Pathogenesis of Subtype IE Venezuelan Equine Encephalitis Virus

描述(申请人提供)：应聘者：亚当斯博士在德克萨斯A&M大学以优异成绩完成了DVM学位，并在康奈尔大学道格拉斯·安特扎克博士的实验室完成了免疫学博士学位，研究胎儿逃避母体免疫反应的机制。亚当斯博士将运用她作为免疫学家的技能来追求她的长期目标，即了解甲型病毒性脑炎的发病机制，这些知识最终将被用于开发有效的疫苗/疗法。拟议的项目旨在为她从一名讲师过渡到比较医学研究的永久学术职位铺平道路。 研究：委内瑞拉马脑炎病毒(VEEV)是一种自然出现的虫媒病毒，威胁整个美洲的人类健康。只有在马群中存在VEEV的有效扩增时，才会发生人类流行病。Veev也被认为是一种潜在的生物恐怖分子，被NIAID归类为B类优先病原体。最近爆发的一种流行性动物亚型IE VEEV突显了VEE在人类群体中出现的持续威胁，以及我们对其在等位基因中扩增和通过突变改变毒力的能力知之甚少。这项建议的目的是剖析流行性乙型脑炎病毒的结构蛋白和非结构蛋白在引起马的有效扩增、神经毒力和免疫调节中的作用。具体目的1：利用家畜/地方病亚型IE VEEV亲本株和嵌合株鉴定VEEV扩增和细胞嗜性的遗传决定因素。作为VEEV感染的早期细胞靶点，马巨噬细胞/树突状细胞(DC)将接受支持病毒复制的能力测试。在早期感染期间，还将检查马外周血白细胞(PBL)体内的病毒复制情况。具体目的2：利用流行性/地方性IEVEEV亲本株和嵌合株鉴定VEEV致病性和免疫原性的遗传决定因素。感染的马巨噬细胞/树突状细胞培养物将通过干扰素生物测定、细胞因子(包括I型干扰素)的定量RT-PCR和用于全球基因调控的基因芯片来测试先天/获得性免疫反应的改变。神经症状将在实验感染的马匹中进行定量评估；神经/神经外组织将进行大体/组织病理损害的定性/定量检查。将测量感染动物的血清中干扰素的诱导，并将使用定量RT-PCR分析PBL和淋巴/非淋巴组织中细胞因子的基因表达变化。总的来说，这些实验将促进对VEE出现机制的理解，并揭示扩增/毒力的自然决定因素。 环境：亚当斯博士的项目是对她提议的赞助商斯科特·韦弗博士目前由美国国立卫生研究院资助的研究的补充。培训的主要地点是德克萨斯大学医学部(UTMB)，拥有一流的设施和设备，以及研究虫媒病毒各个方面的优秀教师，包括比较医学、免疫学和疫苗开发。 公共卫生相关性：委内瑞拉马脑炎病毒(VEEV)是一种发生在整个美洲的重要人畜共患病的病原体，已证明有能力造成影响数十万人和马匹的爆炸性疫情。只有当VEEV在受感染的马、驴和骡子的血液中复制到高滴度，然后通过受感染的蚊子传播给人类时，人类才会发生重大流行病。利用墨西哥最近暴发的VEEV分离株，拟议的研究目的是识别与马的有效复制和神经毒力有关的病毒基因，这对于成功开发未来的干预策略(即疫苗、治疗和其他人类疾病控制措施)至关重要。

DESCRIPTION (provided by applicant): Candidate: Dr. Adams completed her DVM cum laude at Texas A&M University, and she completed her Ph.D. in immunology in Dr. Douglas Antczak's laboratory at Cornell University, studying mechanisms of fetal evasion from maternal immune responses. Dr. Adams will apply her skills as an immunologist to pursue her long-term goal of understanding the pathogenesis of alphaviral encephalitides, knowledge that will ultimately be used for development of effective vaccines/therapeutics. The proposed project is designed to bridge her transition from an Instructor to a permanent academic post in comparative medicine research. Research: Venezuelan equine encephalitis virus (VEEV) is a naturally emerging arbovirus that threatens human health throughout the Americas. Human epidemics only occur when there is efficient amplification of VEEV in equids. VEEV is also considered a potential bioterrorist agent, categorized by the NIAID as a Category B Priority Pathogen. A recent outbreak of an epizootic subtype IE VEEV underscores the continued threat of VEE emergence in human populations and what little we know about its ability to amplify in equids and alter virulence via mutations. The objective of this proposal is to dissect the role of structural and nonstructural proteins of epizootic subtype IE VEEV in causing efficient equine amplification, neurovirulence, and immune modulation. Specific aim 1: Identify genetic determinants of VEEV amplification and cell tropism using parental and chimeric strains of epizootic/enzootic subtype IE VEEV. As early cell targets of VEEV infection, equine macrophages/dendritic cells (DCs) will be tested for their ability to support viral replication. Equine peripheral blood leukocytes (PBLs) will also be examined for viral replication in vivo during early infection. Specific aim 2: Identify genetic determinants of VEEV pathogenicity and immunogenicity using parental and chimeric strains of epizootic/enzootic subtype IE VEEV. Infected equine macrophage/DC cultures will be tested for altered innate/adaptive immune responses using interferon (IFN) bioassays, quantitative RT-PCR of cytokines (including type I IFNs), and cDNA microarrays for global gene regulation. Neurologic symptoms will be quantitatively assessed in experimentally infected equids; neural/extraneural tissues will be qualitatively/quantitatively examined for gross/histopathologic lesions. Sera from infected animals will be measured for IFN induction, and PBLs and lymphoid/non-lymphoid tissues will be analyzed for altered gene expression of cytokines using quantitative RT-PCR. Collectively, these experiments will advance understanding of VEE emergence mechanisms and reveal natural determinants of amplification/virulence. Environment: Dr. Adams' project complements current, NIH-funded research of her proposed sponsor, Dr. Scott Weaver. The primary site of training, the University of Texas Medical Branch (UTMB), has superb facilities and equipment and outstanding faculty who study various aspects of arboviruses, including comparative medicine, immunology, and vaccine development. PUBLIC HEALTH RELEVANCE: Venezuelan equine encephalitis virus (VEEV) is the cause an important zoonotic disease that occurs throughout the Americas, with the demonstrated capacity to cause explosive outbreaks affecting hundreds-of- thousands of people and equids. Major epidemics in humans only occur when VEEV replicates to high titers in the blood of infected horses, donkeys, and mules, which is then transmitted to humans by infected mosquitoes. Using a VEEV isolate from a recent outbreak in Mexico, the purpose of the proposed study is identify viral genes that are responsible for efficient replication and neurovirulence in horses, which is critically important for the successful development of future intervention strategies (i.e., vaccines, therapeutics, and other human disease control measures).