Oropouche orthobunyavirus (OROV) is an emerging public health concern due to its expanding geographic range and increasing case numbers. In Brazil, 13,785 cases were confirmed in 2024, with an additional 3680 reported by January 2025, according to the Ministry of Health. Initially restricted to the Amazon region, OROV has recently been detected in new areas, highlighting the need for enhanced surveillance and vector control strategies. While Culicoides paraensis is the primary vector, the potential role of other species in transmitting the currently circulating OROV strain in Brazil remains unclear. Here, we experimentally assessed the infectivity and dissemination of a recently isolated Oropouche orthobunyavirus (OROV) strain in two widespread mosquito species, Aedes aegypti and Culex quinquefasciatus, collected from diverse regions of Brazil. Our results demonstrated that both mosquito species were refractory to oral infection, suggesting that natural transmission through these vectors is unlikely. However, in artificial systemic infection, Ae. aegypti showed viral replication and immune system activation, indicating its potential to support OROV replication under specific conditions. Additionally, to assess the potential impact of coinfection, we investigated whether Chikungunya virus (CHIKV), an arbovirus that naturally infects Ae. aegypti, could facilitate OROV infection dynamics in this mosquito species. Our results suggest that coinfection does not promote OROV oral infection. Furthermore, we examined whether OROV systemic infection induced an immune response in Ae aegypti. We analyzed the major immune response pathways-RNAi, Toll, IMD, and JAK-STAT-and observed that the RNAi pathway was the most strongly activated in response to OROV infection in Ae. aegypti. These findings highlight the importance of ongoing surveillance and further studies on OROV evolution, vector adaptation, and transmission dynamics, particularly in urban settings where vector populations and viral interactions may facilitate new epidemiological scenarios.

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