title: Identification of newly detected, drug-related HCMV UL97- and UL54-mutations using a modified plaque reduction assay.
abstract: BACKGROUND
  Drug-resistant cytomegalovirus causes major problems in immunocompromised patients and is due to mutations in the UL97-gene (phosphotransferase) and/or the UL54-gene (polymerase).
  
  
  OBJECTIVE
  Three previously unknown UL97-mutations (E596D/Y and I610T), one UL54 single point mutation (D515E) and a UL54 triple mutation (D515E+L516M+I521T) were characterized for drug-resistance by marker transfer analysis using BAC-technology.
  
  
  METHODS
  Mutations were introduced into the bacterial artificial chromosome TB40-BACKL7-UL32EGFP. In addition, mutations M460V (UL97) and I521T (UL54) served as drug-resistant control. Phenotypic resistance testing was performed by a modified plaque reduction assay using a mixture of infected fibroblasts and uninfected ARPE-19 cells which improved formation of clearly definable plaques considerably.
  
  
  RESULTS
  Resistance testing showed ganciclovir (GCV)-resistance for UL97-mutations I610T and E596Y while mutation E596D was drug-sensitive. UL54-mutation D515E was resistant to GCV. The virus strain containing the UL54 triple mutation conferred cross-resistance to GCV and cidofovir (CDV). None of the mutations interfered with normal growth kinetics of the virus.
  
  
  CONCLUSIONS
  New mutations in the UL97- and UL54-gene of HCMV are still detected continuously. Furthermore, several mutations occurring in the same codon often show divergent phenotypes and the accumulation of multiple mutations in one virus strain may lead to increased or decreased drug-resistance. Therefore, characterization of newly detected mutations by marker transfer analysis is essential to confirm that genotypically detected mutations can produce phenotypic resistance. These results allow reliable interpretation of fast genotypic methods generally used in diagnostics.
authors.affiliation: Institute of Medical Virology and Epidemiology of Viral Diseases, University Hospital Tübingen, 72076 Tübingen, Germany.
authors.forename: Lena
authors.initials: L
authors.lastname: Fischer
authors.affiliation: Institute of Virology, University Hospital Ulm, 89081 Ulm, Germany.
authors.forename: Kerstin Laib
authors.initials: KL
authors.lastname: Sampaio
authors.affiliation: Institute of Medical Virology and Epidemiology of Viral Diseases, University Hospital Tübingen, 72076 Tübingen, Germany.
authors.forename: Gerhard
authors.initials: G
authors.lastname: Jahn
authors.affiliation: Institute of Medical Virology and Epidemiology of Viral Diseases, University Hospital Tübingen, 72076 Tübingen, Germany.
authors.forename: Klaus
authors.initials: K
authors.lastname: Hamprecht
authors.affiliation: Institute of Medical Virology and Epidemiology of Viral Diseases, University Hospital Tübingen, 72076 Tübingen, Germany. Electronic address: katharina.goehring@med.uni-tuebingen.de.
authors.forename: Katharina
authors.initials: K
authors.lastname: Göhring
mesh_terms: D000328:Adult; D000998:Antiviral Agents / Q000494:pharmacology; D002460:Cell Line; D002648:Child; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics; D000077404:Cidofovir; D003587:Cytomegalovirus / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D003586:Cytomegalovirus Infections / Q000188:drug therapy / Q000821:virology*; D003596:Cytosine / Q000031:analogs & derivatives / Q000494:pharmacology; D004259:DNA-Directed DNA Polymerase / Q000235:genetics*; D024921:Drug Resistance, Multiple, Viral; D005260:Female; D015774:Ganciclovir / Q000494:pharmacology; D005838:Genotype; D006801:Humans; D007223:Infant; D008297:Male; D009154:Mutation; D063065:Organophosphonates / Q000494:pharmacology; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics*
chemical_list: D000998:Antiviral Agents; D063065:Organophosphonates; C109367:UL54 protein, Human herpesvirus 5; D014764:Viral Proteins; D003596:Cytosine; D017853:Phosphotransferases (Alcohol Group Acceptor); C075365:ganciclovir kinase; D004259:DNA-Directed DNA Polymerase; D000077404:Cidofovir; D015774:Ganciclovir
keywords: Antiviral resistance; Cidofovir; Cytomegalovirus; Foscarnet; Ganciclovir; Stem-cell transplantation