We have used the nested polymerase chain reaction (PCR) combined with fluorescence-activated cell sorting to define sites of latency of human cytomegalovirus (HCMV) in the peripheral blood of healthy subjects. Peripheral blood mononuclear (PBM) cells were separated into T cell or non-T cell populations and monocytes, and were then analysed by PCR for the presence of HCMV DNA. In five of six seropositive subjects, HCMV was found predominantly in the non-T cell population. Further analysis suggested that the virus was present in adherent cells and CD 14 + cells. In three of nine seronegative subjects we could demonstrate HCMV DNA, which we do not believe was due to contamination, reproducibly by PCR. In one of these seronegative subjects, HCMV DNA was present predominantly in the non-T cell fraction of PBM cells. No HCMV DNA was detectable in the remaining six seronegative subjects. We conclude that, within the PBM cells of normal asymptomatic seropositive and some seronegative subjects, HCMV is present predominantly in the monocyte fraction. In addition, the detection of HCMV sequences in seronegative subjects may indicate that infection with HCMV is more widespread than conventional seroepidemiology suggests.

Human cytomegalovirus (HCMV) persists as a subclinical, lifelong infection in the normal human host, but reactivation from latency in immunocompromised subjects results in serious disease. Latency and reactivation are defining characteristics of the herpesviruses and are key to understanding their biology; however, the precise cellular sites in which HCMV is carried and the mechanisms regulating its latency and reactivation during natural infection remain poorly understood. Here we present evidence, based entirely on direct analysis of material isolated from healthy virus carriers, to show that myeloid dendritic cell (DC) progenitors are sites of HCMV latency and that their ex vivo differentiation to a mature DC phenotype is linked with reactivation of infectious virus resulting from differentiation-dependent chromatin remodeling of the viral major immediate-early promoter. Thus, myeloid DC progenitors are a site of HCMV latency during natural persistence, and there is a critical linkage between their differentiation to DC and transcriptional reactivation of latent virus, which is likely to play an important role in the pathogenesis of HCMV infection. P rimary infection of healthy individuals with human cytomegalovirus (HCMV) is often asymptomatic and results in lifelong persistence in the host, a characteristic of all herpes viruses. However, primary infection and reactivation of latent HCMV causes serious disease in immunosuppressed transplant recipients and in advanced HIV infection (1-3). Transfusionmediated HCMV disease can be prevented by leukocyte depletion (4) of blood, but infectious virus cannot be detected in the blood of healthy carriers, suggesting that HCMV is transmitted as latent virus in the peripheral blood leukocyte population. Accumulating evidence has shown that HCMV is carried latently in mononuclear cells of the myeloid lineage during lifelong latency in naturally infected individuals (5-8). Differentiation of monocytes to macrophages in vitro is reported to induce immediate-early (IE) lytic gene expression from latent virus (9), and groups have intermittently reported that infectious virus can be recovered after differentiation of monocytes to macrophages through explant culture (10) and, more recently, by allogeneic T cell stimulation (11), suggesting that reactivation of latent virus is associated with both the differentiation and activation state of myeloid cells.The major IE genes of HCMV, driven by the viral major IE promoter͞enhancer (MIEP), are the two most abundantly transcribed genes at IE times of virus lytic infection (12), and their proteins play a critical role in control of viral early and late gene expression (for review, see ref. 12). Consistent with the differentiation-dependent induction of IE gene expression observed above, there is a clear correlation between the differentiation state of the cell and the regulation of HCMV IE gene expression in vitro. Thus, in transfection assays, the MIEP is transcriptionally repressed in undifferentiated but transcriptionally active ...

Cytotoxic T lymphocytes (CTL) appear to play an important role in the control of human cytomegalovirus (HCMV) in the normal virus carrier: previous studies have identified peripheral blood CD8 ؉ CTL specific for the HCMV major immediate-early gene product (IE1) and more recently, by bulk culture and cloning techniques, have identified CTL specific for a structural gene product, the lower matrix protein pp65. In order to determine the relative contributions of CTL which recognize the HCMV proteins IE1, pp65, and glycoprotein B (gB) to the total HCMV-specific CTL response, we have used a limiting-dilution analysis system to quantify HCMV-specific CTL precursors with different specificities, allowing the antigenic specificity of multiple shortterm CTL clones to be assessed, in a group of six healthy seropositive donors. All donors showed high frequencies of HCMV-specific major histocompatibility complex-restricted CTL precursors. There was a very high frequency of CTL specific for pp65 (lower matrix protein); IE1-specific CTL were also detectable at lower frequencies in three of five donors, while CTL directed to gB were undetectable. A pp65 gene deletion mutant of HCMV was then used to estimate the contribution of pp65-specific CTL to the total HCMV-specific CTL response; this showed that between 70 and 90% of all CTL recognizing HCMV-infected cells were pp65 specific. Analysis of the peptide specificity of pp65-specific CTL showed that some donors have a highly focused response recognizing a single peptide; the T-cell receptor V␤ gene usage in these two donors was shown to be remarkably restricted, with over half of the responding CD8 ؉ T cells utilizing a single V␤ gene rearrangement. Other subjects recognized multiple pp65 peptides: nine new pp65 CTL peptide epitopes were defined, and for five of these the HLA-presenting allele has been identified. All four of the HLA A2 donors tested in this study recognized the same peptide. This apparent domination of the CTL response to HCMV during persistent infection by a single structural protein, irrespective of major histocompatibility complex haplotype, is not clearly described for other persistent virus infections, and the mechanism requires further investigation.

Human cytomegalovirus (HCMV) is a frequent cause of major disease following primary infection or reactivation from latency in immunocompromised patients. Infection of non-permissive mononuclear cells is used for analyses of HCMV latency in vitro. Using this approach, it is shown here that repression of lytic gene expression following experimental infection of CD34 Primary infection of healthy individuals with human cytomegalovirus (HCMV) is often asymptomatic and results in lifelong persistence of the virus within the host, a common feature of all herpesvirus infections. However, primary infection and reactivation of latent HCMV are responsible for serious disease in both immunosuppressed transplant recipients and during advanced human immunodeficiency virus infection (Adler, 1983;Rubin, 1990). The health threat posed by the reactivation of latent HCMV has resulted in considerable efforts from a number of laboratories to identify the sites of latency and potential mechanisms responsible for controlling the progression of HCMV latency to reactivation. These studies include the study of naturally infected cells ex vivo and experimentally infected model systems in vitro.Analysis of peripheral blood mononuclear cells identified monocytes as one site of HCMV carriage in healthy individuals (Bevan et al., 1991;Taylor-Wiedeman et al., 1991). Monocytes represent a short-lived population of cells subject to constant renewal from myeloid precursors residing in the bone marrow. Analysis of these self-renewing CD34 + mononuclear haematopoietic progenitor cells showed that they also harboured HCMV genomes in naturally infected individuals (Kondo et al., 1996;Mendelson et al., 1996) and thus could represent an important reservoir of HCMV latency in vivo. Haematopoietic progenitors can differentiate into a number of cell types, including monocytes, macrophages, dendritic cells (DCs) and granulocytes, and in a recent analysis we showed that DCs generated ex vivo from healthy seropositive carriers reactivated HCMV and that latency and reactivation in these cells may be controlled by chromatin remodelling of the major immediate-early promoter (MIEP) to regulate lytic gene expression (Reeves et al., 2005). However, overall, the analysis of HCMV latency ex vivo has been complicated severely by the low frequency of genome-positive mononuclear cells, which is predicted to be around only 0?01 % of the total population (Slobedman & Mocarski, 1999). Consequently, many studies have relied on experimental infection of primary cells and of cell lines to try to model the events that occur during latency and reactivation of HCMV in the myeloid lineage.A number of these studies have shown that experimental infection of CD34 + cells is non-permissive and that a 'latent infection' is established during extended culture of these cells (Kondo et al., 1994;Minton et al., 1994;Movassagh et al., 1996;Sindre et al., 1996). Such non-permissive infection is defined by the absence of lytic gene expression; however, it is worth noting that a small numbe...