Negative interactions between Toscana virus and Leishmania infantum limit coinfection in sand flies

BACKGROUND: Phlebotomine sand flies transmit a wide range of human and veterinary pathogens, including Leishmania spp. and Toscana virus (TOSV). Both pathogens co-circulate extensively in the Mediterranean basin and may share hosts and vectors, raising the possibility of mixed infections with epidemiological relevance. While previous studies have suggested interactions between TOSV and Leishmania in mammalian hosts and in vitro systems, evidence from natural vectors is still lacking. Understanding these interactions is essential for predicting transmission outcomes in areas of pathogen overlap. METHODS: We investigated coinfection dynamics of TOSV and Leishmania infantum in their natural vector, Phlebotomus tobbi. Female sand flies were experimentally challenged with both pathogens through blood feeding. We measured infection rates, dissemination rates, and infection intensity levels at days 4 and 8 postinfection (p.i.) and compared the coinfected groups with the control harboring a single infection. RESULTS: At day 4 (D4) p.i., the coinfection resulted in significant suppression of both pathogens: TOSV infection rates decreased, as did L. infantum infection rates. However, neither infection intensity nor viral dissemination showed significant differences between groups. By day 8 (D8) p.i., L. infantum maintained a negative effect on TOSV infection, while TOSV did not alter L. infantum development. Dissemination and parasite load remained unaffected. CONCLUSIONS: Our findings suggest competitive interactions between TOSV and L. infantum in sand flies, providing the first experimental indication of pathogen-pathogen interference within a natural vector. Such competition likely contributes to the rarity of coinfected sand flies in field surveys and highlights the importance of considering vector-level interactions when assessing transmission risks in endemic regions.