bromodeoxycytidine and Herpes-Simplex

A rapid screening test for resistance to acyclovir, mediated by a lack of thymidine kinase (TK) activity in herpes simplex virus (HSV), was developed by utilizing the uptake of [125I]iododeoxycytidine (IdC) by infected Vero cells. Cells infected with TK+ virus demonstrate uptake of IdC within 3 hr of infection. The assay can detect as few as 690 pfu of virus. Cells infected with TK virus have an uptake of IdC similar to that of uninfected cells, whereas cells infected with TK+ generally have an uptake greater than 10 times that of uninfected cells if tested when obvious viral cytopathic effect is present. All 19 clinical HSV isolates tested were correctly identified as TK+. Of 17 blinded HSV isolates tested, all six TK- isolates were correctly identified. A single strain with an ID50 of 3.4 micrograms/ml and an altered TK substrate specificity was incorrectly classified as TK+. The assay is a useful, rapid screening test for viral TK activity.

Topics: Acyclovir; Animals; Bromodeoxycytidine; Cell Line; Chlorocebus aethiops; Deoxycytidine; Drug Resistance, Microbial; Herpes Simplex; Humans; Kidney; Microbial Sensitivity Tests; Simplexvirus; Thymidine Kinase

A plaque autoradiography assay to detect and quantitate thymidine kinase (TK) mutants of herpes simplex virus type 1 (HSV-1) and HSV-2 in clinical samples is described. This method utilizes the selective incorporation of [125I]iododeoxycytidine, a pyrimidine analog selectively phosphorylated by the HSV TK. Only cells infected with TK-competent virus will efficiently incorporate iododeoxycytidine and are the only cells detected by autoradiography. Furthermore, this assay discriminates between TK+ virus (TK competent) and TKA virus (TK altered or reduced). This ability to differentiate TK+ from TKA virus is enhanced when infected cells are labeled with [14C]thymidine in tandem with iododeoxycytidine labeling. Reconstruction experiments with mixtures of TK+ (HSV-1 Patton) virus and TK-deficient (TK-) (B2006) or TKA (IUDRr) mutants were performed to determine the limits of detection of this technique. Ten percent TK- or TKA virus was the lower limit for the detection of TK mutants in a mixed population, whereas 1 in 1,000 TK+ virus revertants could be detected in a TK- virus population. In reconstructed populations and 45 clinical samples, a good correlation existed between the increase in 50% inhibitory dose for acyclovir and the percent TK mutant virus present. Similarly, the results of this technique correlated well with the acyclovir phosphorylating activity of extracts from cells infected with isolates or reconstructed mixtures. Plaque autoradiography with [125I]iododeoxycytidine was able to distinguish mixed populations of TK+ and TK- virus and homogeneous populations of TKA virus. The tandem use of [125I]iododeoxycytidine and [14C]thymidine readily identified TKA virus, which appeared as TK+ virus when labeled with [14C]thymidine alone. This technique provides a sensitive screen for antiviral resistance due to alterations in the viral TK and can be used to analyze clinical samples.

Topics: Acyclovir; Autoradiography; Bromodeoxycytidine; Deoxycytidine; Herpes Simplex; Humans; Mutation; Simplexvirus; Thymidine; Thymidine Kinase; Viral Plaque Assay

Topics: Adenosine; Bromodeoxycytidine; Cells, Cultured; Deoxycytidine; DNA, Viral; Herpes Simplex; Humans; Lesch-Nyhan Syndrome; Orotic Acid; Thioguanine; Ultraviolet Rays; Virus Replication; Xeroderma Pigmentosum

Topics: Bromodeoxycytidine; Cell Line; Deoxycytidine; Deoxycytidine Kinase; Herpes Simplex; Humans; Phosphorylation; Phosphotransferases

Topics: Animals; Bromodeoxycytidine; Deoxycytidine; Herpes Simplex; Mice; Simplexvirus; Virus Replication