ENDOTHELIAL CELL TROPISM IN THE PATHOGENESIS AND HOST RESPONSE AGAINST INFLUENZA VIRUSES - RESUBMISSION 01

摘要 甲型流感病毒对人类健康构成严重威胁，造成季节性流行，偶尔 在世界范围内导致严重发病率和死亡率的大流行。沙门氏菌的毒力和致病性 流感病毒株差异很大，通常由病毒基因星座决定。虽然是季节性的 流感病毒会在健康的高致病性禽类中引起轻微的上呼吸道感染 H5N1亚型流感病毒(HPAIV)在下呼吸道复制，导致致命病毒 以肺水肿和血管渗漏为特征的肺炎。此外，H5N1感染是 与宿主免疫反应的失控激活有关，这导致了严重的 疾病。病毒和宿主因素在增强毒力和肺免疫病理中的作用 HPAIV感染的特征尚不清楚。为此，我们设计了具有限制性的H5N1病毒 通过将microRNA(MiRNA)靶点整合到病毒基因组中而产生的趋向性。具体来说，我们 产生了携带内皮细胞特异性miR-126靶点的H5N1病毒(H5N1-126T)，使得病毒 内皮细胞的复制被取消，而不会阻碍其他细胞类型的复制。在我们最近的 研究表明，感染H5N1-126T的小鼠和雪貂的毒力和血管渗漏显著降低。 肺与对照H5N1-ScrbT病毒感染组相比，尽管肺部的病毒载量相似 老鼠的鼻水和雪貂的鼻水。据我们所知，这是第一次证明 内皮细胞对H5N1病毒致病机制的趋向性。目前的提案将(1)确定以下结果： 内皮细胞感染对屏障完整性和功能的影响，(2)探讨内皮细胞趋向性在其中的作用 协调免疫反应，以及(3)评估病毒嗜性对其他疾病发病的贡献 包括H7N7、1918 H1N1和H7N9在内的高毒力毒株在小鼠和雪貂身上。从以下方面获得的知识 这些研究将有助于阐明强毒流感病毒导致严重疾病的机制。 并帮助开发针对强毒流感病毒株的新治疗策略。此外， 这些研究将揭示病毒嗜性如何调节宿主的抗病毒反应，并将为 新型限向性减毒活疫苗的合理设计。

ABSTRACT Influenza A viruses pose a serious threat to human health, causing seasonal epidemics and occasional pandemics that result in significant morbidity and mortality worldwide. The virulence and pathogenic nature of influenza virus strains vary greatly, and is often determined by the constellation of viral genes. While seasonal influenza viruses cause a mild upper respiratory infection in healthy individuals, highly pathogenic avian influenza viruses (HPAIV) of the H5N1 subtype replicate in the lower respiratory tract, causing fatal viral pneumonia characterized by pulmonary edema and vascular leakage. In addition, H5N1 infections are associated with an uncontrolled activation of host immune responses, which contributes to the severity of disease. The role of viral and host factors in the enhanced virulence and pulmonary immunopathology characteristic of HPAIV infections remain unknown. To this end, we engineered H5N1 viruses with restricted tropism through the incorporation of microRNA (miRNA) target sites into the viral genome. Specifically, we generated an H5N1 virus carrying endothelial cell specific miR-126 target sites (H5N1-126T), such that viral replication was abrogated in endothelial cells without impeding replication in other cell types. In our recent studies, H5N1-126T infected mice and ferrets showed significantly reduced virulence and vascular leakage in the lungs as compared to the control H5N1-ScrbT virus infected group, despite similar viral loads in the lungs of mice and nasal washes of ferrets. To our knowledge, this is the first study to demonstrate the importance of endothelial cell tropism to H5N1 pathogenesis. The current proposal will (1) determine the consequence of endothelial cell infection to barrier integrity and function, (2) investigate the role of endothelial cell tropism in orchestrating immune responses, and (3) assess the contribution of viral tropism to the pathogenesis of other highly virulent strains including H7N7, 1918 H1N1, and H7N9 in mice and ferrets. The knowledge gained from these studies will help to elucidate the mechanism by which virulent influenza viruses cause severe disease and aid in the development of novel therapeutic strategies against virulent influenza strains. Furthermore, these studies will reveal how viral tropism modulates host antiviral responses and will pave the way for the rational design of novel live attenuated vaccines with restricted tropism.