US7/US8蛋白影响鸭瘟病毒生命周期和毒力的机制解析

鸭瘟病毒(DPV)是严重危害鸭、鹅及野生水禽最重要的病原之一。我们在前期研究中发现，单独敲除DPV的US7或US8基因与同时敲除US7/US8双基因都能使DPV的毒力显著下降、US7/US8蛋白可互作且US7会影响US8蛋白的糖基化进而影响病毒的二次囊膜包被等。为解析US7/US8蛋白是如何影响DPV的生命周期和毒力及其机制，本项目拟利用基因敲除和点突变、免疫共沉淀、糖基化和透射电镜检测等在细胞上探究US8与US7蛋白互作的关键区域(胞内域/胞外域/跨膜区/保守区等)和位点(糖基化、磷酸化和保守位点等)及其对病毒吸附、进入、复制、装配和释放各生命周期阶段的影响；最后通过鸭体感染、显微病理组织学观察、病毒在鸭体内分布及载量等实验确定相关区域和位点对DPV毒力的影响，为DPV基因缺失疫苗、禽类活载体多价疫苗提供遗传背景清楚的病毒株，对阐明DPV毒力致弱的分子机制提供可靠依据，具有重要科学意义。

Duck plague virus (DPV), which belongs to herpesvirus, is one of the most important pathogens that endanger the waterfowl industry and has the prospect of being a multivalent live vaccine vector. It is of theoretical and practical significance to find out the molecular mechanism of its virulent attenuation. In the early stage of the project, it was found that the deletion of US7 or US8 alone or US7 and US8 at the same time could reduce DPV virulence. Based on the following studies including that US8 protein interacted with US7 protein as well as that US7 protein affected viral final envelopment process by influencing the glycosylation of US8 protein, we further explored the interaction regions (intracellular domain/extracellular domain/transmembrane region/conserved region) and sites (glycosylation site/phosphorylation site/conserved site) of US8 protein and US7 protein by using gene deletion, base-point mutation, CO-IP, glycosylation examination, and transmission electron microscope for finger out their effects on viral life cycle stages including viral absorption, entry, replication, assembly, and release. Finally, we determined the influence of the regions and sites on DPV virulence through experiments such as duck infection, microscopic histopathological assay as well as the examination of viral distribution and load in ducks. It will provide a viral strain with a clear genetic background for DPV gene deletion vaccines and poultry live multivalent vaccines and it will also provide a reliable basis for elucidating the molecular mechanisms of virulent attenuation of herpesvirus or DPV.