Lifelong latency of cytomegalovirus (CMV) provides a reservoir that contributes to disease risk, whether transferred with cells from the donor or reactivated within the bone marrow (BM) recipient. Little is known of the parameters that determine risk of transmission or reactivation. Over the pst four years investigations into the nature of the human CMV:hematopoietic progenitor cell interactions have led to the discovery and characterization of latent gene products. This new project will focus on investigating the expression of latent gene products in the natural host and will assess expression as a predictor of CMV transmission and reactivation. First, this work will identify the cell type(s) in which CMV resides during latency in the natural host, following CD34+ and CD33+ hematopoietic cells most closely. BM mononuclear cells from BM of health seropositive donors (or peripheral blood mononuclear cells from GM-CSF mobilized from BM) will be subjected to FACS analysis to determine which cell type(s) most frequently harbor viral genomes, contain latent transcripts and express the latency-associated protein. Second, to complement studies on the latent cell type, the project will assess the pattern of gene expression as a predictor of CMV transmission and as an indicator of reactivation during allograft transplantation. BM and peripheral blood mononuclear cells will be evaluated for transcription patterns which distinguish between acute and latent infection. Sera will be evaluated for antibody to and lymphocytes will be evaluated for ability to be stimulated by proteins expressed during latent and productive infection. Expression patterns in seropositive donors will be correlated with outcome in the seronegative recipient, and patterns in the seropositive recipient will be correlated with the likelihood they undergo reactivated infection. Third, this project will assess reactivation in BM progenitors cultured from healthy seropositive donors using culture conditions that we have found to reactivate experimental latent CMV infection in cultured CD33+, CD15+, CD10+, CD1A+ cells. Cells that are capable of yielding reactivated virus will be identified through FACS separation for viral antigen-positive cells, by analysis for presence of transcripts consistent with productive infection and by plaque assay for production of virus. The events that we propose to study and the cell types on which we propose to focus appear to contribute to the lifelong persistence and latency of CMV in the naturally infected host, and this reservoir may predispose BM transplant recipients to reactivation and dissemination of active CMV infection
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