Replication enhancer elements within the open reading frame of tick-borne encephalitis virus and their evolution within the Flavivirus genus

Tuplin, A.; Evans, D. J.; Buckley, A.; Jones, I. M.; Gould, E. A.; Gritsun, T. S.

MFold was used to test the ability of these regions to form SL6-like structures within each MBFV group and the stem and loop elements of these SL6-like structures were superimposed onto the TBFV/MBFV alignment (Supplementary Figure S1A) . This comparison revealed that structures predicted within each MBFV group show not only sequence but also structural homology with SL6 of the TBFV group. This alignment was further annotated with RNA structures predicted by the ALIDOT-based analysis of entire flavivirus genomes of 11 000 nt (28), i.e. JE2, JE3 and JE4 for JEV; DV2 and DV3 for DENV and YF4 for YFV (Supplementary Figure S1A) . For all MBFVs with the exception of YFV the MFold predictions were somewhat different from those made using ALIDOT, most likely due to the shorter length of the regions (60-80 nt) used for the MFold analysis. Additional statistical methods, SSSV and STRUCTURE_DIST were used to assess the conservation of the SL6 homologous structures for each of the major MBFV groups (Figure 2) .