Presence of Epstein-Barr virus DNA in tonsillar tissues

Sera from 48 tonsillar carcinoma (TC) patients, 48 matched controls and 16 recurrent exudative tonsillitis (RET) patients were examined for the presence of Epstein-Barr virus (EBV) associated nuclear antigen (EBNA), early antigen (EA) and virus capsid antigen (VCA). Higher prevalence and significantly higher antibody titres against all three EBV-associated antigens were observed in TC patients in comparison with controls and RET patients. Patients suffering from anaplastic TC had higher titres of antibodies against VCA and EA than TC patients with other histological diagnoses. Five out of 11 TC biopsies obtained from 9 patients were positive for EBV DNA at levels of 0.17, 4 to 5, 15 to 18 and in two cases 3 EBV genome equivalents per cellular genome. Among 16 RET patients, 4 were found positive at levels not exceeding 2.17 EBV genome equivalents per cellular genome. Higher titres of antibody against all EBV antigens were found in TC and RET patients with EBV DNA-positive tonsillar tissue than in those with EBV DNA-negative tonsillar tissue.     

Characteristics of viral protein expression by Epstein-Barr virus-infected B cells in peripheral blood of patients with infectious mononucleosis.

The frequency of Epstein-Barr virus (EBV) antigen-positive B cells in the peripheral blood of patients with infectious mononucleosis compared with that for latently EBV-infected individuals was examined by immunocytochemistry. B cells positive for Epstein-Barr nuclear antigen (EBNA) 1, EBNA2, and latent membrane protein were frequently found in all peripheral B lymphocyte preparations from 25 patients suffering for 3 to 28 days from infectious mononucleosis by using monoclonal antibodies and the alkaline phosphatase anti-alkaline technique. There was a significant decrease in the number of positive B cells during the course of disease. EBNA1-positive B cells were detected in 0.01 to 2.5% of total B cells (median, 0.8%), EBNA2-positive B cells were detected in 0.01 to 4.5% of total B cells (median, 0.9%), and latent membrane protein-positive B cells were detected in 0.01 to 1.8% of total B cells (median, 0.5%), depending on the duration of clinical signs. In contrast, we did not find any EBNA1- or EBNA2-positive B cells in 2 x 10(6) peripheral blood B lymphocytes of 10 latently EBV-infected individuals, whereas aliquots of the same cell preparations were EBV DNA positive by a PCR assay. Therefore, it appears to be possible to detect infectious mononucleosis by immunocytochemical determination of latent EBV products, which might be of relevance for the diagnosis of EBV reactivations in immunosuppressed patients.     

Evaluation of four commercial systems for the diagnosis of Epstein-Barr virus primary infections

To compare the performance of four diagnostic commercial systems for Epstein-Barr virus (EBV) serology (for IgM and IgG virus capsid antigen [VCA] and EBV nuclear antigen [EBNA] antibodies), a collection of 125 samples from clinically suspected infectious mononucleosis cases was studied. Indirect immunofluorescence (IIF) for VCA IgM and IgG antibodies and anticomplement immunofluorescence for EBNA antibodies (Meridian Bioscience Inc.) were used as reference methods. By these methods, the cases were classified EBV primary infection (presence of IgM to VCA or IgG to VCA in the absence of EBNA antibodies; n = 82), EBV past infection (presence of VCA IgG and EBNA antibodies in the absence of VCA IgM; n = 26), or no infection (negative for the three markers; n = 17). The following systems were tested: two chemiluminescent immunoassays (CLIAs; the Liason [CLIA-L; DiaSorin] and the Immulite 2000 [CLIA-I; Siemens]), immunofiltration (IF; All.Diag), and an enzyme-linked immunosorbent assay (ELISA; DiaSorin). In the IgM assays, sensitivities ranged from 67.1% (ELISA) to 92.2% (CLIA-L) and specificities ranged from 93.8% (CLIA-L) to 100% (IF). In the VCA IgG assays, sensitivities varied from 79.4% (IF) to 94.4% (CLIA-I) and specificities varied from 94.4% (IF and CLIA-L) to 100% (CLIA-I and ELISA). In EBNA assays, sensitivities ranged from 78.1% (IF) to 93.8% (CLIA-I) and specificities ranged from 32.3% (CLIA-L) to 91.4% (IF). In relation to EBV profiles, the corresponding figures for sensitivity (in detecting primary infection) for IF, CLIA-L, CLIA-I, and ELISA were 92.7%, 93.8%, 89%, and 89.6%, respectively, and those for specificity (to exclude primary recent infection) were 90.7%, 94.6%, 97.7%, and 95.2%, respectively. Although there were limitations in some individual markers, especially CLIA-L for EBNA IgG, the systems evaluated appear to be useful for diagnosis of EBV infection.     

A peptide-based inhibitor for prevention of B cell hyperproliferation induced by Epstein-Barr virus

Epstein-Barr virus (EBV) infects and transforms resting B lymphocytes in vitro. The virus can also cause B cell lymphomas in immunosuppressed humans. Indeed, EBV-mediated post-transplant lymphoproliferative disease causes significant complications in transplant recipients, including loss of the transplanted organ and even death. The limited treatment options include, nonspecific targeting of B cell surface antigens with monoclonal antibodies or withdrawal of immunosuppression. These therapies fail in approximately 50% of patients. Clearly, treatments that specifically target EBV-infected cells are desirable. The EBV antigen EBNA3C regulates cell cycle by targeting critical cellular complexes such as cyclin A/cdk2, SCF(Skp2), and Rb. Here, we use a 20-amino-acid EBNA3C-derived peptide, fused to an HIV TAT tag for efficient delivery, to disrupt cell cycle regulation by EBNA3C. The peptide inhibited hyperproliferation of EBV-infected B cell lines and reduced in vitro immortalization of primary B lymphocytes by EBV. Importantly, the peptide inhibited lymphoblastoid outgrowth from the blood of an EBV-positive transplant patient in vitro.     

No direct role for Epstein-Barr virus in American hepatocellular carcinoma

Epstein-Barr virus (EBV) was recently linked to hepatocellular carcinogenesis in Japanese patients. It is not clear whether EBV infection is also associated with hepatocellular carcinoma (HCC) occurring in American patients. We studied 41 cases of HCC from the Los Angeles area for evidence of EBV infection by in situ hybridization, immunohistochemistry, and polymerase chain reaction methods. Of 41 cases, 16 were seropositive for hepatitis B virus surface antigen (39%), 9 of 29 tested were seropositive for hepatitis C virus antibody (31%); in total, 22 cases were seropositive for hepatitis B virus and/or hepatitis C virus (53%). Of 41 cases, 1 was positive for EBV-encoded small nonpolyadenylated RNA (EBER)-1 (2%) by in situ hybridization. By immunohistochemistry, two cases were positive for EBV nuclear antigen (EBNA)-1 (5%), one was positive for the transactivating immediate early BZLF1 (ZEBRA) (2%), and none was positive for latent membrane protein-1. None of the 41 cases was positive for latent membrane protein-1 and EBV nuclear antigen (EBNA)-4 DNAs by polymerase chain reaction assay. All four positive cases showed rare EBER-1-, ZEBRA-, or EBNA-1- positive cells (<0.1%); in none of these cases was there expression of any other EBV viral genes. In the one case each that was positive for EBER-1 and ZEBRA, both of which occurred in patients of non-Asian ethnicity, the staining was limited to infiltrating small lymphocytes, and tumor cells were negative. In the two cases that were positive for EBNA-1, both of which occurred in patients of Asian ethnicity, the staining was limited to tumor cells, and infiltrating small lymphocytes were negative. Our study indicates that rare cases of American HCC may contain EBV-infected cells, but it is unlikely that EBV plays a major role in the carcinogenesis of HCC.     

Epstein-Barr virus and human immunodeficiency virus serological responses and viral burdens in HIV-infected patients treated with HAART

Epstein-Barr virus (EBV) associated non-Hodgkin lymphoma is recognized as a complication of human immunodeficiency virus (HIV) infection. Little is known regarding the influence of highly active antiretroviral therapy (HAART) on the biology of EBV in this population. To characterize the EBV- and HIV-specific serological responses together with EBV DNA levels in a cohort of HIV-infected adults treated with HAART, a study was conducted to compare EBV and HIV serologies and EBV DNA copy number (DNAemia) over a 12-month period after the commencement of HAART. All patients were seropositive for EBV at baseline. Approximately 50% of patients had detectable EBV DNA at baseline, and 27/30 had detectable EBV DNA at some point over the follow-up period of 1 year. Changes in EBV DNA copy number over time for any individual were unpredictable. Significant increases in the levels of Epstein-Barr nuclear antigen (EBNA) and Epstein-Barr early antigen (EA) antibodies were demonstrated in the 17 patients who had a good response to HAART. Of 29 patients with paired samples tested, four-fold or greater increases in titers were detected for EA in 12/29 (41%), for EBNA in 7/29 (24%), for VCA-IgG in 4/29 (14%); four-fold decreases in titers were detected in 2/29 (7%) for EA and 12/29 (41%) for EBNA. A significant decline in the titer of anti-HIV antibodies was also demonstrated. It was concluded that patients with advanced HIV infection who respond to HAART have an increase in their EBV specific antibodies and a decrease in their HIV-specific antibodies. For the cohort overall, there was a transient increase in EBV DNA levels that had declined by 12 months.     

Infection of human B lymphocytes with high multiplicities of Epstein-Barr virus: kinetics of EBNA expression, cellular DNA synthesis, and mitosis

We studied the time course of early events during the transformation of human B lymphocytes by Epstein-Barr virus (EBV). EBV nuclear antigen (EBNA) was first detectable 6–8 hr after infection in 1–10% of the cells. Even though cells were exposed to virus under conditions which would maximize the chances of synchronous infection, the proportion of EBNA (+) cells increased until 32–36 hr when a maximum level was reached. DNA synthesis in infected cultures increased above control levels between 20 and 36 hr after exposure to virus and thereafter increased progressively. The first cell divisions observed in EBNA (+) cells occurred between 36 and 48 hr after infection and the mitotic index of EBNA (+) cells increased with time reaching 25% of the EBNA (+) cells by 96 hr. However, the total cell number and the percentage of EBNA (+) cells remained unchanged after 36 hr, suggesting that the rate of cell death in EBNA (+) cells was equivalent to the rate of proliferation. Adenine arabinoside (Ara A) and cytosine arabinoside (Ara C), at concentrations which inhibit VCA expression in EBV producer cell lines, did not affect EBNA expression during the first 24 hr after infection. These data together with the very early appearance of EBNA after inoculation show that EBNA synthesis is independent of viral or cellular DNA synthesis and support the hypothesis that synthesis of EBNA is one of the crucial earliest events in lymphocyte transformation by EBV.     

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.     

Evaluation of 12 commercial tests for detection of Epstein-Barr virus-specific and heterophile antibodies

Ten microbiological departments in Norway have participated in a multicenter evaluation of the following commercial tests for detection of Epstein-Barr virus (EBV)-specific and heterophile antibodies: CAPTIA Select viral capsid antigen (VCA)-M/G/EBNA (Centocor Inc.), Enzygnost anti-EBV/immunoglobulin M (IgM) and IgG (Dade Behring), Vironostika EBV VCA IgM/IgG/EBNA enzyme-linked immunosorbent assay (ELISA) (Organon Teknika), SEROFLUOR immunofluorescence assay and EBV Combi-Test (Institute Virion Ltd.), anti-EBV recombinant IgM- and IgG-early antigen/EBNA IgG ELISA (Biotest Diagnostics), EBV IgM/IgG/EBNA ELISA (Gull Laboratories), Paul-Bunnell-Davidsohn test (Sanofi Diagnostics Pasteur), Monosticon Dri-Dot (Organon Teknika), Avitex-IM (Omega Diagnostics Ltd.), Alexon Serascan infectious mononucleosis test (Alexon Biomedical Inc. ), Clearview IM (Unipath Ltd.), and Cards+/-OS Mono (Pacific Biotech, Inc.). The test panel included sera from patients with primary EBV infection, immunocompromised patients with recent cytomegalovirus infection, healthy persons (blood donors), and EBV-seronegative persons. Among the tests for EBV-specific antibodies the sensitivity was good, with only small differences between the different assays. However, there was a greater variation in specificity, which varied between 100% (Enzygnost) and 86% (Biotest). Tests for detection of heterophile antibodies based on purified or selected antigen (Avitex, Alexon, Clearview IM, and Cards+/-OS Mono) were more sensitive than the Paul-Bunnell-Davidsohn and Monosticon tests.     

Diagnostic value of measuring Epstein-Barr virus (EBV) DNA load and carcinoma-specific viral mRNA in relation to anti-EBV immunoglobulin A (IgA) and IgG antibody levels in blood of nasopharyngeal carcinoma patients from Indonesia

Nasopharyngeal carcinoma (NPC) is a prevalent malignancy in Southeast Asia and is strongly associated with Epstein-Barr virus (EBV). We investigated the primary diagnostic value of circulating EBV DNA and anti-EBV immunoglobulin G (IgG) and IgA levels in Indonesian NPC patients (n = 149). By a 213-bp Epstein-Barr virus nuclear antigen 1 (EBNA1)-based real-time LightCycler PCR, 72.5% of patients were positive for EBV DNA in whole blood, with 29.5% having levels above a previously determined clinical cutoff value (COV) of 2,000 EBV DNA copies/ml, the upper level in healthy carriers. In a 99-bp LightCycler PCR, 85.9% of patients were positive and 60.4% had levels above the COV. This assay quantified a significantly higher EBV load than the 213-bp PCR assay (P < 0.0001), suggesting that circulating EBV DNA is fragmented. Using data from 11 different studies, we showed a significant inverse correlation between PCR amplicon size and the percentage of patients positive for circulating EBV DNA (Spearman's rho = -0.91; P < 0.0001). EBV DNA loads were unrelated to anti-EBV IgG or IgA levels, as measured by VCA-p18 and EBNA1-specific synthetic peptide-based enzyme-linked immunosorbent assays. The presence of circulating tumor cells was assessed by amplification of BamHI-A rightward frame 1 (BARF1) mRNA, a viral oncogene abundantly expressed in EBV-carrying carcinomas but virtually absent from EBV-associated lymphomas. Despite high EBV DNA loads and the presence of EBNA1 and human U1A small nuclear ribonucleoprotein mRNA, BARF1 mRNA was never detected in blood. We conclude that amplicon size significantly influences EBV DNA load measurement in NPC patients. The circulating EBV DNA load is independent of serological parameters and does not reflect intact tumor cells. The primary diagnostic value of the EBV DNA load for the detection of NPC is limited.     

IgA antibodies against the Epstein‐Barr nuclear antigen1 as a valuable biomarker for the diagnosis of nasopharyngeal carcinoma in Tunisian patients

Serological tests for Epstein‐Barr virus (EBV) have been used for many years as diagnostic predictors of nasopharyngeal carcinoma. It has been shown previously that the conventional immunofluorescence assay has a limited diagnostic value, especially in young patients from North African area. In the search for more reliable immunoglobulin (Ig) G or IgA antibody markers for the diagnosis of nasopharyngeal carcinoma, immunoblot analysis was performed using a full spectrum of EBV proteins. Sera were collected from 108 patients with nasopharyngeal carcinoma and three control groups composed of 18 patients with lymphoma, 18 other patients with autoimmune diseases and 55 healthy EBV carriers. It was observed that the IgA Epstein‐Barr nuclear antigen 1 (EBNA1), IgA early antigen (EA)‐p138 and IgG EA‐p138 antibodies represent the most specific anti‐EBV responses in either young or older patients with nasopharyngeal carcinoma which yield higher positive rates compared to the three control groups. Since the IgA EBNA1 response showed the highest sensitivity value for the detection of nasopharyngeal carcinoma, a novel enzyme‐linked immunosorbent assay (ELISA) was established using a GST‐EBNA1 protein expressed in bacteria, containing the P‐threonine EBNA1 subtype cloned from DNA EBV sequence of C15 xenograft cells. Detection rates were 85.7% and 94.9% in young and older patients with nasopharyngeal carcinoma respectively, while only 3.6%, 11.1%, and 16.6% in healthy EBV carriers, patients with lymphoma and patients with autoimmune diseases, respectively. Thus, IgA EBNA1 ELISA may be useful for early diagnosis and mass screening of nasopharyngeal carcinoma in Tunisia even in young patients. J. Med. Virol. 81:1412–1421, 2009. © 2009 Wiley‐Liss, Inc.     

Epstein-Barr virus nuclear antigen 1 (EBNA1) induced cytotoxicity in epithelial cells is associated with EBNA1 degradation and processing

Epstein-Barr virus nuclear antigen 1 (EBNA1) has a central role in the maintenance and segregation of the Epstein-Barr virus (EBV) episome and by virtue of a glycine-alanine repeat domain is prevented from being endogenously processed for recognition by HLA class I restricted cytotoxic T lymphocytes (CTLs). We found that EBNA1 expression resulted in growth inhibition and a G2/M arrest in human squamous epithelial cell lines (SCC12F, SVK) but not epithelial cell lines of glandular origin (Hela, Ad/AH). The cytotoxicity of EBNA1 was associated with EBNA1 degradation and both these effects were blocked in SCC12F cells expressing either the anti-apoptotic bcl-2 protein or the EBV homolog of bcl-2, BHRF1. The endogenous degradation of EBNA1 in SVK epithelial cells was associated with specific CTL recognition, an effect not evident in EBNA1-expressing Hela cells. Consistent with the inability of SVK cells to tolerate EBNA1 expression, studies with a recombinant EBV demonstrated that SVK cells are unable to maintain stable virus infection, whereas Hela cells are able to efficiently establish latent EBV infection. These data have important implications for both the cellular requirements necessary to sustain a stable EBV infection and for the possible role of CTL responses in controlling EBV infection of epithelial cells.     

Transformation of lymphocytes by Epstein-Barr virus requires only one-fourth of the viral genome

Inactivation studies were performed to measure the target sizes of three Epstein-Barr virus (EBV) -associated functions: (i) transformation of lymphocytes; (ii) stimulation of cell DNA synthesis in lymphocytes; and (iii) induction of the EBV nuclear antigen (EBNA) expression in lymphocytes. The target size of the plaque-forming function of herpes simplex virus type 1 (HSV-1) was studied in parallel and was used as a standard for comparison with the measured target sizes. When ionizing irradiation was used as an inactivating agent, the target size of EBV transforming function was found to be as large as that determined for the plaque-forming function of HSV-1. A similar size was measured for the EBV function required to stimulate cell DNA synthesis. In contrast, the target size of the viral function needed for induction of EBNA expression was found to be only 35% of that observed for HSV-1 plaque-forming function. Inactivation studies were also performed using the chemical mutagen, N-acetoxyacetylaminofluorene. The target size measured using this agent for the transforming function of EBV was about 25% of that determined for plaque-forming function of HSV-1. This result indicates that only a portion of the viral DNA encodes information necessary for the initiation and maintenance of transformation by EBV.     

Epstein-Barr virus and multiple sclerosis: interaction with HLA

Epstein-Barr virus (EBV) infection, history of infectious mononucleosis (IM) and HLA-A and DRB1 have all been proposed as risk factors for multiple sclerosis (MS). Our aim was to analyse possible interactions between antibodies against Epstein-Barr virus nuclear antigen 1 (EBNA1) or EBNA1 fragments, presence of DRB1*15 and absence of A*02. The study population includes newly diagnosed cases and matched controls. Interaction on the additive scale was calculated using attributable proportion due to interaction (AP), which is the proportion of the incidence among individuals exposed to two interacting factors that is attributable to the interaction per se. IM showed association with MS, odds ratio (OR)=1.89 (1.45-2.48% confidence interval (CI)), as did raised EBNA1 IgG OR=1.74 (1.38-2.18 95%CI). All EBNA1 fragment IgGs were associated with MS risk. However, EBNA1 fragment 385-420 IgG levels were more strongly associated to MS than total EBNA1 IgG, OR=3.60 (2.75-4.72 95%CI), and also interacted with both DRB1*15 and absence of A*02, AP 0.60 (0.45-0.76 95%CI) and AP 0.39 (0.18-0.61 95%CI), respectively. The observed interaction between HLA class I and II genotype and reactivity to EBV-related epitopes suggest that the mechanism through which HLA genes influence the risk of MS may, at least in part, involve the immune control of EBV infection.     

Specific IgA antibody to Epstein-Barr viral capsid antigen: a better marker for screening nasopharyngeal carcinoma than EBV-DNA detection by polymerase chain reaction

Nasopharyngeal carcinoma (NPC) is strongly associated with Epstein-Barr virus (EBV) infection. To assess whether EBV DNA detection by polymerase chain reaction (PCR) or presence of specific serum antibody to viral capsid antigen (VCA) was a better marker for screening NPC, nasopharyngeal tissues and blood samples from 58 NPC patients and 24 non-NPC patients (23 with laryngotracheal stenosis and 1 with chronic tonsillitis) were tested for the presence of EBV DNA and serum specific VCA antibodies, respectively. EBV DNA was detected in 56 (96.5%) of NPC patients and 15 (62.5%) of non-NPC controls, with predominantly EBV type A in both groups. On the other hand, specific VCA IgA antibody was detected in the majority of NPC patients: 52 (89.7%) while only 4 (16.7%) were detected in non-NPC controls. Therefore, specific VCA IgA antibody may serve as a better marker for screening NPC than EBV DNA detected by PCR.     

Epstein-Barr virus interactions with human lymphocyte subpopulations: virus adsorption, kinetics of expression of Epstein-Barr virus-associated nuclear antigen, and lymphocyte transformation.

In order to further understand Epstein-Barr virus (EBV)-lymphocyte interactions, we investigated a chain of events including: (i) EBV binding to human lymphocyte subpopulations; (ii) the earliest appearance of EBV-determined nuclear antigen (EBNA) in the lymphocytes after EBV infection; and (iii) establishment of continuous lymphoblastoid cell lines (LCL) by infecting with EBV different types of lymphocyte preparations from the same as well as from different donors. By using direct membrane immunofluorescence assay, we found that only a small fraction of human peripheral blood and cord blood lymphocytes (CBL), and possibly less than 31% of the T cell-depleted lymphocyte population, carry receptors for P3HR-1 strain of EBV. The number of cells carrying receptors for EBV did not vary considerably among different blood lymphocyte populations from several normal donors. EBV adsorption on lymphocyte subpopulations showed that purified thymus-dependent (T) cells and thymocytes did not adsorb EBV, in contrast to T cell-depleted lymphocyte populations and lymphoid cells from fetal liver and spleen. In CBL infected with EBV strain B95-8, EBNA was detected by anti-complement immunofluorescence as early as 18 h after infection. This indicates that EBNA is the earliest detectable EBV-determined intracellular antigen to appear after infection and before or during lymphocyte transformation by EBV. Transformation was observed only in lymphocyte cultures containing detectable thymus-independent B cells but not in cultures of purified T cells. With one exception (es-b-1), all the EBV-transformed LCL from different origins carried surface-bound immunoglobulins (a B cell marker). These included also the 10 LCL obtained by infecting cultures of adherent cells from different donors. With regard to its surface markers, ES-B-1 appeared to be an exceptional EBV genome-carrying line, and it also lacked the ability to form spontaneous rosettes with sheep erythrocytes (a T cell marker). Therefore, it is possible that ES-B-1 was derived from an atypical B cell or B cell precursor or from a so-called "null cell" transformed by EBV.     

Single-assay combination of Epstein-Barr Virus (EBV) EBNA1- and viral capsid antigen-p18-derived synthetic peptides for measuring anti-EBV immunoglobulin G (IgG) and IgA antibody levels in sera from nasopharyngeal carcinoma patients: options for field screening

Assessment of immunoglobulin A (IgA) antibody responses to various Epstein-Barr virus (EBV) antigen complexes, usually involving multiple serological assays, is important for the early diagnosis of nasopharyngeal carcinoma (NPC). Through combination of two synthetic peptides representing immunodominant epitopes of EBNA1 and viral capsid antigen (VCA)-p18 we developed a one-step sandwich enzyme-linked immunosorbent assay (ELISA) for the specific detection of EBV reactive IgG and IgA antibodies in NPC patients (EBV IgG/IgA ELISA). Sera were obtained from healthy donors (n = 367), non-NPC head and neck cancer patients (n = 43), and biopsy-proven NPC patients (n = 296) of Indonesian and Chinese origin. Higher values of optical density at 450 nm for EBV IgG were observed in NPC patients compared to the healthy EBV carriers, but the large overlap limits its use for NPC diagnosis. Using either EBNA1 or VCA-p18 peptides alone IgA ELISA correctly identified 88.5% and 79.8% of Indonesian NPC patients, with specificities of 80.1% and 70.9%, whereas combined single-well coating with both peptides yielded sensitivity and specificity values of 90.1 and 85.4%, respectively. The positive and negative predictive values (PPV and NPV, respectively) for the combined EBNA1 plus VCA EBV IgA ELISA were 78.7% and 93.9%, respectively. In the Indonesia panel, the level of EBV IgA reactivity was not associated with NPC tumor size, lymph node involvement, and metastasis stage, sex, and age group. In the China panel the sensitivity/specificity values were 86.2/92.0% (EBNA1 IgA) and 84.1/90.3% (VCA-p18 IgA) for single-peptide assays and 95.1/90.6% for the combined VCA plus EBNA1 IgA ELISA, with a PPV and an NPV for the combined EBV IgA ELISA of 95.6 and 89.3%, respectively. Virtually all NPC patients had abnormal anti-EBV IgG diversity patterns as determined by immunoblot analysis. On the other hand, healthy EBV carriers with positive EBV IgA ELISA result showed normal IgG diversity patterns. By using EBV IgG immunoblot diversity as confirmation assay for EBV IgA ELISA-positive samples, the sensitivity and specificity for NPC diagnosis increased to 98% and 99.2%, respectively, in the Indonesian NPC samples. The use of these combined methods for seroepidemiological screening studies is proposed.     

Correlation of the expression of Epstein-Barr virus latent membrane protein and in situ hybridization with biotinylated BamHI-W probes in Hodgkin''s disease.

The detection of Epstein-Barr virus (EBV) nucleic acids by in situ hybridization (ISH) with biotinylated BamHI-W probes was correlated with the expressions of EBV latent membrane protein (LMP) and EB nuclear antigen 2 (EBNA2), in 107 cases of Hodgkin's disease (HD) of different immunomorphologic subtypes. Epstein-Barr virus nucleic acids were present and restricted to the pathogenic cells in 4 of 40 (10%) cases of nodular sclerosis (NS) and 33 of 55 (60%) cases of mixed cellularity (MC), but were undetectable in other subtypes. Of the 37 cases positive for EBV nucleic acids, 35 (95%) showed the expression of LMP. Epstein-Barr virus nucleic acids and LMP were restricted to Reed-Sternberg cells and variants. Only 1 case (MC) showed LMP expression in the absence of EBV detection. The correlation was strengthened by the finding of LMP expression at first diagnosis in 6/7 EBV positive cases at relapse (14-126 months) (5/5 EBV negative cases at relapse were LMP negative at first diagnosis). EBNA2 was absent in all 13 (NS, 2; MC, 11) EBV+ and LMP+ cases tested. Both LMP and EBNA2 were expressed in control EBV-positive tissues and cell lines. EBV serology in MC HD was indicative of latent EBV infection, but neither serology nor clinical parameters correlated with the presence or the absence of EBV, over a short-term follow-up (median, 20 months). The findings, although not proving EBV as the etiologic agent of HD, suggest that: 1) LMP expression alone may be adequate for identifying EBV-associated HD, 2) the MC subtype has a stronger relation with EBV presence, and 3) the regulation of EBV genes in HD is different from other EBV-associated disorders. The clinical implications still remain to be discovered.     

Epstein-Barr virus-related antibody patterns in ataxia-telangiectasia.

Epstein-Barr virus-related antibody titres were determined in twenty-seven patients with ataxia-telangiectasia (AT) and twenty-two healthy members of their families, in twenty-two patients with other diseases, among them ten with Behçet''s disease and ten with various primary immune deficiencies, and fifteen healthy members of their families, and in twenty-three unrelated healthy controls. The AT patients showed an increased incidence (55.6%) of high antibody titres (greater than or equal to 1:320) to viral capsid antigen (VCA), and also a high incidence (48.2%) of antibodies to Epstein-Barr virus (EBV) induced early antigens (EA), but low titres (less than 1:10) of antibodies to the EBV-associated nuclear antigen (EBNA) in 44.2% of the cases. The geometric means of anti-VCA were three-to four-fold higher, and of anti-EBNA six-fold lower, than those of the control groups. The patients with the other diseases did not differ significantly from the controls except for a higher incidence of anti-EBNA titres of less than 1:10 (38.1% vs 4--5%). AT patients with low anti-EBNA titres tended to have more advanced T cell deficiencies than AT patients with moderate anti-EBNA titres, as detected by counts of total lymphocytes and E-rosetting cells, and skin test responses. The results support the hypothesis that a functioning T cell system is required to release EBNA from EBV genome-carrying cells for initial and maintained production of anti-EBNA.