Construction and validation of recombinant SAV3 with a double subgenomic promoter. (A) The full-length SAV3 was engineered to contain an additional ORF encoding the EGFP protein under the control of a SAV specific promoter. The construct was obtained by AscI/NotI cleavage and ligation of a PCR amplified fragment encoding SAV3 structural proteins and the SAV3 subgenomic promoter, into a modified pmSAV3 plasmid. (B) The obtained construct was transfected into BF2 cells and expression of EGFP was visualized by fluorescence microscopy. Presence of infectious virus containing the EGFP ORF was verified by serial passage of supernatant from transfected cells onto naïve CHSE cells followed by fluorescence microscopy of live cells.