Macrophages are the major reservoir of latent murine gammaherpesvirus 68 in peritoneal cells

B cells have previously been identified as the major hematopoietic cell typ e harboring latent gammaherpesvirus 68 (gamma HV68) (N. P. Sunil-Chandra, S . Efstathiou, and A. A. Nash, J. Gen. Virol. 73:3275-3279, 1992). However, we have shown that gamma HV68 efficiently establishes latency in B-cell-def icient mice (K. E. Week, M. L. Barkon, L. I. Yoo, S. H. Speck, and H. W. Vi rgin, J. Virol. 70:6775-6780, 1996), demonstrating that B cells are not req uired for gamma HV68 latency. To understand this dichotomy, we determined w hether hematopoietic cell types, in addition to B cells, carry latent gamma HV68. We observed a high frequency of cells that reactivate latent gamma H V68 in peritoneal exudate cells (PECs) derived from both B-cell-deficient a nd normal C57BL/6 mice. PECs were composed primarily of macrophages in B-ce ll-deficient mice and of macrophages plus B cells in normal C57BL/6 mice. T o determine which cells in PECs from C57BL/6 mice carry latent gamma HV68, we developed a limiting-dilution PCR assay to quantitate the frequency of c ells carrying the gamma HV68 genome in fluorescence-activated cell sorter-p urified cell populations. We also quantitated the contribution of individua l cell populations to the total frequency of cells carrying latent gamma HV 68. At early times after infection, the frequency of PECs that reactivated gamma HV68 correlated very closely with the frequency of PECs carrying the gamma HV68 genome, validating measurement of the frequency of viral-genome- positive cells as a measure of latency in this cell population. F4/80-posit ive macrophage-enriched, lymphocyte-depleted PECs harbored most of the gamm a HV68 genome and efficiently reactivated gamma HV68, while CD19-positive, B-cell-enriched PECs harbored about a 10-fold lower frequency of gamma HV68 genome-positive cells. CD4-positive, T-cell-enriched PECs contained only a very low frequency of gamma HV68 genome-positive cells, consistent with pr evious analyses indicating that T cells are not a reservoir for gamma HV68 latency (N. P. Sunil-Chandra, S. Efstathiou, and A. A. Nash, J. Gen. Virol. 73:3275-3279, 1992). Since macrophages are bone marrow derived, we determi ned whether elicitation of a large inflammatory response in the peritoneum would recruit additional latent cells into the peritoneum. Thioglycolate in oculation increased the total number of PECs by about 20-fold but did not a ffect the frequency of cells that reactivate gamma HV68, consistent with a bone marrow reservoir for latent gamma HV68. These experiments demonstrate gamma HV68 latency in two different hematopoietic cell types, F4/80-positiv e macrophages and CD19-positive B cells, and argue for a bone marrow reserv oir for latent gamma HV68.