BHV-1在牛胚气管细胞中的增殖规律研究

为研究牛疱疹病毒Ⅰ型(BHV-1)在牛胚气管细胞(EBTr)中的生长特性和增殖规律,参考文献设计合成特异性引物和探针,建立了TaqMan实时荧光定量PCR方法,检测BHV-1感染EBTr细胞6、12、24、48、72、96、120、144h后病毒的增殖规律,并观察对应时间点的细胞病变(CPE)。结果显示,用100TCID_(50)的BHV-1感染EBTr细胞,48h后开始出现细胞病变,72h细胞病变明显,120h细胞大部分变圆,开始脱落,144h细胞大面积脱落、崩解。实时荧光定量PCR检测结果表明,BHV-1感染EBTr在6h~24h内,病毒缓慢增殖,48h~96h,病毒增殖速度加快,拷贝数呈对数增长,120h~144h,BHV-1的含量仍然呈现升高的趋势,但增长速度变慢。结果证明,BHV-1能够在EBTr中产生CPE,而且CPE程度与病毒DNA增殖规律一致,该结果可以为深入研究BHV-1对EBTr细胞的致病机理提供基础资料。

Study on Proliferation Profile of BHV-1 in Bovine Embryonic Tracheal Cells

To reveal the proliferation profile of bovine herpesvirus 1 (BHV-1)in bovine embryonic tracheal (EBTr)cells,the specific primers and probe were designed and TaqMan real-time PCR method was devel-oped in this study.The proliferation of the virus in EBTr cells infected with BHV-1 were detected at 6,12, 24,48,72,96,120,144 h and cytopathic effect (CPE)was observed in the corresponding time points.The results showed that CPE were observed when the EBTr cells were inoculated by 100 TCID50 BHV-1 at 48 h post infection (PI).CPE became clear at 72 h PI,most of the cells became round and began to fall off from flasks at 120 h PI,most of the cells were disintegrated at 144 h PI.The results of real time PCR assay showed that BHV-1 were proliferated slowly when the cells infected within 24 hours,and rapidly during 48 to 96 hours.The load of BHV-1 was still increased,but the growth rate became slowed down during 120 h-144 h.In conclusion,not only BHV-1 can produce CPE in EBTr cells,but also the CPE was consistent with the proliferation profile of viral DNA.The results can provide basic data for further study of the pathogenic mechanism of BHV-1 in EBTr cells.