猪传染性胃肠炎病毒四川株的分离鉴定与一步生长曲线的测定

Isolation and identification of porcine transmissible gastroenteritis virus in Sichuan Province and determination of one-step growth curve

为研究猪传染性胃肠炎病毒的生长规律,利用PK-15细胞从四川仔猪腹泻样品中分离得到1株病毒,经PCR检测、病毒理化试验与微量中和试验证实,该分离株为猪传染性胃肠炎病毒(TGEV),被命名为SC-T。以SC-T分离株感染PK-15细胞,感染后不同时间分别收集感染细胞与上清,利用TGEV荧光定量检测方法对不同样品中病毒RNA进行定量分析,绘制TGEV一步生长曲线。结果显示,感染后第2～8小时,细胞内病毒RNA含量迅速增长,第8～32小时细胞内病毒RNA含量变化不大,维持在较高水平。细胞外病毒RNA含量呈"S型"曲线增长,感染后第0～6小时为潜伏期,病毒RNA含量维持在较低水平;第6～28小时为突破期,病毒RNA含量呈对数增长;感染后第28～32小时增长速度减缓,维持在较高水平,逐步进入稳定期。结果表明,TGEV感染PK-15细胞,病毒在细胞内增殖到一定数量以后,便逐步向胞外释出。

To study the growth dynamics of porcine transmissible gastroenteritis virus（TGEV）,a strain of virus（SC-T） was isolated from diarrhea piglets samples in Sichuan by cultivation in PK-15 cells,and the isolated virus was proved to be TGEV through PCR detection,the physical and chemical properties experiment,and microneutralization test.The SC-T virus was used to infect PK-15 cells,and the infected cells and supernatant were collected at different time points post-infection.Fluorescent quantitative PCR was used to analyse TGEV RNA quantity in different samples,and the one-step growth curve of SC-T was determined according to the results.In result,at hour 2-8 post-infection,TGEV RNA increase rapidly in cells,and at hour 8-32 post-infection,virus RNA maintained at a high level.TGEV RNA in supernatant was in an S shaped growth curve.At 0-6 h post-infection,there was a period of incubation,and virus RNA maintained at a low level.At 6-28 h post-infection,there was a stage of breakthrough,and virus RNA was undertaken a logarithmic growth.At hour 28-32 post-infection,the growth rate slowed down,and virus RNA maintained at a high level and stepped into stationary phase gradually.In conclusion,virion was released from the infected cells while TGEV multiplied to a certain number in TGEV-infected PK-15 cells.