Culex Flavivirus (CxFV) is a classical insect-specific virus, which has aroused interest after the first indication that it can produce in nature superinfection exclusion of viruses of medical interest such as West Nile. Despite the detection of CxFV in different regions, CxFV ecology and the influence of co-circulation of arboviruses remains poorly understood. Therefore, our primary goals are to observe the occurrence of CxFV infection in mosquitoes trapped in an urban area of Rio de Janeiro, Brazil, characterize the virus circulating, and provide isolates. A prospective study was carried out for eight months on the campus of the Federal University of Rio de Janeiro, trapping adult mosquitoes. The CxFV minimum infection rates were determined in this period, and the virus isolation process is fully described. Samples from this study were grouped into genotype 2, along with CxFV sequences from Latin America and Africa.

The Oropouche virus (OROV) has been circulating in the Amazon region since the 1960s, with a progressive increase in outbreaks and human cases reported in Brazil and neighboring countries. In the Rio de Janeiro state, there has been a significant rise in suspected cases of arboviruses, with only 30% of laboratory tests confirming infections with dengue, Zika, or chikungunya viruses. The investigation of OROV virus circulation in the Rio de Janeiro state was initiated and confirmed in April 2024. Our study aimed to retrospectively investigate OROV infections in dengue-suspected cases with inconclusive diagnosis in order to better understand the temporal and geographic introduction of OROV in the Rio de Janeiro state. Municipalities from Rio de Janeiro with arbovirus-like fever cases but a low percentage of dengue-positive RT-PCR test confirmations were identified in the laboratory database. Samples were selected for testing OROV infections using real-time RT-PCR as recommended by the Brazilian Ministry of Health. Municipalities in the Middle Paraíba region of the state showed 93% negative tests results for dengue, Zika, and chikungunya starting in September 2023. A total of 118 positive cases of Oropouche were recorded in the state of Rio de Janeiro between March and July 2024. Moreover, by genome sequencing of eight strains, it was shown that OROV circulating in Rio de Janeiro belongs to recently emergent M₁L₂S₂ lineage. Our findings retrospectively revealed a concentration of cases in the Middle Paraíba region and an outbreak in the rural village of Cacaria, located in the municipality of Piraí. According to our data, this region is the first area with sustained transmission in the Rio de Janeiro state.