Detection of Varicella-Zoster Virus DNA in the Oropharynx and Blood of Patients with Varicella

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Varicella-zoster virus (VZV) is an alphaherpesvirus with the characteristic neurotropism of this group, but VZV also infects T cells productively and downregulates major histocompatibility complex (MHC) class I expression on infected T cells, as shown in the SCID-hu mouse model. T-cell tropism is likely to be critical for the cell-associated viremia associated with primary VZV infection. In these experiments, we found that VZV infects human tonsillar CD4؉ T cells in culture, with 15 to 25% being positive for VZV proteins as detected by polyclonal anti-VZV immunoglobulin G (IgG) staining and monitored by flow cytometry analysis. RNA transcripts for VZV gE, a late gene product, were detected in T-cell populations that expressed VZV surface proteins, but not in the VZV-negative cell fraction. Exposure to phorbol myristate acetate resulted in an increase in VZV-positive T cells, indicating that viral DNA was present within these cells and that VZV gene expression could be induced by T-cell activation. By immune scanning electron microscopy, VZV virions were detected in abundance on the surfaces of infected tonsillar T cells. The predominant CD4؉ T-lymphocyte subpopulations that became infected were activated CD69 ؉ T cells with the CD45RA ؊ memory phenotype. Subsets of CD4؉ T cells that expressed skin homing markers, cutaneous leukocyte antigen, and chemokine receptor 4 were also infected with VZV. By chemotaxis assay, VZV-infected T cells migrated to SDF-1, demonstrating that skin migratory function was intact despite VZV infection. The susceptibility of tonsil T cells to VZV suggests that these cells may be important targets during the initial VZV infection of upper respiratory tract sites. Viral transfer to migrating T cells in the tonsils may facilitate cell-associated viremia, and preferential infection of CD4 T cells that express skin homing markers may enhance VZV transport to cutaneous sites of replication.Varicella-zoster virus (VZV) is a human alphaherpesvirus that has a linear double-stranded DNA genome consisting of approximately 125,000 bp and encoding at least 69 unique proteins (34). VZV is a highly contagious pathogen. Primary infection leads to varicella (chickenpox) in susceptible individuals and establishment of latency in sensory ganglia; VZV may reactivate as herpes zoster (shingles) (3). Healthy individuals acquire VZV through contact with skin lesions of an infected person, followed by inoculation of respiratory mucosa, or by direct inoculation of virus in respiratory secretions. By analogy with mousepox, VZV has been presumed to be transferred to regional lymph nodes, where it is thought to initiate a primary viremic phase within a few days after inoculation (12). The mechanism of VZV transport from respiratory mucosa to regional lymph nodes is not known. A secondary viremia has been demonstrated to occur at the end of a 10-to 21-day incubation period, accompanied by the appearance of skin lesions. VZV has been recovered from cultured mononuclear cells obtained from 5 days before until 2 days...

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confidence: 99%

Tropism of Varicella-Zoster Virus for Human Tonsillar CD4⁺T Lymphocytes That Express Activation, Memory, and Skin Homing Markers

Ku¹,

Padilla²,

Grose³

et al. 2002

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129

“…VZV was isolated from 1 to 83 per 10 7 PBMC from healthy children with varicella before the onset of their rash (4). Virus was isolated from T cells in 43% of the patients and from B cells in 33% of the patients.PCR has been used to detect VZV in PBMC and polymorphonuclear leukocytes during varicella (13,21). More recent studies have quantitated the PBMC infected with VZV during varicella by using immunofluorescence with monoclonal antibody to glycoprotein E (gE) and confocal microscopy.…”

mentioning

confidence: 99%

“…PCR has been used to detect VZV in PBMC and polymorphonuclear leukocytes during varicella (13,21). More recent studies have quantitated the PBMC infected with VZV during varicella by using immunofluorescence with monoclonal antibody to glycoprotein E (gE) and confocal microscopy.…”

mentioning

confidence: 99%

Infection of Human T Lymphocytes with Varicella-Zoster Virus: an Analysis with Viral Mutants and Clinical Isolates

Soong

Schultz

Patera

et al. 2000

Varicella-zoster virus (VZV) disseminates in the body in peripheral blood mononuclear cells during chickenpox. Up to 1 in 10,000 mononuclear cells are infected during the viremic phase of the disease. We developed an in vitro system to infect human mononuclear cells with VZV by using umbilical cord blood. In this system, 3 to 4% of T cells were infected with VZV. VZV mutants unable to express certain genes, such as open reading frame 47 (ORF47) or ORF66, were impaired for growth in T cells, while other mutants showed little difference from parental virus. VZV unable to express ORF47 was even more impaired for spread from umbilical cord blood cells to melanoma cells in vitro. Early-passage clinical isolates of VZV infected T cells at a similar rate to the Oka vaccine strain; however, the clinical isolates were more efficient in spreading from infected T cells to melanoma cells. This in vitro system for infecting human T cells with VZV should be useful for identifying cellular and viral proteins that are important for virus replication in T cells and for the spread of virus from T cells to other cells.Varicella-zoster virus (VZV) is the etiologic agent of chickenpox and zoster. Humans are infected when the virus contacts the mucosa of the upper respiratory tract or conjunctiva. The virus then disseminates in the bloodstream in peripheral blood mononuclear cells (PBMC). Virus is transferred from PBMC to epithelial cells, resulting in infection of the skin and the characteristic rash of varicella (2). Virus also disseminates in PBMC to other organs including the nervous system, which results in a lifelong latent infection.Using in situ hybridization, it has been estimated that 1 to 10 per 10 5 PBMC from healthy persons with acute varicella contain VZV DNA or RNA (14). The cells containing VZV nucleic acid were thought to be lymphocytes but could not be identified definitively. VZV has also been cultured from PBMC, including monocytes and lymphocytes, from 5 days before to 1 day after the onset of the rash of varicella (3,19). VZV was isolated from 1 to 83 per 10 7 PBMC from healthy children with varicella before the onset of their rash (4). Virus was isolated from T cells in 43% of the patients and from B cells in 33% of the patients.PCR has been used to detect VZV in PBMC and polymorphonuclear leukocytes during varicella (13,21). More recent studies have quantitated the PBMC infected with VZV during varicella by using immunofluorescence with monoclonal antibody to glycoprotein E (gE) and confocal microscopy. From 1 to 10 in 10 5 PBMC expressed VZV gE on their surface (15). About two-thirds of the cells were T cells and one-third were B cells and monocytes.Previous attempts to infect primary lymphocytes in vitro have had limited success. Infection of primary B cells did not result in detectable VZV replication (14). Only 0.01% of T cells were productively infected with VZV in vitro when assayed by in situ hybridization. We have developed an in vitro system to infect mononuclear cells obtained from human umbilical co...

“…Studies have detected VZV in specific sites at different stages of infection. VZV DNA is present in the oropharynx (27) and in peripheral blood mononuclear cells (PBMCs) of patients with chickenpox (3,16,20). Virus DNA, the glycoproteins gE and gB, and the immediate-early protein 63 (IE63p) are found in skin biopsy samples obtained from patients with chickenpox or zoster (1,(23)(24)(25).…”

mentioning

confidence: 99%

Varicella-Zoster Virus Proteins in Skin Lesions: Implications for a Novel Role of ORF29p in Chickenpox

Annunziato

Lungu

Panagiotidis

et al. 2000

Skin biopsy samples from varicella-zoster virus (VZV)-infected patients examined by immunohistochemistry demonstrated VZV replication in nonepithelial cell types. ORF29p, a nonstructural nuclear protein, was found in nerves of two of six patients with chickenpox. In tissue culture, ORF29p was secreted by VZV-infected fibroblasts. Extracellular ORF29p can be taken up through endocytosis by human neurons, implying a novel role for this protein in pathogenesis.Varicella-zoster virus (VZV) infects dorsal root ganglia (DRG), enters latency, and may later reactivate to cause zoster. Studies have detected VZV in specific sites at different stages of infection. VZV DNA is present in the oropharynx (27) and in peripheral blood mononuclear cells (PBMCs) of patients with chickenpox (3,16,20). Virus DNA, the glycoproteins gE and gB, and the immediate-early protein 63 (IE63p) are found in skin biopsy samples obtained from patients with chickenpox or zoster (1,(23)(24)(25). VZV is found in keratinocytes, antigen-presenting cells, and endothelial cells during acute zoster (23,25) and in keratinocytes and inflammatory cells during chickenpox (1). VZV is present in neurons and satellite cells of DRG years following primary infection (6-8, 12, 17, 22) and has been observed by electron microscopy in sensory nerves during zoster (10). Other details of VZV pathogenesis remain speculative, including how the virus spreads from respiratory epithelial cells to PBMCs, keratinocytes, and DRG. Because PBMCs, sensory nerves, and epithelial cells are in close proximity in the dermis and epidermis, the skin is likely the site where virus enters the nervous system.By analogy with herpes simplex virus (HSV), it is thought that VZV transcription is temporally regulated. Immediateearly (IE) genes are expressed first, followed by early (E) genes and lastly late (L) genes (5). Some VZV proteins encoded by IE and L genes are incorporated in the virion, including transactivators such as IE63p and structural proteins such as gC (14, 15). ORF29p (for open reading frame 29 protein), the major VZV DNA binding protein, is encoded by a putative E gene and is not detected in purified virions (13). During latency, VZV exhibits limited gene expression (6-9, 22), with the accumulation of specific IE and E gene-encoded proteins in neurons (18,19). During reactivation, all kinetic classes of VZV genes are expressed in neurons (18). Whether VZV is in the lytic or latent state is reflected by the localization of expressed VZV gene products. VZV IE and E proteins that are present in both the nucleus and cytoplasm during productive infection are detected only in the cytoplasm of neurons during latency (18).Early observations suggested that there were inclusion bodies in endothelial cells present in varicella lesions (29); however, there was no known association between histology and viral etiology at that time. To determine if, during primary infection, as in zoster, VZV infects endothelial cells and nerves in the dermis and to characterize the inflammatory cells ...