EAV genome replication is not signi®cantly affected by leader TRS and body TRS mutations To dissect EAV RNA synthesis, we routinely use a fulllength cDNA clone (van Dinten et al., 1997) , from which infectious EAV RNA is in vitro transcribed. Following transfection of the RNA into baby hamster kidney (BHK-21) cells, intracellular RNA is isolated and analysed by northern blot hybridization and RT±PCR (van Marle et al., 1999a) . Due to differences in transfection ef®ciency, the total amount of virus-speci®c RNA (genomic RNA and sg mRNA) isolated from transfected cell cultures is somewhat variable. Thus, the accurate quantitation of sg mRNA synthesis by TRS mutants requires an internal standard for transfection ef®ciency. The amount of viral genomic RNA can be this standard, but only if its ampli®cation is not dramatically affected by the TRS mutations. To prove that this is the case, we used the previously described mutants L4, B4 and LB4 (van Marle et al., 1999a) , in which ®ve nucleotides of the TRS (5¢-UCAAC-3¢) were replaced by the sequence 5¢-AGUUG-3¢, either in the leader TRS (L4), RNA7 body TRS (B4) or both TRSs (LB4).