鱼类神经坏死病毒B2蛋白线粒体定位对病毒的复制调控

Fish nervous necrosis virus (NNV) can cause great loss of fish culture in China，but currently there is no effective prevention strategy. This virus possess a B2 protein which can binds double-stranded RNA（dsRNA） and locates to the mitochondrial membrane. Protein B2 binds to virus-derived dsRNA and prevents its cleavage by cellular factors. Therefore B2 is essential for efficient replication of NNV. On the other hand, B2 can locate to the mitochondrial membrane mediated by a specific signal peptide that targets mitochondria and impairs cellular ATP synthesis, resulting in the necrosis of the infected cell due to the depletion of cellular ATP. However, the relationship between mitochondrial localization and viral replication remains obscure. Base on the established reverse genetic system as well as site-mutation method, the critical amino acids required for mitochondria localization of B2 will be mutated, but the viral RNA dependent RNA polymerase will still be intact. The mutated B2 will lose the mitochondria localization ability. The obtained viral genomic RNAs will be transfected into the susceptible cells to produce recombinant virus which contains the mutated B2. The relationship between virus replication and B2 mitochondria localization will be characterized by the infection of the recombinant virus in susceptible fish cells and zebra fish. This study will shed new light on the virus replication and pathogenicity, and will also pave a new way for the development of high effective strategy for the prevention of NNV infection.

鱼类神经坏死病毒感染导致我国鱼类养殖重大损失，目前尚无有效防治措施。鱼类神经坏死病毒B2蛋白有双重特性：RNA结合和线粒体膜定位特性。B2结合病毒RNA，使其免受细胞因子的降解。因此B2蛋白对病毒高效复制是必须的。通过线粒体膜定位信号介导作用，B2蛋白定位在线粒体膜上，阻断线粒体ATP合成，导致感染细胞因ATP枯竭而坏死。到目前为止，B2蛋白线粒体定位和病毒复制两者关系尚未阐明。本研究利用反向遗传系统作为平台，结合点突变技术，在不改变病毒聚合酶前提下，对B2蛋白线粒体膜定位信号关键位点进行点突变，使其丧失进入线粒体能力。转染病毒基因组RNA到敏感细胞，以制备没有线粒体定位能力的重组病毒。用该重组病毒感染敏感细胞和斑马鱼，研究重组病毒的复制，以期揭示病毒复制和B2线粒体膜定位的关系，进一步了解病毒复制机制与致病性，为开展高效预防神经坏死病毒提供理论依据。

本项目开展了鱼类神经坏死病毒感染、复制研究，研究表明：B2蛋白在病毒的复制过程起到重要的作用，其突变会导致重组病毒的复制受阻，是一种致死突变；对病毒感染和宿主miRNA图谱分析，发现了220种miRNA，其中18种是新的miRNA种类，并进一步研究四种miRNA对病毒复制的促进作用；对神经坏死病毒感染GF-1细胞和SSN-1细胞的转录组分析，发现Engdo G参与诱导细胞凋亡，是新的凋亡调控途径；神经坏死病毒高效复制需要谷氨酰胺，而且依赖三羧酸循环途径；建立了一种对神经坏死病毒敏感的细胞株；对神经坏死病毒感染宿主范围调查研究，发现其可以感染多种养殖和野生鱼类；神经坏死病毒主要通过鳃作为感染途径，肠道估计不是主要途径。这些研究成果加深了我们对神经坏死病毒感染和复制的认识，为进一步防控神经坏死病毒的防控提供新的依据。