外泌体介导坦布苏病毒非受体依赖感染途径的分子机制研究

As a member of genus Flavivirus and the family Flaviviridae, Tembusu virus (TMUV) was the causative agent of severely decreased egg production and/or neurological disorders in ducks. However, the mechanism of exosome-mediated TMUV receptor-independent infection was still unreported. A virus-free exosome purification assay was developed in our previous studies and the higher number of exosomes in TMUV infected HEK293 cells was observed in comparison to the uninfected HEK293 cells. Furthermore, the TMUV-induced exosomes was able to infect and replicate on HEK293 cells which caused TMUV typical cytopathic effect (CPE). Base on above research findings, we proposed to characterize the types of TMUV-induced exosomes and analyze the viral components and non-viral components exosomes to figure out the mechanism of exosome-mediated TMUV transmission using high-throughput sequencing. To investigate the molecular basis of exosome-mediated TMUV transmission, the endocytosis route and key protein was screened and determined using blocking antibodies and inhibitors. By analyzing the virus characterization of infection and replication ability on exosome-release-inhibitor treated HEK293 cells and ducks, the important role of exosomes in TMUV infection and transmission was further determined.

坦布苏病毒(TMUV)可导致鸭严重的产蛋下降和神经症状，但作为黄病毒属成员之一，其外泌体介导的非受体依赖的感染途径及相关机制一直未能阐明。申请者在前期工作中建立了TMUV感染细胞的无病毒外泌体纯化方法，明确了TMUV感染对HEK293细胞外泌体分泌量的上调作用，证实了TMUV感染HEK293细胞后产生的外泌体具备感染、复制和致细胞病变能力。本项目拟在此基础上，通过高通量测序方法研究TMUV感染引起HEK293细胞外泌体种类的变化，解析病毒组分外泌体和非病毒组分外泌体对TMUV感染的调控作用，明确TMUV非受体依赖感染的发生机制；通过筛选外泌体进入HEK293细胞的途径及关键作用蛋白，确定外泌体介导TMUV感染的分子基础；通过分析外泌体释放抑制剂对TMUV在HEK293细胞和雏鸭体内感染、复制的干扰效果，进一步明确外泌体在TMUV对宿主感染、传播过程中的重要作用。

坦布苏病毒(TMUV)可导致鸭严重的产蛋下降和神经症状，但作为黄病毒属成员之一，其外泌体介导的非受体依赖的感染途径及相关机制通过本项目进行了研究与验证。通过高通量测序检测外泌体中TMUV核酸成分与种类，确定了外泌体中携带的病毒核酸功能与丰度，分析出了外泌体中的TMUV基因组片段、sgRNA、反义 RNA 等核酸成分在外泌体介导 TMUV 感染中的重要作用，并进一步研究了相关过程的分子机制；通过转录组学分析、非编码 RNA 测序分析、多组学关联网络分析等方法得出在感染过程中，TMUV NS4A 蛋白可通过上调Rab27a的表达从而促进包裹有TMUV核酸与蛋白成分的外泌体释放；同时，通过多种内吞途径抑制剂与释放抑制剂实验，分析并阐明了 TMUV 组分外泌体的内吞释放机制并通过雏鸭体内实验进行验证。