一株源于商品胎牛血清的BVDV-2型的分离鉴定及基因组序列分析

doi:10.11843/j.issn.0366-6964.2020.02.013

摘要/Abstract

摘要： 牛病毒性腹泻病毒（BVDV）是引起牛病毒性腹泻/黏膜病的重要病原，也是牛血清及其制品污染中的常见病原体。本研究从商品化胎牛血清中分离到一株致细胞病变（CP）型BVDV（GS2018株），利用电镜观察、免疫荧光检测、分子鉴定、基因组测序及遗传进化分析对其进行鉴定。结果表明，GS2018株接种MDBK细胞后可见明显的细胞病变（CPE），培养第4天病毒滴度达1×10^6.2·0.1 mL^-1。病毒粒子呈圆形，直径为50~60 nm，间接免疫荧光检测为阳性；其5'UTR扩增片段与BVDV-2相应序列高度相似，病毒基因组包含12 235个核苷酸（nt）。5'UTR、N^pro及E2基因系统进化分析发现，GS2018株与美国BVDV-2 USMARC-60764分离株核苷酸一致性达98%，证明其属于CP型BVDV-2a亚型。但GS2018株在NS2/3区无核酸片段插入，这与大多数CP型BVDV-2明显不同，说明BVDV致细胞病变可能涉及更复杂的机制。

关键词: BVDV-2, 致细胞病变, 基因组, 遗传进化分析

Abstract: Bovine viral diarrhea virus (BVDV) is an important pathogen causing bovine viral diarrhea/mucosal disease, and it is also the common source of contaminant in bovine serum and related biological products. In this study, a cytopathogenic (CP) type BVDV (GS2018 strain) was isolated from commercial FBS and identified by transmission electron microscopy (TEM), molecular identification, immunofluorescence assay, complete genome sequencing and genetic evolution analysis respectively. The results showed that MDBK cells presented obvious cytopathic effect (CPE) when inoculated with the BVDV GS2018 strain, and the virus titer was up to 10^6.2·0.1 mL^-1. The purified virus was detected by transmission electron microscope (TEM), round-shaped virions were observed with the diameter ranging from 50 to 60 nm, and specific positive fluorescence signal was found clearly by immunofluorescence assay. Meanwhile, the complete genome of GS2018 was sequenced as 12 235 nucleotides (nt), and BLAST result of 5'UTR region revealed that the strain had high homology with the reference sequences of BVDV-2. The phylogenetic evolutionary analysis of 5'UTR, N^pro and E2 genes showed that the GS2018 strain was classified into BVDV-2a subtype obviously, sharing the highest homology (up to 98%) with BVDV-2a representative strain USMARC-60764. Moreover, an interesting phenomenon was found that no nucleic acid fragment insertion was present in the NS2/3 region with further analysis of the genome, unlike most of CP type BVDV-2. The diversity of BVDV2 genome suggested that the mechanism of BVDV induced cytopathic effect is complicated and further research of genomic variation will contribute to answering this question.

Key words: BVDV-2, cytopathic effect, complete genome, phylogenetic evolution analysis

中图分类号:

S852.659.6

李艳萍, 王立群, 李慧霞, 梁盼红, 才学鹏, 毛立, 骆学农. 一株源于商品胎牛血清的BVDV-2型的分离鉴定及基因组序列分析[J]. 畜牧兽医学报, 2020, 51(2): 320-328.

LI Yanping, WANG Liqun, LI Huixia, LIANG Panhong, CAI Xuepeng, MAO Li, LUO Xuenong. Identification and Complete Genome Sequencing Analysis of a Cytopathic BVDV-2 from Commercial Fetal Bovine Serum[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(2): 320-328.

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