甘肃、青海和宁夏地区牛病毒性腹泻病毒Erns基因的变异分析

doi:10.11843/j.issn.0366-6964.2021.011.017

畜牧兽医学报 ›› 2021, Vol. 52 ›› Issue (11): 3157-3164.doi: 10.11843/j.issn.0366-6964.2021.011.017

甘肃、青海和宁夏地区牛病毒性腹泻病毒E^rns基因的变异分析

高闪电^1*, 王锦明¹, 田占成¹, 独军政¹, 王建东³, 常惠芸¹, 关贵全¹, 李有全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, 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:2021-02-08 出版日期:2021-11-23 发布日期:2021-11-24
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:
国家重点研发计划项目（2017YFD0500904）；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）；农业科技创新工程（ASTIP）

Genetic Variation of E^rns Gene of Bovine Viral Diarrhea Virus in Gansu, Qinghai Provinces and Ningxia Hui Autonomous Region

GAO Shandian^1*, WANG Jinming¹, TIAN Zhancheng¹, DU Junzheng¹, WANG Jiandong³, CHANG Huiyun¹, GUAN Guiquan¹, LI Youquan¹, YIN Hong^1,2*

1. State Key Laboratory of Veterinary Etiological Biology, 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:2021-02-08 Online:2021-11-23 Published:2021-11-24

摘要/Abstract

摘要： 分析2013—2019年中国西北部分省区不同基因亚型牛病毒性腹泻病毒（BVDV）抗原基因E^rns的分子特征，了解其遗传演化规律。从甘肃、青海、宁夏规模化牛场送检的疑似牛病毒性腹泻发病牛150份EDTA抗凝血提取总RNA，利用RT-PCR扩增病毒基因组E^rns-E1区，克隆测序后比对，构建系统进化树进行遗传演化关系分析。利用牛肾细胞MDBK对检出的不同基因亚型BVDV进行分离，并鉴定其生物型。RT-PCR扩增结果表明，BVDV总体阳性率为37.33%，其中甘肃省、青海省、宁夏回族自治区BVDV阳性率分别为37.68%、35.71%、40.00%。获得56份E^rns-E1 DNA，克隆测序获得33条不同的E^rns序列，长度均为681 bp，分析表明流行株分属10个BVDV基因亚型：BVDV-1a （2株）、BVDV-1b （5株）、BVDV-1c （1株）、BVDV-1d （3株）、BVDV-1m （11株）、BVDV-1o （1株）、BVDV-1p （4株）、BVDV-1q （4株）、BVDV-1v （1株）、BVDV-2a （1株）。分离获得BVDV-1a亚型、BVDV-1b亚型、BVDV-1v亚型、BVDV-2a亚型分离株各1株，BVDV-1 d亚型分离株2株，均为非致细胞病变型。各亚型株间E^rns基因核苷酸相似性以BVDV-1a～1d经典亚型株（79.8%～85.9%）或1m～1q及1v新亚型株（81.0%～87.3%）较高，以BVDV-1 m和BVDV-1p流行株亚型间相似性最高（87.3%）。各亚型株E^rns基因编码蛋白的RNA酶活性位点以及双链RNA作用基序（₁₃₉KKGK₁₄₂）保守，但E^rns第26位糖基化位点（26 NRSL）在1m～1q、1v亚型株移位（24 NVSR）。首次以E^rns核苷酸序列构建系统进化树，结果显示1m～1q及1v等亚型BVDV株在进化上关系较为密切。本研究首次选用E^rns靶标基因对甘肃、青海、宁夏部分省区牛源BVDV株进行同源性及系统进化分析，发现10个基因亚型流行株，以1m亚型株最为普遍，1m～1q及1v等亚型株亲缘关系密切。

关键词: 牛病毒性腹泻病毒, E^rns基因, 遗传演化

Abstract: The study focused on analysis of the molecular characteristics and genetic evolution of the antigen gene E^rns of different subgenotypes of bovine viral diarrhea virus (BVDV) in northwest China from 2013 to 2019. Total RNA was extracted from 150 EDTA-anticoagulated blood samples sent for BVDV detection, which were collected from cattle suspected of BVDV infection on large-scale farms in Gansu, Qinghai provinces and Ningxia Hui Autonomous Region. The RNA samples were submitted for RT-PCR with primers targeting at the E^rns-E1 region of the viral genome, followed by cloning, sequencing, and genetic evolution analysis by constructing phylogenetic trees. The virus of individual BVDV subgenotype identified in the samples was isolated with Madin-Darby bovine kidney (MDBK) cells, followed by biotype determination. Results were as follows: The overall BVDV positive rate was 37.33% by RT-PCR, with positive rate of 37.68%, 35.71% and 40.00% in Gansu Province, Qinghai Province and Ningxia Hui Autonomous Region respectively. E^rns-E1 DNA was obtained by RT-PCR from 56 blood samples and 33 different E^rns coding sequences with 681 bp in length were harvested by cloning and sequencing. Sequence analysis showed that the epidemic strains belonged to 10 BVDV genotypes: two within BVDV-1a, five within BVDV-1b, one within BVDV-1c, three within BVDV-1d, 11 within BVDV-1m, one within BVDV-1o, four within BVDV-1p, four within BVDV-1q, one within BVDV-1v, and one within BVDV-2a. One non-cytopathogenic BVDV isolate of individual subgenotype of BVDV-1a, BVDV-1b, BVDV-1v, BVDV-2a and two isolates of BVDV-1d susgenotype were obtained. Higher nucleotide similarity of E^rns gene among different strains within individual subgenotype was observed in the classical subgenotype BVDV-1a to 1d (79.8%-85.9%) or in BVDV-1m to 1q and the novel subgenotype 1v (81.0%-87.3%), with the highest nucleotide similarity shared by strains within BVDV-1m and BVDV-1p (87.3%). The RNase active sites and the dsRNA-interacting motif (₁₃₉KKGK₁₄₂) were conserved among strains within individual subgenotype while the N-glycosylation site at position N26 (26 NRSL) was translocated (24 NVSR) in strains within the 1m to 1q and 1v subgenotype. For the first time, a phylogenetic tree based on the nucleotide sequences of E^rns gene was constructed, illustrating the close genetic relatedness in the evolution among strains within subgenotypes 1m to 1q and 1v. For the first time, the E^rns gene was selected as a target for homology and phylogenetic analysis of BVDV of bovine origin from Gansu, Qinghai provinces and Ningxia Hui Autonomous Region in northwest China. Epidemic strains within 10 subgenotypes were detected, with the BVDV-1m subgenotype being the most prevalent and close genetic relationship shared by BVDV strains within BVDV-1m to 1q and 1v subgenotypes.

Key words: bovine viral diarrhea virus, E^rns gene, genetic evolution

中图分类号:

S852.659.6

高闪电, 王锦明, 田占成, 独军政, 王建东, 常惠芸, 关贵全, 李有全, 殷宏. 甘肃、青海和宁夏地区牛病毒性腹泻病毒E^rns基因的变异分析[J]. 畜牧兽医学报, 2021, 52(11): 3157-3164.

GAO Shandian, WANG Jinming, TIAN Zhancheng, DU Junzheng, WANG Jiandong, CHANG Huiyun, GUAN Guiquan, LI Youquan, YIN Hong. Genetic Variation of E^rns Gene of Bovine Viral Diarrhea Virus in Gansu, Qinghai Provinces and Ningxia Hui Autonomous Region[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(11): 3157-3164.

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甘肃、青海和宁夏地区牛病毒性腹泻病毒E^rns基因的变异分析

Genetic Variation of E^rns Gene of Bovine Viral Diarrhea Virus in Gansu, Qinghai Provinces and Ningxia Hui Autonomous Region

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甘肃、青海和宁夏地区牛病毒性腹泻病毒Erns基因的变异分析

Genetic Variation of Erns Gene of Bovine Viral Diarrhea Virus in Gansu, Qinghai Provinces and Ningxia Hui Autonomous Region

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甘肃、青海和宁夏地区牛病毒性腹泻病毒E^rns基因的变异分析

Genetic Variation of E^rns Gene of Bovine Viral Diarrhea Virus in Gansu, Qinghai Provinces and Ningxia Hui Autonomous Region