Expression of the transgenes and <i>in vitro</i> growth properties.

<p>(A) RK13 cells were infected with parental rRacH1, rH_VP2 or rH_VP2_5 at an m.o.i of 0.0001. Two days post infection, cells were fixed and incubated with anti-VP2 mAb 13C10 or anti-EHV-1 gp2 mAb 3B12, followed by Alexa Fluor 568-conjugated goat anti-mouse IgG. Fluorescence signal was inspected under the inverted fluorescence microscope. Bar indicates 50 µm. (B) Cell lysates infected by rRacH1, rH_VP2, rH_VP2_5 or BTV-8 were separated by 10% SDS-PAGE and analysed by Western blot. Expression of VP2 and VP5 was detected using primary antibody 13C10 and sheep anti-BTV-8 hyperimmune sera, respectively. EHV-1 MCP was used as a control and detected with mAb 3G4. (C) RK13 cells were infected by the individual virus at an m.o.i of 0.0001 and overlaid. Three days post infection, plaques were photographed and the areas were measured. For each virus, at least 50 plaques were measured. The relative plaque area was compared to that of rRacH, which was set as 100%. * <i>P</i><0.001. (D) The single-step growth kinetics of these viruses was analysed. RK13 cells were infected by the viruses at an m.o.i of 5. Extracellular and intracellular virus titres were determined at the indicated time points.