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[目的]分析杭州地区手足口病的病原谱构成,并对肠道病毒(EV)71型的VP1基因进行核苷酸和氨基酸的序列的比对分析,了解杭州地区EV71的基因特征。[方法]采集98份手足口病确诊病例的粪便标本,用EV71和柯萨奇病毒A组(CoxA)16型特异性引物进行逆转录聚合酶链反应(RT-PCR)鉴定,并选择6例EV71检测结果阳性的标本进行病毒分离并进行VP1基因的特异性扩增并进行测序,结合EV71各亚型的代表株构建系统进化树,并对VP1蛋白的氨基酸序列进行分析。[结果]通过RT-PCR特异性检测,发现EV71阳性结果22份,阳性率为22.4%;CoxA16阳性结果27份,阳性率为27.6%。测序结果表明6株EV71之间的VP1基因核苷酸同源性为92.2%～98.5%,氨基酸同源性为99.0%～100%。通过与各代表亚型进行核苷酸同源性比较后发现此次分离得到的EV71病毒都属于C4亚型。6株EV71与部分亚型代表株间在BCloop区域的氨基酸序列存在着差别,而SP70区域的氨基酸序列则完全一致。[结论]在手足口病患者中,由EV71感染引起的比例越来越大;此外,此次在杭州地区分离的EV71病毒都属于C基因型的C4亚型,尚没有证据说明此次在杭州地区分离的6株EV71毒株的毒力有增强。
[Objective] To analyze the pathogenic spectrum of HFMD in Hangzhou and to compare the nucleotide and amino acid sequences of the VP1 gene of enterovirus 71 (EV) in Hangzhou and to understand the gene characteristics of EV71 in Hangzhou. [Method] A total of 98 stool samples from HFMD patients were collected and identified by reverse transcriptase-polymerase chain reaction (RT-PCR) using EV71 and CoxA type 16 primers. Six patients EV71 was used for virus isolation and VP1 gene specific amplification and sequencing. The phylogenetic tree was constructed based on the representative strains of EV71 subtypes, and the amino acid sequence of VP1 protein was analyzed. [Results] Twenty-two EV71 positive results were detected by RT-PCR. The positive rate was 22.4%. The positive rate of CoxA16 was 27.6%. Sequencing results showed that the nucleotide homology of the VP1 gene of the six EV71 strains was 92.2% -98.5%, and the amino acid homology was 99.0% -100%. By comparing the nucleotide homology with the subtypes of each representative, it was found that the EV71 virus isolated in this time belonged to the C4 subtype. There were differences in amino acid sequence of BCloop between 6 strains of EV71 and some representative subtypes, but the amino acid sequence of SP70 was exactly the same. [Conclusion] In the HFMD patients, EV71 infection caused more and more. In addition, the EV71 virus isolated in Hangzhou area belonged to the C4 subtype of C genotype. There is no evidence to show that this EV71 virus in Hangzhou The virulence of 6 strains of EV71 isolated in the area was enhanced.