牛轮状病毒的分离与细胞培养特性

探讨牛轮状病毒(BRV)的细胞培养方法,为研发诊断试剂盒和疫苗提供依据.将RT-PCR阳性样品在MA-104细胞及Vero细胞上进行培养,盲传4代后MA-104细胞出现病变(CPE),主要表现为细胞脱落,出现拉网现象,部分出现死亡.这表明轮状病毒能够在MA-104细胞上生长,并引起细胞病变;而Vero细胞无病变.用胰酶处理的病毒接种MA-104细胞,用含2μg/mL、3μg/mL和5μg/mL三种(A、B和C)不同浓度胰酶的培养液在MA-104和Vero细胞对犊牛轮状病毒传代培养.将分离培养的病毒经聚丙烯酰胺凝胶电泳,结果表明BRV在C组培养液中分离培养最好,聚丙烯酰胺凝胶电泳图显示病毒核酸呈4∶2∶3∶2排列,与参考株NCDV相似,呈典型A群轮状病毒的特征.

Researches of Isolation and Cell Cultivation of Bovine Rotavirus in Vero and MA-104 Cells

The aims of the present experiments was to study the methods of cell cultivation for bovine rotavirus(BRV),and to provide the experimental bases for developing diagnostic kits and vaccines production.Bovine rotavirus(BRV),which was got from the RV positive samples of calves,was cultivated in MA-104 and Vero cells with three medium that contained 2 μg/mL,3 μg/mL and 5 μg/mL concentrations of trypsin.The isolated BRV were electrophoresed using polyacrylamide gel.The results showed that the cytopathic effects(CPE) was not observed in Vero cells on the fourth generation after blind preach.MA-104 cells appeared significant CPE on the fourth generation after blind preach with the clearest in C medium(5 ug/mL trypsin),CPE rate was 100%,indicating that MA-104 cells was the susceptible cells for isolating and culturing rotavirus.Gel electrophoresis diagram illustrated the BRV nucleic acids were 4∶2∶3∶2 arrangement,similar with NCDV(reference strains),had typical features of rotavirus group A.In conclusion,MA-104 cells were susceptible cells for isolating and culturing rotavirus,trypsin treatment could enhance the efficacy of rotavirus cell culture.The VP7 homology of BRV GSB01 strain was 86.5%,and VP7 was an unstable hydrophobic protein.