Quantitative detection of viral gene expression in populations of Epstein-Barr virus-infected cells in vivo.

The method described in this chapter uses limiting dilution analysis in conjunction with RT-PCR to determine quantitatively what percentage of EBV-infected cells within a given population are expressing the viral genes EBNA-1 Q-K, EBNA-2, LMP-1, LMP-2, BZLF-1, and the EBERs. Because this technique involves limiting dilution analysis, it is possible to define which viral transcription programs are being used at the single-cell level. This assay takes 3-4 d to complete and involves the following steps: (1) sample preparation and isolation of the cell population of interest; (2) DNA-PCR limiting dilution analysis to determine the frequency of infected cells within the cell population; (3) RNA isolation; (4) cDNA synthesis; (5) PCR; (6) visualization of PCR products by Southern blotting; and (7) calculations. As an example, we have used PBMCs from the blood of an acute infectious mononucleosis patient. However, this technique can be applied to other cell populations, such as B cells, and other patient groups, such as healthy long-term virus carriers and immunosuppressed organ transplant recipients.