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By site-directed mutagenesis, we investigate the role of six mutations of herpes simplex virus type 1 thymidine kinase (TK) gene in the acquisition of resistance to acyclovir (ACV). TK activity was not impaired by substitutions located at codons 17, 161 and 374 and these mutations were thus related to TK gene polymorphism. Mutations His105Pro, Leu364Pro and Asp162Ala lead to the loss of TK activity that could result in ACV-resistance.