Biodefense Against an Aerosolized Ebola Threat

描述（由申请方提供）：埃博拉病毒（EBOV）是丝状病毒家族中的一种有包膜、不分节段、负链RNA病毒，可在人类和非人灵长类动物中引起致死性出血性发热疾病，可杀死高达90%的感染者。 目前没有有效的抗病毒剂可用于治疗EBOV感染，也没有开发出适当的疫苗用于人类疾病预防。 2001年9月11日的事件和随后发生的吸入性炭疽生物恐怖主义事件突出了人们的关切，即EBOV也可能被用作生物恐怖主义制剂，可通过气溶胶途径传播。 然而，关于EBOV定殖于人下呼吸道并随后诱导全身性病毒性出血热综合征的能力知之甚少。 目前还没有公开的体外模型用于研究EBOV对人肺细胞的影响。 我们的初步数据表明，EBOV可以复制正常人支气管上皮细胞（HBEC）保持在空气/液体界面原代培养。 在该R21申请中，我们提出：吸入的EBOV气溶胶能够通过在支气管上皮中建立持续感染来定殖肺部，导致随后的肺泡巨噬细胞感染。 特异性假设是EBOV进入支气管上皮与这些细胞中的质膜相关小窝有关。 此外，一旦进入发生，支气管上皮内的病毒复制（其需要EBOV RNA依赖性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（考克斯-2）和诱导型一氧化氮合酶（iNOS）的上调，这又促进病毒进一步复制并抑制支气管上皮细胞凋亡。 为了在体外和体内验证这一假设，我们提出了以下四个具体目标：（1）确定HBEC是否允许EBOV进入和复制而不诱导细胞凋亡和/或细胞毒性;（2）确定细胞进入HBEC是否需要小窝;（3）确定IL-1介导的考克斯-2和iNOS表达的上调是否阻止HBEC中的凋亡并促进HBEC中的EBOV复制;（4）确定RNA干扰（RNAi）是否通过诱导EBOV GP和L基因的转录后沉默来阻止或抑制病毒在HBEC内的复制和出芽。这些研究预计将提供新的信息，如何进入，复制，并在HBEC的EBOV的出芽可以通过多种针对雾化EBOV的生物防御方法调制。

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.