Stepwise characterization of non-synonymous mutations in the HSV-1 thymidine kinase gene by different functional assays.

•Thymidine kinase (TK) mutants of herpes simplex virus type 1 were generated.•Activity to bromodeoxyuridine (BrdU), acyclovir and deoxythymidine was evaluated.•Seven resistance-related amino acid substitutions resulted in no activity to BrdU.•Eleven natural gene polymorphisms led to high activity to BrdU.•The N244H mutant protein was verified as TK with altered substrate specificity.