SVCV genome and infectious cDNA construct.

A. Whole-genome SVCV phylogenetic analysis. The complete genomic sequences of 16 SVCV isolates (Sprivivirus cyprinus) and Pike Fry Rhabdovirus (Sprivivirus esox) were obtained from NCBI Genbank. The SVCV sequence highlighted in red corresponds to the Fijan strain (Genbank #AJ318079.1) and was used to establish the SVCV reverse genetics system. Accession numbers are provided in each branch of the phylogenetic tree and in Table 1. The genomes were aligned using MUSCLE and the choice of substitution model was performed using MEGA X. A Maximum-Likelihood phylogenetic tree was inferred from the alignment using PhyML with the GTR+G substitution model and 1000 bootstrap replicates. Numbers at nodes indicate percent bootstrap support. The tree was drawn to scale using FigTree 1.4.4 and rooted using Pike fry rhabdovirus. Branch lengths correspond to the number of substitutions per site. The host species, date and country of origin are shown when found in the sequence metadata. B. Construction of the pSVCV plasmid encoding the complete antigenomic RNA of the SVCV Fijan strain. Five overlapping cDNA fragments (numbered 1 to 5) covering the complete Fijan strain antigenome were generated by RT-PCR with primers described in Table 2 and were assembled in pBluescript SK− following appropriate restriction enzyme digestion. In the final construct, named pSVCV, the leader end of the antigenome cDNA is flanked by the T7 promoter (T7), and the trailer end is fused to the hepatitis delta virus ribozyme followed by a terminator for T7 RNA polymerase (T7t). The complete antigenomic cDNA was designed to create XhoI, SmaI and EagI unique restriction sites in the M-G, G-L intergenic and trailer regions, respectively (see S1 Fig). In addition, this genome differs from the genome of the Fijan reference strain by three nucleotide substitutions in N and G genes.