Logarithmically growing BHK-21 cells were infected with BTV-8 (1pfu/cell) for 24 hours then harvested and washed once with PBS. Nuclear extracts were prepared from 2.5610 7 cells using the NE-PER nuclear and cytoplasmic extraction reagent kit (Pierce), as directed by the manufacturer. The nuclear extract was mixed volume to volume with sample denaturation buffer and analysed by SDS-PAGE using a 4-20% gradient polyacrylamide gel. Resolved proteins were electro-blotted on 0.2 mm nitrocellulose membrane as described above, blocked with 5% skimmed milk, in TBS-Tween-20 and incubated over night with a dilution of 1/300 anti-BTV-8 NS4 rabbit antiserum. The membranes were washed three times with TBS-Tween-20 and further incubated with monoclonal, anti-rabbit, peroxydase conjugate (Sigma), diluted at 1/750 in 5% skimmed milk. After 2 hours the membranes were washed three times with TBS-Tween-20 then incubated with Lumilight plus (Roche) chemiluminescent detection reagent, as described by the manufacturer. X-Omat radiographic films (Kodak) were exposed for 10 minutes to membranes then developed as described by the manufacturer.