树鼩H5亚型高致病性禽流感病毒感染模型创建及应用

In recent years, H5 highly pathogenic avian influenza viruses (HPAIV) continue their antigenic drift or shift and inter-species transmission which might result in human infection, even death. Currently no effective vaccine and drug against it completely has posed a challenge for the safety of our public health. it will be highly profound to strengthen the basic research on why or how the viruses infect the animals or human beings. For the laboratory animals instead of humans, monkey is so far a best model for influenza although occasionally it can not produce a similar symptom to human case varying to the different influenza strains. Now, the Chinese tree shrew is considered as an alternative animal model instead of the non-human primate since it has a closest genetic trait with the primate. It was reported in 2013 that the tree shrew could be more susceptible to viruses because of the lack of DXX58 gene in it. Therefore, the tree shrew has a huge potential to be an infection model for influenza. Our current project will plan to establish and optimize the tree shrew model infected by H5 HPAIV and standardize the parameters of the assessment according to the clinical cases. Based on the established infection model, we will try to identify new pathogenic genes of the viruses and antiviral genes from the tree shrew, and confirm their biological functions at the specific cells or organs of the tree shrew via the reverse genetic system of the influenza virus, CRISPR technology, RNA-seq, proteomics and bio-information analysis. Eventually, we anticipate understanding the pathogenesis of H5 HPAIV, gene regulation network and key signaling transduction pathways in the tree shrew. Our study will provide the real and reliable data for the vaccine or drug development against human bird influenza viruses.

禽流感病毒跨种间传播引起人类发病死亡，治疗难度大，解析其发病机制有助于药物的研发。猴是流感病毒的最佳动物模型，但对不同毒株的易感性有差异，时有临床症状不典型。树鼩是和灵长类动物亲缘关系最近的小型哺乳动物，其作为实验动物研究已引起了广泛的兴趣。近期树鼩基因组解析结果显示，树鼩具有对病毒易感的遗传特征，具备成为流感疾病模型的优势。本研究通过大规模毒株筛选获取H5亚型高致病性禽流感代表毒株，感染树鼩并建立感染模型，制定评价参数，以期达到行为表象一致性和药物治疗预见性。在细胞和组织水平，运用病毒反向遗传学、CRISPR基因操作、RNA-seq测序、蛋白质组学和生物信息学分析等技术，分析不同H5亚型流感病毒诱导的基因表达谱、基因调控网络和天然免疫信号通路的差异，挖掘新的病毒毒力蛋白和宿主抗病毒因子，阐明H5流感病毒致病和关键信号通路应答的机理同源性机制，为人禽流感药物创制提供更加真实可靠的数据支持。

流感病毒跨种间传播引起人类发病，治疗难度大，解析其发病机制有助于药物的研发。树鼩(Tupaia belangeri chinensis)在生理结构和遗传学上与灵长类动物关系密切，这使其成为人类疾病的潜在动物模型。在本项目中，我们以树鼩为动物模型，系统研究了甲型H1N1流感大流行毒株、禽源H5N1毒株和早期人源H7N9毒株对中国树鼩的致病性、感染性、抗病毒反应。我们发现，流感病毒在原代树鼩细胞和树鼩动物中均可有效复制。感染树鼩的肺脏在接种后第3天出现严重的病理病变。pdmH1N1和H7N9病毒可通过直接接触在树鼩之间传播，并且H7N9病毒可以从树鼩跨越种间障碍传播到豚鼠。树鼩接种病毒后可产生很强的体液免疫反应，并提供对同源病毒的保护。此外，奥司他韦给药可以降低三种病毒对树鼩肺脏的损伤以及病毒在树鼩肺部的复制水平。本研究结果表明，树鼩可以作为研究流感病毒致病性和传播的哺乳动物模型，也可以作为研究人流感疫苗和抗病毒药物的评价平台。. 此外，项目组研究发现树鼩中存在两种功能不同的STING转录本。全长树鼩STING（tSTING-FL）直接参与DNA病毒激活的天然免疫信号通路，发挥抗病毒作用。而截短型树鼩STING（tSTING-mini）能够激活RNA病毒诱发的抗病毒信号。在静息状态下，tSTING-mini与上下游分子tMDA5/tLGP2、tIRF3存在相互作用。RNA病毒感染激活tMDA5/tLGP2介导的信号通路，最终导致I型干扰素的高效表达。同时，通路激活后，tSTING-mini在tIRF3帮助下，转位入核，入核后的tSTING-mini在E3泛素连接酶tDTX3L-tPARP9复合体的作用下迅速降解，展现出天然免疫信号通路的精确调控机制。研究结果揭示了树鼩天然免疫信号通路的特异性，为树鼩作为具有潜在的病毒感染模型提供理论基础。