Epstein-Barr Virus Variants Research Articles

DbEBV: A database of Epstein-Barr virus variants and their correlations with human health

Since Epstein-Barr virus (EBV) was discovered in 1964, it has been reported to be associated with various malignancies as well as benign diseases, and the pathogenicity of EBV has been widely studied. Several databases have been established to provide comprehensive information on the virus and its relation to diseases and introduce convenient analysis tools. Although they have greatly facilitated the analysis of EBV at the genome, gene, protein, or epitope level, they did not provide enough insight into the genomic variants of EBV, which have been suggested as relevant to diseases by multiple studies. Here, we introduce dbEBV, a comprehensive database of EBV genomic variation landscape, which contains 942 EBV genomes with 109,893 variants from different tissues or cell lines in 24 countries. The database enables the visualization of information with varying global frequencies and their relationship with the human health of each variant. It also supports phylogenetic analysis at the genome or gene level in subgroups of different characteristics. Information of interest can easily be reached with functions such as searching, browsing, and filtering. In conclusion, dbEBV is a convenient resource for exploring EBV genomic variants, freely available at http://dbebv.omicsbio.info.

Comparative and Phylogenetic Analysis of Epstein–Barr Virus Isolates in Gastric Cancer and Chronic Gastritis Patients in Iran

Aim: To investigate the correlation between Epstein–Barr virus (EBV) variants and EBV-associated gastric cancer (EBVaGC) in Iran. Materials & methods: Biopsies from 21 gastric cancer (4 positive EBER-ISH [EBVaGC] and 17 negative EBER-ISH) and 11 gastritis patients were analyzed using nested PCR and sequencing. Results: Genotype 1 (75%) was the predominant EBV genotype. The P-thr variant was the most common in all groups (positive EBER-ISH 75%, negative EBER-ISH 76.4%, and gastritis 81.8%), followed by P-ala. Additionally, the predominant LMP1 strain in all groups was Med. Sequencing analysis revealed a strong correlation between the P-thr and type 1. Conclusion: Since no preferential correlations were observed between distinct EBV variants and the study groups, these results might indicate geographic polymorphisms.

Evaluation of a Quantitative Dual-Target EBV DNA Test on a Fully Automated Molecular Testing System.

The measurement of Epstein-Barr virus (EBV) deoxyribonucleic acid (DNA) is key to diagnosing and managing EBV-associated complications in transplant recipients. The performance of the new Conformité Européenne (CE) and Food and Drug Administration (FDA)-cleared quantitative Roche cobas EBV real-time PCR assay was determined by using EDTA-plasma dilution panels and clinical samples that were spiked with either the World Health Organization's EBV international standard or high-titer EBV lambda stock. Correlation with the Abbott Realtime EBV assay was assessed in clinical specimens and conducted at two independent laboratories. An in silico analysis revealed that the dual-target test (EBNA1 and BMRF2) was 100% inclusive for the known diversity of EBV. The overall limit of detection (LoD) was 16.6 IU/mL for genotype 1 (GT1). GT2 LoD was verified at 18.8 IU/mL. The linear ranges were from 1.40 × 101 to 2.30 × 108 IU/mL and from 2.97 × 101 to 9.90 × 107 IU/mL for GT1 and GT2, respectively. Accuracy was confirmed across the linear range (mean difference not exceeding ±0.18 log10). Precision was not influenced by the factors analyzed (standard deviation of 0.02 to 0.17 log10), including the presence of potentially interfering endogenous or exogenous substances. Plasma samples were stable under several conditions (variable time points, storage, and freeze/thaw cycles). In clinical EBV DNA-positive samples, correlation between the cobas EBV test and the comparator was high (n = 126 valid results; R2 = 0.96) with a 0.1 mean log10 titer difference. The cobas EBV test is an accurate, sensitive, specific, and reproducible assay for the detection of EBV DNAemia in plasma. In general, high levels of automation and calibration to the international standard will lead to improvements in the harmonization of quantitative EBV DNA test results across laboratories.

Comprehensive Profiling of EBV Gene Expression and Promoter Methylation Reveals Latency II Viral Infection and Sporadic Abortive Lytic Activation in Peripheral T-Cell Lymphomas.

Epstein-Barr virus (EBV) latency patterns are well defined in EBV-associated epithelial, NK/T-cell, and B-cell malignancies, with links between latency stage and tumorigenesis deciphered in various studies. In vitro studies suggest that the oncogenic activity of EBV in T-cells might be somewhat different from that in EBV-tropic B lymphoid cells, prompting us to study this much less investigated viral gene expression pattern and its regulation in nine EBV+ peripheral T-cell lymphoma (PTCL) biopsies. Using frozen specimens, RT-PCR showed 6/7 cases with a latency II pattern of EBV gene expression. Analyses of EBNA1 promoter usage and CpG methylation status in these six cases showed that only Qp was used, while Cp, Wp, and Fp were all silent. However, the remaining case showed an exceptionally unique latency III type with lytic activation, as evidenced by EBV lytic clonality and confirmed by the full usage of Cp and Qp as well as weakly lytic Fp and Wp, fully unmethylated Cp and marginally unmethylated Wp. Further immunostaining of the eight cases revealed a few focally clustered LMP1+ cells in 7/8 cases, with rare isolated LMP1+ cells detected in another case. Double immunostaining confirmed that the LMP1+ cells were of the T-cell phenotype (CD3+). In 6/8 cases, sporadically scattered Zta+ cells were detected. Double staining of EBER-ISH with T-cell (CD45RO/UCHL1) or B-cell (CD20) markers confirmed that the vast majority of EBER+ cells were of the T-cell phenotype. Predominant type-A EBV variant and LMP1 30-bp deletion variant were present, with both F and f variants detected. In summary, the EBV gene expression pattern in PTCL was found to be mainly of latency II (BART+EBNA1(Qp)+LMP1+LMP2A+BZLF1+), similar to that previously reported in EBV-infected nasopharyngeal epithelial, NK/T-cell, and Hodgkin malignancies; however, fully lytic infection could also be detected in occasional cases. Rare cells with sporadic immediate-early gene expression were commonly detected in PTCL. These findings have implications for the future development of EBV-targeting therapeutics for this cancer.

Viral cfDNA Profiling Reveals Distinct EBV Subtypes and Stratifies Risk in Hodgkin Lymphomas

Introduction: Approximately 10-40% of classic Hodgkin Lymphoma (cHL) tumors are Epstein-Barr virus (EBV) positive, and immunodeficiency can predispose to cHL, suggesting that viral infections are important in cHL pathogenesis. We hypothesized that circulating viral genomic and fragmentomic features would inform pathogenicity and evolutionary selection of EBV in cHL and potentially allow for risk stratification of cHL subtypes. We developed Viral CAncer Personalized Profiling by deep Sequencing (VirCAPP-Seq), a novel capture-based method for sequencing viral cell-free DNA (cfDNA). Using VirCAPP-Seq, we surveyed the composition of the circulating virome in cHL patients, and identified EBV mutations under positive selection in cHL. Methods: 352 peripheral blood plasma specimens were collected from 315 cHL patients (mean age = 32, SD = 16) and 44 controls, including cord blood donors and healthy adults (mean age = 43, SD = 16). cfDNA was extracted from plasma and processed as previously described (Newman et al, 2016). Libraries were subjected to hybrid capture with a custom VirCAPP-Seq viral capture panel spanning 180 viral species, including EBV types 1 and 2, and other common oncotropic viruses. Human sequencing reads were subtracted before alignment to a composite reference genome of targeted viral species. Read coverage and plasma cfDNA concentration were used to calculate absolute viral abundance, and expressed as genome copies per mL plasma. Additional EBV cfDNA sequencing data were analyzed from 55 nasopharyngeal carcinoma patients and 24 EBV+ controls (Lo et al, PNAS 2018). EBV variants were called with GATK HaplotypeCaller (v4.1.2). Results: 48% of cHL plasma samples profiled by VirCAPP-Seq had detectable EBV cfDNA above the background level in healthy controls. Plasma EBV load (PEL) was significantly higher in cHL patients with Epstein-Barr-encoded small RNA (EBER) positive tumors (N = 24, mean = 3810, SD = 8083 copies / mL plasma) compared to healthy controls (N = 29, mean = 0.1, SD = 0.25) (p < 0.001, Wilcoxon T-test). PEL strongly predicted tumor EBER status with an accuracy of 94% at a threshold of 32 copies EBV / mL plasma. Unexpectedly, patients with EBER-neg tumors also demonstrated elevated detectable PEL (N = 137, mean = 11, SD = 81 copies / mL plasma). PEL was correlated with International Prognostic Score (IPS) (r = 0.42, p < 0.001), and patients with "high" pre-treatment PEL (≥ 10 copies / mL plasma) had a significantly lower 5-year PFS (73%) compared to patients with "low" pre-treatment PEL (90%) (p = 0.03, Figure A). In contrast, tumor EBER status showed no association with PFS. Hierarchical clustering of circulating viral load for 180 targeted viral species identified subclusters of cHL patients bearing distinct metaviromic signatures, including 18% of cHL patients with elevated plasma Anellovirus burden, an observation associated with compromised immune function (De Vlaminck et al, Cell 2013). Mutation analysis performed on 92 EBV isolates from cHL patients revealed non-synonymous variants in BARF0, BHLF1, and EBNA-1 under strong positive selection by dN / dS analysis. Fragment length analysis showed that EBV cfDNA molecules were, on average, longer in EBER-pos cHL patients (median = 157 bp, IQR = 154 - 158 bp) compared to healthy controls (median = 147 bp, IQR = 143 -149 bp) (Figure B). Strikingly, EBV cfDNA fragment length profiles were similar between EBER-pos and EBER-neg cHL patients (Median = 155 bp, IQR = 151 - 161 bp), suggesting that a significant proportion of EBV cfDNA may yet be tumor-derived in patients with EBER-neg tumors, reflecting either the limited sensitivity of clinical EBER ISH or, perhaps, "hit-and-run" viral oncogenesis. Conclusions: VirCAPP-Seq enables ultrasensitive detection, quantification, and genotyping of EBV cfDNA in cHL patients, and identifies viral variants under strong evolutionary selection. Our findings provide a proof-of-concept for viral cfDNA sequencing as a powerful tool for discovering prognostic biomarkers. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Epstein-Barr Virus in Burkitt Lymphoma in Africa Reveals a Limited Set of Whole Genome and LMP-1 Sequence Patterns: Analysis of Archival Datasets and Field Samples From Uganda, Tanzania, and Kenya.

Epstein-Barr virus (EBV) is associated with endemic Burkitt lymphoma (eBL), but the contribution of EBV variants is ill-defined. Studies of EBV whole genome sequences (WGS) have identified phylogroups that appear to be distinct for Asian versus non-Asian EBV, but samples from BL or Africa, where EBV was first discovered, are under-represented. We conducted a phylogenetic analysis of EBV WGS and LMP-1 sequences obtained primarily from BL patients in Africa and representative non-African EBV from other conditions or regions using data from GenBank, Sequence Read Archive, or Genomic Data Commons for the Burkitt Lymphoma Genome Sequencing Project (BLGSP) to generate data to support the use of a simpler biomarker of geographic or phenotypic associations. We also investigated LMP-1 patterns in 414 eBL cases and 414 geographically matched controls in the Epidemiology of Burkitt Lymphoma in East African children and minors (EMBLEM) study using LMP-1 PCR and Sanger sequencing. Phylogenetic analysis revealed distinct genetic patterns of African versus Asian EBV sequences. We identified 281 single nucleotide variations (SNVs) in LMP-1 promoter and coding region, which formed 12 unique patterns (A to L). Nine patterns (A, AB, C, D, F, I, J, K and L) predominated in African EBV, of which four were found in 92% of BL samples (A, AB, D, and H). Predominant patterns were B and G in Asia and H in Europe. EBV positivity in peripheral blood was detected in 95.6% of EMBLEM eBL cases versus 79.2% of the healthy controls (odds ratio [OR] =3.83; 95% confidence interval 2.06-7.14). LMP-1 was successfully sequenced in 66.7% of the EBV DNA positive cases but in 29.6% of the controls (ORs ranging 5-11 for different patterns). Four LMP-1 patterns (A, AB, D, and K) were detected in 63.1% of the cases versus 27.1% controls (ORs ranges: 5.58-11.4). Dual strain EBV infections were identified in WGS and PCR-Sanger data. In conclusion, EBV from Africa is phylogenetically separate from EBV in Asia. Genetic diversity in LMP-1 formed 12 patterns, which showed promising geographic and phenotypic associations. Presence of multiple strain infection should be considered in efforts to refine or improve EBV markers of ancestry or phenotype.Lay SummaryEpstein-Barr virus (EBV) infection, a ubiquitous infection, contributes to the etiology of both Burkitt Lymphoma (BL) and nasopharyngeal carcinoma, yet their global distributions vary geographically with no overlap. Genomic variation in EBV is suspected to play a role in the geographical patterns of these EBV-associated cancers, but relatively few EBV samples from BL have been comprehensively studied. We sought to compare phylogenetic patterns of EBV genomes obtained from BL samples in Africa and from tumor and non-tumor samples from elsewhere. We concluded that EBV obtained from BL in Africa is genetically separate from EBV in Asia. Through comprehensive analysis of nucleotide variations in EBV’s LMP-1 gene, we describe 12 LMP-1 patterns, two of which (B and G) were found mostly in Asia. Four LMP-1 patterns (A, AB, D, and F) accounted for 92% of EBVs sequenced from BL in Africa. Our results identified extensive diversity of EBV, but BL in Africa was associated with a limited number of variants identified, which were different from those identified in Asia. Further research is needed to optimize the use of PCR and sequencing to study LMP-1 diversity for classification of EBV variants and for use in epidemiologic studies to characterize geographic and/or phenotypic associations of EBV variants with EBV-associated malignancies, including eBL.

A Systematic Review of Epstein-Barr Virus Latent Membrane Protein 1 (LMP1) Gene Variants in Nasopharyngeal Carcinoma.

Nasopharyngeal carcinoma (NPC) is an aggressive tumor with a complex etiology. Although Epstein–Barr virus (EBV) infection is known environmental factor for NPC development, the degree to which EBV naturally infects nasopharyngeal epithelium and the moment when and why the virus actively begins to affect cell transformation remains questionable. The aim of this study was to explore the association between LMP1 gene variability and potential contribution to NPC development. A systematic review was performed through searches of PubMed, Web of Science (WoS) and SCOPUS electronic databases. Additionally, meta-analysis of the difference in the frequency of seven LMP1 gene variants in NPC and control individuals was accomplished. The results from this study give a proof of concept for the association between 30 bp deletion (OR = 3.53, 95% CI = 1.48–8.43) and Xhol loss (OR = 14.17, 95% CI = 4.99–40.20) and NPC susceptibility when comparing biopsies from NPC and healthy individuals. Otherwise, 30 bp deletion from NPC biopsies could not distinguish NPC from EBV-associated non-NPC tumors (OR = 1.74, 95% CI = 0.81–3.75). However, B95-8, China1 and North Carolina variants were uncommon for NPC individuals. Much more efforts remains to be done to verify the biological significance of the differences observed, define so-called “high-risk” EBV variants and make it available for clinical application.

Association of Epstein-Barr virus latently expressed genes with multiple sclerosis

BackgroundDespite mounting evidence supporting an etiologic role for Epstein-Barr virus (EBV) in multiple sclerosis (MS), the exact mechanisms through which the virus may contribute to disease development are still unknown. The aim of this study was to analyze seven highly polymorphic EBV latently expressed genes in individuals diagnosed with MS in comparison to healthy controls (HC), to investigate the possible association of EBV variants with an individual's risk towards MS. MethodsB-lymphocytes were isolated from MS patients (n = 30) and HC (n = 33) for the isolation of EBV genomic DNA. Sanger sequencing was employed to analyze EBV latent gene regions. ResultsA total of 26 variants were detected in our cohort, 17 of which were significantly associated with the MS group while nine were significantly associated with HC. Following the designation of EBV alleles based on these variants, MS risk was found to be significantly associated with the presence of the EBNA3B2.1 allele (p = 0.0008) and LMP1.1 allele (p = 0.01), whereas the EBNA1.3 allele (p = 0.005), EBNA2.1 allele (p = 0.001) as well as the EBNA3B2.2 allele (p = 0.0003) appeared to provide a protective role. ConclusionsThis study indicates a marked association between EBV genetic variants and MS, lending further support towards possible molecular mechanisms through which EBV may contribute to disease development.

Molecular characterization of Epstein-Barr virus variants detected in the oral cavity of adolescents in Cali, Colombia

ResumenIntroducción. El virus de Epstein-Barr (EBV) es un virus ubicuo y oncogénico, asociado con el desarrollo de enfermedades como la mononucleosis infecciosa, el linfoma de Burkitt, el carcinoma nasofaríngeo y otras neoplasias. Actualmente, se reconocen dos subtipos: EBV-1 y EBV- 2, que tienen diferencias genéticas con sus antígenos nucleares (Epstein-Barr Nuclear Antigens, EBNA). Debido a la gran heterogeneidad y variabilidad encontradas en la proteína LMP1 del virus, se han descrito variantes asociadas con ciertas enfermedades o con regiones geográficas específicas.Objetivo. Identificar y caracterizar molecularmente las variantes del EBV detectadas en la cavidad oral de 84 adolescentes de Cali, Colombia.Materiales y métodos. Se hizo la amplificación por reacción en cadena de la polimerasa (Polymerase Chain Reaction, PCR) convencional, así como la purificación y la secuenciación del gen EBNA3C se realizó para subtipificar el virus y del dominio C-ter de la proteína LMP1 para identificar variantes. Además, se llevó a cabo un análisis filogenético y de variantes nucleotídicas de las secuencias obtenidas comparadas con variantes patogénicas y geográficas reportadas en el GenBank (National Center for Biotechnology Information, NCBI). Resultados. El subtipo viral predominante fue el EBV-1 (79 %); el 72,6 % se agrupó con la variante patogénica Raji, derivada de linfocitos B de un paciente con linfoma de Burkitt; el 13,7 % se relacionó con una variante de origen geográfico del Mediterráneo y otro 13,7 % no se agrupó con ninguna de las variantes de referencia.Conclusiones. Este es el primer estudio que reporta variantes del gen LMP1-EBV en Cali, Colombia. Se requieren nuevos estudios para caracterizar la variante sin identificar y determinar si es patogénica o si es una variante geográfica presente exclusivamente en la ciudad.

Epstein-Barr virus molecular epidemiology and variants identification in head and neck squamous cell carcinoma.

Epstein-Barr virus (EBV) is known as one of the most widespread oncogenic viruses. Head and neck squamous cell carcinoma (HNSCC) is triggered by various risk factors. The aim of the present study was to determine the EBV infection rate, genotyping and variants frequency in HNSCC patients. In this cross-sectional study, 156 patients with HNSCC were enrolled. Formalin fixed paraffin embedded (FFPE) tissue samples were selected from hospitals affiliated to Iran University of Medical Sciences, Tehran, Iran. The EBV EBNA-3C, EBNA-1 and LMP-1 genes were amplified by PCR and then analyzed and confirmed by nucleotide sequencing. CLC work bench 5, MEGA6 and SPSS v.21 software were used for analysis the raw data. The mean age ± SD (years) of the all patients (n = 156) was 60.5 ± 12.6, in which of 121(77.6%) males it was 60.7 ± 11.9 and of 35 (22.4%) females it was 59.7 ± 14.9. Totally, 20 samples (12.8%) were found to be infected with EBV genome. The EBV genotypes 1 and 2 were calculated 90% (18/20) and 10% (2/20), respectively. vLMP-1 found in 40% (4/10) of all LMP-1 tested samples. Furthermore, the EBNA-1 predominant variants were P-ala followed by P-thr and also there were three P-ala-v2 sub variants. Statistics could not find any significant associations although there were some potentials. By our preliminary study in Iran, it revealed that EBV-1 is the predominant Epstein-Barr virus genotype in head and neck squamous cell carcinoma patients. vLMP-1 isolates showed lower survival rate than others. EBNA-1 variants had no significant association with any specific disease complication.

DETECTION OF EPSTEIN-BARR VIRUS GENOME IN ORAL CAVITY SQUAMOUS CELL CARCINOMA SAMPLES OF RUSSIAN PATIENTS

Oral cavity squamous cell carcinoma (OC-SCC) is the most common and aggressive malignancy of the oral cavity. Recent studies have revealed infections with human papilloma virus (HPV) as an additional risk factor for oral squamous cell carcinoma development, while distinguished role of Epstein-Barr virus (EBV) remains still uncertain. However, the evidence for association between virus infection and risk of oral squamous cell carcinoma is controversially and varies significantly by geographic regions and race. The aim of the present study was to elucidate the prevalence of HPV and EBV in OC-SCC samples of Russian patients from Moscow region. We investigated fresh-frozen tumor tissue fragments obtained from 11 patients with OC-SCC. DNA was extracted and the viral genome was examined by quantitative PCR assays with highrisk type-specific HPV and EBV specific markers followed by sequencing-based analysis. No HPV infection in analyzed OC-SCC samples was observed, while EBV was identified in 70.0% (7/10) of patients. Further based on Q-PCR amplification of the EBV targets including BamHI-W, EBNA1 and C-terminal fragment of LMP1 gene, EBV infection and measurement of virus load in the tumor samples was assessed. Sequencing LMP1-positive products revealed that the most samples (5/6) contained variants LMP1 with Cao deletion characterized by an increased transforming potential. These data suggest that prevalence of EBV infections is common and may influence cancer development, although detected LMP1 variants of EBV are not necessarily associated with the pathogenesis of OC-SCC. Further studies are necessary to determine the potential role of EBV and its possible importance as an infection factor in OC-SCC.

Genome sequencing analysis identifies Epstein-Barr virus subtypes associated with high risk of nasopharyngeal carcinoma.

Epstein-Barr virus (EBV) infection is ubiquitous worldwide and is associated with multiple cancers, including nasopharyngeal carcinoma (NPC). The importance of EBV viral genomic variation in NPC development and its striking epidemic in southern China has been poorly explored. Through large-scale genome sequencing of 270 EBV isolates and two-stage association study of EBV isolates from China, we identified two non-synonymous EBV variants within BALF2 strongly associated with the risk of NPC (odds ratio (OR) = 8.69, P=9.69×10−25 for SNP 162476_C; OR = 6.14, P=2.40×10−32 for SNP 163364_T). The cumulative effects of these variants contributed to 83% of the overall risk of NPC in southern China. Phylogenetic analysis of the risk variants revealed a unique origin in Asia, followed by clonal expansion in NPC-endemic regions. Our results provide novel insights into NPC endemic in southern China and also enable the identification of high-risk individuals for NPC prevention.

Gene Expression Profiles of Proteins Involved in Cladribine Metabolism and their Possible Correlation with Epstein-Barr Virus Variants (P2.2-096)

Gene Expression Profiles of Proteins Involved in Cladribine Metabolism and their Possible Correlation with Epstein-Barr Virus Variants (P2.2-096)

High risk Epstein-Barr virus variants characterized by distinct polymorphisms in the EBER locus are strongly associated with nasopharyngeal carcinoma.

Whether certain variants of Epstein-Barr virus (EBV) are linked to the pathogenesis of nasopharyngeal carcinoma (NPC), which shows a marked geographic restriction, remains an unresolved issue. We performed a case-control study comparing genomic sequences of EBV isolated from saliva samples of 142 population carriers with those from primary tumour biopsies derived from 62 patients with NPC of Hong Kong. Cluster analysis discovered five EBV subgroups 1A-C and 2A-B amongst the population carriers in contrast to the predominance of 1A and -B in the majority of NPC. Genome-wide association study (GWAS) identified a panel of NPC-associated single nucleotide polymorphisms (SNPs) and indels in the EBER locus. The most significant polymorphism, which can be found in 96.8% NPC cases and 40.1% population carriers of Hong Kong, is a four-base-deletion polymorphism downstream of EBER2 (EBER-del) from coordinates 7188-7191 (p = 1.91 × 10-7 ). In addition, the predicted secondary structure of EBER2 is altered with likely functional consequence in nearly all NPC cases. Using the SNPs and indels associated with NPC, genetic risk score is assigned for each EBV variant. EBV variants with high genetic risk score are found to be much more prevalent in Hong Kong Chinese than individuals of other geographic regions and in NPC than other EBV-associated cancers. We conclude that high risk EBV variants with polymorphisms in the EBER locus, designated as HKNPC-EBERvar, are strongly associated with NPC. Further investigation of the biological function and potential clinical application of these newly identified polymorphisms in NPC and other EBV-associated cancers is warranted.

Epstein-Barr virus infection and variants of Epstein-Barr nuclear antigen-1 in synovial tissues of rheumatoid arthritis.

ObjectiveThe objective of the present study was to investigate Epstein-Barr virus (EBV) infection as an environmental factor for the development of rheumatoid arthritis (RA).MethodsSynovial tissues were collected during surgery from 128 RA and 98 osteoarthritis (OA) patients. DNA was extracted from synovial tissues. The EBV gene was assessed by nested PCR for the amplification of EBV nuclear antigen-1 (EBNA-1). The nucleotide sequence of the PCR product was elucidated. HLA-DRB1 genotyping was also performed.ResultsEBV DNA was more frequently detected in the synovial tissues of RA patients (32.8%) than OA patients (15.3%) (p<0.01). The frequency of EBNA-1 variants did not significantly differ between RA and OA (RA: 17%, OA: 13%). The population with the HLA-DRB1 shared epitope (SE) was significantly higher in RA patients (70.3%) than in OA patients (44.9%) (p<0.001). In RA patients, the presence of EBV DNA was similar among SE-positive and -negative patients (SE-positive: 34.4%, -negative: 28.9%). The population with the EBNA-1 variant did not significantly differ between SE-positive and -negative patients (SE-positive: 12.9%, -negative: 27.3%).DiscussionThe present results indicate that EBV infection contributes to the onset of RA and chronic inflammation in synovial tissues. The frequency of EBNA-1 gene variants was low and not significantly different between RA and OA, suggesting that EBNA-1 gene variants are not a risk factor for RA. HLA-DRB1 with SE is a genetic risk factor for the development of RA. However, neither the presence of EBV nor EBNA-1 gene variants differed between SE-positive and -negative RA patients. Therefore, these two risk factors, SE and EBV, may be independent.ConclusionEBV infection may be an environmental risk factor for the development of RA, while nucleotide variants of EBNA-1 do not appear to contribute to its development.

657. Epstein–Barr Virus Genetic Diversity in Blood vs. Saliva Samples From Patients with Infectious Mononucleosis

BackgroundThe Epstein–Barr virus (EBV) is associated with several diseases, including infectious mononucleosis as well as malignant disorders. The relationship between strains of the virus and disease manifestation or illness severity is of interest. Such strains have been defined by genetic variations in the major viral genes. As a first step toward a better understanding of the relationship between strains and clinical outcomes, data are required on the patterns of genetic diversity of the virus in different populations. In this study, we examined the genetic diversity of the BZLF-1 gene, which is a major lytic gene of the virus.MethodsWe sequenced the BZLF-1 gene of EBV following amplification from DNA that was extracted from blood and saliva from previously healthy Canadian children and young adults with infectious mononucleosis. Sequencing was done by Sanger methodology (dideoxy DNA sequencing) and the sequences were aligned with a reference strain of EBV using Geneious software. The variant burden and types of single nucleotide variants were compared in blood and saliva samples.ResultsTwenty-six samples were obtained from 24 patients less than 24 years of age (16 saliva and 10 blood samples). Two subjects provided paired blood and saliva samples at the same visit. Among 36 single nucleotide variations (SNVs), 22% were common to both blood and saliva samples. There was a nonstatistically significant trend for more SNVs among blood compared with saliva samples (median 6 and 1, ranges 0–8 and 0–9, respectively). Of the 3 exons of BZLF-1, exon 1 had the greatest frequency of SNVs compared with exons 2 and 3. Among the paired samples of blood and saliva, there were different genetic variants of the BZLF-1 gene in the blood compared with the saliva samples obtained from patients with infectious mononucleosis.ConclusionAmong patients with infectious mononucleosis, different genetic variants of EBV may be present in blood compared with saliva. Blood samples revealed viral strains with a tendency for more genetic diversity compared with saliva. The potential compartmentalization of strains is of relevance in sample selection for the evaluation of the potential clinical impact of the genetic diversity of EBV. In addition, the potential impact on disease pathogenesis is of interest.Disclosures All authors: No reported disclosures.

Distribution of the Epstein-Barr virus in the normal stomach and gastric lesions in Thai population.

The Epstein-Barr virus (EBV) is one of the infectious agents found in stomach tissue. Recently, EBV-associated gastric carcinoma (EBVaGC) was classified as a new subtype of gastric carcinoma. To date, there is a lack of knowledge about the distribution and prevalence of EBV infection in both the normal stomach and various gastric lesions, including EBVaGC, in the Thai population. In this study, we detected EBV in the normal stomach (NS; n = 19), chronic gastritis (CG; n = 36), intestinal metaplasia (IM; n = 40), gastric dysplasia (GD; n = 15), and gastric adenocarcinoma (GC; n = 33) by polymerase chain reaction (PCR) amplification of the latent membrane protein (LMP1) gene of EBV. EBV-PCR amplification was positive in 42.1%, 36.1%, 22.5%, 13.3%, and 33.3% of NS, CG, IM, GD, and GC, respectively. For further clarification in EBVaGC, we performed EBV-encoded small RNA in situ hybridization (EBER-ISH) in PCR-positive cases of GD and GC. Four GC cases were EBER-ISH positive (12.1%), while both GD cases were EBER-ISH negative. In addition, we determined the distribution of the EBV strain (type A or B) based on EBNA3C sequence and EBV variants based on LMP1 variation (wild-type and 30-bp deletion variants; wt-LMP1 or del-LMP1). The results showed that type A and wt-LMP1 were the most prevalent in all lesions. In conclusion, EBV is common in both the NS and gastric lesions, and the frequency of EBVaGC was 12.1% in Thai patients.

Analysis of Epstein-Barr Virus Genomes and Expression Profiles in Gastric Adenocarcinoma.

Epstein-Barr virus (EBV) is a causative agent of a variety of lymphomas, nasopharyngeal carcinoma (NPC), and ∼9% of gastric carcinomas (GCs). An important question is whether particular EBV variants are more oncogenic than others, but conclusions are currently hampered by the lack of sequenced EBV genomes. Here, we contribute to this question by mining whole-genome sequences of 201 GCs to identify 13 EBV-positive GCs and by assembling 13 new EBV genome sequences, almost doubling the number of available GC-derived EBV genome sequences and providing the first non-Asian EBV genome sequences from GC. Whole-genome sequence comparisons of all EBV isolates sequenced to date (85 from tumors and 57 from healthy individuals) showed that most GC and NPC EBV isolates were closely related although American Caucasian GC samples were more distant, suggesting a geographical component. However, EBV GC isolates were found to contain some consistent changes in protein sequences regardless of geographical origin. In addition, transcriptome data available for eight of the EBV-positive GCs were analyzed to determine which EBV genes are expressed in GC. In addition to the expected latency proteins (EBNA1, LMP1, and LMP2A), specific subsets of lytic genes were consistently expressed that did not reflect a typical lytic or abortive lytic infection, suggesting a novel mechanism of EBV gene regulation in the context of GC. These results are consistent with a model in which a combination of specific latent and lytic EBV proteins promotes tumorigenesis.IMPORTANCE Epstein-Barr virus (EBV) is a widespread virus that causes cancer, including gastric carcinoma (GC), in a small subset of individuals. An important question is whether particular EBV variants are more cancer associated than others, but more EBV sequences are required to address this question. Here, we have generated 13 new EBV genome sequences from GC, almost doubling the number of EBV sequences from GC isolates and providing the first EBV sequences from non-Asian GC. We further identify sequence changes in some EBV proteins common to GC isolates. In addition, gene expression analysis of eight of the EBV-positive GCs showed consistent expression of both the expected latency proteins and a subset of lytic proteins that was not consistent with typical lytic or abortive lytic expression. These results suggest that novel mechanisms activate expression of some EBV lytic proteins and that their expression may contribute to oncogenesis.

Epstein-Barr virus strains and variations: Geographic or disease-specific variants?

The Epstein-Barr Virus (EBV) is associated with the development of several diseases, including infectious mononucleosis (IM), Burkitt's Lymphoma (BL), Nasopharyngeal Carcinoma, and other neoplasias. The publication of EBV genome 1984 led to several studies regarding the identification of different viral strains. Currently, EBV is divided into EBV type 1 (B95-8 strain) and EBV type 2 (AG876 strain), also known as type A and type B, which have been distinguished based upon genetic differences in the Epstein-Barr nuclear antigens (EBNAs) sequence. Several other EBV strains have been described in the past 10 years considering variations on EBV genome, and many have attempted to clarify if these variations are ethnic or geographically correlated, or if they are disease related. Indeed, there is an increasing interest to describe possible specific disease associations, with emphasis on different malignancies. These studies aim to clarify if these variations are ethnic or geographically correlated, or if they are disease related, thus being important to characterize the epidemiologic genetic distribution of EBV strains on our population. Here, we review the current knowledge on the different EBV strains and variants and its association with different diseases. J. Med. Virol. 89:373-387, 2017. © 2016 Wiley Periodicals, Inc.

Nasopharyngeal carcinoma risk prediction via salivary detection of host and Epstein-Barr virus genetic variants.

Genetic susceptibility and Epstein-Barr virus (EBV) infection are important etiological factors in nasopharyngeal carcinoma (NPC). In this study, in southern China, where NPC is endemic, a single nucleotide polymorphism (SNP) in the EBV-encoded RPMS1 gene (locus 155391: G > A [G155391A]) and seven host SNPs (rs1412829, rs28421666, rs2860580, rs2894207, rs31489, rs6774494, and rs9510787) were confirmed to be significantly associated with NPC risk in 50 NPC cases versus 54 hospital-based controls with throat washing specimens and 1925 NPC cases versus 1947 hospital-based controls with buffy coat samples, respectively. We established a strategy to detect the NPC-associated EBV and host SNPs using saliva samples in a single test that is convenient, noninvasive, and cost-effective and displays good compliance. The potential utility of this strategy was tested by applying a risk prediction model integrating these EBV and host genetic variants to a population-based case-control study comprising 1026 incident NPC cases and 1148 controls. Receiver operating characteristic (ROC) curve analysis revealed an area under the curve of the NPC risk prediction model of 0.74 (95% CI: 0.71−0.76). Net reclassification improvement (NRI) analysis showed that inclusion of the EBV SNP significantly improved the discrimination ability of the model (NRI = 0.30, P < 0.001), suggesting the promising value of EBV characteristics for identifying high-risk NPC individuals in endemic areas. Taken together, we developed a promising NPC risk prediction model via noninvasive saliva sampling. This approach might serve as a convenient and effective method for screening the population with high-risk of NPC.