摘要
上海市农业科学院畜牧兽医研究所在上海郊区分离到一株猪流行性腹泻病毒（PEDV）。以人工攻毒发病猪的小肠粘膜无菌处理后接种已长成单层的VERO（非洲绿猴肾）传代细胞，并在维持液中加入胰酶50μg／mL和2％的小牛血清。盲传第4代时，于接种后的第3天出现致细胞病变作用（CPE）。自第8代开始，CPE于接种后16h开始出现，以后就一直有规律地稳定出现。CPE的主要表现为细胞的折光度降低、细胞圆缩、颗粒变性、细胞融合而形成多核巨细胞。至40～48h，CPE明显，细胞部分脱落，收获。同时，将人工攻毒发病猪的小肠粘膜无菌处理后的病毒与经胰酶消化分散的VERO一起培养。并在生长液中加入胰酶50μg／mL，3～4d长成单层。如此经盲传若干代，可用荧光抗体检测到在细胞浆内有特异性的荧光颗粒出现。目前，该带毒的VERO细胞已传至102代。
The Animal Husbandry and Veterinary Research Institute,Shanghai Acade-my of Agricultural Sciences, had isolated one porcine epidemic diarrhea virus strain in a Shanghai suburb. In this study, the aseptically treated small intestine mucosa of newborn piglet diseased by artificial challenge were inoculated onto passaged cells of African green monkey kidney(VERO) cells which had grown into monolayers, 5O μg/mL trypsin and 2% calf serum were added to the maintenance medium. When four blind passages were done,the cytopathic effects (CPU)occurred on the third day after inoculation. From the passage 8 on,the CPU began to occur 16 hours after inoculation and thereafter occurred stably and regular-ly. The main expressions of CPE consisted in decreased cell diopter,rounding reduced cells,modified particles and formation of multinuclear large cells due to cell fusion. 4O^8O hoursafter inoculation,the CPE became obvious,partial cells broke away from glass and were col-lected. In the meantime,the aseptically treated viruses from small intestine mucosa of new-born piglet diseased by artificial challenge were cocultivated with trypsin-digested and dis-persed VERO cells and 5O μg/mL trypsin was added to the growth medium. The monolayers formed within 3~4 days. When some blind passages were done,specific fluorescent particles were detected in cell plasm by fluorescent antibody assays. At present,the passage 1O2 of the viruliferous VERO cells is already done.
出处
《上海农业学报》
CSCD
1999年第2期41-44,共4页
Acta Agriculturae Shanghai
关键词
猪
流行性腹泻病毒
分离培养
鉴定
病毒
Porcine Epidemic Diarrhea virus (PEDV )
Isolation
Identification
Blind passage
