1株牛源冠状病毒的分离鉴定及遗传进化分析

doi:10.16431/j.cnki.1671-7236.2024.07.034

中国畜牧兽医 ›› 2024, Vol. 51 ›› Issue (7): 3100-3108.doi: 10.16431/j.cnki.1671-7236.2024.07.034

• 预防兽医 • 上一篇

1株牛源冠状病毒的分离鉴定及遗传进化分析

刘文锴¹, 汪萍^2,3,4, 金映红^2,3,4, 薛晶^2,3,4, 李月^2,3,4, 梁纤纤⁵, 李晓卓⁵, 韩翔舒¹, 郑启铭⁵, 蒋松¹, 夏俊^2,3,4

1. 石河子大学动物科技学院, 石河子 832061;
2. 新疆畜牧科学院兽医研究所(新疆畜牧科学院动物临床医学研究中心), 乌鲁木齐 830000;
3. 农业农村部草食动物疫病防控重点实验室(省部共建), 乌鲁木齐 830000;
4. 新疆动物疫病研究重点实验室, 乌鲁木齐 830000;
5. 新疆农业大学动物医学学院, 乌鲁木齐 830000

收稿日期:2024-01-02 发布日期:2024-07-03
通讯作者: 蒋松, 夏俊 E-mail:9781837@qq.com;1065291747@qq.com
作者简介:刘文锴,E-mail:937965791@qq.com。
基金资助:
天山英才-青年科技拔尖人才项目(2022TSYCCX0046);自治区重大科技专项——新疆畜禽疫病防控体系质量提升工程(2023A02007)

Isolation,Identification and Genetic Evolution Analysis of a Strain of Bovine Coronavirus

LIU Wenkai¹, WANG Ping^2,3,4, JIN Yinghong^2,3,4, XUE Jing^2,3,4, LI Yue^2,3,4, LIANG Qianqian⁵, LI Xiaozhuo⁵, HAN Xiangshu¹, ZHENG Qiming⁵, JIANG Song¹, XIA Jun^2,3,4

1. College of Animal Science and Technology, Shihezi University, Shihezi 832061, China;
2. Institute of Veterinary Medicine, Xinjiang Academy of Animal Husbandry Sciences (Animal Clinical Medical Research Center, Xinjiang Academy of Animal Husbandry Sciences), Urumqi 830000, China;
3. The Key Laboratory for Prevention and Control of Herbivore Diseases of the Ministry of Agriculture and Rural Affairs (Jointly Established by the Ministry of Agriculture and Rural Affairs), Urumqi 830000, China;
4. Xinjiang Key Laboratory of Animal Disease Research, Urumqi 830000, China;
5. College of Veterinary Medicine, Xinjiang Agricultural University, Urumqi 830000, China

Received:2024-01-02 Published:2024-07-03

摘要/Abstract

摘要： 【目的】获得新疆地区牛冠状病毒(Bovine coronavirus,BCoV)毒株,探究BCoV体外分离的最佳条件,了解BCoV新疆株的遗传进化情况,为BCoV疫苗候选株提供研究基础。【方法】通过RT-PCR对来自新疆地区7个牛场的腹泻犊牛小肠及其内容物(n=185)进行BCoV检测,将阳性样本经过处理后接种HCT-8细胞,通过间接免疫荧光试验(IFA)、Western blotting和透射电镜对分离的病毒进一步鉴定,通过Reed-Muench法测定分离株的半数组织培养感染剂量(TCID₅₀),将分离毒株进行全基因组测序,并对测序结果进行遗传进化分析。【结果】腹泻样品BCoV检出率为64.9%,接种HCT-8细胞后成功分离到1株BCoV,命名为XJ-SHZ-37(GenBank登录号:OR750853);IFA可观察到孵育病毒的细胞有绿色的特异性荧光,而对照组没有;Western blotting检测结果显示,该分离株与BCoV N蛋白多克隆抗体发生特异性反应;透射电镜可观察到呈皇冠状的病毒颗粒;Reed-Muench法测定分离株的TCID₅₀为10^-7.9/0.1 mL;基因组测序及遗传进化结果表明,XJ-SHZ-37与从牛中分离到的冠状病毒科冠状病毒属的BCoV分离株ZJNB2106相似性最高。【结论】新疆地区7个牛场有较高的BCoV检出率,成功分离出1株能稳定遗传且能与BCoV N蛋白多克隆抗体特异性结合的分离株,该分离株有较高的病毒滴度,且通过遗传进化关系表明与中国牛源毒株ZJNB2106株进化亲缘关系最近。

关键词: 牛冠状病毒(BCoV); 分离鉴定; 全基因测序; 遗传进化

Abstract: 【Objective】 The experiment was aimed to obtain the Bovine coronavirus (BCoV) strain in Xinjiang,explore the best conditions for in vitro isolation of BCoV,understand the genetic evolution of BCoV Xinjiang strain,and provide candidate strain research basis for BCoV vaccine.【Method】 The small intestine and the contents (n＝185) of diarrhea calves from 7 cattle farms in Xinjiang were detected for BCoV by RT-PCR.The positive samples were inoculated into HCT-8 cells after treatment.The isolated virus was further identified by indirect immunofluorescence assay (IFA),Western blotting and transmission electron microscopy.Median tissue culture infective dose (TCID₅₀) was determined by Reed-Muench method.The isolated virus was subjected to whole genome sequencing,and the sequencing results were subjected to genetic evolution analysis.【Result】 The detection rate of BCoV in diarrhea samples was 64.9%.After inoculation with HCT-8 cells,a strain of BCoV was successfully isolated and named as XJ-SHZ-37 (GenBank accession No.:OR750853). The green specific fluorescence of the cells incubated with the virus could be observed by IFA,however,the control group did not.Western blotting test results showed that the isolated strain had a specific reaction with BCoV N protein polyclonal antibodies.The crown-like virus particles could be observed by transmission electron microscopy.The TCID₅₀ of the isolates measured by Reed-Muench method was 10^-7.9/0.1 mL.The results of genome sequencing and genetic evolution showed that XJ-SHZ-37 had the highest similarity with ZJNB2106,a BCoV isolate from the genus Coronaviruses of the Coronaviruses family,isolated from cattle.【Conclusion】 There was a high detection rate of BCoV in 7 cattle farms in Xinjiang.A strain of virus that could be stably inherited and specifically bind to the polyclonal antibody of BCoV N protein was successfully isolated.The isolate had a high virus titer,and the genetic evolution relationship showed that it had the closest evolutionary relationship with the Chinese bovine strain ZJNB2106.

Key words: Bovine coronavirus (BCoV); isolation and identification; whole gene sequencing; genetic evolution

中图分类号:

S852.65⁺3

刘文锴, 汪萍, 金映红, 薛晶, 李月, 梁纤纤, 李晓卓, 韩翔舒, 郑启铭, 蒋松, 夏俊. 1株牛源冠状病毒的分离鉴定及遗传进化分析[J]. 中国畜牧兽医, 2024, 51(7): 3100-3108.

LIU Wenkai, WANG Ping, JIN Yinghong, XUE Jing, LI Yue, LIANG Qianqian, LI Xiaozhuo, HAN Xiangshu, ZHENG Qiming, JIANG Song, XIA Jun. Isolation,Identification and Genetic Evolution Analysis of a Strain of Bovine Coronavirus[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(7): 3100-3108.

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1株牛源冠状病毒的分离鉴定及遗传进化分析

Isolation,Identification and Genetic Evolution Analysis of a Strain of Bovine Coronavirus

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