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Analysis of genetic characteristics of H9N2 subtype avian influenza viruses in the plateau wetlands of Lashihai, Yunnan

JI Jia;ZHANG Zhen-xing;XUE Xiao-yan;WANG Wen-hui;HAN Lian-xian;DAI Hong-yang;LI Gao-yin;DUAN Bo-fang;ZHANG Wen-dong;LIU Qiang;LI Su-hua;SONG Jian-ling;College of Life Sciences/National Plateau Wetland Research Center, Southwest Forestry University;Yunnan Academy of Animal Husbandry and Veterinary, Yunnan Tropical and Subtropical Animal Virus Diseases Laboratory;The Management Bureau of Huize Black Necked Crane National Nature Reserve;Department of Animal Health Quarantine, Animal Disease Inspection and Supervision Institution of Zhaoyang District;Yunnan Center for Animal Disease Control and Prevention;  
To understand the transmission characteristics of avian influenza virus(AIV) in wild birds and domestic poultry,1807 samples, including 1367 feces of wild birds, 420 cloacal swabs of poultry and 20 environmental water samples, were collected from March 2020 to March 2021 in Lashihai Plateau. One H9N2 AIV strain originated from bar-headed goose and 2strains from poultry were isolated by SPF chicken embryo inoculation and RT-PCR identification. Whole genome sequences of the three strains were obtained by applying Illumina Miseq platform, and genetic evolution and mutations at positions associating with viral pathogenicity and transmissibility were analyzed. Phylogenetic analysis showed that the strain from wild bird shared the same genetic evolution characteristics with the isolations from poultry. All the three H9N2 AIV strains belong to h9.4.2.5 branch of the Eurasian lineage. They are likely to be reassortants, with surface protein segments HA and NA from Y280-like subgroup, PB2 and M segments from G1-like subgroup, and PA, PB1, NP and NS segments from F/98-like subgroup. This recombination pattern is consistent with the prevailing S genotype of H9N2 AIV in China. Furthermore, mutational sites analysis demonstrated that all the strains possess the similarity in the amino acid mutations associating with cross-species transmission and viral pathogenicity of AIV in HA, NA, PB2, PB1, M1 and NS1 proteins. Interestingly, all the wild bird-origin virus and domestic poultry-origin viruses possess N166D substitution in HA and 4 amino acids deletion at positions 63-65 of NA, whereas the N383D in PA was absent in the wild-bird-origin virus. The above results illustrated that the H9N2 AIV form wild bird in this study may originate form poultry and some adaptive mutations occurred in response to the mild replication pattern in the natural host. Overall, this study for the first time systematically analyzed the genetic characteristics of H9N2 AIVs from wild birds and poultry in Lashihai plateau wetland, and provided basic data for prevention and control of avian influenza.
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