基于单颗粒示踪技术的猪流行性腹泻病毒侵染宿主细胞的分子机制研究

Porcine epidemic diarrhea virus can cause pigs of different ages to suffer from the highly contagious intestinal infectious disease, which is characterized by vomiting, diarrhea and dehydration. Since the first outbreak in the UK in 1971, the disease caused by the virus showed a staged outbreak, and has brought huge economic losses to the pig industry. Single-particle tracking technology, as an effective method to study the mechanism of virus infection, can finely analyze the characteristics, trajectories and dynamics of virus-cell interaction through the real-time monitoring of a single virus-infected cell process, so that the understanding of the mechanism of virus infection is deepened. This project aims to use single particle tracking technique, through fluorescently labeled live virus and cells, at the subcellular level, the interaction between porcine epidemic diarrhea virus and host cells is visualized from three aspects: invasion, migration and release of the virus. Based on these studies, we try to obtain an in-depth understanding of the infection mechanism of porcine epidemic diarrhea virus, and these fundamental investigations will provide important methodological and theoretical basis for the prevention and treatment of viral diseases.

猪流行性腹泻病毒可引起不同年龄阶段的猪产生以呕吐、腹泻、脱水等为特征的高度接触性肠道传染病。自1971年在英国首次爆发以来，该病毒引发的疾病呈现阶段性的大爆发流行，给养猪业带来了巨大的经济损失。单颗粒示踪技术通过对单个病毒感染细胞过程的可视化监测，精细分析病毒与宿主细胞相互作用的特点、运行轨迹及动力学规律，加深对病毒侵染细胞机制的理解，已成为病毒侵染机制研究的有效手段。本项目拟利用单颗粒示踪技术，通过对活病毒和活细胞的荧光标记，在亚细胞水平，从病毒入侵、迁移、释放三个方面，从时间与空间上立体解析猪流行性腹泻病毒与宿主细胞的相互作用。通过以上研究，深入阐明猪流行性腹泻病毒的侵染机制，为预防和治疗病毒性疾病奠定重要的方法学和理论学基础。

本项目在单细胞水平对单个病毒粒子进行实时示踪，成功解析了猪流行性腹泻病毒（PEDV）早期入胞、转运以及核酸释放的时空动态机制。通过单颗粒示踪技术，对PEDV早期感染后与宿主入胞相关蛋白相互作用轨迹进行精细追踪，解析了PEDV利用网格蛋白或小窝蛋白侵染宿主细胞的动态过程，首次揭示了PEDV依赖小窝蛋白和网格蛋白协同介导的入胞动态分子机制。进一步，实时解析了PEDV入胞后通过内吞体进行胞内转运和在成熟晚期内体中进行核酸释放的动态过程，并发现动力蛋白和驱动蛋白协同介导PEDV沿微管进行5种不同类型的运动模式，从而实现了在亚细胞水平上对病毒入胞、转运和核酸释放等方面立体示踪，发现了冠状病毒PEDV与宿主细胞相互作用的新路径和新机制。研究成果累计发表SCI论文8篇，申请发明专利3项，培养博士研究生3人，硕士研究生4人。项目按照原定计划开展了实验研究，研究目标全部按计划完成。