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目的研究2012年青海省手足口病(hand-foot-and-mouth disease,HFMD)病例中分离的肠道病毒71型(enterovirus 71,EV71)的VP1区基因特征。方法采集青海省HFMD患者的咽拭子标本共56份,进行病毒分离,经逆转录-聚合酶链反应(reverse transcription polymerase chain reaction,RT-PCR)方法对分离到的EV71进行VP1编码区扩增,并对扩增产物进行核苷酸序列测定和分析,与其他参考毒株序列片段构建基因亲缘性关系树,并进行核苷酸和氨基酸同源性分析。结果在56份临床标本中,共分离到8株阳性病毒分离物,均鉴定为EV71。对8株EV71进行VP1区核苷酸序列测定,核苷酸同源性为95.2%～100.0%,氨基酸同源性为96.6%～100%。与其它39株各基因型和基因亚型的EV71代表株构建的亲缘进化树中显示,青海省2012年EV71分离株属于C4基因亚型C4a进化分支。结论 2012年青海省从HFMD病例中分离到的EV71为C4基因型中的C4a进化分支,加强对EV71的分子流行病学监测,了解EV71的基因特征,对预防和控制EV71的暴发具有重要意义。
Objective To investigate the VP1 gene characteristics of enterovirus 71 (EV71) isolated from hand-foot-and-mouth disease (HFMD) in 2012 in Qinghai Province. Methods A total of 56 throat swabs from HFMD patients in Qinghai Province were collected for virus isolation. The isolated EV71 was amplified by reverse transcription-polymerase chain reaction (RT-PCR) The nucleotide sequence of the amplified product was determined and analyzed. The phylogenetic tree was constructed with the sequences of other reference strains and nucleotide and amino acid homology analysis. Results Of the 56 clinical samples, 8 positive isolates were identified, all of which were identified as EV71. The nucleotide sequences of VP1 region of eight EV71 strains were determined. The nucleotide homology was 95.2% ~ 100.0%, and the amino acid homology was 96.6% ~ 100%. The phylogenetic tree constructed with EV71 strain of 39 other genotypes and subgenotypes showed that the EV71 isolate of Qinghai province belonged to the C4 clade C4a evolutionary branch. Conclusion EV71 isolated from HFMD cases in Qinghai province in 2012 was the C4a evolutionary branch of C4 genotype. It is of great importance to enhance the molecular epidemiological surveillance of EV71 and to understand the genetic characteristics of EV71, which can prevent and control EV71 outbreak.