基于纳米孔测序技术对新型重组H3N2禽流感病毒的测序鉴定及其基因特征分析

The identification of a novel reassortant H3N2 avian influenza virus based on nanopore sequencing technology and genetic characterization

目的利用纳米孔测序技术对新型重组H3N2禽流感病毒进行测序鉴定并分析病毒的基因特征。方法对2021年广州市某农贸市场外环境H3N2禽流感阳性样本进行鸡胚培养,联合病毒全基因组序列靶向扩增和纳米孔测序技术进行病毒高通量测序。通过生物信息学软件,分析病毒基因特点。结果系统发育进化树显示,该毒株各基因片段均属于欧亚进化分支,宿主来源均为禽源。其HA基因与我国H3N6禽流感病毒亲缘关系较近。NA基因与2017-2020年我国H3N2禽流感病毒亲缘关系较近。PB1基因与2016-2022年我国广西壮族自治区、福建省H5N6禽流感病毒亲缘关系较近,与广州市H5N6禽流感流行株PB1基因亲缘关系较远。其余内部基因片段来源复杂,具有明显的遗传多样性。分子特征显示,该毒株具有典型的低致病性禽流感病毒分子特征,倾向结合禽源受体。生物特性相关重要蛋白位点上,该毒株发生PB2-L89V、PB1-L473V、NP-A184K、M1-N30D/T215A、NS1-P42S/N205S等致病性增强突变。结论本研究通过纳米孔测序鉴定一株新型重组H3N2禽流感病毒,分析发现该病毒与H5N6禽流感病毒发生了基因重组。目前该病毒跨种传播能力较低,但有必要密切关注H3亚型禽流感病毒的流行和变异。

Objective To identify a novel reassortant H3N2 avian influenza virus using nanopore sequencing technology and analyze its genetic characteristics.Methods The positive samples of the H3N2 avian influenza virus,collected from the external environment in the farmers'market of Guangzhou,were cultured in chicken embryos.The whole genome was sequenced by targeted amplification and nanopore sequencing technology.The genetic characteristics were analyzed using bioinformatics software.Results The phylogenetic trees showed that each gene fragment of the strain belonged to the Eurasian evolutionary branch,and the host source was of avian origin.The HA gene was closely related to the origin of the H3N6 virus.The NA gene was closely related to the H3N2 avian influenza virus from 2017 to 2020.The PB1 gene was closely related to the H5N6 avian influenza virus in Guangxi Zhuang Autonomous Region and Fujian Province from 2016 to 2022 and was not related to the PB1 gene of the H5N6 avian influenza epidemic strain in Guangzhou.The other internal gene fragments had complex sources with significant genetic diversity.Molecular characteristics indicated that the strain exhibited the molecular characteristics of a typical low pathogenic avian influenza virus and tended to bind to the receptors of avian origin.On important protein sites related to biological characteristics,this strain had mutations of PB2-L89V,PB1-L473V,NP-A184K,M1-N30D/T215A,and NS1-P42S/N205S.Conclusions This study identified a novel reassortant H3N2 avian influenza virus by nanopore sequencing,with the PB1 gene derived from the H5N6 avian influenza virus.The virus had a low ability to spread across species,but further exploration was needed to determine whether its pathogenicity to the host was affected.