Determinants of yellow fever pathogenesis in humans

PROJECT SUMMARY/ABSTRACT Yellow fever (YF) is a deadly disease transmitted by the bite of infected Aedes aegypti mosquitoes. Although an effective vaccine for YF was invented in the 1930s, it is not amenable to large-scale production and has a high rate of adverse events (including death). As a result, YF continues to kill approximately 30,000 people each year. Moreover, due to the re-emergence of Aedes mosquito vectors, substantial portions of Southeast Asia, Oceania, and even Europe and North America are at increasing risk for introduction of the YF virus (YFV), which would be a public health disaster. In humans, YF presents as a classic viral hemorrhagic fever (VHF), causing severe coagulation abnormalities (i.e. coagulopathy) and end-organ damage. This contrasts with what happens in mice, where YFV infection is largely benign and characterized by a distinct lack of coagulopathy/hemorrhagic features. The host factors that determine the development of VHF in YF in humans are unknown. This project aims to identify these factors at both cellular and molecular levels. Currently, VHF in YF is thought to be driven by the infection of hepatocytes. However, unpublished data collected from Brazilian YF patients suggests that hepatocyte destruction alone is insufficient to explain the coagulopathy observed in YF. Aim 1 will test the hypothesis that the infection of immune cells plays a major (and previously underappreciated) role in the development of VHF in YF. This Aim will utilize YFV infection of transgenic mice that have been engrafted with human hepatocytes or human immune cells - two cell populations that are thought to play a major role in the VHF disease process - allowing the study YFV infection of these cell populations in isolation in this otherwise YF-resistant host. A range of coagulation tests will be performed on infected mice to determine the mechanism by which coagulopathy develops during YFV infection. Blood samples drawn daily from YFV-infected macaques - the gold-standard animal model for studying VHF in YF - also will be analyzed, as this will be essential for determining the timing and causal relationships between key events such as clot formation, clotting factor depletion, and liver damage. In Aim 2, a CRISPR-Cas9 genome wide knock-out screen will be performed utilizing human and mouse, hepatocyte and Kupffer cell lines, to identify the factors that render humans (and primates) uniquely susceptible to YFV. The factors that restrict YFV replication in the murine host - hypothesized to be an interferon-stimulated gene (ISG) based on preliminary data - also will be identified. 'Hits' from this screen will be validated using targeted gene knock-out and trans-complementation in vitro. This will identify host factors that are essential in the YFV life cycle, and may lead to the generation of new YF-susceptible mouse models. This research will generate information and biological tools that can be used to combat YF specifically, and VHF more generally. This award will also help the principal investigator, Dr. Adam Bailey, MD, PhD, establish himself as an independent researcher and enable him to pursue high-reward projects that would otherwise not be feasible.