2019-2021年安徽省外环境H9N2亚型禽流感病毒分子进化分析

Molecular evolution of H9N2 subtype avian influenza virus in external environment in Anhui,2019−2021

目的分析安徽省2019—2021年外环境监测中H9N2亚型禽流感病毒的遗传进化特征。方法选取禽流感外环境病原学监测H9N2亚型禽流感病毒阳性样本383份,使用鸡胚分离培养收获21株病毒,采用实时荧光定量PCR方法扩增后进行全基因组测序,用生物信息学软件构建基因进化树分析其遗传进化特征。结果21株H9N2亚型禽流感病毒均为G57基因型,血凝素(HA)基因片段属于JN/99基因型,神经氨酸酶(NA)基因属于G9基因型,基质蛋白(MP)基因属于G1基因型,内部核蛋白(NP)、非结构蛋白(NS)、酸性聚合酶蛋白(PA)、碱性聚合酶蛋白1(PB1)基因属于F/98基因型,碱性聚合酶蛋白2(PB2)基因片段属于ST/7488基因型毒株。HA裂解位点显示低致病性分子特征,存在重要氨基酸位点突变,HA蛋白发生了Q226L、H183N、T189D、A190T氨基酸突变;NA蛋白茎部均发生了62~64位点缺失;大部分毒株未发生PB2-E627K突变,所有毒株均未发生D701N突变;所有毒株PA蛋白具有356R和409N氨基酸突变,M2蛋白均发生S31N氨基酸突变。结论2019—2021年安徽省外环境分离得到的H9N2亚型禽流感病毒为多源重配病毒,且具有逐渐适应哺乳动物宿主相关的分子特征,需密切关注病毒的病原学演化。

Objective To analyze the genetic evolution characteristics of H9N2 subtype avian influenza virus in external environment in Anhui province from 2019 to 2021.Methods A total of 383 positive samples of H9N2 subtype avian influenza virus detected in the etiological surveillance for avian influenza in external environment in Anhui were used,and 21 strains of virus were isolated and cultured in chicken embryos.The whole genome was sequenced after one-step ordinary real-time PCR amplification,and a phylogenetic tree was constructed with bioinformatics software to analyze its genetic evolution characteristics.Results The 21 H9N2 virus strains all belonged to G57 genotype,the hemagglutinin(HA)gene fragment evolved from JN/99 genotype,the neuraminidase(NA)gene originated from G9 genotype,the matrix protein(MP)gene originated from G1 genotype,the nuclear protein(NP),non-structural protein(NS),polymerase acid prorein(PA),polymerase basic protein 1(PB1)gene originated from F/98 genotype,and the polymerase basic protein 2(PB2)gene fragment evolved from ST/7488 genotype.HA cleavage site showed low pathogenic molecular characteristics,and there were important amino acid site mutations.Q226L,H183N,T189D,A190T amino acid mutations occurred in HA protein.62-64 deletion occurred in the stem of NA protein.Most strains had no PB2-E627K mutation,and all the strains had no D701N mutation.The PA protein of all the strains had 356R and 409N amino acid mutations,and the M2 protein of all the strains had S31N amino acid mutations.Conclusion Avian influenza A(H9N2)virus isolated from external environment in Anhui from 2019 to 2021 belonged to a multiple source reassortant strain,and has the molecular characteristics related to the gradual adaptation to mammalian hosts.It is necessary to pay close attention to the pathogenic evolution of the virus.