Biodefense Against an Aerosolized Ebola Threat

描述(申请人提供)：埃博拉病毒(EBOV)是丝状病毒家族中一种包膜的、非分段的负链RNA病毒，可在人类和非人类灵长类动物中引起致命的出血热疾病，高达90%的感染者死亡。目前还没有有效的抗病毒药物可用于治疗EBOV感染，也没有开发出适当的疫苗来预防人类疾病。2001年9月11日的事件和随之而来的吸入性炭疽生物恐怖主义事件突出了人们的担忧，即EBOV也可能被用作生物恐怖主义剂，可能通过气雾剂路线传播。然而，关于EBOV定植于人类下呼吸道并随后引发系统性病毒性出血热综合征的能力，人们知之甚少。目前还没有研究EBOV对人肺细胞影响的体外模型。我们的初步数据表明，EBOV可以在维持在气液界面原代培养的正常人支气管上皮细胞(HBEC)中复制。在R21的应用中，我们提出：吸入EBOV气雾剂能够通过在支气管上皮建立持续感染，导致随后的肺泡巨噬细胞感染而在肺内定植。具体的假设是，EBOV进入支气管上皮与这些细胞中的质膜相关小凹有关。此外，一旦进入，病毒在支气管上皮内的复制(这需要EBOVRNA依赖的RNA聚合酶L和糖蛋白GP基因)诱导白细胞介素1(IL-1)、环氧合酶-2(COX-2)和诱导型一氧化氮合酶(INOS)的上调，这反过来又促进病毒的进一步复制和抑制支气管上皮细胞的凋亡。为了在体外和体内验证这一假说，我们提出了以下四个特定目标：(1)确定HBEC是否允许EBOV进入和复制而不诱导细胞凋亡和/或细胞毒；(2)确定细胞进入HBEC是否需要小凹；(3)确定IL-1介导的COX-2和iNOS表达上调是否能阻止HBEC内的细胞凋亡和促进EBOV复制；(4)确定RNA干扰(RNAi)通过诱导EBOV GP和L基因转录后沉默，是否可以阻止或抑制病毒在HBEC内的复制和萌发。这些研究有望为EBOV在HBEC中的进入、复制和萌发提供新的信息，即针对雾化EBOV的多种靶向生物防御方法如何调节EBOV在HBEC中的进入、复制和萌发。

DESCRIPTION (provided by applicant): Ebola virus (EBOV) is an enveloped, nonsegmented, negative-strand RNA virus within the filovirus family, which can cause a lethal hemorrhagic febrile disease in humans and nonhuman primates, killing up to 90% of those, infected. There are currently no effective antiviral agents available for the treatment of EBOV infections nor have appropriate vaccines been developed for human disease prevention. The events of September 11, 2001 and the ensuing inhalation anthrax bioterrorism incidents have highlighted concerns that EBOV also might be used as a bioterrorism agent that could be disseminated via the aerosol route. However, little is known about the capacity of EBOV to colonize the human lower respiratory tract and to subsequently induce a systemic viral hemorrhagic fever syndrome. There are currently no published in vitro models for studying the effects of EBOV on human lung cells. Our preliminary data indicate that EBOV can replicate in NORMAL HUMAN BRONCHIAL EPITHELIAL CELLS (HBEC) maintained in air/liquid interface primary culture. In this R21 application, we propose that: inhaled EBOV aerosols are able to colonize the lungs by establishing a persistent infection in bronchial epithelium, leading to subsequent infection of alveolar macrophages. The SPECIFIC HYPOTHESIS is that EBOV entry into the bronchial epithelium is associated with plasma membrane-associated caveolae in these cells. Furthermore, once entry has occurred, viral replication within the bronchial epithelium (which requires the EBOV RNA-dependent RNA polymerase L and glycoprotein GP genes) induces up-regulation of interleukin-1 (IL-1), cyclooxygenase-2 (COX-2), and the inducible form of nitric oxide synthase (iNOS), which, in turn, facilitate further viral replication and inhibit bronchial epithelial apoptosis. To test this hypothesis both in vitro and in vivo, we propose the following four SPECIFIC AIMS: (1) to determine whether HBEC are permissive for EBOV entry and replication without inducing apoptosis and/or cytotoxicity; (2) to determine whether caveolae are required for cellular entry into HBEC; (3) to determine whether IL-1-mediated up-regulation of COX-2 and iNOS expression in HBEC prevent apoptosis and facilitate EBOV replication within HBEC; (4) to determine whether RNA interference (RNAi), by inducing post-transcriptional silencing of EBOV GP and L genes, can prevent or inhibit the replication and budding of virus within HBEC. These studies are expected to provide new information as to how the entry, replication, and budding of EBOV in HBEC could be modulated by multiple targeted biodefense approaches against aerosolized EBOV.