云南省G10P[11]基因型牛轮状病毒的分离鉴定及其遗传特征研究

doi:10.16431/j.cnki.1671-7236.2025.10.038

中国畜牧兽医 ›› 2025, Vol. 52 ›› Issue (10): 4930-4940.doi: 10.16431/j.cnki.1671-7236.2025.10.038

• 预防兽医 • 上一篇

云南省G10P[11]基因型牛轮状病毒的分离鉴定及其遗传特征研究

黄洁^1,2, 朱沛², 李占鸿², 王金萍², 何于雯², 喻建荣³, 徐心明⁴, 沙丽东⁵, 陈培富¹, 姚俊²

1. 云南农业大学动物医学院, 昆明 650201;
2. 云南省畜牧兽医科学院, 云南省热带亚热带动物病毒病重点实验室, 昆明 650224;
3. 云南省农业职业技术学院, 昆明 650212;
4. 云南省楚雄州禄丰市动物疫病预防控制中心, 禄丰 651200;
5. 昆明市富民县动物疫病预防控制中心, 富民 650400

收稿日期:2025-02-21 发布日期:2025-09-30
通讯作者: 陈培富, 姚俊 E-mail:cltwins2003@163.com;yaojun_joshua@hotmail.com
作者简介:黄洁，E-mail:1520092187@qq.com。
基金资助:
云南省科技厅乡村振兴科技专项(202304BI090003)；云南省科技厅重点研发计划(202303AK140031)；云南益民养殖示范基地有限公司姚俊专家基层科研工作站；澜沧重牧农业科技有限公司姚俊专家基层科研工作站

Isolation,Identification and Genetic Characteristics Analysis of Genotype G10P[11] Bovine Rotavirus in Yunnan Province

HUANG Jie^1,2, ZHU Pei², LI Zhanhong², WANG Jinping², HE Yuwen², YU Jianrong³, XU Xinming⁴, SHA Lidong⁵, CHEN Peifu¹, YAO Jun²

1. College of Veterinary Medicine, Yunnan Agricultural University, Kunming 650201, China;
2. Key Laboratory of Tropical and Subtropical Animal Viral Diseases of Yunnan Province, Yunnan Academy of Animal Husbandry and Veterinary Sciences, Kunming 650224, China;
3. Yunnan Vocational College of Agriculture, Kunming 650212, China;
4. Animal Disease Prevention and Control Center of Lufeng City, Chuxiong Prefecture, Yunnan Province, Lufeng 651200, China;
5. Animal Disease Prevention and Control Center of Fumin County, Kunming City, Fumin 650400, China

Received:2025-02-21 Published:2025-09-30

摘要/Abstract

摘要： 【目的】A型牛轮状病毒(Bovine rotavirus，BRV)是引起新生犊牛严重肠炎的重要病原，试验旨在了解云南省规模化牛场BRV流行毒株的基因型及分子特征，为牛场病毒性腹泻防控提供理论依据。【方法】采用RT-PCR方法对云南某规模肉牛场7份西门塔尔犊牛腹泻粪便样品进行病原检测，取BRV核酸呈阳性的样品通过MA-104细胞系对病毒进行连续传代分离；利用间接免疫荧光试验(IFA)检测病毒增殖情况，绘制病毒生长曲线；进一步对分离毒株全基因组进行测序，利用在线比对软件进行基因组分型，利用Mega 7.0软件分别绘制VP4、VP7基因遗传进化树，使用GeneDoc软件分析氨基酸突变情况。【结果】7份腹泻样品检测均为BRV阳性，分离获得1株BRV毒株，命名为YNLL-2023，其基因型为G10-P[11]-I2-R2-C2-M2-A3-N2-T6-E2-H2。分离毒株感染MA-104细胞12 h出现明显的细胞病变效应(CPE)。RT-PCR扩增获得分离毒株VP6基因约300 bp的特征性片段；IFA显示分离毒株感染MA-104细胞后胞浆内出现特异性绿色荧光，生长曲线显示病毒滴度从接毒后6～54 h持续上升，达到峰值10^5.57 TCID₅₀/100 μL。遗传进化分析表明，分离毒株VP4基因核苷酸序列与RVA/Yak-tc/CHN/HB-3/2021/G10P[11]株(登录号：ON711387.1)处于同一分支，VP7基因核苷酸序列与RVA/Bovine-wt/CHN/JS31/2020/G10株(登录号：PQ332932.1)和RVA/Bovine-wt/CHN/SD1/2023/G10株(登录号:PQ332943.1)处于同一分支。氨基酸序列分析显示，分离株VP4氨基酸序列与P[11]型羊源疫苗株在中和表位区域存在7个氨基酸位点突变，VP7氨基酸序列与G10型人-牛重组疫苗株在中和表位区域存在2个氨基酸位点突变。【结论】本试验通过MA-104细胞成功分离获得1株G10P[11]型BRV，该毒株与中国牛源多个毒株相似性较高，但与同基因型疫苗株的中和表位存在差异。

关键词: 牛轮状病毒(BRV); G10P[11]基因型; 生物学特性; 遗传特征

Abstract: 【Objective】 Bovine rotavirus (BRV) type A is an important pathogen causing severe enteritis in newborn calves.The experiment aimed to understand the genotypes and molecular characteristics of the prevalent BRV strains in large-scale cattle farms in Yunnan province,providing a theoretical basis for the prevention and control of viral diarrhea in cattle farms.【Method】 Pathogen detection was carried out on 7 fecal samples of Simmental calf diarrhea from a large-scale beef cattle farm in Yunnan by RT-PCR method.Samples positive for BRV nucleic acid were subjected to continuous passage and isolation of the virus through the MA-104 cell line.The virus proliferation was detected by indirect immunofluorescence assay (IFA),and the virus growth curve was plotted.The whole genomes of the isolated strain were further sequenced.The genotypes were analyzed using online comparison software.The genetic evolutionary trees of VP4 and VP7 genes were drawn using Mega 7.0 software, respectively,and the amino acid mutations were analyzed using GeneDoc software.【Result】 7 diarrhea samples were all positive for BRV,from which a BRV strain was isolated and named YNLL-2023,with being genotyped as G10-P[11]-I2-R2-C2-M2-A3-N2-T6-E2-H2.MA-104 cells infected with the isolated strain showed obvious cytopathic effect (CPE) for 12 h.The characteristic fragment of approximately 300 bp of the VP6 gene of the isolated strain was obtained by RT-PCR amplification.IFA showed that specific green fluorescence appeared in the cytoplasm of MA-104 cells after the isolated strain infected.The growth curve showed that the virus titer continuously increased from 6 to 54 h after inoculation,reaching a peak of 10^5.57 TCID₅₀/100 μL.Genetic evolution analysis indicated that the nucleotide sequence of VP4 gene of the isolated strain was in the same branch as the RVA/Yak-tc/CHN/HB-3/2021/G10P[11] strain (accession No.:ON711387.1).The nucleotide sequence of VP7 gene was in the same branch as the RVA/Bovine-wt/CHN/JS31/2020/G10 strain (accession No.:PQ332932.1) and the RVA/Bovine-wt/CHN/SD1/2023/G10 strain (accession No.：PQ332943.1).Amino acid sequence analysis demonstrated that there were 7 amino acid mutations in VP4 between the isolate and the P[11] type sheep vaccine strain,and 2 amino acid mutations in VP7 between the isolate and the G10 type human-bovine recombinant vaccine strain in neutralizing epitope region.【Conclusion】 In this experiment,one strain of type G10P[11] BRV was successfully isolated from MA-104 cells.This strain was highly similar to several strains of bovine origin in China，but there were differences in neutralizing epitopes with vaccine strains of the same genotype.

Key words: Bovine rotavirus (BRV); genotype G10P[11]; biological properties; genetic characteristics

中图分类号:

S852.65⁺3

黄洁, 朱沛, 李占鸿, 王金萍, 何于雯, 喻建荣, 徐心明, 沙丽东, 陈培富, 姚俊. 云南省G10P[11]基因型牛轮状病毒的分离鉴定及其遗传特征研究[J]. 中国畜牧兽医, 2025, 52(10): 4930-4940.

HUANG Jie, ZHU Pei, LI Zhanhong, WANG Jinping, HE Yuwen, YU Jianrong, XU Xinming, SHA Lidong, CHEN Peifu, YAO Jun. Isolation,Identification and Genetic Characteristics Analysis of Genotype G10P[11] Bovine Rotavirus in Yunnan Province[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(10): 4930-4940.

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云南省G10P[11]基因型牛轮状病毒的分离鉴定及其遗传特征研究

Isolation,Identification and Genetic Characteristics Analysis of Genotype G10P[11] Bovine Rotavirus in Yunnan Province

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