The recovery of virus from the two plasmids representing the infectious clone has been described previously (34) (35) (36) . Briefly, plasmid pGGVs 660 (or mutated derivatives) was digested with PspAI, dephosphorylated with Shrimp Alkaline Phosphatase (SAP; USB) and, after heatinactivation of SAP, digested with AgeI. Similarly, pGGVs 660-10927 was digested with NotI, dephosphorylated and then digested with AgeI. The excised linker DNA fragments from pGGVs 660 and pGGVs 660-10927 were removed using MicroSpin TM S-400 columns (Pharmacia Biotech) and ligated at the AgeI site generating full-length cDNA which was linearized with SmaI and used as a template for SP6 transcription (34) . In vitro-synthesized RNA was inoculated intracerebrally into suckling mice to recover the mutant viruses which were not passaged further prior to phenotype evaluation (35) . Recovered viruses were amplified by RT-PCR between nucleotides 1-940 (5 0 -UTR-C-prM region of the TBEV genome) and 10206-10927 (3 0 -UTR), and sequenced to validate the presence of the introduced mutations and to exclude extraneous mutations at the 5 0 -UTR and 3 0 -UTR (36) .