Quantitative Cytophotometry Evaluated as a Method for

INFECTION AND IMMUNITY, May 1976, P. 1503-1509 Copyright X) 1976 American Society for Microbiology Vol. 13, No. 5 Printed in U.SA. Quantitative Cyto...
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INFECTION AND IMMUNITY, May 1976, P. 1503-1509 Copyright X) 1976 American Society for Microbiology

Vol. 13, No. 5 Printed in U.SA.

Quantitative Cytophotometry Evaluated as a Method for Analyses of Herpes Simplex Viral Deoxyribonucleic Acid Synthesis LYNN R. TRUSAL, ADAM ANTHONY, AND JOHN J. DOCHERTY* Department of Biology and Department of Microbiology,* The Pennsylvania State University, University Park, Pennsylvania 16802

Received for publication 28 October 1975

Feulgen deoxyribonucleic acid (F-DNA) microspectrophotometry was evaluated as a potential tool for quantification of herpes simplex virus type 1 (HSV-1) and 2 (HSV-2) DNA synthesis in single cells. Since HSV DNA synthesis has been extensively studied using incorporation of radioactive precursors into viral and cellular DNA, microspectrophotometric measures were correlated with biochemical data obtained using tritium-labeled thymidine ([3H]TdR). It was established that: (i) viral-induced increase in F-DNA can be cytophotometrically detected between 4 and 6 h postinfection (p.i.), which corresponds to the initial incorporation of [3H]TdR into viral DNA; (ii) peak F-DNA levels occurred 8 h p.i., which corresponds to the time when there is maximal uptake of [3H]TdR into viral DNA; (iii) HSV-2-infected nuclei exhibited a faster initial (between 4 and 6 h p.i.) elevation of F-DNA than HSV-1-infected nuclei (between 6 and 8 h p.i.), which supported cytological observations of a more rapid development of an inclusion body in HSV-2-infected nuclei. Despite the fact that F-DNA cytophotometry is unable to distinguish between cell and viral DNA, the overall study supports the existence of a good correlation between data obtained using microspectrophotometric and conventional isotope methods. Furthermore, cytophotometry complements biochemical evaluations in that it permits analyses of DNA changes on a single-cell basis or the detection of infection in very small numbers of cells. It has been reported that microscope cytophotometry is sensitive enough to detect virus replication in individual cells infected with adenoviruses, simian vacuolating viruses, and poliomyelitis virus (1, 2, 7, 8, 1I). However, a review of relevant literature revealed no studies combining biochemical and cytochemical analyses of viral deoxyribonucleic acid (DNA) in herpes-infected cell cultures. The major aim of the present study was to evaluate microspectrophotometry as a valid method for detecting and quantifying herpes simplex virus (HSV) DNA synthesis in a very small number of cells (

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