湖南省首例人感染H3N8亚型禽流感病例的病毒分离及分子进化分析  

Viral isolation and molecular evolution analysis of the first humancase of H3N8 subtype avian influenza infection in Hunan Province

作　　者：裴瑞青[1] 张如胜[1] 叶文[1] 欧新华[1] 姚栋[1] 李灵之[1] 肖姗[1] 黄政[1] PEI Rui-qing;ZHANG Ru-sheng;YE Wen;OU Xin-hua;YAO Dong;LI Ling-zhi;XIAO Shan;HUANG Zheng(Changsha Center for Disease Control and Prevention,Changsha 410004,China)

机构地区：[1]长沙市疾病预防控制中心,长沙410004

出　　处：《中国人兽共患病学报》2023年第4期364-375,共12页Chinese Journal of Zoonoses

基　　金：湖南省自然科学基金项目(No.2022JJ70131);湖南省卫生健康委科研项目(No.202212064383);长沙市自然科学基金项目(No.kq2202040)。

摘　　要：目的本研究从流感样病例样本中分离出湖南省首例人感染H3N8亚型AIV毒株,并对该毒株进行了分子进化分析,为人感染H3N8亚型禽流感病毒(Avian influenza virus,AIV)的防控提供实验室依据。方法通过实时荧光RT-PCR方法检测甲型流感病毒核酸、接种MDCK细胞进行病毒培养并通过血凝试验进行初步鉴定,继而对分离到的AIV进行高通量二代基因测序,并将测序结果进行相似性比较、基因特征和进化分析。结果鉴定出人感染H3N8亚型AIV分离株(A/Changsha/1000/2022(H3N8),相似性分析显示HA和NA基因序列与来自河南省的人感染AIV株A/Henan/4-10CNIC/2022(H3N8)相似性最高,6个内部基因(MP、NS、PB1、PB2、NP、PA)均来源于H9N2亚型AIV。HA蛋白裂解位点为PEKQTR/GLF,未出现连续重复碱性氨基酸,符合低致病性AIV的特征,NA基因出现了耐药位点I312V突变,可能对奥司他韦有耐药性改变。PB2基因出现E627V宿主适应性突变,M2蛋白出现S31N金刚烷胺耐药性位点突变。分子进化显示HA基因属于欧亚分支,NA基因属于北美分支,NS、PB2、MP和PB1基因与湖南省内来源的H9N2毒株亲缘关系密切。结论此次新发现的人源A/Changsha/1000/2022(H3N8)毒株所有基因片段均来源于禽类,属于新型重组低致病性AIV,有可能突破物种屏障发生禽到人之间的传播,建议完善流感/AIV监测网络系统,增加对H3亚型AIV的监测。This study was aimed at isolating the first human H3N8 subtype Avian influenza virus(AIV)strain in Hunan Province from an influenza-like case sample and performing molecular evolutionary analysis of the strain,to provide a laboratory basis for the prevention and control of human H3N8 subtype AIV infection.Avian influenza virus nucleic acid was detected by real-time fluorescence RT-PCR,inoculated with MDCK cells,and initially identified with hemagglutination assays,followed by high-throughput second-generation gene sequencing of the isolated AIV to assess similarity,and genetic characterization and evolutionary analysis.Finally,the human H3N8 subtype AIV isolate(A/Changsha/1000/2022(H3N8)was identified,and similarity analysis indicated that the HA and NA gene sequences had the highest similarity to the human AIV strain A/Henan/4-10CNIC/2022(H3N8)from Henan Province.Six internal genes(MP,NS,PB1 PB2,NP,and PA)were all derived from AIV subtype H9N2.The HA protein cleavage site,PEKQTR/GLF,did not show consecutive repeats of basic amino acids,in agreement with low pathogenicity AIV.The NA gene showed an I312V mutation in the resistance site,which might have altered resistance to oseltamivir.The PB2 gene showed an E627V host-adapted mutation,and the M2 protein showed a mutation in the S31N amantadine resistance locus.Molecular evolution demonstrated that the HA gene belonged to the Eurasian branch;the NA gene belonged to the North American branch;and the NS,PB2,MP,and PB1 genes were closely related to those of the H9N2 strain of Hunan provincial origin.In conclusion,the newly identified human-derived A/Changsha/1000/2022(H3N8)strain,in which all gene fragments are of avian origin,is a novel recombinant low-pathogenic AIV with the potential to break the species barrier and achieve avian-to-human transmission.Thus,the influenza/AIV surveillance network system should be improved,and surveillance of H3 subtype AIV should be increased.

关 键 词：禽流感 H3N8亚型 分子进化 突变 

分 类 号：R373.1[医药卫生—病原生物学]