一株羊冠状病毒的分离鉴定及全基因组序列分析

doi:10.11843/j.issn.0366-6964.2025.09.038

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (9): 4604-4614.doi: 10.11843/j.issn.0366-6964.2025.09.038

一株羊冠状病毒的分离鉴定及全基因组序列分析

余秋蓉¹^,²(), 蔡旭航², 何艺³, 李基棕², 毛立²^,*(), 许信刚¹^,*(), 李彬²

1. 西北农林科技大学动物医学院, 杨凌 712100
2. 江苏省农业科学院兽医研究所农业农村部兽用生物制品工程技术重点实验室, 南京 210014
3. 阜康市城关镇农业(畜牧业)发展服务中心, 阜康 831500

收稿日期:2024-10-14 出版日期:2025-09-23 发布日期:2025-09-30
通讯作者: 毛立,许信刚 E-mail:yuqiurong2233@163.com;mao-li@live.cn;tiger2003@nwsuaf.edn.cn
作者简介:余秋蓉(2001-)，女，云南昆明人，硕士生，主要从事预防兽医学研究，E-mail：yuqiurong2233@163.com
基金资助:
国家重点研发计划(2023YFD1801302)；江苏省重点研发计划(BE2022330)；兽医生物技术国家重点实验室开放基金(SKLVBF202106)

Identification and Isolation of a Caprine Coronavirus and Analysis of the Complete Genome Sequence

YU Qiurong¹^,²(), CAI Xuhang², HE Yi³, LI Jizong², MAO Li²^,*(), XU Xingang¹^,*(), LI Bin²

1. College of Veterinary Medicine, Northwest A&F University, Yangling 712100, China
2. Key Laboratory of Engineering Technology for Veterinary Biological Products of Ministry of Agriculture and Rural Affairs, Institute of Veterinary Science, Nanjing 210014, China
3. Chengguan Agriculture (Animal Husbandry) Development Service Center of Fukang City, Fukang 831500, China

Received:2024-10-14 Online:2025-09-23 Published:2025-09-30
Contact: MAO Li, XU Xingang E-mail:yuqiurong2233@163.com;mao-li@live.cn;tiger2003@nwsuaf.edn.cn

摘要/Abstract

摘要：

冠状病毒在全世界广泛流行，能够引起人和动物呼吸道和消化道疾病，动物感染冠状病毒对养殖业造成巨大经济损失。为进一步丰富我国草食动物冠状病毒的流行病学资料，笔者拟对新疆腹泻山羊样品进行羊冠状病毒(caprine coronavirus，cpCoV)分离研究，并开展生物学特性鉴定。用RT-PCR方法从新疆昌吉某羊场腹泻山羊肛拭子样品中检测到cpCoV阳性，成功分离一株羊冠状病毒，命名为cpCoV/XJCJ2301G。该毒株接种细胞后可观察到明显的细胞病变，IFA试验、RT-PCR检测均为cpCoV阳性，电镜观察可见直径100 nm，周围呈皇冠状的特异病毒颗粒。病毒的遗传演化结果表明，XJCJ2301G株处于β-CoVs Embecovirus分支，与现有羊冠状病毒共同组成独立进化分支，独立于牛冠状病毒的四个亚型。经分析NS4a-NS4b基因序列，发现在两段区域内有75 nt缺失突变，导致非结构蛋白截短或延长。S基因序列比对分析发现，cpCoV与BCoV的差异主要集中于CTD区域。本研究首次从新疆地区分离到的羊冠状病毒，丰富了我国冠状病毒的流行病学资料，其NS4a-NS4b碱基数量在牛、羊和兔冠状病毒之间形成阶梯式依次缺失，提示该毒株可能是动物冠状病毒演化过程中的节点，在草食动物冠状病毒的遗传演化过程中具有重要意义。

关键词: 羊冠状病毒, 分离鉴定, 全基因组测序, 进化分析

Abstract:

Coronavirus is one of the most important pathogen causing respiratory and digestive diseases in humans and animals. In order to enrich the epidemiological data of coronaviruses in China, we conducted this study on the isolation of caprine coronavirus (cpCoV) from diarrheal goat samples in Xinjiang, and further identified the biological characteristics systemly. Rectal swab samples of goats suffering diarrhea from Xinjiang were tested positive by RT-PCR for epidemiological investigation, and the positive samples were treated and inoculated with HRT-18G cells. A novel cpCoV, named as cpCoV/XJCJ2301G, was isolated and identified with obvious cytopathogenic effect. The virus was detected positively by IFA, the purified virus was observed as the diameter of 100 nm with a crown-like surrounding by transmission electron microscopy. According to the genetic evolution analysis, the XJCJ2301G strain was belong to Embecovirus of β-CoVs, which formed an independent evolutionary cluster together with the present cpCoVs. The NS4a and NS4b gene sequences of XJCJ2301G strain have a 75 nt deletion, resulting in truncation or elongation of non-structural proteins. Through sequence alignment analysis of the S gene, it was found that the differences between cpCoV and BCoV mainly present in the CTD region. CpCoV was isolated from Xinjiang for the first time, and this strain enriched the epidemiological data of animal coronavirus. Notably, there was a stepwise pattern deletion between NS4a and NS4b among bovine coronavirus, caprine coronavirus, cpCoV/XJCJ2301G and rabbit coronavirus. Strain XJCJ2301G should be a key node and play a vital role in the evolution of herbivore coronaviruses.

Key words: caprine coronavirus, isolation and identification, whole genome sequencing, evolutionary analysis

中图分类号:

S852.659.6

余秋蓉, 蔡旭航, 何艺, 李基棕, 毛立, 许信刚, 李彬. 一株羊冠状病毒的分离鉴定及全基因组序列分析[J]. 畜牧兽医学报, 2025, 56(9): 4604-4614.

YU Qiurong, CAI Xuhang, HE Yi, LI Jizong, MAO Li, XU Xingang, LI Bin. Identification and Isolation of a Caprine Coronavirus and Analysis of the Complete Genome Sequence[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(9): 4604-4614.

图/表 8

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引物名称 Primer name	引物序列(5′→3′) Primer sequence	扩增产物大小/bp Product size
cpCoV-S1-F	TGCTGTTATAGGAGATTTAAAGT	1 403
cpCoV-S1-R	GCTTTAAAACAATGTTGTGCATA	1 403
cpCoV-S2-F	GCTGCTAATGTTTCTGTTAGC	1 517
cpCoV-S2-R	TCCAGTGCAATTATTATAAGCC	1 517
cpCoV-S3-F	GATCTGCTATAGAGGATTTAC	1 330
cpCoV-S3-R	TAATTACTAACTCCTGGTGTC	1 330
cpCoV-NS4ab-F	TCTTCATATGCTGTTGTACAGG	1 038
cpCoV-NS4ab-R	TCCACATCAAGAACTGGTGGT	1 038

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一株羊冠状病毒的分离鉴定及全基因组序列分析

Identification and Isolation of a Caprine Coronavirus and Analysis of the Complete Genome Sequence

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