RNA virome comparison between sylvatic and urban-interface mosquitoes from Southeastern Brazil

Introduction Mosquitoes (Diptera: Culicidae) are primary vectors of public health pathogens, yet their core viromes remain poorly characterized, particularly in Neotropical sylvatic lineages. This study investigated the RNA virome of multiple mosquito species across urban-to-forest gradients in São Paulo State, Brazil, including neglected sylvatic taxa such as Sabethes, Psorophora, Shannoniana , and Wyeomyia . Methods The RNA virome of multiple mosquito species was investigated across urban-to-forest gradients in São Paulo State, Brazil. Ecological analyses were performed to assess the effects of host taxonomy and environment on virome composition. Network analysis was conducted to investigate virus–host associations and viral sharing across ecological interfaces. Results Our analysis identified 919 viral contigs across 217 viral species and 37 distinct families, revealing a substantial fraction of “viral dark matter” with low amino acid identity (median < 40%) in predominantly sylvatic mosquito species. Although viral families containing known arboviruses, such as Flaviviridae, Phenuiviridae, and Peribunyaviridae , were detected, no high-consequence human pathogens were identified within the sensitivity limits of our sampling and sequencing depth. Ecological analyses demonstrated that virome composition was strongly structured by host taxonomy and environment (R 2 =0.570, p =0.001), with host species explaining 32.9% of the unique variance (PERMANOVA, R 2 =0.329, p =0.001), whereas ecotope played a secondary role (R 2 =0.029, p=0.001). Network analysis revealed a highly modular virus–host structure dominated by host-restricted specialists, with a limited number of bridge species facilitating viral sharing across ecological interfaces. Discussion These findings indicate that intrinsic mosquito biology is the main driver of viral community structure, while environmental gradients play a secondary role, and highlight the importance of host-associated processes in shaping viral diversity at the Neotropical forest–urban interface.