The aim of this study is to analyze the evolutional and molecular characteristics of Hemagglutinin (HA),Neuraminidase(NA)and non-structural (NS) genes of H5N1 avian influenza virus (AIV) from sewage in live bird markets (LBMs) in Changsha,2014.Five hundred and one specimens were collected from environment in LBMs in Changsha,2014,and real-time RT-PCR was used for influenza A typing and subtyping (H5,H7 and H9) detection.Sequencing were used for the positive of single H5.The sequence homology of HA,NA and NS genes of the viruses were analyzed with the online Basic Local Alignment Search Tool (BLAST).The phylogenetic trees for HA,NA and NS genes and the ClustalW Multiple alignments of amino acids were constructed using MEGA 5 and BioEdit software,respectively.Results showed that of 501 environmental samples,177 (35.33 ％) samples were positive for influenza A viruses and H5 subtype.Eight H5N1 subtype AIV were confirmed by sequencing from the samples of the positive of single H5.Phylogenetic analysis indicated that most of HA genes of the H5N1 subtype AIV strains isolated in Changsha city were located in 2.3.2 and clustered into new subclade,and the most of NA and NS genes in this study were clustered into subclade 2.3.2.1b.QSG of the HA protein of the receptor binding site were found in these H5N1 viruses,and the characteristics was shown to be associated with increased affinity of HA to the glycan-receptors of AIV.In strains from this study,we did not found amino acid substitutions of the NA protein at H275Y and N295S,and sensitive to neuraminidases,and the high pathogenicity molecular characteristics of HA,NA and NS genes were showed in these viruses.In conclusion,molecular characteristics of the HA,NA and NS of these H5N1 subtype viruses in this study showed high pathogenicity,but that may not facilitate human infection.So,the prevalence and genetic evolution of this virus should be closely monitored.

We analyzed the evolutional and molecular characteristics of Hemagglutinin(HA),Neuraminidase(NA) and nonstructural(NS) genes of avian influenza A(H9n2) viruses from environment in poultry markets in Changsha,China,2014,providing laboratory data for prevention and control of human infection with avian influenza A(H9N2) virus.Five hundred and one specimens (263 poultry drinking water specimens,226 poultry sewage specimens and 17 others specimens) were collected from environment in poultry markets in Changsha,2014,and real-time RTPCR was used for influenza A typing and subtyping (H5,H7 and H9) detection.HA and NA universal primer sets for conventional RT-PCR and sequencing were used for the positivity of single H9.The sequence homology of HA,NA and NS genes of the viruses were analyzed with the online Basic Local Alignment Search Tool (BLAST).The ClustalW multiple alignments of amino acids and the phylogenetic trees for HA,NA and NS genes were constructed using the BioEdit and MEGA 5 software,respectively.Results showed that among 501 environmental samples,350 samples were positive for influenza A virus,191 (38.12％) for H9 subtype,177 (35.33％) for H5 subtype,11 (2.20％) for H7 subtype and 68 (13.57％) for H5 and H9 subtypes co-detection.Twenty-three H9N2 subtype AIV were confirmed by conventional RT-PCR and sequencing from the samples of the positivity of single H9.Phylogenetic analysis revealed that most of HA,NA and NS genes of the H9N2 subtype AIV isolated in Changsha City had gene constellations of genotype S,and these virues might have acquired their HA,NA and NS from A/Chicken/Shanghai/F/1998-like (H9N2).L235 (correspond to H3 numbering 226) of the HA protein of the receptor binding site (RBS) were found in these H9N2 viruses,and the characteristics was shown to be associated with increased affinity of HA to the glycan-receptors of human influenza virus,and the low pathogenicity molecular characteristics of HA,NA and NS genes were showed in these viruses.The positive rate of nucleic acid of the H9 subtype of avian influenza virus from environment was the highest in poultry markets in Changsha,2014,and molecular characteristics of the HA,NA and NS of these H9N2 subtype AIV showed low pathogenicity,but that may facilitate human infection.So,the prevalence and genetic evolution of this virus should be closely monitored.