The loss of HA activity in TBEV has only previously been reported in relation to selective adaptation of TBEV to ticks. One substitution, E87K was generated during the propagation of a WE Figure 1 . Phylogenetic analysis of Yar strains. MEGA version 4 [43] was used to align E genes (between nucleotide positions 1114-2223 of Vs virus genome) of SIB TBEV strains (accession numbers are specified). Tree topology was reconstructed by Neighbor-Joining. The Tamura-Nei model was used for estimation of evolutionary distances [44] . Bootstraps were based on 1000 replications; values below 90% are hidden. The scale bar shows the number of nucleotide substitutions per site. Geographic origins of SIB strains and clusters I, II and III are shown on the right hand side of the tree. Yar strains are highlighted using triangles. KFDV was used as the outgroup. doi:10.1371/journal.pone.0007295.g001 strain to I. ricinus ticks [30] and two substitutions, E122G and T426I respectively, followed a few SIB TBEV passages in H. marginatum ticks [31] . In support of our observations, the E protein surface in these independently reported HA deficient viruses, is predicted to be of either positive or neutral charge, as we have described for the substitutions, D67G, E122G and D277A respectively.