鲤疱疹病毒Ⅱ型对异育银鲫背鳍细胞的显微形态与免疫基因表达水平的影响

The expression analysis of immune genes and microscopic morphology of CCF cells in response to Cyprinid herpesvirus 2 infection

  • 摘要: 为研究鲤疱疹病毒Ⅱ型(CyHV-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25 cm2培养瓶的底部;经CyHV-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/mL;在病毒感染6 d后出现典型的细胞病变效应;CyHV-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:PNP5aMPOMHCⅠ、LYZ-CIL-11、ITLNPNP5aDUSP,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CyHV-2病毒的细胞模型,为深入研究CyHV-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。

     

    Abstract: The aims of the current study were to investigate the primary culture of dorsal fin from Prussian carp, Carassius auratus gibelio and its susceptibility to Cyprinid herpesvirus 2 due to the lack of commercial cell line supporting CyHV-2 infection. The cells detached from the tissues in 10 days and fully covered the bottom of 25 cm2 culture bottle in 3 weeks. Cyprinid herpesvirus 2 (CyHV-2) continuously propagated in the cultured cells, as confirmed by a quantitive real time PCR, with the highest viral concentration of 106 copies/mL at 3 days post infection. In addition, cell death was observed in the dorsal fin cells upon CyHV-2 infection. Cytopathic effects (CPE) were observed as early as 6 days post-inoculation, and as the infection progressed, CPE became more apparent with cell debris and cellular exudates in inoculated cultures. The expression levels of 8 immune important genes including IL-11, ITLN, PNP5a, MPO, KRT-8, DUSP-1, MHCⅠ and LYZ-C were significantly different after viral challenge, which were determined to be involved in host response to CyHV-2 infection previously. The results above should be helpful in further studies on CyHV-2, including elucidation of pathogenesis, host-pathogen interaction and development of anti-viral drugs that can ultimately lead to prevention of this viral disease.

     

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