EBV-determined Nuclear Antigen Research Articles

Potential Role of Epstein-Barr Virus in Oral Potentially Malignant Disorders and Oral Squamous Cell Carcinoma: A Scoping Review.

Though the oral cavity is anatomically proximate to the nasal cavity and acts as a key reservoir of EBV habitation and transmission, it is still unclear whether EBV plays a significant role in oral carcinogenesis. Many studies have detected EBV DNA in tissues and exfoliated cells from OSCC patients. However, very few studies have investigated the expression of functional EBV proteins implicated in its oncogenicity. The most studied are latent membrane protein 1 (LMP-1), a protein associated with the activation of signalling pathways; EBV determined nuclear antigen (EBNA)-1, a protein involved in the regulation of gene expression; and EBV-encoded small non-polyadenylated RNA (EBER)-2. LMP-1 is considered the major oncoprotein, and overexpression of LMP-1 observed in OSCC indicates that this molecule might play a significant role in oral carcinogenesis. Although numerous studies have detected EBV DNA and proteins from OSCC and oral potentially malignant disorders, heterogeneity in methodologies has led to discrepant results, hindering interpretation. Elucidating the exact functions of EBV and its proteins when expressed is vital in establishing the role of viruses in oral oncogenesis. This review summarises the current evidence on the potential role of EBV in oral oncogenesis and discusses the implications as well as recommendations for future research.

Epstein-Barr virus-positive gastric cancer involves enhancer activation through activating transcription factor 3.

Epstein‐Barr virus (EBV) is associated with particular forms of gastric cancer (GC). We previously showed that EBV infection into gastric epithelial cells induced aberrant DNA hypermethylation in promoter regions and silencing of tumor suppressor genes. We here undertook integrated analyses of transcriptome and epigenome alteration during EBV infection in gastric cells, to investigate activation of enhancer regions and related transcription factors (TFs) that could contribute to tumorigenesis. Formaldehyde‐assisted isolation of regulatory elements (FAIRE) sequencing (‐seq) data revealed 19 992 open chromatin regions in putative H3K4me1+ H3K4me3− enhancers in EBV‐infected MKN7 cells (MKN7_EB), with 10 260 regions showing increase of H3K27ac. Motif analysis showed candidate TFs, eg activating transcription factor 3 (ATF3), to possibly bind to these activated enhancers. ATF3 was considerably upregulated in MKN7_EB due to EBV factors including EBV‐determined nuclear antigen 1 (EBNA1), EBV‐encoded RNA 1, and latent membrane protein 2A. Expression of mutant EBNA1 decreased copy number of the EBV genome, resulting in relative downregulation of ATF3 expression. Epstein‐Barr virus was also infected into normal gastric epithelial cells, GES1, confirming upregulation of ATF3. Chromatin immunoprecipitation‐seq analysis on ATF3 binding sites and RNA‐seq analysis on ATF3 knocked‐down MKN7_EB revealed 96 genes targeted by ATF3‐activating enhancers, which are related with cancer hallmarks, eg evading growth suppressors. These 96 ATF3 target genes were significantly upregulated in MKN7_EB compared with MKN7 and significantly downregulated when ATF3 was knocked down in EBV‐positive GC cells SNU719 and NCC24. Knockdown of ATF3 in EBV‐infected MKN7, SNU719, and NCC24 cells all led to significant decrease of cellular growth through an increase of apoptotic cells. These indicate that enhancer activation though ATF3 might contribute to tumorigenesis of EBV‐positive GC.

Genome-Wide Analysis of Epstein-Barr Virus Isolated from Extranodal NK/T-Cell Lymphoma, Nasal Type.

Extranodal natural killer (NK) cell/T-cell lymphoma (NKTCL), a rare type of non-Hodgkin's lymphoma, has strongly been associated with Epstein-Barr virus (EBV) infection. However, there are no EBV genomes isolated from NKTCL, and the roles the variations of EBV strains play in the pathogenesis of NKTCL are still unclear. In this study, whole EBV genomes from eight primary NKTCL biopsy specimens were obtained using next-generation sequencing, designated NKTCL-EBV1 to NKTCL-EBV8. Compared with the six mostly referenced EBV strains, NKTCL-EBVs closely resemble the GD1 strain but still harbor 2,072 variations, including 1,938 substitutions, 58 insertions, and 76 deletions. The majority of nonsynonymous mutations were located in latent and tegument genes. Moreover, the results from phylogenetic analysis of whole NKTCL genomes and specific genes demonstrated that all the NKTCL-EBVs were related to Asian EBV strains. Based on the amino acid changes in certain residues of latent membrane protein 1 (LMP1) and EBV-determined nuclear antigen 1 (EBNA1), all the NKTCL-EBVs were sorted to China 1 and V-val subtype, respectively. Furthermore, changes in CD4+ and CD8+ T-cell epitopes of EBNA1 and LMP1 may affect the efficacy for a cytotoxic T lymphocyte (CTL)-based therapy. This is the first large study to our knowledge to obtain EBV genomes isolated from NKTCL and show the diversity of EBV genomes in a whole genome level by phylogenetic analysis. In this study, the full-length sequence of Epstein-Barr virus (EBV) isolated from eight patients with nasal natural killer/T-cell lymphoma (NKTCL) was determined and further compared with the sequences previously reported isolated from other malignancies. Phylogenetic analysis showed that NKTCL-EBV strains are close to other Asian subtypes instead of non-Asian ones, leading to the conclusion that EBV infections are more likely affected by different geographic regions rather than particular EBV-associated malignancies. Therefore, these data have implications for the development of effective prophylactic and therapeutic vaccine approaches targeting the personalized or geographic-specific EBV antigens in these aggressive diseases.

Abstract B049: Immunotherapy of EBV associated gastric cancer using EBV specific T cells

Abstract Epstein-Barr virus (EBV)-associated gastric cancer (EBVaGC) is mediated by EBV-infected malignant cells in gastric intestinal tract. About 10% of gastric carcinoma are EBVaGC worldwide, and over 85,000 patients are developed EBVaGC annually. EBVaGC shows several different aspects with EBV-negative gastric cancer such as clinicopathology, CpG island methylation status and expression of EBV-related genes. Therefore, immunotherapy to treat EBVaGC would be a good approach because expression pattern of EBVaGC is evidently different with that of EBV-negative cells in stomach. Adoptive T cell transfer has been effective for treatment of EBV-positive nasopharyngeal carcinoma (NPC) and lymphomas, which express similar EBV antigens to those of EBVaGC cells. Thus, we wanted to use system of NPC clinical trials to EBVaGC. Targeted EBV antigens expressed both in NPC and EBVaGC were latent membrane proteins (LMPs)-1,2 and EBV-determined nuclear antigen (EBNA) 1. To generate EBV-specific cytotoxic T lymphocytes (EBV-CTLs), we firstly isolated peripheral blood mononuclear cells (PBMCs) from healthy human donor blood by using Ficoll and sorted dendritic cells (DCs) to deliver LMP2A and EBNA1 expressing-vectors by nucleofection into DCs. Nucleofected DCs were cocultured with PBMCs in order to cross-present EBV antigens toward CD4+, CD8+ T cells in DC maturation condition. Generated EBV-CTLs were incubated with IL-2, 7, 15-supplemented CTL media for inducing memory-like cells and expanded for 14 days to apply to adoptive transfer of EBVaGC model. Cytotoxicity of EBV-CTLs were specific for EBV-expressing gastric cancer cell line (SNU 719) in vitro, not EBV-negative gastric cancer cell line (MKN45). To confirm efficacy of EBV-CTLs in mouse model, we developed EBVaGC mouse model using SNU719 and injected 1 × 107 cells/mouse once a week for three times. EBV-CTLs had an effect on the reduction of tumor size of EBVaGC and effectively migrated into tumor site. Also, administered EBV-CTLs stably existed in blood and tumor tissue even over 3 days analyzed by FACS and immunohistochemistry. In conclusion, EBV-CTLs represented improved anti-tumor activity to treat EBVaGC and specific lysis to EBV-expressing cells. Also, any side effects or autoimmune diseases were not detected. Therefore, EBV-CTLs would be likely to play important roles in targeting many EBV-associated cancers and be applied to clinical trials. Note: This abstract was not presented at the conference. Citation Format: Jae Seung Moon, Jung Ho Kim, Sang Kyou Lee. Immunotherapy of EBV associated gastric cancer using EBV specific T cells [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B049.

The clinical significance of serum Epstein-Barr virus-determined nuclear antigen 1 (EBNA1)/latent membrane protein 1 (LMP1) assay in patients with nasal type extranodal NK/T-cell lymphoma

To explore the clinical significance of the serum Epstein-Barr virus-determined nuclear antigen 1 (EBNA1)/latent membrane protein 1 (LMP1) in patients with extranodal NK/T-cell lymphoma, nasal type (ENKL). The serum EBNA1 and LMP1 were detected by real-time PCR in 36 ENKL patients hospitalized in Beijing Tongren Hospital from August 2010 to August 2013. Twenty healthy volunteers were recruited as controls. The median serum EBNA1 was 1.9×10(4) (ranged from 0 to 11.0×10(4)) copies/µl in ENKL patients and 8.0 (ranged from 0 to 43.8) copies/µl in healthy volunteers. The median serum LMP1 was 3.9×10(3) (ranged from 118.3 to 24.0×10(3)) copies/µl in ENKL patients and 3.3 (ranged from 0 to 33.3) copies/µl in healthy volunteers. Both EBNA1 and LMP1 were higher in ENKL patients than healthy volunteers (all P < 0.01). The median EBNA1 and LMP1 in ENKL patients posttreatment were 1.0×10(3) (ranged from 0 to 2.0 × 10(3)) copies/µl and 300.8 (ranged from 0 to 825.7) copies/µl respectively, which were both significantly decreased than pretreatment (all P < 0.05). The EBNA1 and LMP1 were decreased in effective treatment group versus ineffective treatment group (P < 0.05). The serum EBNA1 and LMP1 were positively correlated with lactic dehydrogenase (LDH) level (r = 0.364,0.546; P = 0.040,0.012). (1) The measurement of EBNA1/LMP1 may be useful in evaluating the therapeutic effect. (2) The serum EBNA1/LMP1 may reflect the tumor load in ENKL patients.

Presence of Epstein–Barr virus in cerebrospinal fluid from patients with aseptic meningitis appears to be common

We identified coinfection with Epstein–Barr virus (EBV) and echovirus type 6 as the cause of aseptic meningitis in two of 28 patients diagnosed with the disease at our hospital from June through October 2011. Benjamin et al. [1] have already reported that EBV is frequently detected in the cerebrospinal fluid (CSF) of human immunodeficiency virus (HIV)-positive patients with viral meningitis. Our report is the first to suggest that EBV is commonly present in the CSF of immunocompetent patients with viral meningitis. We diagnosed aseptic meningitis on the grounds of pleocytosis in a 4-year-old boy (patient 1) and a 9-year-old boy (patient 2), both of whom had been previously healthy. EBV was detected by means of polymerase chain reaction (PCR) in the CSF of both patients. Neither had mononucleosis-like symptoms, i.e., generalized lymphadenopathy, splenomegaly, hepatomegaly, or pharyngitis with tonsillar enlargement. Two other members of patient 1’s family had been concurrently diagnosed with enterovirus meningitis, but patient 2 had no family history. The intervals between fever onset and the detection of EBV in the CSF were 24 h in patient 1 and 144 h in patient 2. The CSF white cell counts were 68 cells/mm and 166 cells/mm, respectively. Both patients’ sera were positive for antibodies against EBV-determined nuclear antigens (EBNAs) and negative for anti-EBV IgM, demonstrating reactivation patterns in both patients. Echovirus type 6 was detected in a throat swab from patient 2. To clarify the impact of EBV reactivation on the clinical course of aseptic meningitis, we compared the clinical data on enterovirus meningitis between these two patients and 11 other patients with enterovirus but without EBV in the CSF. Although there was no difference in the duration of fever or hospitalization between the two groups, the interval between fever onset and the detection of EBV in the CSF was longer in patients 1 and 2 (84 h vs. 17.7 h). On the basis of these findings, we propose the following characteristics of EBV coinfection in aseptic meningitis: (1) it is relatively common in normal clinical situations; (2) EBV reactivation develops as a sequela of aseptic meningitis; (3) EBV coinfection has little impact on the prognosis of aseptic meningitis. The family history of enterovirus meningitis in patient 1 and the detection of echovirus type 6 in patient 2 indicated that both patients were coinfected with EBV and echovirus. PCR tests for enterovirus in the CSF were negative, but this is not surprising, especially in patient 2, because it is known that the diagnostic yield of the tests is lower when CSF specimens are obtained 2 days or more after clinical onset [2]. In the case of patient 1, his family history made it highly unlikely that any virus other than echovirus led to T. Kimiya presented the findings of this study at the 8th Congress of the Asian Society for Pediatric Research, Seoul, Korea, May 2012.

Hodgkin Disease–Like Posttransplantation Lymphoproliferative Disorder of Donor Origin in a Renal Allograft Recipient

Posttransplantation lymphoproliferative disorder (PTLD) develops in 1.6% of renal allograft recipients. More than 90% are of recipient origin. There are only a few reports of Hodgkin disease-like PTLD in allograft patients. We report the case of a Hodgkin disease-like PTLD of donor origin in a 16-year-old renal allograft recipient. Fourteen months after transplantation, an increasing inhomogeneous structure in the hilar region of the transplanted kidney became apparent and was excised. Histological examination showed Hodgkin- and Sternberg-Reed-like cells. Immunostaining showed CD20-positive and CD15-negative cells and Epstein-Barr virus (EBV) involvement (EBV-encoded small nonpolyadenylated RNA and EBV-determined nuclear antigen 2). DNA fingerprinting analysis proved the lymphoma to be of donor origin. Treatment consisted of nephrectomy, discontinuation of immunosuppression therapy, and local radiation. Three years after lymphoma removal, the patient was still without relapse and underwent retransplantation with stable function of the second allograft for more than 2 years now.

Epstein-Barr virus infection of rat lymphocytes expressing human CD21 results in restricted latent viral gene expression and not in immunoblastic transformation.

Transgenic rats expressing human CD21 gene (hCD21) driven by the mouse immunoglobulin enhancer were generated. hCD21 was expressed in lymphoid tissues, especially in the spleen and in the brain. Flow cytometric analysis indicated that about 20% of spleen cells, most having a B-lymphocyte marker, expressed hCD21. After Epstein-Barr virus (EBV) infection of spleen cells, EBV-determined nuclear antigen (EBNA) was first detected on Day 4 and reached a maximum of 0.3% on Day 5, but the infection was abortive and was not followed by blastogenesis, cellular DNA synthesis or proliferation. Reverse transcription-polymerase chain reaction (RT-PCR) analyses demonstrated that EBV-infected spleen cells expressed EBNA1 and EBV-encoded small RNA (EBER), but not other latent EBV products. EBNA promoter analysis by RT-PCR indicated that the Q promoter was active, whereas C and W promoters were not active. The present findings indicate that human and rat lymphocytes respond to EBV infection differently in vitro.

Evidence of lytic infection of Epstein-Barr virus (EBV) in EBV-positive gastric carcinoma.

Twenty-one cases of gastric carcinoma were tested for the presence of Epstein-Barr virus (EBV) genome by polymerase chain reaction (PCR) analysis. EBV genome was detected in 3 (14%) of the 21 cases. In situ hybridization for EBV-encoded small RNA 1 showed that EBV genomes were present in almost all carcinoma cells of the 3 cases. Southern hybridization for terminal repeats of the EBV-DNA revealed that the cases carried an individual monoclonal EBV genome. The analysis demonstrated the presence of linear form of EBV-DNA indicating lytic EBV infection in one of the cases. The expression of EBV genes in the cases was analyzed by reverse transcription-PCR. The mRNA for EBV-determined nuclear antigen (EBNA) initiating from the BamHI-Q promoter was detected, while both BamHI-W and -C promoters were not detected. EBNA2 and latent membrane protein (LMP) 1 mRNAs were not detected in all cases, while LMP2A mRNA was detected in 2 cases. The transcripts of EBV immediate-early genes, BZLF1 and/or BRLF1 were detected in 2 of the cases. The transcripts of late lytic genes (BcLF1 and BLLF1) were detected partly in the 3 cases. Our results indicate that lytic EBV infection occurs in EBV-positive gastric carcinomas.

Epstein-Barr virus-encoded poly(A)(-) RNA supports Burkitt's lymphoma growth through interleukin-10 induction.

Akata and Mutu cell lines are derived from Burkitt's lymphoma (BL) and retain the in vivo phenotype of Epstein-Barr virus (EBV) expression that is characterized by expression of EBV-determined nuclear antigen 1 (EBNA1), EBV-encoded RNAs (EBERs) and transcripts from the BAM:HI A region (BARF0). We found that EBV-positive Akata and Mutu cell clones expressed higher levels of interleukin (IL)-10 than their EBV-negative subclones at the transcriptional level. Transfection of an individual EBV latent gene into EBV-negative Akata cells revealed that EBERs were responsible for IL-10 induction. Recombinant IL-10 enabled EBV-negative Akata cells to grow in low (0.1%) serum conditions. On the other hand, growth of EBV-positive Akata cells was blocked by treatment either with an anti-IL-10 antibody or antisense oligonucleotide against IL-10. EBV-positive BL biopsies consistently expressed IL-10, but EBV-negative BL biopsies did not. These results suggest that IL-10 induced by EBERs acts as an autocrine growth factor for BL. EBERs, EBER1 and EBER2, are non-polyadenylated RNAs and are 166 and 172 nucleotides long, respectively. The present findings indicate that RNA molecules could regulate cell growth.

CD21-mediated entry and stable infection by Epstein-Barr virus in canine and rat cells.

We developed an adenovirus vector for transduction of the human CD21 gene (Adv-CD21), the Epstein-Barr virus (EBV)-specific receptor on human B lymphocytes, to overcome the initial barrier of EBV infection in nonprimate mammalian cells. Inoculation of Adv-CD21 followed by exposure to recombinant EBV carrying a selectable marker resulted in the successful entry of EBV into three of seven nonprimate mammalian cell lines as evidenced by expression of EBV-determined nuclear antigen (EBNA). The EBV-susceptible cell lines included rat glioma-derived 9L, rat mammary carcinoma-derived c-SST-2, and canine kidney-derived MDCK. Subsequent selection culture with G418 yielded drug-resistant cell clones. In these cell clones, EBV existed as an episomal form, as evidenced through the Gardella gel technique. Among the known EBV latency-associated gene products, EBV-encoded small RNAs, EBNA1 and transcripts from the BamHI-A rightward reading frame (BARF0), and latent membrane protein 2A were expressed in all EBV-infected cell clones. The viral lytic events could be induced in these cell clones by simultaneous treatment with 12-O-tetradecanoylphorbol-13-acetate and n-butyric acid, but they were abortive, and infectious virus was not produced. These results indicate that once the initial barrier for attachment is overcome artificially, EBV can establish a stable infection in some nonprimate mammalian cells, and they raise the possibility that transgenic animals with the human CD21 gene could provide an animal model for EBV infection.

Epstein-Barr virus-specific antibodies in Epstein-Barr virus-positive and -negative gastric carcinoma cases in Japan.

We examined Epstein-Barr virus (EBV)-specific antibodies in serum samples from 64 and 59 patients with EBV-positive and -negative gastric carcinomas, respectively, and 73 healthy controls using immunofluorescence assays. EBV capsid antigen (VCA) IgG and EBV-determined nuclear antigen (EBNA) IgG were detected in all 196 subjects. The geometric mean titer (GMT) of VCA-IgG, but not EBNA-IgG, was higher in EBV-positive carcinoma cases than in EBV-negative carcinoma cases (P < 0.001). The seroprevalence rates of VCA-IgA and EBV early antigen (EA) IgG were higher in EBV-positive carcinoma cases than in EBV-negative carcinoma cases. Odds ratios (ORs) comparing seroprevalence rates between EBV-positive and -negative carcinoma cases were 3.4 (95% confidence interval [CI] = 1.3-8.8) and 6.6 (95% CI = 2.7-16.3) for VCA-IgA and EA-IgG, respectively. These results suggest that EBV reactivation occurs in vivo, since more than 90% of Japanese are infected with EBV in early childhood. The GMT of VCA-IgG in EBV-negative carcinoma cases was higher than that of healthy controls (P = 0.028). The seroprevalence rates of EA-IgG were greater in EBV-negative carcinoma cases than in healthy controls (OR = 4.9, 95% CI = 1.2-19. 7). VCA-IgA was the only antibody that showed a significantly high seroprevalence and GMT in EBV-positive carcinoma cases, but not in EBV-negative carcinoma cases. Thus, VCA-IgA can be a marker of immune response to EBV in EBV-positive carcinoma cases. Our findings support the hypothesis that if EBV is involved in the development of EBV-positive gastric carcinoma, the EBV reactivation occurs in vivo.

Polymorphism analysis of Epstein-Barr virus isolates from patients with cutaneous natural killer/T-cell lymphoproliferative disorders: A possible relation to the endemic occurrence of these diseases in Japan.

Certain forms of cutaneous lymphomas in Asia are associated frequently with Epstein-Barr virus (EBV) infection, whereas such cases are less common in western countries. The virus-related peptides, EBV-determined nuclear antigen (EBNA)-2 and the latent membrane protein (LMP)-1, play an essential role in cell transformation. The polymorphisms of these EBV genes may be related to their transforming abilities. In order to clarify the viral subtype that may be involved in the incidence of EBV-associated lymphomas, we analyzed the EBNA-2 and LMP-1 gene polymorphisms and mutations in healthy adults and in patients with EBV-associated cutaneous natural killer(NK)/T-cell lymphoproliferative disorders in Japan. In EBV-related cutaneous lymphoproliferative disorders, EBV subtype 1 was found in all 15 cases, and 1 sample contained a dual infection with subtypes 1 and 2. All EBV isolates from our patients lost a Xho-1 site in exon 1 of the LMP-1 gene, and 7 of 13 cases had a Nco-1 site within the promoter region. All isolates without the LMP-1-Xho-1 site had a 30 bp deletion in the carboxy terminus of the LMP-1 gene, except for the isolate from a patient with angioimmunoblastic lymphadenophathy-like T-cell lymphoma in which a novel Nco-1 site was present in exon 1. Eleven of fourteen throat washings from healthy adults which contained EBV-DNA harbored EBV subtype 1, and the EBNA2 region was not amplified in the other 3 samples. The Xho-1 site was lost in 12 (86%) of 14 isolates and the 30 bp deletion was present in 11 (78%) of 14 isolates from the throat washings. The findings indicate that the predominant EBV isolate from Japanese healthy adults and patients with cutaneous NK/T-cell lymphoproliferative disorders is subtype 1 with a 30 bp deletion and loss of a Xho-1 site in the LMP-1 gene. Since previous data indicated that either subtype 1 or the 30 bp deletion variant possesses high tumorigenic activity, the prevalence of subtype 1 containing these mutations might be responsible for the high incidence of EBV-associated lymphoproliferative disorders in Japan.

Detection of Epstein–Barr Virus (EBV) in Hepatocellular Carcinoma Tissue: A Novel EBV Latency Characterized by the Absence of EBV-Encoded Small RNA Expression

In this study, we investigated the presence of Epstein–Barr virus (EBV) in liver tissue from 35 patients with hepatocellular carcinoma (HCC). EBV DNA was detected in 13 patients (37%) by Southern blot hybridization. In 10 of these patients, EBV DNA was present in tumor tissue only, whereas in the other 3, it was detected in both tumor and nontumor tissues. The quantity of EBV DNA detected was equivalent to 1–10 viral DNA molecules/100 cells. EBV-determined nuclear antigen was detected in 7–13% of the carcinoma cells in three tumor tissue samples that contained approximately one copy of the EBV genome/10 cells. A single terminal fragment of EBV DNA was identified in these tissues, suggesting that the EBV-infected cells in HCC represent clonal proliferation. Western blotting and reverse transcription–polymerase chain reaction analyses demonstrated that these three tumor tissue specimens were positive for EBV-determined nuclear antigen 1 and BamHI A transcripts but were negative for the other latent EBV products, including EBV-encoded small RNA. The results indicated that there is a high EBV load in HCC tissue and that all of the HCC tissue examined showed a novel pattern of EBV latency characterized by absence of EBV-encoded small RNA expression.

Epstein-Barr virus promotes epithelial cell growth in the absence of EBNA2 and LMP1 expression.

We attempted to infect primary gastric epithelia (PGE) with recombinant Epstein-Barr virus (EBV) carrying a selectable marker that made it possible to select EBV-infected cells. Cells dually positive for EBV-determined nuclear antigen (EBNA) and cytokeratin were detected in 3 of 21 primary cultures after 3 days of EBV inoculation. From one culture, EBV-infected cell clones were repeatedly obtained at a frequency of 3 to 5 cell clones per 10(6) cells. EBV-infected clones had enhanced population doubling and grew to attain a highly increased saturation density, together with acquisition of marked anchorage independence. The infected clones retained the ultrastructural morphology characteristic of gastric mucosal epithelium and have been growing stably for more than 18 months (corresponding to at least 300 generations) so far, in clear contrast to the parental PGE cells, which ceased growth after 60 generations. The p53 gene of the parental PGE cells was found to be overexpressed, perhaps thereby conferring the basal potential for long-term survival in vitro. Moreover, EBV infection accelerated, to a significant extent, the growth rate and agar clonability of NU-GC-3 cells, an established EBV-negative but EBV-susceptible human gastric carcinoma cell line. Both EBV-converted PGE and NU-GC-3 clones, like EBV-positive gastric carcinoma biopsy specimens, expressed a restricted set of EBV latent infection genes characterized by the absence of EBNA2 and latent membrane protein 1 (LMP1) expression. These results indicate that EBV infection causes a transformed phenotype on PGE in the setting of possible unregulated cell cycling and renders even established gastric carcinoma cells more malignant via a limited spectrum of viral latent-gene expression. This study may reflect an in vivo scenario illustrating multiphasic involvement of EBV in carcinogenesis of gastric or other epithelial cancers.

Epstein-Barr virus contributes to the malignant phenotype and to apoptosis resistance in Burkitt's lymphoma cell line Akata.

In the present study, we established an in vitro system representing the Burkitt's lymphoma (BL)-type Epstein-Barr virus (EBV) infection which is characterized by expression of EBV-determined nuclear antigen 1 (EBNA-1) and absence of EBNA-2 and latent membrane protein 1 (LMP1) expression. EBV-negative cell clones isolated from the EBV-positive BL line Akata were infected with an EBV recombinant carrying a selectable marker, and the following selection culture easily yielded EBV-infected clones. EBV-reinfected clones showed BL-type EBV expression and restored the capacity for growth on soft agar and tumorigenicity in SCID mice that were originally retained in parental EBV-positive Akata cells and lost in EBV-negative subclones. Moreover, it was found that EBV-positive cells were more resistant to apoptosis than were EBV-negative cells. EBV-infected cells expressed the bcl-2 protein, through which cells might become resistant to apoptosis, at a higher level than did uninfected cells. This is the first report that BL-type EBV infection confers apoptosis resistance even in the absence of expression of LMP1 and BHRF1, both of which are known to have an antiapoptotic function. Surprisingly, transfection of the EBNA-1 gene into EBV-negative Akata clones could not restore malignant phenotypes and apoptosis resistance, thus suggesting that EBNA-1 alone was not sufficient for conferring them. Our results suggest that the persistence of EBV in BL cells is required for the cells to be more malignant and apoptosis resistant, which underlines the oncogenic role of EBV in BL genesis.

Cell-to-cell contact as an efficient mode of Epstein-Barr virus infection of diverse human epithelial cells.

We show clear evidence for direct infection of various human epithelial cells by Epstein-Barr virus (EBV) in vitro. The successful infection was achieved by using recombinant EBV (Akata strain) carrying a selective marker gene but without any other artificial operations, such as introduction of the known EBV receptor (CD21) gene or addition of polymeric immunoglobulin A against viral gp350 in culture. Of 21 human epithelial cell lines examined, 18 became infected by EBV, as ascertained by the detection of EBV-determined nuclear antigen (EBNA) 1 expression in the early period after virus exposure, and the following selection culture easily yielded a number of EBV-infected clones from 15 cell lines. None of the human fibroblasts and five nonhuman-derived cell lines examined was susceptible to the infection. By comparison, cocultivation with virus producers showed approximately 800-fold-higher efficiency of infection than cell-free infection did, suggesting the significance of direct cell-to-cell contact as a mode of virus spread in vivo. Most of the epithelial cell lines infectable with EBV were negative for CD21 expression at the protein and mRNA levels. The majority of EBV-infected clones established from each cell line invariably expressed EBNA1, EBV-encoded small RNAs, rightward transcripts from the BamHI-A region of the virus genome, and latent membrane protein (LMP) 2A, but not the other EBNAs or LMP1. This restricted form of latent viral gene expression, which is a central issue for understanding epithelial oncogenesis by EBV, resembled that seen in EBV-associated gastric carcinoma and LMP1-negative nasopharyngeal carcinoma. The results indicate that direct infection of epithelial cells by EBV may occur naturally in vivo, and this could be mediated by an unidentified, epithelium-specific binding receptor for EBV. The EBV convertants are viewed, at least in terms of viral gene expression, as in vitro analogs of EBV-associated epithelial tumor cells, thus facilitating analysis of an oncogenic role(s) for EBV in epithelial cells.

Establishment of Epstein-Barr virus-positive human gastric epithelial cell lines.

We have established two Epstein‐Barr virus (EBV)‐positive cell lines, GT38 and GT39, derived from human gastric tissues of two patients bearing gastric carcinoma. Both cell lines were positive for cytokeratin, an epithelial marker, but not for lymphocyte‐related markers. Unlike GT39 cell line, GT38 cells lacked the property of contact inhibition. EBV genome was detected in both cell lines. The cell lines were positive for latent membrane protein 1, and EBV‐determined nuclear antigen 1 (EBNA1). EBNA2 was also detected in GT38. These cell lines should be useful for studying the interaction of EBV with gastric epithelial cells and its role in gastric carcinogenesis.

Epstein-Barr virus infection in non-carcinomatous gastric epithelium

Gastric tissue specimens from 20 patients with chronic atrophic gastritis, one of whom also had an early gastric carcinoma, were studied for evidence of Epstein-Barr virus (EBV) infection by Southern blot analysis, DNA and RNA in situ hybridization, and immunohistochemistry for the presence of the EBV-determined nuclear antigen 1 (EBNA-1) and the latent membrane protein 1 (LMP-1). EBV DNA was detected in two cases with chronic atrophic gastritis and in the case with early gastric carcinoma by Southern blot hybridization. DNA in situ hybridization showed EBV genomes in the epithelial cells of two other cases with chronic atrophic gastritis and in non-carcinomatous and carcinomatous epithelial cells of the early gastric carcinoma case. EBNA-1 was detected in all cases. LMP-1 was detected in areas of intestinal metaplasia in eight patients with chronic atrophic gastritis. EBV-encoded small RNA 1 (EBER-1) expression was limited to carcinoma cells. These results show that gastric epithelium is frequently infected with EBV and suggest that prolonged EBV persistence may contribute to the development of gastric carcinoma.

Epstein-Barr virus (EBV) hypersensitivity of peripheral B lymphocytes in patients with EBV genome-positive Burkitt's lymphoma

Peripheral blood mononuclear cells (PBMC) from four Japanese patients with Epstein-Barr virus (EBV) genome-positive Burkitt's lymphoma (BL) during remission were exposed to the B95-8 strain of EBV. Maximum concentrations of the EBV-determined nuclear antigen (EBNA) before cellular DNA synthesis were similar to those of healthy counterparts. Subsequently, EBV-immortalised cell lines were established. These immortalised lymphoblastoid cells were treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) and superinfected with the P3HR-1 strain of EBV. EBV early antigens (EA) and viral capsid antigen (VCA) were expressed in approximately 3-10 fold higher concentrations by these lymphoblastoid cells than by those from patients with other types of malignant neoplasia including EBV genome-negative BL and from healthy counterparts. Moderate to extremely high IgG antibody titres to EBV VCA as well as IgG antibodies to EA were demonstrated in these patients during the study. These results suggest that defective underlying cellular mechanisms for regulating the replication of EBV may be present in patients with EBV genome-positive BL.