BAKOUMBA

Dengue viruses (DENV-1, -2, -3 and -4) and their main mosquito vector Aedes aegypti are responsible for the most rapidly advancing arboviral disease of humans. Mosquito susceptibility to DENV infection is a quantitative trait that varies between and within species, however the genes that make mosquitoes resistant or susceptible to DENV in nature have remained elusive. This project aims to elucidate the genetic basis of DENV resistance/susceptibility in Ae. aegypti using genome-wide gene mapping and functional validation by genetic engineering. It will take advantage of the natural resistance phenotype of a wild-type Ae. aegypti strain recently isolated from Bakoumba (Gabon). The Bakoumba strain displays strong resistance against DENV-1 and intermediate resistance against DENV-2 and DENV-4. We propose to carry out a genetic cross between the Bakoumba strain and a DENV-1 susceptible strain from Cairns (Australia) to map the underlying genomic loci. Our specific aims are to (1) identify mosquito candidate genes by genome-wide genetic mapping and (2) functionally validate the most promising candidate genes by genetic engineering in vivo. Our genetic mapping strategy is based on bulk segregant analysis, whereby individuals from a segregating population are genotyped in large pools with contrasted phenotypes (resistant vs. susceptible). In a pilot study, we obtained preliminary evidence that segregating single-nucleotide polymorphisms associated with DENV-1 infection can be detected at the gene level in a laboratory cross between the Bakoumba and Cairns strains. We will use reverse genetics to functionally test the candidate genes in the Bakoumba and Cairns genetic backgrounds. Our approach of reverse genetics includes transgenic overexpression and CRISPR/Cas9-mediated swapping of putative resistance/susceptible alleles between their parental genetic backgrounds of origin. Unlike gene knockout or gene knockdown, our reverse genetics strategy will be able to capture allelic effects. Moreover, it will be possible to extend the functional characterization of the allelic variants identified with DENV-1 to the other DENV types. Partners of the project combine expertise in quantitative genetics and mosquito transgenesis, respectively. Overall, this project will shed light on the molecular determinants of mosquito vector competence for arboviruses by identifying and characterizing novel restriction factors and antiviral mechanisms. In addition, it will contribute to expand the molecular toolbox to genetically engineer mosquitoes. Ultimately, results from the project will support the development of novel entomological strategies to fight emerging arboviral diseases.