Conformational changes to the apical loop of SL6. Mutations C10, C11, C15, C21, C22 and C23 changed the shape of the loop and base-paired stem within SL6 (Figure 4) . Replication of mutant C10 (with an enlarged loop and shortened stem) and C17 (restored wild type conformation due to the second compensatory mutation) was delayed in the early stage of the infection cycle; C17 caused slightly reduced cpe but the plaque morphology of both was equivalent to that of the parental pGGVs virus ( Figure 5 and Table 1 ). The minor phenotypic changes resulting from these mutations could be explained by the accompanying amino acid substitutions N 35 !K and Q 32 !P imitating POWV ( Figure 3 ). However, a silent substitution A 234 !G that also enlarged the apical loop of mutant C11 (Figure 4 ) Table 1 . Affect of mutations within the SL6 on TBEV phenotype Plaque size for each mutant was defined as large (5-6 mm), medium (3-4 mm), small (1-2 mm) or pinpointed (>1 mm). Some plaques, in comparison with parent Vs virus, were described as turbid. The cpe produced by each mutant in comparison to the wild-type virus was evaluated on a scale of 0-4 where 0 indicates no cpe and 4 is maximum cpe (i.e. 80% cell lysis as observed for the control pGGVs virus) in five repeated experiments, each in quadruplicates. Nt/AA* -Nucleotide/amino acid substitutions. caused similar biological effects; it did not affect virus plaque size or level of cpe, but reduced virus replication rate early after infection ( Figure 5 and Table 1 ).