Epstein-Barr Virus Sequences Research Articles

Meta-analysis of Epstein-Barr virus genomes in Southern Chinese identifies genetic variants and high risk viral lineage associated with nasopharyngeal carcinoma.

Genetic variants in Epstein-Barr virus (EBV) have been strongly associated with nasopharyngeal carcinoma (NPC) in South China. However, different results regarding the most significant viral variants, with polymorphisms in EBER2 and BALF2 loci, have been reported in separate studies. In this study, we newly sequenced 100 EBV genomes derived from 61 NPC cases and 39 population controls. Comprehensive genomic analyses of EBV sequences from both NPC patients and healthy carriers in South China were conducted, totaling 279 cases and 227 controls. Meta-analysis of genome-wide association study revealed a 4-bp deletion downstream of EBER2 (coordinates, 7188-7191; EBER-del) as the most significant variant associated with NPC. Furthermore, multiple viral variants were found to be genetically linked to EBER-del forming a risk haplotype, suggesting that multiple viral variants might be associated with NPC pathogenesis. Population structure and phylogenetic analyses further characterized a high risk EBV lineage for NPC revealing a panel of 38 single nucleotide polymorphisms (SNPs), including those in the EBER2 and BALF2 loci. With linkage disequilibrium clumping and feature selection algorithm, the 38 SNPs could be narrowed down to 9 SNPs which can be used to accurately detect the high risk EBV lineage. In summary, our study provides novel insight into the role of EBV genetic variation in NPC pathogenesis by defining a risk haplotype of EBV for downstream functional studies and identifying a single high risk EBV lineage characterized by 9 SNPs for potential application in population screening of NPC.

DETECTION OF EPSTEIN-BARR VIRUS GENOME IN PEDIATRIC PATIENTS WITH LEUKEMIA AND HODGKIN LYMPHOMA: VIRAL ETIOLOGY IN PEDIATRIC CANCERS IN TÜRKİYE

Objective: Cancer is a pervasive disease characterized by its widespread occurrence and challenging treatment process. While numerous agents, including viruses, have been identified as potential causes of cancer in both adults and children, the complete pathogenesis of cancers remains incompletely elucidated. This study aimed to examine the presence of four viral agents, namely Human T-cell Lymphotropic Virus Type 1 (HTLV-1), Epstein-Barr Virus (EBV), Kaposi's Sarcoma- Associated Herpesvirus (KSHV), and Human Parvovirus B19 (HPV B19), in blood samples obtained from pediatric patients (n=64) diagnosed with B cell acute lymphoblastic leukemia (ALL), T cell ALL, Hodgkin lymphoma, and patients with relapsed leukemia and lymphoma. 
 Material and Methods: The whole blood samples collected from the patients during the pre-treatment and post-treatment periods underwent polymerase chain reaction (PCR) and real- time PCR to identify the presence of the viral genomes of HTLV-1, EBV, KSHV, and HPV B19. The samples that tested positive were subsequently subjected to Sanger sequencing, followed by phylogenetic analysis. 
 Results: Among a total of 64 samples analyzed, HTLV-1, KSHV, and HPV B19 were found to be negative. However, EBV genome was detected in six samples (9.37%) from patients with ALL and Hodgkin lymphoma, comprising both pre- treatment (n=3) and post-treatment (n=3) cases. Subsequent sequencing and alignment of the positive EBV samples with other EBV sequences deposited in GenBank revealed a high degree of similarity. 
 Conclusion: Our findings suggest that EBV may be one of the viral agents implicated in pediatric cancer cases involving leukemia and Hodgkin lymphoma. Therefore, it is recommended to consider testing for the presence of EBV genome in these patient populations within the context of Türkiye. This information contributes to a better understanding of the viral etiology underlying pediatric cancers, enabling the development of targeted diagnostic and therapeutic strategies in the future.

Molecular and genetic characteristics of Nizhny Novgorod Regionepstein–Barr virus isolates in children with infectious mononucleosis and healthy virus carriers

Numerous foreign studies evidence about a pronounced heterogeneity of the Epstein-Barr virus (EBV) populationcirculating throughout the world. Various EBV classifications have been proposed. The attention of Russian researchers has focused on the study of the structural and functional polymorphism of the EBV LMP-1 oncogene in the context of oncological diseases in adulthood. The aim of the work was to assess EBV molecular genetic diversity in children with EBV infection in the Nizhny Novgorod region. There were analyzed blood leukocyte and saliva specimens from children aged 117 years with EBV-infectious mononucleosis (n = 69) and sex- and age-matched healthy virus carriers of (n = 32). A total of 178 EBV isolates were studied. For differential detection of EBV-1/EBV-2, we used an optimized one-round PCR variant with electrophoretic detection of amplification products in agarose gel. Nucleotide sequences of the LMP-1gene C-terminal fragment were determined by Sanger sequencing. Bioinformatics data analysis was performed using MEGA X software. As a result, during EBV-infectious mononucleosis, only the EBV-1 type was detected in all children, among healthy virus carriers EBV-1 (93.84.3%) and EBV-2 (6.24.3%). Based on the EBV classification according to R.H. Edwards et al. the strain affiliation of EBV isolates was determined. A total of five variants of LMP-1 were identified, namely B95-8, China 1, Med, NC and Alaskan, among which B95-8 dominated. The LMP-1 Med+, China 2, and China 3 variants were not found in any of the studied samples. It has been shown that the region of tandem repeats makes a significant contribution to the genetic diversity of the EBV population. A total of 100 amino acid substitutions were identified, of which the most common in the Nizhny Novgorod region EBV isolates are G212S, S366T, E328Q and S309N. A comparative analysis showed that strains, deletions, repeats, amino acid substitutions in EBV isolates from biological samples in children with infectious mononucleosis had common characteristics with a group of healthy virus carriers. In the active form of EBV infection, the appearance of structurally heterogeneous EBV sequences isolated from blood leukocytes and saliva from a single source was noted. Thus, for the first time, the molecular genetic diversity of EBV in children with various forms of EBV infection was assessed, which is the basis for the prospective development of clinical and epidemiological studies of EBV infection at a new methodological level.

Value of metagenomic next-generation sequencing in children with hemophagocytic syndrome with central nervous system involvement

To study the value of metagenomic next-generation sequencing (mNGS) in detecting intracranial Epstein-Barr virus (EBV) infection in children with hemophagocytic syndrome (HPS) with central nervous system involvement. A retrospective analysis was performed for the cerebrospinal fluid mNGS results of 30 HPS children with central nervous system involvement, which were compared with the results of cerebrospinal fluid EBV-DNA detection and serum EBV antibody profile. The change in serum EBV-DNA copy number after treatment was used to evaluate the efficacy of targeted therapy. The positive rate of EBV in cerebrospinal fluid determined by mNGS was significantly higher than that of EBV-DNA in cerebrospinal fluid (100% vs 10%, P<0.001) and had no significant difference from the positive rate of serum EBV antibody profile (100% vs 93%, P>0.05). The median number of sequences determined by mNGS was 2 400, and serum EBV-DNA copy number before treatment was moderately positively correlated with the number of EBV sequences (rs=0.693, P<0.001). The multiple linear regression analysis showed that the number of sequences determined by mNGS in cerebrospinal fluid increased with the increase in serum EBV-DNA copy number before treatment (P<0.05). EBV-associated HPS often results in EBV-infected viral encephalitis, and mNGS can significantly increase the detection rate of EBV in cerebrospinal fluid, which may help with clinical diagnosis.

Etiological Analysis of Viral Encephalitis in Children in Zhejiang Province from 2018 to 2019.

Objective: To investigate the common pathogens of viral encephalitis (VE) in children, and to provide guidance for the empirical diagnosis and treatment of patients with VE. Methods: A total of 227 cerebrospinal fluid (CSF) samples were collected from pediatric patients with VE in Zhejiang province from January 2018 to December 2019. The samples were tested using multiplex and singleplex Reverse Transcription-Polymerase Chain Reaction (RT-PCR) with primers specific to enterovirus (EV), varicella-zoster virus (VZV), mumps virus (MuV), cytomegalovirus (CMV), herpes simplex virus type 1 (HSV-1)/type 2 (HSV-2), Epstein–Barr virus (EBV), and human herpesvirus 6 (HHV-6). The data of the two analyses were compared and then verified using Sanger sequencing. Results: Of the 227 CSF samples, 90 were shown to be positive for multiplex RT-PCR with a positivity rate of 39.65% and a 95% confidence interval (33.2%, 46.1%). EV was the most common cause of VE, followed by EBV, HHV-6, MuV, CMV, VZV, and HSV-1. Most included cases occurred in summer, accounting for 49.78% of all cases. For EV, EBV, and HSV-2, multiplex RT-PCR showed a positivity rate of 34.36%, which was not statistically different from that of 30.4% shown by singleplex RT-PCR. The sequences of EV, EBV, VZV, MuV, CMV, HSV-1, HHV-6, and HSV-2 were confirmed by sequencing the PCR products obtained from multiplex and singleplex PCR. Conclusions: In children, VE is more prevalent in the summer than in other seasons in Zhejiang province, and EV may be the most common causative pathogen.

Assessment of the Serologic Status in Epstein-Barr Virus in Patients Qualified for Lung Transplantation in the First Half of 2021

The aim of the study was to assessment serologic status of Epstein-Barr virus (EBV) infections in patients qualificated for lung transplantation in the first half of 2021. The study included 72 patients qualified for lung transplantation from January to June 2021. The youngest patient was aged 14 years and the oldest was aged 65 years. The study group consisted of 36 women and 36 men. In the serum of patients, a multi-parameter, comprehensive diagnosis of EBV infections was performed using the IIFT BIOCHIP EBV sequence tests. This test is based on a combination of several substrates, enabling the simultaneous evaluation of antibodies against capsid antigens (anti-CA antibodies), both in the IgG and IgM class, early antigens (anti-EA), nuclear antigens and the assessment of the avidity of anti-CA antibodies. The analysis of all diagnostically significant antibodies specific for EBV infections, including the avidity of anti-CA antibodies, increases the diagnostic accuracy in differentiating active and past infection with EBV. In the studied group it was shown that 58 had past EBV infection (80.6%). Twelve patients (16.6%) have anti-EA antibodies, which indicate that the virus is reactivated. Only 2 patients (2.8%) had no antibodies to EBV. Comprehensive assessment of antibodies against various EBV antigens in patients qualified for lung transplantation is important in the management and further diagnosis of this infection, especially after transplantation, due to the risk of developing post-transplant lymphoproliferative disease.

Genomic landscape of Epstein-Barr virus in familial nasopharyngeal carcinoma.

To better understand the genomic characteristics of Epstein-Barr virus (EBV) in familial nasopharyngeal carcinoma (NPC), we sequenced the EBV genomes by whole-genome capture in 38 unrelated patients with NPC family history in first-degree relatives and 47 healthy controls, including 13 with family history and 34 without. Compared with type 1 reference genome, mutation hotspots were observed in the latent gene regions of EBV in familial NPC cases. Population structure analysis showed that one cluster has a higher frequency in familial cases than in controls (OR=5.33, 95 % CI 2.50-11.33, P=1.42×10-5), and similar population structure composition was observed among familial and sporadic NPC cases in high-endemic areas. By genome-wide association analysis, four variants were found to be significantly associated with familial NPC. Consistent results were observed in the meta-analysis integrating two published case-control EBV sequencing studies in NPC high-endemic areas. High-risk haplotypes of EBV composed of 34 variants were associated with familial NPC risk (OR=13.85, 95 % CI 4.13-46.44, P=2.06×10-5), and higher frequency was observed in healthy blood-relative controls with NPC family history (9/13, 69.23 %) than those without family history (16/34, 47.06%). This study suggested the potential contribution of EBV high-risk subtypes to familial aggregation of NPC.

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.

The Right Treatment of the Right Patient: Integrating Genetic Profiling Into Clinical Decision Making in Advanced Gastric Cancer in Asia.

Gastric cancer is a major global health burden, especially when patients are diagnosed with recurrent or metastatic gastric cancer. Despite recent advances in treatment options with palliative chemotherapy, the median overall survival of patients with gastric cancer remains within 1 or 2 years after the diagnosis of metastatic disease. Gastric cancer is significantly more prevalent in eastern Asia (e.g., Japan and Korea). Next-generation sequencing (NGS) is rapidly being adopted as part of clinical practice in Korea and Japan, especially in patients with gastric cancer. Approximately 10% to 15% of the patients with gastric cancer who undergo NGS of their tumor specimen are allocated to target-matched clinical trials in Japan and Korea. In Japan and Korea, a cell-free DNA NGS panel is also actively being investigated as an alternative NGS test for patients with gastric cancer, which may reflect the tumor heterogeneity of gastric cancer. In Japan and Korea, multiple biomarkers, such as HER2, mismatch repair, Epstein-Barr virus, PD-L1 (combined positive score), EGFR, FGFR2, and CLDN18.2, are routinely assessed through immunohistochemistry or in situ hybridization before initiation of the first-line treatment in all patients with gastric cancer. Most tertiary cancer centers in Korea routinely perform HER2, mismatch repair, Epstein-Barr virus, and PD-L1 NGS before palliative chemotherapy in patients with gastric cancer. Biomarker evaluation for all patients with metastatic gastric cancer enables clinicians to identify available biomarker-based clinical trials early during the course of treatment, which expands treatment opportunities while patients are medically fit for clinical trials, if available. Comprehensive genomic profiling using a tissue or circulating tumor DNA NGS panel is considered necessary during second-line or subsequent treatment. It is hoped that a comprehensive molecular profiling strategy will facilitate greater use of precision medicine through molecularly targeted therapies for patients with gastric cancer in the near future.

Genetic Patterns Found in the Nuclear Localization Signals (NLSs) Associated with EBV-1 and EBV-2 Provide New Insights into Their Contribution to Different Cell-Type Specificities.

Simple SummaryThe Epstein–Barr virus (EBV) has been implicated in several human neoplastic diseases. The EBV-1 can transform B cells into LCL more efficiently than EBV-2, and EBV-2 preferentially infects T-cell lymphocytes. The EBNA3A oncoprotein has an essential role in B-cell transformation. The six peptide motifs called nuclear localization signals (NLSs) from EBNA3A ensure nucleocytoplasmic protein trafficking. Multiple NLSs have been suggested to enhance EBNA3 function or different specificities to different cell types; however, a comprehensive assessment of their genetic variability has not been addressed. Our objective was to study the NLSs’ variability and their relationship with EBV types. Based on a comprehensive analysis of over a thousand EBNA3A sequences from different clinical manifestations and geographic locations, we found that EBNA3A from EBV-2 has two of the six NLSs altered, and genetic patterns in the NLSs are associated with EBV-1 and EBV-2.The Epstein–Barr virus (EBV) is a globally dispersed pathogen involved in several human cancers of B-cell and non-B-cell origin. EBV has been classified into EBV-1 and EBV-2, which have differences in their transformative ability. EBV-1 can transform B-cells into LCL more efficiently than EBV-2, and EBV-2 preferentially infects T-cell lymphocytes. The EBNA3A oncoprotein is a transcriptional regulator of virus and host cell genes, and is required in order to transform B-cells. EBNA3A has six peptide motifs called nuclear localization signals (NLSs) that ensure nucleocytoplasmic protein trafficking. The presence of multiple NLSs has been suggested to enhance EBNA3 function or different specificities in different cell types. However, studies about the NLS variability associated with EBV types are scarce. Based on a systematic sequence analysis considering more than a thousand EBNA3A sequences of EBV from different human clinical manifestations and geographic locations, we found differences in NLSs’ nucleotide structures among EBV types. Compared with the EBNA3A EBV-1, EBNA3A EBV-2 has two of the six NLSs altered, and these mutations were possibly acquired by recombination. These genetic patterns in the NLSs associated with EBV-1 and EBV-2 provide new information about the traits of EBNA3A in EBV biology.

The influence of human genetic variation on Epstein\u2013Barr virus sequence diversity

Epstein–Barr virus (EBV) is one of the most common viruses latently infecting humans. Little is known about the impact of human genetic variation on the large inter-individual differences observed in response to EBV infection. To search for a potential imprint of host genomic variation on the EBV sequence, we jointly analyzed paired viral and human genomic data from 268 HIV-coinfected individuals with CD4 + T cell count < 200/mm3 and elevated EBV viremia. We hypothesized that the reactivated virus circulating in these patients could carry sequence variants acquired during primary EBV infection, thereby providing a snapshot of early adaptation to the pressure exerted on EBV by the individual immune response. We searched for associations between host and pathogen genetic variants, taking into account human and EBV population structure. Our analyses revealed significant associations between human and EBV sequence variation. Three polymorphic regions in the human genome were found to be associated with EBV variation: one at the amino acid level (BRLF1:p.Lys316Glu); and two at the gene level (burden testing of rare variants in BALF5 and BBRF1). Our findings confirm that jointly analyzing host and pathogen genomes can identify sites of genomic interactions, which could help dissect pathogenic mechanisms and suggest new therapeutic avenues.

Epstein-Barr Virus (EBV) Genome Sequence Variations, Virus Strain Classifications and EBV-Associated Tumors

The Epstein-Barr virus (EBV), a member of the Herpesviridae family, belonging to the Gammaherpesvirinae subfamily, has been associated with several nonmalignant and malignant diseases, including Burkitt’s lymphoma, gastric cancer, Hodgkin’s lymphoma, and nasopharyngeal carcinoma. In recent years numerous EBV genome sequences have been analyzed to answer the question of whether EBV genome variations are tumor-specific, ethnically or geographically determined. A clear answer regarding the "specialization" of EBV strains has not yet been received; however, attempts are being made to identify high-risk EBV strains. This mini-review analyzes the most well-known EBV classifications, EBV sequence variations and their association with EBV-associated human pathologies.

Nonhepatosplenic γδ T-Cell Lymphoma: A Subset of Cytotoxic Lymphomas With Mucosal or Skin Localization

AbstractHuman γδ T lymphocytes represent a minor subset of T cells in the peripheral blood, which exhibit a limited diversity and a tissue-restricted repertoire in contrast to their broad specificity. Most postthymic neoplasms that arise from this T-cell subpopulation belong to the hepatosplenic γδ lymphoma entity. Only a few cases of nonhepatosplenic γδ lymphomas have been described in detail previously. This study presents the clinicopathologic features of 11 consecutive cases of nonhepatosplenic γδ lymphoma. All were characterized by mucosal or skin initial involvement: nasal cavity (n = 3), gastrointestinal tract (n = 3), skin (n = 3), lung (n = 1), larynx (n = 1). Most patients presented with B symptoms (eight of 11), without peripheral lymphadenopathy and bone marrow involvement. A past history of chronic antigen exposure was noted in six cases, and four patients had features of immune deficiency. On histology, they were classified as pleomorphic tumors. Features of epitheliotropism and angiocentrism was observed in most cases. Tumor cells had a CD2+, CD3+, T-cell receptor (TCR)δ−1+, βF1− phenotype. They were CD5− (9 of 10) and CD4−/CD8− (9 of 10) or CD8+(1 of 10). A clonal γ-chain gene rearrangement was detected in all tested cases (9/9). All cases had an activated cytotoxic T-cell intracellular antigen-1 (TIA-1)+, Granzyme B+ phenotype. Epstein-Barr virus (EBV) sequences were detected in six cases by in situ hybridization (ISH). Despite an aggressive clinical course, complete remission was obtained in three patients, and one of the latter required a peripheral blood stem-cell transplantation. Nonhepatosplenic γδ peripheral T-cell lymphoma can be regarded as a model of activated cytotoxic lymphoma, occurring in mucosae or skin. These appear to be derived from the subpopulation of tissue-restricted γδ lymphocytes, which are involved in the host epithelial surface surveillance. The role of chronic antigen exposure in the pathogenesis of these rare lymphomas can be suggested, in view of the past history observed in at least some patients.

Analysis of latent T-cell epitopes in Epstein-Barr virus isolated from extranodal nasal-type natural killer/T-cell lymphoma in Taiwanese population

Extranodal nasal-type natural killer (NK)/T-cell lymphoma (NKTCL) is an aggressive lymphoma that is prevalent among East Asian and South American populations. Although Epstein-Barr virus (EBV) is commonly detected in NKTCL, there are limited studies that have analyzed the EBV genomic variations in NKTCL. In this study, 8 EBV latent genes were analyzed using targeted gene sequencing in 23 formalin-fixed paraffin-embedded tissues derived from 18 patients with NKTCL. Five cases with paired samples were comparatively analyzed. The consistency of EBV sequencing data between tissue samples was high (96.3%–98.7%), whereas that of variant calling among the tissue samples and plasma samples (74.3%–79.2%) was low. The highest densities of non-synonymous variants were detected in the EBNA3B gene. Among the 74 known T-cell epitopes, 363 non-synonymous variants were identified in 32 (43.2%) epitopes. Additionally, the AVFDRKSDAK (A1S/P and V2F/M/L) and YHLIVDTDSL (I4L and L10R/V/G/H) epitopes were associated with 5 patterns of amino acid changes in EBNA3B and EBNA-2, respectively. The frequency of variation in the human leukocyte antigen (HLA)-restricted epitopes with corresponding HLA types common among Taiwanese population was significantly low (P = 0.011), whereas that in anchor residues was significantly high (P = 0.012). In conclusion, this study demonstrated the genomic diversity of EBV in NKTCL and its correlation with the HLA-restricted epitope variations in Taiwanese population. The findings of this study provide useful insights for the development of novel therapeutic strategies for NKTCL.

Genomic and Transcriptomic Characterization of Natural Killer T Cell Lymphoma

Natural killer/T cell lymphoma (NKTCL) is an aggressive and heterogeneous entity of non-Hodgkin lymphoma, strongly associated with Epstein-Barr virus (EBV) infection. To identify molecular subtypes of NKTCL based on genomic structural alterations and EBV sequences, we performed multi-omics study on 128 biopsy samples of newly diagnosed NKTCL and defined three prominent subtypes, which differ significantly in cell of origin, EBV gene expression, transcriptional signatures, and responses to asparaginase-based regimens and targeted therapy. Our findings thus identify molecular networks of EBV-associated pathogenesis and suggest potential clinical strategies on NKTCL.

Human Mammary Tumor Virus, Human Papilloma Virus, and Epstein-Barr Virus Infection Are Associated With Sporadic Breast Cancer Metastasis

Background:Viral cause of sporadic breast cancer (SBC) has been suggested based on the experimental murine model of mammary tumor caused by mouse mammary tumor virus (MMTV), Epstein-Barr virus (EBV), and human papillomavirus (HPV). While some studies have demonstrated the presence of viral sequences of MMTV, HPV, and EBV in breast cancer cells, others failed. These contradictions may be attributed to the geographical distribution of breast cancer incidence and/or technical variations. In the current study, we aimed to investigate the correlation of MMTV, HPV, and EBV infections with the development of breast cancer in Jordanian patients.Methods:One hundred SBC tissue samples were subjected to laser capture microdissection for the selection of tumor cells populations. Fluorescence polymerase chain reaction (PCR) was used to detect the presence of the MMTV env-like sequences. Real-time PCR was used for HPV and EBV detection, and EBV was further confirmed by chromogen in situ hybridization (CISH).Results:Mouse mammary tumor virus, HPV, and EBV were detected in SBC in 11%, 21%, and 23%, respectively. Only 3 of 52 (5.7%) positive cases demonstrated multiple virus infections. However, 49 of 52 (94%) of the positive cases revealed the presence of 1 type of viral sequences. Consequently, 52% of the studied breast cancer cases were infected with at least 1 type of the aforementioned viruses.Conclusions:The current cohort suggests that MMTV, HPV, and EBV have a potential role in the development of breast cancer and adding more reasons to proceed with the quest of a possible viral origin of breast cancer.

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

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

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.

Genome-wide profiling of Epstein-Barr virus integration by targeted sequencing in Epstein-Barr virus associated malignancies.

Rationale: Epstein-Barr virus (EBV) is associated with multiple malignancies with expression of viral oncogenic proteins and chronic inflammation as major mechanisms contributing to tumor development. A less well-studied mechanism is the integration of EBV into the human genome possibly at sites which may disrupt gene expression or genome stability.Methods: We sequenced tumor DNA to profile the EBV sequences by hybridization-based enrichment. Bioinformatic analysis was used to detect the breakpoints of EBV integrations in the genome of cancer cells.Results: We identified 197 breakpoints in nasopharyngeal carcinomas and other EBV-associated malignancies. EBV integrations were enriched at vulnerable regions of the human genome and were close to tumor suppressor and inflammation-related genes. We found that EBV integrations into the introns could decrease the expression of the inflammation-related genes, TNFAIP3, PARK2, and CDK15, in NPC tumors. In the EBV genome, the breakpoints were frequently at oriP or terminal repeats. These breakpoints were surrounded by microhomology sequences, consistent with a mechanism for integration involving viral genome replication and microhomology-mediated recombination.Conclusion: Our finding provides insight into the potential of EBV integration as an additional mechanism mediating tumorigenesis in EBV associated malignancies.

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.