Sand fly-borne phleboviruses in France (including Corsica): Detection, isolation, and genetic characterization contributing to Phlenuiviridae phylogenetics

Sand fly-borne phleboviruses, transmitted by Phlebotomus and Sergentomyia species, are emerging pathogens in the Mediterranean basin, with several species causing febrile illnesses and neurological diseases in humans. While Toscana virus and Sandfly fever Sicilian virus are well-documented, the diversity and distribution of other phleboviruses remain poorly understood. This study aimed to characterize the circulation and genetic diversity of sand fly-borne phleboviruses in mainland France and Corsica, within the CLIMOS project framework.Sand flies were collected in 2023–2024 using CDC light traps in 15 sites across mainland France and Corsica. A total of 5,679 specimens were pooled by species, sex, trapping date, and location, and screened for phleboviruses. Positive samples were inoculated on Vero E6 cells, and isolated samples’ complete genome sequence was obtained using NGS. Phylogenetic analyses were performed RNA-dependent RNA polymerase sequences.Thirteen pools out of 802 (1.6%) were positive for phleboviruses, including Punique virus (PUNV) (n=4), Massilia virus (MASV) (n=3), Arbia virus (ARBV) (n=1), Provincia virus (n=1), Olbia virus (n=3), and Arrábida virus (n=1).Viruses were successfully isolated from five pools, with PUNV (named Orta virus), MASV, and ARBV (named Tumboni virus) strains showing >98% amino acid similarity with L segment to reference sequences. Notably, viruses belonging to the Phlebovirus puniqueense species were detected for the first time outside North Africa, and a virus most closely related to ARBV was identified outside Italy, suggesting broader geographic distributions than previously recognized.This study provides evidence of active co-circulation and considerable genetic diversity of phleboviruses in southern France.