The morbidity and mortality of viral hemorrhagic fevers are largely caused by vascular leakage and its ensuing complications. The mechanisms that lead to the vascular leakage have not been well characterized. This project will pursue the hypothesis that dengue and hantavirus-induced vascular leakage is caused by a temporal and differential responsiveness of virus-stimulated endothelium to T-cell derived inflammatory and angiogenic mediators. The first aim will delineate the early innate immune signaling pathways activated in virus-stimulated human microvascular endothelial cells that lead to Type I IFN production. Type I IFN mediates the enhancement of endothelial barrier function and anti-angiogenic effects. Barrier function will be assessed by measuring the permeability of human microvascular endothelial monolayers to macromolecules (70 kDa dextran) and small molecule solutes (3 kDa dextran) in a transwell assay system. Early angiogenic effects on human microvascular endothelium will be measured by a cell/matrix invasion assay. The second aim will delineate key anti-viral T-cell responses and phenotypes that interact with endothelium and lead to augmented permeability. Dengue virus and hantavirus-specific CD4+ and CD8+ T-cells will be activated under a variety of conditions and examined for their abilities to modulate vascular endothelial growth factor signaling, permeability, and cell invasion in the in vitro assays noted above. The third aim will measure inflammatory and angiogenic markers in blood samples from patients with hantavirus infections. Multiplex protein immunoassays for selected biomarkers will be performed on serial daily plasma and urine samples collected from patients with Puumala virus (Old World hantavirus) infections. The objective is to develop a biomarker "angiogenic profile" that can predict disease severity and development of vascular leakage. The effects of this "angiogenic profile" on virus-infected microvascular endothelium permeability will be tested in vitro. A better understanding of vascular leakage in dengue and hantavirus hemorrhagic fever pathogenesis will lead to new diagnostic, therapeutic, and preventive approaches to these pathogens.