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Biodefense Against an Aerosolized Ebola Threat

针对雾化埃博拉威胁的生物防御

基本信息

批准号：
6781871
负责人：
ELLIOTT KAGAN
金额：
$ 29.94万
依托单位：
HENRY M. JACKSON FDN FOR THE ADV MIL/MED
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-08-01 至 2007-03-25
项目状态：
已结题

项目摘要

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.

描述（由申请人提供）：埃博拉病毒（EBOV）是线状病毒家族中的一种包膜、非节段、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。目前没有治疗EBOV感染的有效抗病毒药物，也没有开发出预防人类疾病的适当疫苗。2001年9月11日的事件和随后发生的吸入性炭疽生物恐怖事件突出了人们的担忧，即EBOV也可能被用作可通过气溶胶途径传播的生物恐怖主义剂。然而，EBOV在人类下呼吸道定植并随后诱发全身性病毒性出血热综合征的能力尚不清楚。目前还没有发表的体外模型来研究EBOV对人肺细胞的影响。我们的初步数据表明，EBOV可以在空气/液界面原代培养的正常人支气管上皮细胞（HBEC）中复制。在这项R21应用中，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植肺部，导致随后的肺泡巨噬细胞感染。特异性假说认为EBOV进入支气管上皮与这些细胞中的质膜相关小泡有关。此外，一旦进入支气管上皮，病毒复制（这需要EBOV RNA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，进而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是否需要小泡；(3)确定il -1介导的上调HBEC中COX-2和iNOS的表达是否能阻止HBEC细胞凋亡并促进EBOV在HBEC内的复制；(4)确定RNA干扰（RNA interference, RNAi）能否通过诱导EBOV GP和L基因转录后沉默，阻止或抑制HBEC内病毒的复制和出芽。这些研究有望为EBOV在HBEC中的进入、复制和出芽如何通过针对雾化EBOV的多种靶向生物防御方法来调节提供新的信息。