Insertion of a transcription cassette encoding tracer proteins in the SVCV genome.

A. As described in the Materials and methods, an expression cassette encoding mCherry, firefly luciferase (ffLUC) or akaluciferase (akaLUC) was inserted in the M-G and G-L intergenic regions flanked by additional GS and GE transcription signals of SVCV using the unique XhoI and SmaI restriction enzyme sites, respectively, leading to the final construct pSVCV-mCherry M/G, pSVCV-ffLUC M/G, pSVCV-mCherry G/L, pSVCV-ffLUC G/L and pSVCV-akaLUC G/L (not to scale). Together with an expression cassette encoding GFPmax between M and G genes, a second expression cassette, as described above, encoding mCherry was inserted in the G-L intergenic region using the unique SmaI restriction enzyme site, leading to the final construct pSVCV-GFPmaxCherry that encodes simultaneously both green and red fluorescent proteins. B-C. EPC cells were infected with rSVCV-mCherry (B) or rSVCV-GFPmaxCherry (C) at a final MOI of 0.1. The cells were incubated at 25°C for 24 hours. Live cell monolayers were then visualized with a UV-light microscope after nuclei staining with Hoechst solution in the cell culture medium. 63× objective. Scale bars, 10 μm. D. EPC cells were infected with rSVCV-ffLUC at a MOI of 0.1. At 10 hours post-infection (hpi) and 17 hpi live cells were washed three times with sterile Phosphate Buffer Saline (PBS) and the D-luciferin substrate was added at a concentration of 250 μM. The luminescence was measured using the IVIS Spectrum BL imaging system.