Thus, we hypothesised that each of these three amino acid substitutions could individually abolish HA deficiency in TBEV. To test this hypothesis experimentally we used a TBEV infectious clone to engineer three mutant viruses IC-D67G, IC-E122G and IC-D277A (See Methods) that simulate the wild-type viruses Yar 46-2, Yar 71/Yar 114 and Yar 48 ( Table 1 ). The introduction of any one of the three mutations into the parent HA positive pGGVs virus rendered it HA negative (Fig. 3 ).