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目的测定浙江省丽水市1株H7N9禽流感病毒8个基因序列,从分子水平分析其基因特征和遗传变异特点。方法对人感染H7N9禽流感确诊病例标本在P3实验室进行犬肾传代细胞培养;反转录-聚合酶链反应扩增病毒8个RNA片段后测定基因序列;通过生物信息学软件将8个序列与GISAID和Gen Bank数据库下载的序列进行多重比对和匹配,发现基因特征,分析病毒关键位点的分子变异情况,最大似然法绘制系统进化树用于基因进化分析。结果从病例标本中分离到病毒:A/Zhejiang/LS01/2014(H7N9),测序获得8个基因核苷酸全序列。HA裂解位点只包含2个碱性氨基酸,HA蛋白受体结合关键氨基酸位点发生A134T、G186V和Q226L变异;在PB2蛋白上发现E627K突变;M2蛋白上发现耐药位点S31N。LS01株HA基因与A/chicken/Zhejiang/DTID-ZJU01/2013(H7N9)的同源性最高(98.90%),NA基因与A/chicken/Changzhou/C08/2013(H9N9)的同源性最高(98.60%),LS01株其他6个编码内部蛋白的基因均与禽源H9N2株高度同源(99.10%~99.89%)。LS01株HA和NA基因与沪苏浙一带流行的H7N9禽流感病毒株位于同一亚分支,有着直接的进化关系,在相近地域有进化关系较近的禽类流感病毒存在。结论 A/Zhejiang/LS01/2014(H7N9)株8个基因序列均符合禽源H7N9亚型禽流感病毒特征,其病原基因均为禽类来源。HA裂解位点具有低致病性禽流感病毒基因特点,Q226L和E627K位点发生变异可能与其对人具有高致病力相关。
Objective To determine the sequence of eight H7N9 avian influenza viruses in Lishui City, Zhejiang Province, and to analyze their genetic characteristics and genetic variation at the molecular level. Methods The pathogen inoculated with H7N9 bird flu were cultured in canine kidney of P3 in laboratory. The 8 RNA fragments were amplified by reverse transcription-polymerase chain reaction (PCR-RT) and sequenced. Eight sequences were sequenced by bioinformatics software. Multiple alignment and matching were performed with sequences downloaded from GISAID and Gen Bank databases. Genomic features were found, and molecular variations at key viral sites were analyzed. Maximum likelihood method was used to map phylogenetic tree for phylogenetic analysis. Results The virus was isolated from the specimens: A / Zhejiang / LS01 / 2014 (H7N9), and sequenced to obtain the complete nucleotide sequence of 8 genes. HA cleavage site contains only two basic amino acids, A134T, G186V and Q226L mutations occur at the binding site of HA protein receptor; E627K mutation is found on PB2 protein; and S31N is found on M2 protein. The HA gene of LS01 strain had the highest homology (98.90%) with A / chicken / Zhejiang / DTID-ZJU01 / 2013 (H7N9) and the NA gene had the highest homology with A / chicken / Changzhou / C08 / 2013 98.60%). The other 6 genes coding for the internal protein of LS01 were highly homologous (99.10% -99.89%) to avian H9N2. The HA and NA genes of LS01 strain are closely related to the H7N9 avian influenza virus strains prevailing in the Jiangsu, Jiangsu and Zhejiang provinces in the same sub-branch and have a direct evolutionary relationship with the avian influenza virus having a close evolutionary relationship in similar regions. CONCLUSION: The 8 A / Zhejiang / LS01 / 2014 (H7N9) strains all have the characteristics of avian H7N9 subtype avian influenza virus, and their pathogenic genes are all from poultry sources. HA cleavage sites have the characteristics of low pathogenic avian influenza virus genes, and the variation of Q226L and E627K sites may be related to their high virulence in humans.