Aedes(Ae.) aegypti and Ae. albopictus are two major mosquito species transmitting dengue, Yellow fever and Zika viruses in tropical and subtropical regions. Dengue viruses consist of four serotypes (DENV1-4) that cause ~390 million infected patients and ~25,000 deaths globally each year. There is neither an effective medicine nor licensed vaccine against DENV infection; however there is a global initiative to eradicate these mosquito populations by artificially transinfecting Dorosophia-originated Wolbachia, wMel or wMelPop strain to both Ae. mosquitoes. These intracellular bacteria have been shown to reduce the mosquito population and shorten the mosquito lifespans, enabling possible reduction of DENV transmission. Furthermore, it has been revealed that wMel or wMelPop induce strong antiviral protection to Ae. agypti. In contrast, the data on Wolbachia’s effect on Ae. albopictus is still limited. We have focused on the mechanism of DENV2 replication in wMelPop-transinfected C6/36 cell line (Ae. albopictus). After infecting DENV2 to naïve C6/36 cells or wMelPop-C6/36 cells, cellular transcripts by RNAseq analysis revealed that wMelPop suppressed DENV2-induced cellular gene expressions. In the supernatants, the virus copy numbers were not significantly altered by the presence or absence of wMelPop, although the plaque assay in LLC/MK2 cells with the recovered viruses from wMelPop-C6/36 cells were significantly reduced. Immunofluorescent staining to monitor virus replication showed that the virus from wMelPop-C6/36 cells replicated well in naïve C6/36 cells, but delayed replication in BHK-21 cells. Sequence results by RNAseq revealed that wMelPop caused seven amino acid alterations at E (Glu202Gly), NS2A (Leu201Phe), NS2B (Thr94Ile, Thr101Ile, Ile114Thr), NS4B (Tyr99Cys), and NS5Pol (Leu716Val) in DENV2. Five of these mutations occurred at or close to transmembrane domains (TMDs) in NS2A, NS2B and NS4B, all of which are necessary to form replication compartment in the endoplasmic reticulum (ER). It was observed that this mutant virus kept all these mutations through replication in naïve C6/36 cells, although in BHK-21 cell, it was reverting these mutations to WT sequence except at NS5 and further acquired mutations at NS3 (Val97Ile in protease and Ile212Val in helicase domains). These results suggest that there must be a structural or functional difference of ER between mosquito and mammalian cells and that wMelPop-induced mutations function poorly in mammalian cells and require further mutations at NS3. In Aim 1, we will expand the effect of wMelPop in replication efficiency and mutation induction to all DENV serotypes as well as Zika virus. We will create the infectious clones having the individual mutations and examine the correlations among TMD-NS proteins, E, NS3 and NS5. In Aim 2, we will investigate if the mutated TMD-NS proteins are expressed and localized at ER to form replication compartment and interact with NS3. In Aim 3, we will identi...