The ligation in vitro of two overlapping plasmids, pGGVs H and pGGVs 660-1982del produces full-length infectious clone of TBEV strain Vs [28, 29] . The megaprimer-mediated domain swapping mutagenesis technique [47] was utilized to introduce point mutations in pGGVs 660-1982H (Fig. 6 ). For the Hi-Fi PCR, a pair of primers was used to amplify regions of about 200 nucleotides (megaprimer); one primer contained the appropriate point mutation within the E gene. Subsequently, the megaprimer was used to amplify the pGGVs 660-1982 H template in 15 cycles of circular PCR at 95uC for 30 sec, at 60uC for 30 sec and at 72uC for 5 min. Each cycle of the PCR produced nicked dsDNA molecules, with nascent (circular) DNA strand originating from template bacterial dsDNA and the other one (nicked) from newly amplified PCR product. Following 15 cycles, the accumulation of amplified linear complementary ssDNA strands resulted in the formation of annealed circular (twice nicked) molecules. To facilitate screening, the dammethylated bacterial (template) DNA was digested with 40 U of DpnI (New England Biolabs) at 37uC for 1 h. Following this, PCR products were electroporated into AbleK bacterial cells (Stratagene) and selected clones were completely sequenced.