1株肠道病毒71型毒株的分离与鉴定

Isolation and identification of one Enterovirus 71 strain

目的探讨手足口病病原体肠道病毒71型(EV71)的分离与鉴定方法。方法采集手足口病疑似病例的咽拭子标本1份接种于人横纹肌肉瘤细胞(rhabdomyosarcoma cell,RD细胞),通过观察病毒的细胞病变效应(cytopathiceffect,CPE)来分析EV71的分离效果。分别利用荧光定量聚合酶链反应(RT-PCR)及免疫印迹分析(Western Blotting)方法来鉴定EV71病毒特异性核酸和蛋白。扩增EV71VP1区全长基因,对其序列进行同源性比较及遗传进化分析。结果手足口病疑似病例咽拭子标本接种RD细胞传代至第2代后出现CPE。RT-PCR和Western Blotting检测结果均显示,感染组RD细胞中有EV71病毒特异性条带,暂命名为EV71分离株2016SHYP001。序列分析结果显示,2016SHYP001株病毒的VP1区核苷酸与C型代表株的同源性较高,为93.1%~98.3%;其中与C4a亚型代表株的同源性最高,为98.3%。进化树分析发现本地流行株与深圳地区流行株的亲缘较近。结论成功分离出一株新的EV71病毒株2016SHYP001,其株型为C4a亚型。

Objective To study the isolation and identification methods of enterovirus 71（EV71） in patients with hand-foot-mouth disease. Methods The throat swab specimen taken from one suspected case with hand-foot-mouth disease was inoculated into human rhabdomyosarcoma（RD） cells.The cytopathic effect（CPE）, real time-polymerase chain reaction（RT-PCR） and Western blotting assay were used to detect the effect of EV71 isolation and pathogenesis.The VP1 region of EV71 was amplified.Homology analysis on VP1 region and phylogenetic tree were carried out based on nucleotide sequence.Results The cytopathic effect was found on the second generation RD cells which were inoculated from the throat swab specimen which was taken from the suspected case. Furthermore, the results of RT-PCR and western blotting revealed that the nucleic acid and protein of EV71 were enriched by these assays in the infected group and tentatively named 2016SHYP〈001.The nucleic acid sequence homology analysis of EV71 strain 2016SHYP〈001 showed the isolated strain was close to C strains（the homology was 93.1%-98.3%）, especially close to C4a reference strains（the homology was 98.3%）. According to the genetic relationship tree, we found the local epidemic strain was close to strains from Shenzhen in genomic structure.Conclusion A new EV71 strain 2016SHYP〈001 was successfully isolated, which belongs to subgenotype C4a.