To identify amino acid(s) responsible for the loss of HA-activity, we sequenced Yar viruses and aligned them with 290 available TBEV E sequences ( Fig. 2A) . One amino acid 175N in the E protein was common to all HA-deficient Yar-viruses, in contrast with the highly conserved 175T. Therefore, the substitution T for N at amino acid position 175 was introduced into a TBEV infectious clone (IC) designated pGGVs [28, 29] to generate mutant virus IC-T175N that produced HA titres similar to parent Vs virus and also control pGGVs virus rescued from the infectious clone (Table 1 ). Since no other common amino acids were identified that distinguished Yar-viruses from the other strains, we hypothesised that individual non-shared amino acid substitutions might be responsible for the HA-deficient phenotype. The alignment in Fig. 2A revealed 3 non-conserved mutations, each of which was unique to one of the Yar strains, i.e. D67G (Yar 46-2), E122G (Yar 71 and Yar 114) and D277A (Yar 48). When compared with the parent pGGVs virus, each of these mutations increased net charge and hydrophobicity of the E protein and was surface orientated, mapping on the most protruding loops of the E protein in its native dimeric conformation (Fig. 2B) .