BackgroundThe mosquitoAedes aegyptitransmits two of the most serious mosquito-borne viruses, dengue virus (DENV) and Zika virus (ZIKV), which results in significant human morbidity and mortality worldwide. The quickly shifting landscapes of DENV and ZIKV endemicity worldwide raise concerns that their co-circulation through theAe. aegyptimosquito vector could greatly exacerbate the disease burden in humans. Recent reports have indicated an increase in the number of co-infection cases in expanding co-endemic regions; however, the impact of co-infection on viral infection and the detailed molecular mechanisms remain to be defined.MethodsC6/36 (Aedes albopictus) cells were cultured in Dulbecco's modified Eagle medium/Mitsuhashi and Maramorosch Insect Medium (DMEM/MM) (1:1) containing 2% heat-inactivated fetal bovine serum and 1× penicillin/streptomycin solution. For virus propagation, the cells were infected with either DENV serotype 2 (DENV2) strain 16681 or ZIKV isolate Thailand/1610acTw (MF692778.1). Mosquitoes (Ae. aegyptiUGAL [University of Georgia Laboratory]/Rockefeller strain) were orally infected with DENV2 and ZIKV through infectious blood-feeding.ResultsWe first examined viral replication activity in cells infected simultaneously, or sequentially, with DENV and ZIKV, and found interspecies binding of viral genomic transcripts to the non-structural protein 5 (NS5). When we challengedAe. aegyptimosquitos with both DENV2 and ZIKV sequentially to probe similar interactions, virus production and vector susceptibility to infection were significantly enhanced.ConclusionsOur results suggest that DENV2 and ZIKV simultaneously establishing infection in theAe. aegyptimosquito vector may augment one another during replication. The data also implicate the homologous NS5 protein as a key intersection between the flaviviruses in co-infection, highlighting it as a potential target for vector control.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13071-023-05778-1.