长沙市活禽市场外环境H9N2监测与患者分离株HA基因序列的比较研究

Surveillance of H9N2 avian influenza virus in live poultry markets in Changsha and the homology of HA gene with virus isolated from human

目的了解长沙市活禽市场外环境H9N2禽流感病毒(avian influenza viras,AIV)污染情况及病毒血凝素基因(hermagglutinin,HA)的遗传进化和分子特征,并与长沙市分离的2株人感染H9N2毒株相比较。方法采集长沙市活禽市场监测点外环境样本,实时荧光RT-PCR检测禽流感病毒FluA及H5、H9亚型核酸;H9阳性样本采用鸡胚分离毒株,血凝实验及实时荧光RT-PCR鉴定毒株的型别;普通逆转录PCR扩增病毒HA基因全长并测序,生物信息学软件对序列进行比对和遗传进化分析。结果 2015—2017年共采集活禽市场环境样本1 519份, FluA核酸阳性率为78.80%(1 197/1 519);其中H5阳性率为4.34%(66/1 519)、H9阳性率为41.74%(634/1 519)、H5+H9阳性率为20.28%(308/1 519)。3例环境来源和2份人感染H9N2毒株样本HA基因核苷酸序列同源性为96.31%~98.73%,氨基酸同源性为97.02%~99.15%;系统进化树显示,人源与禽源HA基因均属于欧亚分支中的Y280亚群。序列分析结果显示,5株病毒HA裂解位点均为RSSR↓GLF,符合低致病力病毒特征;受体结合位点均出现Q234L突变,具有结合哺乳动物α-2,6唾液酸受体的能力;糖基化位点均为7个;个别氨基酸位点人源与禽源毒株存在差异(第297、510位氨基酸)。结论长沙市活禽市场H9亚型禽流感病毒污染严重,病毒HA基因与从人体中分离的毒株同源性高,存在跨种感染人的风险。

Objective To understand the prevalence of H9 N2 avian influenza virus(AIV)in live poultry markets(LPMs)in Changsha and the molecular characteristics of hemagglutinin(HA)gene,to describe the homology with 2 strains of H9 N2 virus isolated from human cases.Methods Environmental samples were collected from LPMs in Changsha and real time RT-PCR was used to identify FluA,H5,H9.Viruses were isolated from H9 positive samples using specific-pathogen-free chicken embryos and the genotypes were determined by hemagglutination test and real time RT-PCR.HA genes of the isolated strains were then amplified and sequenced,molecular characteristics and evolution relationship were analyzed using bioinformatic software.Results A total of 1519 environmental samples were collected in LPMs in Changsha during 2015 and 2017,the overall positive rate of FluA was 78.80%(1197/1519),with the positive rate of 4.34%(66/1519)for H5,41.74%(634/1519)for H9,and 20.28%(308/1519)for both H5 and H9.The nucleotide homology of HA gene of three environmental strains and two human H9 N2 virus ranged from 96.31%to 98.73%and amino acid homology ranged from 97.02%to 99.15%.Phylogenetic analysis of HA gene showed that all the above five strains belonged to Y280 subtype.The HA cleavage site sequence of the five strains was RSSR↓GLF,representing the esenteavage sit of the low pathogenic avian influenza virus.The mutation of Q234 Lin receptor-binding domain of AIV enabled its binding to hending tα-2,6 sialic acid receptor.There were seven glycosylation sites identified,and several amino acid sites such as the 297 th,510 th were different between strains isolated from environmental samples and human cases.Conclusions Changsha,the LPMs are severely contaminated by AIV,especially H9 subtype,and the virus HA gene is highly homologous with virus isolated from human cases,indicating apotential risk of transmission from birds to human.