Molecular Basis of Host-Filovirus Interactions in Pathogenesis

发病机制中宿主-丝状病毒相互作用的分子基础

• 批准号: 8336345
• 负责人: Hideki Ebihara
• 金额: $ 69.22万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8336345
• 关键词: Animal Model; Biological; Blood Coagulation Disorders; Case Fatality Rates; Cavia; Cell Culture Techniques; Cellular Tropism; Clinical; Clinical Data; Coagulation Process; Communicable Diseases; Complementary DNA; Democratic Republic of the Congo; Development; Disease; Disease Outbreaks; Disseminated Intravascular Coagulation; Ebola Hemorrhagic Fever; Evaluation; Family; Fibrinogen; Filoviridae; Filovirus; Genome; Hamsters; Human; Immune; Impairment; In Vitro; Infection; Intervention; Length; Licensing; Life Cycle Stages; Macaca mulatta; Mesocricetus auratus; Modeling; Molecular; Mus; Pathogenesis; Pathology; Process; Protein C; Proteins; Recombinants; Research; Rodent; Rodent Model; Role; Syndrome; System; Testing; Therapeutic; Time; Vaccines; Viral; Viral Hemorrhagic Fevers; Viral Proteins; Virulence; Wild Type Mouse; Work; base; effective therapy; in vivo; innovation; member; mutant; nonhuman primate; positional cloning; prophylactic; protein function; protein protein interaction; response; systems research; therapeutic vaccine; tool

Viral hemorrhagic fevers caused by Ebolavirus (EBOV) and Marburgvirus (MARV), both members of the Filoviridae family, are known to be among the most severe infectious diseases in human and nonhuman primates (NHPs), and no licensed vaccines or effective therapeutics are currently available. Zaire ebolavirus (ZEBOV), in particular, has been responsible for multiple Ebola hemorrhagic fever (EHF) outbreaks with case-fatality rates ranging from 65 to 90%. Studies with animal models and limited clinical data from EHF outbreaks suggest that interdependent pathogenic processes, including both the host immune and pathophysiological responses, induced by EBOV infection trigger severe hemorrhagic syndrome. However, in order to develop effective treatments for EHF, it is necessary to better understand the mechanisms of viral and host interactions at the molecular and cellular levels and how these interactions contribute to the in vivo pathogenic process. Therefore, our research is focused on elucidating the roles of viral proteins in the viral replication cycle and pathogenesis. To accomplish this, we have three ongoing projects: (1) the development of a small animal model that recapitulates EHF, (2) the development of efficient reverse genetics systems for generating recombinant ZEBOV from cDNA, and (3) the development of molecular tools for identifying host cellular factors essential for ZEBOV replication. (1) The development of a small animal model that recapitulates EHF. While the NHP model is used to evaluate the efficacy of EBOV vaccines and therapeutics because it accurately recapitulates disease, rodent models (mice and guinea pigs) are convenient and suitable for elucidating the roles of specific viral proteins in the pathogenic process and have been widely used in numerous aspects of EBOV research. However, rodent models produce only limited and inconsistent coagulation abnormalities, which are a hallmark clinical feature of EHF. Therefore, we attempted to develop an EHF rodent model that displays severe coagulation impairment, specifically one that presents with disseminated intravascular coagulation (DIC), a key aspect of EBOV pathology in humans and NHPs. Initially, we demonstrated that rhesus macaques infected with ZEBOV showed severe coagulopathy, as indicated by prolonged coagulation times and decreased fibrinogen and protein C levels. Next, we examined the virulence of mouse-adapted Zaire Ebolavirus (MA-ZEBOV) in Syrian golden hamsters, since MA-ZEBOV, unlike wild-type ZEBOV, produces lethal illness in mice and guinea pigs. Hamsters infected with MA-ZEBOV succumbed to infection and displayed coagulation abnormalities as well as other clinical features that resembled EHF seen in rhesus macaques and humans. The hamster will represent an innovative and powerful rodent model for the evaluation of EHF prophylactic and post-exposure interventions prior to testing in NHPs. Detailed characterization of ZEBOV pathogenesis in this hamster model is ongoing. (2) The development of efficient reverse genetics systems for generating recombinant ZEBOV from cDNA. We have been developing a more efficient rescue system to generate recombinant ZEBOV. We have developed full-length ZEBOV genome clones for wild-type and mouse-adapted ZEBOV. These clones will be used to elucidate the functions of viral proteins and their roles in the viral replication cycle and pathogenesis. (3) The development of molecular tools for identifying host cellular factors essential for ZEBOV replication. We have established various ZEBOV viral protein mutants possessing tandem-tags and fluorescent protein-tags to examine protein-protein interactions among viral and cellular proteins and to identify those cellular interaction partners that are essential for the ZEBOV replication cycle. We have made significant progress in understanding the molecular basis underlying ZEBOV pathogenesis, and we have established or initiated several additional research systems to study host-ZEBOV interaction. We have already developed a small animal model for accurately recapitulating EHF, and we are currently developing a more efficient reverse genetics system as well as various molecular tools for studying ZEBOV. Our work has produced, and will continue to produce, powerful tools for dissecting filovirus pathogenesis and revealing the molecular details behind host-filovirus interactions.

已知由埃博拉病毒（EBOV）和马尔堡病毒（MARV）（均为丝状病毒科成员）引起的病毒性出血热是人类和非人灵长类动物（NHP）中最严重的传染病之一，目前尚无获得许可的疫苗或有效的治疗方法。特别是扎伊尔埃博拉病毒（ZEBOV）是造成多起埃博拉出血热（EHF）暴发的原因，病死率为65%至90%。动物模型研究和EHF暴发的有限临床数据表明，EBOV感染诱导的相互依赖的致病过程，包括宿主免疫和病理生理反应，引发严重出血综合征。然而，为了开发EHF的有效治疗方法，有必要更好地了解病毒和宿主在分子和细胞水平上相互作用的机制，以及这些相互作用如何促进体内致病过程。因此，我们的研究重点是阐明病毒蛋白在病毒复制周期和发病机制中的作用。为了实现这一目标，我们有三个正在进行的项目：（1）开发一个重现EHF的小动物模型，（2）开发有效的反向遗传学系统，用于从cDNA产生重组ZEBOV，以及（3）开发分子工具，用于识别ZEBOV复制所必需的宿主细胞因子。 (1)流行性出血热小动物模型的建立。虽然NHP模型用于评估EBOV疫苗和治疗剂的功效，因为它准确地再现了疾病，但啮齿动物模型（小鼠和豚鼠）方便且适合于阐明特定病毒蛋白在致病过程中的作用，并且已广泛用于EBOV研究的许多方面。然而，啮齿动物模型仅产生有限且不一致的凝血异常，这是EHF的标志性临床特征。因此，我们试图开发一种表现出严重凝血障碍的EHF啮齿动物模型，特别是表现出弥散性血管内凝血（DIC）的EHF啮齿动物模型，这是人类和NHP中EBOV病理学的一个关键方面。最初，我们证明了感染ZEBOV的恒河猴表现出严重的凝血障碍，如凝血时间延长和纤维蛋白原和蛋白C水平降低所示。接下来，我们检查了小鼠适应的扎伊尔埃博拉病毒（MA-ZEBOV）在叙利亚金黄仓鼠中的毒力，因为MA-ZEBOV与野生型ZEBOV不同，在小鼠和豚鼠中产生致命的疾病。感染MA-ZEBOV的仓鼠死于感染，并显示凝血异常以及其他临床特征，类似于在恒河猴和人类中观察到的EHF。仓鼠将代表一种创新和强大的啮齿动物模型，用于在NHP中进行测试之前评估EHF预防和暴露后干预措施。在该仓鼠模型中ZEBOV发病机制的详细表征正在进行中。 (2)开发用于从cDNA产生重组ZEBOV的有效反向遗传学系统。我们一直在开发更有效的拯救系统来产生重组ZEBOV。我们已经开发了野生型和小鼠适应性ZEBOV的全长ZEBOV基因组克隆。这些克隆将用于阐明病毒蛋白的功能及其在病毒复制周期和发病机制中的作用。 (3)开发用于鉴定ZEBOV复制所必需的宿主细胞因子的分子工具。我们已经建立了具有串联标签和荧光蛋白标签的各种ZEBOV病毒蛋白突变体，以检查病毒和细胞蛋白之间的蛋白质-蛋白质相互作用，并鉴定那些对ZEBOV复制周期至关重要的细胞相互作用伴侣。 我们在理解ZEBOV发病机制的分子基础方面取得了重大进展，并且我们已经建立或启动了几个额外的研究系统来研究宿主-ZEBOV相互作用。我们已经开发了一种小动物模型来精确重现EHF，目前我们正在开发一种更有效的反向遗传学系统以及各种研究ZEBOV的分子工具。我们的工作已经产生，并将继续产生强大的工具，用于解剖丝状病毒的发病机制，并揭示宿主-丝状病毒相互作用背后的分子细节。