Varicella-Zoster virus (VZV) is a herpesvirus that becomes latent in sensory neurons after primary infection (chickenpox) and subsequently may reactivate to cause zoster. The mechanism by which this virus maintains latency, and the factors involved, are poorly understood. Here we demonstrate, by immunohistochemical analysis of ganglia obtained at autopsy from seropositive patients without clinical symptoms of VZV infection that viral regulatory proteins are present in latently infected neurons. These proteins, which localize to the nucleus of cells during lytic infection, predominantly are detected in the cytoplasm of latently infected neurons. The restriction of regulatory proteins from the nucleus of latently infected neurons might interrupt the cascade of virus gene expression that leads to a productive infection. Our findings raise the possibility that VZV has developed a novel mechanism for maintenance of latency that contrasts with the transcriptional repression that is associated with latency of herpes simplex virus, the prototypic alpha herpesvirus.Latency has been defined as the reversible nonproductive infection of a cell by a replication-competent virus (1). Several viruses have developed strategies to establish latency in the infected host to prevent their elimination by the host immune response. Varicella-Zoster virus (VZV) is an alpha herpesvirus that becomes latent in dorsal root ganglia (DRG) after primary infection and subsequently may reactivate to cause zoster. It is essential to understand the molecular mechanisms governing VZV latency and reactivation, as approximately 15% of the human population will develop zoster (2, 3) and possibly experience postherpetic neuralgia, a debilitating pain syndrome associated with zoster (4).A controversy regarding the localization of latent VZV (5-8) was resolved by the demonstration that VZV DNA is present both in neurons and satellite cells (9). The percentage of cells within an affected ganglion that are latently infected with VZV has been reported to range from 0.01% to 30% (5, 7, 9-11).Despite a wealth of data indicating that the virus immediate early (IE) proteins IE62, IE63, IE4, and the putative IE gene product ORF61p, are involved in the regulation of VZV gene expression during productive infection (12-17), little is known about the behavior of the virus during latency and the conditions that cause its reactivation. Others have shown that transcription of some VZV genes occurs in human ganglia harboring latent virus as evidenced by the presence of virus specific transcripts for ORFs 21,29, 62, and 63 (7,8,[18][19][20]. Although there is some uncertainty whether VZV latencyassociated transcription takes place in nonneuronal satellite cells or in neurons, it is clear that both IE and putative early (E) VZV genes are transcribed during latency (5-8). One of the IE gene transcripts, that for ORF63, is translated during latency, and the IE63 protein has been detected in the cytoplasm of latently infected human neurons (21) and in the cytoplasm a...

Detection of novel DNA sequences in Kaposi's sarcoma (KS) and AIDS-related body cavity-based, non-Hodgkin's lymphomas suggests that these neoplasms are caused by a previously unidentified human herpesvirus. We have characterized this agent using a continuously infected B-lymphocyte cell line derived from an AIDS-related lymphoma and a genomic library made from a KS lesion. In this cell line, the agent has a large episomal genome with an electrophoretic mobility similar to that of 270-kb linear DNA markers during clamped homogeneous electric field gel electrophoresis. A 20.7-kb region of the genome has been completely sequenced, and within this region, 17 partial and complete open reading frames are present; all except one have sequence and positional homology to known gammaherpesvirus genes, including the major capsid protein and thymidine kinase genes. Phylogenetic analyses using both single genes and combined gene sets demonstrated that the agent is a gamma-2 herpesvirus (genus Rhadinovirus) and is the first member of this genus known to infect humans. Evidence for transient viral transmission from infected to uninfected cells is presented, but replication-competent virions have not been identified in infected cell lines. Sera from patients with KS have specific antibodies directed against antigens of infected cell lines, and these antibodies are generally absent in sera from patients with AIDS without KS. These studies define the agent as a new human herpesvirus provisionally assigned the descriptive name KS-associated herpesvirus; its formal designation is likely to be human herpesvirus 8.

Varicella zoster virus (VZV) has been demonstrated to infect guinea pig enteric neurons in vitro. Latent infection of isolated enteric neurons is established when the cultures predominantly consist of neurons and they are exposed to cell-free VZV. Neurons harboring latent infection survive for weeks in vitro and express mRNA encoding ORFs 4, 21, 29, 40, 62, and 63, but not 14(gC) or 68 (gE) (although DNA encoding the glycoproteins is present). The expressed proteins are the same as those that are also expressed in human sensory neurons harboring latent VZV. In addition to mRNA, the immunoreactivities of ORFs 4, 21, 29, 62, and 63 can be detected. ORF 62 and 29 proteins are cytoplasmic and not intranuclear. VZV does not preferentially infect and/or become latent in intrinsic enteric primary afferent neurons indicating that the virus is latent in these neurons. Lytic infection occurs when mixed cultures of neurons and non-neuronal cells of the bowel wall are exposed to cell-free VZV or when isolated enteric neurons are exposed to cell-associated VZV. When lytic infection occurs, enteric neurons die within 48 hr. Prior to their death, neurons express VZV glycoproteins, including gE and gB, and ORF 62 and 29 proteins are intranuclear. This new animal model should facilitate studies of VZV latency and the efficacy of therapies designed to prevent VZV infection, latency, and reactivation.

Ganglia obtained at autopsy were examined by in situ hybridization from one patient with zoster (also called herpes zoster or shingles), two varicella-zoster virus (VZV)-seropositive patients without clinical evidence of zoster, one VZV-seronegative child, and one fetus. Ganglia positive for VZV had a hybridization signal in both neuronal and nonneuronal satellite cells. Ganglia obtained from the fetus and from the seronegative infant were consistently negative for VZV. Two striking observations were evident regarding the presence of VZV DNA in ganglia obtained from the individual with zoster at the time of death. First, ganglia innervating the sites of reactivation and ganglia innervating adjacent sites yielded strongly positive signals in neurons and satellite cells, whereas ganglia from distant sites were rarely positive. Second, VZV DNA was found in both the nuclei and the cytoplasm of neurons innervating areas of zoster. However, in neurons innervating zoster-free areas, VZV DNA was found only in the nucleus of neurons and their supporting satellite cells. Im Understanding the cellular and viral factors governing VZV latency and reactivation is essential to achieve this goal. Determining the location of persistent and reactivated virus is essential for defining the molecular basis of VZV pathogenesis.Electron microscopy has demonstrated VZV in sensory ganglia of patients with zoster (5, 6), and polymerase chain reaction (PCR) and Southern blot hybridization have detected VZV DNA in latently infected dorsal root ganglia (7,8). Studies utilizing in situ hybridization methods have identified several different VZV transcripts in affected ganglia (7,(9)(10)(11)(12)(13).These studies have led to controversy as to the nature of the cell harboring the latent VZV genome. VZV transcripts have been localized exclusively in neurons (10, 13, 14) or in nonneuronal satellite cells (9, 12) depending on the in situ hybridization technique used.In the current study, the cellular and intracellular location of the VZV genome in latently infected human dorsal root ganglia and in ganglia with clinical evidence of reactivated virus was determined by in situ hybridization. Evidence for virus replication in affected ganglia was provided by polymerase chain reaction (PCR) and immunohistochemistry. These results demonstrate that latent VZV resides in both neurons and their supporting satellite cells and that latent virus can reactivate in neurons. MATERIALS AND METHODSTissue Specimens. Dorsal root ganglia from two VZVseropositive patients without clinical evidence of zoster, one VZV-seronegative child and one fetus without a maternal history of varicella, were obtained at autopsy. Ganglia from an 85-year-old man with malignant glioma of the thoracic spinal cord who had a zosteriform rash in the distribution of the right Ti1, T12, and Li sensory nerves at the time of death were also obtained. Prior to developing zoster, the patient had radiation therapy to the spinal cord at the levels of T4 through Li. At autopsy, he had a ve...