Epstein-Barr virus and associated diseases. Course of Medical Virology, Institut Pasteur, 1995/1996

The Epstein-Barr virus (EBV) is an ubiquitous virus infecting nearly the entire adult human population. The EBV is closely associated with rhinopharyngeal cancer in Southern China and Northern Africa. Three geographic subtypes of EBV have been identified to date. They differ by their nuclear antigene EBNA2. The EBNA2 AC strains predominate in Asia; EBNA2 AD strains predominate in the United States; EBNA2 B strains have all been identified in black Africa. Burkitt's lymphoma is the most frequent tumor in children aged 5 to 9 years in equatorial Africa. A prospective study in 42,000 children in Ouganda demonstrated that children who develop Burkitt's lymphoma have severe EBV infection during the first months of life. Very early EBV infection observed in North or equatorial Africa increases the risk of Burkitt's lymphoma by 20-times that in Europe. Hyperendemic malaria observed in the equatorial zone increases the incidence of tumors by a factor of 20. An association between EBV and rhinopharyngeal cancer is a constant feature only in South China, in North and East Africa, as well as in arctic regions as cases of carcinoma not associated with EBV infection have been reported in Greece. Surveys in the Democratic Republic of China concerning several hundred thousand persons have shown that serum IgA/VCA allows early diagnosis of cancer. It is estimated that the risk of rhinopharyngeal cancer is 20% in Chinese with high levels of IgA/VCA.     

CD4+ T cells inhibit growth of Epstein-Barr virus-transformed B cells through CD95-CD95 ligand-mediated apoptosis

Greater than 90% of the human population acquire Epstein-Barr virus (EBV) in infancy and retain a lifelong latent infection without any clinical consequences. Nevertheless EBV has been identified as the causal agent of infectious mononucleosis, and is associated with several tumours including endemic Burkitt's lymphoma and B cell lymphomas in immunosupressed patients. B cells infected with EBV are transformed in vitro and grow continuously as lymphoblastoid cell lines. The growth of EBV-transformed B cells in vivo is controlled by the immune system. Studies on immunity to EBV have mainly focused on MHC class I-restricted CD8+ cytotoxic T cells specific for viral latent antigens. Here it is reported that in vitro stimulation of peripheral blood lymphocytes by autologous EBV-infected B cells, which have been induced to express lytic cycle antigens, gives rise to a predominantly CD4+ T cell response. Furthermore, the growth of EBV-infected B cells can also be regulated by these activated CD4+ T cells through apoptosis mediated by CD95-CD95 ligand (CD95L). CD95-CD95L-mediated apoptosis is an important mechanism of normal B cell growth regulation. As EBV-transformed B cells remain susceptible to this mechanism, the control of EBV in vivo may be not only by virus-specific CD8+ cytotoxic T cell immunity but also by normal mechanisms of immune regulation of B cell growth.     

Illness-induced hyperalgesia is mediated by a spinal NMDA-nitric oxide cascade

Epstein-Barr virus (EBV) is associated with the development of several B cell malignancies including Burkitt's lymphoma (BL), post-transplant lymphoproliferative disease (PTLD), and AIDS-related lymphomas. The latter two diseases result from EBV-driven B cell proliferation in the absence of normal immunosurveillance and as such, represent a heterogenous family of lymphoproliferative disorders. This article reviews studies on EBV gene expression and antibody development in PTLD and introduces recent information on the levels of EBV+ peripheral blood lymphocytes to discuss possible mechanisms of pathogenesis under varying conditions of immunosuppression.     

Detection of EBV RNA (EBER-1 and EBER-2) in malaria lymph nodes by in situ hybridization

Acute Plasmodium falciparum malaria in African children allows expansion of latent Epstein-Barr virus infection, leading to colonization of lymph nodes by virus-infected lymphoblasts in 60% of cases as demonstrated by in situ hybridization for the detection of EBER-1 and EBER-2 RNA. This probably arises against a background of malaria-induced immunosuppression to EBV and concurrent lymphoid activation. The relevance of the results to the pathogenesis of African endemic Burkitt's lymphoma is discussed.     

Genetic basis of acquired immunodeficiency syndrome-related lymphomagenesis

The molecular pathogenesis of systemic acquired immunodeficiency syndrome (AIDS)-related non-Hodgkin's lymphomas (AIDS-NHL) is a complex process involving both host factors and the accumulation of genetic lesions within the tumor clone. On the basis of the pattern of molecular lesions involved in these tumors, several distinct pathogenetic pathways can be presently identified in AIDS-related lymphomagenesis. These pathways selectively associate with the different clinicopathologic variants of AIDS-NHL. AIDS-related Burkitt's lymphoma is characterized by activation of c-MYC in all cases, disruption of p53 in 60% of the cases, and infection by Epstein-Barr virus (EBV) in 30% of the cases. AIDS-related diffuse large-cell lymphoma harbor frequent EBV infection (80%) and, in 20% of the cases, BCL-6 rearrangements. Finally, the pathogenesis of AIDS-related body cavity-based lymphoma involves infection by human herpesvirus-8 in all cases and frequently also the co-infection by EBV.     

Enantioselective high-performance liquid chromatography assay of (+)R- and (-)S-alpha-lipoic acid in human plasma

The Epstein-Barr virus (EBV) genome was detected by polymerase chain reaction (PCR) in mononuclear cells from bone marrows with diverse types of hematopoietic malignancies. Viral repeated sequences (BamHI-W region) were detected in 42 of 82 (51%) hematopoietic malignancies, including polycythemia vera, but not in nonneoplastic cases. EBV-positive cases were found to consist of various histological types. We did not detect any EBV PCR product in the peripheral blood. The EBV BamHI-Y, -H region, encoding EBV nuclear antigen 2 DNA, which is a single-copy gene in the viral genome, was detected in only 13 of 42 BamHI-W-positive cases, suggesting that the copy number of the EBV genome differed in each case. In all cases, the PCR band was verified by Southern blot hybridization using specific EBV probes. Whether the infected virus is an etiologic agent of the malignancy or merely a latent infection cannot be determined by the PCR assay performed under these conditions. These results, however, suggest that a novel form of EBV latent infection is present in the bone marrow of patients with hematopoietic malignancies.     

Clinical manifestations and therapies of AIDS associated tumors

We are giving an overview over the clinical features and different therapeutic options of HIV associated malignancies. There are three AIDS-defining malignancies: - Kaposi's sarcoma - Non-Hodgkin's lymphoma (NHL) - cervical cancer. In Kaposi sarcoma there is a broad therapeutic spectrum from cryotherapy to systemic chemotherapy depending on the site and stage of the Kaposi sarcoma. In NHL early therapeutic intervention is necessary because of the fast progress of the tumor. The cervical cancer in HIV-infected women seems to be more aggressive than in non-infected and also needs early therapeutic intervention. Many other tumors seem to occur more frequently in patients with HIV infection: anorectal cancer, malignant testicular tumors, lung cancer, Hodgkin's lymphoma, basal cell carcinoma, squamous cell carcinoma, and even malignant melanoma. The cancer incidence in HIV-patients seems to be higher among nonblacks. Most of the immunodeficiency associated tumors are virus induced and they are accompanied by a persistent viral infection, including HHV-8 in Kaposi's sarcoma; Epstein Barr virus (EBV) in NHL; and human papillomavirus (HPV) in cervical cancer. But there are also types of virus induced tumors which are not frequently associated with HIV-infection like the primary hepatocellular carcinoma in patients with hepatitis B virus infection.     

Epstein-Barr virus infection is associated with increased apoptosis in untreated and phorbol ester-treated human Burkitt's lymphoma (AW-Ramos) cells

Several papers have recorded that Epstein-Barr virus (EBV) infection can protect B-cells from apoptosis. In the present paper, we record an increase in apoptosis in an EBV-infected human Burkitt's lymphoma cell line (AW-Ramos), compared with its virus free counterpart (Ramos). The viral-infected cells died more rapidly by apoptosis during normal growth in culture: at 72h, Ramos cells had 8% apoptosis and AW-Ramos had 27% apoptosis. The EBV-infected cells were particularly sensitive to treatment with phorbol-12, 13-dibutyrate (PDBu, 0.05 microgram/ml): at 72h, Ramos cells had 12% apoptosis and AW-Ramos had 42% apoptosis. DNA gel electrophoresis supported the morphological findings. Our results serve as a caveat against generalizations that may be made about prevention of apoptosis by EBV infection.     

Epstein-Barr virus contributes to the malignant phenotype and to apoptosis resistance in Burkitt's lymphoma cell line Akata

In the present study, we established an in vitro system representing the Burkitt's lymphoma (BL)-type Epstein-Barr virus (EBV) infection which is characterized by expression of EBV-determined nuclear antigen 1 (EBNA-1) and absence of EBNA-2 and latent membrane protein 1 (LMP1) expression. EBV-negative cell clones isolated from the EBV-positive BL line Akata were infected with an EBV recombinant carrying a selectable marker, and the following selection culture easily yielded EBV-infected clones. EBV-reinfected clones showed BL-type EBV expression and restored the capacity for growth on soft agar and tumorigenicity in SCID mice that were originally retained in parental EBV-positive Akata cells and lost in EBV-negative subclones. Moreover, it was found that EBV-positive cells were more resistant to apoptosis than were EBV-negative cells. EBV-infected cells expressed the bcl-2 protein, through which cells might become resistant to apoptosis, at a higher level than did uninfected cells. This is the first report that BL-type EBV infection confers apoptosis resistance even in the absence of expression of LMP1 and BHRF1, both of which are known to have an antiapoptotic function. Surprisingly, transfection of the EBNA-1 gene into EBV-negative Akata clones could not restore malignant phenotypes and apoptosis resistance, thus suggesting that EBNA-1 alone was not sufficient for conferring them. Our results suggest that the persistence of EBV in BL cells is required for the cells to be more malignant and apoptosis resistant, which underlines the oncogenic role of EBV in BL genesis.     

Cell block preparation of a Burkitt's lymphoma cell line as a positive control for in situ hybridization for Epstein-Barr virus

OBJECTIVE: To examine the feasibility of the use of a cell block preparation of Epstein-Barr virus (EBV)-infected Burkitt's lymphoma cell line (EB-3) as a positive control for in situ hybridization (ISH) for EBV-encoded RNA (EBER). STUDY DESIGN: EB-3 cells were processed into cell block and cytocentrifuge preparations. ISH for EBER was performed, and the results were compared with a commercially available EBV positive control slide (cytocentrifuge). RESULTS: The cost of preparing the cell block and cytocentrifuge sample was only a fraction of the price of commercial control slides. ISH for EBER was strongly stained in all preparations with similar intensity of staining but with a much higher number of positive cells in the cell block. Although the cellular details in the cell block were considerably inferior to those on the cytocentrifuge and commercial control slide, with distortion of nuclei and smudging of chromatin, the interpretation of the ISH results was unaffected. The cell block was stable at room temperature when examined after one year. CONCLUSION: The cell block preparation of an EBV-infected Burkitt's lymphoma cell line serves as a reliable, stable and a relatively inexpensive control, with good preservation of cellular details of cellular RNA for ISH study for EBER in the detection of latent infection with EBV.     

Lung cancer: intermittent irradiation synchronized with respiratory motion--results of a pilot study

Pyothorax-associated lymphoma (PAL) is a newly-described entity developing several decades after artificial pneumothorax treatment for pulmonary or pleural tuberculosis. It is known to be associated with Epstein-Barr virus (EBV) with constant expression of the two latent membrane proteins: latent membrane protein (LMP)-1 and EBV-associated nuclear antigen (EBNA)-2. We are reporting three new cases of PAL. All of the tumours were of B-cell lineage and classified as large-cell diffuse lymphomas according to the International Working Formulation for the Classification of Lymphomas. The EBV genome was detected in two of the cases with LMP-1 and EBNA-2 expression. No EBV could be detected in the third case suggesting that different mechanisms may be involved in the pathogenesis of the disease. Body cavity-based high grade lymphomas (BCBL) represent a new disease, developing mainly in human immunodeficiency virus (HIV) infected patients: the tumoural cells often contain both human herpes virus (HHV)-8 (or Kaposi's sarcoma herpes virus) and EBV genomes, suggesting that these viruses might co-operate in the pathogenesis of the disease. The pleural location and the association of EBV have led to speculation that PAL could also be related to HHV-8 infection. However, no HHV-8 genome could be detected in any of the 14 tested cases already reported in the literature nor in the two cases we studied (one EBV-positive and one EBV-negative), suggesting that PAL and BCBL are two different entities.     

Epstein-Barr virus in malignancies in renal transplant recipients in Japan

A role for Epstein-Barr virus (EBV) in the development of malignancies including lymphomas, and carcinoma of the stomach, nasopharynx, thymus and salivary gland is suggested. It is indicated that EBV evokes polyclonal-B-cell-proliferative diseases in immunocompromised hosts, such as transplant patients, which results in monoclonal malignant lymphomas. The suppression of immune functions in these patients is thought to lead to incomplete elimination of the cells expressing EBV latent infection genes. To examine the etiological role of EBV in the development of malignancies following renal transplant in Japan, 42 malignancies in 1744 cases of renal transplant were studied for the presence and type of EBV. The polymerase chain reaction revealed that 5 malignancies were positive for EBV, all type A: 2 of 2 cases of non-Hodgkin's lymphoma (NHL), 2 of 8 cases of gastric adenocarcinoma of the common type, and 1 of 2 cases of gastric plasmacytoma. In situ hybridization revealed positive signals in the nucleus of tumor cells in 2 cases of NHL and 1 of plasmacytoma. Positive signals were found in the small lymphoid cells but not in the tumor cells in 2 cases of gastric carcinoma. On the basis of these findings, a role for EBV in the development of malignancies in renal transplant patients is unlikely except for lymphoid neoplasias.     

An Epstein-Barr virus-associated superantigen

More than 90% of adults are latently infected with Epstein-Barr virus (EBV), the causative agent of infectious mononucleosis, a self-limiting lymphoproliferative disease characterized by extensive T cell activation. Reactivation of this herpesvirus during immunosuppression is often associated with oncogenesis. These considerations led us to analyze the early events that occur after exposure of the immune system to EBV. Strong major histocompatibility complex (MHC) class II-dependent but not MHC-restricted, T cell proliferation was observed in vitro in response to autologous, lytically infected EBV-transformed B cells. By measuring the appearance of the early activation marker CD69 on individual T cell V beta subsets, we could demonstrate selective activation of human V beta 13- T cells. This was confirmed with murine T cell hybridomas expressing various human BV genes. While EBV- Burkitt's lymphoma cells were nonstimulatory, they induced V beta-restricted T cell activation after EBV infection. EBV specific activation was also demonstrated in cord blood cells, excluding a recall-antigen response. Thus, all of the characteristics of a superantigen-stimulated response are seen, indicating that induction of the EBV lytic cycle is associated with the expression of a superantigen in B cells. A model is presented proposing a role for the superantigen in infection, latency, and oncogenesis.     

Targeting Epstein-Barr virus nuclear antigen 1 (EBNA1) through the class II pathway restores immune recognition by EBNA1-specific cytotoxic T lymphocytes: evidence for HLA-DM-independent processing

Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is the only viral protein consistently expressed in all malignancies associated with EBV and there is now convincing evidence to suggest that EBNA1 is not recognized by MHC class I-restricted cytotoxic T lymphocytes (CTL). The lack of recognition of EBNA1 has been attributed to a cis-acting inhibitory effect of glycine-alanine repetitive (G-Ar) sequences on the endogenous processing of this antigen through the class I pathway. In the present study we have explored the possibility of targeting EBNA1 through an alternative mechanism using the MHC class II pathway. Using purified EBNA1 protein, we demonstrate here that CD4+ CTL can efficiently recognize EBV-transformed B cells and Burkitt's lymphoma cells following exogenous sensitization with this antigen, and this immune recognition is not affected by the G-Ar domain within EBNA1. Analysis of the processing mechanism revealed that intracellular loading of class II molecules with an EBNA1 epitope occurs through an HLA-DM-independent pathway. These results highlight a novel mechanism for immune recognition of EBNA1 and also demonstrate that the G-Ar-mediated protection from processing can be overridden if this antigen is presented through the class II pathway.     

Epstein-Barr virus in pyothorax-associated pleural lymphoma

Pleural B-cell lymphoma was found in five patients with a history of pyothorax that was the sequelae of tuberculosis 35 to 47 years previously. Epstein-Barr virus (EBV) DNA was detected in all five pleural tumors by polymerase chain reaction and Southern blot hybridization. The lymphoma cells were shown to express the latent membrane protein-1 and the EBV-encoded nuclear antigen-2 by immunocytochemistry and EBV-encoded small RNA by in situ hybridization. Three cases were shown to be EBV subtype A, whereas the remaining two were subtype B, as determined by differences in the EBV-encoded nuclear antigen-2 nucleotide sequence. The patients also had high titers of antibodies against EBV. These findings suggest that EBV is causally associated with the pleural lymphomas that originate at the site of chronic inflammation and fibrosis with a latent period of more than 40 years.     

Spontaneous loss of viral episomes accompanying Epstein-Barr virus reactivation in a Burkitt's lymphoma cell line

Life-long viral persistence is a hallmark of human herpesvirus infection. In the Epstein-Barr virus (EBV)-positive Burkitt's lymphoma (BL) cell line, Mutu, spontaneous loss of all viral episomes accompanied productive viral DNA replication. The molecular configuration of intracellular EBV DNA evolved from monoclonal episomes in cells retaining the original tumor phenotype to predominantly replicating linear DNA and, subsequently, only integrated forms in BL cells that had acquired the lymphoblastoid cell phenotype. Transient appearance of deleted, rearranged WZhet EBV DNA capable of disrupting viral latency, along with the integration of viral DNA into human chromosomes, indicates a genetic instability in the host cell which, if duplicated in vivo, may affect configuration and persistence of the viral genome in expanding malignant cell clones.