PB1378 - Endothelial cell activation after Yellow Fever virus infection

Background: Yellow fever (YF) is an acute disease endemic in parts of South America and Africa caused by the YF virus and transmitted by mosquitoes. The course of the disease in humans ranges from asymptomatic infection to fatal organ failure and internal bleeding in severe cases. The role of the endothelium in the pathogenesis of YF is not yet well understood.

Aims: To study the effects of YFV infection on endothelial cell activation using in vitro models.

Methods: Human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (HMEC) were infected with the vaccine strain YFV-17DD. Susceptibility to infection was analyzed by determining viral infectivity titers in the cell supernatant and the presence of intracellular viral RNA by immunofluorescence. Cell viability was determined by staining with a fixable viability dye and Annexin-V. Activation responses included expression of E-selectin and ICAM-1 by flow cytometry, secretion of IL-1b and von Willebrand factor (VWF) by ELISA, transcript levels of endothelial nitric oxide synthase (eNOS) and tissue factor (TF) by RT-qPCR, and various signaling pathways by Western blot. Results were analyzed by Student's t-test or ANOVA followed by Bonferroni test.

Results: Detection of a significant increase in viral titer and intracellular viral RNA by an anti-dsRNA antibody indicated that HUVEC and HMEC were susceptible to YFV infection. Infection of HUVEC resulted in apoptotic cell death, expression of E-selectin and ICAM-1, and secretion of IL-1b and VWF. In addition, the infected cells showed decreased eNOS and increased TF mRNA levels (Figure 1). Mechanistically, infection led to activation of the NF-κB and MAPK- ERK1/2 signaling pathways (Figure 2).

Conclusion(s): Our results suggest that YFV infection triggers pro-inflammatory, pro-coagulant, and pro-thrombotic responses in endothelial cells which may contribute to YFV pathogenesis. Viral infection activates the ERK1/2 and NF-κB signaling pathways, suggesting that these molecules are potential targets for novel therapeutic strategies.