To recover engineered viruses, the mutated plasmids, constructed on the basis of pGGVs 660-1982 H, were ligated with pGGVs 660-1982del to restore the full-length cDNA of TBEV as described previously [29] . Each mutant full-length clone was subsequently linearised by Sma I and used for SP6 transcription to produce full-length RNA. The SP6-transcribed RNA was transfected into PS cells using Lipofectin reagent (Invitrogen) according to the manufacturer's protocols. Infectious supernatant medium was collected on day 5 pi. The presence of virus in infected cells was confirmed by immunofluorescence microscopy using monoclonal antibodies specific for flavivirus E proteins [46] and by RT-PCR. The entire E-gene was sequenced to ensure no additional substitutions appeared during the genetic manipulation or initial virus replication in PS cells.