Site-directed mutagenesis, RNA transfections and immuno¯uorescence analysis Site-directed mutagenesis of EAV leader and body TRSs was carried out as described by van Marle et al. (1999a) , and all mutant constructs were sequenced. Following in vitro transcription from infectious cDNA clones, full-length EAV RNA was introduced into BHK-21 cells by electroporation, as described by van Dinten et al. (1997) . Immuno¯uorescence assays with EAV-speci®c antisera were performed at 14 h posttransfection as described by van der Meer et al. (1998) . To visualize the nuclei for cell counting, nuclear DNA was stained with 5 mg/ml Hoechst B2883 (Sigma). Cells were counted using the Scion Image software (Scion Corporation) and the percentage of transfected cells was calculated on the basis of the number of cells positive for the EAV replicase component nsp3 (Pedersen et al., 1999) .