牛病毒性腹泻病毒LN-1株的分离鉴定及基因组序列分析

doi:10.16431/j.cnki.1671-7236.2023.03.029

摘要/Abstract

摘要： 【目的】查明并掌握辽宁地区牛病毒性腹泻病毒(Bovine viral diarrhea virus,BVDV)的流行特性及生物学特点,为BVDV的防治提供理论基础和技术支持。【方法】采集辽宁省某养牛场疑似发生BVDV感染的病死牛脾脏组织,用牛传染性鼻气管炎病毒(Infectious bovine rhinotracheitis virus,IBRV)、牛呼吸道合胞体病毒(Bovine respiratory syncytial virus,BRSV)、牛副流感病毒3型(Bovine parainfluenza virus type 3,BPIV3)及BVDV基因特异性引物进行RT-PCR鉴定,将组织液接种MDBK细胞分离病毒。对细胞进行细胞病变效应(CPE)观察后再通过电镜观察、间接免疫荧光试验(IFA)鉴定病毒,PCR扩增病毒全基因组序列并对其进行遗传进化分析。【结果】BVDV特异性引物RT-PCR鉴定结果为阳性,在280 bp处有条带,与预期条带大小相符,其余病原特异性引物未扩增出条带,证明未感染其他病原;分离得到的毒株在MDBK细胞中培养未出现明显细胞病变效应;病毒纯化后,经电镜观察可见直径约为50 nm的病毒粒子;免疫荧光试验结果显示,在接种分离毒株的MDBK细胞内可观察到亮绿色荧光,而对照细胞没有荧光,将分离得到的病毒命名为LN-1株;全基因组扩增序列大小为12 269 bp,遗传进化分析发现,LN-1株与XC、SD-15及ZM-95株在同一分支,与XC株核苷酸相似性为96.2%,属于BVDV-1m型。【结论】本研究成功分离出1株1m型BVDV毒株,对后续疫苗研发、流行病学调查及致病机理等方面研究具有重要意义。

关键词: 牛病毒性腹泻病毒(BVDV); 全基因组测序; 病毒分离; 免疫荧光鉴定; 遗传进化分析

Abstract: 【Objective】 The purpose of this experiment was to identify and master the epidemiological characteristics and biological characteristics of Bovine viral diarrhea virus (BVDV) in Liaoning province,and to provide theoretical basis and technical support for the prevention and treatment of BVDV.【Method】 The tissues suspected of BVDV infection from a cattle farm in Liaoning province were collected,Infectious bovine rhinotracheitis virus (IBRV),Bovine respiratory syncytial virus (BRSV),and Bovine parainfluenza virus type 3 (BPIV3) and BVDV gene-specific primers were identified by RT-PCR,and MDBK cells were inoculated with interstitial fluid to isolate the virus.After cytopathic observation of the cells,the virus was identified again by electron microscopy,indirect immunofluorescence assay (IFA),and the whole genome sequence of the virus was amplified by PCR and analyzed for genetic evolution.【Result】 The identification result of BVDV specific primer RT-PCR was positive.There was a band at 280 bp,which was consistent with the expected band size.The other pathogen specific primers did not amplify the band,which proved that other pathogens were not infected.The isolated strain did not show obvious cytopathic effect in MDBK cells.After virus purification,virus particles with a diameter of about 50 nm were observed by electron microscopy.The results of IFA showed that bright green fluorescence could be observed in the MDBK cells inoculated with the isolated virus strain,while no fluorescence was observed in the control cells.The isolated virus was named LN-1 strain.The size of the whole genome amplification sequence was 12 269 bp.Genetic evolution analysis showed that LN-1 strain was in the same branch as XC,SD-15 and ZM-95 strains,and the nucleotide similarity with XC strain was 96.2%,belonging to BVDV-1m type.【Conclusion】 In this study,one strain of 1m BVDV strain was successfully isolated,which was of great significance for the follow-up vaccine research and development,epidemiological investigation and pathogenesis research.

Key words: Bovine viral diarrhea virus (BVDV); whole genome sequencing; virus isolation; immunofluorescence identification; genetic evolution analysis

中图分类号:

S852.65^＋3

刘可欣, 王超, 张傲, 刘佳利, 谭斌, 张淑琴. 牛病毒性腹泻病毒LN-1株的分离鉴定及基因组序列分析[J]. 中国畜牧兽医, 2023, 50(3): 1150-1159.

LIU Kexin, WANG Chao, ZHANG Ao, LIU Jiali, TAN Bin, ZHANG Shuqin. Isolation,Identification and Genome Sequence Analysis of Bovine Viral Diarrhea Virus LN-1 Strain[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 1150-1159.

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