Epstein-Barr Virus Variants Research Articles

Highly Efficient CRISPR/Cas9-Mediated Cloning and Functional Characterization of Gastric Cancer-Derived Epstein-Barr Virus Strains.

The Epstein-Barr virus (EBV) is etiologically linked to approximately 10% of gastric cancers, in which viral genomes are maintained as multicopy episomes. EBV-positive gastric cancer cells are incompetent for progeny virus production, making viral DNA cloning extremely difficult. Here we describe a highly efficient strategy for obtaining bacterial artificial chromosome (BAC) clones of EBV episomes by utilizing a CRISPR/Cas9-mediated strand break of the viral genome and subsequent homology-directed repair. EBV strains maintained in two gastric cancer cell lines (SNU719 and YCCEL1) were cloned, and their complete viral genome sequences were determined. Infectious viruses of gastric cancer cell-derived EBVs were reconstituted, and the viruses established stable latent infections in immortalized keratinocytes. While Ras oncoprotein overexpression caused massive vacuolar degeneration and cell death in control keratinocytes, EBV-infected keratinocytes survived in the presence of Ras expression. These results implicate EBV infection in predisposing epithelial cells to malignant transformation by inducing resistance to oncogene-induced cell death. Recent progress in DNA-sequencing technology has accelerated EBV whole-genome sequencing, and the repertoire of sequenced EBV genomes is increasing progressively. Accordingly, the presence of EBV variant strains that may be relevant to EBV-associated diseases has begun to attract interest. Clearly, the determination of additional disease-associated viral genome sequences will facilitate the identification of any disease-specific EBV variants. We found that CRISPR/Cas9-mediated cleavage of EBV episomal DNA enabled the cloning of disease-associated viral strains with unprecedented efficiency. As a proof of concept, two gastric cancer cell-derived EBV strains were cloned, and the infection of epithelial cells with reconstituted viruses provided important clues about the mechanism of EBV-mediated epithelial carcinogenesis. This experimental system should contribute to establishing the relationship between viral genome variation and EBV-associated diseases.

Epstein-Barr Virus Infection in Gastric Remnant Carcinoma and Recurrent Gastric Carcinoma in Qingdao of Northern China

BackgroundEpstein-Barr virus (EBV) is associated with a subset of gastric carcinoma which was defined as EBV associated gastric carcinoma (EBVaGC). The proportion of EBVaGC in gastric remnant carcinoma (GRC) which occurs in the intact stomach five or more years after gastric surgery for benign disease is significantly higher than that in conventional gastric carcinoma (CGC). The infection of EBV in recurrent gastric carcinoma (RGC) with local anastomotic recurrence is poorly understood.Methods53 cases of GRC and 58 cases of RGC were analyzed for the presence of EBV, and the variants of EBV Encoded RNAs (EBER), EBV Nuclear Antigen 1 (EBNA1) and Latent Membrane Protein 1 (LMP1) gene in both groups were investigated.ResultsThirteen (24.5%) out of 53 GRC cases and 3 (5.2%) out of 58 RGC cases were identified as EBVaGCs. In 17 paired RGC cases, only one case was classified as EBVaGC in both times specimen. Another one case was identified as EBVaGC in the primary gastroectomy specimen while the recurrent gastric cancer was not. The third EBVaGC in RGC was identified while the primary gastric cancer was not EBVaGC. In GRC and RGC cases, type 1, type F, EB-6m, V-val subtype, del-LMP1 were predominant type or variants, accounting for 10(76.9%) and 2(66.7%), 13(100%) and 3(100%), 13(100%) and 3(100%), 9(69.2%) and 3(100%), 12(92.3%) and 3(100%), respectively. However, Type C was the predominant type in GRC accounting for 9(69.2%) cases while type D was the predominant one accounting for 2(66.7%) cases in RGC.ConclusionsThe prevalence of EBVaGc in GRC and RGC was significantly different. The distributions of these variants were similar to each other in the two groups which indicated that there were no more aggressive EBV variants in EBVaGC in GRC compared with that in RGC.

Intrafollicular Epstein-Barr virus-positive large B cell lymphoma. A variant of "germinotropic" lymphoproliferative disorder.

Germinotropic lymphoproliferative disorders were previously described as localized disorders associated with coinfection by human herpes virus 8 and Epstein-Barr virus and characterized by good clinical outcome. We report the clinical, morphological, phenotypical, and molecular features of three cases of a hitherto unreported variant of Epstein-Barr virus (EBV)-positive, human herpes virus 8 (HHV8)-negative large B cell lymphoma with exclusive intrafollicular localization. All cases occurred in elderly individuals (63, 77, and 65years old; one male, two females) without obvious immunedeficiency, who presented with high stage disease. Lymph nodes showed an effaced nodular architecture with abnormal B follicles colonized by EBV+ large, pleomorphic atypical cells, including Reed-Sternberg-like cells, showing an activated B cell phenotype (CD10-FOXP1-Bcl6-IRF4+ or CD10-FOXP1+Bcl6+IRF4+) and intense expression of CD30. No monoclonal light-chain restriction was detected by immunohistochemistry or in situ hybridization, and IGH rearrangement was polyclonal; notably, EBV clonality was detectable in one case. Lymphoma cells in all cases showed diffuse expression of the c-Myc protein, while Bcl2 was dim or negative; moreover, the strong expression of phosphorylated-STAT3 in tumor cell nuclei suggested activation of the JAK-STAT pathway. FISH analysis was performed in two cases and showed no translocations of BCL2, BCL6, MYC, and PAX5 genes. Response to treatment was poor in 2/3 patients: one died after 18months, one is alive with disease after 12months. The intrafollicular EBV-positive large B cell lymphoma expands the spectrum of EBV-associated lymphoproliferative disorders in immunocompetent individuals.

Killer B lymphocytes and their fas ligand positive exosomes as inducers of immune tolerance.

Induction of immune tolerance is a key process by which the immune system is educated to modulate reactions against benign stimuli such as self-antigens and commensal microbes. Understanding and harnessing the natural mechanisms of immune tolerance may become an increasingly useful strategy for treating many types of allergic and autoimmune diseases, as well as for improving the acceptance of solid organ transplants. Our laboratory and others have been interested in the natural ability of some B lymphocytes to express the death-inducing molecule Fas ligand (FasL), and their ability to kill T helper (TH) lymphocytes. We have recently shown that experimental transformation of human B cells by a non-replicative variant of Epstein-Barr virus (EBV) consistently resulted in high expression of functional FasL protein. The production and release of FasL+ exosomes that co-expressed major histocompatibility complex (MHC) class II molecules and had the capacity to kill antigen-specific TH cells was also observed. Several lines of evidence indicate that FasL+ B cells and FasL+MHCII+ exosomes have important roles in natural immune tolerance and have a great deal of therapeutic potential. Taken together, these findings suggest that EBV-immortalized human B lymphoblastoid cell lines could be used as cellular factories for FasL+ exosomes, which would be employed to therapeutically establish and/or regain immune tolerance toward specific antigens. The goals of this review are to summarize current knowledge of the roles of FasL+ B cells and exosomes in immune regulation, and to suggest methods of manipulating killer B cells and FasL+ exosomes for clinical purposes.

Whole Genome Sequences of Epstein-Barr Viruses Associated with Burkitt Lymphoma Tissues

Epstein-Barr virus (EBV), first discovered from Burkitt’s lymphoma (BL), is a class 1 carcinogen that is now associated with a wide range of hematologic and epithelial cancers, including lymphoma nasopharyngeal carcinoma (NPC), gastric cancer, Hodgkin lymphoma and some AIDS-associated B cell lymphoma. Although almost all BL cases from Africa and NPC in China are EBV-positive, consistent with a direct role of EBV in tumor causation, the precise nature of the mechanisms of causation remains to be elucidated.Of interest, EBV is ubiquitous and causes asymptomatic lifelong infection. Up to 95% of developing world population is infected at an early age. In contrast, the geographical patterns of EBV-associated cancers and their peaks age-incidences vary. For example, BL incidence is highest in equatorial Africa, where peak risk occurs in children aged 5-9 years. By contrast, NPC incidence is highest in Southern China and also parts of northern Africa; with peak risk in the elderly. These variations have led to speculation about presence of EBV variants with different penetration and expression. Previous studies attempting to examine this question have focused on genetic variation in one or only a few EBV genes at a time, precluding firm conclusions about genetic variation.Whole EBV genome analysis in tumor and non-diseased tissue from the same individual as well from healthy individuals in at risk populations may facilitate discovery of sequence heterogeneity that might be associated with cancer risk. Since the first genome of EBV was published, 23 whole EBV genomes have been sequenced, including from 3 BL cell lines, 5 immortalized B cells of normal blood donors (B95-8 plus WT-EBV), and 13 NPC biopsies. No EBV genomes have however been sequenced to date directly from BL biopsies and from healthy individuals from the same region.The goal of this study is to obtain a comprehensive assessment of EBV genome in BL tissue, and to determine how these sequences differ from EBV genomes in matched non-tumor reservoir of same individuals and from EBV genomes in healthy individuals from same regions.We have available 50 BL biopsies, 37 representing endemic BL from Africa, 13 from South America, and normal tissue from healthy individuals from the same region. We are reporting preliminary data obtained from whole genome sequences of EBV genomes from six BL biopsies from West Africa and South America obtained using high-throughput sequencing (HTS) Illumina MiSeq platform. Using the WT-EBV as a reference, EBV genomes in the BL biopsies showed considerable variation ranging from 550-1200 variations per genome (Fig 1). Most were single nucleotide variations. Insertions and deletions ranged between 15 and 51 per genome. As much as one third of variations resulted in amino acid changes. Surprisingly some of the BL biopsies contained EBV genomes with heterozygous reads, suggesting that ongoing mutations in the EBV genome occurred after clonal expansion of BL cells. Novel variations were observed in BZLF1 suggesting a possible influence of variation on regulation of EBV lytic cycle. Using an in-house EBV genome database prepared for comparative analysis that contained genomic DNA sequences of the 23 published EBV genomes, sequence comparison and phylogenetic analysis showed a much greater sequence diversity among EBV sequences from BL biopsies on a whole-genome level. Based upon these results, we are proposing to expand EBV genome wide sequencing from the remaining BL biopsies as well as from paired normal subjects to determine variations commonly associated with BL and to understand how these EBV genomic variations contribute to BL pathogenesis in different geographic areas. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A cancer-associated Epstein-Barr virus variant with epithelial tropism

A recent study has identified an Epstein-Barr virus variant from nasopharyngeal carcinoma with unusually high tropism for epithelial cells and low tropism for B cells, as well as a propensity to enter the lytic cycle in both cell types. The results suggest the existence of EBV variants with increased cancer risk.

Epstein-barr virus diversity in immunocompetent healthy persons: Reassessment of the distribution of genetic variants

Epstein-Barr virus (EBV) has many strains; however, it remains unclear whether a causal relationship exists between different regions and viral genetic variants in healthy persons. This study was designed to examine the relationship between EBV strains in tonsils and adenoids and peripheral blood lymphocytes of the same individuals using different measurements of EBV strain polymorphism. This study examined whether EBV contains two or three copies of a tandem repeat sequence in the first intron of the BZLF-1 gene. The genotype of the virus from P3HR-1, designated Z*, yielded a 415-bp product, and this was distinguished from the smaller, 386-bp product obtained with the B95-8 virus, designated the Z genotype. Simultaneous sequence infections with Z and Z* genotypes were also detected in one of the tonsils examined, suggesting that more than one strain or variant of EBV genotype may be present in a specimen from the same subject. Co-infection with Z and Z* was recognized in two subjects, so variation of the EBV gene may be seen in at least two different strains of EBV. It was seen that Z and Z* strain-infected cells are constantly in flux through lymph nodes and/or the blood stream in healthy persons; therefore, these results indicated that EBV genome variants probably show no specific tissue distribution.

A Spectrum of Basaloid Morphology in a Subset of EBV-Associated “Lymphoepithelial Carcinomas” of Major Salivary Glands

Nasopharyngeal carcinomas of the undifferentiated or lymphoepithelial type are most commonly seen in South East Asians. Identical tumors have also been described at a variety of other sites including lung, skin and salivary gland and have been referred to by a number of names including lymphoepithelial carcinoma (LEC). LECs of major salivary gland are extremely rare. They are particularly common amongst the Inuit populations of the arctic region including Greenland (Denmark), Canada and Alaska, as well as South East Asians. Within the Inuit group, this tumor represents the majority of all salivary gland carcinomas. Amongst primary LEC of major salivary gland, most cases reported in the literature have represented typical nasopharynx-like tumors. Variants of Epstein-Barr Virus (EBV) associated LEC have not been described previously, to the best of our knowledge. In this report, we describe 4 EBV-associated major salivary gland LECs with prominent basaloid morphology, which represent 22% of a cohort of 18 salivary LECs from an Inuit population in Greenland. The features described in these cases raise a differential diagnosis of other basaloid tumors, particularly in light of the salivary gland location. A basaloid variant of LEC in major salivary gland should be recognized, especially in highly prone populations, to avoid misdiagnosis of other more common salivary tumors.

Distinctive Epstein-Barr Virus Variants Associated with Benign and Malignant Pediatric Pathologies: LMP1 Sequence Characterization and Linkage with Other Viral Gene Polymorphisms

Distinctive Epstein-Barr Virus Variants Associated with Benign and Malignant Pediatric Pathologies: LMP1 Sequence Characterization and Linkage with Other Viral Gene Polymorphisms

Epitope mapping of gp350/220 conserved domain of epstein barr virus to develop nasopharyngeal carcinoma (npc) vaccine.

Nasopharyngeal carcinoma (NPC) is a malignant tumor in the nasopharyngeal epithelial cells that caused by many factors, one of which is the viral infection of EBV (Epstein Barr Virus). The standard treatments to cure NPC still have not been encouraging. The prevention through vaccination is an effective way to stop the disease. However, EBV vaccine being able to cover all variants of virus is still not available yet. Therefore, we identified the conserved region of glycoprotein 350/220 of EBV which has immunogenic and antigenic properties. The glycoprotein 350/220 is viral surface protein responsible to bind CR2 receptor, mediated EBV to enter the host cell. The conserved domain is crucial for EBV in infecting host cells. Further, by blocking CR2 binding domain of gp350/220 using antibody will inhibit EBV's spreading, and provoke an immune system to eliminate the virus in a patient. Glycoprotein 350/220 from all variants of Epstein-Barr virus was retrieved from NCBI. The conserved domain of gp350/220 was identified by aligning the protein sequences and structures. The polymorphic structure was used as a template for docking analysis to identify the resemblance of amino acid from polymorphic variants of gp350/220 that binds CR2. The epitope mapping of gp350/220 was done by Discotope BepiPred method. The result revealed that the conserved region of gp350/220 was predicted to have an epitope, QNPVYLIPETVPYIKWDNC residue, and it does not have any similarities to the human's cell surface protein. Therefore, it can be used as a reference to develop vaccine to prevent NPC.

Distinctive Epstein-Barr Virus Variants Associated with Benign and Malignant Pediatric Pathologies: LMP1 Sequence Characterization and Linkage with Other Viral Gene Polymorphisms

The ubiquitous Epstein-Barr virus (EBV) is related to the development of lymphoma and is also the etiological agent for infectious mononucleosis (IM). Sequence variations in the gene encoding LMP1 have been deeply studied in different pathologies and geographic regions. Controversial results propose the existence of tumor-related variants, while others argued in favor of a geographical distribution of these variants. Reports assessing EBV variants in IM were performed in adult patients who displayed multiple variant infections. In the present study, LMP1 variants in 15 pediatric patients with IM and 20 pediatric patients with EBV-associated lymphomas from Argentina were analyzed as representatives of benign and malignant infections in children, respectively. A 3-month follow-up study of LMP1 variants in peripheral blood cells and in oral secretions of patients with IM was performed. Moreover, an integrated linkage analysis was performed with variants of EBNA1 and the promoter region of BZLF1. Similar sequence polymorphisms were detected in both pathological conditions, IM and lymphoma, but these differ from those previously described in healthy donors from Argentina and Brazil. The results suggest that certain LMP1 polymorphisms, namely, the 30-bp deletion and high copy number of the 33-bp repeats, are associated with EBV-related pathologies, either benign or malignant, instead of just being tumor related. Additionally, this is the first study to describe the Alaskan variant in EBV-related lymphomas that previously was restricted to nasopharyngeal carcinomas from North America.

Characteristics of epstein barr virus variants associated with gastric carcinoma in Southern Tunisia

BackgroudEBV-associated Gastric Carcinoma (EBVaGC) has a distinct clinical features and its prevalence is variable worldwide.ResultsTo determine the prevalence of EBVaGC in Tunisia, EBV-encoded small RNA (EBER) expression was assessed in 81 gastric carcinoma (GC) specimens. The nuclear EBER expression was detected in 12 out of 81 GC cases (14.81%) and concordance between the score range of EBER staining and the number of EBV DNA copies as estimate by QPCR is observed. On the other hand, we found that EBVaGC strongly correlated with age at diagnosis, and weakly with tumor differentiation and venous invasion.Furthermore, the EBVaGC specimens were subjected to determine the EBV DNA polymorphisms. Our results show a unique genetic profile of the EBV strains regarding the A and D types, the F prototype, the retention of XhoI restriction site and the 30 bp del-LMP1 variant. According to our previous studies on nasopharyngeal carcinoma (NPC), we suggested that EBV strains associated to GC and NPC shared some similarities in Tunisian patients.ConclusionThe prevalence of EBVaGC is of 14.81% in the southern Tunisia and that common EBV strain are associated with both NPC and GC which are likely to differ from Asian strains. Our findings support therefore a certain geographical distribution of EBV strains which is not restricted to EBV-associated malignancies.

Epstein-Barr virus genome polymorphisms of Epstein-Barr virus-associated gastric carcinoma in gastric remnant carcinoma in Guangzhou, southern China, an endemic area of nasopharyngeal carcinoma

Epstein-Barr virus (EBV) is associated with a subset of gastric carcinoma which was defined as EBV-associated gastric carcinoma (EBVaGC). The proportion of EBVaGC in gastric remnant carcinoma (GRC) was apparently higher than that in conventional gastric carcinoma (CGC) which occurs in the intact stomach. To clarify the possible mechanisms, 26 GRC cases from Guangzhou were investigated for the presence of EBV, and the EBV genome polymorphisms of EBVaGC in GRC were analyzed. Besides, the clinicopathologic characteristics, EBV latency pattern of EBVaGC in GRC were also investigated. Eight (30.8%) out of 26 cases were identified as EBVaGCs. Type A strain, prototype F, type I, mut-W1/I1, XhoI− and del-LMP1 variants were predominant among EBVaGC patients, accounting for 7 (87.5%), 7 (87.5%), 8 (100%), 6 (75%), 5 (62.5%) and 8 (100%) cases, respectively. All EBVaGC cases were male and with the histology of diffuse-type carcinoma. The tumor cells expressed EBNA1 (87.5%) and LMP2A (62.5%) but not LMP1, EBNA2 and ZEBRA. Thus, the EBV latency pattern was latency I. These were similar to those in CGC, except for the significantly higher proportion of EBVaGC in GRC than in CGC, suggesting that there is no more aggressive EBV variant in EBVaGC in GRC, and the injuries of gastric mucosa and/or changes of the microenvironment within the remnant stomach may be involved in the development of EBVaGC in GRC. This, to our knowledge, is the first study concerning about the EBV genome polymorphisms of EBVaGC in GRC in the world.

The association of genomic variation of Epstein–Barr virus BamHI F fragment with the proliferation of nasopharyngeal carcinoma

To investigate the f variant of Epstein-Barr virus (EBV) in nasopharyngeal carcinogenesis, we detected the f variant in primary nasopharyngeal carcinoma (NPC), metastatic carcinoma of the lymph node (LN), and chronic inflammation of the nasopharynx from the Guangdong region. Meanwhile, we analyzed the relationship between the f variant of EBV and LMP1, Fascin, pStat3, p53, Bcl-2, and Ki-67 expression in NPC. The results showed that the f variant of EBV was found in 11 cases of primary NPCs with LN metastasis, 12 LN metastases, and 18 primary NPCs without LN metastasis. However, only one demonstrated the F/f variant in 50 cases of chronic inflammation of the nasopharynx. The expression rate of LMP1, Fascin, pStat3, p53, Bcl-2, and Ki-67 in NPC with the f or F/f variant was higher than that with the F prototype. Furthermore, there was a significantly positive correlation between the f variant of EBV and Ki-67 expression (p < 0.05). Our study suggests that the f variant of EBV may be closely related to nasopharyngeal carcinogenesis.

Epstein–Barr virus strains defined by the latent membrane protein 1 sequence characterize Thai ethnic groups

Epstein-Barr virus (EBV) is ubiquitous in the human population and seroepidemiological studies have revealed that more than 90% of adults are infected with the virus in Thailand. It has been suggested that latent membrane protein 1 (LMP1) variants may differ in their tumorigenicity and geographical localization. The distribution of LMP1 variants of EBV in the Thai population was studied. A total of 259 LMP1 sequences from ten Thai ethnic groups (Lahu, Lisu, Shan, Red Karen, White Karen, Hmong, Akha, Mlabri, Moken and Urak Lawoi) were studied using direct PCR sequencing. Nucleotide sequences corresponding to the C terminus of the LMP1, including previously published sequences from central and southern Thais, were used in the phylogenetic analysis. Five strains--the B95-8 prototype, China 1, China 2, Mediterranean (Med) and SEA 2--were identified in ethnic groups in Thailand. The major strain and the distribution pattern differed by group and location. When the ethnic groups were classified by linguistic group, the prevalence of the SEA 2 strain was significantly different between Austro-Thais and other linguistic groups (P=0.0001), whereas, among Tibeto-Burman linguistic groups, the prevalence of the Med strain was different between matrilocal and patrilocal groups (P=0.0245). The distribution of LMP1 strains in ethnic minorities in Thailand is associated with ethnogeographical factors and the social/marriage system. This study thus provides evidence for the importance of interactions between populations in virus diversity.

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.

Epstein-Barr virus genetic variation in Vietnamese patients with nasopharyngeal carcinoma: full-length analysis of LMP1

Genetic variation in tumor virus genes and its impact on function might contribute to the understanding of geographic differences in risks for virus-associated tumors. This is particularly true for the genes known to contribute to the biology of the tumor. It is has been proposed that Epstein-Barr virus (EBV) gene variation has a role in the high risk of nasopharyngeal carcinoma (NPC) in South-East Asia. NPC is among the five most common cancers in Vietnam. EBV-NPC cells always express EBV nuclear antigen 1 (EBNA1) and also frequently latent membrane protein 1 and 2 (LMP1 & LMP2). To investigate EBV gene variation in Vietnamese NPC patients we analyzed the full length of LMP1 gene including its promoter region, and the N-termini of both EBNA1 and LMP2A genes from five NPC biopsies. We detected two EBV variants V1 and V2 based on the LMP1 nucleotide sequence pattern compared with the prototype B95-8 and some available sequences including Chinese variants. The V1 variant shows strong similarity to a variant dominant in Southern China (China 1), while the V2 variant is similar to a Thai variant SEA 2 and partly identity with GD1 in the C-terminus. The promoter region and transmembrane domain of the SEA 2-like samples contained some specific differences compared with previously published variants. In contrast, analysis of EBNA1 N- and LMP2A N-termini only revealed minor changes. Our findings reinforces that the polymorphisms of whole LMP1 sequence should be considered in future EBV molecular epidemiology studies in different geographic populations.

Analysis of Epstein-Barr virus strains and variants in classical Hodgkin's lymphoma by laser microdissection.

Epstein-Barr virus (EBV) seems to have an etiological role in the pathogenesis of classical Hodgkin's lymphoma (cHL). Studies of whole tissue DNA by polymerase-chain reaction (PCR) have shown a considerable number of cHL cases with co-infections by different EBV strains and variants, which apparently contradict the clonality of EBV in cHL previously demonstrated by Southern blot analysis. Due to the paucity of HRS cells in HL tissues, studies on single cell DNA are necessary to identify the specific cellular location (HRS cells and/or bystander B lymphocytes) of the EBV strains and variants present in tissue specimens. In the current study, the presence of EBV was determined by PCR of the 3' end of the LMP-1 gene and EBNA-3C gene in whole tissue and, consecutively, in isolated cells from 26 cases of cHL: 10 HIV-positive and 16 sporadic cHL cases. EBV EBERs were present in all but 2 sporadic cHL cases, which were used as negative controls. At isolated cell level, EBNA-3C gene PCR was more sensitive. Indeed, from the cHL cases in which dual-infection was present, it was observed that, in most of them, HRS cells were infected by type 1 virus, and B lymphocytes were co-infected by both types, which points towards EBV infection occurring early in cHL development. Moreover, the finding of 2 cases with dual-infection in HRS may suggest that, in a small percentage of cHL cases, HRS cells derive from different neoplastic clones, or that HRS cells are superinfected by other viral types after the establishment of the neoplastic clone.

Epstein Barr virus genotypes and LMP‐1 variants in HIV‐infected patients

Two Epstein Barr virus (EBV) genotypes: EBV-1 and EBV-2 have been described. A 30-bp deletion in latent membrane protein-1 gene (del-LMP-1) has been identified in various pathologies. The aim of this study was to determine EBV genotypes and 30-bp deletion frequency in HIV-infected patients from Argentina. The study was performed on 258 individuals: 144 HIV-infected patients that included: (a) 7 AIDS patients with primary central nervous system lymphoma (PCNSL), (b) 62 AIDS patients, and (c) 75 asymptomatic HIV-infected patients. 114 HIV-negative individuals. EBV genotypes and variants in LMP-1 gene were detected by polymerase chain reaction (PCR)-Southern blot on DNA extracted from peripheral blood mononuclear cells and brain biopsies. In PCNSL, the presence of EBV was confirmed by EBER RNA in situ hybridization, and DNA sequencing of 3' end LMP-l gene of PCR products was performed. In HIV-infected patients, EBV-1 was detected in 48.6%, EBV-2 in 18.8%, and co-infection with both genotypes in 32.6%. In control group, EBV-1 was present in 74.3%, EBV-2 in 12.4%, and co-infection in 13.3%. Del-LMP-1 was found in 44.4% of HIV-infected patients samples (20.7% alone and 23.7% co-infection with non-deleted form) while it was found in 25.3% (6.3% alone and 19% with co-infection) in HIV-negative individuals. In HIV-infected patients EBV-2, co-infection and 30-bp deletion are more prevalent than in control group. In all, PCNSL brain biopsies samples, del-LMP-1 always was detected with EBV-2, but more cases would have to be included to draw definitive conclusions.

Polymorphism analysis of Epstein-Barr virus isolates of nasopharyngeal carcinoma biopsies from Tunisian patients

Many studies suggest that the focal distribution of nasopharyngeal carcinoma (NPC) may be influenced not only by host genetics, diet and environments but also by interplay with Epstein-Barr virus (EBV) genetics. Specific EBV gene variants (the A and C types, the BamHI f configuration, a C terminal 30 bp deletion and a N terminal loss of an XhoI site in the BNLF1 gene) have been explored in high incidence areas in southern Asian NPC patients. In contrast, in Tunisia where NPC represents the most frequent type of Head and Neck cancer the distribution of these polymorphisms remains poorly investigated. In order to characterize the epidemiology of EBV variants in Tunisian NPC patients, we have investigated the A or B type of the EBV nuclear antigen (EBNA)2 gene, the C or D type of the BamHI W1/I1 region, the F/f variants of the BamHI F region and the presence or the absence of the XhoI site, 30 bp deletion and Taq1 site in the BNLF1 gene in 47 NPC biopsies, 12 being younger than 30 and 35 older than 30. Our results show a unique genetic profile of the tumor EBV strains regarding the A and D types, the prototype F and retention of the XhoI restriction site in the N terminal region of BNLF1 gene. With regard to the C terminal region of this gene, four genetic profiles were detected: (1) the occurrence of the 30 bp deletion in association with the Taq1 site in 39 cases (83%), (2) the presence of the Taq1 site by itself in 5 cases, (3) the occurrence of the 30 bp deletion by itself in 2 cases and (4) the occurrence of a new deletion of 81 bp covering the 30 bp deletion in association with the Taq1 site in one case. With the exception of the 81 bp deletion, which has not been previously described in the literature, the summarized results have shown the same genetic profile in Tunisian NPC tumor isolates as tumor isolates from other North African and Mediterranean countries. Hence, the observed EBV polymorphisms are not fully specific of to the Tunisian NPCs. Nevertheless, the notion of a divergence between North African and Asian tumor EBV isolates is reinforced by this study.