Understanding innate immune responses of insect vectors to intracellular pathogens.

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This proposal will address the molecular interactions between Aedes aegypti, the insect vector of Dengue viruses (DENv) to humans. Specifically we will look at the immune immune processes used by mosquitoes to eliminate DENv, and how the viruses circumvent these responses. Recent studies have addressed the role of molecules and pathways that regulate or determine the intracellular development of DENv in mosquitoes, but the majority of these studies have used lab strains of mosquitoes that are susceptible to DENv. With collaborators in Colombia we have identified field-collected Ae. aegypti that are naturally resistant to DENv. These have been maintained in the lab and further selected by exposure to DENv, resulting in selected lines with differential susceptibility to DENv. Our initial data indicate that the 2 resistant strains use apoptosis, a form of cell death, to eliminate DENv. With these data in hand we propose to: 1: Compare the genes expressed in the resistant (Cali-MIB) and susceptible (Cali-S) insects after exposure to DENv using high throughput sequencing (RNA-seq); 2: Confirm and quantify, using Real Time quantitative PCR, the differential expression of selected genes after exposure to Dengue virus. 3) Use gene knockdown strategies (RNAi) to determine/confirm the role of specific molecules in determining the phenotype, and 4: Evaluate the gene expression and apoptotic responses of field collected insects to DENv to determine if our lab-based hypotheses represent what truly happens in the field, and evaluate the immune responses of our selected lab strains to all 4 DENv serotypes. The results of these studies will identify those genes and proteins that determine the phenotype in field populations and will pave the way to understanding the interplay and molecular interactions between the genomes of vector and pathogen.