Persistence of Epstein-Barr virus in Reed-Sternberg cells throughout the course of Hodgkin's disease.

Non-isotopic in situ hybridization employing digoxigenin-labelled DNA probes has been used to localize Epstein-Barr virus (EBV) in 55 cases of Hodgkin's disease (HD). The virus was found in Reed-Sternberg (RS) and mononuclear Hodgkin's cells in nine patients (16 per cent). Further samples taken at different times from three patients also showed the presence of EBV in the malignant cell population. Estimations of the number of EBV genomes present per cell suggested wide variations between different patients, but relatively constant amounts in different samples from the same patient. These findings are compatible with a stable infection of the neoplastic cells and support the notion that EBV may play a role in the development of HD in these patients. We also found evidence for the presence of EBV in a small percentage of non-neoplastic cells in 8 of the 55 samples. This suggests that isolation of EBV from HD tissue does not always signify a pathogenetic role for the virus. Furthermore, it is apparent that a high percentage of HD tissues do not contain demonstrable EBV, and the virus is therefore unlikely to be a causative agent for all cases of HD.     

P53 overexpression and Epstein-Barr virus infection in undifferentiated and squamous cell nasopharyngeal carcinomas

We have analysed 22 nasopharyngeal carcinomas (NPCs) for expression of the small nuclear Epstein-Barr virus (EBV)-encoded RNAs (EBERs) and for immunohistologically detectable overexpression of p53. in situ hybridization demonstrated expression of the EBERs in 13 undifferentiated NPCs while nine squamous cell NPCs were EBER-negative. These results therefore confirm our previous DNA-DNA in situ hybridization studies and demonstrate that in the nasopharynx EBV is exclusively associated with undifferentiated but not with squamous cell carcinomas. p53 overexpression was demonstrated by immunohistology in 5 of 9 squamous cell NPCs and in 9 of 13 undifferentiated NPCs. Thus, there appears to be no correlation of p53 overexpression with EBV infection. These results are unexpected in the light of previous studies demonstrating that the p53 gene in primary undifferentiated NPC is consistently in the wild-type configuration. By contrast, analyses of squamous cell carcinomas of the head and neck have demonstrated that p53 overexpression in these cases is the result of p53 gene mutation. Whilst more detailed genetic analysis is required, our results suggest that mechanisms other than mutation of the p53 gene may be responsible for the stabilization of the protein in cases of undifferentiated NPC. It is tempting to speculate that an EBV-encoded protein may be involved.     

Epstein-Barr virus-associated carcinomas: facts and fiction

The Epstein-Barr virus (EBV) is associated with several human tumours including lymphoid and epithelial malignancies. Most EBV-associated tumours are rare or occur at higher incidence only in certain geographical regions. The recently reported detection of EBV in gastric, breast, and hepatocellular carcinomas raises the possibility of involvement of the virus in the pathogenesis of common cancers. This article reviews the evidence linking EBV infection to epithelial tumours. It is concluded that at present, there is no convincing evidence to suggest that breast carcinoma and hepatocellular carcinoma are EBV-associated tumours.     

A20 RNA expression is associated with undifferentiated nasopharyngeal carcinoma and poorly differentiated head and neck squamous cell carcinoma

A20 is an anti-apoptotic gene that can be induced in human epithelial cell lines in response to expression of the Epstein–Barr virus (EBV) gene product latent membrane protein 1 (LMP1). EBV is a ubiquitous, persistent human herpesvirus that is consistently associated with undifferentiated nasopharyngeal carcinoma (NPC), in which antigen expression includes LMP1. Consistent with a potential role in the development of NPC, LMP1 has profound effects on epithelial cell growth. A20 may be a key downstream effector of LMP1 in NPC, as LMP1-induced A20 blocks p53-mediated apoptosis in H1299 epithelial cells and most NPCs have wild-type p53. Moreover, the potential role of A20 in the development of epithelial malignancies may extend to tumours not associated with EBV. The purpose of this study was to develop an in situ hybridization assay to assess expression of A20 RNA in undifferentiated NPC and in non-EBV-associated poorly differentiated head and neck squamous cell carcinomas (SCCs) and well-differentiated SCCs of the skin. A20 RNA expression was also examined in normal samples of oral mucosa and skin. Expression of A20 was demonstrated in 76 per cent of undifferentiated NPCs and in 80 per cent of poorly differentiated head and neck SCCs, suggesting a role for A20 in the pathogenesis of these epithelial malignancies. By contrast, A20 RNA was not detected in well-differentiated SCCs of the skin, or in any normal samples of squamous epithelial tissue. The pathway leading to A20 expression in non-EBV-associated poorly differentiated head and neck SCCs is clearly LMP1-independent. LMP1 expression was demonstrated in 29 per cent of NPC biopsies, suggesting an LMP1-independent pathway to A20 induction in undifferentiated NPC. Copyright © 1999 John Wiley & Sons, Ltd.     

Epstein-Barr virus infection in colorectal neoplasms associated with inflammatory bowel disease: detection of the virus in lymphomas but not in adenocarcinomas

Epstein-Barr virus (EBV) is associated with several lymphoid and epithelial human malignancies. The latter include gastric adenocarcinomas, while sporadic colorectal adenocarcinomas (CRCs) have been reported to be EBV-negative. Recently, increased numbers of EBV-infected B lymphocytes have been detected in intestinal mucosal samples affected by ulcerative colitis (UC) and, to a lesser extent, Crohn's disease (CD). Both CRC and colorectal non-Hodgkin's lymphoma (NHL) are recognized complications of inflammatory bowel disease (IBD), but it is unclear to what extent EBV contributes to the development of these neoplasms. Seventeen cases of IBD-associated CRC and nine cases of IBD-associated colorectal NHL were therefore studied for the presence of EBV by in situ hybridization. EBV-positive cases were further studied for the expression of the EBV-encoded nuclear antigen (EBNA) 2 and the latent membrane protein (LMP) 1 of EBV by immunohistochemistry. Four out of seven cases of colorectal NHL associated with UC were shown to be EBV-positive. In addition, two of two colorectal NHLs developing in patients with CD were EBV-positive. Of the EBV-positive lymphomas, three displayed a pattern of EBV latent gene expression consistent with type I latency (EBNA2(-)/LMP1(-)), two a type II pattern (EBNA2(-)/LMP1(+)), and one a type III pattern (EBNA2(+)/LMP1(+)). These findings suggest that EBV infection is involved in the pathogenesis of a proportion of colorectal NHLs developing in IBD. Iatrogenic immunosuppression may contribute to the development of these lymphomas. By contrast, all 17 IBD-associated CRCs were EBV-negative, including a case of CRC occurring synchronously with an EBV-positive NHL. In conjunction with previous reports on sporadic CRCs, this suggests that EBV is not involved in the pathogenesis of CRC.     

Biclonal expansion of T cells infected with monoclonal Epstein-Barr virus (EBV) in a patient with chronic,active EBV infection

Recent studies have suggested that a high percentage of Epstein-Barr virus (EBV)-infected lymphocytes in peripheral blood of patients with chronic, active EBV infection (CAEBV) is of T cell origin. Although T cells are expanded oligoclonally in CAEBV, it is not clear whether the restricted diversity of T cells arise from immune reaction against EBV-related antigens or from proliferation of EBV-infected cells. We experienced a patient with CAEBV who had biclonal expansion of peripheral blood T cells. We identified clonotypes of these two T cell clones in detail and purified the T cell clones. EBV infected mainly the two T cell clones, whereas the viral loads in peripheral blood cells other than these T cell clones were low or undetectable. The EBV strains infecting the two T cells clones were indistinguishable from each other by a series of genotype analyses of the virus. These results suggest that the two T cell clones infected with the same monoclonal EBV proliferated in peripheral blood of the patient.     

鼻咽刷片细胞学检查和EB病毒检测在鼻咽癌诊断中的意义

目的：探讨鼻咽细胞学检查结合DNA核型判断和细胞EB病毒检测在可疑鼻咽癌病人筛查中的作用。方法：分别对66例可疑鼻咽癌就诊病人作鼻咽刷片细胞学诊断和应用CAS200图像分析仪测定涂片细胞DNA含量,细胞学癌阳性病例同时作EB病毒编码RNA（EBERs)原位杂交。结果：与组织学诊断相比,细胞学诊断和DNA非二倍体诊断癌的敏感性分别为66％和55％,两者结合判断不能提高敏感性,并且假阴性率高;细胞学癌阳性病例的癌细胞核EBERs阳性率92．1％,其中6例DNA二部体核型病例均呈EBERs阳性,可诊断为鼻咽癌。结论：鼻咽细胞学检查结合DNA核型判断不能提高可凝鼻咽癌病人的诊断率,不适用于鼻咽癌筛查。细胞涂片的EB病毒原位杂交方法,在鼻咽癌可疑病人的诊断和鉴别诊断上具有重大实用价值,在鼻咽癌筛查的作用有待进一步研究。     

Analysis of Epstein-Barr virus (EBV) receptor CD21 on peripheral B lymphocytes of long-term EBV- adults.

Primary infections with EBV are rarely observed after the age of 20. Some individuals even remain seronegative all their lives. Previously, a lack of EBV receptors on B cells of persistently EBV- adults was described as a reason for long-term EBV-seronegativity. The present study examined the CD21 receptor status of 20 repeatedly EBV- healthy adults and 32 EBV+ volunteers by means of flow cytometry. CD21 molecules on the surface of CD19+ B cells were quantified using anti-IgG-coated microbeads. The percentage of CD19+/CD21+ B lymphocytes was slightly lower in the peripheral blood of EBV- donors, but the CD21 antibody binding capacity on CD19+ B cells showed no significant differences between EBV- and EBV+ adults. In vitro studies showed an equally good EBV transformability of peripheral B lymphocytes of EBV- and EBV+ donors. Since HLA-DR was recently described as a co-receptor for EBV infection of B cells, we also determined HLA-DRB1 alleles in the EBV- group. We found a significant negative association of EBV-seronegativity with HLA-DR13 in comparison with 111 healthy blood donors. In summary, a biologically significant lack of the EBV receptor CD21 on peripheral B lymphocytes of persistently EBV- adults was excluded as a reason for long-term EBV-seronegativity.     

Detection of Epstein-Barr virus genome within thymic epithelial tumours in Taiwanese patients by nested PCR,PCR in situ hybridization,and RNA in situ hybridization

Epstein-Barr virus (EBV) is known to be associated with a variety of tumours, including Burkitt's lymphoma, nasopharyngeal carcinoma, and some carcinomas of other organs with similar lymphoepithelioma-like features. The association between EBV and thymic epithelial tumours is inconclusive, as reports in this regard are not entirely consistent and the methods employed are of different sensitivity and specificity. This study examined 78 thymomas and 21 thymic carcinomas in Taiwanese patients, to detect the viral genome at both DNA and RNA levels. The tissue blocks were first screened by nested polymerase chain reaction (PCR) targeting on the first tandem internal repeats. The positive cases were further submitted for viral localization by in situ PCR insitu hybridization (ISH) and Epstein-Barr-encoded RNA-1 (EBER-1) ISH. None of the thymomas showed a detectable EBV genome. Eight thymic carcinomas were positive for EBV by nested PCR, of which six displayed nuclear signals within the tumour cells by in situ PCR ISH and/or RNA ISH, one displayed signals within the lymphocytes, and one showed no discernible in situ signals. Most of them exhibited a lymphoepithelioma-like morphology. These results show that nested PCR is a sensitive method for screening the EBV genome in thymic epithelial tumours. In situ PCR ISH is reliable for localization of the virus, in addition to EBER-1 RNA ISH. Thymomas are not related to EBV, even in this endemic area. Thymic carcinomas, especially the lymphoepithelioma-like thymic carcinomas, are more often associated with the virus.     

Elevated Epstein-Barr virus seroreactivity among unaffected members of families with nasopharyngeal carcinoma

Serum antibodies to Epstein–Barr virus (EBV) antigens can be used to predict the risk of nasopharyngeal carcinoma (NPC). To investigate whether EBV seropositivity rates were higher among healthy family members from multiplex and sporadic families with NPC (i.e., families with multiple or single cases) compared to the general population, a study was conducted on 2,665 unaffected individuals from 140 multiplex and 413 sporadic families. The titers of the IgA antibody to the EBV capsid antigen (VCA‐IgA) were compared to those of 904 controls from the general population. The VCA‐IgA titer was correlated among sibling pairs to a high significance in both family types (P < 0.0001 and P = 0.0005 for the multiplex and the sporadic families, respectively); parent–offspring pairs also showed significant correlation (P < 0.0001 and P = 0.0002, respectively); and spouse pairs were correlated, but at lower significance levels (P = 0.0790 and P = 0.0040, respectively). When compared to the controls, among first‐degree relatives in the multiplex families, the age‐ and gender‐adjusted odds ratio (OR) was 2.06 (95% confidence interval 1.56–2.71), 3.55 (2.24–5.64), and 2.25 (1.57–3.23) for siblings, parents, and children, respectively. In the sporadic families, the adjusted OR was 1.55 (1.21–2.00) and 2.08 (1.51–2.86) for siblings and parents, respectively. The adjusted P‐value of spouses lost significance in the multiplex families, but remained significant in the sporadic families (P = 0.0146). In conclusion, EBV seropositivity rates were elevated among unaffected family members in both multiplex and sporadic families with NPC. J. Med. Virol. 83:1792–1798, 2011. © 2011 Wiley‐Liss, Inc.     

Prevalence of Epstein–Barr virus in oral squamous cell carcinoma

Forty-six samples of oral squamous cell carcinoma (OSCC) were evaluated for the prevalence of Epstein–Barr virus (EBV) infection by the polymerase chain reaction (PCR), Southern blot hybridization, and in situ hybridization (ISH). EBV DNA was detected in 7 (15·2 per cent) out of 46 samples by a combination of PCR and Southern blot hybridization methods. All seven positive samples showed well-differentiated carcinoma, thus suggesting a possible relationship between EBV infection and the degree of differentiation of carcinoma tissue. Latent infection membrane protein 1 (LMP1) was detected immunohistochemically in six of the EBV-positive OSCCs. However, no signal of the EBV-encoded small RNA (EBER)-1 was demonstrated by the ISH method. No significant relationship was observed between EBV infection and lymph node metastasis. A follow-up study (range from 4·4 to 79 months; mean 34·9 months) showed no recurrence or death to occur in the EBV-positive patients, which thus suggested a good prognosis for EBV-positive OSCC patients. Copyright © 1999 John Wiley & Sons, Ltd.     

The association of squamous cell carcinomas of the nasopharynx with Epstein–Barr virus shows geographical variation reminiscent of Burkitt's lymphoma

Nasopharyngeal carcinoma (NPC) is rare in most parts of the world but occurs with high incidence in certain regions, such as South-East Asia. Two major histological types of NPC are recognized, non-keratinizing carcinoma and squamous cell carcinoma. Non-keratinizing NPCs, which include undifferentiated NPC, are invariably associated with Epstein–Barr virus (EBV) infection, regardless of the geographical or ethnic origin of the patients. By contrast, conflicting results have been published concerning a possible association of squamous cell NPC with the virus. To address this question, squamous cell NPCs have been collated from an area where NPC is endemic, Hong Kong, and from two regions where NPC occurs with a lower incidence, Chengdu, PR China, and Birmingham, United Kingdom. In situ hybridization for the detection of the small EBV-encoded nuclear RNAs (EBERs) demonstrated that all 22 cases from Hong Kong were EBV-positive. By contrast, EBV was detectable in 7 of 19 cases from central China, and in 3 of 7 cases from the U.K. Expression of the virus-encoded latent membrane protein 1 (LMP1) was detected in 3 of 32 EBV-positive squamous cell NPCs. These results indicate that the association of squamous cell NPCs with EBV shows geographical variability in a manner which is reminiscent of the situation encountered in Burkitt's lymphoma. This suggests that squamous cell NPCs are a pathogenetically heterogeneous group of tumours distinct from non-keratinizing NPCs. © 1997 John Wiley & Sons, Ltd.     

Establishment and characterization of an in vivo model for Epstein-Barr virus positive gastric carcinoma

Research regarding the role of the Epstein-Barr virus (EBV) in gastric carcinogenesis has been hampered by the absence of a suitable model system. SNU-719 is a gastric carcinoma cell line naturally infected with EBV. This cell line developed tumors in nude mice approximately 40-56 days after inoculation. SNU-719 also showed low serum dependency and anchorage independent growth in vitro. The developed tumors expressed EBERs, EBNA1, and LMP2A but not other EBV latent genes. Additionally, Qp was active and either mono- or bi-clonal EBV genome was observed in the tumor tissues. Because the developed tumors retained characteristics of EBV-associated gastric cancer, this cell line could serve as a useful in vivo system to investigate the tumorigenesis mechanism and treatment methods for this type of tumor.     

肠道淋巴瘤与EB病毒相关性研究

目的：探讨EB病毒（Epstein-Barr virus,EBV)感染在肠道淋巴瘤发病中的意义。方法：采用EBV的DNA原位杂交及S－P法免疫组化技术（第一抗体为EBV、CD3、CD20、CD43、CD45、CD45RO、CD74等），观察24例肠道淋巴瘤患者（8例肠病相关T细胞淋巴瘤、16例黏膜相关淋巴组织B细胞淋巴瘤）EBV感染情况。以20例慢性结肠炎作为对照。结果：患者年龄21－92岁（平均52．8岁），男女之比为3．8：1。临床上均以腹痛、腹胀或便血就诊。组织病理学：T细胞淋巴瘤细胞多形性、核大，不规则，嗜血管性及大片坏死；B细胞淋巴瘤细胞中等大小，多呈圆形、椭圆形、胞质较少淡染，核稍大，核分裂象多见，可见“淋巴上皮病变”。24例淋巴瘤中检出（原位杂交及免疫组化）EBV－DNA 14例（检出率为58．3％），其中T细胞淋巴瘤EBV的检出率为75％，B细胞淋巴瘤EBV的检出率为50％（P＜0．01）。结论：肠道淋巴瘤的发生与EBV的感染有明显的相关性。     

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.     

Association of Epstein-Barr virus infection with p53 protein accumulation but not bcl-2 protein in nasopharyngeal carcinoma

AIMS: The aim of this study was to investigate the association of Epstein-Barr virus (EBV) infection with status of p53 protein expression in nasopharyngeal carcinoma (NPC). The expression of EBV gene and gene product, p53 protein and bcl-2 protein in NPC was histopathologically studied. METHODS AND RESULTS: In-situ hybridization using oligonucleotide probe to EBV-encoded small RNAs (EBERs) and immunohistochemistry using monoclonal antibodies against EBV latent membrane protein 1 (LMP1), p53 protein and bcl-2 proteins were performed in 56 primary NPCs. EBERs were detected in 46 (82%) cases and LMP1 in 17 (30%) cases. While 30 of 32 (94%) cases in differentiated nonkeratinizing carcinoma (NKC, WHO type 2) and 16 of 17 (94%) cases in undifferentiated carcinoma (UC, WHO type 3) showed EBERs expression, neither five cases of keratinizing squamous cell carcinoma (KSCC, WHO type 1) nor two cases of adenocarcinoma showed EBERs. bcl-2 protein was detected in 50 (89%) cases, but its expression did not depend on expression of LMP1. p53 protein was detected in 31 (55%) cases, and there was a correlation between expression of EBERs and p53 protein (P < 0.05) but not between LMP1 and p53 protein. CONCLUSION: In this study, close association of NKC and UC but not KSCC with the latent infection with EBV was demonstrated. The induction of bcl-2 protein by LMP1, as shown in vitro, was not demonstrated. The association between overexpression of p53 protein and the presence of EBV suggests that some EBV-encoded protein, which may be different from LMP1, may play a role for nuclear accumulation of p53 protein.     

Characterization of the expanded T cell population in infectious mononucleosis: apoptosis, expression of apoptosis-related genes, and Epstein-Barr virus (EBV) status

Infectious mononucleosis (IM), a manifestation of primary infection with EBV, is characterized by a massive expansion of the T cell population. In this study we examined this expanded T cell population regarding its EBV status, its proliferative and apoptotic activity, and its expression of apoptosis-related genes. Whereas previous studies were performed on ex vivo cultures or on peripheral blood, our investigations included in vivo analysis of IM tonsillectomy specimens (14 cases) by in situ hybridization for viral RNA (EBERs) combined with immunohistochemistry (IHC; CD3, CD45RO, CD20, CD79a, Ki-67, Bcl-2, Bax, Fas, FasL) and the TUNEL method. Of the EBER+ cells 50-70% showed expression of the B cell markers CD20/CD79a. The remainder of the EBER+ cells expressed neither B nor T cell antigens. No co-expression of EBERs and T cell antigens was detected in any of the specimens. In accordance with a high rate of apoptosis (up to 2.37%) within the expanded T cell population, Bcl-2 expression was drastically reduced and FasL expression remarkably increased. The levels of Bax and Fas expression showed no or moderate up-regulation. In conclusion, the massive expansion of IM T cells is not caused by EBV infection of these cells but merely represents an intense immune reaction. Through altered expression of Bcl-2/Bax and Fas/FasL, the activated T cells are subject to enhanced apoptosis while residing within the lymphoid tissue, which eventually allows the efficient silencing of this potentially damaging T cell response.     

Humoral immune responses to Epstein-Barr virus encoded tumor associated proteins and their putative extracellular domains in nasopharyngeal carcinoma patients and regional controls

Epstein-Barr virus (EBV) latency proteins EBNA1, LMP1, LMP2, and BARF1 are expressed in tumor cells of nasopharyngeal carcinoma (NPC). IgG and IgA antibody responses to these non-self tumor antigens were analyzed in NPC patients (n=125) and regional controls (n=100) by three approaches, focusing on the putative LMP1, LMP2 extracellular domains. Despite abundant IgG and IgA antibody responses to lytic antigens and EBNA1, patients had low titer (1:25-1:100) IgG to LMP1 (81.2%), LMP2 (95.6%), and BARF1 (84.8%), while immunoblot showed such reactivity in 24.2%, 12.5%, and 12.5% at 1:50 dilution, respectively. Few IgA responses were detected, except for EBNA1. Controls only showed IgG to EBNA1. ELISA using peptides from different domains of LMP1, LMP2, and BARF1 also yielded mostly negative results. When existing, low level IgG to intracellular C-terminus of LMP1 (62.9%) prevailed. Rabbit immunization with peptides representing extracellular (loop) domains yielded loop-specific antibodies serving as positive control. Importantly, these rabbit antibodies stained specifically extracellular domains of LMP1 and LMP2 on viable cells and mediated complement-driven cytolysis. Rabbit anti-LMP1 loop-1 and -3 killed 50.4% and 59.4% of X50/7 and 35.0% and 35.9% of RAJI cells, respectively, and 22% of both lines were lysed by anti-LMP2 loop-2 or -5 antibodies. This demonstrates that (extracellular domains of) EBV-encoded tumor antigens are marginally immunogenic for humoral immune responses. However, peptide-specific immunization may generate such antibodies, which can mediate cell killing via complement activation. This opens options for peptide-based tumor vaccination in patients carrying EBV latency type II tumors such as NPC.     

Native early antigen of Epstein-Barr virus, a promising antigen for diagnosis of nasopharyngeal carcinoma

The Epstein-Barr virus (EBV) early antigen (EA) complex consists of multiple proteins with relevance for diagnosis of acute, chronic and malignant EBV related diseases, including nasopharyngeal carcinoma (NPC). In a recent study, it was found that the molecular diversity of EBV-specific IgG and IgA antibody responses in NPC patients and demonstrated that these reflect independent B-cell triggering leading to distinct EBV antigen-recognition profiles. The fine-specificity of NPC-related IgG and IgA responses was explored further against defined recombinant and synthetic EBV-EA antigens using immunofluorescence, immunoblot and ELISA techniques and determined their diagnostic value in a large panel of sera from NPC (n = 154), non-NPC tumor patients (n = 133), acute mononucleosis patients (n = 70) and healthy EBV carriers (n = 259). Individual recombinant EBV-EA markers yielded sensitivity/specificity values not exceeding 86%, whereas selected EA-specific peptide epitopes were rather poorly recognized by IgG and IgA antibodies in NPC sera. Surprisingly, we found that a "low salt" native EA-protein extract reproducibly prepared from purified nuclei of EA-induced HH514 cells, and containing characteristic EA(D)-polypeptides, such as p47-54 (BMRF1), p138 (BALF2), p55-DNAse (BGLF5), and p65-TK (BXLF1), but without viral capsid (VCA) or nuclear antigen (EBNA) reactivity, gave highest sensitivity (90.4%) and specificity (95.5%) values for NPC diagnosis in both IgG and IgA ELISA. The data support further the notion that EBV-EA reactive IgG and IgA antibodies in NPC patients are directed against distinct conformational and-in part-linear epitopes on EBV-specific proteins, barely recognized in other EBV-related syndromes. The use of a defined native EBV EA-specific antigen opens the way to further improve serological diagnosis of NPC.