Expression of Epstein-Barr virus replicative proteins in AIDS-related non-Hodgkin's lymphoma cells.

Epstein-Barr virus (EBV) infection in lymphoproliferative lesions has been assumed to be strictly latent. In order to investigate the possible occurrence of EBV replication in AIDS-related lymphoma (ARL) cells, we studied 13 cases by immunohistology using monoclonal antibodies to the EBV-encoded switch-protein BZLF1, early antigens (EAs), late replicative proteins [virus capsid antigens (VCAs) and membrane antigens (MAs)], and to the latent proteins EB nuclear antigen 2 (EBNA 2) and latent membrane protein (LMP). EBV genomes were detected by in situ hybridization. EBV genomes and/or gene products were demonstrated in ten cases, including all immunoblast-rich lymphomas, two Burkitts lymphomas, and a T-cell anaplastic large-cell lymphoma. The BZLF1 protein, which disrupts latency in B cells, was identified in six (60 per cent), and EAs in four (40 per cent) of the EBV-positive ARL. Only one lymphoma (10 per cent) expressed VCAs and MAs. EBNA 2 and LMP were detected in three (30 per cent) and eight (80 per cent) of EBV-positive cases, respectively. EBV DNA was detected in lymphoma cells in 7 of 12 (58 per cent) cases. The most important finding of this study was frequent spontaneous activation of latent EBV in ARL. Production of complete virus, however, was either aborted, or tumour cells expressing late productive cycle proteins (VCA, MA) were rapidly cleared from tissues. It is suggested that host factors that normally inhibit replication of EBV are deficient in AIDS patients.     

IN SITU DETECTION OF THE EPSTEIN–BARR VIRUS-ENCODED NUCLEAR ANTIGEN 1 IN ORAL HAIRY LEUKOPLAKIA AND VIRUS-ASSOCIATED CARCINOMAS

A new monoclonal antibody has been used to examine immunohistochemically the expression of the Epstein–Barr virus (EBV)-encoded nuclear antigen (EBNA) 1 in virus-associated epithelial lesions. EBNA1 was detected in the tumour cell nuclei of 10/13 undifferentiated nasopharyngeal carcinomas and of 10/10 EBV-associated gastric carcinomas. EBNA1 was also detected in 13 of 16 oral hairy leukoplakia (HL) samples, where its expression was confined to nuclei in the upper epithelial cell layers whilst basal epithelial cells were negative. This observation is in agreement with previous studies demonstrating the absence of latent EBV infection in the basal cell compartment of HL and suggests an essential role for EBNA1, not only in latent EBV infection but also in virus replication.     

Detection of Epstein-Barr virus-infected mucosal lymphocytes in nasal polyps.

Primary nasal lymphomas of T or NK cell origin are known to be associated with Epstein-Barr virus (EBV). However, it is not known whether EBV is normally present in nasal mucosa as distinct to nasopharyngeal tissue. This study investigates the prevalence of EBV infection in 13 cases of nasal polyps. EBV DNA was detected in 2 of 13 (15%) by Southern blot hybridization and in 9 of 13 (69%) by polymerase chain reaction. In situ hybridization for EBV-encoded small nuclear RNAs (EBER) was positive in 11 of 13 (85%) cases; the virus was present in stromal lymphocytes only and not in the epithelial cells. Immunohistochemistry for EBV proteins in 7 cases revealed EBV nuclear antigen (EBNA)-2, latent membrane protein (LMP)-1, and ZEBRA (the switch protein encoded by gene BZLF1) expression in rare isolated stromal lymphocytes in 3 cases. Double immunostaining in 1 case showed that the LMP-1+ cells were B or T cells. Immunohistochemistry for EBV lytic proteins showed very rare viral capsid antigen (VCA)+ and membrane antigen (MA)+ cells in 1 case and very rare diffuse early antigen (EA-D)+ and VCA+ cells in 1 other case. The expression of ZEBRA, EA-D, VCA, and MA suggested a disruption of latency in very rare stromal lymphocytes leading to a productive cycle. Although the incidence of EBV positivity in nasal polyps in our population is high (85%), very low numbers of EBV+ cells are found in each case. Nevertheless, they indicate that nasal mucosa could be one of the sites of EBV persistence through a low level of infection of the resident lymphocytes and thereby provide a possible setting for the emergence of virally associated tumors in this site.     

A survey of Epstein-Barr virus gene expression in sporadic non-Hodgkin''s lymphomas. Detection of Epstein-Barr virus in a subset of peripheral T-cell lymphomas.

This study analyzes the association of Epstein-Barr virus (EBV) with non-Hodgkin's lymphoma (NHL) arising in patients without pre-existing overt immunodeficiency. The authors examined 201 lymphomas (105 high-grade B-cell, 82 peripheral T-cell, 7 high-grade non-B-cell, non-T-cell, and 7 hairy-cell leukemia) for EBV gene expression by immunohistologic procedures using monoclonal antibodies to EBV latent, immediate early, and replicative infection antigens. Transformation-associated EBV latent membrane protein 1 (LMP 1) was detected in 13 (6%) NHL, comprising 4 (4%) high-grade B-cell, 8 (10%) peripheral T-cell, and 1 non-B-cell, non-T-cell lymphomas. Anaplastic large-cell lymphoma of T-cell type was consistently LMP 1-negative. EBV nuclear antigen 2 was demonstrated in only three (1%) cases. Induction of replication as defined by expression of the immediate early BamHI Z leftward reading frame 1 (BZLF1) protein was detected in five cases, but early (EA) and late (VCA and MA) lytic cycle antigens were only found in two cases and in one case, respectively. The presence of EBV was confirmed by in situ DNA hybridization in 9 of 11 EBV antigen-positive lymphomas. This study shows the surprisingly frequent presence of EBV in peripheral T-cell NHL in European patients without pre-existing overt immunodeficiency. Interestingly, most sporadic B-cell NHL are not associated with the virus. Furthermore, the usefulness of selected monoclonal antibodies for the routine immunohistological diagnosis of EBV infection was confirmed.     

Evidence for lytic infection by Epstein-Barr virus in mucosal lymphocytes instead of nasopharyngeal epithelial cells in normal individuals.

Normal nasopharyngeal tissues from 23 individuals who died of causes unrelated to the upper respiratory system and had no evidence of Epstein-Barr virus (EBV)-related diseases were studied using in situ hybridisation (ISH) and immunohistochemistry for the detection of EBV RNA and expression of EBV proteins, respectively. ISH using ³⁵S-labelled riboprobe for EBV EBER RNA showed occasional to a few EBER + lymphocytes in the stroma of nasopharyngeal mucosa in 14/16 cases with available paraffin-embedded tissues. In addition, very rare intraep-ithelial EBER+ lymphocytes were also detected in 3/16 cases. However, in none of these cases was EBER detected in the epithelial cells. Similar results were obtained using a nonradioactive ISH method for EBER (Dako). In 3/23 cases, im-munostaining using monoclonal antibodies for EBV proteins on cryostat sections showed occasional cells in the stroma expressing EBV nuclear antigen 2 (EBNA2), latent membrane protein-1 (LMP), and switch protein encoded by BZLF1 gene (ZEBRA) in two cases and only very rare LMP+ and ZEBRA+ cells in one other case. Double immuno-staining combining alkaline phosphatase anti-alkaline phosphatase (APAAP) for CD markers and indirect immunofluorescence for LMP showed that the LMP+ cells were either CD19+ or less frequently CD3+, but none were CD68+. These results show that both B and T lymphocytes harbouring EBV can be found in the normal nasopharyngeal tissues. Interestingly, EBV proteins associated with lytic viral replication—diffuse early antigen (EA-D), viral capsid antigen (VCA), or membrane antigen (MA)—were also detected in rare cells in the stroma in one case, and in another case only one MA+ cell was detected. These results provide evidence for a lytic type of infection by EBV in the normal nasopharynx involving a very small proportion of stromal lymphocytes and support the view that nasopharyngeal lymphocytes can act as a reservoir for EBV in normal individuals. © 1995 Wiley-Liss, Inc.     

EPSTEIN–BARR VIRUS (EBV) INFECTION IN INFECTIOUS MONONUCLEOSIS: VIRUS LATENCY,REPLICATION AND PHENOTYPE OF EBV-INFECTED CELLS

Primary Epstein–Barr virus (EBV) infection may manifest itself as a benign lymphoproliferative disorder, infectious mononucleosis (IM). EBV infection has been characterized in lymphoreticular tissues from nine patients with IM using the abundantly expressed EBV-encoded nuclear RNAs (EBERs) as a marker of latent infection. Expression of the virus-encoded nuclear antigen (EBNA) 2 and of the latent membrane protein (LMP) 1 was seen in variable proportions of cells in all cases. Double labelling revealed heterogeneous expression patterns of these proteins. Thus, in addition to cells revealing phenotypes consistent with latencies I (EBNA2⁻/LMP1⁻) and III (EBNA2⁺/LMP1⁺), cells displaying a latency II pattern (EBNA2⁻/LMP1⁺) were observed. Cells expressing EBNA2 but not LMP1 were also detected; whilst this may represent a transitory phenomenon, the exact significance of this observation is at present uncertain. EBER-specific in situ hybridization in conjunction with immunohistochemistry revealed expression of the EBERs mainly in B-lymphocytes, many of which showed features of plasma cell differentiation. By contrast, convincing evidence of latent EBV infection was not found in T-cells, epithelial or endothelial cells. Double-labelling immunohistochemistry revealed expression of the replication-associated BZLF1 protein in small lymphoid cells, often showing plasmacytoid differentiation. There was no unambiguous expression of this protein in other cell types. These results suggest that B-cells are the primary target of EBV infection and that plasma cells may be a source of infectious virus found in the saliva of IM patients. © 1997 John Wiley & Sons, Ltd.     

Epstein–Barr virus can infect rabbits by the intranasal or peroral route: An animal model for natural primary EBV infection in humans

Epstein–Barr virus (EBV) is spread universally in humans, and it causes infectious mononucleosis and sometimes induces serious EBV‐associated disease. The detailed mechanism of primary infection in humans has remained unclear, because it is difficult to examine the dynamics of EBV in vivo. In this study, a natural EBV‐infection rabbit model by intranasal or peroral inoculation is described. Ten male rabbits were examined for EBV‐DNA or mRNA expression and anti‐EBV antibodies in blood. Four of 10 rabbits showed the evidence of EBV infection; detection of EBV‐DNA or EBV‐related genes mRNA in peripheral blood mononuclear cells, increased EBV antibodies in the plasma, and the presence of lymphocytes expressing EBER1 and EBV‐related gene proteins in the lymphoid tissues of a rabbit. Three of four infected rabbits were detected transiently EBV‐DNA and/or mRNA of EBV‐related genes such as EBNA1, EBNA2, BZLF1, and EA in blood, while in one of four, EBV‐DNA and/or mRNA were detected for more than 200 days after viral inoculation. The level of EA‐IgG increased and its level was maintained in all infected rabbits, whereas those of VCA‐IgM and VCA‐IgG increased transiently, and EBNA‐IgG was not elevated. Pathological examination of a rabbit infected transiently revealed some scattered lymphocytes expressing EBER1, LMP1, and EBNA2 in the spleen and lymph nodes. EA expression was also observed in the spleen. These findings suggest that EBV can infect the rabbit by the intranasal or peroral route, and that this rabbit model is useful for examining the pathophysiology of natural primary EBV infection in humans. J. Med. Virol. 82:977–986, 2010. © 2010 Wiley‐Liss, Inc.     

Epstein-Barr virus infection and its gene expression in gastric lymphoma of mucosa-associated lymphoid tissue

The role of Epstein-Barr virus (EBV) in the pathogenesis of gastric lymphoma of mucosa-associated lymphoid tissue (MALT) has not been well understood. The aim of the study was to investigate EBV infection and its gene expression in this tumor in order to understand its role in the pathogenesis. EBV infection was screened by in situ hybridization for EBV-encoded nonpolyadenylated RNA (EBER ISH) in 79 cases of gastric MALT lymphoma of nonimmunocompromised patients. The expression of EBV proteins [LMP1 (latent membrane protein 1), EBNA2 (EBV nuclear antigen 2), ZEBRA (switch protein encoded by BZLF1 gene)] was studied by immunohistochemistry in EBER-positive cases. EBV was detected with EBER ISH in 15 (19%) of the 79 cases. EBV was found in virtually all tumor cells in 2 cases of high-grade MALT lymphoma (2.5%) (EBV-associated), and was found only in occasional large or small lymphoid cells in 13 cases (16.5%). False positive EBER signal was detected in the mucinous glandular epithelial cells of gastric antrum with FITC-labeled oligonucleotide probe but not with digoxigenin or ³⁵S-labeled riboprobes. Type II latency (EBER+LMP1+ EBNA2-) was detected in both EBV-associated cases. Type III latency (EBER+LMP1+EBNA2+) was also identified in one EBV-associated case besides latency II. Double labeling showed coexpression of LMP1 and EBNA2 in a small number of tumor cells, indicating the presence of type III latency in single cell level. In cases with only occasional EBER-positive large or small lymphoid cells, LMP1 and EBNA2 were not detected. ZEBRA was negative in all the cases. These findings suggest that EBV may contribute to the pathogenesis of a small proportion of high-grade MALT lymphoma, where virtually all tumor cells harbored EBV and the oncogenic viral protein LMP1 was expressed. Moreover, latency III of EBV infection may exist in nonimmunocompromised patient. J. Med. Virol. 56:342–350, 1998 . © 1998 Wiley-Liss, Inc.     

Detection of Epstein–Barr virus‐specific memory CD4+ T cells using a peptide‐based cultured enzyme‐linked immunospot assay

Approaches to evaluate T‐cell responses to Epstein–Barr virus (EBV) include enzyme‐linked immunospot (ELISPOT), which quantifies cells capable of immediate interferon‐γ secretion upon antigen stimulation. However, evaluation of expandable EBV‐specific memory T cells in an ELISPOT format has not been described previously. We quantified EBV‐specific T‐cell precursors with high proliferative capacity by using a peptide‐based cultured interferon‐γ ELISPOT assay. Standard and cultured ELISPOT responses to overlapping peptide pools (15‐mers overlapping by 11 amino acids) covering the lytic (BZLF1 and BMRF1) and latent (EBNA1, EBNA3a, EBNA3b, EBNA3c, LMP1 and LMP2) EBV proteins were evaluated in 20 healthy subjects with remote EBV infection and, for comparison, in four solid organ transplant recipients. Cultured ELISPOT responses to both lytic and latent EBV antigens were significantly higher than standard ELISPOT responses. The distribution of EBV‐specific T‐cell responses detected in healthy virus carriers showed more consistent cultured ELISPOT responses compared with standard ELISPOT responses. T‐cell responses quantified by cultured ELISPOT were mainly mediated by CD4⁺ T cells and a marked pattern of immunodominance to latent‐phase antigens (EBNA1 > EBNA3 family antigens > LMP2 > LMP1) was shown. Both the magnitude and distribution of EBV‐specific T‐cell responses were altered in solid organ transplant recipients; in particular, cultured ELISPOT responses were almost undetectable in a lung‐transplanted patient with EBV‐associated diseases. Analysis of T‐cell responses to EBV by ELISPOT assays might provide new insights into the pathogenesis of EBV‐related diseases and serve as new tools in the monitoring of EBV infection in immunocompromised patients.     

Distribution and phenotype of Epstein-Barr virus-infected cells in human pharyngeal tonsils.

Although Epstein-Barr virus (EBV) is often found in human tonsils, it remains to be precisely determined in what cells and microenvironment the virus is present. Although generally regarded as a B lymphotropic virus, EBV is associated with non-B-cell tumors, for example, NK/T-cell lymphoma, carcinoma, and leiomyosarcoma. To provide a basis for understanding the origin and biology of EBV-infected non-B cells, the immunophenotype of all EBV-infected cells in reactive human tonsils was determined by subjecting tonsil sections to dual/triple EBER in situ hybridization and immunohistochemistry with monoclonal antibodies to T cells (CD3, CD4, CD8, CCR3), B cells (CD20), plasma cells (CD138), natural killer (NK) cells (PEN5), and epithelial cells (cytokeratin), as well as frozen section immunostaining with antibodies to EBV latent proteins EBNA1, EBNA2, LMP1, and EBV early protein BZLF1. Most tonsils contained nearly equal numbers of EBNA1- and LMP1-positive cells (latency program) while only a few contained EBNA2-positive cells (growth program). More than 1000 EBER-positive cells from six tonsils were detected in the interfollicular zone (59%), tonsillar crypts (26%), and follicles (15%). Most (82%) EBER-positive cells are CD20-positive B cells, 7% are CD3-positive T cells, and 11% are cells of indeterminate lineage, often with plasmacytoid morphology. However, no EBER-positive plasma cells were identified. Rare EBER-positive NK cells and EBER/BZLF1-positive epithelial cells were identified. The direct demonstration of EBV within rare T cells, NK cells, and epithelial cells in reactive human tonsils provide a basis for further understanding of the origin of EBV-associated tumors of non-B-cell type.     

The seroepidemiology of infection due to Epstein-Barr virus in southern India

We investigated the seroepidemiology of infection due to Epstein-Barr virus (EBV) in 181 south Indian subjects aged 0-25 years using the indirect immunofluorescence method to titrate antibodies to viral capsid antigen (VCA), nuclear antigen (EBNA), and early antigen (EA). The age-specific prevalence of IgG antibodies to VCA rose rapidly to 90% by the age of 5 years. The prevalence of VCA-specific IgM and the geometric mean titre of VCA-specific IgG antibodies were highest between the ages of 6 months and 2 years, the median age of primary infection being 1.4 years. Thus primary EBV infection occurs early in life. EA antibody prevalence was highest (55%) in the third year of life and remained between 30% and 40% thereafter. This pattern of EA antibody prevalence suggests that the latent EBV infection that persists lifelong after primary infection may be reactivated in many individuals. EBNA antibody prevalence was low until the age of 2 years but rose to 80% in the fourth year. Geometric mean titres of antibodies to EA and EBNA were low and stable at all ages. These results are similar to data from areas where EBV-associated Burkitt's lymphoma is endemic and indicate a high EBV infection load early in life.     

Characterization of the Epstein-Barr virus glycoprotein BMRF-2

Epstein-Barr virus (EBV) BMRF-2 protein interaction with the beta1 family of integrins plays an important role in EBV infection of polarized oral epithelial cells. In this work, we characterized BMRF-2 protein expression in EBV-infected B lymphoblastoid and polarized oral epithelial cells, and in hairy leukoplakia (HL) epithelium. BMRF-2 expression in B cells and polarized oral epithelial cells was associated with the EBV lytic infection. In these cells, BMRF-2 is efficiently transported to the cell membrane and its integrin binding Arg-Gly-Asp (RGD) motif is exposed on the cell surface. BMRF-2 is highly expressed in HL epithelium and accumulates at the lateral border of oral keratinocytes. In EBV-infected polarized oral epithelial cells, this protein is transported to the basolateral membranes and co-localized with beta1 integrin. These data suggest that BMRF-2 may play an important role in cell-to-cell spread of EBV within the oral epithelium. BMRF-2 is glycosylated through O-linked oligosaccharides; it forms oligomers and is associated with the virion envelope. Its C-terminal tail is localized in the cytoplasm. We found that beta1, alpha5, and alpha3 integrins are present in purified EBV virions. We show that BMRF-2 is a ligand for beta1, alpha5, alpha3, and alphav integrins and our data are consistent with a role for BMRF-2 in viral lytic infection.     

Infection of the human T-cell-derived leukemia line Molt-4 by Epstein-Barr virus (EBV): induction of EBV-determined antigens and virus reproduction

We have reported previously that human T-cell-derived Molt-4 cells become susceptible to Epstein-Barr virus (EBV) infection after implantation of functional EBV receptors into the plasma membranes of Molt-4 cells (Volsky et al., 1980). In the present work, we expand this finding by analyzing the following: (i) Virus adsorption vs viral penetration—using [³H]thymidine-labeled EBV, we demonstrate that the virus could adsorb to both the untreated and the receptor-implanted Molt-4 cells. However, only the altered cells became susceptible to EBV penetration followed by the viral antigen synthesis; (ii) EBV substrain specificity of infection—EBV P3HR-1 virus induced the nuclear (EBNA), early (EA), and virus capsid (VCA) antigens, whereas EBV B-95-8 virus induced only EBNA; (iii) virus reproduction—in situ hybridization was used to demonstrate the EBV-DNA synthesis in the P3HR-1 virus-infected cells. In addition, immature herpes-like particles were observed in electron micrographs of infected cells. It is concluded that EBV infection of human T-cell-derived Molt-4 cells may lead to a full viral-lytic cycle in a portion of infected cells. The results suggest, however, that the primary EBV productive infection may not necessarily involve any immunofluorescence-detectable EBNA synthesis.     

Studies of an Epstein-Barr virus-induced DNA polymerase.

Epithelial/Burkitt hybrid cells (D98/HR-1) typically express the Epstein-Barr-associated nuclear antigen (EBNA). Synthesis of early antigen (EA) and virus capsid antigen (VCA) is repressed. Following treatment with 5-iodo-2′-deoxyuridine (IUdR), however, Epstein-Barr virus (EBV) EA and late virus-specific products, including EBV DNA and complete virions, are induced. This report demonstrates that phosphonoacetic acid inhibits expression of VCA, but has no effect on EA. Cells treated with IUdR were also found to express a new DNA polymerase which was susceptible to stimulation by salt and was not present in uninduced cells. This salt-stimulated DNA polymerase resembles other herpesvirus-induced DNA polymerases in its requirements for maximal activity and its ability to use synthetic templates in DNA synthesis. Data suggest that this DNA polymerase is induced by EBV and is necessary for the productive synthesis of virus-specific DNA.