In a previous study using MFold-simulated RNA structures for a limited number of mTBFV species, we predicted the existence of SL6 in the C-coding region of TBFV. A conserved hexanucleotide UGCCAA in the apical loop and compensatory mutations in the duplex stem of the SL6 implied the formation of the stable RNA structure in ORF of the TBFV genomes (26, 27) . However, contradicting these findings a deletion within the C-coding region, which included SL6, did not prevent recovery of viable, albeit attenuated, TBEV (42) . In this study, we employed a variety of complementary phylogenetic and thermodynamic methods to examine the evolutionary conservation of SL6 using a much larger sample of significantly divergent TBFVs, including new members of the mTBFV, sTBFV and Kadam subgroups (37) . The viruses in the other ecological groups, namely MBFV, NKV and PABV were also included in this analysis to trace the evolution of SL6 throughout the entire genus Flavivirus. In addition, we used a reverse genetic system (34, 36) to engineer TBEV strains with mutated SL6 to reveal the biological significance of this structure.