猫疱疹病毒1型毒株的分离与鉴定  

Isolation and Identification of a Feline Herpesvirus-1 Strain

作　　者：杨丁凡 孟媛 郝媛婕 陈冰洁[2] 张欣珂[1,2] 杜涛峰[1,2] YANG Ding-fan;MENG Yuan;HAO Yuan-jie;CHEN Bing-jie;ZHANG Xin-ke;DU Tao-feng(College of Veterinary Medicine,Northwest A&F University,Yangling,Shaanxi,712100,China;Northwest A&F University Teaching Animal Hospital,Xi an,Shaanxi,710065,China)

机构地区：[1]西北农林科技大学动物医学院,陕西杨陵712100 [2]西北农林科技大学西安动物医院,陕西西安710065

出　　处：《动物医学进展》2025年第3期138-144,共7页Progress In Veterinary Medicine

基　　金：西北农林科技大学校企合作计划项目(202200523100410)。

摘　　要：从西北农林科技大学西安教学动物医院某病猫采集眼鼻口分泌物,PCR初步鉴定为猫疱疹病毒1型(FHV-1)与猫杯状病毒(FCV)混合感染。利用血清中和试验,将处理的样品与实验室前期制备的FCV兔阳性血清孵育后接种于猫肾细胞(CRFK)进行FHV-1的分离,连续传代3次。通过荧光定量PCR、病毒形态学、生长动力学、同源性分析以及遗传进化分析等试验,对分离毒株进行鉴定。结果显示,用FCV阳性血清中和后的样品荧光定量PCR检测结果为FHV-1阳性,FCV阴性;将血清中和后的病毒液接种CRFK细胞出现圆缩、脱落、成葡萄串样聚集等细胞病变;表明分离到FHV-1临床株,并将其命名为F2023SX1;经氯化铯密度梯度离心得到纯化的FHV-1病毒粒子,透射电镜下可观察到球形、有囊膜、结构清晰、直径约150~200 nm的病毒粒子,符合FHV-1形态特征。病毒生长动力曲线测定显示,分离株感染细胞后,上清中病毒滴度呈现先上升后下降最后趋于平稳的趋势,病毒滴度在接种48 h时最高。采用PCR扩增分离株的gD基因,经序列比对分析,与国内外FHV-1流行毒株同源性100%。论文采用分离培养结合血清中和试验成功分离到1株FHV-1。A nasal swab sample was collected from a sick cat at Xi'an Teaching Animal Hospital of Northwest A&F University,and PCR initially identified as a mixed infection of feline herpesvirus(FHV-1)and feline calicivirus(FCV).The serum neutralization test was used to isolate FHV-1 from sample.Briefly,a rabbit polyclonal serum specific for FCV(prepared in our laboratory)was mixed with the treated sample and inoculated into feline renal cells(CRFK).After three consecutive passages,the viruses-inoculated cells were identified by fluorescence quantitative PCR,viral morphology,growth kinetics and genetic evolution analysis.The results showed that the serum-neutralized samples were positive for FHV-1 and negative for FCV by quantitative fluorescence PCR.The FHV-1-inoculated CRFK cells showed cytopathic effects,such as rounding,detachment,and grapevine cluster-like aggregation.These results suggested that an FHV-1 clinical strain was successfully isolated and named F2023SX1.The FHV-1 virus particles were purified by density gradient centrifugation using cesium chloride.Several spherical enveloped virus particles of approximately 150-200 nm in diameter were observed under transmission electron microscopy,which is consistent with the characteristic morphology of FHV-1.The growth kinetics curve of the virus showed that the viral titer in the supernatant increased initially,followed by a decrease,and eventually reached a stable trend.The viral titer is highest at 48 h post infection.The gD gene of the isolated FHV-1 strain was amplified by PCR.Gene sequencing and homology analysis revealed that gD gene of the isolated FHV-1 strain was 100%homology to reported gD gene of other FHV-1 strains.These results could provide a biological material for the development of vaccines and therapeutic drugs to combat FHV-1 infection.

关 键 词：猫疱疹病毒 猫杯状病毒 GD基因 分离与鉴定 

分 类 号：S858.293[农业科学—临床兽医学]