Innate immune pathways act synergistically to constrain RNA virus evolution in Drosophila melanogaster

Host-pathogen interactions impose recurrent selective pressures that lead to constant adaptation and counter-adaptation in both competing species. Here, we sought to study this evolutionary arms-race and assessed the impact of the innate immune system on viral population diversity and evolution, using D. melanogaster as model host and its natural pathogen Drosophila C virus (DCV). We first isogenized eight fly genotypes generating animals defective for RNAi, Imd and Toll innate immune pathways and also pathogen sensing and gut renewal pathways. Wild-type or mutant flies were then orally infected and DCV was serially passaged ten times. Viral population diversity was studied after each viral passage by high-throughput sequencing, and infection phenotypes were assessed at the beginning and at the end of the passaging scheme. We found that the absence of any of the various immune pathways studied increased viral genetic diversity and attenuated the viruses. Strikingly, these effects were observed in both host factors with antiviral properties and host factors with antibacterial properties. Together, our results indicate that the innate immunity system as a whole, and not specific antiviral defense pathways in isolation, generally constrains viral diversity and evolution.