2023年河南省外环境H7N9禽流感病毒的基因组序列分析

Genome sequences of H7N9 avian influenza virus in poultry-related environment in Henan Province in 2023

目的分析活禽市场相关环境中检出的H7N9禽流感病毒(avian influenza virus,AIV)的遗传进化和分子特征。方法采集禽类粪便、污水,以及脱毛机和案板擦拭拭子等标本,实时荧光定量PCR鉴定甲型流感病毒和H7N9亚型。对于H7N9阳性标本以甲型流感病毒通用引物扩增病毒全基因组并测序,利用BLAST和MEGA X软件进行序列比对、系统进化和分子特征分析。结果2023年2月采集于许昌市活禽市场的7份外环境标本中检出4份H7N9 AIV阳性,3份H7N9 AIV阳性标本测序成功,其各基因核苷酸一致性较高(98.37%~100.00%)。BLAST分析显示主要与我国2020—2021年国内禽中分离的H7N9毒株一致性最高。遗传进化分析显示3株病毒株聚在同一分支,与最近环境分离株相聚较近,而与最近人/禽感染病毒株关系较远。通过与各时期的代表性病毒株序列比对发现,本研究检出的病毒株呈禽高致病性,裂解位点处插入了4个氨基酸KRAA,病毒株血凝素受体结合位点为QSG,属于禽结合受体,血凝素出现G186I位点突变。聚合酶碱性蛋白2未出现哺乳动物适应性E627K突变。未检测到与神经氨酸酶抑制剂(奥司他韦)和聚合酶酸性蛋白抑制剂(巴洛沙韦)耐药相关的R292K和I38T位点突变,提示病毒对上述药物敏感性未降低。M2蛋白出现S31N突变,提示对烷胺类药物耐药。结论从活禽市场检出的3株H7N9病毒株呈禽高致病性,与既往感染人/禽代表株相比,未明显增加人受体结合力、哺乳动物致病性、病毒传播力和耐药相关分子位点。

Objective To analyze the genetic evolution and molecular characteristics of H7N9 avian influenza virus(AIV)isolated in a live poultry market.Methods Samples such as poultry feces,sewage,and hair removal machine and chopping board swabs were collected.Real-time fluorescent quantitative PCR was used to detect influenza A virus and H7N9 AIV in the samples.The whole genome of H7N9 AIV was amplified with influenza A virus universal primers and sequenced.BLAST and MEGA X were used for sequence alignment,phylogenetic analysis and molecular characterization.Results Seven poultry-related environment samples were collected in the live poultry market in Xuchang city in February 2023,and four were positive for H7N9 AIV.The whole genome sequences of three H7N9 AIV isolates were successfully obtained,and the isolates shared high nucleotide identity in different genes(98.37%-100.00%).BLAST analysis showed they were highly identical to H7N9 strains isolated from domestic poultry in China from 2020 to 2021.Genetic evolution analysis showed that the three isolates clustered in the same branch and were closer to the recent environmental isolates than to the recent strains isolated from human or avian.Through comparison with the sequences of the representative strains in different periods,it was found that the isolated strains in this study showed high avian pathogenicity with four amino acids KRAA inserted at the cleavage site;the hemagglutinin receptor-binding site was QSG,which was an avian binding receptor;there was a G186I mutation in hemagglutinin.Mammalian-adaptive mutation E627K was not detected in polymerase basic protein 2.Mutations(R292K and I38T)associated with drug resistance to neuraminidase inhibitor(oseltamivir)and polymerase acidic protein inhibitor(baloshavir)were not detected,suggesting that these isolates remained susceptible to these drugs.A S31N mutation was found in M2 protein,indicating they were resistant to alkamines.Conclusions The three H7N9 AIV strains isolated in the live poultry market have high avian pathogenicity,but there are no significant increase in mutations related to the binding ability to human receptors,mammalian pathogenicity,viral transmissibility,or drug resistance as compared with previous representative strains causing human or avian infection.