title: Recombinant herpes simplex virus type 1 strains with targeted mutations relevant for aciclovir susceptibility.
abstract: 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.
authors.affiliation: Institute of Infection Medicine, Christian-Albrecht University Kiel and University Medical Center Schleswig-Holstein, Kiel, Germany.
authors.forename: Anne-Kathrin
authors.initials: AK
authors.lastname: Brunnemann
authors.affiliation: Institute of Virology and Antiviral Therapy, Consulting Laboratory for HSV and VZV, Jena University Hospital, Jena, Germany.
authors.forename: Kristin
authors.initials: K
authors.lastname: Liermann
authors.affiliation: Institute of Medical Microbiology, Jena University Hospital, Jena, Germany.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Deinhardt-Emmer
authors.affiliation: Institute of Infection Medicine, Christian-Albrecht University Kiel and University Medical Center Schleswig-Holstein, Kiel, Germany.
authors.forename: Gregor
authors.initials: G
authors.lastname: Maschkowitz
authors.affiliation: Institute of Virology, Hannover Medical School, Hannover, Germany.|German Center for Infection Research (DZIF), Hannover, Germany.
authors.forename: Anja
authors.initials: A
authors.lastname: Pohlmann
authors.affiliation: Institute of Virology, Hannover Medical School, Hannover, Germany.|German Center for Infection Research (DZIF), Hannover, Germany.
authors.forename: Beate
authors.initials: B
authors.lastname: Sodeik
authors.affiliation: Institute of Infection Medicine, Christian-Albrecht University Kiel and University Medical Center Schleswig-Holstein, Kiel, Germany.
authors.forename: Helmut
authors.initials: H
authors.lastname: Fickenscher
authors.affiliation: Institute of Virology and Antiviral Therapy, Consulting Laboratory for HSV and VZV, Jena University Hospital, Jena, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Sauerbrei
authors.affiliation: Institute of Infection Medicine, Christian-Albrecht University Kiel and University Medical Center Schleswig-Holstein, Kiel, Germany.
authors.forename: Andi
authors.initials: A
authors.lastname: Krumbholz
mesh_terms: D000212:Acyclovir / Q000494:pharmacology*; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D002522:Chlorocebus aethiops; D024882:Drug Resistance, Viral / Q000187:drug effects / Q000235:genetics*; D018259:Herpesvirus 1, Human / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics*; D007700:Kinetics; D009154:Mutation / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic / Q000235:genetics*; D013937:Thymidine Kinase / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014709:Vero Cells; D019562:Viral Load / Q000235:genetics; D014779:Virus Replication / Q000187:drug effects
chemical_list: D000998:Antiviral Agents; D013937:Thymidine Kinase; D000212:Acyclovir