对虾白斑综合症病毒WSV152在线粒体凋亡途径中的作用机制

Infection of white spot syndrome virus (WSSV) causes massive apoptotic cells in the infected tissue of Litopenaeus vannamei, along with some pathological changes in the mitochondria of shrimps. However, the underlying mechanism remains largely unknown. In the previous study, we have identified a mitochondrial-localized protein encoded by WSSV, named WSV152. Results showed that WSV152 was expressed throughout the virus replication cycle and able to induce apoptotic S2 cells. Thus, WSV152 may be the point to explain why WSSV infection causes apoptosis. To illuminate the role of WSV152 played in virus infection, its effect on virus replication, virus-induced shrimp mortality and apoptosis will be analyzed in this study. Besides, we will also analyze the mitochondrial damage in WSV152-konckdown cells, as well as the host mitochondrial proteins combined with WSV152, which contributes to reveal the mechanism of apoptosis and mitochondrial damage during WSSV infection. In addition, we will explore the possibility of protecting L. vannamei against WSSV infection via oral administration of WSV152 dsRNA-chitosan complex nanoparticle. In conclusion, we intend to investigate the mechanism of virus-induced mitochondrial damage and apoptosis, as well as evaluate the feasibility of WSV152 dsRNA-chitosan nanoparticles as a novel antiviral oral agent, which provides new ideas for virus research and practical significance in disease control.

白斑综合症病毒（WSSV）感染凡纳滨对虾后，病虾组织中出现大量凋亡细胞，线粒体呈现器质性病变，推测WSSV可通过线粒体凋亡途径引发细胞凋亡，但具体机制尚不清楚。我们的前期研究表明，WSSV编码的WSV152蛋白在病毒复制过程中保持高水平表达，是一种定位于线粒体外膜的促凋亡蛋白，可能在病毒感染及线粒体凋亡途径中有着重要作用。因此，阐明WSV152的功能可能是解释WSSV引发细胞凋亡现象的关键突破点。本项目拟通过研究WSV152对病毒复制、线粒体损伤、caspase-3表达调控及血细胞凋亡率的影响，并鉴定WSV152的互作蛋白，系统解析WSV152在线粒体凋亡途径中的作用机理，为探讨WSSV引发对虾线粒体损伤及细胞凋亡的机制提供理论依据。同时，本项目拟制备壳聚糖-WSV152 dsRNA纳米颗粒，并评估其作为新型抗病毒生物制剂的可行性，为对虾的健康养殖及病害防控提供策略。

1. 项目背景：. 凡纳滨对虾（Litopenaeus vannamei）是我国养殖对虾的主要品种，白斑综合症病毒（white spot syndrome virus，WSSV）是全球对虾养殖业所面临的危害性最大的病害之一，感染对虾死亡率在3~10天内可达90~100%。WSSV感染可引起大量对虾细胞凋亡，但具体机制尚不清楚。在前期的研究中，我们基于WSSV的全基因组序列，发现WSSV的第152个开放性阅读框所编码的蛋白与线粒体相关，命名为WSV152。WSV152在病毒复制过程中保持高水平表达，是一种定位于线粒体外膜的促凋亡蛋白。因此，阐明WSV152的功能可能是解释WSSV引发细胞凋亡现象的关键突破点。.2. 主要研究内容：. 1）WSV152对WSSV感染复制的影响. 2）WSV152对线粒体功能的影响. 3）WSV152对对虾凋亡相关基因的调控. 4）鉴定与WSV152相互作用的线粒体蛋白. 5）壳聚糖-WSV152 dsRNA纳米颗粒的抗病毒效应.3. 取得的主要研究进展、重要结果、关键数据等及其科学意义或应用前景：. 1）解析了WSV152在对虾线粒体凋亡途径中的作用机理，阐明了WSSV引发病虾线粒体损伤及细胞凋亡的机制。WSV152对病毒复制有促进作用，可通过上调ROS含量，降低细胞内ATP含量，上调凋亡相关基因如细胞色素C、凋亡诱导因子和caspase-3的表达等途径促使细胞凋亡。鉴定了宿主与WSV152相互作用的线粒体蛋白，发现对虾中定位于线粒体上的蛋白COX5a可作为WSV152作用于宿主线粒体的靶点，从而促进细胞凋亡。. 2）评估壳聚糖-WSV152 dsRNA纳米颗粒作为抗WSSV生物制剂的可行性。实验结果显示病虾口服壳聚糖-WSV152 dsRNA纳米颗粒后，与对照组相比，死亡率没有显著变化，初步表明WSV152不适用于制备抗WSSV的生物制剂。