Pathogen blocking in Wolbachia-infected Aedes aegypti is not affected by Zika and dengue virus co-infection

Background Wolbachia's ability to restrict arbovirus transmission makes it a promising tool to combat mosquito-transmitted diseases. Wolbachia-infected Aedes aegypti are currently being released in locations such as Brazil, which regularly experience concurrent outbreaks of different arboviruses. A. aegypti can become co-infected with, and transmit multiple arboviruses with one bite, which can complicate patient diagnosis and treatment. Methodology/principle findings Using experimental oral infection of A. aegypti and then RT-qPCR, we examined ZIKV/DENV-1 and ZIKV/DENV-3 co-infection in Wolbachia-infected A. aegypti and observed that Wolbachia-infected mosquitoes experienced lower prevalence of infection and viral load than wildtype mosquitoes, even with an extra infecting virus. Critically, ZIKV/DENV co-infection had no significant impact on Wolbachia's ability to reduce viral transmission. Wolbachia infection also strongly altered expression levels of key immune genes Defensin C and Transferrin 1, in a virus-dependent manner. Conclusions/significance Our results suggest that pathogen interference in Wolbachia-infected A. aegypti is not adversely affected by ZIKV/DENV co-infection, which suggests that Wolbachia-infected A. aegypti will likely prove suitable for controlling mosquito-borne diseases in environments with complex patterns of arbovirus transmission. Author summary Wolbachia is an endosymbiotic bacterium that infects insects. It has been artificially transferred into Aedes aegypti, a mosquito species that can transmit medically important viruses including dengue, chikungunya, and Zika. Wolbachia in A. aegypti limits infection with these viruses, making the mosquitoes much less capable of transmitting them to people. In tropical areas, where these viral pathogens are commonly found, it is not unusual for outbreaks of different viruses to occur at the same time, which can complicate diagnosis and treatment for those afflicted. Mosquitoes with Wolbachia are currently being released into these areas to reduce transmission of these diseases. In our study, we assessed whether Wolbachia infection in A. aegypti mosquitoes could still effectively inhibit the dengue and Zika viruses if the mosquitoes were fed both viruses at the same time. We found that Wolbachia was still very effective at inhibiting the replication of both viruses in the mosquito, and likewise still greatly reduced the chance of transmission of either virus. Our results suggest that Wolbachia-infected mosquitoes should be able to limit infection with more than one virus, should they encounter them in the field.