Mesenchymal stem cells isolated from human fetal bone marrow harbor latent KSHV. Following extraction from human fetal bone marrow, hematopoietic cells were incubated with a stem cell enrichment media and maintained in tissue culture under optimal conditions for mesenchymal stem cell growth (“Study design”). (A) Phase contrast microscopy (original magnification × 40, Nikon Eclipse TE2000-E, Improvision Software, Improvision, Lexington, MA) reveals confluent layers of elongated cells with reduced nuclear-cytoplasmic ratios. (B) Flow cytometric staining demonstrates that greater than 99% of the cells expressed cell surface CD29, CD73, and CD105 (dark histograms) compared to isotype controls (light histograms). In contrast, cell surface expression of CD31, CD34, and CD45 was absent. This staining pattern identified the population of cells isolated as MSCs (“Results and discussion”) and remained unchanged for at least 3 weeks after infection (not shown). MSCs were incubated with concentrated KSHV (C-D), induced BCBL-1 cells (E-F), UV-inactivated KSHV (G-H), or uninduced BCBL-1 cells (I-J). Forty-eight hours later, indirect IFA for the latency-associated nuclear antigen (LANA) revealed KSHV infection with either route of infection (D, F), giving rise to the punctate intranuclear staining pattern (green dots) characteristic of LANA expression. The controls (H, J) lacked LANA reactivity. Of note, infection with concentrated KSHV led to a higher rate of infection than did BCBL-1 coculture at the donor-to-target-cell ratios employed in this experiment (compare D to F). Omission of polybrene in cell-free experiments also led to infection but with approximately one third the efficiency (data not shown). Nuclei were counterstained with DAPI (4′6-diamino-2-phenylindole) (C, E, G, I).