梅迪-维斯纳病毒在绵羊4种细胞中的增殖特性  

Proliferative properties of maedi-visna virus in four cell types of sheep

作　　者：方卉 史晓娜[1,2,3] 陈思旭 刘淑英 FANG Hui;SHI Xiaona;CHEN Sixu;LIU Shuying(College of Veterinary Medicine,Inner Mongolia Agricultural University,Huhhot 010018,China;Key Laboratory of Animal Clinical Diagnosis and Treatment Technology,Ministry of Agriculture and Rural Affairs,Huhhot 010018,China;Key Laboratory of Basic Veterinary Medicine,Huhhot 010018,China)

机构地区：[1]内蒙古农业大学兽医学院,内蒙古呼和浩特010018 [2]农业农村部动物临床诊疗技术重点实验室,内蒙古呼和浩特010018 [3]内蒙古自治区基础兽医学重点实验室,内蒙古呼和浩特010018

出　　处：《中国兽医科学》2024年第6期833-844,共12页Chinese Veterinary Science

基　　金：国家自然基金面上资助项目(32072819);内蒙古科技重大专项计划资助项目(2021ZD0010);牛羊疫病防控与发育工程创新团队资助项目(2200001130);内蒙古草原英才创新团队资助项目(20151031)。

摘　　要：为了解梅迪-维斯纳病毒(MVV)感染绵羊不同组织细胞的偏好性及其在不同细胞中的增殖情况,将MVV A2毒株接种绵羊成纤维细胞(OAR-L1)、绵羊脉络丛细胞(SCP)、绵羊滋养层细胞(STCs)、羊胎肾细胞(FLK-BLV),通过间接免疫荧光试验、PCR、Western-blot试验观察不同时间点细胞病变特征及检测MVV感染细胞后CA蛋白相对表达量,判断病毒在细胞中增殖情况。结果显示,SCP、OAR-L1这2种细胞在接毒组中可持续增殖,SCP出现多核合胞体细胞、拉丝等病变,并且细胞随着病毒感染时间的延长死亡程度增加;OAR-L1出现拉丝且细胞随着病毒感染时间延长死亡明显,未出现典型病变多核合胞体,对照组及空白组细胞状态良好,未见显著差异。FLK-BLV、STCs这2种细胞接毒组、对照组及空白组均未见明显变化。间接免疫荧光结果显示,SCP、OAR-L1这2种细胞接毒组出现绿色荧光,而FLK-BLV、STCs接毒组与对照组、空白组均未出现绿色荧光。SCP、OAR-L1细胞随着病毒感染时间延长,绿色荧光面积逐渐扩大,SCP接毒9 d后绿色荧光面积达到90%以上,为最佳感染时间;OAR-L1接毒12 d后绿色荧光面积达到90%以上,为最佳感染时间。Western-blot结果显示在25 ku处有明显条带,经ImageJ灰度值分析,CA蛋白相对表达量水平随着接毒时间的延长逐渐上调,与对照组和空白组有极显著差异(P<0.0001)。综上,本研究发现SCP、OAR-L1这2种细胞在感染MVV A2毒株后随着感染时间的延长病毒蛋白表达量逐渐升高,MVV在以上2种细胞中可持续感染,并找到1株细胞系可以持续感染生产慢病毒。In order to understand the tropism of Maedi-Visna virus(MVV)infection in different tissues of sheep and its proliferation in different cells,the MVV A2 strain was inoculated into OAR-L1,SCP,STCs,and FLK-BLV.The cytopathic features of cells and the relative expression level of the MVV infection-causing CA protein were observed through indirect immunofluorescence assay and Western-blot assay at different time points to determine the viral proliferation in cells.The results showed that SCP and OAR-L1 cells exhibited sustained proliferation in the inoculation group.SCP cells showed cytopathic changes such as syncytia,and the degree of cell death increased with the duration of viral infection,OAR-L1 cells showed significant cell death with prolonged viral infection,but typical cytopathic changes such as multinucleated giant cells were not observed.The control and blank groups of cells remained in good condition without significant differences.FLK-BLV and STCs cells in the inoculation group,control group,and blank group did not show significant changes.Indirect immunofluorescence results showed green fluorescence in SCP and OAR-L1 cells in the inoculation group,while FLK-BLV and STCs cells in the inoculation group did not show green fluorescence compared to the control and blank groups.The green fluorescence area gradually expanded with the prolongation of viral infection in SCP and OAR-L1 cells.The green fluorescence area reached over 90%after 9 days of inoculation in SCP cells,which was considered the optimal infection time.The green fluorescence area reached over 90%after 12 days of inoculation in OAR-L1 cells,which was considered the optimal infection time.Western-blot results showed a clear band at 25 ku,and the relative expression level of CA protein gradually increased with the prolongation of inoculation time,showing extremely significant differences compared to the control and blank groups(P<0.0001)according to Image J grayscale analysis.In conclusion,this study found that the expression level of viral protein

关 键 词：梅迪-维斯纳病毒 细胞培养 增殖特性 致病变特性 

分 类 号：S852.659.3[农业科学—基础兽医学]