FIG. 7.

Peritoneal macrophages from C57BL/6 mice harbor latent γHV68, as detected by an ex vivo reactivation assay. Limiting-dilution analysis was used to quantitate the frequency of cells that reactivate γHV68 by using FACS-sorted PEC populations isolated from γHV68-infected C57BL/6 mice. (A) Limiting-dilution reactivation analysis to determine the frequency of cells that reactivate γHV68 by using PECs from C57BL/6 mice 11 days postinfection (p.i.). PECs were FACS sorted into macrophage- or lymphocyte-enriched populations based on forward and side scatter characteristics (as for Fig. 5A and 6A). (B) Pre- and postsorting Wright’s differential staining analysis of total and fractionated PECs isolated from C57BL/5 mice 11 days postinfection. Cells were categorized by morphological criteria as macrophages (Mac), lymphocytes (Lymph), or monocytes-lymphoblasts (Mono/Blast). (C) Limiting-dilution reactivation analysis to determine the frequency of cells that reactivate γHV68 by using PECs collected from C57BL/6 mice 5 weeks postinfection. Cells were stained with F4/80, and the F4/80-negative and F4/80-positive cell populations were separated by FACS sorting as described in Materials and Methods. (D) Pre- and postsorting Wright’s differential staining analysis of total and fractionated PECs isolate from C57BL/6 mice 5 weeks postinfection. For the limiting-dilution reactivation analyses shown in panels A and C, the percentage of wells that scored positive for viral CPE 3 weeks after plating is plotted as a function of the number of cells plated per well. Twenty-four wells were plated per cell dilution. Each graph represents a single experiment. Cells from 4 to 10 mice were pooled and assayed per experiment.