To exclude the effect of spontaneous mutations in the 5 0 -and 3 0 -UTRs which contain TBFV promoter and enhancer elements (16) that might compensate for the effect of the SL6-mutations, rescued virus was not passaged prior to phenotype evaluation and key regions of the genome (1-940 and 10206-10927) were sequenced following recovery of each SL6-mutated virus. Only the intended substitutions were present, with no reversions or other compensatory mutations were observed. The effect of each mutation (reduction from large wild-type plaques of the pGGVs virus to medium, small or pin pointed) was scored if the SL6-mutated strain contained >90% of plaques with the altered morphology. The presence of a minor plaque population (between 1 and 10%) was considered as the inevitable result of the variation inherent R  1  4  5  3  3  3  6  2  4  5  2  6  3  2  8  2  2  9  0  2  |  |  |  |  |  |  |  |  TBEV  L40361  ACG CGU CAA UCC AGA GUC CAA AUG CCA AAU GGA CUC GUG UUG AUG CGC  TRQSRVQMPNGLVLMR TBEV  C10 . in all RNA viruses, the consequence of a high error rate in the virus RdRp (41) .