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目的:开展2016—2018年长沙市人群感染和活禽市场(live poultry markets, LPMs)环境污染H5N6亚型禽流感病毒(avian influenza virus, AIV)监测，为防控人感染H5N6亚型AIV提供实验室数据。方法:采集2016—2018年长沙市流感样病例和不明原因肺炎病例咽拭子6 909份及LPMs环境样品1 719份，利用实时RT-PCR进行A型、H5、H7、H9和N6亚型AIV核酸扩增，并对82份AIV核酸阳性样品进行高通量核苷酸测序，然后对测序结果进行BLAST比对和氨基酸(amino acids, aa)关键位点分析。结果:从6 909份病例的咽拭子样品中检出H5N6亚型AIV核酸阳性1份，从1 719份LPMs环境样品中检出A型AIV核酸阳性927份(53.93%)，H5N6亚型AIV核酸阳性193份(11.23%)。高通量核苷酸测序获得14株H5N6亚型AIV的基因组序列，aa关键位点分析显示病毒血凝素(hemagglutinin, HA)蛋白连接肽第338～347位出现6个碱性aa，对禽表现为高致病性的分子特征，受体结合位点(receptor binding site, RBS)第238～240位aa(对应H3型流感病毒第226～228位编码aa)为QSG或QRG，受体特征为禽源。神经氨酸酶(neuraminidase, NA)蛋白第290位耐药基因位点aa未出现R290K突变现象，对NA抑制剂(达菲/磷酸奥司他韦)敏感；病毒PB2蛋白发生E627K和D701N(I)突变，表明病毒致病力强。结论:长沙市人群感染H5N6亚型AIV为偶发，LPMs环境H5N6亚型AIV污染较重，需要进一步加强LPMs环境AIV监测。“,”Objective:To carry out the monitoring of H5N6 subtype avian influenza virus (AIV) in the population infection and live poultry markets (LPMs) environmental pollution in Changsha city from 2016 to 2018. Laboratory data were provided to prevent human infection with H5N6 subtype AIV.Methods:A total of 6 909 throat swab specimens from the population and 1 719 environmental specimens from LPMs were collected in Changsha city from 2016 to 2018. The real-time RT-PCR method was used for the detection of influenza virus in type A, H5, H7, H9 and N6 subtypes. Eighty-two AIV nucleic acid-positive specimens were subjected to high-throughput nucleotide sequencing, and then BLAST similarity and amino acid (aa) key site analyses were performed on the sequencing results.Results:One H5N6 subtype AIV nucleic acid was detected from 6 909 population throat swab specimens, 927 (53.93%) positive specimens of A-type AIV nucleic acids and 193 (11.23%) positive specimens of both H5 and N6 subtypes were detected from 1 719 LPMs environmental specimens. Fourteen genomic sequences of H5N6 subtype AIV were obtained with high-throughput nucleotide sequencing, and aa key site analyses showed that there were 6 basic aa in 338-347 aa of viral HA protein-linked peptide, which was highly pathogenic to birds. Receptor binding site (RBS) 238-240 aa (corresponding to H3 type influenza virus 226-228 aa) is QSG or QRG, and the receptor is characterized by avian origin. The 290th resistance gene locus of NA protein did not show R290K mutation, and was sensitive to NA inhibitor (Duffy/osoterosine phosphate). The E627K and D701N(I) mutations in the viral PB2 protein indicate that the virus is pathogenic.Conclusions:Human infected with H5N6 subtype AIV in Changsha city is sporadic, and the H5N6 subtype AIV of LPMs is seriously polluted. It is necessary to further strengthen the AIV surveillance of LPMs.