肠道病毒71型Lanzhou01株的分离及其生物学特性

目的对肠道病毒71型(Enterovirus 71,EV71)Lanzhou01株进行分离、鉴定,并分析其生物学特性,为EV71疫苗候选毒株的筛选奠定基础。方法从兰州市手足口病患者的粪便样本中分离EV71,经蚀斑克隆传代后,RT-PCR法检测其特异性;Vero细胞甲基纤维素蚀斑法分析病毒的毒力;克隆病毒的VP1基因,进行序列测定及分析;扫描电镜观察病毒的形态;以不同的MOI接种Vero细胞,观察细胞病变,并绘制病毒的增殖曲线;连续传代,分析VP1基因和氨基酸序列,并测定毒力,检测其遗传稳定性。结果经细胞传代和蚀斑克隆,获得增殖性能稳定的分离株,RT-PCR扩增产物经1%琼脂糖凝胶电泳分析可见约250bp的特异性条带;经VP1基因测序与进化树鉴定为EV71,其与浙江、深圳等地的EV71分离株的核苷酸序列同源性均大于99%,属于C4基因亚型;电镜下观察病毒颗粒为球形,直径20～30nm;该病毒在Vero细胞上培养后产生典型的细胞病变,毒力为6.8LgPFU/ml;经Vero细胞连续传30代,不同代次的病毒VP1基因序列的核苷酸序列同源性大于或等于99.6%,氨基酸序列几乎没有明显变异,毒力也无明显改变。结论已成功分离1株EV71,其生物学特性良好,为灭活疫苗的研制及疫苗候选株的筛选奠定了基础。

Isolation and Biological Characteristics of Enterovirus 71 Lanzhou01 Strain

Objective To isolate and identify enterovirus 71(EV71) Lanzhou01 strain,analyze its biological characteristics and lay a foundation of screening candidate virus strain of EV71 vaccine.Methods EV71 Lanzhou01 strain was isolated from the fecal specimens of patients with hand-foot-and mouth disease in Lanzhou City,China for plaque cloning and subculture,then determined for specificity by RT-PCR and for virulence by Vero cell methyl cellulose plaque method.The VP1 gene of the isolated strain was cloned and sequenced,the virulence was determined,and the morphology was observed by scanning electron microscopy.The strain was inoculated to Vero cells at various MOIs,and the CPEs were observed,based on which a virus proliferation curve was plotted.The isolated strain was subcultured continuously and analyzed for VP1 gene and amino acid sequences to evaluate the genetic stability.Results A isolated strain was obtained by subculture in cells and plaque cloning,of which the RT-PCR product showed a specific band at length of about 250 bp on 1% agarose gel electrophoretic profile.The strain was identified as EV71 of C4 subtype by VP1 gene sequencing and phylogenetic analysis,of which the homologies of nucleotides to the isolated EV71 strains in other regions including Zhejiang and Shenzhen were not less than 99%.The virus particles under electron microscope were in spherical forms,at diameters of 20 ～ 30 nm.The strain caused typical CPE of Vero cells,of which the virulence was 6.8 LgPFU/ml.After subculture in Vero cells for 30 passages,the nucleotide homologies of VP1 gene of various passages were more than 99.6%.However,no significant variation of amino acid sequence or significant change of virulence was observed.Conclusions An EV71 strain with good biological characteristics was isolated,which laid a foundation of developing inactivated vaccine and screening candidate vaccine virus strain.