一株猫疱疹病毒Ⅰ型的分离与鉴定  

Isolation and Identification of a Feline Herpesvirus-1 Strain

作　　者：许鑫燕 郑亚婷 刘迪 马波[1] 刘家森[2] 曲连东[2] XU Xinyan;ZHENG Yating;LIU Di;MA Bo;LIU Jiasen;QU Liandong(College of Veterinary Medicine,Northeast Agricultural University,Harbin 150030,China;State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute,CAAS,Harbin 150069,China)

机构地区：[1]东北农业大学动物医学学院,哈尔滨150030 [2]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室,哈尔滨150069

出　　处：《畜牧兽医学报》2023年第4期1713-1720,共8页ACTA VETERINARIA ET ZOOTECHNICA SINICA

摘　　要：采集哈尔滨某猫舍中表现为上呼吸道感染的患病猫眼、鼻拭子,PCR初步确诊为猫杯状病毒(FCV)和猫疱疹病毒(FHV-1)混合感染。利用猫肾细胞(CRFK)对样本进行病毒分离,前两代细胞培养物的核酸检测结果为FHV-1和FCV阳性,第三代开始只有FCV阳性,由此分离出FCV。制备此株FCV的鼠源多克隆抗体,并中和混合培养物中的FCV,从而分离出FHV-1,命名为HRB2019株。通过病毒粒子形态观察、间接免疫荧光试验(IFA)和基因序列分析等方法鉴定HRB2019株,并研究其体外生长动力学。结果显示,HRB2019株可在CRFK细胞上产生典型病变,测得其第四代病毒滴度为1×10^(8.43) TCID 50·mL^(-1);电镜下可观察到球形、有囊膜、直径约为200 nm的病毒粒子;IFA结果显示,分离株可与FHV-1阳性血清结合,出现特异性荧光;扩增分离株的gD基因,其与国内外流行株高度同源。病毒一步生长曲线表明,HRB2019株感染细胞12 h后开始复制增殖,12~36 h为快速增殖期,48~72 h进入增殖平台期且滴度达到高峰,84 h病毒滴度开始下降。本研究首次从FCV与FHV-1混合感染的病料中成功分离出FHV-1,为FHV-1的流行病学调查和病原学研究提供了重要数据,为混合感染中生长弱势毒株的分离提供了方法学依据。Ocular and nasal swabs were collected from cats with upper respiratory tract infections in a cattery in Harbin,and the initial diagnosis was a mixed infection of feline herpesvirus-1(FHV-1)and feline calicivirus(FCV)by PCR.CRFK cells were used to isolate viruses from the swabs of infected cats.Nucleic acid assay of the first two generations of cell cultures showed that FHV-1 and FCV were both positive,and only FCV was isolated from the third generation.FHV-1 was isolated by serum neutralization using the prepared mouse-derived polyclonal antibody of the isolated FCV,which named HRB2019 strain.HRB2019 was identified by morphological observation of viral particles,indirect immunofluorescence assay(IFA)and gene sequence analysis.And the growth kinetics in vitro of HRB2019 was studied.The results showed that HRB2019 could produce typical cytopathic effect on CRFK cells and viral titer of the fourth generation of virus culture reached 1×10^(8.43) TCID 50·mL^(-1).Electron microscopy examination showed spherical virus particles with vesicle membrane,and its diameter was about 200 nm.IFA results showed that HRB2019 could bind to FHV-1 positive serum and appeared specific fluorescence.The gD gene of the isolate was amplified and the nucleic acid sequence analysis showed that the isolate was highly homologous with the domestic and foreign prevalent strains.The one-step growth curve of the virus showed that the virus began to replicate and proliferate from 12 h after inoculation,and the rapid proliferation period was from 12 to 36 h.The viral titers peaked at 48 to 72 h and began to decline at 84 h.In this study,FHV-1 was successfully isolated for the first time from samples infected with a mixture of FCV and FHV-1,which provided important data for epidemiological investigation and pathogenetic study of FHV-1 and provided methodological basis for isolation of growth-vulnerable strains in mixed infection.

关 键 词：猫疱疹病毒 猫杯状病毒 分离 鉴定 

分 类 号：S852.659.1[农业科学—基础兽医学]