山东地区1株牛病毒性腹泻病毒的分离鉴定及遗传进化分析

doi:10.16431/j.cnki.1671-7236.2024.10.029

摘要/Abstract

摘要： 【目的】了解并掌握山东地区牛病毒性腹泻病毒(Bovine viral diarrhea virus，BVDV)流行毒株的生物学特性和遗传变异情况，为BVDV防控提供理论依据和技术支持。【方法】采集养牛场疑似BVDV感染样品制备组织上清液，利用RT-PCR方法进行病原鉴定，接种MDBK细胞进行病毒分离培养，采用间接免疫荧光试验(IFA)和电镜观察对分离毒株进行鉴定，对分离毒株全基因组进行分段扩增、克隆和测序，并采用Lasergene等软件分别对全基因组序列、N^pro和E2基因进行比对和遗传演化分析。【结果】病料样品BVDV特异性引物RT-PCR扩增得到大小约504 bp的目的条带，与预期大小一致。IFA结果显示特异性绿色荧光；电镜下可见直径约50 nm的病毒粒子，表明分离到1株非致细胞病变型BVDV毒株，命名为SDNF9株。分离株基因组全长为12 232 nt，与BVDV参考毒株的全基因组核苷酸相似性为79.1%～93.8%，其中，与中国分离毒株22AH-1和澳大利亚毒株Bega-like的相似性最高，为93.8%；N^pro和E2蛋白氨基酸存在多个位点变异；SDNF9分离毒株与BVDV2型毒株和BVDV3型毒株处于遗传演化的不同分支，而与BVDV 1型毒株处于同一分支，属于BVDV 1c基因型。【结论】本研究成功分离到1株BVDV 1c流行毒株，并进行基因组和遗传演化分析，与中国分离毒株22AH-1亲缘关系最近，为山东地区牛病毒性腹泻的防控和疫苗研制提供数据和材料支持。

关键词: 牛病毒性腹泻病毒(BVDV); 分离; 鉴定; 序列分析; 遗传进化分析

Abstract: 【Objective】 This study was aimed to understand and master the biological characteristics and genetic variation of Bovine viral diarrhea virus(BVDV) epidemic strains in Shandong province，and provide theoretical basis and technical support for the prevention and control of BVDV.【Method】 The tissue supernatant was prepared from suspected BVDV-infected samples of a cattle farm.RT-PCR method was used for pathogen identification，MDBK cells were inoculated for virus isolation and culture，and then subjected to indirect immunofluorescence assay(IFA) and electron microscopy for identification，and the whole genome of the isolated strain was segmented and amplified，cloned and sequenced.The alignment and analysis of genetic evolution of the whole genome，N^pro and E2 genes were done by Lasergene and other softwares.【Result】 The RT-PCR results of BVDV-specific primers showed BVDV-specific bands of approximately 504 bp，which was consistent with the expected band size.The IFA results showed specific green fluorescence,and virus particles about 50 nm in diameter were visible by electron microscopy，indicating that a non-cytopathogenic BVDV strain named SDNF9 was isolated.The total sequence of the strain was 12 232 nt.Comparing to BVDV reference strains，the sequence similarity was 79.1% to 93.8%，with the highest homology of 93.8% observed with the 22AH-1 strain from China and the Bega-like strain from Australia.Multiple amino acid variations were found in the N^pro and E2 proteins.Phylogenetic analysis showed that SDNF9 was in a different branch of genetic evolution from the BVDV2 and BVDV3 strains，while it was in the same clade as BVDV1 and belonged to the BVDV 1c genotype.【Conclusion】 In this study，an epidemic strain of BVDV 1c was successfully isolated and genomic and genetic evolution analysis was carried out，it was closely related to Chinese isolated 22AH-1，which provided data and material support for the prevention，control and vaccine development in the region.

Key words: Bovine viral diarrhea virus (BVDV); isolation; identification; sequence analysis; phylogenetic analysis

中图分类号:

S852.65⁺3

毕峻铭, 董雅琴, 崔进, 沙洲, 顾丛丛, 郑辉, 魏荣, 孙福亮, 张锋, 张志宏. 山东地区1株牛病毒性腹泻病毒的分离鉴定及遗传进化分析[J]. 中国畜牧兽医, 2024, 51(10): 4485-4499.

BIJunming, DONG Yaqin, CUIJin, SHA Zhou, GU Congcong, ZHENG Hui, WEI Rong, SUN Fuliang, ZHANG Feng, ZHANG Zhihong. Isolation，Identification and Genetic Evolution Analysis of a Strain of Bovine Viral Diarrhea Virus in Shandong Province[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(10): 4485-4499.

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