Assay for Epstein-Barr virus based on stimulation of DNA synthesis in mixed leukocytes from human umbilical cord blood

Abstract

Relationships between the rate of DNA synthesis in cultured human umbilical cord leukocytes and the multiplicity of added Epstein-Barr virus (EBV) were studied. At low multiplicities of approximately 0.1 transforming units/cell (approximately 10 physical particles/cell), inoculated cultures demonstrated increased rates of DNA synthesis, by comparison to uninoculated cultures, 3 days after inoculation. Stimulation of DNA synthesis was evident of progressively longer intervals after inoculations of 10-fold dilutions of virus. The rate of DNA synthesis, determined by short [-3H]thymidine pulses, reflected as small as twofold changes in multiplicity and thus can serve as a quantitative assay for the virus. Changes in the rate of DNA synthesis were evident before increases in cell number or alteration in morphology. Stimulation of DNA synthesis in umbilical cord leukocytes was inhibited by treatment of EBV with antibody and also in graded fashion, by progressive doses of UV irradiation to the virus. Induction of DNA synthesis by EBV was serum dependent. Estimates of the number of cells transformed were obtained by extrapolation from a standard curve relating known numbers of transformed cells to [-3H]thymidine incorporation and also by cloning cells after exposure to virus. At the low multiplicities of infection used in these experiments approximately 0.04 to 0.002 of the total cellular population was transformed. The high efficiency of cell transformation by EBV by comparison to other DNA tumor viruses is emphasized.