Toscana virus (TOSV) is a Phlebovirus in the family, found in the countries surrounding the Mediterranean. It is unusual within the genus for exhibiting tropism for the central nervous system: TOSV is one of the top causes of seasonal acute meningitis/encephalitis within its range. However, little progress has been made in the study of TOSV, largely due to the lack of a reliable reverse genetics system. We used RNA sequencing to determine the sequence of Toscana strain 1500590, a lineage A virus obtained from an infected patient (Marseille, 2007). This data was used to construct cDNA plasmids encoding the viral L, M and S antigenomic sequences under control of the T7 RNA promoter to recover recombinant viruses when expressed in BSR-T7/5 CL21 cells. By sequencing amplified viral cDNA, we identified two single base pair mismatches in the original TOSV reference genome (NC_006318, 19, 20), which when corrected restored functionality to the polymerase. We were then able to recover infectious recombinant TOSV (rTOSV) from cDNA clones, as well as establishing a minigenome system. Using reverse genetics, it has been possible to produce the non-structural gene (NSs) deletion mutants (a known interferon antagonist). These strains are in the process of characterisation and are to be used in infection studies in Phlebotomine flies. TOSV vaccine development has been severely hindered by the failure of previous efforts to develop reverse genetics systems for this virus. With a system now in place, it will hopefully be possible to design novel attenuated vaccine candidates.

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