Using multiple approaches to identify the factors that determine if Aedes aegypti will transmit or eliminate Dengue viruses

Aedes aegypti is the principal vector of Dengue viruses (DENV) globally. In the city of Cali, Colombia, however, ~30% of female Ae. aegypti collected in the field are refractory to all 4 dengue serotypes. We have used targeted (suppressive subtractive hybridization) and general (RNA-seq) approaches to identify differentially expressed genes that might help DENV replication in susceptible females or impede DENV in refractory mosquitoes. We proposed that increased apoptosis in refractory strains reduced DENV transmission but knocking down inhibitors of apoptosis in susceptible strains was lethal. We have used RNAi to evaluate the role of specific genes in determining the phenotype, and reduced, but did not eliminate, the prevalence of DENV in susceptible mosquitoes. We compared the microbiome diversity between the two strains but identified no significant differences. Ultimately, none of these approaches has identified single or combined factors or genes that explain the difference in vector competence between the phenotypes. Metabolic and lipidomic approaches comparing the milieu of midgut molecules that may interact with DENV have identified many molecules that differ between the strains, including putative antiviral compounds. These complex molecular interactions between vector and virus are mediated through the innate immune system of the mosquito vectors. Understanding the basis of refractoriness will advance our understanding of basic invertebrate immunity and host-pathogen interactions. Ultimately, we wish to exploit this knowledge and use CRISPR-Cas gene editing tools to generate stable DENV-refractory lines of Ae. aegypti that can contribute to the global reduction in transmission of DENV.