Dysfunction of the central nervous system (CNS) is a prominent feature of the acquired immune deficiency syndrome (AIDS). Many of these patients have a subacute encephalitis consistent with a viral infection of the CNS. We studied the brains of 12 AIDS patients using in situ hybridization to identify human immunodeficiency virus [HIV, referred to by others as human T-cell lymphotropic virus type III (HTLV-III), lymphadenopathy-associated virus (LAV), AIDS-associated retrovirus (ARV)] nucleic acid sequences and immunocytochemistry to identify viral and cellular proteins. Nine patients had significant HIV infection in the CNS. In all examined brains, the white matter was more severely involved than the grey matter. In most cases the infection was restricted to capillary endothelial cells, mononuclear inflammatory cells, and giant cells. In a single case with severe CNS involvement, a low-level infection was seen in some astrocytes and neurons. These results suggest that CNS dysfunction is due to indirect effects rather than neuronal or glial infection.

In this investigation, we studied the ability of human cytomegalovirus to infect peripheral blood mononuclear cells. With monoclonal antibody technology, we demonstrated that cytomegalovirus could infect human lymphocytes of T-and B-cell (PBM). Infectious CMV has occasionally been found in buffy-coat preparations (4-6) obtained from patients with clinical CMV infection; only rarely has the virus been isolated from healthy donors (7). Neither the exact cell involved in the leukocyte fraction nor the state of the virus in such cells has been established. When transformed lymphoblastoid and erythroleukemia cell lines were studied for the ability to support CMV replication, some researchers found evidence of short-term virus replication (8, 9), but rarely was infectious virus produced (10, 11). There have been occasional reports of expression of CMV antigens on B-lymphoblastoid cell lines (12). Attempts to infect normal PBM from healthy donors with laboratory-adapted strains of CMV have failed (11, 13).In the cascade regulation of the CMV genome (14), immediate-early protein synthesis precedes early polypeptide synthesis, both of which set the regulatory state for late polypeptide synthesis and production of mature virions. The early class polypeptides appear to have predominantly regulatory functions; the late polypeptides have mainly structural functions. We reasoned that if lymphocytes were abortively infected, techniques such as cocultivation assays and probes for late CMV gene products would be negative. Hence, we looked for CMV expression in peripheral blood lymphocytes (PBL) with monoclonal antibodies specifically produced to detect polypeptides relevant to major epochs in the cascade regulation of the viral genome. Prompted by observations of biological differences between strains of virus recently derived from infected patients and the fibroblastadapted strains of virus (15), we also studied both forms of this virus. MATERIALS AND METHODSLymphocyte Infection. PBM were removed from the blood of healthy human donors by density gradient centrifugation on Ficoll-Paque and infected at a multiplicity of 0.01-1.0 with CMV recently isolated from patients with various CMV syndromes (Table 1), or with stocks of plaque-purified laboratory-strain AD-169. The recent isolates were propagated in human foreskin fibroblasts (Flow Laboratories) for <12 passages. Because low-passage isolates of human virus are associated predominantly with the cell matrix (15), infected or mock-infected fibroblasts were sonicated, and this material was cultured with PBM for up to 6 days. Thereafter, the PBM were washed 3 times and prepared for immunofluorescence studies by air-drying and fixing the cells with acetone on glass slides. In some experiments, T-lymphocytes were positively selected from the PBM cultures by erythrocyterosetting techniques (17) or by sorting on a fluorescence-activated cell sorter (16).Immunofluorescence Techniques. Monoclonal antibodies were raised against CMV-infected fibroblasts with standard techniques...

In situ hybridization was used to detect human cytomegalovirus (HCMV) in the peripheral blood mononuclear cells of some naturally infected (seropositive) individuals. A subpopulation of cells hybridized specifically to a portion of the HCMV genome that is heavily transcribed during the immediate-early period of infection. The hybridization signal was markedly reduced by base hydrolysis and ribonuclease, and therefore the probe appears to be detecting viral RNA. A fluorescence-activated cell sorter was used to select lymphocytes bearing the OKT4 and OKT8 markers. Hybridization with the HCMV probe revealed a higher proportion of positive cells in the OKT4 than in the OKT8 subset. This observation specifically identifies lymphocytes as a cell population involved in natural HCMV infection and suggests that lymphocytes may be a reservoir for maintaining infection and may also serve as a vehicle for its spread by blood transfusion.

Monocytes are one of the predominant cell types in the peripheral blood that are infected by human cytomegalovirus (HCMV). Although virus can be detected in these cells in vivo, HCMV replication in cultured monocytes has been unsuccessful. In this study, we demonstrate efficient HCMV replication in cultured monocytes. HCMV permissiveness in these cells was dependent on nonadherent cell-induced stimulation of the monocyte, with subsequent morphological differentiation into macrophages. Approximately 40% of the cells infected by virus were detected by immunofluorescent staining with both immediate-early and late antibodies. In addition, viral plaque assays demonstrated signfficant productive infection of macrophages. These observations are consistent with the suggestion that the monocyte/macrophage serves as a source of viral amplification and dissemination.