猪源牛病毒性腹泻病毒JLS-01株的分离鉴定及致病性研究

doi:10.16431/j.cnki.1671-7236.2019.02.027

摘要/Abstract

摘要：

为了解猪源牛病毒性腹泻病毒（bovine viral diarrhea virus，BVDV）的分子特征及致病性，本研究利用RT-PCR从吉林省某猪场出现严重腹泻症状的仔猪病料中检测到BVDV核酸阳性，将处理后的BVDV阳性样品接种于MDBK细胞，分离到1株病毒，命名为BVDV JLS-01。通过免疫荧光检测、5'UTR与N^pro RT-PCR扩增对其分子进化特征进行分析。结果显示，该分离毒株在MDBK细胞上盲传至8代未出现细胞病变，在免疫荧光试验中呈阳性荧光信号。RT-PCR扩增获得大小分别为280和735 bp的5'UTR和N^pro片段。BVDV JLS-01株5'UTR与N^pro序列遗传进化分析表明，其与LN-1和ZM-95亲缘性最近，与牛源毒株LN-1基因同源性达99.3%，提示该毒株可能来源于牛源毒株。将BVDV JLS-01株F8代细胞培养液人工感染BVDV和猪瘟病毒（CSFV）抗体阴性猪，感染猪未表现出明显的体温升高，但白细胞数量下降，并在感染猪的白细胞提取物中分离到该毒株，表明该毒株具有一定的致病性。该毒株的成功分离对进一步开展BVDV流行病学调查及致病机理等方面的研究具有重要意义。

关键词: 猪源牛病毒性腹泻病毒(BVDV); 进化分析; 分离鉴定; 致病性

Abstract:

In order to understand the molecular characteristics and pathogenicity of bovine viral diarrhea virus (BVDV),in this study,RT-PCR was used to detect BVDV nucleic acid positive in piglets with severe diarrhea symptoms in a pig farm in Jilin province.The treated BVDV positive samples were inoculated into MDBK cells,and one strain of virus was isolated and designated as BVDV JLS-01.The molecular evolution characteristics were analyzed by immunofluorescence detection and 5'UTR and N^pro RT-PCR amplification.The results showed that the isolated strain was blindly transmitted to MDBK cells for 8 generations without cytopathic effect,and showed a positive fluorescent signal in the immunofluorescence assay.5'UTR and N^pro fragments of 280 and 735 bp in size were obtained by RT-PCR amplification.The genetic evolution analysis of the 5'UTR and N^pro sequences of BVDV JLS-01 strain showed that it was closely related to LN-1 and ZM-95,and the homology with the LN-1 of bovine source strain was 99.3%,suggesting that the strain might be derived from a bovine strain.BVDV and classical swine fever virus (CSFV) antibody negative pigs were artificially infected with BVDV JLS-01 strain F8 cell culture medium,the infected pigs did not show significant elevation of body temperature,but the number of white blood cells decreased,and the virus strain was isolated from the leukocyte extract of infected pigs,indicating that the virus strain had certain pathogenicity.The successful isolation of this strain was of great significance for further research on the epidemiological investigation and pathogenesis of BVDV.

Key words: bovine viral diarrhea virus (BVDV) from porcince; evolutionary analysis; isolation and identification; pathogenicity

中图分类号:

S852.65⁺3

张淑琴, 谭斌, 刘泽余, 王超, 郭利, 孙娜, 程世鹏. 猪源牛病毒性腹泻病毒JLS-01株的分离鉴定及致病性研究[J]. 《中国畜牧兽医》, 2019, 46(2): 557-564.

ZHANG Shuqin, TAN Bin, LIU Zeyu, WANG Chao, GUO Li, SUN Na, CHENG Shipeng. Isolation,Identification and Pathogenicity Analysis of Bovine Viral Diarrhea Virus JLS-01 Strain from Porcine[J]. , 2019, 46(2): 557-564.

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