Flow cytometric evaluation of anti‐herpes drugs

Abstract

A rapid means of screening drugs for toxicity and anti-herpes simplex virus activity was developed based on the flow cytometric detection of HSV induced changes in cellular DNA content. Subconfluent monolayers of human diploid fibroblasts (HEL 299) were assayed for DNA content with propidium iodide 24 h after infection with HSV-1 (multiplicity of infection 1-10) and treatment with the drug to be tested. Infection was detected by a broadening of the normal diploid and tetraploid peaks and presence of greater than 4-n DNA staining. Inhibition of viral DNA synthesis and maintenance of the normal growth pattern of control cells was indication of antiviral activity. Toxicity of the compound was indicated by the loss of S phase and tetraploid cell populations. Using this assay, we evaluated the activities of one experimental and two established antiviral agents.

Applications of Flow Cytometry to Clinical Microbiology

“…Since the pioneer works of Rosenthal et al (280) and Pauwels et al (247) on the use of FCM to study the effect of antiviral agents on herpesvirus and HIV, FCM has been used by several groups, particularly those studying these viruses and CMV. The detection and quantification of viral antigens and viral nucleic acids in combination with antibodies or nucleic acid probes, respectively, or any other cellular parameter associated with viral infection have been used in FCM protocols for the in vitro evaluation of antiviral-drug activities.…”

“…The method was based on HSV-induced changes in cellular DNA content measured by the binding of PI to the DNA. The lower the antiviral activity, the larger was the DNA amount and the more intense was the PI fluorescence (280). The activity of antiviral drugs against HSV-1 (300), HHV-6 (276) and varicella-zoster virus (295,296) has also been assayed by FCM by measuring the inhibition of specific viral antigen expression for each drug with antibodies against virus antigens.…”

Analysis of DNA synthesis in herpes simplex virus infected cells by dual parameter flow cytometry

“…The increase of viral DNA synthesis in the HSV-2-infected CaSki cells was clearly demonstrated in all cell-cycle compartments by BrdUrd incorporation. The application of one-parameter DNA-flow cytometry for the evaluation of the effectivity of antiherpes drugs [17] may give false results, because changes in the proportions of S-phase cells only can be detected. IdUrd and BrdUrd are incorporated into viral DNA as thymidine analogues and although they may make the progeny virus DNA fragile [23] they do not seem to interfere with the HSV-2 DNA replication during the first replicative cycle of the virus.…”

Uses of flow cytometry in virology

“…Uninfected cells gave a normal DNA profile consisting of GJG1, S, and G2/M peaks. herpesvirus group since this broadening of the DNA profile in herpesvirus-infected cells seems to be a general phenomenon (54,124,165,191).…”

“…Flow cytometry was used to study the effects of antiviral drugs on HSV infection (165). Cellular DNA in HSV-infected and uninfected cells was stained by PI binding, and the DNA profiles were detected by flow cytometry.…”