Fig 4. Detection of rCDV double- and triple-infected cells.

(A) Virus isolation from WBC by titration on Vero-cCD150 cells revealed syncytia that were simultaneously positive for two reporter proteins (i.e. Venus/dTom [yellow], dTom/TagBFP [purple] or Venus/TagBFP [cyan]) or all three reporter proteins (Venus/dTom/TagBFP [white]). (B, upper panel) Direct confocal analysis of the trachea from ferret D2 showed that single infected cells (Venus, dTom or TagBFP) were dominant, but sparse Venus/TagBFP and Venus/dTom double-positive cells were detected as cyan (*) or yellow (#), respectively. (B, lower panel) Direct confocal analysis of the spleen from ferret D2 revealed the presence of single-infected and all possible combinations of double-infected cells. (C) Direct flow cytometry of WBC collected from ferret D2 on 8 DPI indicated that single-, double- and triple-positive cells were present in peripheral blood. Similar results were obtained at different time-points. (C, upper panel) When Venus⁺ cells were selected (as indicated by red gate), dTom^-/TagBFP^- (single-infected cells), dTom⁺/TagBFP^- or dTom^-/TagBFP⁺ (double-infected cells) and dTom⁺/TagBFP⁺ (triple-infected) cells were detected. (C, middle and lower panel) Similar results were obtained when the analysis was performed by initially gating dTom⁺ or TagBFP⁺ cells. Percentages of positive cells are indicated in the plots. (D) Canine lymphoma B-cells (CLBL-1) were inoculated with rCDV^SHVenus(6) and/or rCDV^SHTagBFP(6) in the presence of infection-enhancing lipopeptide Pam3CSK4. Green and blue fluorescence levels were determined 24 hours post infection by flow cytometry. Venus and TagBFP single positive cells are shown in green and blue, respectively, while double-positive cells are shown in cyan.