Characterization of Epstein-Barr virus–infected B cells in patients with posttransplantation lymphoproliferative disease: disappearance after rituximab therapy does not predict clinical response

Blood ◽  
2000 ◽  
Vol 96 (13) ◽  
pp. 4055-4063 ◽  
Author(s):  
Jie Yang ◽  
Qian Tao ◽  
Ian W. Flinn ◽  
Paul G. Murray ◽  
Linda E. Post ◽  
...  
Keyword(s):  
Viral Load ◽  
B Cells ◽  
Epstein Barr Virus ◽  
Viral Gene Expression ◽  
Lymphoproliferative Disease ◽  
Viral Gene ◽  
Healthy Controls ◽  
Viral Loads ◽  
Barr Virus ◽  
Epstein Barr

Abstract Post-transplantation lymphoproliferative disease (PTLD) is associated with Epstein-Barr virus (EBV). Quantitative and qualitative differences in EBV in peripheral blood mononuclear cells (PBMCs) of PTLD patients and healthy controls were characterized. A quantitative competitive polymerase chain reaction (QC-PCR) technique confirmed previous reports that EBV load in PBMCs is increased in patients with PTLD in comparison with healthy seropositive controls (18 539 vs 335 per 106 PBMCs, P = .0002). The average frequency of EBV-infected cells was also increased (271 vs 9 per 106 PBMCs, P = .008). The distribution in numbers of viral genome copies per cell was assessed by means of QC-PCR at dilutions of PBMCs. There was no difference between PTLD patients and healthy controls. Similarly, no differences in the patterns of viral gene expression were detected between patients and controls. Finally, the impact of therapy on viral load was analyzed. Patients with a past history of PTLD who were disease-free (after chemotherapy or withdrawal of immunosuppression) at the time of testing showed viral loads that overlapped with those of healthy seropositive controls. Patients treated with rituximab showed an almost immediate and dramatic decline in viral loads. This decline occurred even in patients whose PTLD progressed during therapy. These results suggest that the increased EBV load in PBMCs of PTLD patients can be accounted for by an increase in the number of infected B cells in the blood. However, in terms of viral copy number per cell and pattern of viral gene expression, these B cells are similar to those found in healthy controls. Disappearance of viral load with rituximab therapy confirms the localization of viral genomes in PBMCs to B cells. However, the lack of relationship between the change in viral load and clinical response highlights the difference between EBV-infected PBMCs and neoplastic cells in PTLD.

Characterization of Epstein-Barr virus–infected B cells in patients with posttransplantation lymphoproliferative disease: disappearance after rituximab therapy does not predict clinical response

Blood ◽  
2000 ◽  
Vol 96 (13) ◽  
pp. 4055-4063 ◽  
Author(s):  
Jie Yang ◽  
Qian Tao ◽  
Ian W. Flinn ◽  
Paul G. Murray ◽  
Linda E. Post ◽  
...  
Keyword(s):  
Viral Load ◽  
B Cells ◽  
Epstein Barr Virus ◽  
Viral Gene Expression ◽  
Lymphoproliferative Disease ◽  
Viral Gene ◽  
Healthy Controls ◽  
Viral Loads ◽  
Barr Virus ◽  
Epstein Barr

Post-transplantation lymphoproliferative disease (PTLD) is associated with Epstein-Barr virus (EBV). Quantitative and qualitative differences in EBV in peripheral blood mononuclear cells (PBMCs) of PTLD patients and healthy controls were characterized. A quantitative competitive polymerase chain reaction (QC-PCR) technique confirmed previous reports that EBV load in PBMCs is increased in patients with PTLD in comparison with healthy seropositive controls (18 539 vs 335 per 106 PBMCs, P = .0002). The average frequency of EBV-infected cells was also increased (271 vs 9 per 106 PBMCs, P = .008). The distribution in numbers of viral genome copies per cell was assessed by means of QC-PCR at dilutions of PBMCs. There was no difference between PTLD patients and healthy controls. Similarly, no differences in the patterns of viral gene expression were detected between patients and controls. Finally, the impact of therapy on viral load was analyzed. Patients with a past history of PTLD who were disease-free (after chemotherapy or withdrawal of immunosuppression) at the time of testing showed viral loads that overlapped with those of healthy seropositive controls. Patients treated with rituximab showed an almost immediate and dramatic decline in viral loads. This decline occurred even in patients whose PTLD progressed during therapy. These results suggest that the increased EBV load in PBMCs of PTLD patients can be accounted for by an increase in the number of infected B cells in the blood. However, in terms of viral copy number per cell and pattern of viral gene expression, these B cells are similar to those found in healthy controls. Disappearance of viral load with rituximab therapy confirms the localization of viral genomes in PBMCs to B cells. However, the lack of relationship between the change in viral load and clinical response highlights the difference between EBV-infected PBMCs and neoplastic cells in PTLD.

scholarly journals Identification of Host Biomarkers of Epstein-Barr Virus Latency IIb and Latency III

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Joshua E. Messinger ◽  
Joanne Dai ◽  
Lyla J. Stanland ◽  
Alexander M. Price ◽  
Micah A. Luftig
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
Epstein Barr Virus ◽  
Single Cell Analysis ◽  
Viral Gene Expression ◽  
Molecular Pathogenesis ◽  
Viral Gene ◽  
Barr Virus ◽  
Ebv Infection ◽  
Epstein Barr

ABSTRACTDeciphering the molecular pathogenesis of virally induced cancers is challenging due, in part, to the heterogeneity of both viral gene expression and host gene expression. Epstein-Barr virus (EBV) is a ubiquitous herpesvirus prevalent in B-cell lymphomas of immune-suppressed individuals. EBV infection of primary human B cells leads to their immortalization into lymphoblastoid cell lines (LCLs), serving as a model of these lymphomas. In previous studies, reports from our laboratory have described a temporal model for immortalization with an initial phase characterized by expression of Epstein-Barr nuclear antigens (EBNAs), high levels of c-Myc activity, and hyperproliferation in the absence of the latent membrane proteins (LMPs), called latency IIb. This is followed by the long-term outgrowth of LCLs expressing the EBNAs along with the LMPs, particularly NFκB-activating LMP1, defining latency III. However, LCLs express a broad distribution of LMP1 such that a subset of these cells express LMP1 at levels similar to those seen in latency IIb, making it difficult to distinguish these two latency states. In this study, we performed mRNA sequencing (mRNA-Seq) on early EBV-infected latency IIb cells and latency III LCLs sorted by NFκB activity. We found that latency IIb transcriptomes clustered independently from latency III independently of NFκB. We identified and validated mRNAs defining these latency states. Indeed, we were able to distinguish latency IIb cells from LCLs expressing low levels of LMP1 using multiplex RNA-fluorescencein situhybridization (RNA-FISH) targeting EBVEBNA2orLMP1and humanCCR7orMGST1. This report defines latency IIb as a bona fide latency state independent from latency III and identifies biomarkers for understanding EBV-associated tumor heterogeneity.IMPORTANCEEBV is a ubiquitous pathogen, with >95% of adults harboring a life-long latent infection in memory B cells. In immunocompromised individuals, latent EBV infection can result in lymphoma. The established expression profile of these lymphomas is latency III, which includes expression of all latency genes. However, single-cell analysis of EBV latent gene expression in these lymphomas suggests heterogeneity where most cells express the transcription factor, EBNA2, and only a fraction of the cells express membrane protein LMP1. Our work describes an early phase after infection where the EBNAs are expressed without LMP1, called latency IIb. However, LMP1 levels within latency III vary widely, making these states hard to discriminate. This may have important implications for therapeutic responses. It is crucial to distinguish these states to understand the molecular pathogenesis of these lymphomas. Ultimately, better tools to understand the heterogeneity of these cancers will support more-efficacious therapies in the future.

Epstein-Barr Virus-Associated Haemophagocytic Lymphohistiocytosis in Adults Characterised by High Viral Genome Load within Circulating Natural Killer Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3672-3672
Author(s):  
Christopher P Fox ◽  
Claire Shannon-Lowe ◽  
Philip Gothard ◽  
Bhuvan Kishore ◽  
Jeffrey R. Neilson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Nk Cells ◽  
Peripheral Blood ◽  
Epstein Barr Virus ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Barr Virus ◽  
Ebv Infection ◽  
Epstein Barr ◽  
Restricted Pattern

Abstract Abstract 3672 Poster Board III-608 Introduction Epstein Barr virus (EBV) is predominantly B lymphotrophic both in-vitro and in vivo, where in immunocompetent individuals the virus persists asymptomatically in the B lymphoid compartment under host T cell control. EBV's association with B cell malignancies, such as Hodgkin and Burkitt lymphoma, can be viewed as rare accidents of the virus' lifelong interaction with the B cell system. By contrast, EBV infection of NK and T cells is considered a rare event but is nonetheless strongly associated with a spectrum of rare lymphoproliferations: EBV-associated haemophagocytic lymphohistiocytosis (EBV-HLH), chronic active EBV infection (CAEBV), aggressive NK leukaemia and NK/T lymphoma all characterised pathogenically by the presence of monoclonal EBV in the T and/or NK cells. The mechanism of viral entry and its contribution to lymphoproliferation in these cell lineages remains to be clearly defined. The majority of reported cases of EBV-HLH occur in the context of primary EBV infection in children or adolescents, some of whom have a defined inherited immune defect. Adult cases of EBV-HLH occur extremely rarely and appear to be more frequent in individuals of East Asian origin. Furthermore, the vast majority of analysed cases of EBV-HLH have identified CD8+ lymphocytes as the predominant virus-bearing cell. To-date, EBV infection of (CD3-CD56+) circulating NK cells has not been reported and the pattern of viral gene expression remains unclear. Methods/Patients We analysed peripheral blood from three consecutive cases of EBV-HLH, referred to our laboratory between 2007-2009, to identify the predominant virus-harbouring cell. All three cases occurred in adults (mean age 44yrs), with no history of inherited immunodeficiency, who presented with clinical and laboratory features consistent with a diagnosis of HLH; fever, hepatosplenomegaly, pancytopenia, markedly elevated serum ferritin and lactate dehydrogenase and EBV copy number of 105-106 per millilitre of whole blood. Haemophagocytosis was unequivocally present on tissue biopsy from two patients. Mononuclear cells were separated using the MoFlo™ cell sorter into pure populations. Patient 1 and 2: CD19+CD3 −CD56−, CD3+CD19−CD56−, and CD56+CD3−CD19−. Patient 3: CD19+CD3 –CD16−, CD3+CD19−CD16−, CD16+CD3−CD19− and CD3−CD19−CD16−. DNA was subsequently extracted from each population and assayed by quantitative PCR, expressed as genome copies per million cells. Results In all three cases we found the predominant EBV load within the non-B, non-T lymphocyte populations; definitively shown to be the CD56+CD3− cell fraction in 2 cases and for case 3 within CD3−CD19−CD16− lymphocytes likely to represent CD56+CD16- NK cells (a minority population in normal peripheral blood). A representative figure is shown: We then quantitatively examined latent and lytic viral gene transcripts by real-time PCR and, in contrast to previously published data, we found a tightly restricted pattern of EBV gene expression with extremely high levels of EBER (EBV-encoded RNA) transcripts present. Lymphocytes derived from tonsillar tissue and peripheral blood, from both healthy and immunosuppressed individuals, served as control samples and demonstrated the predominant EBV genome load in the CD19+ B-cells but not the T or NK fractions. Conclusion This novel finding of high EBV genome copy numbers and a restricted pattern of viral gene expression, within circulating natural killer cells in the context of adult EBV-HLH, is both pathogenically intriguing and importantly, has relevance for the investigation of targeted therapies for this aggressive disease. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Infection Caused by Herpes Simplex Virus, Varicella-Zoster Virus and Epstein-Barr Virus in Organ Transplant Recipients, and their Diagnosis

1993 ◽  
Vol 4 (suppl c) ◽  
pp. 33-40
Author(s):  
F Diaz-Mitoma
Keyword(s):  
Herpes Simplex ◽  
B Cells ◽  
Epstein Barr Virus ◽  
Dna Amplification ◽  
Lymphoproliferative Disease ◽  
Lymphoproliferative Disorders ◽  
Diagnostic Methods ◽  
Transplant Recipients ◽  
Barr Virus ◽  
Epstein Barr

Immunosuppressed patients are at risk of severe herpesvirus infections. Herpes simplex (HSV), varicellazoster (VZV) and Epstein-Barr virus (EBV) infections are associated with characteristic syndromes in this population. Typically I-ISV and VZ:V infections cause mucocutaneous lesions; diagnosis is usually con finned by tissue culture or nuorescent microscopy. The availability of effective antiviral agents, and accurate techniques for laboratory diagnosis have improved the management of I-ISV and VZ:V infections. Antibody assays to demonstrate I-ISVorVZ:V infections are of limited value in immunocompromised patients, because the presence of antibodies does not indicate a decreased risk for HSV, varicella or zoster, but indicates susceptibility for reactivated infection. EBV is associated with lymphoproliferative disorders in transplant recipients. Infection of lymphocytes by EBV is a necessary step in achieving B cell transformation and immortalization. The lack of immunosurveillance against EBV-transformed B cells predisposes patients to developing invasive infiltration of transformed B cells . Diagnostic methods for EBV infections include lymphocyte transfom1ation, serology, and detection of DNA by direct hybridization or by DNA amplification. Quantitative oropharyngeal EBV shedding is a good marker for the development of lymphoproliferative disease in transplant recipients. Patients experiencing primary EBV infection are at the highest risk for lymphoproliferative disorders. Prophylactic antiviral therapy may be of benefit in preventing EBV replication and therefore in decreasing the risk for lymphoproliferation.

scholarly journals Genetic Evidence that EBNA-1 Is Needed for Efficient, Stable Latent Infection by Epstein-Barr Virus

1999 ◽  
Vol 73 (4) ◽  
pp. 2974-2982 ◽  
Author(s):  
May-Ann Lee ◽  
Margaret E. Diamond ◽  
John L. Yates
Keyword(s):  
B Cells ◽  
Cell Lines ◽  
Epstein Barr Virus ◽  
Frameshift Mutation ◽  
Latent Infection ◽  
Replication Initiation ◽  
Viral Gene ◽  
Circular Chromosome ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT Replication and maintenance of the 170-kb circular chromosome of Epstein-Barr virus (EBV) during latent infection are generally believed to depend upon a single viral gene product, the nuclear protein EBNA-1. EBNA-1 binds to two clusters of sites at oriP, an 1,800-bp sequence on the EBV genome which can support replication and maintenance of artificial plasmids introduced into cell lines that contain EBNA-1. To investigate the importance of EBNA-1 to latent infection by EBV, we introduced a frameshift mutation into the EBNA-1 gene of EBV by recombination along with a flanking selectable marker. EBV genomes carrying the frameshift mutation could be isolated readily after superinfecting EBV-positive cell lines, but not if recombinant virus was used to infect EBV-negative B-cell lines or to immortalize peripheral blood B cells. EBV mutants lacking almost all of internal repeat 3, which encode a repetitive glycine and alanine domain of EBNA-1, were generated in the same way and found to immortalize B cells normally. An EBNA-1-deficient mutant of EBV was isolated and found to be incapable of establishing a latent infection of the cell line BL30 at a detectable frequency, indicating that the mutant was less than 1% as efficient as an isogenic, EBNA-1-positive strain in this assay. The data indicate that EBNA-1 is required for efficient and stable latent infection by EBV under the conditions tested. Evidence from other studies now indicates that autonomous maintenance of the EBV chromosome during latent infection does not depend on the replication initiation function of oriP. It is therefore likely that the viral chromosome maintenance (segregation) function of oriP and EBNA-1 is what is required.

scholarly journals The Role of Epstein-Barr Virus in Adults With Bronchiectasis: A Prospective Cohort Study

2020 ◽  
Vol 7 (8) ◽  
Author(s):  
Chun-Lan Chen ◽  
Yan Huang ◽  
Miguel Angel Martinez-Garcia ◽  
Jing-Jing Yuan ◽  
Hui-Min Li ◽  
...  
Keyword(s):  
Epstein Barr Virus ◽  
Quantitative Polymerase Chain Reaction ◽  
Induced Sputum ◽  
Chronic Obstructive ◽  
Healthy Controls ◽  
Viral Loads ◽  
Barr Virus ◽  
Opportunistic Bacteria ◽  
Ebv Dna ◽  
Epstein Barr

Abstract Background Epstein-Barr virus (EBV) is implicated in the progression of chronic obstructive pulmonary disease. We aimed to determine whether EBV correlates with bronchiectasis severity, exacerbations, and progression. Methods We collected induced sputum in healthy controls and spontaneous sputum at 3–6-month intervals and onset of exacerbations in bronchiectasis patients between March 2017 and October 2018. EBV DNA was detected with quantitative polymerase chain reaction. Results We collected 442 sputum samples from 108 bronchiectasis patients and 50 induced sputum samples from 50 healthy controls. When stable, bronchiectasis patients yielded higher detection rates of EBV DNA (48.1% vs 20.0%; P = .001), but not viral loads (mean log10 load, 4.45 vs 4.76; P = .266), compared with controls; 64.9% of patients yielded consistent detection status between 2 consecutive stable visits. Neither detection rate (40.8% vs 48.1%; P = .393) nor load (mean log10 load, 4.34 vs 4.45; P = .580) differed between the onset of exacerbations and stable visits, nor between exacerbations and convalescence. Neither detection status nor viral loads correlated with bronchiectasis severity. EBV loads correlated negatively with sputum interleukin-1β (P = .002), CXC motif chemokine-8 (P = .008), and tumor necrosis factor–α levels (P = .005). Patients initially detected with, or repeatedly detected with, EBV DNA had significantly faster lung function decline and shorter time to next exacerbations (both P < .05) than those without. Detection of EBV DNA was unrelated to influenza virus and opportunistic bacteria (all P > .05). The EBV strains detected in bronchiectasis patients were phylogenetically homologous. Conclusions Patients with detection of EBV DNA have a shorter time to bronchiectasis exacerbations. EBV may contribute to bronchiectasis progression.

Epstein-Barr virus colonization of tonsillar and peripheral blood B-cell subsets in primary infection and persistence

Blood ◽  
2009 ◽  
Vol 113 (25) ◽  
pp. 6372-6381 ◽  
Author(s):  
Sridhar Chaganti ◽  
Emily M. Heath ◽  
Wolfgang Bergler ◽  
Michael Kuo ◽  
Maike Buettner ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Primary Infection ◽  
Epstein Barr Virus ◽  
Quantitative Polymerase Chain Reaction ◽  
Viral Loads ◽  
Barr Virus ◽  
Memory Pool ◽  
Epstein Barr ◽  
B Cell Subsets

AbstractEpstein-Barr virus (EBV) persists in the immune host by preferentially colonizing the isotype-switched (IgD−CD27+) memory B-cell pool. In one scenario, this is achieved through virus infection of naive (IgD+CD27−) B cells and their differentiation into memory via germinal center (GC) transit; in another, EBV avoids GC transit and infects memory B cells directly. We report 2 findings consistent with this latter view. First, we examined circulating non–isotype-switched (IgD+CD27+) memory cells, a population that much evidence suggests is GC-independent in origin. Whereas isotype-switched memory had the highest viral loads by quantitative polymerase chain reaction, EBV was detectable in the nonswitched memory pool both in infectious mononucleosis (IM) patients undergoing primary infection and in most long-term virus carriers. Second, we examined colonization by EBV of B-cell subsets sorted from a unique collection of IM tonsillar cell suspensions. Here viral loads were concentrated in B cells with the CD38 marker of GC origin but lacking other GC markers CD10 and CD77. These findings, supported by histologic evidence, suggest that EBV infection in IM tonsils involves extrafollicular B cells expressing CD38 as an activation antigen and not as a marker of ectopic GC activity.

Expression of Epstein-Barr Virus BamHI-A Rightward Transcripts in Latently Infected B Cells From Peripheral Blood

Blood ◽  
1999 ◽  
Vol 93 (9) ◽  
pp. 3026-3032 ◽  
Author(s):  
Honglin Chen ◽  
Paul Smith ◽  
Richard F. Ambinder ◽  
S. Diane Hayward
Keyword(s):  
B Cells ◽  
Peripheral Blood ◽  
Epstein Barr Virus ◽  
Open Reading Frames ◽  
Viral Gene ◽  
Barr Virus ◽  
Restricted Form ◽  
Latently Infected ◽  
Epstein Barr

In addition to the Epstein-Barr virus (EBV) EBNA and LMP latency genes, there is a family of alternatively spliced BamHI-A rightward transcripts (BARTs). These latency transcripts are highly expressed in the EBV-associated malignancies nasopharyngeal carcinoma and Burkitt’s lymphoma, and are expressed at lower levels in latently EBV-infected B-cell lines. The contribution of the BARTs to EBV biology or pathogenesis is unknown. Resting B cells have recently been recognized as a reservoir for EBV persistence in the peripheral blood. In these cells, EBV gene expression is tightly restricted and the only viral gene known to be consistently expressed is LMP2A. We used cell sorting and reverse-transcriptase polymerase chain reaction (RT-PCR) to examine whether BARTs are expressed in the restricted form of in vivo latency. Our results demonstrated that RNAs with splicing diagnostic for transcripts containing the BART RPMS1 and BARFO open-reading frames (ORFs) were expressed in CD19+ but not in CD23+ B cells isolated from peripheral blood of healthy individuals. The product of the proximal RPMS1 ORF has not previously been characterized. The RPMS1 ORF was shown to encode a 15-kD protein that localized to the nucleus of transfected cells. Expression of the BARTs in peripheral blood B cells suggests that the proteins encoded by these transcripts are likely to be important for maintenance of in vivo latency.

scholarly journals Fatty Acid Synthase Expression Is Induced by the Epstein-Barr Virus Immediate-Early Protein BRLF1 and Is Required for Lytic Viral Gene Expression

2004 ◽  
Vol 78 (8) ◽  
pp. 4197-4206 ◽  
Author(s):  
Yuling Li ◽  
Jennifer Webster-Cyriaque ◽  
Christine C. Tomlinson ◽  
Marielle Yohe ◽  
Shannon Kenney
Keyword(s):  
Gene Expression ◽  
Fatty Acid ◽  
Epstein Barr Virus ◽  
Fatty Acid Synthase ◽  
Viral Gene Expression ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Viral Gene ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT The Epstein-Barr virus (EBV) immediate-early (IE) protein BRLF1 (R) is a transcription factor that induces the lytic form of EBV infection. R activates certain early viral promoters through a direct binding mechanism but induces transcription of the other EBV IE gene, BZLF1 (Z), indirectly through cellular factors binding to a CRE motif in the Z promoter (Zp). Here we demonstrate that R activates expression of the fatty acid synthase (FAS) cellular gene through a p38 stress mitogen-activated protein kinase-dependent mechanism. B-cell receptor engagement of Akata cells also increases FAS expression. The FAS gene product is required for de novo synthesis of the palmitate fatty acid, and high-level FAS expression is normally limited to liver, brain, lung, and adipose tissue. We show that human epithelial tongue cells lytically infected with EBV (from oral hairy leukoplakia lesions) express much more FAS than uninfected cells. Two specific FAS inhibitors, cerulenin and C75, prevent R activation of IE (Z) and early (BMRF1) lytic EBV proteins in Jijoye cells. In addition, cerulenin and C75 dramatically attenuate IE and early lytic gene expression after B-cell receptor engagement in Akata cells and constitutive lytic viral gene expression in EBV-positive AGS cells. However, FAS inhibitors do not reduce lytic viral gene expression induced by a vector in which the Z gene product is driven by a strong heterologous promoter. In addition, FAS inhibitors do not reduce R activation of a naked DNA reporter gene construct driven by the Z promoter (Zp). These results suggest that cellular FAS activity is important for induction of Z transcription from the intact latent EBV genome, perhaps reflecting the involvement of lipid-derived signaling pathways or palmitoylated proteins. Furthermore, using FAS inhibitors may be a completely novel approach for blocking the lytic form of EBV replication.

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