草鱼呼肠孤病毒Ⅱ型在不同鱼类细胞中的增殖情况  

The study on the proliferation of GCRVⅡ in different fish cell lines

作　　者：李贤[1,2] 曾伟伟[1] 王庆[1] 王英英[1] 李莹莹[1] 宋新建[1] 黄琦雯[1] 石存斌[1] 吴淑勤[1] 

机构地区：[1]中国水产科学研究院珠江水产研究所,农业部渔药创制重点实验室,广东省水生动物免疫重点实验室,广东广州510380 [2]上海海洋大学水产与生命学院,上海201306

出　　处：《水产学报》2016年第8期1249-1257,共9页Journal of Fisheries of China

基　　金：“十二五”国家科技支撑计划(2013BAD12B02);江西省科技计划项目(20152ACF60021)~~

摘　　要：草鱼呼肠孤病毒Ⅱ型（GCRVⅡ）是当前导致草鱼出血病流行和暴发的主要基因型,本研究拟通过分析GCRVⅡ代表株HZ08在不同鱼类细胞中的增殖情况来筛选GCRVⅡ的敏感细胞系。首先以不同浓度的HZ08株接种草鱼吻端成纤维细胞（PSF）和草鱼鳔细胞（GSB）,以确定病毒接种的最佳浓度;然后以最佳接种浓度同时感染PSF、GSB、草鱼肝细胞（L8824）、草鱼卵巢细胞（CO）等10种鱼类细胞,接毒后每天观察细胞状态,用荧光定量PCR（q PCR）方法定量分析HZ08在各种细胞系中的增殖量,并在感染5 d后用间接免疫荧光定性分析病毒在各种细胞中的增殖情况。结果显示,HZ08感染细胞的最佳接种浓度为1.0×10^4拷贝/μL,该毒株接种的10种鱼类细胞均无明显的细胞病变效应（CPE）;q PCR分析病毒感染5~8 d后的结果显示,HZ08在GSB、L8824、PSF、草鱼鳍条细胞（CF）、CO、草鱼脑细胞（CIB）、锦鲤吻端细胞（KS）7种细胞中均能增殖,其中在GSB、L8824和PSF细胞中的增殖量较大,最大分别为1.14×10^7、5.90×10^6和6.30×10^4拷贝/μL。而在鲤上皮细胞（EPC）、锦鲤脑细胞（KB）、鲫脑细胞（Cc B）中不增殖,免疫荧光定性检测结果与荧光定量检测结果相吻合,在GSB、L8824和PSF中的荧光信号较多、较强,其他细胞中则较弱或没有荧光信号。研究表明,GSB、L8824和PSF是GCRVⅡ较为敏感的细胞系,该研究结果对今后Ⅱ型GCRV的研究和防控产品开发具有重要意义。Currently, grass carp reovirus genotype Ⅱ（GCRVⅡ） are the leading cause of grass carp haemorrhage disease. To determine the optimum concentration of virus inoculation, various concentrations of HZ08 strain was inoculated with proboscis snout into fibers（PSF） cell of grass carp（PSF） and swim bladder cell of grass carp（GSB）. Then we infected 10 kinds of fish cell lines with the optimum inoculation concentration, including liver cell of PSF, GSB, grass carp（L8824）, ovary cell of grass carp（CO）, etc. After inoculation, we observed the state of the cells with microscope daily, and real-time fluorescence quantitative PCR（q PCR） was used to analyze the proliferation of HZ08 in various cell lines. We also used indirect immunofluorescence to further qualitatively analyze the proliferation of HZ08 in various cell lines five days after infection. The results showed that the optimum inoculation concentration of HZ08 infected cells was 1.0×10^4 copies/μL. There was no significant cytopathic effect（CPE） observed in 10 kinds of fish cells inoculated with the HZ08 strain. q PCR analysis showed that HZ08 strain can proliferate in GSB, L8824, PSF, CF, CO, CIB, KS, 7 kinds of cells, which proliferated better in GSB, L8824 and PSF cells. The titers can reach 1.14×10^7, 5.90×10^6 and 6.30×10^4 copies/μL respectively. The result of immunofluorescence are coincided with the results of q PCR, there were strong fluorescence signals in GSB, L8824 and PSF cell lines and weak or even no fluorescence signals were observed in other cells. In conclusion, GSB, L8824 and PSF, these 3 kinds of cells are more sensitive cell lines for the proliferation of GCRVⅡ. In conclusion, this research is of great significance to the research of GCRVⅡ and the development of prevention and control product for GCRVⅡ.

关 键 词：草鱼 呼肠孤病毒 基因Ⅱ型 细胞系 增殖情况 

分 类 号：S941.41[农业科学—水产养殖]