绍兴市手足口病肠道病毒71型基因特征研究

A study on genetic characteristics of Enterovirus type 71 of hand- foot- mouth disease in Shaoxing city

目的研究绍兴市2012—2013年手足口病患者的肠道病毒71型（EV 71）病毒分离株的基因特征。方法采集手足口病患者粪便、疱疹液和咽拭子标本,采用逆转录聚合酶链反应（RT-PCR）特异性扩增进行EV 71鉴定,阳性的样本进行病毒分离,对10株分离株分A、B、C、D和E五段进行分段PCR,测序后并拼接成全长,以EV 71 A、B和C各基因型的参考毒株和以往中国EV 71的分离毒株为参考株,基于核苷酸序列和氨基酸序列对VP1区分别进行同源性分析和系统发生树构建。结果 RT-PCR结果证实分离的10株病毒均为EV 71,与C4a亚型亲缘关系最近,它们之间核苷酸和氨基酸同源性分别为95.06%-97.87%和93.60%-98.99%;与亲缘关系最远的参考毒株Cox A16的核苷酸和氨基酸同源性分别为61.17%-62.74%和68.00%-71.38%。在系统发生树上,这10株EV 71均在C基因型中,与属C4a亚型的病毒株属同一分支。结论绍兴地区手足口病患者中分离的EV 71毒株属C基因型的C4a亚型进化分支。

Objective To undersyand the genetic characteristics of Enterovirus Type 71（ EV 71） isolated from clinical specimens of children with hand- foot- mouth disease（ HFMD） in Shaoxing city from 2012 to 2013. Methods RNA was extracted from specimens collected from stool,throat swab and vesicle from children with HFMD,and EV 71 were identified by reverse transcriptase polymerase chain reaction（ RT- PCR）. Ten positive samples were isolated and were amplified by segmented PCR,then,they were spliced into a full- length after sequenced according to the reference strains.Homology analysis on region VP1 and phylogenetic tree were carried out based on nucleotide sequence and amino acid sequence. Results The results indicated that the ten isolated strains were all EV 71 positive. Sequence alignment of VP1 showed that the nucleotide and amino acid homogeneity of these 10 strains were related to C4 a genotypes,with 95. 06%-97. 87% and 93. 60%- 98. 99% respectively. Phylogenetic tree based on VP1 indicated that these 10 strains all belonged to C4 a genotype. Conclusion The identified EV 71 strains isolated from HFMD children in Shaoxing city belong to genotype C4 a.