【摘要】 目的分离甲型H1N1流感病毒,分析福建省首例病毒分离株全基因组序列和遗传特征,为研究病毒进化、致病性、流行规律提供科学依据。方法采用MDCK细胞和Real-time PCR法进行病毒分离、鉴定;提取病毒RNA,通过RT-PCR扩增其8个基因片段,测定核苷酸序列,利用生物信息软件拼接全基因组序列;分析重要基因位点,利用GENBANK中相关序列对首例病毒分离株A/Fujian/01/2009(H1N1)进行基因进化树分析。结果从82例甲型H1N1流感确诊病例标本中分离出50株甲型H1N1流感病毒,第一代分离阳性率60.98%。在福建省首次获得甲型H1N1流感病毒株及全基因组序列。基因组序列分析证明:该毒株与2009年大流行株高度同源,其基因组存在四源重组现象;氨基酸位点分析其对达菲药物敏感,对金刚烷胺类药物耐药;相对于猪流感代表株A/Swine/Iowa/15/1930(H1N1)存在6个HA抗原决定簇位点变异。结论MD-CK细胞对甲型H1N1流感病毒具有较高敏感性;福建省首例甲型H1N1流感病例分离病毒株与北美流行株高度同源;相对于以往古典型猪流感代表株出现了HA蛋白抗原性漂移;为今后进一步开展甲型H1N1流感病毒分子生物学研究奠定基础。更多还原

【Abstract】 Influenza type A H1N1 virus was isolated from the confirmed cases with influenza and the complete genome sequence was analyzed from the virus isolated from the first confirmed case with influenza A H1N1 (A/Fujian/01/2009 (H1N1). The virus isolation and identification were performed by means of inoculation of the throat swab specimens of patients into MDCK cells and the real-time PCR assay. The viral RNA was extracted from MDCK cell culture inoculated with throat swab,and then transcribed. Genome fragments were amplified by PCR,and the PCR products were sequenced. The complete genome sequence was obtained and analyzed via bioinformatics. It was found that 50 strains of influenza type A H1N1 virus were isolated from 82 throat swab specimens of the confirmed cases with influenza. As demonstrated by bioinformatics,this new influenza A virus was highly homologous with that of in 2009 influenza pandemic. It also showed that the virus was a quadruplex reassortment with classical swine flu genes,eurasia swine flu genes,avian flu genes and human flu genes. In comparison with the representative strain of swine influenza A/swine/Iowa/15/1930,six HA antigenic determinant focus variations were found. Analysis of amino acid sequences indicated that this virus possessed oseltamivir sensitivity and amantadine resistibility. Phylogenetic analysis further confirmed that each of the 8 genes of virus strain was closely related to current new H1N1 virus originated in the influenza pandemic in North America,2009.更多还原