Abstract
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 and encephalitis within its range. Here, we determined the full sequence of the TOSV strain 1500590, a lineage A virus obtained from an infected patient (Marseille, 2007) and used this in combination with other sequence information to construct functional cDNA plasmids encoding the viral L, M, and S antigenomic sequences under the control of the T7 RNA promoter to recover recombinant viruses. Importantly, resequencing identified two single nucleotide changes to a TOSV reference genome, which, when corrected, restored functionality to the polymerase L and made it possible to recover infectious recombinant TOSV (rTOSV) from cDNA, as well as establish a minigenome system. Using reverse genetics, we produced an NSs-deletant rTOSV and also obtained viruses expressing reporter genes instead of NSs. The availability of such a system assists investigating questions that require genetic manipulation of the viral genome, such as investigations into replication and tropism, and beyond these fundamental aspects, also the development of novel vaccine design strategies.
Highlights
Toscana virus (TOSV) belongs to the genus Phlebovirus of the Phenuiviridae family, first isolated in 1971 from Phlebotomus perniciosus and P. perfiliewi sand flies in Italy [1,2]
BSR and BSRT7/5 CL21 cells were grown in GMEM (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS), 10% tryptose phosphate broth (TPB), pencillin/streptomycin with the BSRT7/5 CL21 cells supplemented with 0.25 mg/mL G418
All available complete nucleotide sequences relating to TOSV were downloaded from NCBI [50] and 2,856,292 paired end reads retained after QC and preprocessing were mapped to the downloaded viral nucleotide sequences using Bowtie2 aligner [51]
Summary
Toscana virus (TOSV) belongs to the genus Phlebovirus of the Phenuiviridae family (order Bunyavirales), first isolated in 1971 from Phlebotomus perniciosus and P. perfiliewi sand flies in Italy [1,2]. This and related viruses are widely spread around the Mediterranean basin [3]. We generated viruses no longer expressing the type I interferon antagonist NSs [45], either deleted or replaced with reporter genes These novel tools could assist studies on this emerging pathogen by allowing direct manipulation of the virus
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