Novel phenotype of Wolbachia strain wPip in Aedes aegypti challenges assumptions on mechanisms of Wolbachia-mediated dengue virus inhibition

The bacterial endosymbiontWolbachiais a biocontrol tool that inhibits the ability of theAedes aegyptimosquito to transmit positive-sense RNA viruses such as dengue and Zika. Growing evidence indicates that whenWolbachiastrainswMel orwAlbB are introduced into local mosquito populations, human dengue incidence is reduced. Despite the success of this novel intervention, we still do not fully understand howWolbachiaprotects mosquitoes from viral infection. Here, we demonstrate that theWolbachiastrainwPip does not inhibit virus infection inAe.aegypti. We have leveraged this novel finding, and a panel ofAe.aegyptilines carrying virus-inhibitory (wMel andwAlbB) and non-inhibitory (wPip) strains in a common genetic background, to rigorously test a number of hypotheses about the mechanism ofWolbachia-mediated virus inhibition. We demonstrate that, contrary to previous suggestions, there is no association between a strain's ability to inhibit dengue infection in the mosquito and either its typical density in the midgut or salivary glands, or the degree to which it elevates innate immune response pathways in the mosquito. These findings, and the experimental platform provided by this panel of genetically comparable mosquito lines, clear the way for future investigations to define howWolbachiapreventsAe.aegyptifrom transmitting viruses. Author summary Dengue virus, transmitted by theAedes aegyptimosquito, is one of the fastest-growing infectious diseases, causing an estimated 390 million human infections per year worldwide. Vaccines have limited efficacy and there are no approved therapeutics. This has driven the rise of novel vector control programs, in particular those that use the bacterium,Wolbachia, which prevents transmission of dengue and other human pathogenic viruses when stably introduced intoAe.aegyptipopulations. Although this is proving to be a highly effective method, the details of how this biocontrol tool works are not well understood. Here we characterise a newWolbachiastrain,wPip, and find thatAe.aegypticarryingwPip are still able to transmit dengue similar to mosquitoes that do not carryWolbachia. This finding has allowed us to begin a rigorous program of comparative studies to determine which features of aWolbachiastrain determine whether it is antiviral. Understanding these mechanisms will enable us to predict the risk of viral resistance arising againstWolbachiaand facilitate preparation of second-generation field release lines.