Genetic Characteristics And Phylogenetic Analysis Of Avian Influenza Virus In Order Anseriformes In Three Wintering Areas,2014-2015

Avian influenza virus(AIV)is negative-sense,single-stranded,segmented RNA virus belonging to the Influenza A virus(IAV)of Orthomyxoviridae,which is highly mutable and has the ability to infect birds and mammals.As the natural reservoir of AIV,wild birds,especially Order Anseriformes,play an important role in the transmission of AIV.From October 2014 to March 2015,656 samples of oropharyngeal and cloacal swabs and 1473 samples of feces were collected from Pudong Dongtan wetland in Shanghai,East Dongting Lake wetland in Hunan province and Caohai wetland in Guizhou province.The viral RNA was extracted using the MagMAX? Pathogen RNA/DNA kit(Applied Biosystems,Foster City,CA,USA)on the Magmax-96 Express(Applied Biosystems)according to the manufacturer's instructions.The viruses were confirmed by real-time reverse transcription-PCR on ABI 7500(Applied Biosystems)targeting the matrix gene.Eight gene segments of each virus were amplified with PrimeScript? One-step RT-PCR kit Ver.2 kit(TaKaRa,Biotechnology [Dalian] Co.,Ltd,Dalian,China).The genome amplification was performed with MightyAmp? DNA Polymerase Ver.3 kit.PCR products were purified with MinElute? Gel Extraction Kit(Applied Biosystems)and sent to the sequencing company for next-generation sequencing.Host species were identified by using primers for the mitochondrial cytochrome-oxidase I gene.Through the data analysis,the virus infection status in different sample collection sites,pathogenicity,nucleotide homology and phylogenetic tree of representative strains were sorted out,and the relationship between wild bird migration route and influenza virus transmission was discussed.The main results were as follows:1.Sample collection and virus infectionFrom October to November 2014,326 waterfowl of 11 species were collected from Pudong Dongtan wetland,656 samples of oropharyngeal and cloacal swabs were obtained,with a positive rate of 40.80%.The subtypes of the virus included H5N2,H6N1 and H4,and the hosts included 8species of Order Anseriformes such as spot-billed duck(Anas poecilorhyncha),falcated teal(Anas falcata),and Green-winged Teal(Anas crecca).In December 2014 and February 2015,921 fresh fecal samples were collected from the East Dongting Lake wetland in Hunan province,with a positive rate of 4.13%.The virus subtypes included H6N2,H9N2,H11N8 and H12N2,and the hosts included Greater White-fronted Goose(Anser albifrons),bean goose(Anas fabalis),swan goose(Anser cygnoides)and falcated teal.Besides,H11N8 AIV was first detected from wild birds in Asian and H12N2 AIV was first detected from wild birds in China.In March 2015,552 fresh fecal samples were collected from Caohai wetland in Guizhou province,with a positive rate of 0.72%.The virus subtype was H8N4,and the hosts included falcated teal and spotted-billed duck.In addition,H8N4 AIV was first identified from wild birds in mainland China.2.Pathogenicity analysis9 representative strains were selected in this study,of which one H5N2 virus collected from Pudong Dongtan wetland was highly pathogenic avian influenza virus(HPAIV),and the other 8 strains were all low pathogenic avian influenza virus(LPAIV).The variation sites on amino acid sequence showed that these viruses had good affinity for avian ?-2,3 Sialic acid(SA)receptors,and their potential replication ability and toxicity in mammals might be enhanced,but they have not developed resistance to ion channel blockers and were also sensitive to neuraminidase inhibitors.3.Nucleotide homology analysisThe 8 genes of DT-767(H9N2)were probably all from wild birds,while the other 8 viruses had complex gene sources,some of which were from poultry and some of which from wild birds.The reassortment among the 9 viruses was observed.4.Phylogenetic analysisThe HA gene of PD-1415(H5N2)was located in Clade 2.3.4.4 of HPAI H5.HA genes of all three H6 viruses fell into AHL221-like under HN573-like.The HA gene of DT-767(H9N2)wasbelonged to the Y439-like.In addition,DT-767(H9N2)was a transcontinental reassortant with PA gene belonged to the North American lineage and the other 7 genes belonged to the Eurasian lineage.All genes of the other 8 viruses fell into the Eurasian lineage.From the perspective of wild bird migration routes,the viruses collected in this study might have gene exchange with viruses on the Central Asia Flyway and Black Sea/Mediterranean Flyway.