Synthetic peptides deduced from the amino acid sequence of Epstein-Barr virus nuclear antigen 6 (EBNA 6): Antigenic properties,production of monoreactive reagents,and analysis of antibody responses in man

Studies on the antibody responses to various Epstein-Barr virus (EBV) antigens have been instrumental in the understanding of the seroepidemiology and diagnosis of this viral infection and the subsequent carrier state. While antibodies to the viral capsid antigen (VCA), early antigen (EA), and nuclear antigens 1 and 2 (EBNA 1 and 2) have been well characterized, the antibody response to the other nuclear antigens is not well understood. EBNA 6 is expressed by lymphoblasts during acute EBV infection and may be an important antigen for diagnosis and evaluation of the immune response. In order to analyze the antibody response to EBNA 6, ten peptides (20–21 amino acids), deduced from the EBNA 6 coding region, were synthesized and evaluated for antigenicity by ELISA. One peptide (p-63; PA-PQAPYQGYQEPPAPQAPY) derived from the amino acid repeats showed the highest specific reactivity with human sera. This peptide was evaluated further for detection of human EBNA 6-reactive antibodies. Forty-two of forty-nine (86%) EBV-seropositive healthy donors had p-63- specific IgG reactivity, while none of 50 EBV seronegative patients reacted with the p-63 peptide. Twenty-two of fifty-one (43%) patients with ongoing primary EBV infection had detectable p-63-specific IgG. Serum samples drawn sequentially from patients during and after primary EBV infection revealed an increase in p-63-reactive IgG over time. A similar pattern was found for reactivity with an EBNA I-specific peptide (p-107), in contrast to the EBNA 2 (polyproline) response, which decreased over time. Some EBV seropositive individuals who had no detectable IgG against peptide p-63 did have antibodies against the native EBNA 6 by anticomplement immunofluorescence to EBNA 6 transfected cells. Rabbit antiserum raised against p-63 reacted specifically with native EBNA 6 by an immunofluorescence assay and by immunoblotting, indicating the EBNA 6-specific antigenicity of the peptide. Thus, the peptide p-63 derived from the amino acid repeats of the EBNA 6 coding region constitutes a predominant, although not exclusive, epitope in the EBNA 6 antibody response. © 1995 Wiley-Liss, Inc.     

Detection of a nuclear antigen 2 (EBNA2)-variant Epstein-Barr virus strain in two siblings with fatal lymphoproliferative disease

An EBV type 1 variant strain was detected in two Turkish siblings (boy and girl), who both suffered and died from similar progressive Epstein-Barr virus (EBV)-associated lymphoproliferative disease. Molecular characterisation of this EBV isolate revealed a 51bp-deletion and six nucleotide changes within the Epstein-Barr nuclear antigen 2 (EBNA2). Both isolates contained EBV type 2 sequences in the Epstein-Barr virus-encoded small RNAs (EBER), which are 40 kb proximal to EBNA2. Sequencing of the EBV isolates in a region of Epstein-Barr nuclear antigen 3 (EBNA3a), which is 40 kb distal to EBNA2, revealed the normal EBV type 1 sequence of laboratory strain B95-8. This EBV isolate may represent a distinct wild type EBV strain with altered biological properties. It is suggested that this EBNA2-variant strain may be responsible at least in part for the severe clinical course in both affected children. © 1996 Wiley-Liss, Inc.     

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.     

Epstein-Barr virus nuclear antigen 1 evades direct immune recognition by CD4+ T helper cells

The Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is the only viral protein regularly expressed in EBV-associated malignancies. Immune recognition of EBNA1 by CD8+ T cells is prevented by an internal glycine-alanine repeat (GAr) which blocks proteasomal degradation. To test whether EBV-infected cells could be recognized by T helper cells, human CD4+ T cell clones specific for EBNA1 were isolated from latently EBV-infected individuals. These T cells, however, failed to recognize EBV-positive target cells. To investigate whether endogenous presentation of EBNA1 epitopes on MHC class II was prevented by the GAr domain, a mutant EBV strain with an EBNA1 lacking the GAr (EBNA1DeltaGA) was generated and used to establish an Epstein-Barr virus-immortalized lymphoblastoid B cell line (LCL). The EBNA1DeltaGA LCL were not recognized by the EBNA1-specific T cell clones either, indicating that the GAr domain does not mediate this effect. Immune recognition could be restored by overexpression of EBNA1, for which at least 60-fold higher levels of both EBNA1 or EBNA1DeltaGAr protein were required. These results demonstrate that EBNA1 evades direct recognition by CD4+ T helper cells, since its steady state level is below the threshold required for efficient presentation on MHC class II. These findings have important implications for the design of immunotherapeutic approaches to target EBV-positive malignancies.     

Evaluation of commercial EBV RecombLine assay for diagnosis of nasopharyngeal carcinoma

BACKGROUND: In recent years a number of Epstein-Barr virus (EBV) proteins were defined as being immunodominant for either IgM, IgG or IgA immune responses, yielding promising markers for diagnostic serology. Specific reactivity patterns to these proteins have been described for infectious mononucleosis (IM), nasopharyngeal carcinoma (NPC), various types of lymphoma, and healthy EBV carriers. OBJECTIVES: To compare the NPC-related diagnostic value of EBV RecombLine test (Mikrogen, Germany) with a standardized immunoblot assay [Fachiroh J, Schouten T, Hariwiyanto B, Paramita DK, Harijadi A, Haryana SM, et al. Molecular diversity of Epstein-Barr virus IgG and IgA antibody responses in nasopharyngeal carcinoma: a comparison of Indonesian, Chinese, and European subjects. J Infect Dis 2004;190:53-62] and to define the diagnostic value of individual EBV marker proteins in a population with high incidence of NPC. RESULT: Sera from Indonesian NPC patients taken at primary diagnosis (n=108) were analyzed for IgG and IgA reactivity and compared with regional healthy blood donors (n=62), non-NPC patient controls (n=10) and IM patients (n=10). Most NPC patients and controls showed strong IgG reactivity to VCA-p18, -p23, and EBNA1, limiting their diagnostic use. Few (<20%) healthy donors and patient controls showed IgG reactivity to EA proteins p47/54 and p138, yielding combined sensitivity/specificity and PPV/NPV values of 92.6%/98.3% and 99.0%/88.1%, for diagnosing NPC. NPC sera showed significantly more EBV reactive IgA antibody (>80% positive) than controls (<10% positive), although being less broadly reactive and significantly less strong compared to IgG. For IgA best results were observed for RecombLine EBNA1 with sensitivity/specificity and PPV/NPV values of 92%/89% and 93.4%/85.9%, respectively. CONCLUSION: In high incidence NPC regions with low incidence IM yet high prevalence of EBV infection, both RecombLine IgG and IgA tests provide a useful alternative to the more complex cell-extract based immunoblot assay as confirmation test for NPC diagnosis in particular when using EA and EBNA1 as discriminators in IgG and IgA testing, respectively.     

EB病毒对人胃癌细胞系HSC-39感染的研究

目的 探讨EB病毒（EBV）对胃癌细胞系的感染作用。方法 用Akata和P3HR－1 EBV毒株感染人胃癌印戒细胞系（HSC－39），有限稀释法对感染细胞进行克隆。结果 EBV感染细胞中可检测到EBV编码的核抗原（EBNA)，2种EBV毒株感染的细胞克隆表现有不同的形态学特征及生长方式。EBV感染的亲代细胞及大部分克隆表达EBNA1，但不表达EBNA2、潜伏期膜蛋白（LMP）1和LMP2A；亲代细胞及所有细胞克隆未观察到裂解感染，EBNA启动子Qp表达阳性，而启动子Cp和Wp未见表达。结论 HSC－39对2种EBV毒株均易感，EBV感染可改变HSC－39的细胞表型，且不同EBV毒株对其影响不同，提示印戒细胞癌细胞系可用作EBV感染的靶细胞。     

The Amino Acid Region 248–382 of the Epstein–Barr Virus Nuclear Protein 2 (EBNA2) is Responsible for the EBNA2-Induced EBV Reactivation

We showed previously that Epstein–Barr virus (EBV) latency is disrupted and the virus-replicative cycle is activated after expression of EBNA2 in the Burkitt's lymphoma-derived Akata cells. Here, an EBNA2 deletion mutant lacking the amino acid residues 248–382, including the region responsible for association with RBP-J , was generated and tested for its ability to activate EBV replication in Akata cells. This mutant was shown clearly deficient in inducing the EBV-replicative cycle, suggesting that association with RBP-J is necessary for the EBV activating function of EBNA2. It is thus likely that EBV activation by EBNA2, seemingly in conflict with its involvement in lymphocyte immortalization, is nevertheless based on the standard mechanism of transactivation by the protein.     

Carboxy-terminal sequence variation of LMP1 gene in Epstein-Barr-virus-associated mononucleosis and tumors from Serbian patients

Seven strains of Epstein–Barr virus (EBV) are defined based on C‐terminal sequence variations of the latent membrane protein 1 (LMP1). Some strains, especially those with a 30‐bp deletion, are thought to be related to tumorigenic activity and geographical localization. The aims of the study were to determine the prevalence of different LMP1 strains and to investigate sequence variation in the C‐terminal region of LMP1 in Serbian isolates. This study included 53 EBV‐DNA‐positive plasma and tissue block samples from patients with mononucleosis syndrome, renal transplantation, and tumors, mostly nasopharyngeal carcinoma. The sequence of the 506‐bp fragment of LMP1 C terminus was used for phylogenetic analyses and identification of LMP1 strains, deletions, and mutations. The majority of isolates were non‐deleted (66%), and the rest had 30‐bp, rare 69‐bp, or yet unknown 27‐bp deletions, which were not related to malignant or non‐malignant isolate origin. However, the majority of 69‐bp deletion isolates were derived from patients with nasopharyngeal carcinoma. Less than five 33‐bp repeats were found in the majority of non‐deleted isolates (68.6%), whereas most 69‐bp deletion isolates (75%) had five or six repeats. Serbian isolates were assigned to four LMP1 strains: B95‐8 (32.1%), China 1 (24.5%), North Carolina (NC; 18.9%), and Mediterranean (Med; 24.5%). In NC isolates, three new mutations unique for this strain were identified. EBV EBNA2 genotypes 1 and 2 were both found, with dominance of genotype 1 (90.7%). This study demonstrated noticeable geographical‐associated characteristics in the LMP1 C terminus of investigated isolates. J. Med. Virol. 84:632–642, 2012. © 2011 Wiley Periodicals, Inc.     

Identification of type A and B isolates of Epstein-Barr virus by polymerase chain reaction

A method is described for the identification of type A and type B isolates of Epstein-Barr virus (EBV) by means of the polymerase chain reaction. The use of three pairs of primers specific for genomic sequences coding for the two forms of EBV nuclear antigen (EBNA), 2A and 2B, and for a DNA sequence from the BamZ/BamR region allows the reliable and rapid detection of type A and B viruses in as little as 1000 EBV positive cells.     

Epstein–Barr virus in breast cancer: artefact or aetiological agent?

The Epstein-Barr virus (EBV) has been reported to be associated with a variety of different tumours; for some of these malignancies, including breast cancer, the association remains controversial. This might be due in part to differences in the methodologies used to detect EBV in tissue samples. One approach favoured by many groups is to use immunohistochemistry to detect an EBV protein, EBNA1, which is essential for the maintenance of viral latency in infected cells and therefore should be a good marker for the presence of the virus. However, in this issue of the Journal of Pathology, Hennard and colleagues report that one of the antibodies frequently employed to detect EBNA1 in tissue samples cross-reacts with the MAGE4 protein, a cancer testis antigen expressed in many cancer types. Their observation suggests that reports documenting an EBV association on the basis of reactivity with this antibody must be considered unreliable. It also re-opens the debate about whether breast cancer is an EBV-associated disease.     

Novel method for isolating untouched rat natural killer cells with higher purity compared with positive selection and fluorescence-activated cell sorting

The Epstein–Barr virus (EBV) nuclear antigen 1 (EBNA1) is regularly expressed in all proliferating virus‐infected cells and is therefore an interesting target for immunotherapy. Alleles of the human leucocyte antigen (HLA) ‐A2 family are dominantly expressed in Caucasians so we sought to identify EBNA1‐specific cytotoxic T‐lymphocyte (CTL) responses restricted through this allele. We report on the characterization of the LQTHIFAEV (LQT) epitope. LQT‐specific memory CTL responses were reactivated in three of 14 healthy EBV seropositive donors (21%) whereas responses to HLA‐A2‐restricted epitopes, two derived from LMP2 and one from EBNA3A, were detected in 93%, 71% and 42% of the donors, respectively. The LQT‐specific CTL clones did not lyse EBV‐carrying lymphoblastoid cell lines and Burkitt’s lymphoma cell lines nor EBNA1‐transfected Burkitt’s lymphoma cells but specifically released interferon‐γ upon stimulation with HLA‐matched EBNA1‐expressing cells and this response was enhanced by deletion of the Gly‐Ala repeat domain that inhibits proteasomal degradation. The poor presentation of the endogenously expressed LQT epitope was not affected by inhibition of peptidases that trim antigenic peptides in the cytosol but full presentation was achieved in cells expressing a trojan antigen construct that releases the epitope directly into the endoplasmic reticulum. Hence, inefficient proteasomal processing appears to be mainly responsible for the poor presentation of this epitope.     

Long-term carriers generate Epstein-Barr virus (EBV)-specific CD4(+) and CD8(+) polyfunctional T-cell responses which show immunodominance hierarchies of EBV proteins

T cells simultaneously producing multiple cytokines and possessing cytotoxic capacity termed polyfunctional cells (PFCs) are increasingly recognized as the immune correlate of protection against pathogenic viruses. We investigated co‐expression of four cytokines (interferon‐γ, macrophage inflammatory protein 1‐α, tumour necrosis factor‐α and interleukin‐2) and degranulation capacity (CD107a surface expression) of Epstein–Barr virus (EBV) ‐specific CD4⁺ and CD8⁺ T cells upon stimulation by overlapping peptides of EBV lytic (BZLF1) and latent (EBNA1, EBNA3 and LMP2) proteins, in 20 healthy Chinese long‐term carriers. Two patients with post‐transplant lymphoproliferative disorder (PTLD), who had impaired T‐cell immunity, were studied for comparison. Both EBV‐specific CD4⁺ and CD8⁺ PFCs were readily generated in long‐term carriers and showed immunodominance hierarchies of latent proteins (EBNA1 > EBNA3/LMP2 and EBNA3 > LMP2 > EBNA1 for CD4⁺ and CD8⁺ T cells, respectively), as evidenced by a higher proportion of PFCs generated by immunodominant EBV proteins than by subdominant viral proteins. In contrast, the proportion of EBV‐specific PFCs was markedly decreased in patients with PTLD. The EBV‐specific PFCs produced more cytokine per cell than single‐functional T cells and comprised different subsets. Five‐functional CD4⁺ and CD8⁺ T cells were detected and four‐functional CD4⁺ T cells were mainly CD107a negative and expressed all four cytokines whereas four‐functional CD8⁺ T cells were mainly CD107a positive and expressed three of the four cytokines (interleukin‐2‐negative). We conclude that EBV‐specific PFCs are generated in much higher proportions in the long‐term carriers than in the patients with PTLD and maintain the immunodominant characteristics of the virus.     

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.     

New variations of Epstein–Barr virus‐encoded small RNA genes in nasopharyngeal carcinomas,gastric carcinomas,and healthy donors in northern China

It has been generally believed that the Epstein–Barr virus (EBV)‐encoded small RNA 1 and 2 (EBER1 and EBER2) genes are conserved as two families that correlated with type 1 (B95‐8) and type 2 (AG876 or P3HR‐1) EBV strains. EBER polymorphism and its association with EBV‐associated disease have not received much attention. To explore the variations of EBER genes in different malignancies and healthy donors, the sequences of EBER genes were analyzed in 154 EBV‐positive samples, including 47 nasopharyngeal carcinoma (NPC), 50 EBV‐associated gastric carcinoma (EBVaGC) biopsies and 57 throat washing (TW) samples from healthy donors in northern China, where NPC is non‐endemic. Three main distinct variants of EBER genes, designated as EB‐6m, EB‐8m, and EB‐10m, were identified. EB‐6m had a previously identified EBER sequence identical to P3HR‐1 and was found in 33/47 (70.2%) NPC, 48/50 (96.0%) EBVaGC, and 54/57 (94.7%) TW isolates. EB‐8m and EB‐10m were new EBER variants with more mutations in EBER2 genes. They were found in 13/47 (27.7%) NPC cases, whereas in only 1/50 (2.0%) EBVaGC cases and not found in TW cases. The distributions were significantly different (P < 0.05). Other five isolates (one NPC, one EBVaGC and three TW cases) showed different sequences and could not be assigned to any of the three groups. Type 1 EBV strains showed heterogeneous in terms of EBER variants. These results suggest that EBER locus can be useful to identify different EBV isolates, and it would be interesting to evaluate the association of EBER polymorphisms with EBV‐associated tumors. J. Med. Virol. 82: 829–836, 2010. © 2010 Wiley‐Liss, Inc.     

2005-2010年北京地区儿童原发性EBV感染流行株EBNA1与LMP1基因特征分析

目的 分析2005-2010年北京地区儿童原发性EBV感染流行株EBNA1与LMP1基因特征,为研究EBV变异株与疾病临床表型间是否存在相关性提供背景资料.方法 应用PCR方法扩增EBV的EBNA3C、EBNA1和LMP1基因片段,测序后应用BioEdit 7.0.9和Mega 4.0.2软件进行序列分析.结果 62例进行了EBV分型,以EBV-Ⅰ型为主,检出率为98％.62例EBNA1基因扩增阳性,其中V-val亚型(均是Vvv1变异株)为98％.50例LMP1基因羧基段扩增阳性,以China 1为主,其检出率为90％.Vvv1变异株和China 1变异株在EBV-IM与EBV-HLH中的检出率差异均无统计学意义(P=1.00).40例进行了多基因连锁分析,其中EBV-Ⅰ型、EBNA1-Vvv1变异株和LMP1-China 1变异株高度连锁,其连锁检出率为90％.35例患儿LMP1基因全长扩增阳性,CG1-CG4的检出率分别为85％、6％、6％和3％.结论 北京地区儿童原发性EBV感染疾病中,EBV亚型以EBV-Ⅰ型为主,Vvv1变异株和China 1变异株分别是EBNA1和LMP1变异株中的优势变异株,且二者高度连锁.儿童原发性EBV感染流行株可以分为CG1-4组,其中以CG1为主.     

Association of distinctive Epstein–Barr virus variants with gastric carcinoma in Guangzhou,southern China

To investigate the clinicopathologic features, Epstein–Barr virus (EBV) latency pattern and genome polymorphism of EBV‐associated gastric carcinoma (EBVaGC) in Guangzhou, an endemic area of nasopharyngeal carcinoma (NPC), an in situ hybridization assay of EBV‐encoded small RNA‐1 (EBER‐1) was used to identify the presence of EBV in 676 consecutive gastric carcinoma cases. EBV‐encoded proteins EBNA1, EBNA2, LMP1, and ZEBRA were detected by immunohistochemistry. EBV genome polymorphism was also analyzed by PCR and DNA sequencing. Of the 676 cases, 45 EBV‐positive cases (6.7%) were identified, including 37 (8.5%) male and 8 (3.3%) female cases. EBNA1 was detected in 42 cases (93.3%), while EBNA2, LMP1, and ZEBRA were all negative. In the EBV genome polymorphism analysis, type A strain, prototype F, type I, XhoI?, and del‐LMP1 variants were predominant among EBVaGC patients, accounting for 44 (97.8%), 37 (82.2%), 45 (100%), 34 (75.6%), and 42 (93.3%) cases, respectively. Moreover, a new hotspot mutation in the BamHI‐W1/I1 boundary region (148,972 T → C) was found in 39 (86.7%) of the 45 cases. The predominant EBV variants in EBVaGC in Guangzhou are prototype F, type I, and XhoI?, which are different from those in NPC in this area (predominant variant‐type “f”) and in EBVaGC in Latin American countries (predominant type “i” and XhoI+), suggesting that the EBV variants are not only geographically distributed but also disease restricted, and the pathogenic role of EBV in different EBV associated epithelial malignancies in different areas may be distinct. J. Med. Virol. 82:658–667, 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.