Virological aspects of Epstein-Barr virus infections.

Epstein-Barr virus (EBV) is usually maintained in an asymptomatic and latent form by the host immune system, and primarily by EBV-specific cytotoxic T cells (CTLs). However, EBV has been linked to several refractory diseases such as EBV-associated hemophagocytic syndrome(EBV-AHS) and chronic active EBV infection (CAEBV). In these ectopic diseases, EBV infects T/NK cells, causing severe immunodeficiency with a very high EBV load. In recent years, the laboratory procedure to assess these types of EBV infections has been improved. In particular, real-time polymerase chain reaction (PCR) has been used to quantify the EBV load, and the MHC: peptide tetramer assay has been used to quantitate EBV-specific CTLs; these tests have been employed for the management of the illnesses associated with EBV infection. Here, we have reviewed the recent progress in the clinical application of these assays. The pathogenesis of EBV-infected T/NK cells, and the host immune response to infection, including the roles carried out by innate immunity and inflammatory cytokines, are likely to be revealed in the future.</p

Clonal origin of Epstein-Barr virus-infected T/NK-cell subpopulations in chronic active Epstein-Barr virus infection

Clonal expansion of Epstein-Barr virus (EBV) infected B-cells occasionally occurs in immunocompromized subjects. EBV-infected T/natural killer (NK)-cells proliferate in patients with chronic active EBV infection (CAEBV) that is a rare mononucleosis syndrome. It is classified into either T-cell type or NK-cell type according to the primary target of infection, while the pathogenesis remains unclear. To search the clonal origin of EBV-infected T/NK-cells, virus distribution and clonotype were assessed by using highly purified cell fractions obtained from 6 patients. Patient 1 had a monoclonal proliferation of EBV-infected T-cell receptor V&#x3b4;2/V&#x3b3;9-expressing cells, and carried lower copy number of EBV in &#x3b1;&#x3b2;T-cells. Patients 2 and 3 had a clonal expansion of EBV-infected CD4+T-cells, and lower EBV load in CD56+cells. Patients 4, 5 and 6 had an expansion of CD56+cells with higher EBV load than CD3+cells. EBV-terminal repeats were determined as clonal bands in the minor targeted populations of 5 patients. The size of terminal repeats indicated the same clonotype in minor subsets as in major subsets of 4 patients. However, EBV was not detected in bone marrow-derived lineage negative CD34+cells of patients. These results suggested that EBV could infect T/NK-cells at differentiation stage, but spared bone marrow CD34+hematopoietic stem cells in CAEBV patients

Purification and quantification of recombinant Epstein-Barr viral glycoproteins gp350/220 from Chinese hamster ovary cells

Truncated Epstein-Barr virus (EBV) membrane antigen gp350/220 (EBV-MA) lacking the membrane anchor was expressed and secreted into the medium of recombinant Chinese hamster ovary cells that had been cultured in Plasmapur hollow-fibre modules using defined serum-free medium. The EBV-MA in the medium was concentrated by 70% (w/v) ammonium sulphate precipitation and subsequently purified by immunoaffinity chromatography using an anti-EBV-MA (EBV.0T6) monoclonal antibody (mAb) column. Adsorbed antigen was eluted with 3 M MgCl2 in phosphate-buffered saline, concentrated by Mono Q anion-exchange chromatography and analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, silver staining and Western blotting using EBV-positive serum and anti-EBV-MA specific mAbs. Monospecific polyclonal rabbit antibodies against the purified EBV-MA were raised and purified by protein G affinity chromatography. For the measurement of EBV-MA antigen levels a sandwich enzyme-linked immunosorbent assay using rabbit polyclonal antibodies and a horseradish peroxidase-conjugated anti-MA mAb was developed having a detection level of 10 ng/ml

Epstein-Barr virus lytic infection promotes activation of Toll-like receptor 8 innate immune response in systemic sclerosis monocytes

BACKGROUND: Monocytes/macrophages are activated in several autoimmune diseases, including systemic sclerosis (scleroderma; SSc), with increased expression of interferon (IFN)-regulatory genes and inflammatory cytokines, suggesting dysregulation of the innate immune response in autoimmunity. In this study, we investigated whether the lytic form of Epstein-Barr virus (EBV) infection (infectious EBV) is present in scleroderma monocytes and contributes to their activation in SSc. METHODS: Monocytes were isolated from peripheral blood mononuclear cells (PBMCs) depleted of the CD19+ cell fraction, using CD14/CD16 negative-depletion. Circulating monocytes from SSc and healthy donors (HDs) were infected with EBV. Gene expression of innate immune mediators were evaluated in EBV-infected monocytes from SSc and HDs. Involvement of Toll-like receptor (TLR)8 in viral-mediated TLR8 response was investigated by comparing the TLR8 expression induced by infectious EBV to the expression stimulated by CL075/TLR8/agonist-ligand in the presence of TLR8 inhibitor in THP-1 cells. RESULTS: Infectious EBV strongly induced TLR8 expression in infected SSc and HD monocytes in vitro. Markers of activated monocytes, such as IFN-regulated genes and chemokines, were upregulated in SSc- and HD-EBV-infected monocytes. Inhibiting TLR8 expression reduced virally induced TLR8 in THP-1 infected cells, demonstrating that innate immune activation by infectious EBV is partially dependent on TLR8. Viral mRNA and proteins were detected in freshly isolated SSc monocytes. Microarray analysis substantiated the evidence of an increased IFN signature and altered level of TLR8 expression in SSc monocytes carrying infectious EBV compared to HD monocytes. CONCLUSION: This study provides the first evidence of infectious EBV in monocytes from patients with SSc and links EBV to the activation of TLR8 and IFN innate immune response in freshly isolated SSc monocytes. This study provides the first evidence of EBV replication activating the TLR8 molecular pathway in primary monocytes. Immunogenicity of infectious EBV suggests a novel mechanism mediating monocyte inflammation in SSc, by which EBV triggers the innate immune response in infected cells

Extra-telomeric functions of telomerase in the pathogenesis of Epstein-Barr virus-driven B-cell malignancies and potential therapeutic implications

Abstract The Epstein-Barr virus (EBV) is a ubiquitous human \u3b3-herpesvirus causally linked to a broad spectrum of both lymphoid and epithelial malignancies. In order to maintain its persistence in host cells and promote tumorigenesis, EBV must restrict its lytic cycle, which would ultimately lead to cell death, selectively express latent viral proteins, and establish an unlimited proliferative potential. The latter step depends on the maintenance of telomere length provided by telomerase. The viral oncoprotein LMP-1 activates TERT, the catalytic component of telomerase. In addition to its canonical role in stabilizing telomeres, TERT may promote EBV-driven tumorigenesis through extra-telomeric functions. TERT contributes toward preserving EBV latency; in fact, through the NOTCH2/BATF pathway, TERT negatively affects the expression of BZLF1, the master regulator of the EBV lytic cycle. In contrast, TERT inhibition triggers a complete EBV lytic cycle, leading to the death of EBV-infected cells. Interestingly, short-term TERT inhibition causes cell cycle arrest and apoptosis, partly by inducing telomere-independent activation of the ATM/ATR/TP53 pathway. Importantly, TERT inhibition also sensitizes EBV-positive tumor cells to antiviral therapy and enhances the pro-apoptotic effects of chemotherapeutic agents. We provide here an overview on how the extra-telomeric functions of TERT contribute to EBV-driven tumorigenesis. We also discuss the potential therapeutic approach of TERT inhibition in EBV-driven malignancies

Measurement of Epstein-Barr virus DNA load using a novel quantification standard containing two EBV DNA targets and SYBR Green I dye

BACKGROUND Reactivation of Epstein-Barr virus (EBV) infection may cause serious, life-threatening complications in immunocompromised individuals. EBV DNA is often detected in EBV-associated disease states, with viral load believed to be a reflection of virus activity. Two separate real-time quantitative polymerase chain reaction (QPCR) assays using SYBR Green I dye and a single quantification standard containing two EBV genes, Epstein-Barr nuclear antigen-1 (EBNA-1) and BamHI fragment H rightward open reading frame-1 (BHRF-1), were developed to detect and measure absolute EBV DNA load in patients with various EBV-associated diseases. EBV DNA loads and viral capsid antigen (VCA) IgG antibody titres were also quantified on a population sample. RESULTS EBV DNA was measurable in ethylenediaminetetraacetic acid (EDTA) whole blood, peripheral blood mononuclear cells (PBMCs), plasma and cerebrospinal fluid (CSF) samples. EBV DNA loads were detectable from 8.0 × 10² to 1.3 × 10⁸ copies/ml in post-transplant lymphoproliferative disease (n = 5), 1.5 × 10³ to 2.0 × 10⁵ copies/ml in infectious mononucleosis (n = 7), 7.5 × 10⁴ to 1.1 × 10⁵ copies/ml in EBV-associated haemophagocytic syndrome (n = 1), 2.0 × 10² to 5.6 × 10³ copies/ml in HIV-infected patients (n = 12), and 2.0 × 10² to 9.1 × 10⁴ copies/ml in the population sample (n = 218). EBNA-1 and BHRF-1 DNA were detected in 11.0% and 21.6% of the population sample respectively. There was a modest correlation between VCA IgG antibody titre and BHRF-1 DNA load (rho = 0.13, p = 0.05) but not EBNA-1 DNA load (rho = 0.11, p = 0.11). CONCLUSION Two sensitive and specific real-time PCR assays using SYBR Green I dye and a single quantification standard containing two EBV DNA targets, were developed for the detection and measurement of EBV DNA load in a variety of clinical samples. These assays have application in the investigation of EBV-related illnesses in immunocompromised individuals.The Ausimmune Study is funded by the National Multiple Sclerosis Society of the USA, the National Health & Medical Research Council (Project Grant 316901) and Multiple Sclerosis Research Australia

Identification of Epstein-Barr virus replication proteins in Burkitt’s lymphoma cells

The working model to describe the mechanisms used to replicate the cancer-associated virus Epstein-Barr virus (EBV) is partly derived from comparisons with other members of the Herpes virus family. Many genes within the EBV genome are homologous across the herpes virus family. Published transcriptome data for the EBV genome during its lytic replication cycle show extensive transcription, but the identification of the proteins is limited. We have taken a global proteomics approach to identify viral proteins that are expressed during the EBV lytic replication cycle. We combined an enrichment method to isolate cells undergoing EBV lytic replication with SILAC-labeling coupled to mass-spectrometry and identified viral and host proteins expressed during the OPEN ACCESS Pathogens 2015, 4 740 EBV lytic replication cycle. Amongst the most frequently identified viral proteins are two components of the DNA replication machinery, the single strand DNA binding protein BALF2, DNA polymerase accessory protein BMRF1 and both subunits of the viral ribonucleoside-diphosphate reductase enzyme (BORF2 and BaRF1). An additional 42 EBV lytic cycle proteins were also detected. This provides proteomic identification for many EBV lytic replication cycle proteins and also identifies post-translational modifications

Epstein-Barr virus infections and DNA hybridization studies in posttransplantation lymphoma and lymphoproliferative lesions: The role of primary infection

Fourteen patients who developed B cell lymphomas or lymphoproliferative lesions after kidney, liver, heart, or heart-lung transplantation in Pittsburgh during 1981-1983 had active infection with Epstein-Barr virus (EBV)of the primary (six patients), reactivated (seven patients), or chronic (one patient) type. In transplant patients without tumors, the incidence of EBV infection was 30% (39 of 128). Only three of these patients had primary infections. Thus the frequency of active infection was significantly higher in patients with tumors, and patients with primary infections were at greater risk of developing tumors. Five of 13 tumors tested contained EBV nuclear antigen (EBNA) and nine of 11 contained EBV genomes detected by DNA-DNA hybridization with BamHI K, BamHI W, or EcoRI B cloned probes. All EBNA-positive tumors, except one, were also positive by hybridization. Only one tumor was negative for both EBNA and EBV DNA. These data suggest that EBV plays an etiologic role in the development of these lesions. © 1985 by The University of Chicago

Deciphering the role of Epstein-Barr virus in the pathogenesis of T and NK cell lymphoproliferations

Epstein-Barr virus (EBV) is a highly successful herpesvirus, colonizing more than 90% of the adult human population worldwide, although it is also associated with various malignant diseases. Primary infection is usually clinically silent, and subsequent establishment of latency in the memory B lymphocyte compartment allows persistence of the virus in the infected host for life. EBV is so markedly B-lymphotropic when exposed to human lymphocytes in vitro that the association of EBV with rare but distinct types of T and NK cell lymphoproliferations was quite unexpected. Whilst relatively rare, these EBV-associated T and NK lymphoproliferations can be therapeutically challenging and prognosis for the majority of patients is dismal. In this review, we summarize the current knowledge on the role of EBV in the pathogenesis of these tumours, and the implications for treatment. \ud \u

lHuman cytotoxic T lymphocytes with reduced sensitivity to Fas-induced apoptosis

Effector-memory T cells expressing Fas (Apo-1/CD95) are switched to an apoptotic program by cross-linking with Fas-ligand (FasL). Consequently, tumors that express FasL can induce apoptosis of infiltrating Fas-positive T lymphocytes and subdue any antitumor host immune response. Since Epstein-Barr virus (EBV)-associated tumors such as Hodgkin lymphoma (HL) and nasopharyngeal carcinoma (NPC) express FasL, we determined whether EBV-specific cytotoxic T lymphocytes (EBV-CTLs) could be modified to resist this evasion strategy. We show that long-term down-modulation of Fas can be achieved in EBV-CTLs by transduction with small interfering RNA (siRNA) encoded in a retrovirus. Modified T cells resisted Fas/FasL-mediated apoptosis compared with control cells and showed minimal cleavage of the caspase3 substrate poly(ADP-ribose) polymerase (PARP) protein after Fas engagement. Prolonged Fas stimulation selected a uniformly Fas(low) and FasL resistant population. Removal of responsiveness to this single death signal had no other discernible effects on EBV-CTLs. In particular, it did not lead to their autonomous growth since the modified EBV-CTLs remained polyclonal, and their survival and proliferation retained dependence on antigen-specific stimulation and on the presence of other physiologic growth signals. EBV-CTLs with knocked down Fas should have a selective functional and survival advantage over unmodified EBV-CTLs in the presence of tumors expressing FasL and may be of value for adoptive cellular therapy. (c) 2005 by The American Society of Hematology