Metagenomic sequencing discloses the virome composition of mosquitoes and sandflies from Central-Southern Tuscany, Italy

Mosquitoes and sandfly species are well-known vectors of viral pathogens of public health concern. However, the diversity and ecology of viruses within mosquitoes, including those responsible for clinical and sub-clinical infections in humans, remain poorly understood. In this study, we investigated the presence of phleboviruses and flaviviruses in <i>Aedes albopictus</i> and <i>Culex pipiens</i> mosquitoes, as well as Phlebotominae species, collected from the Siena and Grosseto districts (Tuscany, Italy) during the 2022-2024 summer season. Furthermore, <i>A. albopictus</i> and <i>C. pipiens</i> larvae were reared under laboratory conditions, and adults were collected for further virological analysis. Molecular investigations (reverse-transcription polymerase chain reaction [RT-PCR]) detected phleboviruses and/or flaviviruses in several batches of both field-collected and laboratory-reared flies. Notably, the highest incidence and co-circulation of both viral genera were observed in samples from the 2024 season. Furthermore, metagenomic analysis was performed on only 42 out of 67 RT-PCR-positive pools of mosquitoes and sandflies. This approach aimed to identify wild-type or recombinant viruses and assess the virome of autochthonous arthropods, contributing to knowledge on viral ecology in southern Tuscany and potential threats to humans. The resulting data revealed a wide viral community shared among mosquitoes, spanning over 30 taxonomic virus families, albeit no potential human pathogen virus was identified. Furthermore, our findings confirmed the mosquito specificity of certain endogenous arthropod viruses and provided evidence of their potential transovarial transmission in some cases. The present study provides a comprehensive analysis of the mosquitoes and sandflies virome, contributing to viral surveillance efforts and underscoring the need for enhanced monitoring of arthropod-borne pathogens.IMPORTANCEIn this study, we analyzed the co-circulating phleboviruses and flaviviruses, providing foundational data on the diversity, composition, and transmission of insect-specific and vector-borne viruses in Central-Southern Tuscany, an area increasingly exposed to arbovirus threats due to climate change and globalization. This is the first comprehensive metagenomic study to characterize the virome of <i>Aedes albopictus</i>, <i>Culex pipiens</i>, and <i>Phlebotomine</i> spp. in this region. Furthermore, we identified for the first time Punique virus (PUNV) in Italy, a phlebovirus with potential (though not yet confirmed) human pathogenicity.