一种肠道病毒71型毒株的一般生物学特性检测及分子鉴定

Generally biological characteristics and molecular identification of an enterovirus 71 strain

目的对一种肠道病毒71型(enterovirus 71,EV71)毒株进行一般生物学特性检测及分子鉴定。方法将EV71毒株接种于人横纹肌瘤(rhabdomyosarcoma,RD)细胞进行病毒培养,通过EV71致细胞病变效应(CPE)分析、CCID50法检测子代病毒产量、实时荧光定量PCR(qRT-PCR)检测EV71核酸合成情况以及免疫荧光印迹检测EV71蛋白表达水平,来确认其生物学特性;RT-PCR扩增EV71保守区的基因片段后进行基因测序,并将测序结果经BLAST程序进行同源性比对。结果该毒株接种RD细胞48 h后出现明显的CPE,其可在RD细胞中增殖,最终导致细胞死亡;该毒株在RD细胞中处于快速复制状态,其核酸合成与蛋白表达随时间的延长大量增加;该毒株与EV71/wuhan/3018/2010(KF501389)的核苷酸序列同源性为100%。结论该EV71毒株为EV71/wuhan/3018/2010,在RD细胞中可快速增殖,毒力较强,适合作为抗EV71药物筛选毒株。

Objective To identify an enterovirus 71（EV71） strain and test its generally biological characteristics.Methods EV71 was inoculated onto human rhabdomyosarcoma cells for culture. EV71-mediated cytopathogenic effect（CPE） was determined by plaque assay. The progeny proliferation was analyzed through CCID_（50） virus titration. The expressions of viral RNA and viral proteins were determined by real-time fluorescent quantitative PCR（q RT-PCR） and immunofluorescence assay respectively. The gene sequences of conserved region of EV71 was amplified by RT-PCR and sequenced, and the result was compared with those in Gen Bank by BLAST software. Results Obvious CPE was observed in RD cells 48 h after infection, indicating that the EV71 strain might grow in RD cells and cause the cell death. The strain was reduplicated rapidly in RD cells, of which the expressions of viral RNA and protein increased as time went on.The homology of nucleotide sequence of the strain was 100% to that of EV71 / wuhan / 3018 / 2010 strain. Conclusion The strain was identified as EV71 / wuhan / 3018 / 2010, which was proliferated rapidly in RD cells, showed strong virulence, and was suitable for screening of drugs against EV71.