in patients under 40 years and none after 65 years. There was no difference between clinical symptoms observed in patients infected with H1N1v or with other pathogens. CONCLUSION: Our results highlight the high frequency of non-influenza viruses involved in ILI during the pre-epidemic period of a flu alert and the lack of specific clinical signs associated with influenza infections. Rapid diagnostic screening of a large panel of respiratory pathogens may be critical to define and survey the epidemic situation and to provide critical information for patient management.", "Text: In order to monitor the spread of influenza and alert health handlers, several epidemiological tools have been developed. In France, a network of 1300 general practitioners, ''Réseau Sentinelles'', working throughout the country, provides real-time clinical data used to evaluate regional and national influenza spreading [1, 2] . The criteria used by this network to define clinical influenza-like illness (ILI) are the occurrence of a sudden fever above 39uC with myalgia and respiratory signs. In general no formal viral diagnosis is carried out. The Groupes Régionaux d'Observation de la Grippe (GROG) is a second French network that surveys the emergence and the spread of the influenza viruses [3, 4] . This network is based on clinical surveillance of acute respiratory infections and laboratory analysis of nasal specimens collected from adults and children by volunteer general practitioners and pediatricians.", "According to the sentinel network's criteria, French health authorities proclaimed that flu epidemic level was reached during the second week of September 2009 (week 37) [5, 6] . On the contrary, data provided by the GROG showed only sporadic H1N1v activity until the last week of October (week 44) [6, 7] . Thus, it became rapidly obvious that a variety of viruses were circulating in the community and that an overestimation of myxovirus infection was at stake [8, 9, 10, 11] .", "As a better knowledge of the epidemic status was a key feature for national healthcare organization, hospital preparedness, patient management and disease control, unambiguous viral diagnosis appeared critical. In France, data on viral aetiologies associated with ILI were at best sporadic and correlations with clinical symptoms were often lacking. Extensive molecular assays to screening for respiratory viruses were not available countrywide for routine diagnosis. Therefore the epidemiological pattern of respiratory pathogens with overlapping seasonality was poorly known.\n\nThe aim of the present study was to investigate respiratory pathogens involved in ILI during the early weeks of the 2009-2010 H1N1v diffusion in France (weeks 35 through 44) and describe the associated symptoms in paediatric and adult populations.", "This study was a non-interventional study with no addition to usual proceedures. Biological material and clinical data were obtained only for standard viral diagnostic following physicians' prescriptions (no specific sampling, no modification of the sampling protocol, no supplementary question in the national standardized questionnaire). Data analyses were carried out using an anonymized database. According to the French Health Public Law (CSP Art L 1121-1.1), such protocol does not require approval of an ethics committee and is exempted from informed consent application.", "In the two academic hospitals, Saint-Louis hospital (SLS) in Paris and Tours hospital (TRS), influenza-like illness (ILI) was defined as a patient suffering from at least one general symptom (fever above 38uC, asthenia, myalgia, shivers or headache) and one respiratory symptom (cough, dyspnoea, rhinitis or pharyngitis), in agreement with the guidelines from the French Institut de Veille Sanitaire (InVS), a governmental institution responsible for surveillance and alert in all domains of public health [12] . Criteria for severe clinical presentation were temperature below 35uC or above 39uC despite antipyretic, cardiac frequency above 120/min, respiratory frequency above 30/min, respiratory distress, systolic arterial pressure below 90 mmHg or altered consciousness. Predisposing factors of critical illness were children younger than one year old, pregnant women, diabetes, chronic pre-existing disease (such as respiratory, cardiovascular, neurologic, renal, hepatic or hematologic", "diseases) and immunosuppression (associated with HIV infection, organ or hematopoietic stem cells transplantation, receipt of chemotherapy or corticosteroids) [13, 14] . A cluster of suspected influenza infections was defined as at least three possible cases in a week in a closed community (household, school,…) [15] .", "In the two institutions, the prescription of H1N1v molecular testing was recommended for patients with ILI and with either a severe clinical presentation, an underlying risk factor of complications or a condition which was not improving under antiviral treatment. Investigation of grouped suspected cases was also recommended. From week 35 (last week of August) to 44 (last week of October), 413 endonasal swabs were collected in 3 ml of Universal Transport Medium (Copan Diagnostics Inc, Murrieta, CA) from adults and children seen in emergency rooms for suspected ILI (Table 1 ) and sent to SLS and TRS laboratories for H1N1v detection. The two microbiology laboratories participated in the reference laboratories network for the detection of pandemic influenza H1N1v.", "Clinical data were collected at the time of medical attention and reported by clinicians on a national standardized questionnaire provided by InVS [1, 12] . This questionnaire included the presence or absence of the main general and respiratory symptoms associated with ILI (fever, asthenia, myalgia, shivers, headache, cough, rhinitis, pharyngitis, sudden onset) [12] .\n\nTotal nucleic acid was extracted from 400 mL of Universal Transport Medium using the EasyMag System (Biomérieux, Marcy l'Etoile, France) in SLS or the EZ1 Advanced XL (Qiagen, Courtaboeuf, France) in TRS, according to the manufacturers' instructions (elution volume: 100 mL in SLS or 90 mL in TRS). Before extraction, 5 ml of an Internal Amplification Control (IAC) which contained an encephalomyocarditis virus (EMC) RNA transcript was added into the sample.", "Pandemic H1N1v infection was diagnosed by real-time reverse transcription-PCR (RT-PCR) assay on a 7500 Real Time PCR System (Applied Biosystems, Foster City, CA) according to the protocol of the Centers for Disease Control (CDC) [16] . Other respiratory infections were investigated by a multiplex molecular assay based on the Multiplex Ligation-dependent Probe-Amplification (MLPA) technology (RespiFinder19H, Pathofinder, Maastricht, The Netherlands) that allows the detection and differentiation of 14 respiratory viruses, including influenza virus A (InfA), influenza virus B (InfB), rhinovirus (RHV), parainfluenza viruses 1 to 4 (PIV-1 to PIV-4), human metapneumovirus (hMPV), adenovirus (ADV), respiratory syncytial virus A (RSVA), respiratory syncytial virus B (RSVB) and human coronaviruses 229E, OC43 and NL63 (Cor-229E, Cor-OC43, Cor-NL63) [17] . The test allows also the detection of H5N1 influenza A virus and of four bacteria: Chlamydophila pneumoniae (CP), Mycoplasma pneumoniae (MP),", "Legionella pneumophila (LP) and Bordetella pertussis (BP). The amplified MLPA products were analyzed on an ABI 3100 genetic analyzer (Applied Biosystems, Foster City, CA). Fragment sizing analysis was performed with the GeneMarker software (SoftGenetics, LLC, State College, PA).", "Further testing for H1N1v was carried out with Simplexa TM Influenza A H1N1 (2009) (Focus Diagnostics, Cypress, California) when the CDC real time RT-PCR assay was negative for H1N1 and the RespiFinder19H assay was positive for Influenza A. If this latter assay was negative, H3N2 typing was performed as previously described [18] .\n\nData from our study are summarized as frequencies and percentages for categorical variables. Quantitative variables are presented as medians, 25th and 75th percentiles. To compare those variables according to the viral infection status, Fisher tests", "By using CDC reference assay, H1N1v was detected in 66 samples out of 413 (16.6%), more frequently in SLS (38 samples) than in TRS (28 samples) (p,10 24 ). Overall, weekly percentage of H1N1v positive endonasal swabs remained under 10% until week 41 and increase significantly after (P Trend ,0.0001) ( Figure 1 ). Rate of H1N1v detection reached 30% in SLS at week 42 and in TRS at week 44. Overall, this rate was in agreement with results provided by the GROG network, showing an earlier start of H1N1v epidemic in Paris area [7, 19] .", "All 413 nucleic acid extracts were analyzed using the RespiFinder19H assay ( Figure 2 ). Sixty six patients tested H1N1v positive with CDC real time RT-PCR assay were confirmed with the multiplex assay. Thirteen were also co-infected by one or two other respiratory pathogens (multiple infections) ( Figure 2 ). Three of the 347 H1N1v negative samples could not be studied with the multiplex assay because they contained RT-PCR inhibitors (no amplification of the internal control). Two hundred and fifteen (62.5%) of the remaining 344 H1N1v negative samples were found positive for at least one respiratory pathogen ( Figure 2 ). Two hundred and twelve were positive for non influenza pathogens (189 single infections and 23 mixed infections with two, three or four viruses) and three additional single infections by influenza A were identified in SLS, including two by pandemic H1N1v and one by seasonal H3N2, as determined after molecular typing (data not shown).", "Overall, 68 patients (16.5%) were then positive for H1N1v, one for H3N2 and 212 for non influenza pathogens. There were 245 single infections (55 with H1N1v and 190 with other respiratory pathogens) and 36 mixed infections (13 with H1N1v and 23 without H1N1v) ( Figure 2 ).", "Among H1N1v negative single infections, the most prevalent viruses were rhinovirus (62.6%, 119 patients), followed by parainfluenza viruses 1 to 4 (24.2%, 46 patients), adenovirus (5.3%, 10 patients), human coronavirus 229E, OC43 and NL63 (3.2%, 6 patients) and respiratory syncytial virus A and B (2.6%, 5 patients) (Figure 2 ). In addition, RespiFinder19H assay identified three patients with bacterial infection, two with Mycoplasma pneumoniae (one 25 years old female in SLS and one 39 years old female in TRS) and one with Bordetella pertussis (one 60 years old male in SLS). No single infection by influenza B, hMPV, Chlamydophila pneumoniae or Legionella pneumophila was identified ( Figure 2 To analyze if viral co-infections occurred more frequently for some viruses, we carried out a two by two comparisons, that showed a higher proportion of co-infection only for ADV (p = 0.05).", "Non-influenza respiratory viruses presented a different epidemic profile compared to H1N1v. Overall, in both hospitals, weekly rate of non-H1N1v respiratory viruses whether alone or involved in co-infection increased between week 37 and 39 (from 51.4% to 81.3%) and then consistently decreased ( Figure 3 ). RHV infections that represented nearly half of non-H1N1v viral infections (141 out of 213, 66.2%) were a significant contributing factor. In both hospitals, emergence of H1N1v cases was associated with a rapid decline of RHV rate of infection from 50-60% down to less than 20% with a one to two weeks gap between SLS and TRS.", "Data on age ( In both institutions, 85.5% (106/124) children younger than 15 years of age were infected by at least one respiratory pathogen ( Table 2 ). H1N1v infected patients were not significantly younger than H1N1v non infected patients (27 years old vs. 25 years old, p = 0.80) (Figure 4) . However, 70.6% (48/68) of H1N1v cases were identified in patients under 40 years old (22 in SLS and 26 in TRS) and no case was observed in patients older than 65 years ( Table 2) . PIV infection occurred in very young patients (median (Figure 4) . Consequently, PIV and ADV were more frequently detected in the younger population of TRS versus SLS (p,10 24 and p,10 23 respectively). In contrast, although individuals with RHV infection were slightly younger than individuals without (median age = 24 vs. 29 for patients without RHV, p = 0.05) (Figure 4) , influenza-like illness associated with RHV was more frequent in SLS than in TRS (p = 0.012). Finally, patients with viral multiple infection were", "significantly younger than those with single infection (median, IDR: 4, 2-18.5 vs. 25, 6-43) and rates of mixed infection At the time of medical attention, 383 (92.7%) standardized clinical questionnaires were collected out of 413 patients. Four of them could not be exploited because they were too incomplete. A review of the 379 workable questionnaires showed that 90.8% (344/379) of the patients included in this study fulfilled the criteria of ILI as defined above, and 52.5% had either a severe clinical presentation or an underlying risk factor of complications (45.9%, 174/379), or were in a suspected cluster of grouped cases (6.6%, 25/379).", "Overall, most patients have fever (93.9%) and cough (86.1%) ( Table 3) . Other classical clinical signs associated with ILI such as asthenia, myalgia, shivers, headache, rhinitis or pharyngitis were less frequent. A sudden onset was also described in 59.2% of cases. Only 32.5% of the patients had a temperature above 39uC; the age of these patients ranged from zero to 86 years, with a median age of 32 years and a mean age of 34 years (data not shown).", "In H1N1v infected patients (including single and multiple infections), the main symptoms were also fever (98.2%) and cough (89.5%) ( We then compared clinical characteristics between patients positive for H1N1v, patients positive for other respiratory pathogens and negative for H1N1v and patients without any detection of respiratory pathogens (as detected with RespiFin-der19H) ( Table 3 ). There was no difference between the three groups except for fever, cough, pharyngitis. However for these latter symptoms, the comparison between patients positive for H1N1v and those positive for other respiratory pathogens or between patients positive for H1N1v and those without any detection of respiratory pathogens, showed no difference except for pharyngitis, which was less frequent in patients positive for H1N1v than in patients positive for other respiratory pathogens ( Table 3) .", "As RHV was the most frequent aetiology in ILI, we also compared clinical symptoms observed in patients with a single infection by RHV or by H1N1v (data not shown). There was no difference except that rhinitis and pharyngitis were significantly more frequent in RHV infection (62.7% vs. 34.1% [p = 0.006] and 39.0% vs. 10.0% [p = 0.001], respectively). Viral multiple infection (including samples with H1N1v) was not associated with a different clinical presentation. Fever and cough were observed in over 90% of the patients (90.6% and 90.3%, respectively), but only 33.3% of these patients had a temperature above 39uC, which was not different from patients with single viral infection (28.6%).", "Our results highlight the high frequency of non-influenza viruses involved in acute respiratory infections during the epidemic period of a flu alert as defined by the Réseau Sentinelles according to ILI definition (a sudden fever above 39uC accompanied by myalgia and respiratory signs). These data extent previous observations in Europe reporting high prevalence of RHV infections before seasonal influenza [4, 20] or in 2009, before H1N1v pandemic influenza [1, 8, 9, 11, 21] . We confirm that RHV represent the most frequent aetiology of acute respiratory Table 2 . Age of patients with respiratory samples positive for H1N1v, positive for other respiratory pathogens or negative. infections both in adult and paediatric populations and may represent more than 50% of cases. We show that other viral infections than influenza and RHV may represent up to 30% of aetiologies. We observed differences between the two hospitals, with a higher frequency of parainfluenza and ADV infections in Tours in", "contrast with a higher frequency of RHV in Paris, likely explained by the higher proportion of paediatric samples collected in Tours. However, despite the distance between the two institutions (about 250 km) and differences between the two populations, both presented similar patterns of high frequency of non-influenza viruses in acute respiratory infections before the flu epidemic wave and a decline when influenza reached epidemic levels.", "In the two cities, high frequencies of RHV were seen at the same level with a likely different evolution speed, with sudden increase and decrease in SLS and more progressive variation in TRS. In both institutions, there was a decrease in the proportion and number of RHV diagnoses roughly in parallel with the increase of influenza diagnoses. Indeed, H1N1v exceeds 20% of positive detection's rate only when RHV dropped under 40%. These data are thus consistent with negative interaction of the two epidemics at the population level. It was previously hypothesised that RHV epidemic could interfere with the spread of pandemic influenza [20, 21, 22] . Few in vitro data support this hypothesis. It has been reported that interferon and other cytokines production by RHV infected cells induced a refractory state to virus infection These data include the three patients whose respiratory samples could not be studied with the multiplex assay because of RT-PCR inhibitors. of neighbouring cells [23] .", "Further work is needed to confirm in vitro and in vivo such negative interactions and if viral interference are really translated to a population level. Analysis of rhinovirus and influenza epidemics in previous years should also help to determine if similar interferences were observed with seasonal influenza and to elaborate modelling and prediction of the spread of influenza according to respiratory viruses' circulation. Systematic extensive screening of respiratory viruses at a national level should be implemented for this purpose.", "Very few RSV infections were observed in contrast to usual epidemiology which was characterized the last four past years by a start of epidemics in weeks 44-45 [1] . It has been confirmed by other laboratories and the French InVS that the 2009-10 RSV epidemic was delayed and had a lower impact compared with the previous winter season [1, 24] . Delayed and reduced RSV spread may be due to viral interference between RSV and influenza. Another possible explanation is better prevention behaviour about respiratory infections as recommended by a national campaign including recommendations for hands washing after sneezing and the use of mask [1] .", "Influenza infections were mainly detected in patient under 40 years old and no case was found in patients older than 65. These results corroborate previous data suggesting that past seasonal H1N1 infections or vaccination may give partial crossed protection [10, 13, 25] . We have previously shown that the neutralizing titers against pandemic H1N1v virus correlate significantly with neutralizing titers against a seasonal H1N1 virus, and that the H1N1v pandemic influenza virus neutralizing titer was significantly higher in subjects who had recently been inoculated by a seasonal trivalent influenza vaccine [26] .\n\nViral co-infections were predominantly seen in paediatric patients, as previously described [4, 27, 28, 29] , both in influenza and non-influenza cases at a similar rate. No evidence of more pronounced respiratory impact was seen in these patients.", "Our results showed the lack of specific clinical signs associated with proven H1N1v infections. Clinical characteristics did not differ between influenza infections or other viral infections. In particular, the proportion of patients with fever above 39uC was not higher in H1N1v positive patients. In addition, the patients without any evidence of respiratory viral infections did not have different symptoms. These patients may have been infected with other virus not included in the multiplex assay (human Bocavirus, coronavirus HKU1) [9, 10, 11] or were seen too late at the time of viral shedding was cleared [30] . However, to determine how specific the symptoms are for influenza would require to assess also the distribution of respiratory pathogens (H1N1v and other respiratory viruses) and related symptoms in patients presented at the emergency departments in SLS and TRS with respiratory syndromes, but not tested for H1N1v. In addition, despite some underlying conditions that were", "associated with complications not previously observed in seasonal influenza, most illnesses caused by the H1N1v virus were acute and self-limited [13, 31] . The higher proportion of non influenza viruses reported in ILI in 2009 was thus most likely a consequence of more frequent visits to a doctor for respiratory tract infections than usually observed for fear of the flu pandemic. The general lack of difference in symptoms in the particular context of H1N1v pandemic has therefore to be considered with caution and does not rule out that more significant differences may arise in future influenza epidemics with other influenza viruses. Our data confirm that it may be virtually impossible to recognize symptoms heralding H1N1v infections and virological data should be helpful along with clinical reports to monitor influenza epidemic [10] .", "Molecular multiplex detection has recently emerged as a potent diagnostic tool to determine acute respiratory infections' aetiologies [11, 32, 33] . These data show that sensitive molecular multiplex detection of respiratory viruses is feasible and efficient for the detection of virus involved in acute respiratory infections and provides insights into their epidemic profile. Our results confirm the performance of RespiFinder19H assay to detecting respiratory viruses in the general population as recently shown in transplant patients with ILI [34] . RespiFinder19H confirmed all H1N1 infections detected by the CDC reference assay and was able to identify two additional H1N1 cases suggesting a high sensitivity of this multiplex assay to detect influenza A infections.", "In conclusion, our results highlight that successive and mixed outbreaks of respiratory viral infections may affect influenza epidemiology and can lead to misinterpret the early development of a flu epidemic. Rapid diagnostic screening of a large panel of respiratory pathogens may be critical to define and survey the epidemic situation and to provide critical information for patient management." ]

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