犬细小病毒新乡株的分离鉴定及其VP2 基因的序列分析

徐彦召; 申月华; 董亚楠; 钟秋月; 宋会帅; 谈晓梅; 安志兴; 王 青

文章摘要

徐彦召，申月华，董亚楠，钟秋月，宋会帅，谈晓梅，安志兴，王青.犬细小病毒新乡株的分离鉴定及其VP2 基因的序列分析[J].广东农业科学,2023,50(10):174-180

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Isolation and Identification of Canine Parvovirus Xinxiang Strain and Sequence Analysis of Its VP2 gene

DOI：10.16768/j.issn.1004-874X.2023.10.019

中文关键词: 犬细小病毒分离鉴定透射电镜全基因序列扩增遗传进化分析

英文关键词: canine parvovirus isolation and identification transmission electron microscopy whole gene sequence amplification phylogenetic analysis

基金项目:河南省高等学校重点科研项目（23A230011）；河南省高校国家级大学生创新创业训练计划项目（202210467033）；河南省高校大学生创新创业训练计划项目（202210467013）；河南省科技攻关项目（232102110086）

作者	单位
徐彦召，申月华，董亚楠，钟秋月，宋会帅，谈晓梅，安志兴，王青	河南科技学院动物科技学院，河南新乡 453003

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中文摘要:

【目的】探究河南省新乡地区犬细小病毒（Canine parvovirus，CPV）的流行和遗传变异进化情况，为有效防控区域性 CPV 流行提供参考。【方法】对从新乡地区宠物医院收集的 23 份疑似患犬细小病毒病的病犬粪便样品和眼、鼻、口、肛拭子预处理后进行 PCR 检测。将 PCR 检测结果为阳性的样品采用浸泡、过滤除菌处理后接种于单层猫肾细胞 CRFK 进行病毒增殖培养，将分离的病毒置于透射电子显微镜下观察。参考 GenBank中已收录的国内外 CPV 不同亚型的毒株序列，经 PCR 分段扩增、测序后拼接获得病毒全长基因，利用分子生物学软件对病毒基因进行序列分析和氨基酸序列同源性分析。【结果】从送检的样品中成功分离出 1 株 CPV，命名为 XX-2022 株。该病毒株能使 CRFK 细胞产生明显病变，导致细胞出现变圆、拉网和脱落。通过透射电镜可观察到典型的病毒粒子，呈圆形或六边形，直径约 25 nm，无囊膜。通过 PCR 分段扩增拼接后获得的病毒全基因组序列全为长 4 588 bp，CPV 系统进化分析结果显示，XX-2022 株与 Canine/SH/1/2019（MN840830.1）株的亲缘关系较近。利用 MegAlign 软件对 XX-2022 株 VP2 基因推导的氨基酸序列进行分析，该毒株与 YANJI-5（MW715601）的 VP2 氨基酸序列在同一小分支，氨基酸同源性为 99.7%。根据 CPV 的基因分型原则，最终确定 XX-2022 株 CPV 属于 CPV-2a 型。【结论】从临床病料中成功分离出 1 株 CPV，经分析确定为 CPV-2a 型，研究结果可为新乡地区 CPV 的流行病学、致病性及生物学特性研究提供依据，为 CPV 的区域流行及有效防控提供参考，为新疫苗的研发提供地方种毒。

英文摘要:

【Objective】 The purpose of this study was to explore the prevalence and genetic variation of canine parvovirus (CPV) in Xinxiang City, Henan Province, and provide reference for effective prevention and control of regional CPV epidemics.【Method】In this study, 23 stool samples and eye, nose, mouth and anus swabs of dogs suspected of CPV disease collected from several pet hospitals in Xinxiang were first pretreated and then PCR detected. The corresponding samples with positive PCR detection results were soaked, filtered and sterilized, and then inoculated into feline kidney cell (CRFK) for virus proliferation and culture. The isolated virus was observed under a transmission electron microscopy. Referring to the sequences of different subtypes of CPV both domestically and internationally included in GenBank, PCR was used for segmented amplification, followed by sequencing and splicing to obtain the full length genes of the virus. Molecular biology software was used for sequence analysis and amino acid homology analysis of the virus genes.【Result】One CPV was successfully isolated from the sample sent for examination and designated strain XX-2022. The virus strain was able to produce significant cytopathic changes in the CRFK cells, resulting in cell rounding, pulling and falling off. Transmission electron microscopy can observe typical canine parvovirions in a circular or hexagonal shape with a diameter of approximately 25 nm and no capsule. The full genome sequence of this virus, 4 588 bp in length, was obtained by PCR segmental amplification. Phylogenetic analysis results showed that the XX-2022 strain CPV had a close relationship with the Canine/SH/1/2019 (MN840830.1) strain CPV. The amino acid sequence of the VP2 gene of XX-2022 strain, which shares 99.7% amino acid identity with the VP2 amino acid sequence of YANJI-5 (MW715601), was analyzed using megalign software. Based on the genotyping principles of CPV, it was finally determined that the XX-2022 strain CPV belonged to CPV-2a type. 【Conclusion】In this study, a CPV strain was successfully isolated from clinical material, determined as CPV-2a type, which provide a basis for the study of the epidemiology, pathogenicity, and biological characteristics of CPV in Xinxiang, as well as a reference for the regional prevalence and effective prevention and control of CPV, and to provide an endemic virus for the development of new vaccines.

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