2019−2020年苏中地区某活禽市场H9N2禽流感病毒分子进化

Genetic evolution of avian influenza A(H9N2)virus in a live poultry market in central Jiangsu,China,2019–2020

【背景】20世纪90年代以来,H9N2禽流感病毒成为危害我国养禽业及人类健康和公共卫生的重要病原。【目的】了解苏中地区2019−2020年活禽市场H9N2禽流感病毒的分子进化特征。【方法】通过荧光定量PCR法对标本进行流感病毒分型检测,原始标本用SPF鸡胚进行病毒分离,用特异性引物对病毒分离物进行全基因组测序,利用BLAST、ClustalX和MEGA 6等软件进行序列比对和系统发育分析。【结果】2019−2020年间从苏中地区某农贸市场采集到231份环境和禽类标本,共检出34份甲型流感病毒,其中33份为H9N2亚型。阳性标本接种SPF鸡胚,分离到20株H9N2病毒。对其中11株病毒进行全基因组测序,系统发育分析表明11株病毒的HA和NA都属于H9N2禽流感Y280-like系的G57基因型。根据HA和NA的进化特征,11株病毒可分为5个基因组合(A、B、C、D和E),其中A(n=5)是优势流行基因组合。11株H9N2分离病毒的HA蛋白HA1和HA2亚单位的裂解位点是一个碱性氨基酸R,具有低致病性禽流感病毒的特征。HA蛋白的受体结合部位有4个氨基酸位点(I155T、H183N、A190T/V和Q226L)发生突变,推测这些病毒可能增强了对人SAα2-6Gal受体的结合力。【结论】苏中地区活禽市场H9N2禽流感病毒进化活跃,存在外溢传播人的风险,应该加强对活禽市场禽流感病毒的监测和跨种传播的研究。

[Background]Since the 1990s,H9N2 avian influenza virus has become a major threat to human and animal health.[Objective]To understand the molecular evolutionary characteristics of H9N2 avian influenza virus in a live poultry market in central Jiangsu,2019–2020.[Methods]The specimens were detected by real-time quantitative PCR method,and SPF chicken embryos were used for the isolation of virus strains from the specimens.The whole genome of each isolate was sequenced with specific primers.BLAST,ClustalX and MEGA 6 were used for sequence alignments and phylogenetic analysis.[Results]A total of 231 environmental and poultry specimens were collected from a farmers’market from 2019 to 2020,from which of 34 influenza A virus strains were detected,including 33 strains of H9N2 subtype.Twenty strains of H9N2 virus were isolated with SPF chicken embryos,and the whole genomes of 11 strains were sequenced and aligned.The HA and NA gene sequences indicated that the 11 strains belonged to G57 genotype of Y280-like lineage.According to the evolutionary characteristics of HA and NA,the 11 strains can be divided into 5 gene constellations(A,B,C,D and E),of which constellation A(n=5)was dominant.The cleavage site of HA1 and HA2 subunits of HA protein of the 11 strains was a basic amino acid R,which indicated low pathogenicity.Four mutations(including I155T,H183N,A190T/V and Q226L)were occurred in the receptor-binding region of HA protein,indicating that these viruses had enhanced ability of binding to human SAα2-6Gal receptor.[Conclusion]The H9N2 avian influenza virus in the live poultry market in central Jiangsu is characterized by active evolution and has a risk of transmission to people.Efforts should be made to strengthen the monitoring and the research on cross-species transmission of avian influenza virus in the live poultry market.