传染性脾肾坏死病毒结构蛋白VP71的功能研究

Infectious spleen and kidney necrosis virus (ISKNV), the type species in genus Megalocytivirus, is the clear causative agents to outbreak of highly infectious diseases in mandarin (Siniperca chuatsi) industry in China. Since its first occurrence in China in 1994, huge economic losses have been caused because of this viral disease. Our recent studies confirmed that VP71, a structural viral protein in ISKNV, was found to interact with the major capsid protein (MCP), VP71 itself and voltage-dependent anion channel 2 (VDAC-2), a multifunctional host protein in cellular life and death. All these findings suggest that ISKNV-VP71 may act as an important virulent factor to take part in ISKNV infectivity to host cell. To further reveal its possible function, three parts of experiments are designated in this proposal. Firstly, reciprocal sub-localizations among VP71, MCP and VDAC-2 will be performed to validate their functionally dependent relations. Secondly, MFF-1 cell model or zebrafish embryo model will be used to study the biological effects and their possible signal pathways when overexpression of VP71 and VDAC2 are performed in both models. Finally, CRISPR/CAS9 based knockout method and recombinant virus technology will be applied to construct VDAC-2 knockout stable MFF-1 cell line and VP71 deletion ISKNV mutant recombinant virus, respectively. Then the virus properties of yielded ISKNV under knockout of each or both will be assessed comprehensively. All in all, the proposal tries to reveal the function of VP71 from both views of viral side and host side. The expected results will benefit to explore a possible novel virulent factor from ISKNV and reveal its pathogenic mechanism. It will also be of scientific significance for construction of a novel concept of attenuated live vaccine to prevent ISKNV diseases.

传染性脾肾坏死病毒（ISKNV）是虹彩病毒科肿大细胞病毒属的代表种，是国内养殖鳜暴发性传染病的明确病原。我们最近的研究证实ISKNV结构蛋白VP71与主衣壳蛋白（MCP）、VP71自身及宿主编码电压依赖性阴离子通道2（VDAC-2）存在相互作用，提示VP71可能作为毒力因子参与病毒对宿主的入侵或致病。为揭示VP71的功能，本研究拟通过：（一）VP71、MCP和VDAC-2的交互共定位实验研究三者的功能性主导关系；（二）斑马鱼胚胎模型研究过表达VP71或VDAC-2的生物学效应及其可能信号通路；（三）利用CRISPR/Cas9技术和改良的重组病毒技术分别构建VDAC-2敲除稳定细胞系和VP71缺失重组病毒，研究缺失两者之一或两者共缺失后的病毒学特性。本研究将从宿主和病毒角度揭示VP71的功能，研究结果对于发掘ISKNV的新的毒力基因从而推动其新型减毒活疫苗的研制具有重要科学意义和应用价值。