1株猫嵌杯病毒的分离鉴定及全基因序列分析

doi:10.16431/j.cnki.1671-7236.2023.09.027

摘要/Abstract

摘要： 【目的】从疑似患猫鼻咽拭子和粪便病料中分离猫嵌杯病毒(Feline calicivirus,FCV),为研究FCV的生物学特性及流行分布提供理论依据。【方法】从广东省江门市采集的11份疑似患猫的病料中,以RT-PCR筛选含有FCV的病料,利用F81细胞及病毒空斑纯化试验对其进行纯化和扩繁。电镜下观察纯净化分离病毒的形态。通过间接免疫荧光试验(indirect immunofluorescence assay,IFA)测定病毒的半数组织培养感染剂量(median tissue culture infectious dose,TCID₅₀)及不同收毒时间的病毒滴度,绘制分离病毒的一步生长曲线。对分离病毒全基因序列进行核苷酸序列测定,并对其遗传进化及相似性进行分析。【结果】从疑似患猫的粪便病料中分离获得1株FCV,经空斑纯化后,该毒株会导致F81细胞发生皱缩、变圆、葡萄串样聚集和脱落等细胞病变效应。在电镜下病毒粒子呈圆形、直径35~39 nm、无囊膜。IFA结果显示,分离病毒可与兔源FCV VP1-F蛋白特异性多克隆抗体发生结合,在荧光显微镜下可见明显特异性荧光,病毒滴度为1×10⁹ TCID₅₀/mL。分离病毒基因组全长为7 722 bp,与FCV-SH毒株核苷酸相似性最高,为83.94%,属于同一分支。VP1基因与FCV-SH1902毒株核苷酸相似性最高,为81.32%,与FCV-SH1901毒株氨基酸相似性最高,为89.99%。【结论】本研究成功分离到1株FCV,并对其全基因序列进行分析。该研究为FCV致病机理及疫苗研究奠定了基础。

关键词: 猫嵌杯病毒(FCV); 分离鉴定; 基因测序; 遗传进化分析

Abstract: 【Objective】 This study was aimed to isolate Feline calicivirus (FCV) from nasopharynx swab and fecal samples of suspected cats,and provide theoretical basis and reference for studying the biological characteristics and epidemic distribution of FCV.【Method】 11 samples of suspected cats,collected from Jiangmen city,Guangdong province,were screened by RT-PCR.The positive virus was purified by virus plaque purification test and expanded on F81 cells.The purified virus was observed under electron microscope.The median tissue culture infectious dose (TCID₅₀) of the virus was determined by indirect immunofluorescence assay (IFA).The one-step growth curve of the isolate was drawn by measuring the virus titer at different time points of virus collection.The full nucleotide sequence of the isolate was determined,and its genetic evolution and similarity were analyzed.【Result】 A strain of FCV was isolated from faecal disease samples of suspected cats.After purification by plaque,the strain caused the cytopathic effect (CPE) of F81 cells,including shrinkage,rounded,grape cluster aggregation and shedding.Under the electron microscope,the virus appeared as a round,non-envelope virus particle with the diameter of 35-39 nm.The result of IFA showed that the isolated strain could bind specifically with the polyclonal antibody,rabbit anti-FCV VP1-F protein and obvious specific fluorescence could be seen under the fluorescence microscope,and the virus titer was 1×10⁹ TCID₅₀/mL.The whole genome length of the isolate was 7 722 bp,and the nucleotide similarity with FCV-SH strain was the highest (83.94%),belonging to the same branch.VP1 gene had the highest nucleotide similarity with FCV-SH1902 strain (81.32%) and the highest amino acid similarity with FCV-SH1901 strain (89.99%).【Conclusion】 A strain of FCV was successfully isolated and its whole genome sequence was analyzed.This study laid a foundation for the research of pathogenesis and vaccine of FCV.

Key words: Feline calicivirus (FCV); isolation and identification; gene sequencing; genetic evolution analysis

中图分类号:

S852.65^＋9.6

陈昌毅, 李根, 何红玲, 陈昱彤, 黄学喆, 罗均, 郭霄峰. 1株猫嵌杯病毒的分离鉴定及全基因序列分析[J]. 中国畜牧兽医, 2023, 50(9): 3707-3718.

CHEN Changyi, LI Gen, HE Hongling, CHEN Yutong, HUANG Xuezhe, LUO Jun, GUO Xiaofeng. Isolation,Identification and Whole Genome Sequence Analysis of a Strain of Feline Calicivirus[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(9): 3707-3718.

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