浙江省肠道病毒71型的分离与VP1区域序列分析

Isolation and sequencing of VPl region of enterovirus 71 strains in Zhejiang, China

目的 研究浙江省手足口病患者中肠道病毒(EV)71型分离株的病毒基因特征。方法采集手足口病患者粪便、疱疹液和咽拭子标本，进行病毒分离和逆转录-聚合酶链反应(RT-PCR)特异性扩增进行鉴定，同时选取其中6株EV71分离毒株．对其抗原决定簇部位VP1区进行核苷酸序列测定并参考EV71 A、B、C各基因型的参考毒株和以往中国EV71的分离毒株进行同源性分析和构建系统发生树。结果从14份标本中分离出EV9株，经中和试验和RT-PCR特异性扩增，均证实为EV71。其中6株EV71分离毒株与A、B基因型参考毒株的核苷酸和氨基酸同源性分别为82.9%～85.5%和94.9%～98.0%；与C基因型比较，同源性分别为89.2%～94.1%和97.0%～99.0%。而与C基因型的C1、C2、C3、C4亚型代表株的核苷酸同源性分别为91.0%～92.2%、90.2%～90.3%、89.2%～89.5%、96.7%～96.9%，其中与国内以往分离到的C4亚型毒株的核苷酸同源性可达93.8%～97.1%。在系统发生树上，这6株毒株均在C基因型中，与属CA亚型的11株毒株属同一分支。结论 浙江省手足口病患者中分离的EV71毒株属C基因型的CA亚型。

Objective To study the gene characterization of enterovirus 71 （EV71） virus strains isolated from clinical specimens of children with hand-foot-and-mouth disease （HFMD） in Zhejiang province. Methods Virus were isolated from clinical samples including stool, throat swab and vesicle from patients with HFMD. The EV71 isolates were identified by microneutralization assay and reverse transcriptase PCR （RT-PCR） with specific primer pair for VPI genes of EV71. Complete VPI gene sequences（891 nucleotides） for recent 6 EV71 isolates were determined and compared with that of A,B, C genotype reference EV71 strains and 11 EV71 China isolates available from GeneBank by homogeneity and phylogenetic tree analyses. Results 9 strains of EV were isolated from 14 clinical specimens. Data from microneutralization and RT-PCR results indicated that all the strains belong to EV71. The nucleotide and amino acid homogeneity of these 6 Zhejiang strains with the representative isolates of A and B genotypes were 82.9 % -85.5 % and 94.9 % -98.0 % respectively; with the representative isolates of C were 89.2 % - 94.1% and 97.0 % -99.0 % respectively. There were 91.0 % -92.2 % , 90.2 % -90.3 %, 89.2 % -89.5 %, 96.7 % -96.9 % nucleotide homology with representative strains of CI, C2, C3, CA subgenotypes of EV71. The nucleotide homogeneity of these 6 EV71 isolated strains with 9 previously isolated Chinese strains appeared to be 93.8%-97.1%. These 6 EV71 isolated strains were within genotype C subgenogroup CA in the phylogenetic tree. Conclusion The recently identified EVTI isolates in Zhejiang province belonged to subgenogroup CA.