Rapid phenotypic characterization method for herpes simplex virus and Varicella-Zoster virus thymidine kinases to screen for acyclovir-resistant viral infection.
A rapid phenotypic screening method for herpes simplex virus (HSV) and varicella-zoster virus (VZV) thymidine kinase (TK) genes was developed for monitoring acyclovir-resistant viruses. This method determines the biochemical phenotype of the TK polypeptide, which is synthesized in vitro from viral DNA using a procedure as follows. The TK gene of each sample virus strain is amplified and isolated under the control of a T7 promoter by PCR. The PCR products are transcribed with T7 RNA polymerase and translated in a rabbit reticulocyte lysate. Using this method, enzymatic characteristics and the size of the TK polypeptides encoding HSV and VZV DNA were defined in less than 2 days without virus isolation. The assay should be a powerful tool in monitoring drug-resistant viruses, especially in cases in which virus isolation is difficult.
Suzutani
T
T
Department of Microbiology, Asahikawa Medical College, Asahikawa, Japan. suzutani@asahikawa-med.ac.jp
Saijo
M
M
Nagamine
M
M
Ogasawara
M
M
Azuma
M
M
D000212:Acyclovir / Q000494:pharmacology / Q000627:therapeutic use*; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology / Q000627:therapeutic use*; D017123:Bacteriophage T7; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D004352:Drug Resistance, Microbial; D018259:Herpesvirus 1, Human / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics*; D018258:Herpesvirus 2, Human / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics*; D014645:Herpesvirus 3, Human / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D011817:Rabbits; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D013937:Thymidine Kinase / Q000235:genetics* / Q000378:metabolism; D014709:Vero Cells
D000998:Antiviral Agents; D013937:Thymidine Kinase; D000212:Acyclovir