Abstract: Objective To establish a culture method for Kilham Rat Virus (KRV) using the Rat glioma cell line (C6) instead of primary rat embryo cells (RE). Methods 500 TCID50 KRV was inoculated into C6 (The C6 cells were diluted to 2×105/ml and then cultivated overnight before infection) and the infected cells were cultivated until the CPE (cytopathic effect) reached “++~+++”. Then, the viral antigen was identified by immunofluorescence (FITC) assay and the viral titer in the supernatant of culture was detected by hemagglutination test (HA). In addition, the virus was identified by DNA sequencing of the conserved regions of FRV genome. And the virulence of viral culture (TCID50) was determined by the 96 wells cell culture method. Results The CPE of C6 cells reached “+++” at 4~5 day post infection with KRV. The result of FITC indicated that the viral antigen was positive for KRV, and HA titer of the viral culture reached 1:5,120.The cultivated virus was identified as KRV, as the sequence of it had 97% similarity with KRV, and the virulence of KRV obtained by infection on C6 cells was 104.6 TCID50/0.1mL. Conclusion The infection of KRV on C6 cells and subsequently identification experiments indicated that KRV could be maintained in C6 cells instead of RE cell.

Key words: Kilham Rat Virus, C6 cell, RE cell, FITC assay, Hemagglutination inhibition test

摘要： 目的 建立用大鼠胶质瘤细胞系( Rat glial cell line C6)替代大鼠原代胚细胞(Primary rat embryo cells，RE)来 培养大鼠细小病毒（Kilham Rat Virus，KRV）的方法。方法 将500 TCID50 KRV培养物接种到75T-C6细胞培养瓶 中（ 细胞接种量为2×105/mL）， 培养过夜， 待细胞病变CPE达++～+++时， 分别用免疫荧光(FITC)鉴定所培 养病毒的特异抗原，用血球凝集试验(HA)测定培养物上清的效价，用DNA测序鉴定所培养的病毒，最后用96孔 板培养法测定KRV的TCID50。结果 KRV在接种到C6细胞的第4～5天， 细胞发生明显的病变，CPE可达++++， FITC鉴定呈KRV抗原阳性， 病毒的培养上清中HA效价为1:5 120，测序结果表明， 该病毒序列与NCBI中KRV序 列同源性达98％ ， 确定为KRV。收获的KRV的TCID50为104.6/0.1 mL。结论 通过对C6细胞系培养KRV方法 的标准化和对所培养病毒的一系列鉴定表明，用大鼠胶质瘤细胞系可以替代大鼠原代胚细胞系进行大鼠细小病毒 的培养。

关键词: 大鼠细小病毒, 大鼠胶质瘤细胞, 大鼠原代胚细胞, 免疫荧光试验, 血球凝集试验