We also compared E proteins of TBEV strains isolated from I. ricinus (WE-strains) with those isolated from I. persulcatus (FE-and SIB-strains), to identify group amino acids that might be involved in TBEV adaptation to these two different tick species (Fig. 2A ). Ten amino acid differences were revealed that have previously been localised in hypervariable clusters of the envelope protein [33] and five showed overall increased E protein hydrophobicity in the I. ricinus-transmitted WE-strains in contrast with FE-and SIBstrains ( Fig. 2A) . Four of these ten substitutions were localised on the virion surface, with two (S47A and S88G) increasing the surface hydrophobicity of ''ricinus'' strains and two (D178E and D277E) being conserved (Fig. 2B) . Remarkably, the position of the ''tick''-specific (i.e. I. ricinus or I. persulcatus) D277E amino acid substitution overlapped with the D277A substitution of Yar 48 ( Figs. 2A and 2B) . Two substitutions that also increased the hydrophobicity of strains adapted to I. ricinus were localised either inside the E protein (T115A) or on the membrane-oriented side (S267A), i.e. under the virion surface. Four substitutions (T427A, T431S, V433I and L437V) were located in the transmembrane domain of the E protein, with one (T427A) more hydrophobic for ''ricinus'' strains. Louping ill virus (LIV), a TBEV-related virus which is transmitted by I. ricinus in the UK, also showed the same more hydrophobic pattern and localisation of ''tick-specific'' amino acids as the I. ricinus-transmitted WE TBEV strains ( Fig. 2A) . Discussion TBE is currently reported in more than 30 countries of Eurasia and causes significant outbreaks of encephalitis in 16 countries, including 13 EU Member States (Austria, the Czech Republic, Estonia, Finland, Germany, Greece, Hungary, Latvia, Lithuania, Poland, Slovak Republic, Slovenia, Sweden) and three non-EU Member States (Norway, Russia and Switzerland) [3] . The clinical manifestations of TBE in endemic regions vary widely, from inapparent and febrile infections, with recovery of patients, to debilitating or fatal encephalitis, even in some vaccinated individuals [1, 2] and no adequate explanations have as yet been produced.