Development of a reverse genetics system for Toscana virus (lineage A)

Toscana virus (TOSV) is a Phlebovirus in the Phenuiviridae family, order Bunyavirales, found in the countries surrounding the Mediterranean. TOSV is an important cause of seasonal acute meningitis/encephalitis within its range. Here we determined the full sequence of Toscana strain 1500590, a lineage A virus obtained from an infected patient (Marseille, 2007) and used this in combination with other sequence information to construct cDNA plasmids encoding the viral L, M and S antigenomic sequences under control of the T7 RNA promoter to recover recombinant viruses. Re-sequencing allowed us to identify two single base pair mismatches in the original TOSV reference genome, which when corrected restored functionality to the polymerase L allowing us to recover infectious recombinant TOSV (rTOSV) from cDNA clones, as well as establish a minigenome system. Using reverse genetics, we produced an NSs deletant and also obtained viruses expressing reporter genes instead of NSs. This system can now be used to assess questions relating to TOSV replication and tropism but also for example vaccine design.