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

<p>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 <i>XhoI</i> and <i>SmaI</i> 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 <i>SmaI</i> 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.</p>