Animal and human lentiviruses elude host defences by establishing covert infections and eventually cause disease through cumulative losses of cells that die with activation of viral gene expression. We used polymerase chain reaction in situ double-label methods to determine how many CD4+ lymphocytes are latently infected by human immunodeficiency virus (HIV) in patient lymph nodes and whether the pool of infected cells is large enough to account for immune depletion through continual activation of viral gene expression and attrition of cells responding to antigens. We discovered an extraordinarily large number of latently infected CD4+ lymphocytes and macrophages throughout the lymphoid system from early to late stages of infection, and confirmed the extracellular association of HIV with follicular dendritic cells. Follicular dendritic cells may transmit infection to cells as they migrate through lymphoid follicles. Latently infected lymphocytes and macrophages constitute an intracellular reservoir large enough ultimately to contribute to much of the immune depletion in AIDS, and represent a difficult problem that must be resolved in developing effective treatments and protective vaccine.

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Latent and productive viral infections are at the extremes of the spectrum of virus-cell interactions that are thought to play a major role in the ability of such important human pathogens as human immunodeficiency virus (HIV) to elude host defenses and cause disease. The recent development of PCR-based methods to amplify target sequences in individual cells in routinely fixed tissues affords opportunities to directly examine the subtle and covert virus-ceDl relationships at the latent end ofthe spectrum that are inaccessible to analysis by conventional in situ hybridization techniques. We have now used PCR in situ with in situ hybridization to document latent and permissive HIV infection in routinely fixed and paraffinembedded tissue. In one of the first specimens we examined, a tumor biopsy from an HIV-infected individual, we found many of the lymphocytes and lymphocytes infiltrating the tumor had HIV DNA that was detectable only by PCR in situ. The fraction of positive cells varied regionally, but there were foci where most of the cells contained HIV DNA. Most of these lymphocytes and macrophages are latently infected, as we could detect HIV RNA in fewer than one in a thousand ofthese cells. We also detected HIV RNA, surprisingly, in 6% of the tumor cells, where the number of copies of viral RNA per cell was equivalent to productively infected cell lines. The alternative states of HmV-gene expression and high local concentration of latently infected lymphocytes and monocytes revealed by these studies conceptually supports models of lentiviral pathogenesis that attribute persistence to the reservoir of latently infected cells and disease to the consequences of viral-gene expression in this population. The magnitude of infection of lymphocytes documented in this report is also consistent with the emerging view that HIV infection per se could contribute substantially to depletion of immune cells in AIDS.Human immunodeficiency virus (HIV), the etiological agent of AIDS, can establish productive or latent infections in CD4+ lymphocytes and monocytes in culture, and these alternative states of viral-gene expression could readily account for the devastating consequences of infection -and the formidable difficulties in developing a protective vaccine (1-3). Latently infected cells could escape detection and destruction by host defenses and disseminate infection in and between individuals in the face of natural or vaccine-induced immunity. With activation of viral-gene expression, the immune system will eventually be destroyed, and the individual will succumb to a variety of opportunistic infections and unusual tumors. One critical prediction of this reconstruction of pathogenesis is that in vivo one can show that there is a population of infected cells that harbor the HIV provirus in a transcriptionally silent state with no detectable viral RNA and a second population in which viral transcripts are abundant. We have now used PCR-based technologies with singlecell resolution, originally developed for studies o...