title: Drug resistance of clinical varicella-zoster virus strains confirmed by recombinant thymidine kinase expression and by targeted resistance mutagenesis of a cloned wild-type isolate.
abstract: In this study, approaches were developed to examine the phenotypes of nonviable clinical varicella-zoster virus (VZV) strains with amino acid substitutions in the thymidine kinase (TK) (open reading frame 36 [ORF36]) and/or DNA polymerase (Pol) (ORF28) suspected to cause resistance to antivirals. Initially, recombinant TK proteins containing amino acid substitutions described as known or suspected causes of antiviral resistance were analyzed by measuring the TK activity by applying a modified commercial enzyme immunoassay. To examine the effects of these TK and Pol substitutions on the replication of recombinant virus strains, the method of en passant mutagenesis was used. Targeted mutations within ORF36 and/or ORF28 and an autonomously expressed gene of the monomeric red fluorescent protein for plaque identification were introduced into the European wild-type VZV strain HJO. Plaque reduction assays revealed that the amino acid substitutions with unknown functions in TK, Q303stop, N334stop, A163stop, and the deletion of amino acids 7 to 74 aa (Δaa 7 to 74), were associated with resistance against acyclovir (ACV), penciclovir, or brivudine, whereas the L73I substitution and the Pol substitutions T237K and A955T revealed sensitive viral phenotypes. The results were confirmed by quantitative PCR by measuring the viral load under increasing ACV concentrations. In conclusion, analyzing the enzymatic activities of recombinant TK proteins represent a useful tool for evaluating the significance of amino acid substitutions in the antiviral resistance of clinical VZV strains. However, direct testing of replication-competent viruses by the introduction of nonsynonymous mutations in a VZV bacterial artificial chromosome using en passant mutagenesis led to reliable phenotypic characterization results.
authors.affiliation: Institute for Infection Medicine, Christian-Albrecht University 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, German Consulting Laboratory for HSV and VZV, Jena University Hospital, Friedrich Schiller University, Jena, Germany.
authors.forename: Kathrin
authors.initials: K
authors.lastname: Bohn-Wippert
authors.affiliation: Institute of Virology and Antiviral Therapy, German Consulting Laboratory for HSV and VZV, Jena University Hospital, Friedrich Schiller University, Jena, Germany.
authors.forename: Roland
authors.initials: R
authors.lastname: Zell
authors.affiliation: Institute of Virology and Antiviral Therapy, German Consulting Laboratory for HSV and VZV, Jena University Hospital, Friedrich Schiller University, Jena, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Henke
authors.affiliation: Institute of Virology and Antiviral Therapy, German Consulting Laboratory for HSV and VZV, Jena University Hospital, Friedrich Schiller University, Jena, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Walther
authors.affiliation: Institute for Infection Medicine, Christian-Albrecht University and University Medical Center Schleswig-Holstein, Kiel, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Braum
authors.affiliation: Institute for Infection Medicine, Christian-Albrecht University and University Medical Center Schleswig-Holstein, Kiel, Germany.
authors.forename: Gregor
authors.initials: G
authors.lastname: Maschkowitz
authors.affiliation: Institute for Infection Medicine, Christian-Albrecht University 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, German Consulting Laboratory for HSV and VZV, Jena University Hospital, Friedrich Schiller University, Jena, Germany andreas.sauerbrei@med.uni-jena.de.
authors.forename: Andreas
authors.initials: A
authors.lastname: Sauerbrei
authors.affiliation: Institute for Infection Medicine, Christian-Albrecht University and University Medical Center Schleswig-Holstein, Kiel, Germany.
authors.forename: Andi
authors.initials: A
authors.lastname: Krumbholz
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology*; D002460:Cell Line; D024882:Drug Resistance, Viral / Q000235:genetics; D014645:Herpesvirus 3, Human / Q000187:drug effects* / Q000201:enzymology*; D006801:Humans; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D013937:Thymidine Kinase / Q000235:genetics / Q000378:metabolism*
chemical_list: D000998:Antiviral Agents; D011994:Recombinant Proteins; D013937:Thymidine Kinase