Uncovering determinants of pathogenic outcome versus protective responses in filovirus infections

ABSTRACT The filoviruses Ebola virus (EBOV) and Marburg virus (MARV), cause severe disease in humans with high case fatality rates. Advanced metagenomic analyses led to the discovery of previously unknown filoviruses from a range of animal species. Newly discovered filoviruses include Lloviu virus (LLOV) and Dehong virus (DEHV). While recent work on LLOV suggests that it might not pose a threat to human health, it is not known if DEHV can cause disease in humans. The discovery of novel filoviruses provides an excellent research avenue for determining the molecular correlates that define pathogenicity or protection by comparing closely related pathogenic and potentially nonpathogenic viruses. In this application, we propose to perform comparative studies with pathogenic (EBOV, MARV), likely nonpathogenic (Reston virus, LLOV), and filoviruses of unknown pathogenicity (DEHV). We will analyze potential determinants of filovirus pathogenicity across viruses, including replication kinetics (Aim 1), the magnitude of the virus-induced inflammatory response in macrophages (Aim 2), and virulence in two humanized mouse models (Aim 3). By integrating findings from all three aims, we will be able to define key molecular signatures of filovirus infection that have the potential to inform the assessment of the pathogenic potential of known and newly emerging filoviruses. In Aim 1, we will compare the replication kinetics of the various viruses in distinct cell types, which will inform about the virus-intrinsic and cell-dependent factors determining replication efficiency. We will perform RNA FISH analysis to gain insight into the earliest events of viral transcription and genome replication at single-cell level. Since viral replication kinetics determine the timing and rate of viral RNA production and protein expression, they may play a major role in shaping antiviral host responses and virulence. In Aim 2, we will comprehensively profile the phosphoproteomic/proteomic and transcriptomic changes in filovirus-infected human macrophages to map the differences in the host response signatures induced by pathogenic and nonpathogenic viruses. These analyses will be accompanied by mechanistic studies aimed to dissect the molecular mechanisms of filovirus-induced immune activation using knockdown cells and targeted inhibitor approaches. In Aim 3, we will comparatively assess the ability of the various filoviruses to cause disease in two human immune system (HIS) mouse models. This includes state-of-the-art histopathological analysis of the tissues including spatial transcriptomics. Our multidisciplinary team combines expertise in filovirus biology, virus-induced activation of innate immune cells, omics analysis, humanized mouse models, and histopathology of filovirus-infected tissues. We are therefore well positioned to perform the proposed work.