G6P[1]型牛轮状病毒的分离培养及鉴定

Isolation and identification of G6P[1]bovine rotavirus

目的从轮状病毒阳性的牛粪便标本中,分离出一株G6P[1]型牛轮状病毒(Bovine Rotavirus,BRV),对其进行培养和鉴定。方法用PBS溶液重悬粪便标本并离心,将其上清过滤除菌和胰酶处理后,利用MA104细胞进行分离培养;通过逆转录-聚合酶链式反应(Reverse Transcription-polymerase Chain Reaction,RT-PCR)对样本VP4和VP7基因进行扩增和测序,与GenBank上的参考序列进行同源性分析,构建进化树,分析确定其G/P基因分型。通过聚丙烯酰胺凝胶电泳法(Polyacrylamine Gel Electrophoresis,PAGE)、噬斑实验和电镜(Transmission Electron Microscope,TEM)等技术对分离到的病毒进行鉴定和纯化。绘制病毒生长动力学曲线。结果分离培养了1株BRV毒株,将其命名BLL。VP7和VP4基因测序结果显示此毒株为G6P[1]型轮状病毒。PAGE胶结果显示分离株电泳型为长型,条带呈现A组轮状病毒排列电泳图谱;通过噬斑实验将毒株进行了纯化。电镜检测到典型的轮状病毒颗粒。病毒生长动力学曲线可发现病毒在感染后6 h已经开始复制。结论本研究成功分离到G6P[1]型牛型轮状病毒,为研究G6P[1]型轮状病毒的病原学特征提供实验基础和技术参考。

This study was aimed at isolating a G6P[1]group A bovine rotavirus(BRV)from bovine fecal specimens.Fecal specimens were resuspended in PBS and centrifuged,and the supernatants were filtered and activated with trypsin.The virus was isolated with MA104 cells.The VP4 and VP7 viral genes were analyzed with reverse transcription-polymerase chain reaction(RT-PCR),and sequencing to determine the G and P genotypes.The evolutionary characteristics of the virus were analyzed on the basis of homology with reference sequences in GenBank.The virus was identified with polyacrylamide gel electrophoresis,plaque assays and transmission electron microscopy.Growth curves of the virus were generated.One BRV strain,denoted BLL,was isolated.The sequencing results of the VP7 and VP4 genes indicated that this strain was a G6P[1]rotavirus.PAGE analysis indicated that the BLL strain had a long RNA migration,which was the typical of RVA.The strain was purified with plaque assays.Typical rotavirus particles were detected by transmission electron microscopy.Growth curves demonstrated that the virus began to replicate at 6 hpi.In this study,the G6P[1]strain of bovine rotavirus was successfully isolated,thus providing an experimental basis and technical reference for future studies of the pathogenetic characteristics of G6P[1]rotavirus.