Orthoflavivirus circulation in mosquitoes (Diptera: Culicidae) from an Atlantic Forest remnant and adjacent urban areas, in São Paulo, Brazil.

Orthoflaviviruses cause large epidemics in Brazil, such as those caused by dengue (DENV), yellow fever (YFV), and Zika viruses (ZIKV). Since they are transmitted by mosquitoes, entomo-virological surveillance is recommended in many areas of the country. In addition to medically important orthoflaviviruses, insect-specific viruses (ISVs), which replicate only in insect cell lines, have been detected in mosquitoes collected in this country. Parque Ecológico do Tietê (PET) is an Atlantic forest remnant located within a large urban area in the city of São Paulo, State of São Paulo, Brazil. Since PET provides favorable conditions for the circulation of orthoflaviviruses, the objective of this study was to detect RNA of orthoflaviviruses in mosquitoes collected in PET as well as in adjacent urban and peri-urban areas. Mosquitoes (Diptera: Culicidae) were collected from March 2024 to February 2025 and were screened for the presence of Orthoflavivirus RNA by means of reverse transcription and semi-nested PCR followed by Sanger sequencing. A total of 2,118 specimens were collected and categorized into 584 pools. In total, ten mosquito taxa were identified, highlighting those of public health importance: Aedes aegypti, Aedes albopictus, Aedes scapularis, and Culex (Culex) quinquefasciatus. Nucleotide sequences were retrieved from twenty-five pools of mosquitoes (25/584). Our analyses revealed the presence of DENV-1 and DENV-2 RNA in two distinct pools of Aedes aegypti females collected in an urban area. Additionally, ISV-like sequences related to Culex flavivirus, Mosquito flavivirus, Guapiaçu virus, Long Pine Key virus, and Mansonia flavivirus were detected in eight, five, seven, two, and one pool, respectively. Most of these ISVs have previously been detected in other regions of Brazil, reinforcing they may circulate in different ecological settings. Furthermore, we isolated a virus related to Mosquito flavivirus from one of the positive pools in C6/36 cells of Aedes albopictus and observed a virus-induced cell aggregation, confirming a cytopathic effect. In conclusion, orthoflaviviruses related to Culex flavivirus, Mosquito flavivirus and other putative orthoflaviviruses, might occur in mosquitoes from PET and adjacent areas. Moreover, our findings indicate that DENV-1 and DENV-2 could have been circulating in the adjacent urban areas of PET during the study period, reinforcing the importance of entomo-virological surveillance in this region.