贵州省首例人感染高致病性禽流感H7N9突变株血凝素分子特征与溯源分析

Molecular characteristics and tracing of hemagglutinin of the first highly pathogenic avian influenza A （H7N9） virus mutant strain infection case in Guizhou Province

目的了解贵州省人感染H7N9禽流感病毒血凝素（hemagglutinin, HA）基因的分子特征、溯源和疾病风险，为高致病性禽流感H7N9病毒的预防和控制提供依据。方法采用实时PCR对5株高致病性禽流感H7N9毒株标本（1株人感染鼻咽拭子标本，4株活禽市场环境标本）进行核酸的提取、HA基因的扩增和测序，运用生物信息学软件对H7N9禽流感病毒HA基因的同源性、遗传进化、受体结合关键位点、致病相关位点和糖基化位点变异情况进行分析。结果贵州省威宁县人感染H7N9禽流感病毒HA基因与当地活禽市场环境中H7N9禽流感病毒核苷酸和氨基酸同源性分别为99.8%和99.6%，而4株活禽市场环境中的H7N9禽流感病毒核苷酸和氨基酸同源性分别为100.0%和100.0%。与2017年广西人感染的A/Guangxi/5/2017毒株同源性最高，分别为99.7%～99.9%和99.4%～99.8%；与2016年底广东环境中分离的毒株A/Environment/Guangdong/ C16283222/2016同源性为99.0%～99.2%和98.9%～99.2%，而与WHO推荐的A/Shanghai/2/2013和A/Anhui/1/2013候选疫苗株的同源性为96.8%～97.0%和95.8%～96.2%。与广西毒株A/Guangxi/5/2017遗传进化距离最近。5株毒株HA蛋白裂解位点突变为PEVPKRKRTAR↓GLF，为高致病性禽流感突变株；受体结合关键位点均发生了G186V的突变，均未发生Q226L突变，5株毒株均发生的新突变为A363S，仅感染人的毒株发生的突变是R56K和I297V；5个潜在糖基化位点中仅421NWT和493NNT发生位置后移的变异。结论贵州省威宁县5株H7N9禽流感病毒均为高致病性禽流感突变株，候选疫苗株匹配保护效果可能已经降低，HA蛋白裂解位点、G186V和新的突变位点的变异可能增强了H7N9禽流感病毒对人体的易感性和致病性。

ObjectiveTo investigate the molecular characteristics and tracing of the hemagglutinin （HA） gene, and to analyze the risk of human infection with influenza virus A （H7N9） in Guizhou Province, so that to provide evidence for the prevention and control of highly pathogenic avian influenza A （H7N9）.MethodsNucleic acids of 5 strains of H7N9 including 1 sample of the patient′s nasopharyngeal swab and 4 samples of the live poultry market （LPM） environment were extracted and HA genes were amplified and sequenced. Then the homology, genetic evolution and the pivotal sites related to receptor binding regions, pathogenicity and potential glycosylation of the avian influenza A （H7N9） viruses were analyzed by a series of bioinformatics softwares.ResultsHomology analysis revealed that the homologies of nucleotide and amino-acid of the HA gene of H7N9 strains from the patient and LPM in Weining County, Guizhou Province were 99.8% and 99.6%, respectively, while those of 4 strains from LPM were both 100%. The homologies of nucleotide and amino-acid of the HA gene of H7N9 strains were the highest with the strain of A/Guangxi/5/2017 isolated from a Guangxi infected patient （99.7%-99.9% and 99.4%-99.8%, respectively）, while those with the strain isolated from LPMs environment at the end of 2016 （A/Environment/Guangdong/C16283222/2016） were 99.0%-99.2% and 98.9%-99.2%, respectively. However, the homologies of nucleotide and amino-acid of the HA gene of H7N9 strains with A/Shanghai/2/2013 recommended by world health organization and the candidate vaccine strain A/Anhui/1/2013 were 96.8%-97.0% and 95.8%-96.2%, respectively. Phylogenetic analysis showed that the 5 strains had the nearest genetic distance to the strain A/Guangxi/5/2017. All the 5 strains cleavage site sequences of HA protein showed mutation of PEVPKRKRTAR↓GLF, and they were highly pathogenic avian influenza viruses mutant strains, which all had mutation of G186V at the receptor binding sites of HA gene, while no Q226L mutation was found. All 5 strains had new mutation of A363S, and new mutations of R56K and I297V were only found in the strain isolated from the patient. Among the five potential glycosylation motifs in the HA, only 421NWT and 493NNT had variation of the position post shift.ConclusionsAll the 5 H7N9 strains isolated in Weining County, Guizhou Province are highly pathogenic avian influenza mutative viruses. The current candidate vaccine may not provide a very good protection. The mutations of cleavage site of HA protein, G186V as well as other new mutation sites of HA may enhance the susceptibility and pathogenicity to human beings.