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为研究鲤疱疹病毒Ⅱ型(Cy HV-2)体外感染复制特征以及异育银鲫抗病毒免疫应答反应。本实验采用组织块培养法建立了异育银鲫背鳍细胞的原代培养体系。结果显示,在10 d左右可观察到组织块迁移分离出新的单层细胞,3周左右细胞可覆盖底部面积为25cm2培养瓶的底部;经Cy HV-2悬液感染离体培养的原代细胞,3 d后病毒滴度增殖至106拷贝/m L;在病毒感染6 d后出现典型的细胞病变效应;Cy HV-2感染原代细胞后,分析前期通过鱼体水平实验鉴定出的与该病毒感染相关的免疫基因:PNP5a、MPO、MHCⅠ、LYZ-C、IL-11、ITLN、PNP5a和DUSP,Real-time Rt-PCR结果显示大部分基因在细胞水平均有显著性的上调,与鱼体水平实验结果一致。本研究建立了原代培养的异育银鲫背鳍细胞,用于构建体外感染Cy HV-2病毒的细胞模型,为深入研究Cy HV-2的感染复制规律及其与宿主的相互作用关系,以及细胞水平筛选抗病毒药物实验奠定了基础。
To investigate the in vitro replication characteristics of Cyprinid carp herpesvirus type 2 (Cy HV-2) and the anti-viral immune response of allogynogenetic crucian carp (Carassius auratus gibelio). In this experiment, the tissue culture method was used to establish the primary culture system of dorsal fin cells of allogynogenetic crucian carp. The results showed that at about 10 days, new monolayer cells could be observed in the migration of the tissue mass, and the cells could cover the bottom of the 25cm2 flask with the bottom area of about 3 weeks. The primary cultured in vitro with Cy-HV-2 suspension After 3 days, the virus titer multiplied to 106 copies / ml. After 6 days of virus infection, typical cytopathic effect appeared. After primary infection of Cy-HV-2 cells, the level of The results of real-time Rt-PCR showed that most of the genes were significantly upregulated at the cellular level, and were associated with the immunogenicity of PNP5a, MPO, MHCⅠ, LYZ-C, IL-11, ITLN, PNP5a and DUSP. Fish body level experiment result is same. In the present study, primary cultured dorsal fin cells of Carassius auratus gingivalis were used to construct a cell model of Cy-HV-2 virus in vitro. In order to further study the replication and interaction of Cy-HV-2 and its interaction with host, Cell-level screening of antiviral drugs laid the foundation for the experiment.