A rapid phenotypic assay for detection of acyclovir-resistant varicella-zoster virus with mutations in the thymidine kinase open reading frame

Détails

ID Serval
serval:BIB_746B659784E9
Type
Article: article d'un périodique ou d'un magazine.
Collection
Publications
Titre
A rapid phenotypic assay for detection of acyclovir-resistant varicella-zoster virus with mutations in the thymidine kinase open reading frame
Périodique
Antimicrobial Agents and Chemotherapy
Auteur(s)
Sahli  R., Andrei  G., Estrade  C., Snoeck  R., Meylan  P. R.
ISSN
0066-4804 (Print)
Statut éditorial
Publié
Date de publication
04/2000
Volume
44
Numéro
4
Pages
873-8
Notes
Journal Article
Research Support, Non-U.S. Gov't --- Old month value: Apr
Résumé
Susceptibility assays by cell culture methods are time-consuming and are particularly difficult to perform with varicella-zoster virus (VZV). To overcome this limitation, we have adapted a functional test of the viral thymidine kinase (TK) in TK-deficient (tdk mutant) bacteria to detect ACV-resistant VZV in clinical samples. After PCR amplification, the complete viral TK open reading frame (ORF) is purified from PCR primers, digested with two restriction enzymes, and ligated in an oriented fashion into a bacterial expression vector. The ligation products are then used to transform tdk mutant bacteria. After transformation, an aliquot of the bacteria is plated onto a plate with minimal medium containing (i) ampicillin to select for plasmids carrying the viral TK ORF and (ii) isopropyl beta-D-thiogalactopyranoside (IPTG) to induce its expression. An identical aliquot of bacteria is also plated onto a medium containing, in addition to the components described above, 5-fluorodeoxyuridine (FUdR). Compared to the number of transformants on FUdR-free medium, the number of colonies carrying TK derived from susceptible strains was reduced by 86%, on average, in the presence of FUdR. In contrast, the number of transformants carrying TK from resistant strains with a mutant TK were reduced by only 4%, on average, on FUdR-containing plates. We have assessed the validity of this assay with cell culture isolates and several clinical samples including two cerebrospinal fluid samples from which no virus could be isolated. This colony reduction assay allowed the correct identification of the TK phenotype of each VZV isolate tested and can be completed within 3 days of receipt of the sample.
Mots-clé
Acyclovir/*pharmacology Antiviral Agents/*pharmacology Chickenpox/virology Cloning, Molecular Escherichia coli/drug effects/genetics/metabolism Floxuridine/pharmacology Herpesvirus 3, Human/*drug effects/*genetics Humans Mutation/*drug effects Open Reading Frames/drug effects/*genetics Phenotype Reverse Transcriptase Polymerase Chain Reaction Thymidine Kinase/drug effects/*genetics
Pubmed
Web of science
Création de la notice
24/01/2008 21:51
Dernière modification de la notice
03/03/2018 18:21
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