Fig. 5.

Induction of IFN by PIV5-VΔC correlates with the presence of DI viruses. (A) Equivalent dilutions of PIV5-VΔC were used to infect A549/pr(IFN-β).GFP cells. Cells were fixed at 16 h post-infection and immunostained for NP expression. GFP (green) and NP (red) were visualised by fluorescence microscopy. Culture media were collected from infected cells and subjected to a CPE-reduction bio-assay to estimate the IFN present. Relative units of IFN (RUI) produced and the MOI (PFU/cell) for each virus dilution are shown as insets in the GFP and anti-NP panels, respectively. (B) A549/pr(IFN-β).GFP cells were infected with PIV5-VΔC vM0 and vM2 preparations at 1–2 PFU/cell. At 16 h post-infection, cells were trypsinised, fixed and subjected to flow cytometry analysis to determine GFP expression. The percentage of cells considered to be GFP positive (based on the line gate indicated) is given in the top right hand of each panel. (C) Lysates of A549/pr(IFN-β).GFP cells infected with 5 PFU/cell of PIV5-VΔC vM0 or vM2 for 24 h were subjected to immunoblotting with antibodies specific to phosphorylated IRF3 (p-IRF3), GFP, ISG56, MxA, viral NP and actin. Uninfected (UI) cells were included as a negative control.