Functional advantage of NPC-related V-val subtype of Epstein-Barr virus nuclear antigen 1 compared with prototype in epithelial cell line

Epstein-Barr virus (EBV) is closely associated with nasopharyngeal carcinoma (NPC) and the viral nuclear antigen 1 (EBNA1) plays a crucial role in viral latency. Three EBNA1 subtypes, P-ala, V-thr and V-val have been detected from healthy carriers in Guangzhou area. A close relation of V-val EBNA1 with NPC was suggested by its preference to infect NPC cells. We therefore investigated the functional difference among these three EBNA1 subtypes in human epithelial cell line. The three coding sequences of the EBNA1 subtypes were cloned into the pGFP-C2 vector, and transfected into 293 cells, respectively. Effect of EBNA1 expression on cell proliferation was examined. The maintenance activity and expression level of EBNA1-plasmid in 293 cells were evaluated by using GFP as a reporter. The expression of P-ala, V-thr or V-val EBNA1 had no effect on 293 cell growth, while the relative average intensity of fluorescence after 14-day selection in V-val-EBNA1/293 cells was statistically higher than P-ala-EBNA1/293 (P<0.05, t test). We suggest that V-val EBNA1 with the functional advantage compared with prototype shown in this study might contribute to the tumorigenesis of NPC by increasing the expression of itself or other viral or cellular genes.     

Molecular epidemiology of EBNA-1 substrains of Epstein-Barr virus in posttransplant lymphoproliferative disorders which have infrequent p53 mutations

Posttransplant lymphoproliferative disorders (PTLDs), which are highly associated with Epstein-Barr virus infection, have a low frequency of molecular genetic abnormalities. Recently it has been suggested certain EBV substrains may be associated with specific lymphoma subtypes. The goals of our study were two fold: 1) to determine the prevalence of EBNA-1 substrains and prognostic utility in PTLD and 2) to determine the incidence of p53 gene mutations and p53 protein overexpression in 32 EBV-positive PTLD cases. Tumor DNA was sequenced to identify EBNA-1 substrains at codon 487 and p53 gene mutations in exons 5-8. The PTLD samples contained the following EBNA-1 substrains: P-thr in 17/32 (53%), P-ala in 11/32 (34%), and V-leu in 4/32 (13%). More heterogeneity within major subtypes was seen in the PTLD cases than in the referral group. A second group of 25 referral (non-PTLD) samples including infectious mononucleosis (6) and sequential EBV positive virology samples (19) contained P-thr in 17/25 (68%); P-ala in 2/25 (8%); and V-leu in 6/25 (24%). In the 29 B-cell PTLD the time to presentation was an average of 13.3 months in the P-ala group, 16.6 months in the P-thr group, and 40.6 months in the V-leu group: (p>0.05). There was no difference in survival in patients (median overall--60 months) between the three different substrains of EBNA-1 (Log rank test, p=0.39). One of 31 (4.1%) cases (a diffuse large cell B-cell) had a p53 mutation. Seven of 31 (23%) cases (all B-cell), including the p53 mutated case, had over-expression of p53 protein. We conclude EBNA-1 substrains vary in PTLD and suggest the pattern reflects the geographical incidence of substrains in the region. We also conclude p53 mutations are not a significant molecular genetic abnormality in PTLD.     

The 30-bp deletion variant: a polymorphism of latent membrane protein 1 prevalent in endemic and non-endemic areas of nasopharyngeal carcinomas in China.

Development of nasopharyngeal carcinoma (NPC) is closely associated with Epstein-Barr virus (EBV) infection. However, NPC occurs with a marked geographic and racial distribution, whereas EBV infection is ubiquitous in the world. This leads to a question whether certain subtypes of EBV have a greater potential to induce cell transformation. Latent membrane protein 1 (LMP1) is an EBV-encoded oncogenic protein and its 30-bp deleted variant (del-LMP1) has been reported to be predominant in biopsies of NPC. We have assessed the polymorphism of LMP1 in 47 biopsies of NPC, 107 cases of throat washings (TWs) from NPC patients, and 106 cases of TWs from non-NPC patients in Guangzhou, an endemic area of NPC in southern China, as well as 103 cases of TWs from healthy donors in Haerbin, a non-endemic area of NPC in northern China. Our results found a similar extent of the LMP1 polymorphism between NPC patients and non-NPC patients in Guangzhou, with the del-LMP1 being predominant in both Guangzhou and Haerbin. Sequence analyses showed identical substitutions in other coding regions of the del-LMP1 isolated from Guangzhou and Haerbin. These results indicate that del-LMP1 represents a geographic or race-associated polymorphism rather than an NPC disease phenotype-associated polymorphism.     

V-val突变型EB病毒核抗原1的功能研究

背景与目的:EB病毒核抗原1(Epstein-Barr viral nuclear antigen 1,EBNA 1)对于维持EB病毒的潜伏感染有重要作用。V-val变异型的EBNA1与鼻咽癌有密切的相关性。本研究旨在比较原型和V-val变异型的EBNA1基因在上皮细胞中的功能的差异。方法:用PCR方法扩增出原型和V-val变异型EBNA1基因的全长并克隆到pGFP载体上,转染HEK293细胞,检测两种亚型的EBNA1蛋白的表达对细胞生物学性状的影响。以含有EB病毒增强子FR序列的荧光素酶载体作为报告基因,比较两种亚型的EBNA1基因对质粒的转录激活能力。结果:原型和V-val变异型EBNA1基因的表达对HEK293细胞的生长速度没有明显影响,但表达原型EBNA1的细胞的克隆形成能力明显低于V-val亚型。在裸鼠致瘤实验中,接种表达原型和V-val变异型EBNA1细胞的实验组均未见肿瘤形成。但是在瞬时转染实验中,表达V-val变异型EBNA1基因的HEK293细胞的荧光素酶活性明显高于原型EBNA1基因。结论:原型和变异型EBNA1均未发现有明显的转化细胞的能力,但是V-val变异型EBNA1蛋白与原型相比,其转录激活的功能明显增强。     

Consistent sequence variation of Epstein-Barr virus nuclear antigen 1 in primary tumor and peripheral blood cells of patients with nasopharyngeal carcinoma.

PURPOSE: Nasopharyngeal carcinoma (NPC) has been proven as a cancer associated with Epstein-Barr virus (EBV). This study was performed to examine sequence variations of the EBV nuclear antigen 1 gene (EBNA-1) in primary tumor and peripheral-blood cells of NPC patients from Taiwan. EXPERIMENTAL DESIGN: DNA extracted from freshly frozen tumor tissues and corresponding peripheral-blood cells of 13 previously untreated NPC patients were subjected to PCR and direct sequencing using EBNA-1-specific primers. We compared the sequence data and analyzed the clinical outcomes. RESULTS: We obtained a 100% positive-detection rate of EBV DNA in the primary tumors of all patients irrespective of the degree of differentiation. The EBNA-1 gene of all tumor samples was the "V-val" strain, showing the same clustered point mutations. They included 21 nucleotide exchanges, leading to 14 amino-acid mutations and 6 silent exchanges, relative to B95-8 cell line. Two of 13 tumors exhibited an additional point mutation at codon 585. EBV DNA was also detected in peripheral-blood cells of 9 of 13 patients under our experimental conditions. Direct-sequencing data showed match alterations of EBNA-1 gene between the primary tumor and peripheral-blood cells. Tumor relapse was observed in four of nine patients with detectable EBNA-1 DNA in their peripheral-blood cells, whereas none of the four patients without detectable EBNA-1 DNA in their peripheral-blood cells developed tumor relapse. CONCLUSIONS: Results of the current study represents the first demonstration of consistent sequence variation of EBNA-1 in primary tumors and peripheral-blood cells. Clinical observations support that the presence of EBV DNA in the peripheral-blood cells may arise from disseminated cancer cells, resulting in a higher relapse rate and poor prognosis.     

Serum EBV DNA as a biomarker in primary nasopharyngeal carcinoma of Indian origin

BACKGROUND: Nasopharyngeal carcinoma (NPC) is a unique tumor due to its etiology and endemic distribution. Ethnic and regional factors are found to strongly influence the risk of disease; however, there have been no well-conducted studies on Indian patients. The present study assesses the relationship between Epstein-Barr Virus (EBV) and sporadic Indian NPC and the role of serum EBV DNA in NPC detection. METHODS: Primers directed against non-polymorphic Epstein-Barr nuclear antigen-1 (EBNA-1) gene were used to detect the presence of EBV DNA from fresh tissue and serum in NPC, using PCR. RESULTS: EBV DNA was detected in 69% of the biopsies and 58% of the serum of the NPC patients. With respect to histology, WHO Type III NPC, WHO Type II tumors and WHO I tumors showed 100%, 72.2% and 33% EBV positivity, respectively. EBV positivity was also observed in 23% (6/26) of benign samples. All biopsies of patients with positive serum samples were positive for EBV DNA. CONCLUSION: EBV infection was found in sporadic NPC of South Indian origin, which confirms the etiological role of EBV in NPC. Detection of EBNA-1 in the serum and corresponding tissues of NPC patients suggests that the serum EBV DNA originates from NPC and also indicates the benefit of circulating viral DNA as an early marker in the diagnosis of NPC. Serum DNA-PCR methods can be extrapolated to follow-up studies involving tumor regression or to assess the response to various therapies.     

Expression of Epstein-Barr virus-encoded proteins in nasopharyngeal carcinoma

Expression of the Epstein-Barr virus (EBV) encoded nuclear antigens (EBNA 1 to 6) and membrane-associated protein (LMP) was investigated by immunoblotting in 83 nasopharyngeal carcinoma (NPC) biopsies and 25 other tumor and normal tissue specimens from the head and neck region. Fifty-eight of the 83 NPC biopsies were large enough to yield parallel data on virus DNA and viral expression. All 16 cases of clinically diagnosed and histologically confirmed NPCs from North Africa contained EBV DNA and expressed EBNA-1. Of 31 clinically diagnosed NPCs from China, 29 contained EBV DNA and 25 of these expressed EBNA-1. One control tissue biopsy from the oropharynx of NPC patients contained EBV DNA, but none expressed EBNA-1. The latent membrane protein (LMP) was detected in 22/31 of the Chinese and in 10/16 of the North African NPC biopsies. None of the NPC biopsies or control tissues expressed detectable amounts of EBNA 2 or any of the other 4 nuclear antigens which are invariably expressed in EBV-transformed B cells. A smaller number of tumors from Malaysia and East Africa exhibited a similar pattern of expression. EBV was rescued from a nude-mouse-passaged North African NPC tumor by co-cultivation of the tumor cells with umbilical cord blood lymphocytes. The tumor expressed EBNA 1 and LMP, but not EBNA 2 or the other 4 EBNAs. The resulting LCLs expressed all 6 nuclear antigens, EBNA 1 to 6 and LMP. Our data suggest that expression of the EBV genome is regulated in a tissue-specific fashion.     

Detection of Epstein-Barr virus DNA in the peripheral-blood cells of patients with nasopharyngeal carcinoma: relationship to distant metastasis and survival.

PURPOSE: Nasopharyngeal carcinoma (NPC) has been proved to be an Epstein-Barr virus (EBV)-associated cancer. By use of nested polymerase chain reactions (PCRs), we examined whether the presence of EBV DNA in the peripheral-blood cells (PBC) can serve as a prognostic indicator for NPC. PATIENTS AND METHODS: Peripheral blood from 124 patients with NPC who had no evidence of distant metastasis and 114 healthy volunteers with serologically positive findings for EBV infection was collected prospectively. Plasma and erythrocytes were separated. DNA was extracted from PBCs and analyzed by a nested PCR using primers specific to Epstein-Barr virus nuclear antigen 1 (EBNA-1). All patients were treated by radiotherapy with or without chemotherapy. Clinical parameters and status of EBNA-1 in PBCs were used for survival analysis using the Kaplan-Meier method and the Cox proportional hazards model. RESULTS: Positive rates of EBNA-1 DNA in PBCs of NPC patients and healthy volunteers are 71% and 14%, respectively (P =.001). No significant difference was observed with regard to the clinical characteristics of patients who were EBNA-1-positive (n = 88) and those who were EBNA-1-negative (n = 36). After a median follow-up period of 38 months (range, 24 to 56 months), 29 of 88 EBNA-1-positive patients and only one of 36 EBNA-1-negative patients developed distant metastases (P =.00015). Kaplan-Meier estimates of overall survival (P =.0010), metastasis-free survival (P =.0004), and progression-free survival (P =.0004) were significantly lower for the patients in the EBNA-1-positive group than for those in the EBNA-1-negative group. Multivariate Cox analysis confirmed the same results. CONCLUSION: The presence of EBNA-1 DNA in PBCs is a novel, important risk factor for patients with NPC that indicates a significantly higher risk of developing distant metastasis as well as a lower survival rate.     

Molecular epidemiology of Epstein-Barr virus (EBV) in EBV-related malignancies

The prevalent strain of Epstein-Barr virus (EBV) in EBV-related malignancies and in healthy adults in Southern Japan was examined by means of polymerase chain reaction (PCR) and/or restriction fragment length polymorphism (RFLP) analysis. In EBV-related gastric cancers, 51/73 cases were subtype A, 4 were subtype B and the EBNA-2 region was not amplified in 18 cases. Sixty-three were wild-type F, and only one was variant “f”. Sixty-one cases had type C and 2 type D. EBNA-2 subtype A was found in 10/12 EBV-related T/NK-cell lymphomas, and 11 samples harbored the wild-type F. Neither subtype B nor the “f” variant was detected. Type C EBV was found in 8 cases and type D in 3 specimens. Two Japanese nasopharyngeal carcinomas (NPC) harbored subtype A with wild-type F and type C. Throat washings from healthy adults harbored wild-type F virus in 60/153 cases, and 25 of these samples were EBNA-2 subtype A. Type C viruses were detected in 92% of cases and type D in 7.4%. Therefore, the prevalent strain in EBV-related malignancies in Southern Japan was the same as in the healthy population in this geographical region. Int. J. Cancer 72:72–76, 1997. © 1997 Wiley-Liss Inc.     

Complementary serum test of antibodies to Epstein-Barr virus nuclear antigen-1 and early antigen: a possible alternative for primary screening of nasopharyngeal carcinoma

This hospital-based cohort study evaluated the efficacy of three Epstein-Barr virus (EBV) - associated assays for nasopharyngeal carcinoma (NPC) primary screening and monitoring treatment outcome. Five hundred and seventeen consecutive subjects, including 156 NPC patients, 264 healthy volunteers and 97 patients with head and neck squamous cell carcinoma (HNSCC) were enrolled. The sensitivity and specificity of EBV IgAs to viral capsid antigen (VCA), complementary EBV IgAs to early antigen and nuclear antigen-1 (EA+EBNA-1), and EBV DNA load were examined by immunofluorescent assays, enzyme-linked immunosorbent assays, and quantitative real-time PCR, respectively. After constructing the receiver operating characteristics to demonstrate screening efficacy, EBV EA+EBNA-1 IgA (AUC: 0.952; 95% CI, 0.930-0.974) was proved superior to EBV VCA IgA (AUC: 0.888; 95% CI, 0.854-0.922) or EBV DNA load (AUC: 0.893; 95% CI, 0.854-0.932) in differentiating NPC patients from controls. Comparison of screening efficacy between NPC patients and HNSCC patients revealed EBV EA+EBNA-1 IgA (AUC: 0.964; 95% CI, 0.943-0.985) still outperformed EBV VCA IgA (AUC: 0.884; 95% CI, 0.845-0.923). In subjects with higher serum titer or level equal to or above 1:80 and 6 EU/ml for EBV VCA IgA and EA+EBNA-1 IgA, the specificity reached as high as 99.2% and 95.1%, respectively, in the control groups. However, correlation of these three assays with clinicopathological manifestations of NPC, revealed only EBV DNA load significantly associated with N stage and overall stage in NPC patients. Additionally, EBV DNA load could be used to further raise the specificity of EBV EA+EBNA-1 IgA assays and was also the only assay to be consistently predictive of tumor relapse in post-treatment patients according to serial test results by time frame. Consequently, an EBV EA+EBNA-1 IgA-based protocol is recommended for mass screening, but EBV DNA load should be used solely for post-treatment monitoring for NPC in endemic areas.     

Expression of the c-fgr related transcripts in Epstein-Barr virus-associated malignancies

The proto-oncogene c-fgr is expressed at high levels in cell lines derived from lymphomas which are infected with Epstein-Barr virus (EBV) (Cheah et al., 1986). mRNA extracted directly from biopsies of EBV-infected tissues was analyzed on Northern blots to determine if c-fgr is expressed during lympho-proliferations induced in vivo by EBV and in nasopharyngeal carcinoma (NPC), the epithelial malignancy associated with the virus. Elevated levels of c-fgr expression were detected in all EBV-positive lympho-proliferations in vivo but not in cell lines established by EBV infection in vitro. This indicates that the induction of the c-fgr proto-oncogene is not an essential component of EBV-induced transformation. Although no c-fgr expression was detected in EBV-positive or -negative epithelial cell lines, the 3.0-kb c-fgr mRNA was detected at low levels in mRNA obtained from NPC biopsy specimens. However, NPC tissue, after passage in nude mice (which eliminates infiltrating lymphoid and myeloid cells) did not contain the 3.0-kb c-fgr mRNA. The absence of expression of c-fgr in the malignant epithelial cells infected with EBV contrasts with the elevated level of the proto-oncogene in EBV-infected lymphoma tissue and cell lines established from lymphomas. This suggests differences in the expression of cellular functions in EBV-induced malignancies of these 2 distinct cell types.     

Expression of the Epstein-Barr virus genome in a nasopharyngeal carcinoma epithelial tumor cell line

An epithelial tumor cell line was recently established from a biopsy specimen of a nasopharyngeal carcinoma (NPC), and designated HONE-I. Uncloned (parental) HONE-I and HONE-I clone (C)-40 cells were found to contain latent Epstein-Barr virus (EBV). Expression of the latent EBV genome in HONE-I C-40 cells has been examined. It was possible to detect a small percentage of cells spontaneously synthesizing EBV early antigen (EA) and virus capsid antigen (VCA) by immunofluorescence (IF). In addition, the EBV nuclear antigens (EBNA-I and EBNA-2), as well as the EBV latent membrane protein (LMP) were detected in the HONE-I cells. Attempts were made to induce the latent EBV genome in these cells with iododeoxyuridine (IUdR). We observed a significant increase in the number of EA/VCA-positive cells, an increase in EBV DNA, the synthesis of virus particles, and the rescue of infectious virus after treatment of HONE-I C-40 cells with IUdR. The HONE-I C-40 cells should facilitate studies of the expression and regulation of the EBV genome in NPC epithelial tumor cells, which have not previously been available.     

Association between Epstein-Barr Virus Gene Polymorphism and Breast Cancer Risk among Egyptian Females

Background: Epstein-Barr virus (EBV) has been implicated in the development of breast cancer (BC) since 1995. It is classified into A/B genotypes, C/D subtypes, and F/f variants according to variations in its genome. Aim: To determine the distribution difference of EBV types between BC patients and healthy controls in Egypt and to detect the association between different EBV types and BC characteristics. Methods: Three hundred and sixty-two participants (142 BC patients and 220 controls) were enrolled in this study. All participants were screened for EBV infection by determination of viral-capsid-IgG antibodies in their sera. EBNA-1 gene was detected by PCR in tumor biopsies of seropositive patients and in peripheral blood mononuclear cells of controls. A/B genotyping of EBV was performed by nested-PCR targeting the EBNA-2 gene. C/D subtypes and F/f variants were identified by Restriction fragment length polymorphism at BamHI-I W1/I1 and BamHI-F regions of EBV genome, respectively. Results: Among 362 participants, 300(82.9%) were EBV-seropositive, including 120/142(84.5%) of the BC patients and 180/220(81.8 %) of the controls. EBNA-1 gene was positive in 54(45%) of seropositive BC patients and in 38(21.1%) of seropositive controls. There was a significant association of EBNA-1 gene with breast cancer (OR=3.05, 95%CI=1.84-5.07). Moreover, EBNA-1 gene positivity was significantly associated with the more aggressive tumors. Genotype-A and prototype-F were predominant among patients (90.4%, 100%, respectively) as well as among controls (91.7%, 100%, respectively) with no statistical significant association with BC risk.  However, subtype-D was significantly more frequent in patients (95.6%) than in controls (64.7%) and was significantly associated with a higher BC risk as compared to subtype-C (OR=11.7, 95%CI=2.4-57.08). Subtype-D was significantly associated with higher grades tumors (100% among grade III),  with progesteron receptor-negative tumors and with HER2-positive tumors (100% for each). The combined genotypes that significantly associated with BC risk were ADF (OR=4.9) and BDF (OR=5.5). Conclusions: Subtype-D of EBV could be the only EBV type implicated in BC development among Egyptian females and associated more with poor prognosis.     

Detection of distinct Epstein-Barr virus genotypes in NPC biopsies from southern Chinese and Caucasians.

Using the polymerase chain reaction (PCR) to analyze paraffin sections from 12 Caucasian patients, we detected only the prototype F Epstein-Barr virus (EBV) in 10 specimens from patients with nasopharyngeal carcinoma (NPC). This is in contrast to the higher frequency of association of "f" variants in NPC biopsies from Southern Chinese. The results of EBV genotyping support evidence that the EBV strains associated with NPC in the Southern Chinese population differ from those found in Caucasians. DNA sequencing confirmed that a simple point mutation is responsible for the restriction-fragment-length polymorphism which distinguishes the prototype F virus from the "f" variant.     

Association of EBV strains, defined by multiple loci analyses, in non-Hodgkin lymphomas and reactive tissues from HIV positive and HIV negative patients

Epstein-Barr virus (EBV) associated with lymphoid neoplasms demonstrates preferential association with certain viral strains. Previous subtyping studies have however been confined to analysis of sequence variability within a single locus in EBV. Variations have now been reported for several latently expressed EBV genes, including, EBNAs-1, 2 and LMP-1. Variant EBNA-1 strains have been identified in Burkitt's lymphomas and clustering of subtypes for LMP and EBNA-2 have been associated with either malignancy and/or clinical disease. To investigate the linkage between the variability in these three loci in EBV associated with lymphoid malignancies, we subclassified EBV-associated lymphoproliferations (9 reactive and 24 malignant) from HIV-negative and HIV-positive patients by analysis of the EBNA-1, LMP1, and EBNA-2 genes. Our results demonstrate that (1) EBV identical to the prototype B95.8 strain (Type 1 EBNA-2, wild type EBNA-1 and LMP-1) is very rarely associated with tumors. (2) The EBNA-1 variant V-leucine, restricted to malignant lymphomas in immunocompetent patients, was readily identified in non-malignant lesions in HIV infected patients. (3) Variations of EBNA-1 occur independent of variations at other loci.     

THE INTRACELLULAR FORM OF EPSTEIN-BARR VIRUS GENOME IN NASOPHARYNGEAL CARCINOMA

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Development of EBV-positive T-cell lymphoma following infection of peripheral blood T cells with EBV.

Chronic active Epstein-Barr virus (EBV) infection is manifested clinically by the persistence of infectious mononucleosis-like symptoms or its complications for a prolonged period ranging from one to several years. This syndrome may include severe disease manifestations and can be fatal. The role of EBV in the pathogenesis of chronic active EBV infection has been unclear. We investigated two Japanese patients with severe chronic active EBV infection who subsequently developed EBV-positive T-cell lymphoma. We found that the patients had evidence of EBV infection in the peripheral blood CD4+ T-cells 19 and 3 months, respectively, before the T-cell lymphoma was diagnosed. The lymphomas were infected with monoclonal EBV and expressed the EBV latency genes EBNA-1, LMP-1, and LMP-2A, a virus latency pattern referred to as latency II. Genetic studies showed that the virus detected in the T-cell lymphoma was indistinguishable from the virus in the peripheral blood CD4+ T-cells. These studies support an important pathogenetic role of T-cell infection with EBV in chronic active EBV infection and in the EBV-positive T-cell lymphoma that followed.     

Epstein-barr virus genotypes in NPC biopsies from North Africa

The genotypes of Epstein-Barr virus (EBV) were investigated in North African nasopharyngeal carcinoma (NPC) biopsies, nasopharyngeal chronic inflammation (NCI) biopsies, and saliva of healthy individuals from Algeria and Tunisia where there is an intermediate incidence of NPC. The prevalence of A-type virus in NPC, NCI biopsies and saliva of healthy individuals was found in these regions by means of a PCR assay. Restriction enzyme polymorphism analysis by Southern blotting revealed that all North African EBV variants have a conserved restriction site on BamHI W'-1′ and XhoI LMP gene. No additional BamHI enzyme site on the BamHI-F fragment was observed; however, the presence of an extra BamHI site on the BamHI-H fragment giving 2 H1 and H2 fragment-like EBV M-ABA strains was found. All EBV strains present in NPC or NCI biopsies at all ages were homogeneous in these polymorphisms and no correlation was observed between the EBV genotypes from NPC patients and clinical stages of the cancer. These characteristics revealed a significant difference between the EBV variants common in Chinese NPC and those in North African NPC.     

Epstein-Barr Virus and Breast Cancer: Lack of Evidence for an Association in Iranian Women

Controversies regarding the role of Epstein-Barr virus (EBV) in breast cancer and lack of published literature in this regard in Iran, prompted us to assess EBV presence in 100 breast carcinoma and 42 control biopsies obtained from Iranian women. Breast carcinoma cases were comprised of 81 invasive ductal carcinoma NOS, 9 invasive lobular carcinoma, 1 apocrine carcinoma, 2 cribriform carcinoma, 2 papillary carcinoma and 5 mucinous carcinoma. Control biopsies consisted of 13 fibroadenoma, 9 benign epithelial proliferation (adenosis and sclerosing adenosis), 9 usual ductal hyperplasia, 4 atypical ductal hyperplasia, 4 non-proliferative fibrocystic changes and 3 normal breast tissue. To identify EBV-infected cells we applied immunohistochemical analysis, using monoclonal antibody against Epstein-Barr virus-encoded nuclear antigen 2 (EBNA-2) and latent membrane protein 1 (LMP-1). Further, polymerase chain reaction (PCR) was used to amplify EBV DNA, with primers that cover the EBV encoded RNA (EBER) and BamHIW regions. EBNA-2 and LMP-1 immunohistochemistry were negative in all breast cancer and control specimens. Using PCR, none of the 100 breast cancer samples or the 42 control specimens showed detectable EBV DNA. These results indicate that EBV may not play a significant role in the etiology of breast cancer in Iranian women.     

鼻咽癌组织中EBV潜伏膜蛋白2跨膜区CTL表位序列分析

背景与目的：Epstein-Barr病毒在华南地区鼻咽癌细胞中表达核抗原1（EBNA1）、潜伏膜蛋白1（LMP1）和潜伏膜蛋白2（LMP2）等病毒蛋白质。LMP2 mRNA不仅几乎100％表达于鼻咽肿瘤细胞,而且LMP2蛋白还具有较强的免疫原性,是一个较理想的免疫治疗靶点。本研究分析广州地区来源鼻咽癌组织的LMP2基因跨膜区的CTL表位序列,为设计以LMP2抗原为靶点的鼻咽癌免疫治疗提供依据。方法：收集广州地区鼻咽癌患者鼻咽活检组织20例和正常鼻咽粘膜活检组织3例,提取DNA,半巢式PCR扩增LMP2基因跨膜区．直接测序．分析CTL表位序列。结果：与标准株B95．8相比．鼻咽癌和正常鼻咽粘膜活检组织来源的LMP2基因跨膜区存在14处碱基替换,形成6处氨基酸替换．导致4处CTL表位序列变异（SSC、TYG、CLG和VMS）,其中VMS多态性为初次报道。由于从鼻咽癌组织扩增的LMP2序列与从正常鼻咽粘膜扩增的LMP2序列相同,表明这些变化是地域相关的病毒多态性而非鼻咽癌相关的病毒变异。结论：广州地区来源的Epstein-Barr病毒LMP2基因存在多态性．产生4处CTL表位序列变化。在设计以LMP2为靶点的免疫治疗时,应充分考虑病毒基因多态性的影响。