非结构蛋白NS3/NS3a决定流行性出血病病毒毒力的分子基础

流行性出血病病毒（EHDV）毒力因子的确定是基因工程减毒疫苗研发和疫情防控的必要前提。前期研究发现，NS3/NS3a中存在显著影响病毒复制的区域，筛选到与NS3/NS3a互作的宿主蛋白Annexin 2（A2），EHDV感染导致A2聚集成斑块状与NS3/NS3a共定位，且A2过表达利于病毒复制。由此提出假说：EHDV NS3/NS3a通过相互作用的方式招募宿主细胞蛋白A2参与病毒复制、影响病毒毒力。本项目拟通过以下研究证实这一假说：分析A2在EHDV复制中的作用，验证NS3/NS3a-A2互作并鉴定结合域；缺失结合域序列并拯救互作缺失突变株，分析NS3/NS3a丧失与A2的互作对EHDV复制能力的影响；在干扰素受体缺失小鼠体内评价此互作缺失突变株的致病性及免疫原性。研究结果将确认非结构蛋白NS3/NS3a是EHDV的关键毒力因子，阐明NS3/NS3a决定EHDV复制能力及毒力的分子基础。

The determination of epidemic hemorrhagic disease virus (EHDV) for virulence factors is a necessary prerequisite for the development of genetically engineered attenuated vaccines and the prevention and control of epidemic situation. In the previous studies, we found that some amino acid regions in viral NS3/NS3a have significant effects on virus replication, the Annexin2 (A2) as a host protein interacting with NS3/NS3a was screened, and the intracellular diffuse of A2 to aggregate into plaques and obvious co-localization with NS3/NS3a were caused by EHDV infection. Based on these results, we hypothesize that NS3/NS3a might recruit A2 through interaction, and then affect replication and virulence of EHDV. This study intends to verify this hypothesis through the following research contents: To analyze the functions of A2 in EHDV replication, verify the interaction between NS3/NS3a and A2, and identify the binding region; to analyze the effect of NS3/NS3a on EHDV replication after losing its interaction with A2 by destroying the interaction binding area and rescuing the corresponding viral mutants; to evaluate pathogenicity and immunogenicity of these mutants in interferon receptor-deficient mice. These studies will confirm that NS3/NS3a is the key virulence factor of EHDV, and clarify the molecular basis on virulence and replication ability of EHDV determined by NS3/NS3a.