竹鼠源细小病毒分离鉴定及全基因组序列分析

doi:10.16431/j.cnki.1671-7236.2023.07.024

摘要/Abstract

摘要： 【目的】了解并掌握广西地区竹鼠细小病毒（Bamboo rat parvovirus，BRPV）的生物学特性及遗传变异情况，为BRPV的防控提供理论依据和技术支撑。【方法】用养殖场患病死亡的竹鼠病料制备组织上清液，采用Vero细胞同步接毒法进行病毒分离，通过细胞病变观察、PCR、透射电镜观察及血凝试验等方法鉴定，设计合成特异性扩增引物对BRPV全基因组序列进行测序分析。【结果】分离获得的病毒可在Vero细胞上稳定生长增殖，感染细胞变长、变梭或成拉丝状；病毒纯化后经电镜观察可见呈立体对称、无囊膜、直径20~25 nm的病毒粒子；经PCR鉴定、凝集试验及全基因组测序表明，分离株为BRPV。本研究共获得3株BRPV （GenBank登录号：MF497824、MF497825、MF497826），毒株基因组全长约4 758 bp，全基因组序列比对发现，其与蝙蝠源细小病毒关系较近。BRPV基因编码氨基酸遗传特征与其宿主特异性有很强的相关性，NS1基因编码氨基酸变异与蝙蝠及大鼠源细小病毒更接近，与其处于同一分支，核苷酸相似性为96.9%~97.6%，氨基酸相似性为97.5%~98.4%。其中第257和360位氨基酸属于高突变位点；基于VP2基因编码氨基酸的遗传进化树显示，其进化树处在一个独立的分支上，与大鼠源及犬、猫等动物源细小病毒核苷酸相似性为58.3%~66.8%，氨基酸相似性为51.6%~63.0%。VP2蛋白第8、99、114、118、247和253位氨基酸存在宿主偏好性，第53和386位氨基酸属于高突变位点。【结论】本研究分离获得的BRPV为一种细小病毒，本研究结果明确了中国BRPV遗传特性及病毒粒子形态特征，为BRPV的流行病学调查及防控奠定了基础。

关键词: 竹鼠细小病毒(BRPV); 分离鉴定; 全基因组; 遗传进化分析

Abstract: 【Objective】 This study was aimed to understand and master the biological characteristics and genetic variation of Bamboo rat parvovirus (BRPV) in Guangxi, and to provide theoretical basis and technical support for the control of BRPV.【Method】 The tissue supernatant was prepared from the diseased bamboo mice from farms, and the virus was isolated by Vero cell synchronous inoculation method.Identification was carried out through methods such as cytopathic observation, PCR, transmission electron microscopy observation, and hemagglutination test, and specific amplification primers were designed and synthesized for sequencing analysis of the whole genome sequence of BRPV.【Result】 The isolated virus could grow and proliferate stably on Vero cells, and the infected cells became long, spindle or thread-like.After purification, virions with a diameter of 20-25 nm were observed by electron microscopy with stereosymmetric, membraneless virions.They were identified as BRPV by PCR, agglutination test and whole genome sequencing.In this study, a total of 3 BRPV (GenBank accession No.:MF497824, MF497825 and MF497826), the total length of the genome of this strain was about 4 758 bp, and the whole genome sequence comparison showed that it was closely related to Bat-derived arvovirus.The amino acid genetic characteristics of BRPV gene have strong correlation with its host specificity.The amino acid variation of NS1 gene was more similar to that of bat and Rat parvovirus, which was in the same branch.Nucleotide homology was 96.9% to 97.6%, and amino acid homology was 97.5% to 98.4%.Amino acids 257 and 360 belonged to high mutation sites.The phylogenetic tree of VP2 gene encoded amino acids showed that the phylogenetic tree was located in an independent branch, with the nucleotide homology of 58.3% to 66.8% and the amino acid homology of 51.6% to 63.0% with rat, dog, cat and other animal parvoviruses.Amino acids at sites 8, 99, 114, 118, 247 and 253 of VP2 peotein showed host bias, and amino acids at sites 53 and 386 were highly mutated.【Conclusion】 The BRPV strains isolated in this study were Parvovirus.The results clarified the genetic characteristics and morphological characteristics of BRPV in China, which laid a foundation for the epidemiological investigation and control of BRPV.

Key words: Bamboo rat parvovirus (BRPV); isolation and identification; whole genome; genetic evolutionary analysis

中图分类号:

S852.65

唐海波, 姜佳佳, 陈凤莲, 杨锦兰, 白安斌, 刘金凤, 覃绍敏, 吴健敏. 竹鼠源细小病毒分离鉴定及全基因组序列分析[J]. 中国畜牧兽医, 2023, 50(7): 2843-2853.

TANG Haibo, JIANG Jiajia, CHEN Fenglian, YANG Jinlan, BAI Anbin, LIU Jinfeng, QIN Shaomin, WU Jianmin. Isolation, Identification and Genomic Characterization Analysis of Parvovirus Isolate from Bamboo Rat[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(7): 2843-2853.

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