Objective To detect VP1 and 2A genes of Enterovirus type 71 (EV71) isolated from clinical specimens of patients with light or heavy symptoms and analyze the homogeneity and phylogenetic tree. Methods Fifty clinical specimens of children with hand-foot-and-mouth disease ( HFMD) were dealed with, which were tested by RT-PCR assay with specific primer pairs for EV71. EV71 isolates from patients with light or heavy clinical symptoms were tested by RT-PCR assay with two specific primer pairs for VP1 and 2A genes of EV71 respectively. All of the PCR products were sequenced and compared with that of previously isolated EV71 isolates available from GenBank by homogeneity and phylogenetic tree analyses. Results The RT-PCR results indicated that 30 isolates were EV71, 13 of 30 isolates were from clinical specimens of patients with light symptoms of hand-foot and mouth, the other were from clinical specimens of patients with heavy symptoms of complications. VP1 genes and 2A genes of 10 EV71 isolated strains including 5 light strains and 5 heavy strains were sequenced and compared with that of previously isolated 5 EV71 Chinese isolates available from GenBank (fuyangEU703814.1, xi_anHM003207. 1, shandongEU753418.1, shenzhenFJ607337.1, henanGU366191. 1) by homogeneity and phylogenetic tree analyses. The homogeneity of VP1 and 2A genes of the 10 EV71 isolated strains and 5 previously isolated strains were between 94.7% -99.4% and 93.6% -99.3% respectively, with the representative isolates of A and B genotypes was between 81.0%-84. 6% and 78. 4%-82. 2% respectively. The data suggested that all of the 10 Chinese isolates belong to EV71 genotype C. There were only 87.8% -90.2% homology among these 10 strains and the representative strains of C1, C2, C3 sub-genotypes of EV71 but 96. 8% -99.6% homology among these 10 strains and the representative strains of C4 sub-genotypes of EV71, this suggested that these 10 Chinese isolates composed the C4 sub-genotype, of the C genotype, that formed a single branch in the phylogenetic tree. Conclusion EV71 of sub-genotype C4 distributed in Mainland China, and VP1 genes have close genetic relationship between isolated strains. There is no obvious difference in 2A genes between clinical specimens of patients with light or heavy symptoms by homogeneity and phylogenetic tree analyses.%目的 检测临床分离的肠道病毒71型(EV71)轻型、重型毒株VP1基因、2A基因的核苷酸序列,进行遗传进化途径分析.方法 将50份临床手足口病患者粪便标本处理后分离病毒,用EV71特异性引物进行RT-PCR扩增,获得EV71毒株,针对不同临床背景的标本,分别选取轻型、重型的毒株,用特异性引物分别对VP1基因及2A基因进行RT-PCR扩增,对其产物进行测序及遗传进化分析,并探讨它们之间的差别.结果 50份临床手足口病标本中有30份检测是EV71,其中轻型(手足口)13份,重型(手足口并发脑炎或心肌炎)17份.轻型和重型中各自选取5株对VP1基因和2A基因进行测序和遗传学分析,通过同源性比较和构建系统发生树发现,此10株EV71病毒和中国大陆已发表的5株EV71病毒(fuyangEU703814.1、xi_anHM003207.1、shandongEU753418.1、shenzhen-FJ607337.1、henanGU366191.1)全部属于C基因型,且核苷酸同源性较高,VP1基因和2A基因的同源性分别在94.7%～99.4%和93.6%～99.3%范围内.本次分离的10株EV71病毒与A、B基因型代表株比较,核苷酸同源性分别为81.0%～84.6%和78.4%～82.2%,差异较大.与已知的C1、C2、C3亚型代表株比较,核苷酸同源性在87.8%～90.2%,差异≥10%,与已知的C4亚型代表株比较,核苷酸同源性在96.8%～99.6%,因此认为可将这10株病毒划分为C4亚型.在系统发生树上,这10株病毒形成一个较独立的分支.结论 EV71 C4亚型病毒在中国大陆有较广泛的传播,且毒株之间VP1基因的遗传关系紧密;2A基因在轻型和重型病例毒株之间遗传差异不大.
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