猫疱疹病毒1型的分离鉴定及gD基因序列分析

Isolation and Identification of Cat Herpesvirus Type 1 and Sequence Analysis of gD Gene

为研究猫疱疹病毒1型(FHV-1)的分子流行病学特征,对2016年-2017年从洛阳及天津地区多家宠物医院收集的具有上呼吸道感染症状的病猫眼鼻拭子样品,用FK-81细胞进行病毒分离,通过间接免疫荧光试验、电镜形态观察和gD基因测序分析等方法鉴定所分离的病毒,并进行病毒体外生长动力学研究。结果显示,病料在FK-81细胞上接种后24 h^36 h呈现变圆、融合、局灶性堆积聚团和脱落等细胞病变;分离毒株可被FHV-1单克隆抗体特异性识别;电镜下可观察到病毒粒子呈圆形、有囊膜、直径约130 nm,具有疱疹病毒科的典型形态特征,将所分离到的5株FHV-1分别命名为2016TJ1株、2016TJ2株、2017LY1株、2017LY2株和2017LY3株;病毒一步生长曲线测定结果显示,接种后12 h^36 h病毒快速增殖,40 h^60 h病毒滴度达到高峰,72 h病毒滴度开始下降;5株FHV-1 gD基因序列之间的同源性为100%,核酸序列高度保守且与国内外流行株及疫苗株的同源性极高。研究结果为我国宠物猫群中FHV-1的病原分离、分子流行病学调查等研究积累了资料。

In order to study the molecular epidemiological characteristics of feline herpesvirus type 1(FHV-1),samples of eye and nose swabs of diseased cats with upper respiratory tract infection signs were collected from several pet hospitals in Tianjin and Luoyang from 2016 to 2017.The virus was isolated by FK-81 cells and identified by indirect immunofluorescence assay,electron microscopic morphology observation and gD gene sequence analysis.Also we studied the viral growth kinetics in vitro.The results showed that the infected cells showed cytopathic effects such as roundness,fusion,focal accumulation and exfoliation between 24 and 36 hours after inoculation.The isolates could be specifically recognized by FHV-1 monoclonal antibodies.Under electron microscopy,the virions were round in shape,130 nm in diameter,and with envelopes,which had the morphological characteristics of Herpesviridae.The five FHV isolates were named as 2016 TJ1 strain,2016 TJ2 strain,2017 LY1 strain,2017 LY2 strain and 2017 LY3 strain.According to one step growth curve,the virus grow rapidly between 12 h and 36 h after inoculation,reached the peak at 40 h-60 h,and began to decline at 72 h.The homology of FHV-1 gD gene sequences between five strains was 100%,and the nucleic acid sequence was highly conserved and highly homologous with the domestic and foreign epidemic strains and vaccine strains.This study laid a foundation for pathogen isolation and molecular epidemiological investigation of FHV-1 in pet cats in China.