Impact of temperature on patient-derived dengue virus breakthrough infections in wMel-infected Aedes aegypti

Abstract The w Mel strain of the insect endosymbiont Wolbachia reduces the potential for Aedes aegypti to transmit mosquito-borne viruses such as dengue (DENV). Field trials that have introgressed w Mel into Ae. aegypti populations have shown this approach significantly reduces dengue incidence. In a laboratory setting some w Mel- Ae. aegypti develop infectious saliva following a viremic blood meal. Additionally, studies have demonstrated that exposing w Mel- Ae. aegypti to heat treatment, particularly during the larval stage, reduces w Mel density in key tissues such as the ovaries, midgut and salivary glands. Here we build on these studies, using viremic blood collected from 13 dengue inpatients at the Hospital for Tropical Diseases in Ho Chi Minh City (Viet Nam), to assess how temperature affects the protection afforded to Ae. aegypti by w Mel. We found that, compared to w Mel- Ae. aegypti reared at 28 ± 4°C, those reared at 31 ± 4°C developed infectious saliva more frequently, but the risk of this occurring was still reduced compared to WT mosquitoes reared at the same temperature. Heat treatment reduced the density of w Mel in all tissues tested, decreased the magnitude of w Mel’s protection against DENV replication in the head/thorax, and significantly increased the amount of DENV replication in w Mel- Ae. aegypti . When comparing cohorts of w Mel- Ae. aegypti that did or did not develop infectious saliva, DENV levels in the head/thorax were associated with increased odds of mosquitoes developing infectious saliva, but w Mel density was not. Overall, these findings show that elevated rearing temperatures increase the risk of patient-derived DENV breakthrough infections in w Mel- Ae. aegypti , potentially due to increased DENV replication in these mosquitoes. This limitation suggests it would be prudent to increase surveillance in regions using w Mel for dengue control when daily mean temperatures remain above 30°C for multiday periods. Author Summary The mosquito species Ae. aegypti can be infected with the bacterium Wolbachia ( w Mel strain), reducing its capacity to transmit viruses like dengue (DENV). Wolbachia is now being used as a biocontrol tool to reduce the burden of dengue in communities. However, some mosquitoes with Wolbachia can still transmit DENV. Here we utilised a natural infection model using dengue patient-derived blood to examine how temperature may increase the risk of DENV transmission occurrence in mosquitoes with w Mel. Mosquitoes with w Mel were more likely to transmit virus when reared at an average temperature of 31°C compared to those reared at an average temperature of 28°C but these mosquitoes still had a lower risk of developing infectious saliva compared to their w Mel-free counterparts. Higher temperatures reduced the amount of w Mel in mosquito tissues and increased the amount of DENV replicating in the head/thorax. Increasing levels of DENV RNA in these tissues were found to be associated with increased risk of mosquitoes with w Mel developing infectious saliva. This finding indicates surveillance is warranted in high temperature settings or during heat waves, to monitor for changes in w Mel frequency and DENV infection in Ae. aegypti .