Reverse Genetics as a Key to Understanding Toscana Virus – Sand Fly Dynamics

Abstract

The Toscana virus (TOSV), a member of the genus Phlebovirus Phenuiviridae, Hareavirales ), is a neglected human pathogen that can cause febrile illness, severe infection of the CNS, or remain asymptomatic. It is distributed throughout the Mediterranean area and is transmitted by phlebotomine sand flies. To date, three TOSV lineages (A, B, C) have been identified; however, only two (A, B) have been successfully isolated. Vector competence varies among sand fly species. Although Phlebotomus tobbi and P. sergenti were found susceptible to TOSV B infection, P. papatasi and Sergentomyia schwetzi were refractory. Another tested TOSV strain (TOSV A) was found to be incapable of developing in any of the species tested 1 To explore these differences, we used a reverse reverse-genetic system 2,3 to prepare TOSV, which produces viruses with defined RNA genomes and ensures a more uniform inoculum compared to repeatedly passaged isolates. This approach also enables the manipulation of viral genetic information, such as the reassortment of viral RNA segments (L, M, and S) or theintroduction of targeted mutations. We assessed the susceptibility of P. tobbi to two reverse geneticsgenetics-derived TOSV strains: lineage A (rgTOSV A) and lineage B (rgTOSV B). Furthermore, we examined the infectivity of rgTOSV A reassortments carrying one of theRNA segments derived from rgTOSV B. Our results show that the infection dissemination rate of rgTOSV B in P. tobbi is comparable to the wild wild-type (wt) strain. Unexpectedly, rgTOSV A could establish an infection (infection rate 10%) and disseminate (dissemination rate15%) through the body of the sand fly to the head and salivary glands, unlike its wild wild-type counterpart. Following the successful establishment of rgTOSV production and infectious sand fly feeding, ongoing experiments with rgTOSV A/B reassortments will elucidatewhich segments (and the proteins they encode) are crucial for sand fly infection.