Establishment and characterization of a chronic infectious mononucleosislike syndrome in common marmosets

Epstein-Barr virus (EBV) was inoculated into two species of marmosets. Successful infection was established in the majority of the animals of one species, Callithrix jacchus, as evidenced by the development of high, persistent levels of antibody against virus-specific capsid and early nonstructural proteins. Antibodies also were produced against the major membrane antigen and, in some animals, against EBV nuclear antigen (EBNA) 2 but not against EBNA 1. This is the antibody profile normally noted in individuals with chronic infectious mononucleosis (IM). EBV-induced lymphoproliferation was not seen, and EBV-specific proteins were not detected in the peripheral blood lymphocytes of infected animals. Hence, EBV infection in C. jacchus apparently does not generally include extensive B-cell involvement. However, the marmosets clearly are useful as a model for EBV primary infection and also possibly for chronic IM.     

Performance of the Architect EBV Antibody Panel for Determination of Epstein-Barr Virus Infection Stage in Immunocompetent Adolescents and Young Adults with Clinical Suspicion of Infectious Mononucleosis

The Architect EBV antibody panel is a new chemiluminescence immunoassay system used to determine the stage of Epstein-Barr virus (EBV) infection based on the detection of IgM and IgG antibodies to viral capsid antigen (VCA) and IgG antibodies against Epstein-Barr nuclear antigen 1 (EBNA-1). We evaluated its diagnostic accuracy in immunocompetent adolescents and young adults with clinical suspicion of infectious mononucleosis (IM) using the RecomLine EBV IgM and IgG immunoblots as the reference standard. In addition, the use of the antibody panel in a sequential testing algorithm based on initial EBNA-1 IgG analysis was assessed for cost-effectiveness. Finally, we investigated the degree of cross-reactivity of the VCA IgM marker during other primary viral infections that may present with an EBV IM-like picture. High sensitivity (98.3% [95% confidence interval {CI}, 90.7 to 99.7%]) and specificity (94.2% [95% CI, 87.9 to 97.8%]) were found after testing 162 precharacterized archived serum samples. There was perfect agreement between the use of the antibody panel in sequential and parallel testing algorithms, but substantial cost savings (23%) were obtained with the sequential strategy. A high rate of reactive VCA IgM results was found in primary cytomegalovirus (CMV) infections (60.7%). In summary, the Architect EBV antibody panel performs satisfactorily in the investigation of EBV IM in immunocompetent adolescents and young adults, and the application of an EBNA-1 IgG-based sequential testing algorithm is cost-effective in this diagnostic setting. Concomitant testing for CMV is strongly recommended to aid in the interpretation of EBV serological patterns.     

Appearance and some unusual features of Epstein-Barr virus antibody production in infectious mononucleosis and other health disorders.

The indirect immunofluorescence (IF) test and the complement-fixation reaction (CFR) were used in examination of over 1500 sera obtained from patients with infectious mononucleosis (IM) and other health disorders. The evidence obtained supports a direct aetiological relationship between Epstein-Barr virus (EBV) and IM and points on a relationship of EBV to some other lymphadenopathies and health disorders. The incidence of the IgG type antibody against virus capsid antigen (EB-VCA) and soluble antigen (CF-SA) obtained from EBV genome-positive cells among different age groups of patients is described along with results of long-term examinations of serum samples from IM patients. The appearance and dynamics of production of both types of EBV antibody and their persistence in the organism varied. Long-lasting oscillations, in particular of the EB-VCA antibody levels were found in sera of patients with prolonged health disorders following IM. The diagnosis value of the IF test and the CFR is discussed.     

Detection of an idiotope on a human monoclonal autoantibody by monoclonal anti-idiotypic antibody and its relationship to Epstein-Barr virus-induced autoimmunity.

We have recently described a human IgM monoclonal antibody (mAb), reactive with both self antigens, i.e., cytoskeleton filaments and smooth muscle, and Epstein-Barr virus (EBV)-induced nuclear antigen (EBNA), produced by EBV-transformed B lymphocytes isolated from a patient with infectious mononucleosis (IM). In order to achieve higher antibody secretion in culture supernatant, the mAb-producer cells were fused with ouabain-resistant mouse myeloma cells and a stable human-mouse heterohybrid, coded HY 5488, producing up to 80 micrograms/ml IgM mAb, was isolated after 4 cloning procedures. Purified HY 5844 mAb was used to immunize mice for the production of a murine anti-idiotypic mAb, which was used to probe the expression of the idiotope of HY 5488 mAb (Id 5488) in sera of IM patients and normal controls by ELISA. It was found that Id 5488 is expressed both in IM patients and normal controls, and that Id 5488 expression is significantly higher in IM patients' sera; furthermore, in IM sera a statistically significant correlation between Id 5488 expression and anti-cytoskeleton and anti-smooth muscle autoantibodies was found. It is suggested that at least part of EBV-induced IgM autoantibodies appearing during IM are secreted by B lymphocytes programmed to the production of "natural antibodies" bearing Id 5488-like idiotopes.     

Epstein-Barr virus serology in bone marrow transplantations: a one-year retrospective study with detection of EBV IgM-VCA-specific antibodies

The specific antibody response to Epstein-Barr virus (EBV) antigens of 41 bone marrow transplant recipients with leukemia or aplastic anemia was examined retrospectively by immunofluorescence test (IF) over 1 year. We observed high titers (greater than 640) of IgG-viral capsid antigen (VCA) with emergence of IgG-early antigen (EA) and frequent absence or low levels of Epstein-Barr nuclear antigen (EBNA) antibodies. After absorption to remove rheumatoid factor (RF), five of the 41 recipients had IgM-VCA antibody to EBV, which appeared between weeks 26 and 48 after BMT and persisted for 1-4 months. No heterophil antibodies were detected in these sera, and none of the five recipients had a history of infectious mononucleosis.     

Inosiplex enhances the growth of Epstein-Barr nuclear antigen-positive B-cells from patients with rheumatoid arthritis and infectious mononucleosis

The in vitro effects of inosiplex (INO) on proliferation of Epstein-Barr virus (EBV)-carrying B-cells were evaluated. As measured by 3H-thymidine uptake, INO significantly augmented the proliferation of long-term cultured Epstein-Barr nuclear antigen (EBNA)-positive B-cells which were obtained from patients with rheumatoid arthritis (RA) and infectious mononucleosis (IM). In co-culture experiments, INO and anti-immunoglobulin M antibodies coupled to sepharose (anti-mu) had additive effects on proliferation of EBNA-positive B-cells. We conclude that its direct effects on B-cell function should be considered when INO is used for the treatment of diseases characterized by B-cell hyperreactivity.     

High incidence of elevated antibody titers to Epstein-Barr virus in patients with uveitis

Summary. We assayed Epstein-Barr virus (EBV) antibody titers in patients’ sera using indirect immunofluorescence and tested for the presence of antibody to EBV immediate-early BZLF1 protein ZEBRA by Western blotting to explore the association of EBV infection with uveitis. IgG and IgA antibodies to viral capsid antigen (VCA), IgG antibodies to early antigen (EA), and antibodies to EBV nuclear antigen were detected at higher titers in sera of patients with uveitis than in the sera of healthy controls. Neither IgM antibody to VCA nor EA was detected in the patients’ sera. Anti-ZEBRA-IgG antibodies were detected in most patients’ sera, but not in those of healthy controls. These results suggest that uveitis might be a disease accompanied by EBV reactivation.     

Presence of infective Epstein-Barr virus in the urine of patients with infectious mononucleosis

The presence of Epstein-Barr virus (EBV) in the blood and urine of 20 patients with infectious mononucleosis (IM) was investigated together with the clinical course of the disease, and in 9 patients up to 2–7 months after recovery. EBV DNA, analyzed by the polymerase chain reaction (PCR), was detected in the blood of all 20 patients from the first sample obtained and detected between 3 to 42 days from the beginning of symptoms and up to 2–3 months after recovery. In the urine, EBV DNA was detected in 15 out of 16 (93%) patients in the first sample obtained and detected between 3 to 50 days during the clinical course of the disease. In four patients EBV DNA was detected in the urine up to 3 months after full recovery. Seventeen out of 26 (65%) urine samples including 3 which were obtained 2–7 months after recovery infected B cells as assessed by PCR. Nine out of 12 (75%) urine samples tested induced Epstein-Barr nuclear antigen (EBNA) in the infected B-cell line. In addition to the persistence of EBV in the blood of IM patients, these studies show for the first time the presence of infective EBV in the urine during the clinical course of the disease and up to 7 months after full clinical recovery. © 1994 Wiley-Liss, Inc.     

Unchanged expression of Epstein-Barr virus-determined nuclear antigen-1 in productive virus cycle

The level of the Epstein-Barr virus (EBV)-determined nuclear antigen (EBNA-1) encoded by the Bam HI-K fragment of EBV DNA remained unchanged in P3HR-1 cells after induction of the productive cycle of virus replication by sodium-n-butyrate and 12-o-tetradecanoylphorbol-13-acetate (TPA). This was shown by the same capacity of cell-free extracts from untreated and treated P3HR-1 cells to absorb anti-EBNA-1 antibody from a known human serum.     

Continuous lymphoblastoid suspension cultures from cells of haematopoietic organs of baboons with malignant lymphoma. Report III: Immunological studies.

Lymphoblastoid cultures established from haematopoietic organs of baboons with lymphoma belong to the B-cell type. Cytoplasmic antigen is revealed in 2--6% of suspension culture cells established from lymphomatous baboons by indirect immunofluorescence reaction using serum from human Burkitt's lymphoma patients with antibody titer to capsid antigen of Epstein-Barr virus 1: 320. There is a correlation between the number of antigen-containing cells in these cultures and the number of cells in which herpes virus is revealed electron microscopically. Sera of baboons with lymphoma contain high titers of antibodies to Epstein-Barr (EBV) virus. Specific differences between antigens being produced in cultures by HBV (herpes virus of baboon) and EBV (Epstein-Barr virus) are minimal, that indicates their immunologic similarity.     

Detection of a nuclear antigen 2 (EBNA2)-variant Epstein-Barr virus strain in two siblings with fatal lymphoproliferative disease

An EBV type 1 variant strain was detected in two Turkish siblings (boy and girl), who both suffered and died from similar progressive Epstein-Barr virus (EBV)-associated lymphoproliferative disease. Molecular characterisation of this EBV isolate revealed a 51bp-deletion and six nucleotide changes within the Epstein-Barr nuclear antigen 2 (EBNA2). Both isolates contained EBV type 2 sequences in the Epstein-Barr virus-encoded small RNAs (EBER), which are 40 kb proximal to EBNA2. Sequencing of the EBV isolates in a region of Epstein-Barr nuclear antigen 3 (EBNA3a), which is 40 kb distal to EBNA2, revealed the normal EBV type 1 sequence of laboratory strain B95-8. This EBV isolate may represent a distinct wild type EBV strain with altered biological properties. It is suggested that this EBNA2-variant strain may be responsible at least in part for the severe clinical course in both affected children. © 1996 Wiley-Liss, Inc.     

Production of human monoclonal antibodies against Epstein-Barr virus-specific antigens by the virus-immortalized lymphoblastoid cell lines

The possible production of human monoclonal antibodies against Epstein-Barr virus (EBV) was assessed through the EBV immortalization technique. When individual lymphocyte samples from 50 clinical patients and healthy donors were immortalized by EBV, 4 lymphoblastoid lines yielded antibodies to EBV antigens. These positive lines were cloned and each line yielded cultures that secreted monoclonal antibodies against either viral capsid antigen (VCA) or membrane antigen (MA) component. Above all, a clonal line TAKA-SP-8 produced 5 micrograms MA antibody/10(6) cells/ml for more than 12 months. The culture fluid specifically immunoprecipitated a single polypeptide with a size of 93K from both P3HR-1 and B95-8 cell extracts. FUKA-SP-3, on the other hand, secreted 5 micrograms VCA antibody/10(6) cells/ml for at least 8 months. This antibody recognized two polypeptides with sizes of 123K and 120K, from P3HR-1 and B95-8 cell extracts, respectively. When B95-8 and P3HR-1 EBV were treated with the human MA monoclonal, both nuclear antigen (EBNA) synthesis and early antigen (EA) induction were strongly inhibited. All EBV antibody-producing cultures were exclusively achieved from splenic lymphocytes of patients with autoimmune diseases, but not from other donors.     

Structure of the Epstein-Barr virus nuclear antigen as probed with monoclonal antibodies

Five hybridoma cell lines secreting antibodies directed against an Epstein-Barr virus (EBV) nuclear antigen (BamHI K antigen) have been isolated. All five antibodies detect this antigen in a variety of EBV-positive lymphoblastoid cell lines. The reaction of these antibodies with several mutant forms of this protein has allowed the antibody binding site(s) to be predicted.     

Expression of a germline human kappa chain-associated cross-reactive idiotype after in vitro and in vivo infection with Epstein-Barr virus

The mouse monoclonal antibody 17.109 recognizes a cross-reactive idiotype (CRI) associated with kappa IIIb light chains of human IgM-rheumatoid factor (RF) paraproteins. The 17.109 idiotypic determinant is encoded by one or a group of closely related V kappa genes. The association of the idiotype with IgM- and IgA-rheumatoid factors in certain autoimmune diseases necessitates an understanding of how human B lymphocytes can be induced to express the idiotype. To investigate the cellular expression of the 17.109 CRI, peripheral blood lymphocytes from normal donors were stimulated in vitro with Epstein-Barr virus (EBV) and pokeweed mitogen (PWM). EBV induced greater expression of IgM-associated 17.109 CRI than did PWM. The 17.109 CRI was preferentially associated with IgM rather than with IgG. In vivo EBV infection was studied in college students with infectious mononucleosis and displayed similar elevation of IgM-associated 17.109 CRI in sera obtained at presentation of clinical illness. Later, IgM levels declined while IgG-associated 17.109 CRI rose. The 17.109 idiotype was unrelated to antibodies against the Epstein-Barr virus nuclear antigen and the viral capsid antigen and was probably due to generalized activation of early B cells. These observations support the hypothesis that the 17.109 CRI is expressed by in vitro and in vivo EBV-infected cells. The 17.109 idiotype identifies a highly conserved V kappa gene product, which is expressed preferentially after EBV infection, but not exclusively with RF autoantibodies.     

Chronic active Epstein-Barr virus infection in patients with Chediak-Higashi syndrome

The results of clinical and Epstein-Barr virus (EBV) serological studies on nine Chediak-Higashi syndrome (CHS) patients are reported. Persistently elevated antibodies to the viral capsid antigen (VCA) and the restricted component of the early antigen complex (EA-R) developed in six patients who experienced primary EBV infection which either remained silent or were accompanied by clinical signs of infectious mononucleosis (IM). Hepatosplenomegaly and moderate lymphadenopathy, both clinical signs of the accelerated phase, remained detectable in the six patients for a long period of time after seroconversion. The clinical, serological, and histopathological observations are suggestive of a nonmalignant lymphoproliferative disease and consistent with an immunodeficiency to EBV. The abnormal serological responses to EBV in CHS are therefore considered manifestations of a chronic active EBV infection which may result in lethal lymphoproliferation. The three as yet seronegative CHS patients revealed no signs of the accelerated lymphoproliferative phase of the syndrome.     

Epstein-Barr nuclear antigen (EBNA) carrying lymphocytes in human palatine tonsils.

The presence of Epstein-Barr virus (EBV) antigens in human palatine tonsilderived lymphocytes (TDL) was investigated using the indirect fluorescent antibody (FA) technique. The TDL were screened for the presence of EBV early antigen (EA), virus capsid antigen (VCA), and EBV nuclear antigen (EBNA). In 76% of the patients diagnosed as recurrent exudative tonsillitis, and in 33% diagnosed as recurrent tonsillitis and/or serous otitis media, EBNA was demonstrated in the purified TDLs. No EA- or VCA-producing cells were found in either the glass adsorbed or TDL cell preparations from all of the patients. These data suggest that in our patient sample, the tonsils may serve as a reservoir for EBV carrying lymphocytes and a basis for recurrent disease.     

A novel EBV-related nucleo-cytoplasmic antigen in a null cell-line (HLN-STL-C) reactive to antibodies in the sera from patients with Sj?gren's syndrome

We have established a non-T- and non-B-cell line, HLN-STL-C(STL-C), which harbors the EBV genome, from the lymph node cells of a Japanese ATL patient. This cell line expresses a unique Epstein-Barr virus (EBV)-related nucleo-cytoplasmic (N-C) antigen which is detected by indirect immunofluorescence (IF) with the sera from patients with nasopharyngeal cancer (NPC), infectious mononucleosis (IM) or adult T-cell leukemia (ATL). One of the molecular components of this antigen is proved to be STL-C specific 125 kD molecule by immunoblot analysis (IB). To study the involvement of EBV in Sj?gren's syndrome (SS), we examined the reactivity of the N-C antigen with the sera of SS patients by IF and IB. Among 24 cases examined, the sera of 21 cases (87.5%) positively stained the N-C antigen by IF. The staining patterns were divided into two types. Type I, (seven cases) showed positive staining for only N-C antigen, and Type II, (14 cases) was positive for N-C antigen associated with diffuse nuclear staining due to antinuclear antibodies in the SS patient's sera. Only one out of 11 non-Sj?gren's patients' sera, which were almost all healthy controls, was positive for N-C antigen in this study. By IB, however, only two out of 15 IF-positive SS patients' sera reacted with STL-C specific 125 kD molecule. These results suggested the presence of heterogenous components in the N-C antigen. Our findings may support the hypothetical conception that EBV plays an etiological role in SS.     

Epstein-Barr virus (EBV) antibodies in children with non-Hodgkin's lymphomas

Antibody titres against Epstein-Barr virus (EBV) antigens in children suffering from non-Hodgkin's lymphoma (NHL) were determined. IgG antibody titres against the viral capsid antigen (VCA) and early antigen (EA) exceeded those found in healthy control subjects. On the other hand, antibody titres against EBV-determined nuclear antigen (EBNA complex) were generally lower than in the control group. The most striking phenomenon observed in the patient group was the frequent activation of latent virus infection as revealed by the periodical appearance of anti-EA and IgM class anti-VCA antibodies. Antibody titres against EBV antigens were generally lower among patients with progressing disease than in those with a more favourable course of the illness. The closest relation to EBV based on serological findings, was detected in lymphoblastic lymphomas of Burkitt-type histology, poorly differentiated lymphocytic lymphomas, and in lymphomas localized in the abdomen. The question whether EBV might be involved in a certain proportion of the cases examined is discussed and further approaches to elucidate this problem are suggested.     

Detection of JC virus DNA in the cerebrospinal fluid of patients with progressive multifocal leukoencephalopathy

Four children with infectious mononucleosis (IM) and one with reactivated Epstein-Barr virus (EBV) infection had concomitant central nervous system disorders. Cerebrospinal fluid (CSF) samples from all five patients contained EBV genomic sequences and EBV-specific antibodies in the neurologic stage, but not during convalescence. Cerebrospinal fluid from two non-neurologic IM patients had neither EBV DNA nor EBV antibodies. The EBV-positive CSF of the five with neurological disorders were aseptic in culture and all negative for other human herpesvirus DNAs and antibodies: herpes simplex virus types 1 and 2, cytomegalovirus, varicella-zoster virus, and human herpesvirus 6. Epstein-Barr virus DNA and EBV antibodies were not detected in the CSF of 17 EBV-seropositive patients with mumps meningitis, rubella encephalitis, unknown febrile convulsion, or partial epilepsy. It is suggested that EBV plays a causal role in neurologic manifestations in patients with acute and reactivated EBV infections, through direct viral invasion and immunopathological reactions. © 1993 Wiley-Liss, Inc.     

Anti-human cytomegalovirus nuclear antigen antibodies of different immunoglobulin classes

IgG, IgM and IgA immunoglobulin classes of antibodies to human cytomegalovirus nuclear antigens (CMNA) were studied by the acid-fixed nuclear binding technique (AFNB) and combined anti-complement immunofluorescence (combined ACIF). In acute cases of infectious mononucleosis (IM) of human cytomegalovirus (HCMV) origin and in the so-called double virus infections (HCMV + Epstein-Barr virus), anti-CMNA IgM antibodies were detected. They were absent from both anti-HCMV positive sera of healthy donors and sera of patients suffering of IM caused by EBV used as controls. The presence of anti-CMNA IgM may thus serve as an additional evidence of acute HCMV infection. Non-complement-fixing IgA classes of the anti-CMNA antibodies were not found in some of the sera gathered during the acute phase of IM of EBV origin: in one fourth of the HCMV seropositive donors and in a number of late serum samples. But non-complement-fixing and complement-fixing anti-CMNA components of the IgG class were detected.