柯萨奇病毒B4型江苏地方株的病毒分离与基因进化分析

Virus isolation and gene evolution analysis on Jiangsu local strain of coxsackievirus B4 type

目的建立柯萨奇病毒B4型病毒的分离方法,并对已分离到的江苏地方株进行基因进化分析。方法使用荧光定量PCR方法检测江苏省哨点医院收治的呼吸道感染患者咽拭子样本中肠道病毒感染情况,使用HeLa细胞对阳性样本进行病毒分离,然后进行VP4基因测序与进化树分析。结果荧光定量PCR检测肠道病毒阳性12例,1份样本经病毒分离后呈现细胞病变效应,RT-PCR扩增出特异性的VP4基因,测序显示该病毒株与其他20株CVB4病毒株的VP4核苷酸同源性都在86.56%~93.59%之间,确诊为CVB4江苏地方株。进化树分析显示,该病毒株只与CVB4病毒KY369904株形成一个聚簇。结论本研究采用HeLa细胞分离与鉴定到了1株肠道病毒,VP4基因进化分析判断为CVB4江苏地方株,对预防与控制当地的肠道病毒感染具有重要价值。

Objective To established the isolation method of coxsackievirus B4 (CVB4), and to perform genetic evolution analysis of the isolated Jiangsu local strain.Methods Fluorescence quantitative PCR was used to detect enteroviruses in the throat swabs from patients with respiratory infections admitted to the sentinel hospitals of Jiangsu province. The positive samples were used for virus isolation with HeLa cell line, followed by VP4 gene sequencing and phylogenetic analysis.Results Twelve samples were positive of enterovirus by fluorescence quantitative PCR. Cytopathic effect was observed in one sample during virus isolation. The VP4 gene were amplified by specific RT-PCR. The sequence analysis showed that the similarities of the VP4 gene sequence with the other 20 CVB4 strains ranged from 86.56% to 93.59%, and the strain was identified as a Jiangsu local strain of CVB4. The phylogenetic tree showed that the strain only clustered with CVB4 strain KY369904.Conclusions HeLa cell line was used to isolate and identify one strain of enterovirus in the present study. The virus strain was identified as Jiangsu local strain of CVB4 using evolution analysis of VP4 gene. The work presented important value for the prevention and control of local enterovirus infections.