Possible involvement in oncogenesis of a single base mutation in an internal ribosome entry site of Epstein-Barr nuclear antigen 1 mRNA

It has been reported recently that the U leader exon located within the 5' untranslated region of Epstein-Barr nuclear antigen 1 (EBNA1) gene contains an internal ribosome entry site (IRES) element. Sequence analysis of the U leader exon was undertaken in samples from 19 patients with infectious mononucleosis and 19 patients with lethal lymphoproliferative diseases and in 15 spontaneously established lymphoblastoid cell lines. The sequence was conserved except for a single base substitution (T-C) at position 67,585. Although the mutation was detected in only one case of infectious mononucleosis, it was found in more than half of the lethal lymphoproliferative diseases and all lymphoblastoid cell lines. The results suggest that a mutation in the IRES element affects EBNA1 gene expression at the translational level and provides Epstein-Barr virus (EBV)-infected cells with a growth advantage, leading to immortalization of cells in vitro and to the development of lethal lymphoproliferative diseases in vivo.     

Detection of Epstein-Barr virus by polymerase chain reaction.

The polymerase chain reaction (PCR) was used to study DNA extracted from the blood of 25 transplant patients, 5 patients with infectious mononucleosis, and 13 healthy subjects and autopsy or biopsy tissue from 29 patients with lymphoproliferative disorders. Primers were directed to conserved regions of the Epstein-Barr virus (EBV) genome encoding capsid protein gp220 and Epstein-Barr nuclear antigen 1. Specific EBV amplification was found in the blood of 11 of 25 transplant patients and all patients with infectious mononucleosis. All patients with lymphoproliferative disorders occurring in the presence of immunosuppression (eight organ transplant patients and two patients with acquired immunodeficiency syndrome) had biopsies positive for EBV by PCR. Only 1 of 19 samples from lymphomas or leukemias unrelated to immunosuppression contained EBV. PCR confirmed the very close association of EBV and lymphoproliferative disorders occurring in the presence of immunosuppression. The significance of detecting EBV sequences in the blood of transplant patients, particularly in relationship to lymphoproliferation, requires further study.     

Prospective Follow-up of Epstein-Barr Virus Load in Adult Kidney Transplant Recipients by Semiquantitative Polymerase Chain Reaction in Blood and Saliva Samples

The aim of the study presented here was to set up and standardize a semiquantitative polymerase chain reaction method for monitoring Epstein-Barr virus (EBV) DNA levels in blood and saliva samples from transplant recipients and to determine the value of these levels as an early marker for the development of post-transplant lymphoproliferative disorders. EBV DNA load was prospectively measured in 53 adult kidney transplant recipients. Results were correlated with clinical features and degree of immunosuppression. Healthy blood donors and patients with infectious mononucleosis were used as controls. Levels higher than 500 EBV DNA copies/75,000 peripheral blood mononuclear cells were found in all patients with infectious mononucleosis and in all patients with post-transplant lymphoproliferative disorder but in only 7.5% of transplant recipients without that complication. Electronic Publication     

Reactivation of Epstein-Barr virus in B cells of patients with chronic hepatitis C

Hepatitis C virus (HCV) infection is associated with lymphoproliferative disorders. HCV infection of B cells is a predictive factor for lymphoproliferative disorders in patients with chronic hepatitis C, although its molecular mechanisms remain unknown. Epstein-Barr virus (EBV) is a B cell-tropic virus with the potential to cause lymphoproliferative disorders, and its reactivation is induced by several viruses and cytokines. The possibility that HCV infection triggers reactivation of EBV and induces lymphoproliferative disorders were investigated. Expression of EBV mRNAs was analyzed by RT-PCR in patients infected with HCV and control subjects, and correlations between reactivation of EBV and markers for lymphoproliferative disorders were investigated. BZLF1 mRNA, a starter molecule of reactivation, was detected in peripheral blood mononuclear cells from 12 of 52 (23%), patients infected with HCV and the frequency was higher than in healthy subjects [3 of 43 (9%), P = 0.032]. But the presence of the BZLF1 mRNA was not associated with an abnormality of markers for lymphoproliferative disorders. This study on BZLF1 mRNA expression among lymphoid cell subsets showed that reactivation of EBV was observed specifically in B cells. The BZLF1 mRNA disappeared following anti-viral therapy and remained negative after eradication of HCV in patients with a sustained viral response, while the EBER1 RNA, a marker for persistence of EBV, was detected throughout the therapy. Infection with HCV induces reactivation of EBV in B cells, but this reactivation was not associated directly with lymphoproliferative disorders triggered by HCV.     

EBV latent membrane protein (LMP-1) and bcl-2 protein expression in Reed-Sternberg-like cells in post-transplant lymphoproliferative disorders

An inconsistent association exists between EBV-LMP-1 and bcl-2 protein expression in Reed-Sternberg cells seen in Hodgkin's disease. In fact, many studies have concluded that there is no correlation between EBV-LMP and bcl-2 expression in Hodgkin's disease. We undertook an analysis of post-transplant lymphoproliferative disorders to explore the relationship between EBV-LMP and bcl-2 in Reed-Sternberg-like cells found in this condition, given the strong association between this disorder and EBV. Reed-Sternberg-like cells were found histologically in 11 of 28 cases of renal, heart and heart-lung post-transplant lymphoproliferative disorders. Formalin-fixed, paraffin-embedded sections were stained with monoclonal antibodies to EBV-LMP-1 and bcl-2 proteins. Reed-Sternberg-like cells in all 11 cases co-expressed EBV-LMP and bcl-2. A similar relationship was noted with large, mononuclear cells and occasional small lymphoid cells. The staining pattern seen with both antibodies was of similar intensity and both displayed cytoplasmic Golgi accentuation. In the setting of post-transplant lymphoproliferative disorders, Reed-Sternberg-like cells exhibit strong co-expression of EBV-LMP-1 and bcl-2 proteins, supporting a positive correlation between them. This is in contrast to the findings in Hodgkin's disease. The reason for this discrepancy may be due to the iatrogenic immunosuppression and resultant severe EBV infection, together with other cellular events.     

A mouse model for infectious mononucleosis

Epstein-Barr virus (EBV) is a ubiquitous human gamma-herpesvirus that establishes life-long latency and is associated with lymphoproliferative disorders and the development of several malignancies. EBV infection is frequently, but not always, associated with the development of a syndrome termed infectious mononucleosis. The recent isolation and characterization of a murine gamma-herpesvirus, MHV-68 (gammaHV-68) has provided the first small animal model for studying immunity and pathogenesis of a gamma-herpesvirus in its natural host. MHV-68 has important biological and genetic similarities with the human gamma-herpesviruses. Following intranasal infection of mice with MHV-68, an acute respiratory infection in the lung develops and is cleared, followed by the establishment of latency. Similar to EBV, MHV-68 latency is largely established in B cells, although other cell types can be latently infected. The establishment of latency correlates with a prominent splenomegaly, polyclonal B cell activation with associated autoantibody production, and CD8+ T cell-dominated peripheral blood lymphocytosis, in many aspects mirroring EBV-induced infectious mononucleosis. There are key differences in the MHV-68- and EBV-induced CD8+ T cell responses however. Whereas the expanded CD8+ T cells associated with EBV-induced mononucleosis are largely the outgrowth of T cells responding to lytic viral epitopes elicited during the acute phase of the response, the CD8+ T cell lymphocytosis associated with MHV-68-induced infectious mononucleosis is dominated by an oligoclonal population of T cells expressing Vbeta4+ T cell receptors that are not reactive to acute viral epitopes. The focus of this article will be to highlight the similarities and differences in the infectious mononucleosis syndrome associated with human and murine gamma-herpesviruses.     

An Overview of Epstein-Barr Virus: from Discovery to Future Directions for Treatment and Prevention

Discovered in 1964 by Epstein and Barr, the Epstein-Barr virus (EBV) is widespread in all areas of the world, infecting over 95% of the adult population and earning it the informal name, 'Every Body's Virus'. It was shown to be the causal agent of infectious mononucleosis (IM) in 1968, and many reports followed linking EBV to several other clinical syndromes. In 1970, the virus was shown to be able to immortalize B lymphocytes, which are one of its target cells. This oncogenic potential underlies the role of EBV in Burkitt's lymphoma, post-transplant lymphoproliferative disorders and lymphoma in HIV-infected patients. In addition, EBV can infect epithelial cells in the nasopharynx and is a co-factor for development of nasopharyngeal carcinoma. There is no treatment available for IM to date; however, virus-associated complications may be treated with various modes of immune modulation. Currently, radiotherapy and chemotherapy remain the main treatments for EBV-associated malignancies. Recent evidence has suggested that adoptive therapy through infusions of human leukocyte-associated antigen-matched EBV cytotoxic T cells may form a novel strategy for both prophylaxis and treatment of EBV-induced lymphoproliferative disorders. Additionally, a vaccine based on immunization with a structural antigen, gp350, is under evaluation.     

Dynamics of Epstein-Barr virus DNA levels in serum during EBV-associated disease

A real-time polymerase chain reaction assay for quantitation of Epstein-Barr virus (EBV) DNA in serum was developed. This assay detected EBV DNA in 24 (89%) of 27 sera from patients with infectious mononucleosis, but only in 9 (18%) of 51 sera from EBV carriers (P < 0.001) and in none of the sera from 32 EBV-seronegative individuals. EBV DNA levels were higher in sera from infectious mononucleosis (median 8,000, range 1833-150,069 copies/ml) than from carriers (median < 2, range < 2-2980; P < 0.001). In sera of 36 children with infectious mononucleosis followed prospectively, EBV DNA levels correlated inversely with the duration of symptoms. Among 18 children with tumors including Hodgkin's disease (n = 7), non-Hodgkin's lymphoma (n = 6), Burkitt's lymphoma (n = 1), lymphoproliferative disorder (n = 4), and osteosarcoma (n = 1), EBV DNA was detected in serum from those 9 (100%) expressing EBV in the tumor (Hodgkin's disease, 3; non-Hodgkin's lymphoma, 2; lymphoproliferative disorder, 4), the levels peaking at diagnosis and correlating with disease activity. Quantitation of EBV DNA in serum may offer a simple means of monitoring patients at risk of EBV-associated lymphoproliferation.     

Interferon-gamma in a family with X-linked lymphoproliferative syndrome with acute Epstein-Barr virus infection

A 20-month-old male with fulminant infectious mononucleosis and the X-linked lymphoproliferative syndrome (XLP) was studied. Epstein-Barr virus (EBV)-determined nuclear antigen (EBNA) and EBV DNA were detected in various tissues. Despite a combined treatment with acyclovir, immunoglobulin, and methylprednisolone, the patient deteriorated rapidly. Following treatment with recombinant interferon-gamma (IFN-), defervescence occurred and circulating EBNA-positive cells markedly decreased. IFN- prior to treatment ranged from 10.8 to 24.5 U/ml in the patient's serum and increased linearly post exogenous IFN- treatment. His natural killer (NK)-cell activity remained in the normal range throughout his illness but autologous EBV-infected cells were not killedin vitro by his peripheral blood lymphocytes (PBL). These results suggest that patients with the fatal infectious mononucleosis phenotype of XLP may produce endogenous IFN-. Defective cytotoxic T cells against EBV-infected cells seem to be responsible for the fulminant infectious mononucleosis in this patient.     

Persistent Hypogammaglobulinemia Following Mononucleosis in Boys Is Highly Suggestive of X-Linked Lymphoproliferative Disease—Report of Three Cases

Hypogammaglobulinemia is a common symptom in different immunodeficiencies. It is, however, not usually associated with Epstein-Barr virus (EBV) infections. The X-linked lymphoproliferative disease (XLP) on the other hand shows immunological changes in response to the EBV. Here we report three previously healthy boys, all of which developed persistent hypogammaglobulinemia following severe acute infectious mononucleosis. All three patients revealed T-cell abnormalities including inverted CD4/CD8 and increased CD8(+) T-cell numbers. The number of IFN-gamma-producing T cells were markedly increased in the two patients studied so far. In addition, patient 2 showed mainly T cells, instead of B cells, to be infected with the EBV. Apart from an uncle of patient 3, who died of malignant lymphoma, family history was unremarkable in all cases. All three patients exhibited mutations in the SH2D1A gene, establishing the diagnosis of XLP. Protein expression was found on immunoblot analysis in one patient with a missense mutation. Development of persistent hypogammaglobulinemia after severe primary EBV infection seems to be a specific diagnostic sign for XLP even in males with unremarkable family history.     

Evaluation of families wherein a single male manifests a phenotype of X-linked lymphoproliferative disease (XLP)

The Epstein-Barr virus (EBV)-induced diseases of males with X-linked lymphoproliferative disease (XLP) include fatal infectious mononucleosis (IM), non-Hodgkin lymphoma (ML), agammaglobulinemia, and aplastic anemia. These phenotypes also occur as sporadic cases in families, and EBV seronegative males in these families must be considered at risk for XLP until they seroconvert normally to EBV. Given that 50% of males inheriting the defective XLP gene die following primary EBV infection, it is vital that they be identified pre-EBV infection. Here we report result using molecular genetic techniques to provide information as to the relative risks of EBV negative males and potential carrier females in ten families wherein a single male had died of IM. © 1993 Wiley-Liss, Inc.     

Immunopathology associated with Epstein-Barr virus (EBV) infection: Evidence for interactions with T-lymphocyte EBV receptor CD21

Epstein-Barr virus (EBV), also termed Human Herpes Virus 4, is the causative agent of infectious mononucleosis and may be a cofactor in some human cancers. The virus has also been suggested to play a role in human autoimmune diseases including rheumatoid arthritis, systemic lupus erythematosus, and multiple sclerosis. X-linked lymphoproliferative syndrome caused by the deficiency of the Sarc homology 2 domain protein 1A, also termed signaling lymphocyte activation marker-associated protein, can result in immune dysfunction and death after EBV infection. The EBV-related immunopathology in X-linked lymphoproliferative syndrome and prototypical autoimmune syndromes is summarized in this review. A novel model of viral interaction with complement receptor CD21, which is also the receptor for EBV, is proposed to account for both the immunological abnormalities of X-linked lymphoproliferative syndrome and autoimmune diseases associated with EBV infection. The pathogenesis of both X-linked lymphoproliferative syndrome and EBV-associated immune diseases is proposed to result from increased direct infection of T cells by EBV through the T-cell complement receptor CD21 expressed on T cells. A prediction of this model is that therapy designed to decrease CD21-mediated EBV infection of T lymphocytes could also be beneficial in the treatment of some autoimmune diseases.     

Immune defence against EBV and EBV-associated disease

Epstein-Barr virus (EBV), a B-lymphotropic herpesvirus widespread in the human population and normally contained as an asymptomatic infection by T cell surveillance, nevertheless causes infectious mononucleosis and is strongly linked to several types of human cancer. Here we describe new findings on the range of cellular immune responses induced by EBV infection, on viral strategies to evade those responses and on the links between HLA gene loci and EBV-induced disease. The success of adoptive T cell therapy for EBV-driven post-transplant lymphoproliferative disease is stimulating efforts to target other EBV-associated tumours by immunotherapeutic means, and has reawakened interest in the ultimate intervention strategy, a prophylactic EBV vaccine.     

Epstein-Barr virus and human diseases: recent advances in diagnosis.

Since the discovery of Epstein-Barr virus (EBV) from a cultured Burkitt's lymphoma cell line in 1964, the virus has been associated with Burkitt's lymphoma, nasopharyngeal carcinoma, and infectious mononucleosis. During the recent decade, EBV has been etiologically implicated in a broad spectrum of human diseases. The precise role of this virus in these diseases is not well understood, but clearly, defective immunosurveillance against the virus may permit an uncontrolled proliferation of EBV-infected cells. As a result, a growing number of cases of EBV-associated B-cell proliferative diseases or lymphoma have been noted in patients with primary and acquired immunodeficiencies. These lymphoproliferative diseases and others, such as chronic mononucleosis syndrome, are leading to new areas of investigation which are providing information regarding the pathogenetic mechanisms of EBV-induced diseases. The early accurate diagnosis of EBV infection can be achieved by performing EBV-specific serology, detecting for EBV-determined nuclear antigen in tissues, establishing spontaneous lymphoid cell lines, and using molecular hybridization techniques for demonstrating the presence of viral genome in affected lesions.     

Clinicopathological characteristics of four cases of EBV positive T-cell lymphoproliferative disorders of childhood in China

A new category, “EBV positive T-cell lymphoproliferative disorders (LPD) of childhood”, was proposed in the 2008 World Health Organization’s (WHO) classifications of lymphoma. This series of lymphoproliferative disorders is rare. There are two major types of this series of disorders: systemic EBV positive T-cell LPD of childhood and hydroa vacciniforme-like lymphoma (HVLL). In this study, we describe the distinct features of four cases of EBV positive T-cell LPD of childhood in China. Two were systemic EBV positive T-cell LPD of childhood, one was HVLL and one was chronic active EBV (CAEBV). The main manifestations were lymphadenopathy, fever, hepatosplenomegaly and skin rashes. The structure of the lymph nodes in the patients ranged from preserved to partially or totally destroyed. Small- to medium-sized, atypical T cells had infiltrated the lymph nodes. In HVLL, the neoplastic cells had infiltrated the dermis and subcutaneous region surrounding sweat glands and nerves. All of the cases tested positive for CD8, other T cells, cytotoxic markers and EBV-encoded RNA (EBER) without CD56 expression. Molecular analysis was performed in three cases. All of the three analyses showed a TCRγ rearrangement and one case also had an IGH rearrangement. One of the patients with systemic EBV positive T-cell LPD of childhood experienced rapid evolved and died within five months of onset. CAEBV, systemic EBV-positive T-cell LPD of childhood and HVLL are distinct but overlapping diseases within the category of EBV-positive T-cell LPD of childhood. They constitute a continuous spectrum of EBV-infected associated disorders.     

Progress and problems in understanding and managing primary Epstein-Barr virus infections

Epstein-Barr virus (EBV) is a gammaherpesvirus that infects a large fraction of the human population. Primary infection is often asymptomatic but results in lifelong infection, which is kept in check by the host immune system. In some cases, primary infection can result in infectious mononucleosis. Furthermore, when host-virus balance is not achieved, the virus can drive potentially lethal lymphoproliferation and lymphomagenesis. In this review, we describe the biology of EBV and the host immune response. We review the diagnosis of EBV infection and discuss the characteristics and pathogenesis of infectious mononucleosis. These topics are approached in the context of developing therapeutic and preventative strategies.     

Epstein-Barr virus VCA IgM and EBNA IgG antibodies titered by immunofluorescence in microplates

We describe methods of immunofluorescence in microplates for titration of EBV VCA-IgM and EBNA-IgG antibodies and compare the sensitivity of the methods with glass slides and with microplates, showing the reproducibility of the methods.Results of VCA-IgM and -IgG, EA and EBNA titers obtained in four groups of patients are given, comprising cases of infectious mononucleosis, renal graft recipients, lymphoproliferative diseases and controls.     

Different Epstein-Barr virus expression in lymphomas from immunocompromised and immunocompetent patients.

Eighteen tissue samples from lymphoproliferative lesions and lymphomas in immunodeficiency states were investigated for their content of Epstein-Barr virus (EBV) genome by dot blotting and for the distribution of EBV in tissue sections by in situ hybridization. Fourteen lymphomas from AIDS patients and four children with disorders of the immune system were available. For control reasons, six cases of infectious mononucleosis (IM) and eight Burkitt's lymphomas (BL) from malaria-free regions of Africa were included in the study. Two different patterns of EBV distribution are described: 1) heterogeneous scattered EBV-positive cells, as originally seen in IM and therefore called the IM-type pattern, 2) and a BL-type pattern seen in endemic Burkitt's lymphoma with homogeneous EBV-positive cells all over the tumor. In lymphomas in patients with inborn immunodeficiencies, an IM-type pattern was found. In lymphomas from AIDS patients, the two different patterns were found. There were lymphomas with the IM-type pattern as well as some with the BL-type pattern. In some AIDS-associated lymphomas, both patterns occurred in one tumor. The findings suggest that it is not the disease process that is the distinguishing feature between the two patterns of EBV infection but rather the patient's underlying disease and the extent of this disease.