Resistance reasoning: RNA-seq analysis of changes in gene expression in strains of Aedes aegypti that are susceptible or refractory to Dengue-2 virus

Dengue is the most widespread arbovirus, infecting 50-100 million people each year. It is primarily transmitted by Aedes aegypti and may cause DHF (dengue hemorrhagic fever) or DSS (dengue shock syndrome), which can be fatal. There are currently no vaccine or treatment options available. Current vector control programs rely on insecticides, environmental alteration and transgenic mosquitoes. We have described the situation in Cali, Colombia, where approximately 30% of feral Ae. aegypti are refractory to Dengue virus 2 (DENv2) and reported that there were significant differences in the expression of apoptosis related genes in the Refractory strain. In order to remove our bias we infected Susceptible or Refractory mosquitoes with blood, or blood and DENv2. We used RNA-seq technology to compare differential gene expression in the midguts of Refractory and Susceptible mosquitoes at 24, 36 and 48 hours post-feeding. Gene expression and transcript accumulation were compared across time in each treatment group, between treatments (+/-virus) within the same strain, and between strains with the same treatment (+ virus) using GFOLD, DESeq and Cufflinks. A subset of highly differentially expressed genes was selected for further verification via real time PCR (qPCR) analyses. We identified several of the apoptosis related genes described previously, but also immune related genes linked to other immune pathways. These genes will be selected for Knock-down procedures using RNAi to determine their contribution to the Susceptible or Refractory phenotype.