Dengue fever (DF), dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) are diseases caused by 4 serotypes of dengue virus (DV). Edema and hemorrhagic disease found in DHF/DSS result from vascular leakage, and these severe manifestations are enhanced by preexisting DV antibodies and subsequent infection by a second DV serotype. Thus an immune enhanced disease process contributes to increased endothelial cell (EC) permeability in DHF/DSS cases. DVs infect peripheral human leukocytes, dendritic cells and endothelial cells, and mechanisms by which DVs enhance vascular permeability are just beginning to be define. The ability of DVs to infect immune cells has resulted in many studies on infection of peripheral blood lymphocytes (PBLs) and their chemokine responses. Both are clearly important to viral pathogenesis and are readily studied in blood from humans or DV infected murine disease models. Although ECs are more difficult to study in vivo, they form the primary fluid barrier of the vasculature, and ultimately edema or hemorrhagic disease result from altering barrier functions of the endothelium. Postmortem studies of DV-infected patients and the AG129 mouse model demonstrate that ECs in the liver, lung, and spleen are infected. The ability of DVs to infect ECs provides a means for infection to alter capillary permeability, replicate and induce EC chemokine responses that activate and recruit immune cells to the endothelium. However, the contribution of DV infected ECs to immune enhancement and vascular permeability has yet to be factored into the DV disease process. DV infects ECs in culture, however prior in vitro studies were performed on ECs that were only 2-10% infected. In contrast, our recent studies of DV infected ECs were performed by synchronously infecting >80% of primary human ECs and monitoring viral and cellular responses. We found that DV productively infects primary human ECs, with a rapid increase in viral titer (~105/ml) 1 day p.i. Our findings suggest that DV infected ECs contribute to viremia, viral dissemination and the presentation of DV antigens on ECs that make them targets of antibodies and immune cells. Our analysis of EC responses to DV infection revealed the high level induction of chemokines that direct immune cell recruitment and activation. Cytokines CXCL10, CXCL11, IL-7, RANTES and BAFF are induced 337, 45, 128, 84 and 119 fold, respectively, in synchronously infected ECs by 24 hrs p.i. CXCL10/11 and RANTES secretion recruit and activate leukocytes and T-cells while IL-7 stimulates B, T and NK cell proliferation and T cell maturation. BAFF is a B and T cell activating factor that is released or expressed on the surface of activated ECs. DV infection also induced properdin (Factor P, 34-fold), which activates the alternative pathway (AP) complement system producing C3a/C5a: chemotactic anaphylatoxins which trigger localized inflammation, mast cell degranulation and vascular permeability. Importantly, high levels of C3a are present in severely ill dengue patients and associated with severe disease, plasma leakage and shock. These newly discovered EC responses indicate that ECs are not only targets of DV infection, but release factors that potentiate immune cell chemotaxis and permeability of the infected endothelium. Collectively these findings suggest that DV infected ECs contribute to viremia, enhanced immune responses and vascular permeability which are fundamental components of DHF and DSS. We propose to define EC responses to DV infection that enhance immune responses by activating and recruiting immune cells to the endothelium.

Public Health Relevance

Dengue virus is carried by mosquitos and infects 50-100 million people/ year, causing a vascular leak disorder responsible for edema and hemorrhagic disease in 500,000-1,000,000/ year with a 5-30% mortality rate. Dengue results in an immune enhanced disease process whereby the 2nd infection is more severe from a distinct serotype. DV infects immune as well as endothelial cells that line capillaries and regulate edema and bleeding. We have found that DV infected endothelial cells elicit responses that enhance immune mediated responses that contribute to vascular leakage and contribute to increased virus production and spread. We address this process in endothelial cells in this study.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
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Virology - B Study Section (VIRB)
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Cassetti, Cristina
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State University New York Stony Brook
Schools of Medicine
Stony Brook
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