一株牛病毒性腹泻/黏膜病强毒株的分离与鉴定

Isolation and identification of one high virulence Bovine Viral Diarrhea Virus/Mucosal Disease Virus (BVDV/MDV) strain

关键词:  牛病毒性腹泻/黏膜病病毒  分离  鉴定

Key words:  Bovine Viral Diarrhea Virus (BVDV)  Isolation  Identification

摘要:

从云南某牛场疑似BVDV感染的病料通过接种MDBK细胞分离到1株BVDV，将其命名为BVDV/W。该分离毒株连传15代均不产生CPE，为NCP型BVDV。通过电镜检测可观察到直径为 40～60nm病毒粒子。该分离病毒能被牛病毒性腹泻标准阳性血清中和，且能被BVDV IFA荧光抗体识别；针对常用作BVDV基因分型的5′-UTR设计特异性引物，经RT-PCR可扩增出288bp特异性片段。将所测的目的片段序列与中参考序列进行同源性比较，结果显示与293株和Y2株同源关系较近，分别为90.0%和89.9%，与2014年以来我国报道的分离株同源性在88%左右，具有一定的代表性。5′-UTR遗传进化分析证实该分离毒株为BVDV-1型。动物回归试验显示，该分离毒株可引起出现体温升高、腹泻、粘膜病等典型的BVD/MD症状，表明该毒株为1株BVD强毒株。

Abstract:

One BVDV strain named BVDV/W was isolated from a cattle farm in Yunnan province by cultivated in MDBK cell. It was identified as a NCP biotype since no CPE produced after 15 times passaged, and the virus particles with diameter of 40~60 nm were observed under the electron microscopic.. This virus can be neutralized by the positive serum of bovine viral diarrhea, and also can be identified by BVDV IFA fluorescent antibody. Using 5’-UTR as the specific primer, 288bp of DNA fragment was amplified by RT-PCR. As compared with the 5’-UTR of reference sequences, the identity of this gene sequence was 90.0% and 89.9% to 293 and Y2 virus, respectively. The similarity of this virus was approximately 88% to the isolated virus which reported since 2014 in China. phylogenetic tree of 5’-UTR verified that the strain was affiliated to BVDV1. The animal regression experiment revealed that the infected cattle presented typical BVD/MD symptoms such as fever, diarrhea, and mucosal lesions. All of these results revealed that this virus was a high virulence BVDV strain.