Fig. 1.

(A) Fluorescent protein performance in plate reader assays. HEK 293T cells were transiently transfected with CMV-promoter driven Cerulean, EGFP, Venus, or Cherry plasmids. Fluorescence was measured 24 h post-transfection and normalized to mock-transfected cells. (B) Schematic of the strategy used to generate the single reporter viruses late Cherry (or Red; LR) and late Venus (LV). The canonical late F17R viral promoter was used. The recombination cassette was generated by overlap PCR for homologous, loss-less insertion in the 60 bp intergenic region between Vaccinia J4R and J5L genes. (C) Plate reader fluorescence for Cherry following LR infection, or Venus following LV infection, at 2 and 12 hpi of A549 cells. (D) 10× image of a confluent BHK21 cell monolayer 24 hpi after low MOI co-infection with LR and LV viruses, showing easily identifiable foci of infected cells originating from infection centers with either virus.