The simultaneous circulation of multiple arboviruses, often driven by (re)emergence events, poses challenges to public health systems. In Brazil, the co-circulation of Dengue virus (DENV), Zika virus (ZIKV), Chikungunya virus (CHIKV), and Oropouche virus (OROV), together with the potential urban emergence of Mayaro virus (MAYV), underscores the importance of understanding interactions among these pathogens within their vectors. This study investigated the effects of CHIKV and MAYV coinfection and superinfection on replication dynamics in Aedes aegypti. Mosquitoes were experimentally exposed to CHIKV and MAYV through artificial blood meals under coinfection and superinfection conditions. Infection (IR), dissemination (DR), and transmission (TR) rates, as well as viral loads, were quantified by quantitative reverse transcription PCR (qRT-PCR). To confirm viral replication and assess cytopathic effects, positive saliva samples were inoculated in Vero cells, followed by serial passages and plaque assays for viral titration. The results showed that Ae. aegypti is capable of transmitting both CHIKV and MAYV concurrently during coinfection. However, in superinfection scenarios, prior infection with either virus significantly reduced the transmission efficiency of the subsequently acquired virus, indicating viral interference at the replication level. These findings underscore the complexity of arboviral interactions within vectors and highlight their potential implications for transmission dynamics. Continuous entomo-virological surveillance and targeted research are essential for anticipating and mitigating the impact of arboviral co-circulation in endemic regions.

Here, we present a protocol for infecting adult female Aedes aegypti with arboviruses using intrathoracic microinjection or artificial blood feeding as alternative approaches. We describe steps for rearing mosquitos, virus preparation, intrathoracic microinjection, and artificial blood feeding. We then detail procedures for saliva collection, tissue dissection (midgut and carcass), and assessment of vector competence. The protocol provides practical guidance, critical experimental considerations, and validation steps to improve reproducibility and consistency across laboratory settings.