杭州市肠道病毒71型的分离与VP1区域序列分析

Isolation and sequencing of VP1 region of Enterovirus 71 srains in Hangzhou,Zhejiang,China

目的了解杭州市2008年和2011年肠道病毒71型(EV71)流行株VP1基因特征。方法手足口病患者咽拭子、粪便等样本先通过荧光RT-PCR方法初筛,然后选部分肠道病毒71型阳性样本进行病毒分离、培养;通过逆转录-聚合酶链反应(RT-PCR)鉴定上述培养物,并扩增VP1区全长基因和序列测定,利用生物信息学软件对序列进行分析,构建基因进化树。结果从2008年和2011年手足口样本中分离到EV71阳性毒株各4株,VP1基因全序列测定结果显示其与C4亚型代表株核苷酸和氨基酸的相似性最高,分别为93.8%～95.2%、99.0%～99.7%。结论杭州市手足口病患者感染EV71病毒的流行株属C基因型的C4亚型。

Objective To analyze the genetic characteristics of Enterovirus 71（EV71） strains isolated in 2008 and 2011 epidemics of hand-foot-mouth disease（HFMD） in Hangzhou.Methods The stool and throat swab samples of HFMD patients were tested for EV71 with one step reverse transcription real-time PCR（RT-PCR）.EV71 strains were isolated from RT-PCR positive specimens by cell culture.The VP1 complete gene of EV71 strains were amplified and sequenced.Results Four EV71 strains each were isolated in 2008 and 2011 respectively.The phylogenetic tree of EV71 strains was constructed by aligning the partial VP1 gene sequence of isolated strains with EV71 reference strains.Phylogenetic tree analysis revealed that the selected 8 stains were clustered with reference strains of C4 subgenotype.The homogeneity of nucleotides and amino acids were 93.8%~95.2% and 99.0%~99.7%,respectively.Conclusion The epidemic strains of HFMD in Hangzhou belonged to C4 subgenotype.