Sequence Analysis And Identification For Avian Influenza Viruses Of Migrant Birds From Some Regions Of China In 2014-2015

Avian influenza virus(AIV) belongs to Orthomyxoviridae, the member of Influenza Virus gender, which is consisted with eight segments of negative-standed RNA. As a natural host,migratory birds play an important role in the transmission, infection and rearrangement of AIVs in poulties and humanbings. The surveillance and characterization of AIVs circulating in the migratory birds not only illustrate better the epidemiology of AIVs in their natural hosts but also provide the authentic prediction of the outbreak of influenza in domestic poultry and human. In order to understand the genetic variation and biological characteristics of AIV in wild bird in china, a total number of 7447 samples including swabs and feces were colleted from migratory wild birds in Jilin, Henan, Hebei, Hunan, Qinghai and Inner Mongolia from 2014-2015. 37 strains of AIVs were isolated from these samples, including H4N6, H5N1, H5N6, H5N8, H6N5,H9N2, H9N7, H13N6, H13N8 AIV subtypes. In this study, four AIV strains of H5N1, two AIV strains of H5N6 and one AIV strain of H10N7 subtypes, which were concerned for the public health risk to human, were selected to perform the sequence analysis, the genetic evolution analysis and rearrangement analysis.The sequence analysis of H5N1 subtype strains revealed that the four H5N1 isolates classified into the clade of 2.3.2.1 were located in the same evolution branch, sharing highly homology to each other. The reduced amino acids sequence of HA gene from 4 AIV strains had the cleavage site, R-R-R-K/-/R-G, representing the molecular basis of highly pathogenic AIV stain. Most receptor-binding sites of HA genes were conservative, the variation of individual site indicate that it may have an impact on the range of infected host. Meanwhile, the 4 virus strains had a 20-amino-acid deletion in the NA stalk and 3 the potential glycosylation sites in the NA.The I314 V mutation in the NA protein and the V27 A mutation in the M2 protein, which might cause resistance of neuraminidase inhibitors and M2 protein inhibitors, were found in the four strains. In addition, they had a 5-amino-acid deletion in the NS(residues 80-84) showing that the four H5N1 virus might have the abality to anti-interferon in host.The two strains of H5N6 isolates fall into the novel clade of 2.3.4.4 of H5 AIVs. The substype of A/Peacock/Hunan/HN-1/2015(H5N6) was a rearrangement virus reasserting among H5N6 and H5N8 AIV. The amino acids sequence of HA genes from the two H5N6 strains had the cleavage site R-R-R-K/-/R-G as highly pathogenic AIV stains. The strains ofA/Peacock/Hunan/HN-1/2015(H5N6) had a variation of K205 N and Q208 K in receptor-binding region of HA. There was a mutation of glycosylation site at 209(NPT) in the strain A/Peacock/Hunan/HN-1/2015(H5N6). Meanwhile, all the 2 virus strains had a 19-amino-acid deletion at 56-74 residuse in the NA stalk and 7-amino-acid addition at 13-19 residuse of NA.A/Peacock/Hunan/HN-1/2015(H5N6) had no deletion in the NS(residues 80-84), whereas, the strains of A/Black Swan/Changde/HN-1/2016(H5N6) had a 6-amino-acid deletion in the NS.The V116 A ? I117 V, and I314 V mutation, which determined resistance of neuraminidase inhibitors, were found in NA of the two strains. M2 ion channel inhivitor might have effects on the H5N6 virus because no mutation in the M2 protein was found.H10N7 subtype isolates in our study is belongs to Eurasian lineage. This subtype shared highly similarity to multiple subtypes of influenza virus such as H3N8, H5N3, H10N2, H10N4 and H10N7 subtype AIVs, demonstrating that the H10 substype might reassortant virus. The reduced amino acid sequence of HA genes from the H10N7 strain had the cleavage site NVPEL—QGR/G, which represented the molecular basis of low pathogenic AIV stain. In addition, the HA amino acids at positions 226 and 228 were Gln and Gly, indicating that the virus might retain the ability of binding to the avian like influenza virus receptors. The virus of HA gene had 6 the potential glycosylation sites without deletion and mutation.Our studies showed that obvious rearrangement and variation were found in H5N1, H5N6 and H10N7 subtypes isolated from migratory birds. It is important to strengthen the monitoring of avian influenza in migratory birds for the comprehensive prevention and control of avian influenza.