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Background Since the highly pathogenic H5N1 influenza caused thousands of deaths of wild bird in this area in 2005, Qinghai Lake in China has become a hot spot for study of the influence of avian influenza to migratory wild birds. However, the ecology and evolution of low pathogenic avian influenza virus in this region are limited. This project-based avian influenza surveillance in Qinghai lake region was initiated in year 2012. Method Samples of wild bird feces and lake surface water were collected in Qinghai Lake in year 2012.Virus isolation was conducted on embryonated chicken eggs. The influenza A virus was determined by rRT-PCR. Virus sequences were acquired by deep sequencing. The phylogenetic correlation and molecular characteristics of the viruses were analyzed. The virus growth and infection features, receptor binding preference were studied, and pathogenicity in vitro as well as. Results Two H13N8 subtype influenza viruses were isolated. The viruses are phylogenetically belong to Eurasian lineage. Most of the genes are associated with gull origin influenza virus except PB1 gene, which is most probably derived from Anseriformes virus. The evidence of interspecies reassortment was presented. The two viruses have limited growth capacity on MDCK and A549 cells while grow well in embryonated eggs. The dual receptor binding features of the two viruses was shown up. The low pathogenic features were determined by trypsin dependence plaque formation assay. Conclusions The two H13N8 subtype influenza viruses are highly associated with gull origin. The interspecies reassortment of H13 subtype virus among Anseriforme sand Charadriiformes wild birds emphasizes the importance of strengthening avian influenza surveillance in this region. This study is helpful to understand the ecology, evolution and transmission pattern of H13 subtype influenza virus globally. Electronic supplementary material The online version of this article (10.1186/s12985-017-0842-1) contains supplementary material, which is available to authorized users.

Background Since 2008, avian influenza surveillance in poultry-related environments has been conducted annually in China. Samples have been collected from environments including live poultry markets, wild bird habitats, slaughterhouses, and poultry farms. Multiple subtypes of avian influenza virus have been identified based on environmental surveillance, and an H1N8 virus was isolated from the drinking water of a live poultry market. Methods Virus isolation was performed by inoculating influenza A-positive specimens into embryonated chicken eggs. Next-generation sequencing was used for whole-genome sequencing. A solid-phase binding assay was performed to test the virus receptor binding specificity. Trypsin dependence plaque formation assays and intravenous pathogenicity index tests were used to evaluate virus pathogenicity in vitro and in vivo, respectively. Different cell lines were chosen for comparison of virus replication capacity. Results According to the phylogenetic trees, the whole gene segments of the virus named A/Environment/Fujian/85144/2014(H1N8) were of Eurasian lineage. The HA, NA, PB1, and M genes showed the highest homology with those of H1N8 or H1N2 subtype viruses isolated from local domestic ducks, while the PB2, PA, NP and NS genes showed high similarity with the genes of H7N9 viruses detected in 2017 and 2018 in the same province. This virus presented an avian receptor binding preference. The plaque formation assay showed that it was a trypsin-dependent virus. The intravenous pathogenicity index (IVPI) in chickens was 0.02. The growth kinetics of the A/Environment/Fujian/85144/2014(H1N8) virus in different cell lines were similar to those of a human-origin virus, A/Brisbane/59/2007(H1N1), but lower than those of the control avian-origin and swine-origin viruses. Conclusions The H1N8 virus was identified in avian influenza-related environments in China for the first time and may have served as a gene carrier involved in the evolution of the H7N9 virus in poultry. This work further emphasizes the importance of avian influenza virus surveillance, especially in live poultry markets (LPMs). Active surveillance of avian influenza in LPMs is a major pillar supporting avian influenza control and response. Electronic supplementary material The online version of this article (10.1186/s12879-019-4079-z) contains supplementary material, which is available to authorized users.

H6 avian influenza viruses (AIVs), which are prevalent in domestic and wild birds in Eurasian countries, have been isolated from pigs, a dog and a human. Routine virological surveillance at live poultry markets or poultry farms was conducted in southern China from 2009 to 2011. This study investigated the genetic and antigenic characteristics, analyzed the receptor-binding properties and evaluated the kinetics of infectivity of the AIVs in A549, MDCK and PK15 cells. A total of 14 H6N6 and 2 H6N2 isolates were obtained from four provinces in southern China. Genetic analysis indicated two distinct hemagglutinin lineages of the H6 strains cocirculating in southern China, and these strains facilitated active evolution and reassortment among multiple influenza virus subtypes from different avian species in nature. None of these isolates grouped with the novel Taiwan H6N1 virus responsible for human infection. Receptor-binding specificity assays showed that five H6 AIVs may have acquired the ability to recognize human receptors. Growth kinetics experiments showed that EV/HB-JZ/02/10(H6N2) and EV/JX/15/10(H6N6) initially reproduced faster and achieved higher titers than other viruses, suggesting that enhanced binding to α-2,6-linked sialic acids correlated with increased viral replication in mammalian cells. Overall, the results emphasize the need for continued surveillance of H6 outbreaks and extensive characterization of H6 isolates to better understand genetic changes and their implications.

Clade 2.3.4.4 highly pathogenic avian influenza viruses (H5Nx) have spread from Asia to other parts of the world. Since 2014, human infections with clade 2.3.4.4 highly pathogenic avian influenza H5N6 viruses have been continuously reported in China. To investigate the genesis of the virus, we analyzed 123 H5 or N6 environmental viruses sampled from live-poultry markets or farms from 2012 to 2015 in Mainland China. Our results indicated that clade 2.3.4.4 H5N2/N6/N8 viruses shared the same hemagglutinin gene as originated in early 2009. From 2012 to 2015, the genesis of highly pathogenic avian influenza H5N6 viruses occurred via two independent pathways. Three major reassortant H5N6 viruses (reassortants A, B, and C) were generated. Internal genes of reassortant A and B viruses and reassortant C viruses derived from clade 2.3.2.1c H5N1 and H9N2 viruses, respectively. Many mammalian adaption mutations and antigenic variations were detected among the three reassortant viruses. Considering their wide circulation and dynamic reassortment in poultry, we highly recommend close monitoring of the viruses in poultry and humans. IMPORTANCE Since 2014, clade 2.3.4.4 highly pathogenic avian influenza (H5Nx) viruses have caused many outbreaks in both wild and domestic birds globally. Severe human cases with novel H5N6 viruses in this group were also reported in China in 2014 and 2015. To investigate the genesis of the genetic diversity of these H5N6 viruses, we sequenced 123 H5 or N6 environmental viruses sampled from 2012 to 2015 in China. Sequence analysis indicated that three major reassortants of these H5N6 viruses had been generated by two independent evolutionary pathways. The H5N6 reassortant viruses had been detected in most provinces of southern China and neighboring countries. Considering the mammalian adaption mutations and antigenic variation detected, the spread of these viruses should be monitored carefully due to their pandemic potential.

Summary Background Human infections with different avian influenza viruses—eg, H5N1, H9N2, and H7N9—have raised concerns about pandemic potential worldwide. We report the first human infection with a novel reassortant avian influenza A H10N8 virus. Methods We obtained and analysed clinical, epidemiological, and virological data from a patient from Nanchang City, China. Tracheal aspirate specimens were tested for influenza virus and other possible pathogens by RT-PCR, viral culture, and sequence analyses. A maximum likelihood phylogenetic tree was constructed. Findings A woman aged 73 years presented with fever and was admitted to hospital on Nov 30, 2013. She developed multiple organ failure and died 9 days after illness onset. A novel reassortant avian influenza A H10N8 virus was isolated from the tracheal aspirate specimen obtained from the patient 7 days after onset of illness. Sequence analyses revealed that all the genes of the virus were of avian origin, with six internal genes from avian influenza A H9N2 viruses. The aminoacid motif GlnSerGly at residues 226–228 of the haemagglutinin protein indicated avian-like receptor binding preference. A mixture of glutamic acid and lysine at residue 627 in PB2 protein—which is associated with mammalian adaptation—was detected in the original tracheal aspirate samples. The virus was sensitive to neuraminidase inhibitors. Sputum and blood cultures and deep sequencing analysis indicated no co-infection with bacteria or fungi. Epidemiological investigation established that the patient had visited a live poultry market 4 days before illness onset. Interpretation The novel reassortant H10N8 virus obtained is distinct from previously reported H10N8 viruses. The virus caused human infection and could have been associated with the death of a patient. Funding Emergency Research Project on human infection with avian influenza H7N9 virus, the National Basic Research Program of China, and the National Mega-projects for Infectious Diseases.

Objectives The first classical swine influenza A H1N1 viruses were isolated in Mainland China in 1991. To aid surveillance of swine influenza viruses as part of pandemic preparedness, we sought to identify their origin. Methods We sequenced and phylogenically analyzed 19 swine influenza viruses isolated in 1991 and 1992 in China and compared them with viruses isolated from other regions during the same period. Results All 19 swine influenza viruses analyzed in our study shared the highest similarity with the classical swine influenza virus A/Swine/Maryland/23239/1991 (H1N1). Phylogenetic trees of eight segmented genes exhibited similar topology, with all segments in the cluster of classical swine influenza viruses. In addition, antigenic analysis also indicated that the tested isolated were related to classical swine influenza isolates. Conclusions Classical swine H1N1 influenza viruses were predominant in Beijing pig herds during this period. Since both antibody and virus detections did not indicate the presence of CS H1N1 before 1991 in Mainland China, we combined with the data on pigs imported to and exported from China and concluded that these viruses might spread to China via pigs imported from North America and that they could affect the genetic evolution and transmission dynamics of swine influenza viruses in Hong Kong.

Human infection associated with a novel reassortant avian influenza H7N9 virus has recently been identified in China. A total of 132 confirmed cases and 39 deaths have been reported. Most patients presented with severe pneumonia and acute respiratory distress syndrome. Although the first epidemic has subsided, the presence of a natural reservoir and the disease severity highlight the need to evaluate its risk on human public health and to understand the possible pathogenesis mechanism. Here we show that the emerging H7N9 avian influenza virus poses a potentially high risk to humans. We discover that the H7N9 virus can bind to both avian-type (α2,3-linked sialic acid) and human-type (α2,6-linked sialic acid) receptors. It can invade epithelial cells in the human lower respiratory tract and type II pneumonocytes in alveoli, and replicated efficiently in ex vivo lung and trachea explant culture and several mammalian cell lines. In acute serum samples of H7N9-infected patients, increased levels of the chemokines and cytokines IP-10, MIG, MIP-1β, MCP-1, IL-6, IL-8 and IFN-α were detected. We note that the human population is naive to the H7N9 virus, and current seasonal vaccination could not provide protection.

Infection of poultry with influenza A subtype H7 viruses occurs worldwide, but the introduction of this subtype to humans in Asia has not been observed previously. In March 2013, three urban residents of Shanghai or Anhui, China, presented with rapidly progressing lower respiratory tract infections and were found to be infected with a novel reassortant avian-origin influenza A (H7N9) virus.We obtained and analyzed clinical, epidemiologic, and virologic data from these patients. Respiratory specimens were tested for influenza and other respiratory viruses by means of real-time reverse-transcriptase-polymerase-chain-reaction assays, viral culturing, and sequence analyses.A novel reassortant avian-origin influenza A (H7N9) virus was isolated from respiratory specimens obtained from all three patients and was identified as H7N9. Sequencing analyses revealed that all the genes from these three viruses were of avian origin, with six internal genes from avian influenza A (H9N2) viruses. Substitution Q226L (H3 numbering) at the 210-loop in the hemagglutinin (HA) gene was found in the A/Anhui/1/2013 and A/Shanghai/2/2013 virus but not in the A/Shanghai/1/2013 virus. A T160A mutation was identified at the 150-loop in the HA gene of all three viruses. A deletion of five amino acids in the neuraminidase (NA) stalk region was found in all three viruses. All three patients presented with fever, cough, and dyspnea. Two of the patients had a history of recent exposure to poultry. Chest radiography revealed diffuse opacities and consolidation. Complications included acute respiratory distress syndrome and multiorgan failure. All three patients died.Novel reassortant H7N9 viruses were associated with severe and fatal respiratory disease in three patients. (Funded by the National Basic Research Program of China and others.).

The human respiratory tract is a major site of avian influenza A(H5N1) infection. However, many humans infected with H5N1 present with gastrointestinal tract symptoms, suggesting that this may also be a target for the virus. In this study, we demonstrated that the human gut expresses abundant avian H5N1 receptors, is readily infected ex vivo by the H5N1 virus, and produces infectious viral particles in organ culture. An autopsy colonic sample from an H5N1-infected patient showed evidence of viral antigen expression in the gut epithelium. Our results provide the first evidence, to our knowledge, that H5N1 can directly target human gut tissues.

Background Avian influenza viruses have caused human infection and posed the pandemic potential. Live poultry markets are considered as a source of human infection with avian influenza viruses. Avian influenza routine surveillance of live poultry markets is taken annually in China. We isolated the 2 H11N9 influenza virus from the surveillance program. To better understand the risk caused by these new viruses, we characterize the genetic and pathogenicity of the two viruses. Methods Viral isolation was conducted with specific pathogen-free (SPF) embryonated chicken eggs. Whole genome was sequenced, and phylogenetic analysis was conducted. Results Two H11N9 viruses were identified, with all 8 segments belonging to the Eurasian lineage. The HA, NA, M, NS and PA genes were similar to virus isolates from ducks, and the NP, PB2 and PB1 gene segments were most similar to those viruses from wild birds, indicating that the H11N9 viruses might represent reassortant viruses from poultry and wild birds. The HA receptor binding preference was avian-like, and the cleavage site sequence of HA showed low pathogenic. The NA gene showed 94.6 % identity with the novel H7N9 virus that emerged in 2013. There was no drug resistance mutation in the M2 protein. The Asn30Asp and Thr215Ala substitutions in the M1 protein implied a potentially increased pathogenicity in mice. Both viruses were low-pathogenic strains, as assessed by the standards of intravenous pathogenicity index (IVPI) tests. Conclusion Two reassortant H11N9 avian influenza viruses were detected. These viruses showed low pathogenicity to chickens in the IVPI test. Public health concern caused by the reassortant H11N9 viruses should be emphasized during the future surveillance. Electronic supplementary material The online version of this article (doi:10.1186/s40249-016-0149-2) contains supplementary material, which is available to authorized users.

Highly pathogenic influenza A (H5N1) virus causes a widespread poultry deaths worldwide. The first human H5N1 infected case was reported in Hong Kong Special Administrative Region of China in 1997. Since then, the virus re-emerged in 2003 and continues to infect people worldwide. Currently, over 400 human infections have been reported in more than 15 countries and mortality rate is greater than 60%. H5N1 viruses still pose a potential pandemic threat in the future because of the continuing global spread and evolution. Here, we summarize the epidemiological, clinical and virological characteristics of human H5N1 infection in China monitored and identified by our national surveillance systems.

Summary Background In December, 2007, a family cluster of two individuals infected with highly pathogenic avian influenza A (H5N1) virus was identified in Jiangsu Province, China. Field and laboratory investigations were implemented immediately by public-health authorities. Methods Epidemiological, clinical, and virological data were collected and analysed. Respiratory specimens from the patients were tested by reverse transcriptase (RT) PCR and by viral culture for the presence of H5N1 virus. Contacts of cases were monitored for symptoms of illness for 10 days. Any contacts who became ill had respiratory specimens collected for H5N1 testing by RT PCR. Sera were obtained from contacts for H5N1 serological testing by microneutralisation and horse red-blood-cell haemagglutinin inhibition assays. Findings The 24-year-old index case died, and the second case, his 52-year-old father, survived after receiving early antiviral treatment and post-vaccination plasma from a participant in an H5N1 vaccine trial. The index case's only plausible exposure to H5N1 virus was a poultry market visit 6 days before the onset of illness. The second case had substantial unprotected close exposure to his ill son. 91 contacts with close exposure to one or both cases without adequate protective equipment provided consent for serological investigation. Of these individuals, 78 (86%) received oseltamivir chemoprophylaxis and two had mild illness. Both ill contacts tested negative for H5N1 by RT PCR. All 91 close contacts tested negative for H5N1 antibodies. H5N1 viruses isolated from the two cases were genetically identical except for one non-synonymous nucleotide substitution. Interpretation Limited, non-sustained person-to-person transmission of H5N1 virus probably occurred in this family cluster. Funding Chinese Ministry of Science and Technology; US National Institute of Allergy and Infectious Diseases, National Institutes of Health; China-US Collaborative Program on Emerging and Re-emerging Infectious Diseases.

Human infection with avian influenza A H7N9 virus was first identified in March 2013 and represents an ongoing threat to public health. There is a need to optimize serological methods for this new influenza virus. Here, we compared the sensitivity and specificity of the hemagglutinin inhibition (HI), microneutralization (MN), and Western blot (WB) assays for the detection of human antibodies against avian influenza A (H7N9) virus. HI with horse erythrocytes (hRBCs) and a modified MN assay possessed greater sensitivity than turkey erythrocytes and the standard MN assay, respectively. Using these assays, 80% of tested sera from confirmed H7N9 cases developed detectable antibody to H7N9 after 21 days. To balance sensitivity and specificity, we found serum titers of ≥20 (MN) or 160 (HI) samples were most effective in determining seropositive to H7N9 virus. Single serum with HI titers of 20-80 or MN titer of 10 could be validated by each other or WB assay. Unlike serum collected from adult or elderly populations, the antibody response in children with mild disease was low or undetectable. These combinations of assays will be useful in case diagnosis and serologic investigation of human cases.

Background In the spring of 2013, a novel avian-origin influenza A (H7N9) virus in Eastern China emerged causing human infections. Concerns that a new influenza pandemic could occur were raised. The potential effect of chemical agents and physical conditions on inactivation of the novel avian influenza H7N9 virus had not been assessed. Methods To determine the inactivation effectiveness of the novel avian influenza A (H7N9) virus under various physical conditions and chemical treatments, two H7N9 viruses A/Anhui/1/2013 and A/Shanghai/1/2013 were treated by varied temperatures, ultraviolet light, varied pHs and different disinfectants. The viruses with107.7 EID50 were exposed to physical conditions (temperature, ultraviolet light and pH) or treated with commercial chemical agents (Sodium Hypochlorite, Virkon®-S, and Ethanol) respectively. After these treatments, the viruses were inoculated in SPF embryonated chicken eggs, the allantoic fluid was collected after 72–96 hours culture at 35°C and tested by haemagglutination assay. Results Both of the tested viruses could tolerate conditions under 56°C for 15 minutes or 60°C for 5 minutes, but their infectivity was completely lost under 56°C for 30 minutes, 65°C for 10 minutes, 70°C, 75°C and 100°C for 1 minute. It was also observed that the H7N9 viruses lost their infectivity totally after exposure of ultraviolet light irradiation for 30 minutes or longer time. Additionally, the viruses were completely inactivated at pH less than 2 for 0.5 hour or pH 3 for 24 hours, however, viruses remained infectious under pH treatment of 4–12 for 24 hours. The viruses were totally disinfected when treated with Sodium Hypochlorite, Virkon®-S and Ethanol at recommended concentrations after only 5 minutes. Conclusions The novel avian influenza A (H7N9) virus can be inactivated under some physical conditions or with chemical treatments, but they present high tolerance to moderately acidic or higher alkali conditions. The results provided the essential information for public health intervention of novel H7N9 avian influenza outbreak.

One of the primary efforts in influenza vaccine strain recommendation is to monitor through gene sequencing the viral surface protein haemagglutinin (HA) variants that lead to viral antigenic changes. Here we have developed a computational method, denoted as PREDAC, to predict antigenic clusters of influenza A (H3N2) viruses with high accuracy from viral HA sequences. Application of PREDAC to large-scale HA sequence data of H3N2 viruses isolated from diverse regions of Mainland China identified 17 antigenic clusters that have dominated for at least one season between 1968 and 2010. By tracking the dynamics of the dominant antigenic clusters, we not only find that dominant antigenic clusters change more frequently in China than in the United States/Europe, but also characterize the antigenic patterns of seasonal H3N2 viruses within China. Furthermore, we demonstrate that the coupling of large-scale HA sequencing with PREDAC can significantly improve vaccine strain recommendation for China.

Swine origin 2009 H1N1 influenza virus has spread globally to cause the first influenza pandemic of the 21st century. Serological studies can improve our understanding of the extent of human infection and risk factors associated with the transmission of this pandemic virus. The “gold standard” for serodiagnosis of human influenza virus infection is the detection of seroconversion between acute- and convalescent-stage samples. However, the timing of seroepidemiological investigations often precludes the collection of truly acute-phase sera, requiring development of serological criteria for evaluating convalescent-phase sera that optimize detection of true positives and true negatives. To guide seroepidemiological investigations into the spread of the novel 2009 pandemic H1N1 virus, we characterized serum antibody responses to 2009 H1N1 virus in 87 individuals with confirmed viral infection and 227 nonexposed U.S. individuals using microneutralization (MN) and hemagglutination inhibition (HI) assays. Sensitivity and specificity were determined for each assay alone and in combination for detection of 2009 H1N1 virus-specific antibodies in convalescent-phase sera. Although the HI assay was more specific for detecting antibody to 2009 H1N1, the MN assay was more sensitive, particularly for detecting low-titer seroconversions. A combination of titers (MN ≥ 40 and HI ≥ 20) provided the highest sensitivity (90%) and specificity (96%) for individuals aged

Continued H5N1 virus infection in humans highlights the need for vaccine strategies that provide cross-clade protection against this rapidly evolving virus. We report a comparative evaluation in ferrets of the immunogenicity and cross-protective efficacy of isogenic mammalian cell-grown, live attenuated influenza vaccine (LAIV) and adjuvanted, whole-virus, inactivated influenza vaccine (IIV), produced from a clade 1 H5N1 6:2 reassortant vaccine candidate (caVN1203-Len17rg) based on the cold-adapted A/Leningrad/134/17/57 (H2N2) master donor virus. Two doses of LAIV or IIV provided complete protection against lethal homologous H5N1 virus challenge and a reduction in virus shedding and disease severity after heterologous clade 2.2.1 H5N1 virus challenge and increased virus-specific serum and nasal wash antibody levels. Although both vaccines demonstrated cross-protective efficacy, LAIV induced higher levels of nasal wash IgA and reduction of heterologous virus shedding, compared with IIV. Thus, enhanced respiratory tract antibody responses elicited by LAIV were associated with improved cross-clade protection.

A critical step for avian influenza viruses to infect human hosts and cause epidemics or pandemics is acquisition of the ability of the viral hemagglutinin (HA) to bind to human receptors. However, current global influenza surveillance does not monitor HA binding specificity due to a lack of rapid and reliable assays. Here we report a computational method that uses an effective scoring function to quantify HA-receptor binding activities with high accuracy and speed. Application of this method reveals receptor specificity changes and its temporal relationship with antigenicity changes during the evolution of human H3N2 viruses. The method predicts that two amino acid differences at 222 and 225 between HAs of A/Fujian/411/02 and A/Panama/2007/99 viruses account for their differences in binding to both avian and human receptors; this prediction was verified experimentally. The new computational method could provide an urgently needed tool for rapid and large-scale analysis of HA receptor specificities for global influenza surveillance., National Key Project (2008ZX10004-013), National Institutes of Health (U.S.) (grant AI07443), Singapore-MIT Alliance for Research and Technology, Massachusetts Institute of Technology. International Science and Technology Initiatives Global Seed Fund, National Basic Research Program (973 Program) (2009CB918503), National Basic Research Program (973 Program) (2006CB911002)

Background Adamantane-derived drugs have been used for treatment and prophylaxis of influenza A virus infection for many years worldwide. Rapid surveillance of antiviral drug resistance is important for appropriate clinical guideline development. Here, we retrospectively assessed adamantane resistance among different influenza A subtypes (H1N1, H3N2 and H5N1) over 53 years (1956–2009) in mainland China. Methods A total of 1,451 viruses, including 773 H3N2 viruses, 647 H1N1 viruses and 31 human H5N1 viruses, were analysed by matrix gene sequencing and assayed for drug resistance. Results Our results show that the prevalence of adamantane- resistant H3N2 viruses was low between 1956 and 2002, but substantially increased in 2003 to the extent that since 2006 all H3N2 viruses have been drug resistant. The percentage of adamantane- resistant H1N1 viruses also increased from 50.0% in 2004 to 98.7% in 2007; however, this decreased to 46.7% in 2009. Only three adamantane-resistant H5N1 viruses have been detected since 2003, when the first case of human H5N1 virus infection was detected in mainland China. Phylogenetic analysis demonstrated that the increase of adamantane-resistant isolates was caused by point mutations or intrasubtype reassortment instead of intersubtype reassortment. Conclusions Because of the high percentage of adamantane- resistant H3N2 and H1N1 viruses in mainland China, the use of amantadine and rimantadine drugs for prophylaxis and treatment of current seasonal influenza A infection is not recommended.

Background: Memory CD8 T cells to influenza A viruses are widely detectable in healthy human subjects and broadly crossreactive for serologically distinct influenza A virus subtypes. However, it is not clear to what extent such pre-existing cellular immunity can provide cross-subtype protection against novel emerging influenza A viruses. Methodology/Principal Findings: We show in the mouse model that naturally occurring sequence variations of the conserved nucleoprotein of the virus significantly impact cross-protection against lethal disease in vivo. When priming and challenge viruses shared identical sequences of the immunodominant, protective NP366/D b epitope, strong cross-subtype protection was observed. However, when they did not share complete sequence identity in this epitope, cross-protection was considerably reduced. Contributions of virus-specific antibodies appeared to be minimal under these circumstances. Detailed analysis revealed that the magnitude of the memory CD8 T cell response triggered by the NP366/D b variants was significantly lower than those triggered by the homologous NP366/D b ligand. It appears that strict specificity of a dominant public TCR to the original NP366/D b ligand may limit the expansion of cross-reactive memory CD8 T cells to the NP366/D b variants. Conclusions/Significance: Pre-existing CD8 T cell immunity may provide substantial cross-protection against heterosubtypic influenza A viruses, provided that the priming and the subsequent challenge viruses share the identical sequences of the immunodominant, protective CTL epitopes.

Background. InChina,30humancasesofavianinfluenzaA(H5N1)virusinfectionwereidentifiedthroughJuly 2008. We conducted a retrospective case-control study to identify risk factors for influenza H5N1 disease in China. Methods. A questionnaire about potential influenza H5N1 exposures was administered to 28 patients with influenza H5N1 and to 134 randomly selected control subjects matched by age, sex, and location or to proxies. Conditional logistic regression analyses were performed. Results. Before their illness, patients living in urban areas had visited wet poultry markets, and patients living in rural areas had exposure to sick or dead backyard poultry. In multivariable analyses, independent risk factors for influenza H5N1 were direct contact with sick or dead poultry (odds ratio [OR], 506.6 [95% confidence interval {CI}, 15.7‐16319.6]; P .001),indirect exposure to sick or dead poultry (OR, 56.9 [95% CI, 4.3‐745.6]; P .002), and visiting a wet poultry market (OR, 15.4 [95% CI, 3.0‐80.2]; P .001). Conclusions. To prevent human influenza H5N1 in China, the level of education about avoiding direct or close exposures to sick or dead poultry should be increased, and interventions to prevent the spread of influenza H5N1 at live poultry markets should be implemented. In parallel with the unprecedented epizootic of highly pathogenicavianinfluenzaA(H5N1)virusesamongpoultryandmigratorybirds[1],418confirmedhumancasesof

We investigated avian influenza infections in wild birds, poultry, and humans at Eastern Dongting Lake, China. We analyzed 6,621 environmental samples, including fresh fecal and water samples, from wild birds and domestic ducks that were collected from the Eastern Dongting Lake area from November 2011 to April 2012. We also conducted two cross-sectional serological studies in November 2011 and April 2012, with 1,050 serum samples collected from people exposed to wild birds and/or domestic ducks. Environmental samples were tested for the presence of avian influenza virus (AIV) using quantitative PCR assays and virus isolation techniques. Hemagglutination inhibition assays were used to detect antibodies against AIV H5N1, and microneutralization assays were used to confirm these results. Among the environmental samples from wild birds and domestic ducks, AIV prevalence was 5.19 and 5.32%, respectively. We isolated 39 and 5 AIVs from the fecal samples of wild birds and domestic ducks, respectively. Our analysis indicated 12 subtypes of AIV were present, suggesting that wild birds in the Eastern Dongting Lake area carried a diverse array of AIVs with low pathogenicity. We were unable to detect any antibodies against AIV H5N1 in humans, suggesting that human infection with H5N1 was rare in this region.

Autopsy studies have shown that human highly pathogenic avian influenza virus (H5N1) can infect multiple human organs other than just the lungs, and that possible causes of organ damage are either viral replication and/or dysregulation of cytokines and chemokines. Uncertainty still exists, partly because of the limited number of cases analysed. In this study, a full autopsy including 5 organ systems was conducted on a confirmed H5N1 human fatal case (male, 42 years old) within 18 hours of death. In addition to the respiratory system (lungs, bronchus and trachea), virus was isolated from cerebral cortex, cerebral medullary substance, cerebellum, brain stem, hippocampus ileum, colon, rectum, ureter, aortopulmonary vessel and lymph-node. Real time RT-PCR evidence showed that matrix and hemagglutinin genes were positive in liver and spleen in addition to positive tissues with virus isolation. Immunohistochemistry and in-situ hybridization stains showed accordant evidence of viral infection with real time RT-PCR except bronchus. Quantitative RT-PCR suggested that a high viral load was associated with increased host responses, though the viral load was significantly different in various organs. Cells of the immunologic system could also be a target for virus infection. Overall, the pathogenesis of HPAI H5N1 virus was associated both with virus replication and with immunopathologic lesions. In addition, immune cells cannot be excluded from playing a role in dissemination of the virus in vivo.