猪血凝性脑脊髓炎病毒在PK-15细胞中的增殖特性

Replicative kinetics of porcine hemagglutinating encephalomyelitis virus(HEV) in PK-15 cells

利用光学显微镜观察、间接免疫荧光(IFA)、Real-time PCR和病毒感染滴度(TCID50)等测定方法,分别从病毒抗原分布、病毒基因组复制水平以及病毒感染滴度变化等方面对猪血凝性脑脊髓炎病毒(HEV-67N)在猪肾上皮传代细胞系(PK-15细胞)上的增殖特性进行了研究。使用HEV-67N株感染24孔细胞培养板内的PK-15细胞,接种剂量为400个TCID50(104.37)/孔,间接免疫荧光检测结果显示,在感染后8h即可检测到被荧光抗体标记的感染细胞,且随着感染时间的延长,出现荧光的细胞数量逐渐增多,至感染后32h,几乎所有的细胞均出现有荧光。Real-time PCR检测结果显示,病毒基因组RNA的复制在感染后32～48h呈快速上升趋势,其基因组拷贝数在感染后48h达到最高值,之后增殖速度减慢,至感染后56h细胞出现CPE。TCID50的测定结果显示,HEV感染滴度的变化趋势与基因组RNA含量的变化相一致,在感染后32～48h增殖速度最快,之后逐渐减缓,至72h病毒感染滴度达到最高。

To study the replication characteristics of HEV-67N strain in porcine kidney PK 15 cell lines,indirect im- munofluorescent assay（IFA） ,real-time RT PCR assay and virus titration assay were used to detect HEV protein expression,virus genome RNA replication and assembly of matured virus particles, respectively. The monolayer cells were inoculated with HEV-67N strain at 400 TCID50 per well iri 24-well tissues culture plates. The results showed that the viral antige could be detected as early as 8h p. i,and the cells were fully infected at 32 h p. i. The virus genome RNA synthesized rapidly between 32-48 h p. i,reached its peak at 48 h p. i. The virus-infected cells exhibited obvious CPE at 56 h p. i. The result of virus titration assay and Rreal time RT PCR assay are consistent with each other.