Here, we describe a novel reliable method to assess the significance of individual mutations within the thymidine kinase (TK) gene of herpes simplex virus type 1 (HSV-1) to nucleoside analogue resistance. Eleven defined single nucleotide polymorphisms that occur in the TK gene of clinical HSV-1 isolates and a fluorescence reporter were introduced into the HSV-1 strain 17 + that had been cloned into a bacterial artificial chromosome. The susceptibility of these different strains to aciclovir, penciclovir, brivudin, and foscarnet was determined with a modified cytopathic effect reduction assay. The strains were also tested for their aciclovir susceptibility by measuring the relative fluorescence intensity as an indicator for HSV-1 replication and by quantifying the virus yield. Our data indicate that the amino acid substitutions R41H, R106H, A118V, L139V, K219T, S276R, L298R, S345P, and V348I represent natural polymorphisms of the TK protein, whereas G61A and P84L mediate broad cross-resistance against aciclovir, penciclovir, brivudin, and susceptibility to foscarnet. This method allows the definition of the resistance genotype of otherwise unclear mutations in the TK gene of HSV-1. Thus, it provides a scientific basis for antiviral testing in clinical isolates of patients suffering from serious diseases and will facilitate testing of new antivirals against HSV-1.Herpes simplex virus type 1 (HSV-1) belongs to the genus Simplexvirus of the Alphaherpesvirinae, a subfamily within the Herpesviridae 1 . With its high seroprevalence ranging in Europe from 50% to 90%, this human pathogen is of great medical importance 2 . After onset of primary infection, HSV-1 establishes a lifelong latency followed by endogenous reactivations 2 .Severe HSV-1 infections are mainly observed in immunosuppressed patients 3 who suffer from extensive, disseminated, or persistent herpetic lesions but also from more serious complications such as pneumonia 4,5 , encephalitis 6,7 , or hepatitis 8,9 . Thus, abundant and prolonged antiviral treatment is often inevitable but promotes the selection of resistant HSV-1 variants 3 . Approximately 2% to 10% of the immunosuppressed patients develop an infection or reactivation with HSV-1 variants resistant against antivirals in clinical use 10,11 . The highest rates of up to 45% have been described in patients who have received stem-cell transplantation 12 . So far, aciclovir (ACV) remains the drug of choice for prophylaxis and treatment of HSV infections 3 . Resistance-associated substitutions are predominantly observed within the viral thymidine kinase (TK) that otherwise converts the guanosine analogue to its active monophosphate form 13 . The incorporation of ACV results in DNA chain termination due to the missing hydroxyl group on the acyclic molecule. Accordingly, alterations of the TK-encoding gene UL23 are reported in 95% of clinical isolates resistant to ACV 14,15 . The high degree of UL23 sequence variability includes nucleotide deletions or insertions, which usually result in frame shifts or s...
