| Objective: To analyze the complete genome sequence and phylogenetic structure of H5N6 Avian influenza virus(AIVs)from live poultry markets in Urumqi during2016-2018 and wild bird-origin H1N1 AIVs from the northern Tianshan Mountain.Methods: The swabs of poultry and environmental samples were collected from live poultry markets in Urumqi during 2016-2018,and wild birds’ fresh feces samples were collected from several reservoirs at the northern Tianshan Mountain.AIVs were isolated and identified by inoculation of chicken embryo,hemagglutination inhibition test and RT-PCR with PB1 universal primer,the viral genome 8 fragments were amplified with the universal primers of influenza A virus and the viral whole genome was sequenced,pairwise sequence alignments,phylogenetic and molecular characteristics analysis were performed by BLAST,Meg Align,MEGA 7.0 and Mr Bayes software.Results:(1)In this work,two H5N6 virus strains(named XJ-H5N6/2016)were isolated from the geese in live poultry markets(LPMs)in Urumqi during the winter of2016-2017.Except for the PB2,phylogenetic analysis showed that the other 7 viral genes were clustered together with NX-WB-H5N6 and CQ-EN-H5N6,while the viral PB2 was clustered together with the poultry H9N2/H7N9.The viral HA protein contained multiple basic amino acid residues at the cleavage site,and the NS1 contained D92 E mutation,which represents HPAIV.Additionally,the viral multiple mutations could enhance the viral virulence and the capacity of cross-species transmission.(2)In2017-2018,16 H5N6 AIV strains were isolated from poultry samples,all of which were HPAIV,and the 16 strains were divided into three branches.XJ-H5N6/2017 belongs to2.3.4.4B,except that NA was clustered as a branch with NX-WB-H5N6 and CQ-EN-H5N6,the other 7 gene fragments were closely related to the H5N8 AIV carried by Anseriformes wild birds in many regions at home and abroad,so the virus could be a new reassortant originating from H5N8 and H5N6 AIVs carried by wild birds.XJ-H5N6-017/2017 belongs to 2.3.4.4C,and the 7 gene fragments except PA are consistent with the homology and phyletic evolution relationship of the corresponding gene of XJ-H5N6/2016,while PA was clustered together with the wildbird-origin H5N6/ H5N8.All gene fragments of XJ-H5N6/2018 virus were clustered together with a number of H5N6 AIV strains isolated from Guangdong environment and poultry in Jiangsu and infected humans.And multiple mutations in the virus strains may enhance the viral virulence,pathogenicity,and ability to spread across species.(3)Influenza viruses were isolated and identified from 6 out of 320 samples of the wild bird feces,the positive rate was 1.88%,and one of them was H1N1 AIV,named XJ-H1N1.All 8 genes segments of XJ-H1N1 were derived from AIVs isolated from wild ducks of Anseriformes,both surface genes HA and NA were Eurasian lineages,and derived from H1N1 isolated from Mongolian Anas platyrhynchos and H3N1 isolated from Bangladesh wild duck respectively;the 6 internal genes were derived from H6N8 isolated from Anasstrepera in Siberia,H7N3 isolated from Anasstrepera and teal in Egypt,and H7N5 AIVs isolated from wild birds such as Anas platyrhynchos in the Netherlands respectively.XJ-H1N1 virus is low pathogenic AIVs,and might bind both SA α 2,3-Gal and SA α 2,6-Gal receptors.T200 A and E227 A mutations of HA and P42 S mutation of NS1 can all enhance the replication ability and pathogenicity of viruses in mammalian cells.Conclusion: There were many kinds of H5N6 AIVs in Urumqi live poultry market during 2016-2018,which were derived from the reassortant of wild birds and poultry AIVs.A low pathogenic H1N1 AIV was isolated from wild birds from the northern Tianshan Mountain,which was derived from the multiple reassortments of the AIVs carried by migrating wild ducks. |
