1株进口胎牛血清源牛病毒性腹泻病毒的分离鉴定及小鼠致病性分析  

Isolation,Identification and Mouse Pathogenicity Analysis of a Strain of Bovine Viral Diarrhea Virus from Imported Fetal Bovine Serum

作　　者：刘照 董倩倩 吴澳迪 王凯月 梁成哲 Adnan Ali 盛金良[1] LIU Zhao;DONG Qianqian;WU Aodi;WANG Kaiyue;LIANG Chengzhe;Adnan ALI;SHENG Jinliang(College of Animal Science and Technology,Shihezi University,Shihezi 832000,China)

机构地区：[1]石河子大学动物科技学院,石河子832000

出　　处：《中国畜牧兽医》2024年第9期4052-4059,共8页China Animal Husbandry & Veterinary Medicine

基　　金：兵团研究生创新创业项目;新疆生产建设兵团科技发展专项资金(2017BA044);国家自然科学基金(31960691)。

摘　　要：【目的】对进口胎牛血清中的牛病毒性腹泻病毒(Bovine viral diarrhea virus,BVDV)进行分离鉴定和遗传进化分析。【方法】通过RT-PCR方法检测到实验室购买的某进口胎牛血清中存在BVDV核酸污染,将污染血清接种MDBK细胞,传代培养至第7代,对每一代细胞的培养物进行BVDV 5′-UTR扩增,PCR扩增产物连接pMD19-T载体并测序,对测序结果进行遗传进化分析;利用间接免疫荧光试验(IFA)进行分离株抗原检测和病毒滴度测定;将分离株接种BALB/c小鼠,采集眼眶血进行血常规检测和RT-PCR检测。【结果】该病毒分离株在培养MDBK细胞时未能引起细胞病变效应,细胞培养物RT-PCR扩增为阳性;基于5′-UTR序列的相似性分析表明,该分离株与BVDV UDEC-COY7-17(OL860952.1)毒株5′-UTR序列相似性为99.6%,同属BVDV-1e亚型;IFA检测到感染分离株的MDBK细胞细胞质中出现特异性绿色荧光信号,而对照组无信号;半数培养感染剂量(50%tissue culture infectivedose,TCID 50)为10-4.7/0.1 mL;接种BALB/c小鼠后第7天血常规检测其血液中白细胞(WBC)和淋巴细胞(LYM)数量极显著低于对照组(P<0.01)。提取血液总RNA进行5′-UTR RT-PCR检测,检测结果为阳性,扩增产物大小为298 bp,与预期相符。【结论】本研究在进口胎牛血清中分离得到1株非致细胞病变型BVDV-1e亚型毒株,证明进口胎牛血清中存在BVDV抗原污染,为进一步研究BVDV的相关分子机制提供试验材料。【Objective】The experiment aims to isolate,identify and analyze the genetic evolution of Bovine viral diarrhea virus(BVDV)in imported fetal bovine serum.【Method】The presence of BVDV nucleic acid contamination in the imported fetal bovine serum purchased by the laboratory was detected by RT-PCR,the contaminated serum was inoculated with MDBK cells,and the cells were subcultured to the 7th passage,and the cultures of each generation of cells were amplified by BVDV 5′-UTR,and the PCR amplification product was ligated with the pMD19-T vector and sequenced for genetic evolution analysis.Indirect immunofluorescence assay(IFA)was used to detect the antigen and virus titer of the isolates.BALB/c mice were inoculated with the isolates,and orbital blood was collected for blood routine detection and RT-PCR detection.【Result】The results showed that the virus strain failed to cause cytopathy during MDBK cell culture,and the RT-PCR amplification of cell culture was positive.Analysis based on the similarity of the 5′-UTR sequence indicated that the isolated strain had 99.6%similarity in its 5′-UTR sequence to the BVDV UDEC-COY7-17(OL860952.1)strain,both belonged to the BVDV-1e subtype.IFA result detected specific green fluorescent signals in the cytoplasm of MDBK cells infected with the isolated strain,while no signals were observed in the control group.The 50%tissue culture infectious dose(TCID 50)was determined to be 10-4.7/0.1 mL.Blood routine tests conducted on BALB/c mice on the 7th day after immunization revealed extremely significantly lower counts of white blood cells(WBC)and lymphocytes(LYM)compared with control group(P<0.01).Total RNA extracted from the blood was subjected to 5′-UTR RT-PCR testing,which yielded positive results with an amplified product size of 298 bp,consistent with expectations.【Conclusion】A NCP BVDV-1e subtype strain was isolated in this study.It was proved that there was BVDV antigen contamination in imported fetal bovine serum,and also provided experimental materials for fu

关 键 词：牛病毒性腹泻病毒(BVDV) 胎牛血清 分离 鉴定 致病性 

分 类 号：S852.65[农业科学—基础兽医学]