1株牛病毒性腹泻病毒分离毒株的基因组特征

doi:10.11843/j.issn.0366-6964.2020.08.015

畜牧兽医学报 ›› 2020, Vol. 51 ›› Issue (8): 1913-1922.doi: 10.11843/j.issn.0366-6964.2020.08.015

1株牛病毒性腹泻病毒分离毒株的基因组特征

高闪电^1*, 王锦明¹, 田占成¹, 独军政¹, 张忠辉¹, 王建东³, 康晓冬³, 李有全¹, 关贵全¹, 刘光远¹, 罗建勋¹, 殷宏^1,2*

1. 中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室甘肃省动物寄生虫病重点实验室, 兰州 730046;
2. 江苏省动物重要疫病与人兽共患病防控协同创新中心, 扬州 225009;
3. 宁夏农林科学院动物科学研究所, 银川 750002

收稿日期:2020-01-03 出版日期:2020-08-25 发布日期:2020-08-19
通讯作者: 高闪电,E-mail:gaoshandian@caas.cn;殷宏,主要从事兽医微生物及其分子生物学研究,E-mail:yinhong@caas.cn
作者简介:高闪电(1980-),男,河北石家庄人,副研究员,主要从事家畜分子病毒学研究
基金资助:
国家重点研发计划（2017YFD0500904；2018YFD0502305）；宁夏农林科学院对外合作项目（DW-X-2018020）；国家肉牛牦牛产业技术体系（NBCIS，CARS-37）

Genomic Characteristics of a Bovine Viral Diarrhea Virus Isolate

GAO Shandian^1*, WANG Jinming¹, TIAN Zhancheng¹, DU Junzheng¹, ZHANG Zhonghui¹, WANG Jiandong³, KANG Xiaodong³, LI Youquan¹, GUAN Guiquan¹, LIU Guangyuan¹, LUO Jianxun¹, YIN Hong^1,2*

1. State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China;
2. Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009, China;
3. Institute of Animal Science, Ningxia Academy of Agricultural and Forestry Sciences, Yinchuan 750002, China

Received:2020-01-03 Online:2020-08-25 Published:2020-08-19

摘要/Abstract

摘要： 旨在从宁夏某奶牛群持续感染牛分离牛源牛病毒性腹泻病毒（BVDV），并解析其基因组特征，为研究我国不同地区BVDV分离株遗传演化规律提供理论依据。利用BVDV抗原检测试剂盒检测宁夏回族自治区银川市某示范区的240头高产奶牛间隔两周的双份抗凝血，筛选持续感染牛，分离血液淋巴细胞制备裂解液接种牛肾细胞（MDBK），分离鉴定获得BVDV株，克隆测序获得全基因组序列，比较分析其遗传演化关系。从该示范区高产奶牛筛选获得2头持续感染牛，分离获得1株非致细胞病变型BVDV，命名为NX2019/01。测序获得基因组全序列（12 107 nt），其中ORF长11 703 nt，编码3 898个氨基酸。在基因组水平，NX2019/01株与我国SD-15、ZM-95、XC、LN-1等1m亚型分离株相似性较高（92.17%~93.84%），但E^rns、E1以及E2基因存在较大差异。示范区同群牛急性感染BVDV时，毒株E2蛋白N端编码区核苷酸突变可导致第9位或第67位氨基酸变异。重组分析表明，NX2019/01株E2基因179—288位核苷酸区段以及ZM-95株E1基因168位—E2基因332位核苷酸区段存在相似的重组信号，可能由主要亲本SD-15株与次要亲本LN-1株重组形成，表明NX2019/01株、ZM-95株在演化进程中与SD-15株以及LN-1株或早期流行的高度相似毒株存在密切关联。本研究从持续感染高产奶牛分离获得了牛源BVDV-1m亚型毒株，在基因组水平厘清了BVDV-1m亚型毒株的进化关系，并首次发现同亚型BVDV毒株基因同源重组，为进一步研究BVDV在我国的演化规律奠定了基础。

关键词: 牛病毒性腹泻病毒, 持续感染, 基因组序列, 同源重组

Abstract: The study focused on isolation of the cattle origin bovine viral diarrhea virus (BVDV) from persistently infected dairy cattle in Ningxia and determination of the viral genomic nucleotide sequences, which would provide theoretical basis for the studying the genetic evolution of BVDV strains from various regions of China. Two consecutive anticoagulated blood samples were collected at a two weeks interval from 240 cattle on a demonstration farm of high-yielding dairy cows in Yinchuan, Ningxia Hui Autonomous Region. The samples were tested for the presence of BVDV viral antigen by BVDV antigen detection kit to screen cattle with persistent infection (PI). Lysis of blood lymphocytes was prepared from PI cattle and inoculated onto MDBK monolayers to isolate BVDV. The viral genomic sequence was obtained by cloning and sequencing and compared with referenced viral sequences to analyze the evolutionary relationships between viral strains. A total of two cattle persistently infected with BVDV were screened from cattle on the demonstration farm. A non-cytopathogenic strain of BVDV, NX2019/01, was isolated. A genomic nucleotide sequence of 12 107 nucleotide position (nt) with an 11 703 nt open reading frame (ORF) encoding 3 898 amino acids, was determined by sequencing. At the genomic level, NX2019/01 shared higher similarity ranged from 92.17% to 93.84% with domestic strains SD-15, ZM-95, XC and LN-1 that were segregated into the 1m sub-genotype, but large differences were observed in the E^rns, E1 and E2 genes. In the viruses from acutely infected cattle on the demonstration farm, nucleotide mutations in N terminus coding region of E2 protein led to variations of amino acid at position 9 or 67. Recombination analysis demonstrated similar recombination signals in a fragment between nt 179-288 in E2 gene of NX2019/01 strain with another fragment spanning nt 168 in E1 gene and nt 332 in E2 gene of ZM-95 strain. The signals indicated that NX2019/01 and ZM-95may originated from a recombination between the major parent SD-15 strain and the minor parent LN-1, sharing close relatedness with SD-15, LN-1 strains or highly similar viruses at earlier time during the evolutionary process. The strain of BVDV-1m sub-genotype originated from persistently infected cattle was isolated. The evolutionary relationship of viruses of BVDV-1m subtype was determined at the genomic level and genomic homologous recombination between strains within a same sub-genotype was first reported in this study, which may lay the foundation for further study about evolution analysis of BVDV in China.

Key words: bovine viral diarrhea virus, persistent infection, genome sequence, homologous recombination

中图分类号:

S852.659.6

高闪电, 王锦明, 田占成, 独军政, 张忠辉, 王建东, 康晓冬, 李有全, 关贵全, 刘光远, 罗建勋, 殷宏. 1株牛病毒性腹泻病毒分离毒株的基因组特征[J]. 畜牧兽医学报, 2020, 51(8): 1913-1922.

GAO Shandian, WANG Jinming, TIAN Zhancheng, DU Junzheng, ZHANG Zhonghui, WANG Jiandong, KANG Xiaodong, LI Youquan, GUAN Guiquan, LIU Guangyuan, LUO Jianxun, YIN Hong. Genomic Characteristics of a Bovine Viral Diarrhea Virus Isolate[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(8): 1913-1922.

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1株牛病毒性腹泻病毒分离毒株的基因组特征

Genomic Characteristics of a Bovine Viral Diarrhea Virus Isolate

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