牛A群轮状病毒G10型XJ-2022株分离鉴定及基因型分析

doi:10.16431/j.cnki.1671-7236.2022.09.027

摘要/Abstract

摘要： 【目的】试验旨在对新疆某规模牛场患腹泻疾病的犊牛进行病原学鉴定及基因型分析。【方法】采用抗原诊断试剂盒方法对在新疆某牛场随机采集的15份腹泻犊牛粪便样品进行检测，对抗原检测结果为阳性的样品进行反复冻融和过滤处理，然后将样品接种于Marc-145细胞进行病毒的分离和传代。对分离毒株进行间接免疫荧光试验（IFA）和负染电镜观察进一步确定病原。对分离株的VP6和VP7基因进行PCR扩增测序，并对其进行基因相似性比对和进化树分析。【结果】抗原诊断试剂盒结果显示，3份粪便样品呈牛轮状病毒（Bovine rotavirus，BRV）抗原阳性。将阳性粪便样品分别接种于Marc-145细胞，仅有1份样品连续盲传至第9代出现明显的细胞病变效应（CPE）。IFA结果显示，接种该分离株的Marc-145细胞有亮绿色荧光，对照组未见荧光。电镜观察可见约65 nm的圆形病毒粒子，并命名为XJ-2022株。经PCR扩增获得VP6和VP7基因的目的条带，长度分别为1 356和342 bp。基因相似性比对和遗传进化分析表明，VP6基因与人源A群轮状病毒参考株DB2015-066（LC367318.1）相似性最高且遗传进化亲缘关系最近；VP7基因与牛源G10轮状病毒参考株XJX2（MN937506.1）相似性最高，遗传进化亲缘关系最近，确定该分离株为A群G10型轮状病毒。【结论】试验成功分离到基因型为A群G10型BRV XJ-2022株，该毒株为新疆地区首次发现的多宿主来源的基因重配病毒。

关键词: 牛轮状病毒(BRV); 分离鉴定; 间接免疫荧光试验(IFA); 基因型分析

Abstract: 【Objective】 The purpose of the experiment was to identify the etiology and genotype of calves suffering from diarrhea in a large-scale cattle farm in Xinjiang.【Method】 15 fecal samples were randomly collected from calves suffering from diarrheal diseases in a cattle farm in Xinjiang using the antigen diagnostic kit method.The virus was isolated and passaged in Marc-145 cells.Indirect immunofluorescence assay (IFA) and negative-staining direct electron microscopy were performed on the isolated strains to further identify the pathogen.The VP6 and VP7 genes of the isolates were amplified and sequenced by PCR and genetic evolution analysis.【Result】 The results of the antigen diagnostic kit showed that 3 stool samples were positive for Bovine rotavirus (BRV) antigen.Positive fecal samples were inoculated into Marc-145 cells, and only 1 sample showed obvious cytopathic effect (CPE) in the ninth passage.The IFA results showed that the Marc-145 cells inoculated with this isolate had bright green fluorescence, but no fluorescence was seen in the control group.Circular virus particles of about 65 nm were observed by electron microscope, and were named as XJ-2022 strain.The target bands of VP6 and VP7 genes were obtained by PCR amplification with lengths of 1 356 and 342 bp, respectively.Gene similarity comparison and genetic evolution analysis showed that VP6 gene had the highest similarity and the closest genetic evolutionary relationship with the human group A Rotavirus reference strain DB2015-066 (LC367318.1), and VP7 gene had the highest similarity and genetic evolutionary relationship with bovine G10 Rotavirus reference strain XJX2(MN937506.1), the isolate was identified as group A G10 Rotavirus.【Conclusion】 The genotype of group A G10 BRV XJ-2022 strain was successfully isolated, which was the first multi-host gene reassortment virus discovered in Xinjiang.

Key words: Bovine rotavirus (BRV); isolation and identification; IFA; genotyping

中图分类号:

S852.65^＋3

席静, 易继海, 王月丽, 徐朕宇, 李培东, 张江伟, 陈创夫. 牛A群轮状病毒G10型XJ-2022株分离鉴定及基因型分析[J]. 中国畜牧兽医, 2022, 49(9): 3530-3538.

XI Jing, YI Jihai, WANG Yueli, XU Zhenyu, LI Peidong, ZHANG Jiangwei, CHEN Chuangfu. Isolation, Identification and Genotype Analysis of Group A Bovine Rotavirus G10 Strain XJ-2022[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(9): 3530-3538.

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