摘要
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。
Objectives To understand the genetic characteristics and mutation of 1 avian influenza A（ H7N9） virus strain isolated in Lishui. Methods The sample was collected from a confirmed H7N9 virus infection case and the virus was cultured on M DCK cells in P3 laboratory. By using RT-PCR,the 8 RNA fragments of the cultured virus were sequenced and analyzed with bio-software. The sequences of 8 RNA fragments were multi-aligned with sequences downloaded from GISAID and Gen Bank. The gene characteristics and molecular mutation of key evolution-determined nucleotide sites were analyzed.Phylogenetic analysis was performed based on the phylogenetic trees. Results One human H7N9 virus strain,A / Zhejiang /LS01 /2014（ H7N9）,was successfully isolated and whole genome was sequenced. The results showed that there was only 2basic amino acids in cleavage site of HA,3 mutations（ A134 T,G186V and Q226L） occurred in the crucial sites related with the receptor of HA protein,1 mutation（ E627K） occurred in PB2 protein,and also 1 drug resistance site（ S31N） was found in M2 protein. The LS01 strain＇s HA protein and NA protein shared 98. 90% homology with that of A / chicken / Zhejiang /DTIDZJU01 /2013（ H7N9） strain and 98. 60% homology with that of A / chicken / Changzhou / C08 /2013（ H9N9） strain,respectively. Phylogenetic analyses showed that other proteins were highly homologous（ 99. 10- 99. 89%） with avian H9N2 viruses. The results showed that the isolated LS01 strain was directly evolved from avian H7N9 virus,which had circulated in Shanghai,Zhejiang and Jiangsu. The avian flu viruses with high similarity were also found in adjacent regions. Conclusion The characteristics of 8 RNA fragments of LS01 strain was consistent with avian-originated H7N9 subtype virus. The HA cleavage sites had the properties of lowpathogenic avian influenza virus,but mutations of Q226 L and E627 K might be related with high pathogenicity to human beings.
出处
《疾病监测》
CAS
2016年第2期106-110,共5页
Disease Surveillance
基金
浙江省科技厅公益性技术应用研究计划项目(No.2012C33002)~~
