BHK-21 cells grown in 24 well plates (75% confluence), were transfected in triplicate, with pCI-BTVNS4 or pCI-GIVNS4 (4 mg/well) using Fugene-6 (Roche). At 48 hours post-transfection, the cells were fixed in 4% paraformaldehyde and processed for immuno-fluorescence, using anti-NS4 antibodies, as described above.