猪轮状病毒四川株的分离鉴定及增殖规律

Isolation and identification of porcine rotavirus in Sichuan province and its proliferation profile detected by fluorescence quantitative PCR

为了解猪轮状病毒（PoRV）在MARC-145细胞系上的培养特性及增殖规律，本试验利用MARC-145细胞，从四川仔猪腹泻样品中分离到1株PoRV，并通过PCR检测、病毒理化实验与微量中和实验证实，命名为SC-R株。用含不同浓度胰酶营养液培养SC-R株48h后，分别收集病毒液进行定量分析；同时，以SC-R分离株感染MARC-145细胞，在感染后不同时间分别收集感染病毒液，利用PoRV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PoRV生长曲线。结果表明，用含3％胰酶营养液培养的细胞液中PoRV的RNA含量明显高于其他组；-步生长曲线显示细胞外病毒RNA含量呈“s型”曲线增长，感染后0～8h为潜伏期，病毒RNA含量维持在较低水平；8～36h为突破期，病毒RNA含量呈对数增长；感染48h增长速度减缓，维持在较高水平，逐步进入稳定期。胰酶可增加PoRV对细胞的感染性，3％是本试验最为适用的胰酶浓度；PoRV感染MARC-145后在细胞内增殖并逐步释到细胞外。

To study the culture characteristics and the proliferation regularity of porcine rotavirus in MARC-145 cell,a strain of virus （SC-R） was isolated from diarrhea piglets samples in Sichuan by cultivation in MARC-145 cells. Through PCR detection, physical and chemical properties experiment and microneutralization test confirmed that virus was porcine rotavirus（PoRV）, named SC- R. SC-R were cultured with liquid containing different concentrations trypsin nutrient for 48 h, then collecting virus liquid respectively for the quantitative analysis;after PoRV fluorescent quan titative PCR analysis for the quantitative of the viral RNA in the different samples, the PoRV growth curve was drawn. Meanwhile, the effect of trypsin in cell infection process was quantified. The results showed that RNA content of PoRV cells cultured with 3 % trypsin liquid nutrient was significantly higher than that of other groups;One step growth curve showed that the virus RNA content of extracellular existed ＂S＂ curve,0-8 h after infection was the incubation period,the virus RNA content was maintained at a low level;8-36 h was the breakthrough period,the viral RNA content showed logarithmic growth;48 h after infection the growth maintained a stable period in a higher level. Trypsin can increase infection of viral to the cells, and 3 % is the most suitable concen tration in this research. When infected MARC-145 cells,PoRV proliferated in the cell and released into the extracellular gradually.