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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.

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.

A sensitive reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for rapid visual detection of pandemic influenza A H1N1 virus infection. The reaction was performed in one step in a single tube at 65 degrees C for 60 min with the addition of hydroxynaphthol blue (HNB) dye prior to amplification. The detection limit of the RT-LAMP assay was approximately 60 copies, and no cross-detection was observed. The assay was evaluated further with 50 clinical specimens diagnosed clinically with seasonal influenza or pandemic influenza A H1N1 virus infection. RT-LAMP with HNB dye was demonstrated to be a sensitive and easy assay for rapid detection of pandemic influenza A H1N1 virus.

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.

We report the use of ResPlex III for genotyping influenza A viruses. The performance characteristics of the assay with regard to H5N1 are further evaluated. The ResPlex system incorporates a novel multiplex PCR technology, target-enriched multiplex PCR, to simultaneously amplify multiple molecular targets in one reaction. The ResPlex III assay targets the H1, H2, H3, H5, H7, H9, N1, and N2 genes from the influenza A virus as well as the NS genes from influenza A (NSA) and B (NSB) viruses, providing detection and genotyping of influenza A and B viruses. The analytical sensitivities for detecting the H5, N1, and NSA genes were 1, 10 −1 , and 10 50% tissue culture infectious doses/200 μl/reaction, respectively. A total of 217 sequential clinical samples including 14 samples with human H5N1 infections were tested by the ResPlex III assay, and the results were compared to a reference standard combined with results of viral culture and conventional reverse transcriptase and real-time PCR. The clinical sensitivity and specificity for detecting H5N1 were 93.3% and 100%, respectively, indicating that different subtypes of influenza A virus can be quickly and correctly identified using the ResPlex III genotyping approach.

Background Reverse transcription PCR (RT-PCR) and real time RT-PCR (rRT-PCR) have been indispensable methods for influenza surveillance, especially for determination of avian influenza. The movement of testing beyond reference lab introduced the need of quality control, including the implementation of an evaluation system for validating personal training and sample proficiency testing. Methods We developed a panel with lysates of seasonal influenza virus (H1N1, H3N2 and B), serials of diluted H5N1 virus lysates, and in-vitro transcribed H5 hemaglutinin (HA) and an artificial gene RNAs for RT-PCR and rRT-PCR quality control assessment. The validations of stability and reproducibility were performed on the panel. Additionally, the panel was implemented to assess the detection capability of Chinese human avian influenza networks. Results The panel has relatively high stability and good reproducibility demonstrated by kappa's tests. In the implementation of panel on Chinese human avian influenza networks, the results suggested that there were a relatively low number of discrepancies for both concise and reproducibility in Chinese avian influenza virus net works. Conclusions A quality control panel of RT-PCR and real-time RT-PCR for avian influenza A (H5N1) surveillance network was developed. An availably statistical data, which are used to assess the detection capability of networks on avian influenza virus (H5N1), can be obtained relatively easily through implementation of the panel on networks.

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.

To establish the DNA microarray to detect influenza viruses and avian influenza viruses, and identify their virulence.Hemagglutinin (HA), neuramidinase (NA) and nucleoprotein(NP) genes were chosen simultaneously as targets for designing a microarray used for detection of viruses and identification virulence. The nucleic acid were amplified by single primer amplication (SPA). And then its specificity,sensitivity and reproducibility were evaluated.The microarray was able to specially detect H1N1, H3N2, B influenza viruses and H5N1, H9N2 avian influenza viruses. Their limits were 8HAU, 16HAU, 32HAU, and 8HAU, 8HAU respectively. The limit for virulence was 32HAU. When samples were analyzed by both RT-PCR and microarray in parallel, the results agreed in 83.9% (47/56).The microarray can detect and distinguish five tested viruses, and especially identify virulence. It not only supplies an assistant tool for clinical diagnosis and control of infectious disease, but also is valuable for controlling and preventing outbreak of avian influenza epidemic.

To understand the origin of hemagglutinin (HA), and neuraminidase (NA) gene of swine influenza A (H1N1) viruses isolated in pigs in mainland China in 2002 and reveal the reason of pathogenesis of them in pigs.The target gene amplified by PCR,PCR product was linked with PGEM-T Easy Vector(Promega company, USA) at 4? degrees C, the recombined plasmid was transferred into DH 10B bacteria, positive colonies were selected and identified them with restriction enzyme. Afterwards, they were sent to Liu He Tong company in Beijing for testing nucleotide sequence. Finally, phylogenetic analysis of the sequencing data was performed with MegAlign (Version 1.03)and Editseq (Version 3.69) software.The HA and NA genes of three strains of swine influenza A (H1N1) viruses isolated from pigs in China were closely related to those of swine influenza A (H1N1) virus, but different from those of avian or human influenza A (H1N1) virus. The swine strain of influenza A (H1N1) virus isolated in 2002 was derived from swine influenza A (H1N1) virus circulated in pigs in China in 1991. Since the antigenic drifts of HA and NA proteins of the new isolates occurred, their activity in pigs is increasing and they can cause disease in pigs.The HA and NA genes of three strains of influenza A (H1N1) virus tested were identified to be derived from those of swine influenza A (H1N1) virus. The increased activity and pathogenesis of them in pigs were most likely due to antigenic drifts of HA and NA proteins of the new isolates.

To understand the epidemics and antigenic drift of influenza viruses in China from 2000 to 2001.The viruses were grown in embryonated hen eggs with 9 - 10 days old. The egg allantoic fluids with influenza viruses were used. Virion RNA was transcribed into cDNA by reverse transcriptase while cDNA amplified by PCR. Products of PCR were purified. RNA sequence analysis was then performed. Finally, phylogenetic analysis of the sequencing data was performed with MegAlign (Version 1.03) and Editseq (Version 3.69) software.Data from comparison of amino acid sequence on HA1 domain of HA protein molecule between H1N1 viruses isolated in 2001 and A/Shanghai/7/99 (H1N1) strain indicated that there was only one difference of amino acid located at 190 position (antigenic determinant D). However, phylogenetic analysis showed that there were two distinguishable genetically lineages of H1N1 viruses co-circulating in men in China in 2001. Two antigenically distinct genetic lineages of influenza B viruses were still existing in men in China. Most of influenza B viruses were Yamagata-like strain and there were two different amino acid sequences located at 197 and 199 position on HA1 domain of HA protein molecule, between Victoria-like virus isolated and B/Shandong/7/97 strain. When comparing amino acid sequences on HA1 protein domain of H3N2 viruses isolated in 2000 with those of A/Sydeney/5/97 (H3N2) virus, it was revealed that there were 7 - 8 differences of amino acid sequences between them. However, there were four differences related to amino acid sequences on HA1 protein domain between H3N2 viruses isolated in 2000 and in 2001. These results were further demonstrated by analysis of phylogenic tree.Influenza was not prevalent in China from 2000 to 2001. The antigenic drifts of H3N2 and B/Victoria-like viruses occurred. Two antigenically distinct genetic lineages of influenza B viruses were still co-circulating in men in China. Two genetically distinct lineages of influenza A (H1N1) virus were also co-circulating in men in China.

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.