Molecular Epidemiology of Toscana Virus in Northern and Central Italy Using Metagenomic Next-Generation Sequencing.

Toscana virus (TOSV) is an arthropod-borne virus, transmitted by sandflies, which represents a major cause of aseptic meningitis in Mediterranean countries during summer months. Despite its epidemiological importance, recent genomic data on circulating Italian strains remain limited. We performed comprehensive phylogenetic and genotypic characterization of 34 TOSV isolates (32 obtained from human biological samples and 2 from sandfly homogenates) collected between 2022 and 2025 from Northern/Central Italy. All the sequenced isolates clustered within Lineage A, with strains circulating in Tuscany showing significantly lower intra group genetic divergence (p < 0.05), indicative of compartmentalized local circulation. Both S and M segments exhibited negative selection and identified non-synonymous mutations deserving functional investigation. This study documents stable Lineage A predominance across Italian regions, with Tuscany showing distinct phylogeographic structuring. mNGS proves effective for TOSV genomic surveillance, supporting refined public health strategies, including targeted sandfly control in endemic foci.