Epstein-Barr Virus Integration Research Articles

Non-Random Pattern of Integration for Epstein-Barr Virus with Preference for Gene-Poor Genomic Chromosomal Regions into the Genome of Burkitt Lymphoma Cell Lines

Background: Epstein-Barr virus (EBV) is an oncogenic virus found in about 95% of endemic Burkitt lymphoma (BL) cases. In latently infected cells, EBV DNA is mostly maintained in episomal form, but it can also be integrated into the host genome, or both forms can coexist in the infected cells. Methods: In this study, we mapped the chromosomal integration sites of EBV (EBV-IS) into the genome of 21 EBV+ BL cell lines (BL-CL) using metaphase fluorescence in situ hybridization (FISH). The data were used to investigate the EBV-IS distribution pattern in BL-CL, its relation to the genome instability, and to assess its association to common fragile sites and episomes. Results: We detected a total of 459 EBV-IS integrated into multiple genome localizations with a preference for gene-poor chromosomes. We did not observe any preferential affinity of EBV to integrate into common and rare fragile sites or enrichment of EBV-IS at the chromosomal breakpoints of the BL-CL analyzed here, as other DNA viruses do. Conclusions: We identified a non-random integration pattern into 13 cytobands, of which eight overlap with the EBV-IS in EBV-transformed lymphoblastoid cell lines and with a preference for gene- and CpGs-poor G-positive cytobands. Moreover, it has been demonstrated that the episomal form of EBV interacts in a non-random manner with gene-poor and AT-rich regions in EBV+ cell lines, which may explain the observed affinity for G-positive cytobands in the EBV integration process. Our results provide new insights into the patterns of EBV integration in BL-CL at the chromosomal level, revealing an unexpected connection between the episomal and integrated forms of EBV.

DeepEBV: a deep learning model to predict Epstein-Barr virus (EBV) integration sites.

Epstein-Barr virus (EBV) is one of the most prevalent DNA oncogenic viruses. The integration of EBV into the host genome has been reported to play an important role in cancer development. The preference of EBV integration showed strong dependence on the local genomic environment, which enables the prediction of EBV integration sites. An attention-based deep learning model, DeepEBV, was developed to predict EBV integration sites by learning local genomic features automatically. First, DeepEBV was trained and tested using the data from the dsVIS database. The results showed that DeepEBV with EBV integration sequences plus Repeat peaks and 2-fold data augmentation performed the best on the training dataset. Furthermore, the performance of the model was validated in an independent dataset. In addition, the motifs of DNA-binding proteins could influence the selection preference of viral insertional mutagenesis. Furthermore, the results showed that DeepEBV can predict EBV integration hotspot genes accurately. In summary, DeepEBV is a robust, accurate and explainable deep learning model, providing novel insights into EBV integration preferences and mechanisms. DeepEBV is available as open-source software and can be downloaded from https://github.com/JiuxingLiang/DeepEBV.git. Supplementary data are available at Bioinformatics online.

Characterization of the Blood and Bone Marrow Microbiome of MDS Patients and Associations with Clinical Features

To date, several malignancies of the lymphoid lineage have been linked with microbial infection. Indeed, lymphomas have been associated with the bacteria Helicobacter pylori, and T-cell leukemia and Burkitt's lymphoma have long been known to be commonly caused by viral infections (human T-cell lymphotropic virus 1 and Epstein-Barr virus (EBV), respectively). However, metagenomics research in myeloid malignancies has primarily focused on the roles of gut flora rather than on the tumor site itself (blood and bone marrow). Traditionally, human blood and bone marrow was considered to be normally sterile, and therefore microbiome analysis of these entities would only be performed when deleterious infection was suspected. However, evidence is now accumulating for a normal blood microbiome in healthy individuals. Given the growing number of established relationships in other malignancies between the microbiome at the tumor site and clinical characteristics, we hypothesized roles for the bone marrow microbiome in MDS patients. To this end, we extracted bacterial, fungal, and viral sequence from the bone marrow and blood of 640 MDS patients taken at diagnosis. Briefly, whole-genome sequencing (minimum coverage 100x) was performed on DNA extracted from each patient sample. Reads not aligning to the human genome were aligned to NCBI's database of known microbial reference genomes, and microbial species were assessed for presence and abundance in each sample. As expected, bacterial reads dominated, comprising 99.8% of all microbial reads, while viral reads were 2.73-fold as prevalent as fungal reads. We calculated microbial diversity (Shannon diversity index) on the genus and phylum levels for each patient. As has been reported in solid tumors, higher alpha-diversity is associated with favorable overall survival (age- and sex-adjusted P = 8.6 x 10-3; Figure 1A). Interestingly, individuals with normal karyotype had significantly higher alpha-diversity at the phylum level (Wilcoxon P = 0.002; Figure 1B), while those with deletion of chromosome 5q had significantly lower alpha-diversity (P = 2.2 x 10-5; Figure 1C). Among viral infections, EBV (formally Human gammaherpesvirus 4) was the most common, with 192 patients (30%) harboring EBV DNA sequence. Human cytomegalovirus (HCMV; formally Human gammaherpesvirus 5) and Human betaherpesvirus 6 infections were also observed, in 23% and 2% of patients, respectively. EBV infection was associated with worse overall survival (age- and sex-adjusted P = 0.037; Figure 1D). There was also a substantial dose effect, with higher EBV levels corresponding to poorer outcomes (P = 1.96 x 10-6). Mining the paired-end DNA reads for human-viral chimeras, we found multiple instances of EBV integration into the patient genome, suggesting a possible mechanism for EBV infection impacting patient outcomes. Bacterial composition was also analyzed, showing strong concordance between several pairs of phyla, suggesting synergy between these bacterial communities (Figure 1E). On the other hand, the Proteobacteria and Actinobacteria phyla were strongly discordant with one another (Figure 1E). Multiple studies have shown dominance of Proteobacteria in the circulating bacteriome of healthy individuals. In contrast, the MDS patients in our study showed a continuum between complete dominance of the Proteobacteria phylum and complete dominance of the Actinobacteria phylum (Figure 1F). Intriguingly, lower levels of Actinobacteria was strongly associated with the presence of various chromosomal aberrations, while the opposite was largely true for Proteobacterial abundance. As a result, the ratio of these two phyla abundances was strongly positively associated with normal karyotype and negatively associated with del(5q) (Table 1). Overall, our study provides evidence for roles of the bone marrow microbiome in phenotypes and may even influence outcomes of patients with MDS. Further research is required to determine: a) whether microbial content is cause or consequence of patient characteristics, and b) what the mechanisms are for these microbiome-clinical associations. Disclosures Maciejewski: Alexion, BMS: Speakers Bureau; Novartis, Roche: Consultancy, Honoraria.

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.

Genome-wide Analysis of Epstein-Barr Virus (EBV) Integration and Strain in C666-1 and Raji Cells.

EBV is a key risk factor for many malignancy diseases such as nasopharyngeal carcinoma (NPC) and Burkitt lymphoma (BL). EBV integration has been reported, but its scale and impact to cancer development is remains unclear. C666-1 (NPC cell line) and Raji (BL cell line) are commonly studied EBV-positive cancer cells. A rare few EBV integration sites in Raji were found in previous research by traditional methods. To deeply survey EBV integration, we sequenced C666-1 and Raji whole genomes by the next generation sequencing (NGS) technology and a total of 909 breakpoints were detected in the two cell lines. Moreover, we observed that the number of integration sites was positive correlated with the total amount of chromosome structural variations (SVs) and copy number structural variations (CNVs), and most breakpoints located inside or nearby genome structural variations regions. It suggested that host genome instability provided an opportunity for EBV integration on one hand and the integration aggravated host genome instability on the other hand. Then, we respectively assembled the C666-1 and Raji EBV strains which would be useful resources for EBV-relative studies. Thus, we report the most comprehensive characterization of EBV integration in NPC cell and BL cell, and EBV shows the wide range and random integration to increase the tumorigenesis. The NGS provides an incomparable level of resolution on EBV integration and a convenient approach to obtain viral strain compared to any research technology before.

Integrated genomic analysis suggests MLL3 is a novel candidate susceptibility gene for familial nasopharyngeal carcinoma.

Little is known about genetic factors associated with nasopharyngeal carcinoma (NPC). To gain insight into NPC etiology, we performed whole exome sequencing on germline and tumor DNA from three closely related family members with NPC. The family was ascertained through the Pediatric Familial Cancer Clinic at The University of Chicago (Chicago, IL). The diagnosis of NPC was confirmed pathologically for each individual. For each sample sequenced, 97.3% of the exome was covered at 5×, with an average depth of 44×. Candidate germline and somatic variants associated with NPC were identified and prioritized using a custom pipeline. We discovered 72 rare deleterious germline variants in 56 genes shared by all three individuals. Of these, only three are in previously identified NPC-associated genes, all of which are located within MLL3, a gene known to be somatically altered in NPC. One variant introduces an early stop codon in MLL3, which predicts complete loss-of-function. Tumor DNA analysis revealed somatic mutations and Epstein-Barr virus (EBV) integration events; none, however, were shared among all three individuals. These data suggest that inherited mutations in MLL3 may have predisposed these three individuals from a single family to develop NPC, and may cooperate with individually acquired somatic mutations or EBV integration events in NPC etiology. Our finding is the first instance of a plausible candidate high penetrance inherited mutation predisposing to NPC.

High-throughput RNA sequencing-based virome analysis of 50 lymphoma cell lines from the Cancer Cell Line Encyclopedia project.

Using high-throughput RNA sequencing data from 50 common lymphoma cell culture models from the Cancer Cell Line Encyclopedia project, we performed an unbiased global interrogation for the presence of a panel of 740 viruses and strains known to infect human and other mammalian cells. This led to the findings of previously identified infections by Epstein-Barr virus (EBV), Kaposi's sarcoma herpesvirus (KSHV), and human T-lymphotropic virus type 1 (HTLV-1). In addition, we also found a previously unreported infection of one cell line (DEL) with a murine leukemia virus. High expression of murine leukemia virus (MuLV) transcripts was observed in DEL cells, and we identified four transcriptionally active integration sites, one being in the TNFRSF6B gene. We also found low levels of MuLV reads in a number of other cell lines and provided evidence suggesting cross-contamination during sequencing. Analysis of HTLV-1 integrations in two cell lines, HuT 102 and MJ, identified 14 and 66 transcriptionally active integration sites with potentially activating integrations in immune regulatory genes, including interleukin-15 (IL-15), IL-6ST, STAT5B, HIVEP1, and IL-9R. Although KSHV and EBV do not typically integrate into the genome, we investigated a previously identified integration of EBV into the BACH2 locus in Raji cells. This analysis identified a BACH2 disruption mechanism involving splice donor sequestration. Through viral gene expression analysis, we detected expression of stable intronic RNAs from the EBV BamHI W repeats that may be part of long transcripts spanning the repeat region. We also observed transcripts at the EBV vIL-10 locus exclusively in the Hodgkin's lymphoma cell line, Hs 611.T, the expression of which were uncoupled from other lytic genes. Assessment of the KSHV viral transcriptome in BCP-1 cells showed expression of the viral immune regulators, K2/vIL-6, K4/vIL-8-like vCCL1, and K5/E2-ubiquitin ligase 1 that was significantly higher than expression of the latency-associated nuclear antigen. Together, this investigation sheds light into the virus composition across these lymphoma model systems and provides insights into common viral mechanistic principles. Viruses cause cancer in humans. In lymphomas the Epstein-Barr virus (EBV), Kaposi's sarcoma herpesvirus (KSHV) and human T-lymphotropic virus type 1 are major contributors to oncogenesis. We assessed virus-host interactions using a high throughput sequencing method that facilitates the discovery of new virus-host associations and the investigation into how the viruses alter their host environment. We found a previously unknown murine leukemia virus infection in one cell line. We identified cellular genes, including cytokine regulators, that are disrupted by virus integration, and we determined mechanisms through which virus integration causes deregulation of cellular gene expression. Investigation into the KSHV transcriptome in the BCP-1 cell line revealed high-level expression of immune signaling genes. EBV transcriptome analysis showed expression of vIL-10 transcripts in a Hodgkin's lymphoma that was uncoupled from lytic genes. These findings illustrate unique mechanisms of viral gene regulation and to the importance of virus-mediated host immune signaling in lymphomas.

Genotype instability during long-term subculture of lymphoblastoid cell lines

Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs) promise to address the challenge posed by the limited availability of primary cells needed as a source of genomic DNA for genetic studies. However, the genetic stability of LCLs following prolonged culture has never been rigorously investigated. To evaluate genotypic errors caused by EBV integration into human chromosomes, we isolated genomic DNA from human peripheral blood mononuclear cells and LCLs collected from 20 individuals and genotyped the DNA samples using the Affymetrix 500K SNP array set. Genotype concordance measurements between two sources of DNA from the same individual indicated that genotypic discordance is negligible in early-passage LCLs (<20 passages) but substantial in late-passage LCLs (>50 passages). Analysis of concordance on a chromosome-by-chromosome basis identified genomic regions with a high frequency of genotypic errors resulting from the loss of heterozygosity observed in late-passage LCLs. Our findings suggest that, although LCLs harvested during early stages of propagation are a reliable source of genomic DNA for genetic studies, investigations that involve genotyping of the entire genome should not use DNA from late-passage LCLs.

Chromosomes 2q, 4q and 13q21 Are Preferential Targets of Epstein-Barr Virus (EBV) Integration In Burkitt's Lymphoma Cell Lines

AbstractAbstract 4452The Epstein-Barr virus (EBV) was first described in 1964 as a possible cause of Burkitt's lymphoma (BL), the most common childhood cancer in equatorial Africa. EBV is an oncogenic virus found in about 95% of the endemic BL. In latently infected cells, the EBV DNA can be maintained in episomal form, but integrated EBV could also be present. Data on the exact integration loci of EBV in BL are very rare, however, integration of EBV-DNA into the human genome has been pointed as an important mechanism in malignant cellular transformation. We have investigated the EBV integration loci (EBV-IL) in 20 BL cell lines using fluorescence in situ hybridization (FISH).FISH was performed using, as EBV-DNA probe, a biotinylated 3,000 bp fragment amplified by long distance-PCR from DNA of the EBV-positive BL cell line Raji. FISH on metaphase spreads of the BL cell line Namalwa was performed to validate the quality of the EBV-FISH probe. Negative controls were performed on EBV-negative BL cell lines CA-46, CW698, and Tanoue. Integration of EBV was defined by the presence of symmetrical doublet hybridization signals at the same chromosomal loci in both sister chromatids. A minimum of 15 well-conserved and complete metaphases was evaluated in each case.As expected, Namalwa showed symmetrical doublet hybridization signals of our EBV-FISH probe on chromosome 1p35. EBV-negative cell lines did not show hybridization signals of the probe. We detected 632 EBV-IL in the total of metaphases analyzed in 20 BL cell lines. Integration of EBV was seen in all chromosomal arms except Yp, 8p, 14p, 20q, 21p, and 22p. An analysis of the chromosomal distribution of all EBV-IL revealed a pattern of preferential insertion for EBV-IL on chromosomes 13q (14% of the metaphases), 2q and 4q (12%, respectively), 7q (10,5%), 3q (9%), and 17q (6,5%).Regarding the recurrency of EBV-IL, the BL cell lines were divided in 4 groups. The first group consisted of 8 cell lines with high-recurrent EBV-IL at a specific chromosomal band (75%-100% of the metaphases analyzed). This first group showed specific EBV-IL at a precise chromosomal location on homologous chromatids on 2p23 (Seraphine), 2q21~31 (Switzer), 22q12 (PA-682), 7p22 and 13q21 (AG876), 7q11 and 17q25 (Naliaka), and 7q21 and 13q21 (LY-67). Interestingly, in BL60 and Maku, the unique EBV-IL visualized was localized near the translocation breakpoint junction of a der(19)t(17;19) and a der(13)t(3;13), respectively. The second group contained 5 cell lines with medium-recurrent EBV-IL (27%-50%). In this group, we observed metaphases with 1 to 3 recurrent EBV-IL localized on 2p12 (Rael), 15q11~14 (JI), 15q13~15 (JBL2), 4q21 and 4q32 (BL16), and 3q21~26, 4p12, and 13q21 (Akuba). The third and fourth groups comprised 4 cell lines with low-recurrent EBV-IL (13%-20%) and 3 cell lines with absence of recurrent EBV-IL, respectively. Non-recurrently integrated EBV-DNA signals on additional chromosomes were observed in cell lines of all 4 groups, except in 4 of the high-recurrent group. We further investigated the distribution of the EBV-IL of groups 3 and 4, of those in groups 1 and 2 with a recurrency less than 27% of the metaphases, and of those that were non-recurrently integrated. We observed again that the viral integration targeted preferentially chromosomes 2q (21%), 4q (14%), and 3q (11%).Our results identify for the first time the integration loci of EBV in BL cell lines and that this integration succeed preferentially on chromosomes 2q, 3q, and 4q. Moreover, in 12 cell lines, this integration ocurred non-randomly at a specific chromosomal sites, targeting 13q21 recurrently. Whether the integration of EBV in these loci affects the expression of genes important for the pathogenesis of BL or whether the EBV integration contributes to an enhanced chromosomal instability remains to be elucidated. Disclosures:No relevant conflicts of interest to declare.

Epstein-Barr virus integrates frequently into chromosome 4q, 2q, 1q and 7q of Burkitt's lymphoma cell line (Raji)

Epstein-Barr virus (EBV) integration into a Burkitt's lymphoma (BL) cell line (Raji) was investigated, using polymerase chain reaction (PCR), Southern hybridization, genomic library screening and fluorescence in situ hybridization (FISH). BaMHIW fragments of the EBV genome and DNA sequences of the viral latent membrane protein (LMP)1 and LMP2 genes were detected in Raji cells. BaMHI-digested high-molecular weight DNA from Raji cells generated 4 and 10 kb, 23 kb fragments that hybridized to Probe-1 (EBV genome 13232–16189) and Probe-2 (EBV genome 5–3271). Genomic library for Raji cells was constructed. Plaques (1 × 10 5) were screened with Probe-2, and four positive clones were obtained. Chromosomal integration of EBV DNA was detected in the Raji cell. The viral integration sites included 1p, 1q, 2q, 3p, 3q, 4q, 5q, 6q, 7p, 7q, 9q, 11p, 14q and 15q. Despite this multiplicity of integration sites, integration showed high frequency only at the sites 4q, 2q, 1q and 7q; 64% of the total signals were found in these four chromosomal bands. No viral integration occurred in chromosomes 16–22 or the sex chromosomes (X, Y). This study is the first comprehensive FISH analysis of EBV integration into the chromosomes of the Raji cell line. The findings support the notion that EBV integrates into the Raji cell genome non-randomly.

Integration of Epstein-Barr Virus into Chromosome 6q15 of Burkitt Lymphoma Cell Line (Raji) Induces Loss of BACH2 Expression

Epstein-Barr virus (EBV) initially isolated from cultured Burkitt lymphoma (BL) cells, is a well-known oncogenic virus. The Raji cell line was established from BL tissue and used for research worldwide. Previous study showed that each Raji cell contains an average of 50-60 EBV genome equivalents, and a significant proportion of the EBV genome is linearly integrated into host genome through BamHI-W close to the BamHI-Y fragment. However, a definitive EBV integration site in the chromosome has not been identified as yet. In this study, direct evidence that EBV DNA is integrated into the host genome was provided through cloning of the fragments containing nucleotide sequence of Raji integration sites. Integrated EBV DNA consisted of the BamHI-W fragment at one end and BamHI-D fragment at another end. Both junction sites were highly guanine/cytosine-rich. The BamHI-W fragment and the adjacent part of chromosome 6 showed 70% homology, while no homology was found between the BamHI-D and adjacent host sequences. EBV was present at intron 1 of the BACH2 gene located on chromosome 6q15. BACH2 was not expressed in the Raji cell line. Because BACH2 is a putative tumor suppressor gene, loss of its expression through EBV integration might contribute to lymphomagenesis.

EBV-associated, extranodal NK-cell lymphoma, nasal type of the breast, after heart transplantation

Post-transplantation lymphoproliferative disorders (PTLDs) are predominantly Epstein–Barr virus (EBV)-associated B-cell lymphoproliferations. PTLDs of T-cell lineage are rare, mostly reported after renal transplantation and show less frequent association with EBV. NK-cell lymphomas after transplantation (NK-cell PTLDs) are very rare; only five cases are reported so far in the English literature, all developed after renal transplantation. We describe a case of EBV-associated, extranodal NK-cell lymphoma of nasal type, involving the breast in a cardiac allograft recipient 5 years after transplantation. The neoplastic cells are positive for CD2, cytoplasmic CD3, CD7, CD43, CD56, TIA-1 and p53; and negative for surface CD3 and CD57. Analysis of T-cell receptor beta and gamma genes fails to show clonal rearrangement. EBV studies show clonal episomal integration of EBV and latency II pattern (EBER-1+, LMP-1+, EBNA-1+, EBNA-2-). In conclusion, NK-cell PTLDs are rare complications that arise relatively late after solid organ transplantation, show strong association with EBV, and can follow an aggressive clinical course. To the best of our knowledge, we present the first reported case of NK-cell PTLD after cardiac transplantation and the unifying clinical and diagnostic features of NK-cell PTLDs occurring after solid organ transplantation.

Complex Genomic Rearrangement of ALK Loci Associated with Integrated Human Epstein-Barr Virus in a Post-Transplant Myogenic Liver Tumor

Epstein-Barr virus (EBV) is a ubiquitous viral agent, well known to be associated with lymphoid, epithelial, and smooth-muscle malignancies in immunocompromised individuals. This report describes a 10-year-old patient with an EBV-related liver tumor occurring after kidney transplantation. The neoplasm presented a phenotypic spectrum, ranging from a smooth-muscle tumor to an inflammatory pseudotumor (IPT). The neoplastic cells failed to disclose CD21, CD35, or ALK expression, the latter confirmed by reverse-transcription polymerase chain reaction. Cytogenetic analysis revealed a single clonal cell population showing 46,XY,del (2)(p23),der(3)t (2;3)(p23;q29),der(21) t(Y;21)(q12;p13) karyotype. By metaphase FISH analysis, the neoplastic cells demonstrated the presence of two molecularly different but related aberrant clones, one with the loss of one ALK allele and the second with translocation of the 3'end of ALK kinase domain on the der(3) chromosome. Using FISH with an EBV-specific and 3'end ALK DNA probes, a co-localization of the viral DNA and the ALK sequences was found on the der(3) chromosome. Metaphases with loss of rearranged ALK did not show integrated virus; instead, viral particles together with an associated 3'end ALK domain formed an ex-chromosomal, episomal-like type configuration. The interphase study, using dual-color 5'/3' end ALK FISH assay, revealed 30% of nuclei with only one fused signal, confirming the total loss of one ALK allele in the subset of tumor cells. A combined immunofluorescence and FISH study indicated this separate clonal variant to correspond to desmin-positive smooth-muscle cells. In contrast, desmin-negative myofibroblasts showed the presence of both normal and rearranged ALK alleles. Our results indicate that ALK locus may be a target of EBV integration, a hitherto unreported finding. Although the sustained clonal expansion in EBV-related smooth-muscle tumors/IPTs may depend on functions provided by the EBV oncogenic proteins, the tumor phenotype may be further modified by the secondary genomic rearrangements imposed by the virus during and/or after the integration event. In this respect, the observed phenotypic heterogeneity most likely reflects divergence during neoplastic progression, with the subsequent expansion of morphologically and molecularly distinct but cytogenetically related clones.

Epstein-Barr virus (EBV)-positive pyothorax-associated lymphoma (PAL): chromosomal integration of EBV in a novel CD2-positive PAL B-cell line.

Pyothorax-associated lymphoma (PAL) is a clinico-pathological entity arising in the pleural cavity of patients with long-standing inflammatory pyothorax. PAL is closely associated with Epstein-Barr virus (EBV), but how this virus contributes to the development of the lymphoma is unknown. We have successfully obtained a novel EBV-infected PAL cell line, designated Pal-1. The cell line and its source coexpressed CD2 and CD20 molecules, but other representative B- and T-cell markers such as CD1, CD3, CD5, CD7, CD10 and CD19 were not found. The B-cell origin of Pal-1 cells was proven by rearrangement of the immunoglobulin heavy- and light-chain genes without rearranged T-cell receptor genes. Both the cell line and primary tumour cells carried monoclonal EBV genome. Although EBV genome is known to be maintained as circular extrachromosomal DNA, neither circular nor linear extrachromosomal EBV DNA was detectable in Pal-1 cells by in situ lysis gel analysis. Fluorescence in situ hybridization demonstrated viral integration at a marker chromosome mostly consisting of the centromere region of chromosome 1. The viral integration event may enhance a chromosomal instability at the insertion site. This cell line represents the first example of EBV integration in PAL and could enable the study of the potential role of integrated viral infection in the development of PAL as well as mechanism of the aberrant phenotype expression.

Chromosomal integration of Epstein - Barr virus genomes in nasopharyngeal carcinoma cells

Background Little has been known about whether Epstein-Barr virus (EBV) could persist in nasopharyngeal carcinoma (NPC) cells by chromosomal integration, and no NPC cell line harboring integrated EBV has been reported. In this study, we explored this issue through isolating EBV-infected NPC cell clones generated from an in vitro infection system and examining the configuration of EBV DNA in these cells. Methods and Results EBV genomes were demonstrated in NPC cell clones using polymerase chain reaction and Southern hybridization. Viral nuclear antigens were also detected by use of an anticomplement immunofluorescence assay and an immunoblotting assay. Gardella gel analysis showed that two of the EBV-positive cell clones, H2B4 and H2B17-7, harbored no extrachromosomal form of the viral genome. Restriction analysis of EBV genomic termini indicated that EBV DNA in these two cell clones was not circularized, and the viral genomes were integrated into chromosomes as demonstrated by fluorescence in situ hybridization. Conclusions This is the first in vitro model of EBV persistence in NPC cells by genomic integration, which represents a unique state of virus-cell interaction. Using this model, investigation into the association between EBV integration and chromosomal abnormality in tumor cells will help to reveal the underlying biologic significance. © 2002 John Wiley & Sons, Inc.

Chromosomal integration of Epstein-Barr virus genomes in nasopharyngeal carcinoma cells.

Little has been known about whether Epstein-Barr virus (EBV) could persist in nasopharyngeal carcinoma (NPC) cells by chromosomal integration, and no NPC cell line harboring integrated EBV has been reported. In this study, we explored this issue through isolating EBV-infected NPC cell clones generated from an in vitro infection system and examining the configuration of EBV DNA in these cells. EBV genomes were demonstrated in NPC cell clones using polymerase chain reaction and Southern hybridization. Viral nuclear antigens were also detected by use of an anticomplement immunofluorescence assay and an immunoblotting assay. Gardella gel analysis showed that two of the EBV-positive cell clones, H2B4 and H2B17-7, harbored no extrachromosomal form of the viral genome. Restriction analysis of EBV genomic termini indicated that EBV DNA in these two cell clones was not circularized, and the viral genomes were integrated into chromosomes as demonstrated by fluorescence in situ hybridization. This is the first in vitro model of EBV persistence in NPC cells by genomic integration, which represents a unique state of virus-cell interaction. Using this model, investigation into the association between EBV integration and chromosomal abnormality in tumor cells will help to reveal the underlying biologic significance.

Integrated and episomal forms of Epstein–Barr virus (EBV) in EBV associated disease

Epstein–Barr virus (EBV) is known to be linear in viral particles but EBV circularizes into an episomal form after infection. Recently, the presence of integrated EBV DNA has been reported. We investigated EBV integration into the human genome in EBV-associated disease using Southern blotting. One hundred four cases in which the presence of EBV was confirmed by Southern blotting with EBV-W probes were thus analyzed with left- and right-hand end probes of linear EBV. Integrated EBV was demonstrated in 11 of 104 cases; five of 14 cases with B cell lymphoma (36%), one of 12 cases with nasopharyngeal carcinomas (8%), four of 31 cases with natural killer (NK) leukemia/lymphoma (13%) and one of 11 cases with chronic EBV infection (9%). However, none of the 24 T cell lymphoma, seven Hodgkin's disease, or five acute EBV infection cases showed integrated EBV. In addition, seven of the 11 cases with EBV integration (five B cell lymphoma and two NK leukemia/lymphoma) showed only an integration form, however, the other four (two NK leukemia/lymphoma, one nasopharyngeal carcinoma and one chronic EBV infection) showed both integrated and episomal forms. The integrated form was frequently found in B cell lymphoma and especially in high grade B cell lymphoma. Fluorescence in situ hybridization (FISH) was performed in two cases (NK and B cell lymphoma), which represented integrated EBV in Southern blotting and the integration form was confirmed in both. However, it is still uncertain as to whether or not the EBV integration site is directly associated with chromosomal abnormality.

Integrated Epstein‐Barr virus (EBV) and chromosomal abnormality in chronic active EBV infection

In order to examine the role of Epstein-Barr virus (EBV) in the pathogenesis of chronic active EBV infection (CAEBV), we investigated whether or not EBV integration into the human genome is associated with any chromosonal abnormality. We therefore analyzed 4 cases of CAEBV: 2 cases showed a normal karyotype, while one had an oligo-clonal 6th chromosomal abnormality and the fourth had a clonal 6th deletion (q15q23). In addition, the case with an oligo-clonal abnormality also had oligo-clonal EBV terminal repeat (TR) bands, while the case with a clonal abnormality showed a clonal TR band. In contrast, the 2 cases with a normal karyotype showed no clonal band. Two-color fluorescence in situ hybridization (FISH) was used to detect the integrated EBV and the 6th chromosomal site. The presence of integrated EBV into the 6th chromosome was not frequent in the 2 cases with a normal karyotype, but it was statistically frequent in the case with an oligo-clonal 6th abnormality. In the case with a clonal 6th deletion, integration in the 6th chromosome was also slightly higher than that in the other chromosomes. In CAEBV, integrated EBV might thus be associated both with chromosomal abnormality and with pathogenesis. Int. J. Cancer 71: 943-947, 1997. © 1997 Wiley-Liss Inc.

Integrated Epstein-Barr virus (EBV) and chromosomal abnormality in chronic active EBV infection.

In order to examine the role of Epstein-Barr virus (EBV) in the pathogenesis of chronic active EBV infection (CAEBV), we investigated whether or not EBV integration into the human genome is associated with any chromosonal abnormality. We therefore analyzed 4 cases of CAEBV: 2 cases showed a normal karyotype, while one had an oligo-clonal 6th chromosomal abnormality and the fourth had a clonal 6th deletion (q15q23). In addition, the case with an oligo-clonal abnormality also had oligo-clonal EBV terminal repeat (TR) bands, while the case with a clonal abnormality showed a clonal TR band. In contrast, the 2 cases with a normal karyotype showed no clonal band. Two-color fluorescence in situ hybridization (FISH) was used to detect the integrated EBV and the 6th chromosomal site. The presence of integrated EBV into the 6th chromosome was not frequent in the 2 cases with a normal karyotype, but it was statistically frequent in the case with an oligo-clonal 6th abnormality. In the case with a clonal 6th deletion, integration in the 6th chromosome was also slightly higher than that in the other chromosomes. In CAEBV, integrated EBV might thus be associated both with chromosomal abnormality and with pathogenesis.

Integration of Epstein-Barr virus in Burkitt's lymphoma cells leads to a region of enhanced chromosome instability

Several lymphomas are associated with Epstein-Barr virus (EBV) infection. However EBV is not detectable in 100% of cases using standard staining techniques. It still remains an open question whether in these EBV-negative cases EBV has never infected the cell, whether it has infected the cell and escapes conventional screening methods, or whether it has been lost again after initial infection. The physical status of EBV in the Burkitt's lymphoma cell line BL60-P7 as well as in three somatic cell hybrids between BL60-P7 and its autologous EBV-immortalized lymphoblastoid cell line IARC 277 was analyzed using conventional cytogenetics, Southern blotting, and fluorescence in situ hybridization (FISH). Integration of EBV into the host genome of the lymphoma cell line BL60-P7 leads to an achromatic gap which causes a 'vulnerable site'. In hybrid cells, loss of integrated EBV, together with an adjacent chromosomal fragment, occurs during long-term cultivation. The integrated EBV genome, including genes encoding for LMP and EBER, is partly deleted. We assume that integration of EBV into the host cell genome could be a more common event in lymphoma cells. Partially deleted EBV might escape standard detection assays. The integration might constitute a chromosomal region prone to break events akin to the phenomenon of fragile sites, leading to the loss of viral DNA as well as chromosomal DNA. This observation makes it tempting to speculate that under certain conditions EBV can act in lymphomagenesis by a so-called hit-and-run mechanism.