轮状病毒P[2]G3株在人二倍体细胞KMB17上的适应性

目的探讨轮状病毒P2]G3株在人二倍体细胞KMB17上的适应性。方法轮状病毒P2]G3株在猴肾细胞MA104上经蚀斑筛选纯化后,得到的克隆以不同MOI接种至人二倍体细胞KMB17上。用微量滴定法与蚀斑法平行检测病毒滴度,用免疫荧光法检测病毒的增殖情况,最后进行病毒基因组稳定性检测。结果经MA104细胞培养的轮状病毒P2]G3株在人二倍体细胞KMB17上可产生明显的细胞病变(CPE),但病变出现的时间随病毒传代次数的增加而延迟,抗原性也逐渐减弱。通过蚀斑筛选纯化得到的克隆病毒,可在KMB17细胞上稳定地增殖。以0.05MOI病毒量接种可产生稳定的CPE,时间为72～96h。连续传代至第10代,病毒滴度达105.5PFU/ml,病毒基因组稳定。结论轮状病毒P2]G3株在人二倍体细胞KMB17上表现出毒力及增殖能力的衰退;经蚀斑筛选纯化可获得毒力较强的病毒株,并在KMB17细胞中稳定复制、增殖。

Adaptivity of Rotavirus P[2]G3 Strain in Human Diploid Cell Line KMB17

Objective To explore the adaptivity of rotavirus P2]G3 strain in human diploid cell line KMB17.Methods The clones of rotavirus P2]G3 strain cultured in monkey kidney MA104 cells were purified by plaque screening and inoculated to human diploid cell line KMB17 at various MOI.Determine the virus titer by microtitration and plaque formation test and the virus proliferation by IFA,and perform genomic stability test.Results Obvious CPE was observed in KMB17 cells inoculated with ro-tavirus P2]G3 strain after culture in MA104 cells.The antigenicity of virus decreased with the increasing passage,while the appear-ance of CPE was delayed.The purified P2]G3 strain was proliferated stably in KMB17 cells.Stable CPE was observed in the KMB17 cells 72-96 h after inoculation with P2]G3 strain at MOI of 0.05.The P2]G3 strain after subculture in KMB17 cells for 10 passages reached a titer of 105.5 PFU / ml and showed good genomic stability.Conclusion Both virulence and proliferation level of rotavirus P2]G3 strain cultured in MA104 cells decreased after subculture in KMB17 cells.Virulent P2]G3 strain was pu-rified by plaque screening,and replicated and proliferated stably in KMB17 cells.