南宁市1株人感染高致病H7N9禽流感病毒遗传特性分析

Genetic characterization of a highly pathogenic avian influenza A(H7N9)virus strain isolated in Nanning city

目的通过对广西省南宁市2017年首例人感染高致病H7N9禽流感病毒株血凝素(HA)及神经氨酸酶(NA)基因测序,从分子水平分析毒株的溯源及遗传特性。方法通过RT-PCR扩增H7N9禽流感毒株HA基因和NA基因并测序,经NCBI数据库BLAST比对,利用MEGE 5.1等软件构建进化树及统计蛋白关键位点的变异情况。结果系统进化树表明中国H7N9毒株HA基因与NA基因主要为2个类群,长三角分支和珠三角地区分支,南宁毒株A/Nanning/01/2017(H7N9)HA和NA基因均在珠三角分支上,与广东毒株高度同源。病毒HA蛋白裂解位点插入4个氨基酸由PEIPKGR↓GLF突变为PEVPKRKRTAR↓GLF,含有5个碱性氨基酸,使其具有高致病性禽流感病毒的分子特征;毒力相关位点225由天冬氨酸(D)突变为甘氨酸(G)(D225G),毒力增强;受体结合186位点由甘氨酸(G)突变为缬氨酸(V)(G186V);飞沫传播关键氨基酸位点没有发生突变组合;糖基化位点高度保守。NA蛋白丢失5个氨基酸,毒力可能增强,耐药性位点、糖基化位点均相对保守,未发生突变。结论南宁市人感染H7N9禽流感病毒可能来源于广东省珠三角地区禽类的感染,人传人的可能性不大,对NA抑制剂药类敏感,但毒株已经具有高致病性禽流感病毒的分子特征,毒力增强。南宁市外环境已经检测到H7N9核酸阳性标本,提示需要加强监测有效防控传染源。

Objectives To analyze the traceability and genetic characteristics of a highly pathogenic H7N9 avian influenza virus strain isolated in Nanning city, 2017 through sequencing of hemagglutinin （HA） and neuraminidase （NA） gene of the virus. Methods Both HA and NA gene sequences of the isolated virus were amplified with real-time reverse transcription PCR （RT-PCR） and sequenced with Basic Local Alignment Search Tool （BLAST） database of National Center for Biotechnology Information （NCBI）. The phylogenic tree analysis was drawn with Mega 5.1 and variations in dominant loci for proteins were statistically analyzed. Results The established phylogenetic tree showed that the HA and NA gene sequences of the virus were mainly divided into two branches of Yangtze River Delta and Pearl River Delta; the HA and NA gene of the isolated A/Nanning/01/2017 （H7N9） virus were evolved form the Pearl River Delta Branch and were highly homologous with those of the avian influenza H7N9 virus isolated in Guangdong province. The inserted 4 amino acids at HA protein cleavage site mutated from PEIPKGR↓GLF to PEVPKRKRTAR↓GLF; the mutation involved in 5 basic amino acids and resulted in molecular characteristics of high pathogenicity of avian influenza virus. The amino acid of virulence-related site 225 changed from D-aspartic acid to glycine （D225G）, resulting in enhanced virulence; the amino acid of receptor binding site （RBS） 186 changed from glycine to valine （G186V）. No mutated combination was observed for on droplet propagation-related key amino acid sites and glycosylation-related sites were highly conservative. Five amino acids were absent for NA protein, which may increase virulence of the virus strain. No mutations were observed for loci related to drug resistance and glycosylation. Conclusion The avian influenza A H7N9 virus isolated in Nanning city may be derived from poultry viruses isolated in Guangdong province in Pearl River Delta region and human to human transmission of the virus is unlikely to occur. The virus strain is sensitive to neuraminidase drugs, but is of the molecular characteristics of highly pathogenic avian influenza virus. Contamination of H7N9 avian influenza virus exists in Nanning external environments, suggesting that we need to strengthen surveillance, effective prevention and control of infection source.