1株猪源牛病毒性腹泻病毒的分离与鉴定

畜牧兽医学报 ›› 2012, Vol. 43 ›› Issue (3): 416-423.doi:

1株猪源牛病毒性腹泻病毒的分离与鉴定

邓宇1,2，3，孙春清2，张宏彪2，4，蔺涛2，4，张荣2，龙进学2，黄律2，
曹三杰1，袁世山2，文心田1*，郑浩2*

1.四川农业大学动物医学院，雅安 625014；2.中国农业科学院上海兽医研究所，上海 200241；3.西昌学院动物科学学院，西昌 615000；4.南京农业大学动物医学院，南京 210095

收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2012-03-28 发布日期:2012-03-28
通讯作者: 文心田1*，郑浩2*

Isolation and Identification of a Pig Bovine Viral Diarrhea Virus

DENG Yu1,2,3, SUN Chunqing2，ZHANG Hongbiao2,4, LIN Tao2,4 , ZHANG Rong2, LONG Jinxue2，HUANG Lv2，CAO Sanjie1，YUAN Shishan2, WEN Xintian1*，ZHENG Hao2*

1. College of Veterinary Medicine, Sichuan Agricultural University, Yaan 625014, China; 2. Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China; 3.School of Animal Science, Xichang College, Xichang 615000, China; 4. College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China

Received:1900-01-01 Revised:1900-01-01 Online:2012-03-28 Published:2012-03-28
Contact: WEN Xintian1*，ZHENG Hao2*

摘要/Abstract

摘要： 从BVDV阳性仔猪病料中分离病毒，为开展猪源BVDV病原学研究奠定基础。将处理后BVDV阳性仔猪组织样品接种MDBK细胞，分离到1株猪源BVDV，命名为SD0803株。通过细胞培养、直接免疫荧光、5′UTR与Npro PCR扩增、电镜观察、TCID50测定及对其分子进化特征加以分析。结果表明，该毒株在MDBK细胞上盲传至13代未出现细胞病变。在直接免疫荧光试验中呈阳性荧光信号。PCR扩增分别获得5′UTR与Npro预期大小DNA片段。电镜观察，病毒粒子略呈圆形，有囊膜，直径约50 nm。病毒滴度为10-6.5 TCID50·0.2 mL-1。SD0803 5′UTR、Npro序列进化分析显示，该分离株属于BVDV1，与已知BVDV1各亚型之间同源性较低，单独成一分支。结果表明，成功分离鉴定1株猪源BVDV SD0803，该毒株为非致病变型BVDV1，极有可能为BVDV1新的亚型。

Abstract: The aim of this study was to isolate virus from bovine viral diarrhea virus (BVDV) positive sample of the piglet and to study BVDV originating from pigs. BVDV positive samples from the piglet were inoculated in MDBK cells and a pig BVDV strain (SD0803) was obtained. In the study, cell culture, direct immunofluorescence assay, PCR amplification of 5′UTR，electron microscopy and TCID50 were performed, moreover，bioinformatics software were used to analyze the characteristics of molecular evolution. The results showed that the virus strain hadn’t caused cytopathic effect (CPE) until the 13th passage on MDBK cell. SD0803 infected cells showed strong fluorescent signal. DNA fragment of 307 bp and 428 bp were amplified by PCR from 5′UTR and Npro of SD0803. Under electron microscopy, virus particles were near round and enveloped with size of 50 nm in diameter. Virus titer of SD0803 was 10-6.5 TCID50·0.2 mL-1. The homology comparison and phylogenetic analysis of 5′UTR and Npro between SD0803 and representative BVDV isolates showed that SD0803 belongs to BVDV1. However, SD0803 had an obvious distance from the subtypes of BVDV1 and formed a separate group according to the phylogenetic relationship and this showed that the isolate might be a new subtype of BVDV1. So a pig originated BVDV SD0803 was successfully isolated and identified, and belongs to NCP BVDV1 and maybe a probably new subtype of BVDV1.

邓宇;;孙春清;张宏彪;蔺涛;张荣;龙进学;黄律;曹三杰;袁世山;文心田;郑浩. 1株猪源牛病毒性腹泻病毒的分离与鉴定[J]. 畜牧兽医学报, 2012, 43(3): 416-423.

DENG Yu;;SUN Chunqing;ZHANG Hongbiao;LIN Tao;;ZHANG Rong;LONG Jinxue;HUANG Lv;CAO Sanjie;YUAN Shishan;WEN Xintian;ZHENG Hao. Isolation and Identification of a Pig Bovine Viral Diarrhea Virus[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2012, 43(3): 416-423.

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