Generation of a biologically contained mouse model of Ebola virus infection for mechanistic studies - Project Summary Due to its high mortality rate and persistent re-emergence, the Ebola virus disease (EVD) remains a significant threat to global health. In Central and West Africa, the Ebola virus (EBOV), a member of the Filoviridae family, has caused sporadic outbreaks of a deadly hemorrhagic disease, with a case fatality rate of up to 90%. Patient fatalities result from severe inflammation, multiple organ dysfunction syndrome, endotheliopathy, and hemorrhage. Despite initial descriptions of the hemorrhagic manifestations of EVD nearly five decades ago, the pathobiology of Ebola-associated coagulopathy (EAC) remains a mystery, even after 40 filoviral outbreaks in the subsequent years. While non-human primate (NHP) models of lethal disease have facilitated preclinical evaluations of medical countermeasures, little has been learned about the nature and causes of coagulopathy and bleeding from these severe disease models. Even the limited pathophysiological signals have been incomplete, hindered by a focus on a small number of proteins, concentrating solely on levels rather than function and activity, and facing challenges in assessing complex interactions among cellular (e.g., endothelial cells, platelets) and coagulation protein components of a complex system. The extreme virulence of the virus, which requires biosafety level 4 (BSL-4) containment, has impeded its study, leaving many important questions about the pathophysiology of EAC unanswered. Leveraging our experience in characterizing animal models of viral infection-associated coagulopathies and using the paradigm-shifting preliminary data obtained from longitudinal samples collected from Ebola-infected NHPs, we propose studies aimed at generating a biologically contained EVD mouse model that will permit the investigation of pathophysiological alterations induced by the infection at a BSL2+ level. This will open up a vast array of tools for mouse studies, significantly expanding the potential for investigating this highly pathogenic virus. The experimental effort in our overall Aim will focus on generating a transgenic mouse that ubiquitously expresses the EBOV transactivator protein VP30, utilizing CRISPR/Cas9 technology. We will also engineer and rescue a biologically contained mouse-adapted (MA) EBOV derived from the existing EBOV-ΔVP30 virus, a recombinant virus lacking the VP30 coding sequence whose entire replication is contained in cells expressing the missing viral protein. Subsequently, we will characterize the disease induced by the MA-EBOV-ΔVP30 virus in the newly generated VP30 transgenic mouse, focusing on dissecting the hemostatic system during acute infection, thus providing much-needed mechanistic insights into EAC. The successful completion of these studies will not only yield a mouse model of EBOV infection that can be handled at BSL-2+, greatly expanding accessibility to previously unreachable cutting-edge technologies, but will also enhance our understanding of EAC, provide critical mechanistic insights for key drivers of EAC, and allow for future preclinical testing of several potential pharmacological approaches to mitigate the severity of EVD.
