上海2009甲型H1N1流行性感冒病毒全基因组分析

Whole-genome analysis of 2009 influenza A virus subtype H1N1 isolates in Shanghai

2009年全球暴发2009甲型H1N1流行性感冒(简称流感)疫情,上海于2009年5月出现第1例输入型病例。为了解上海地区输入型2009甲型H1N1流感病毒的生物学特征,以上海较早发现的2例输入型甲型H1N1流感患者作为研究对象,分离出A/Shanghai/37T/2009和A/Shanghai/71T/2009病毒,利用实时定量荧光反转录-聚合酶链反应(RT-PCR)鉴定病毒,通过扫描透射电子显微镜观察、免疫荧光检测、全基因组测序和生物信息软件分析,对这2株流感病毒形态、结构、耐药性、基因特点和病毒型别等进行研究。结果显示,病毒呈现正黏病毒颗粒形态特征;犬肾(MDCK)细胞内的病毒能与患者恢复期血清反应。此2株病毒的全基因核酸序列和氨基酸序列与美国参考株A/California/04/2009(H1N1)有较高同源性,其中第31位氨基酸残基发生改变。对金刚烷胺耐药,而对奥司他韦敏感。基于全基因组的系统发育分析,确认此2株病毒属2009甲型H1N1流感病毒。

A new strain of influenza A virus subtype H1N1 first identified in April 2009 has caused global pandemics. The first confirmed case in Shanghai was in May 2009. In order to study the characteristics of the 2009 influenza A virus subtype H1N1 in Shanghai,the virus stains A/Shanghai/37T/2009 and A/Shanghai/71T/2009,which were identified by real-time reverse transcriptase-polymerase chain reaction （RT-PCR）,were isolated from two imported cases. Then,the characteristics of viral structure,drug resistance,gene and subtyping of the two viruses were studied by electron microscopy,immunofluorescence technique,genome sequencing,and bioinformation software analysis. Virus particles from Madin-Darby canine kidney （MDCK） cell culture showed that it contained numerous enveloped and highly pleomorphic orthomyxovirus particles with a diameter ranging from 60 nm to 80 nm and surrounded by a fringe of well-defined surface projection. The genome nucleotide sequence and amino acid sequence were then compared. The results showed that the two virus strains and the reference strain A/California/04/2009（H1N1） were highly homologous. Amantadine resistance occurred at residue 31（Asn） in M2 protein,while in the neuraminidase protein domain,it was sensitive to oseltamivir. Phylogenetic analysis showed that the two strains were highly genetically similar to those previously reported on the 2009 influenza A virus subtype H1N1 strains,suggesting the same origins for the two Shanghai strains and 2009 influenza A virus subtype H1N1 strains.