title: Expression of herpes simplex virus type 1 recombinant thymidine kinase and its application to a rapid antiviral sensitivity assay.
abstract: Antiviral-resistant herpesvirus infection has become a great concern for immunocompromised patients. Herpes simplex virus type 1 (HSV-1) infections are treated with viral thymidine kinase (vTK)-associated drugs such as acyclovir (ACV), and most ACV-resistance (ACV(r)) is due to mutations in the vTK. The standard drug sensitivity test is usually carried out by the plaque reduction assay-based method, which requires over 10 days. To shorten the time required, a novel system was developed by the concept, in which 293T cells transiently expressing recombinant vTK derived from the test sample by transfection of the cells with an expression vector were infected with vTK-deficient and ACV(r) HSV-1 (TAR), and then cultured in a maintenance medium with or without designated concentrations of ACV, ganciclovir (GCV) and brivudine (BVdU). The replication of TAR was strongly inhibited by ACV, GCV and BVdU in 293T cells expressing recombinant vTK of the ACV-sensitive HSV-1, whereas replication was not or slightly inhibited in cells expressing the recombinant vTK of highly resistant or intermediately resistant HSV-1, respectively. An inverse correlation was demonstrated in the 50% effective concentrations (EC(50)s) and inhibitory effects of these compounds on the replication of TAR among ACV(s) and ACV(r) HSV-1 clones. These results indicate that the EC(50)s of the vTK-associated drugs including ACV can be assumed by measuring the inhibitory effect of drugs in 293T cells expressing recombinant vTK of the target virus. The newly developed antiviral sensitivity assay system for HSV-1 makes it possible to estimate EC(50) for vTK-associated drugs, when whole vTK gene is available for use by gene amplification directly from lesion's samples or from virus isolates.
authors.affiliation: Special Pathogens Laboratory, Department of Virology 1, National Institute of Infectious Diseases, Gakuen 4-7-1, Musashimurayama, Tokyo 208-0011, Japan.
authors.forename: Tomoyuki
authors.initials: T
authors.lastname: Shiota
authors.forename: Wang
authors.initials: W
authors.lastname: Lixin
authors.forename: Mutsuyo
authors.initials: M
authors.lastname: Takayama-Ito
authors.forename: Itoe
authors.initials: I
authors.lastname: Iizuka
authors.forename: Momoko
authors.initials: M
authors.lastname: Ogata
authors.forename: Masanori
authors.initials: M
authors.lastname: Tsuji
authors.forename: Hidekazu
authors.initials: H
authors.lastname: Nishimura
authors.forename: Shuichi
authors.initials: S
authors.lastname: Taniguchi
authors.forename: Shigeru
authors.initials: S
authors.lastname: Morikawa
authors.forename: Ichiro
authors.initials: I
authors.lastname: Kurane
authors.forename: Masashi
authors.initials: M
authors.lastname: Mizuguchi
authors.forename: Masayuki
authors.initials: M
authors.lastname: Saijo
mesh_terms: D000212:Acyclovir / Q000494:pharmacology*; D019943:Amino Acid Substitution; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D001973:Bromodeoxyuridine / Q000031:analogs & derivatives / Q000494:pharmacology; D002522:Chlorocebus aethiops; D024882:Drug Resistance, Viral; D015774:Ganciclovir / Q000494:pharmacology; D015967:Gene Expression Regulation, Viral; D005822:Genetic Vectors; D057809:HEK293 Cells; D018259:Herpesvirus 1, Human / Q000187:drug effects* / Q000235:genetics / Q000502:physiology; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods*; D009154:Mutation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D013937:Thymidine Kinase / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D014709:Vero Cells; D010948:Viral Plaque Assay / Q000379:methods; D014779:Virus Replication
chemical_list: D000998:Antiviral Agents; D011994:Recombinant Proteins; C020235:brivudine; D013937:Thymidine Kinase; D001973:Bromodeoxyuridine; D015774:Ganciclovir; D000212:Acyclovir