In order to resolve the difficulties with design of mutations, three different approaches were adopted ( Figure 3) . First, we introduced all possible silent substitutions, to target the conserved hexanucleotide and the stem (mutants C12, C13, C14, C16 and C33). Second, we introduced mutations (C10, C15, C17, C19 and C34) that mimicked 'natural' amino acid substitutions observed in this region of other mTBFV spp. Third, as a control for mutations that changed amino acids we also introduced compensatory substitutions encoding the same mutated amino acids while restoring the SL6 structure. Accordingly, mutations R32, S31, N 28, V 39, V 39 and P 28 were designed as controls for non-synonymous mutants C22, C23, C27 and C34 (Figure 3) .