All 3 natural HA-negative isolates (Yar71, Yar 46-2, and Yar 48) displayed small-plaque phenotype (1 mm, Fig. 4B ), in . Abbreviated comparative alignment of TBEV E protein sequences (complete version is available on request). TBEV strains are specified by subtype (FE-, SIB-or WE-subtypes) and GenBank accession numbers. HA-disabling mutations are encircled. The ''tick-specific'' amino acids that differentiate I. persulcatus-transmitted viruses (shadowed) from the I. ricinus-transmitted viruses are highlighted in yellow; those that increase hydrophobicity are boxed and surface-faced amino acids are marked with asterisks (*). The fusion peptide is underlined. (B) Mapping of HA-disabling residues onto native dimeric conformation of E protein crystal structure [45] (1SVB.pdb); the monomer is shown as it lies on the virion membrane. The ''persulcatus'' and ''ricinus'' residues are highlighted in orange; fusion peptide is in green and HAdeficient amino acids (arrows) are red. The position of Yar substitution D277A (red coloured) coincides with the position of ''ricinus/persulcatus'' substitution (orange colour is masked). (C) Residues 67, 122 and 277 (purple spheres) are mapped onto the E protein in trimeric post-fusion conformation (1URZ.pdb) [18] . The fusion peptide is highlighted in green and can be seen protruding out of the virion membrane towards the endosomal membrane. Different subunits of the trimer are coloured in red, blue and yellow. doi:10.1371/journal.pone.0007295.g002 comparison with control Vs strain which produced much larger plaques (3 mm) but only two corresponding mutations, E122G and D277A caused plaque size reduction in the engineered viruses (0.7 to 1.5 mm as shown in Fig. 4B ). Clearly, as yet unidentified additional mutations contributed to the small plaque phenotype of Yar 46-2 (D67G). Similarly Yar 48 virus formed smaller plaques (1 mm) than the corresponding IC-D277A mutant (1.5 mm) demonstrating that HA activity and plaque phenotype are not always determined by the same amino acid.