Comparative studies of Epstein-Barr virus strains from Ghana and the United States.

A high incidence of oropharyngeal excretion of Epstein-Barr virus (EBV) has been observed in African children with Burkitt's lymphoma (BL) (48%) and matched controls (45%). This compares with an incidence of 77% in American patients with infectious mononucleosis (IM) and 13% in age-matched controls. Cross-neutralization tests between EBV strains derived from BL and IM patients and their sera failed to detect differences in the major neutralizing antigenic components. Cord-blood lymphocytes transformed by American EBV expressed only early viral functions (EBV nuclear and soluble complement-fixing antigens) and produced no detectable transforming activity. By contrast, cord-blood lymphocytes transformed by African EBV strains contained 0.2-0.3% of cells with EBV capsid and early antigen and produced EBV with transforming activity. These cells contained twice as many copies of EBV homologous DNA as the cells transformed by American EBV strains.     

Association of Human Papillomavirus and Epstein-Barr Virus Infection with Tonsil Cancer in Northeastern Thailand

Background: Human papillomavirus (HPV) and Epstein-Barr virus (EBV) are associated with head and neck cancer, including tonsil cancer (TC) in the oropharyngeal area. Increasing incidence of HPV and EBV infection in different cancer tissues of oropharynx in both epithelial and lymphoid tissues, have been reported. However, little is known about association of these tumor viruses with TC in the Thai population. Here, we investigated the prevalence of HPV and EBV infection in different histology of TC and their association with TC from Thai patients. Methods: Eighty-three exfoliated tonsil cells from non-cancer controls (NCC) and 65 formalin-fixed paraffin-embedded TC tissues (TC) that were histologically classified as tonsillar squamous-cell carcinoma (TSCC) or diffuse large B-cell lymphoma (DLBCL) were studied. Prevalence of HPV and EBV infection was determined by real-time PCR. HPV genotyping was performed by reverse line blot hybridization and HPV genome status was investigated by multiplex qPCR. Localization of EBV infection was determined by EBER in situ hybridization. Results: Infection of HPV and EBV in TC cases was 16.9% and 30.8%, whereas in exfoliated tonsil cells was 1.2% and 66.3% respectively. HPV infection was significantly higher in TSCC (30.6%) than DLBCL samples (13.8%). HPV58 was commonly detected and presented as an integrated form in TSCC, whereas only episomal form was found in DLBCL. EBV infection was significantly higher in DLBCL (44.8%) than TSCC samples (19.4%), and detected in both lower than among exfoliated tonsil cell samples (66.3%). By EBER in situ hybridization in TSCC, EBV infection localized both in epithelial cells and infiltrating lymphocytes. The co-occurrence of HPV and EBV infection was 11.11% and 13.79% of TSCC and DLBCL, respectively, was associated with well-differentiated TSCC. Conclusion: HPV and EBV infection was significantly involved in a specific TC tissue, and associated with a good clinical outcome in TSCC.     

A quantitative analysis of the susceptibility of human leukocytes to transformation by epstein-barr virus

Susceptibility of lymphocyte-enriched cell fractions isolated from human umbilical cord blood and adult peripheral blood to transformation by the B95–8 strain of Epstein-Barr virus (EBV) was investigated quantitatively. Minimum multiplicity of input of virus (50% transforming dose) per cell (MOI) necessary to induce maximum level transformation of cord cells ranged from 0.02 to 0.2. The frequency of initially transformed cells (fraction of transformable cells) in the cord cell samples from two different individuals was estimated to be 2.6 to 6.2%. In this system, the appearance of cells positive for EBV-associated nuclear antigen (EBNA) paralleled the growth curve of transformed cells. About 70% of the latter were EBNA-positive. In adult cell preparations from two individuals, 1.8 and 0.03%, respectively, of the cells were transformable indicating larger individual variations in sensitivity to EBV than in cord cells. The EBV susceptibility was also determined by the transforming efficiency (TE) expressed as the negative log of the virus dilution which induces transformation in 50% of cell cultures infected at an MOI of 0.2. From the TE value, a minimum MOI which induces transformation could be calculated. Also by this test it was shown that the EBV susceptibility of adult cells was not only lower but also much more variable between individuals than that of cord cells. There was no correlation between the susceptibility of cells and the titer of anti-EBV antibody in donors' sera. In cultures of mixed cord cells and adult cells known to have low EBV susceptibility, the minimum MOI increased in proportion to the amount of adult cells.     

Further characterization of a herpesvirus-positive orang-utan cell line and comparative aspects of in vitro transformation with lymphotropic old world primate herpesviruses.

An orang-utan (Pongo pygmaeus) suspension line, CP81, was shown to lack myeloid markers of lysozyme activity an d phagocytosis but to be positive for lymphocytic N-alkaline phosphatase activity, and to release a B-cell-tropic herpesvirus. This herpesvirus, termed Herpesvirus pongo, had 30--40% DNA homology with EBV and was present at 2-3 genome copies per CP-81 cell. Gibbon lymphocytes transformed by H. pongo, Epstein-Barr virus (EBV), and H. papio (of baboon, Papio hamadryas, origin) were found to be virus antigen-positive B cells. Gibbon lymphocytes transformed by H. pongo and EBV and transplanted to nude mice by the intracranial (IC) route (had a 75% and a 45% success rate, respectively), while transplants of similar cells transformed by H. papio were only 10% successful. None of these lines transplanted subcutaneously (SC) nor manifested a high degree of colony formation in 0.33% agarose (less than or equal to 0.5%), Gibbon lymphocytes transformed by H. pongo were hypodiploid while those transformed by EBV or H. papio were diploid. CP-81 cells themselves could be transplanted both IC (100%) and SC (70%) and showed a relatively high degree of colony formation in agarose (6.4-7.6%). B95-8 cells (marmoset, Saguinus oedipus-EBV) could be transplanted IC (66%) but not SC and had a low but significant ability to grow in agarose (1.6%). 594S (baboon, P. hamadryas-H. papio) cells could be transplanted IC (25%) but not SC, and grew to very low levels in agarose (0.1%).     

Human dendritic cells pulsed with autologous Epstein-Barr virus transformed B-cell lymphoblastoid cell (BCL) lysate elicit a BCL specific MHC-class II restricted T-cell response

Epstein Barr Virus (EBV) associated lymphoproliferative disorders (LPD) express EBV latent antigens that are also expressed on normal B-cells transformed with EBV. This could potentially be exploited to develop immunotherapeutic strategies for LPD and other EBV associated malignancies. To this end we investigated the capacity of human monocyte derived dendritic cells (DC) pulsed with lysate from autologous EBV transformed B-cell lymphoblastoid cell (BCL) lysate to elicit an in vitro antitumor response. BCL lysate pulsed DC generate BCL specific cytotoxic lymphocytes, as lymphocytes primed with such DCs induce cytolysis of autologous (>60%) but not allogeneic BCL (<5%). In addition, lymphocytes primed with BCL lysate pulsed DC secrete gamma-IFN (3176 pg/ml). Whereas gamma-IFN production was markedly reduced (>99%) when BCL specific T-cells were stimulated by BCL lysate pulsed DC in the presence of blocking antibodies to HLA-DR, DP and DQ, use of antibodies to MHC class-I resulted in only a minimal reduction in gamma-IFN production (17%). These studies demonstrate that BCL lysate pulsed DC elicit a predominantly BCL specific, MHC class-Il restricted T cell response. This suggests that vaccination with autologous BCL lysate pulsed DC may represent a viable immunotherapeutic approach for the treatment of LPD.     

Colonies of EBNA-positive cells in soft agar from peripheral leukocytes of infectious mononucleosis patients.

Epstein-Barr virus (EBV)-associated nuclear antigen (EBNA)-positive lymphoblastoid cells grew as colonies in soft agar after seeding of leukocytes from the peripheral blood of four patients with infectious mononucleosis serologically determined to be caused by EBV. In individual cases more colonies were obtained from blood specimens during the acute phase of the disease than during the convalescent phase. Incorporation of human umbilical cord serum, which contained neutralizing antibody to EBV, into the agar medium did not reduce the number of colonies developing. Our observations indicate that colony-forming cells were originally present in the blood samples, and that they were not infected and subsequently transformed in vitro. Cells from less than 20% of the EBNA-positive colonies grew to form lymphoblastoid cell lines, which were EBNA-positive and had B lymphocyte surface markers. However, the majority (over 80%) of the EBNA-positive colonies failed to form immortalized cell lines. No colonies were obtained from 91 blood samples from healthy young adults and from five patients with an IM-like disease unrelated to EBV infections. The present results strongly suggest that already transformed cells or cells very easily transformed by EBV are present in the blood of IM patients.     

Detection of Epstein-Barr Virus and Genotyping Based on EBNA2 Protein in Mexican Patients With Hodgkin Lymphoma: A Comparative Study in Children and Adults

BackgroundEpstein-Barr virus (EBV) is a member of the Herpesviridae family and is associated with Hodgkin lymphoma (HL). Isolates of EBV are classified according to sequence variation in the latency genes such as Epstein-Barr virus nuclear antigen (EBNA). EBNA2 contains the most divergent locus and is classified into type 1 and type 2 or EBNA2A and EBNA2B, respectively. We compared the frequency of EBV and the distribution of EBNA genotypes in Mexican children and adults with HL.Patients and MethodsLymph node biopsy specimens from children and adults with HL were embedded in paraffin. EBV was identified by LMP1 amplification and Epstein-Barr–encoded RNA EBER by in situ hybridization (ISH) and genotyped as EBNA2A or EBNA2B using nested polymerase chain reaction (PCR) and specific primers for the detection of subtype.ResultsSixty-six samples were obtained from 3 hospitals—42 (63%) from children and 24 (37%) from adults with HL. Thirty-two of the 42 samples (76.1%) were positive for EBV in children and 16 of 24 (66.6%) samples were positive in adults (P = .41). In both children and adults, EBV was found more frequently in male patients. Thirty-four of 48 cases could be typed (70.8%). EBNA2A was found in 7/21 (33.3%) children and in 4/13 (30.8%) adults (P = 1.0), and EBNA2B was found in 10/21 (47.6%) children and in 9/13 (69.2%) adults (P = .22). A mix of subtypes was found in 4/21 (19%) children.ConclusionEBV was found frequently in both children and adults with HL. EBNA2B was the most frequent subtype, and a high frequency of mixed subtypes was found in children.     

Epstein-Barr virus: transformation of non-human primate lymphocytes in vitro

Continuous lymphoblastoid cell cultures were established from marmoset (Saguinus sp.), squirrel (Saimiri sciureus), owl (Aotus trivirgatus) and cebus (Cebus apella) monkeys after culturing their peripheral lymphocytes with lethally X-irradiated cells carrying Epstein-Barr virus (EBV). Transformation also was achieved by exposing simian lymphocytes to infectious, cell-free EBV derived from the simian lymphoblastoid cell cultures. Simian lymphocytes were not transformed after exposure to cell-free EBV derived from HR-1 cells. The simian cell cultures were similar to cell cultures derived from Burkitt's lymphoma or infectious mononucleosis patients. EBV-induced early, viral capsid and membrane antigens, intranuclear inclusion bodies and herpesvirus virions were demonstrable in most cultures. Seven cultures were insusceptible to superinfection with EBV and treatment of the cultures with halogenated pyrimidines was relatively ineffective for inducing synthesis of early or viral capsid antigens. All cell cultures had B-cell characteristics: they produced immunoglobulins but did not form spontaneous rosettes with sheep erythrocytes. Four of six marmoset monkeys, inoculated with EBV-transformed marmoset lymphocytes, developed antibodies to EB viral capsid antigens and one marmoset inoculated with autochthonous transformed cells also developed heterophile antibodies. Seven marmosets, inoculated with cell-free EBV derived from HR-1 cell cultures, developed no detectable levels of antibodies to EBV-specified antigens or heterophile antibodies. No overt clinical abnormalities were detected in any of the marmosets inoculated with HR-1 or Kaplan EBV but one of five marmosets inoculated with B95hyphen;8 EBV developed a lymphoma.     

Transformation of lymphocytes by Herpesvirus papio.

Cotton-topped (CT) or white-lipped (WL) marmoset lymphocytes were transformed in vitro with herpesvirus papio (HVP) into permanently growing lymphoblastoid cell lines (LCL). Five of 9 HVP-transformed CT cell lines contained cells with antigens reacting with antibodies to Epstein-Barr virus (EBV) capsid antigen (VCA) and/or to EBV-induced early antigens (EA). None of 12 WL LCL revealed such antigen-producing cells. Cells from both groups of cultures failed to react with antibodies to the EBV-specified nuclear antigen (EBNA). Exposure of baboon circulating lymphocytes to X-irradiated HVP or EBV-carring cells, or to suspensions of EBV resulted in establishment of LCL which all contained VCA and/or EA-positive, but no EBNA-positive cells. Nuclear antigens were undetectable also with anti-VCA-positive sera from baboons, chimpanzees, or other non-human primates. DNA-complementary RNA (cRNA) filter hybridization with EBV cRNA showed that with one exception transformed CT or WL marmoset cells contained at least 1-2 virus genome equivalents per cell, while at least 12-25 virus genome equivalents per cell were detected in transformed baboon cells. These data need confirmation by DNA-DNA reassociation kinetics.     

miRNA-200a在EB病毒感染相关的儿童散发性伯基特淋巴瘤中的表达及其意义

目的 探讨miRNA-200a在EB病毒(EBV)阳性和阴性的儿童散发性伯基特淋巴瘤(BL)中的表达及其意义.方法 应用原位杂交对40例BL患儿肿瘤组织及13例慢性扁桃体炎患者扁桃体组织进行EBER检测,应用qRT-PCR方法对Raji和Ramos细胞株、40例BL患儿肿瘤组织及13例慢性扁桃体炎组织进行miRNA-200a检测.结果 40例BL组织中EBER阳性16例,阴性24例,13例扁桃体EBER均阴性;miRNA-200a在EBV阳性BL组织表达量为0.033±0.018,较EBV阴性BL组织(0.504±0.214)和扁桃体组织(0.284±0.153)降低,差异有统计学意义(均P＜0.05);Raji和Ramos比较,miRNA-200a表达显著降低(P=0.000).结论 miRNA-200a的低表达与EBV阳性儿童散发性BL的发病机制密切相关,miRNA-200a可能起着类似于抑癌基因的作用.     

Immunologic cytotoxicity against autologous human lymphocytes transformed or infected by Epstein-Bar virus: role of antibody-dependent cellular cytotoxicity in health individuals

Immunologic cytotoxicity against lymphocytes transformed or infected by Epstein-Barr virus (EBV) was mainly studied in an autologous in vitro system by 51Cr release assay and EBV-determined nuclear antigen (EBNA)-specific trypan blue exclusion method. When the cells of newly established EBV-transformed or spontaneously transformed lines were incubated with unfractionated autologous healthy donor lymphocytes or T-cell-depleted lymphocytes in the presence of EBV-positive autologous or allogeneic serum, the transformed cells were killed with high frequency. Exposure to lymphocytes alone or to EBV-positive serum alone was not effective. The cytotoxic reaction was directed against cells positive for EBV-induced membrane antigens (MA) but not against MA-negative transformed cells. A very small fraction (1 of 200) of healthy donor lymp]hocytes exposed to EBV converted into EBNA-positive and MA-positive cells, and these were also killed by the remaining autologous lymphocytes in the presence of EBV-positive serum. These results indicated that the present cytotoxic reaction represents antibody-dependent cellular cytotoxicity (ADCC), and this particular mechanism probably plays an important role in the immunologic surveillance in the protection against EBV-induced oncogenesis in seropositive individuals. Such ADCC, however, does not seem to function effectively in patients with systemic lupus erythematosus.     

Growth and spread in nude mice of Epstein-Barr virus transformed B-cells from a chronic lymphocytic leukemia patient

Subcutaneous inoculation of Epstein-Barr virus (EBV) transformed peripheral blood B-lymphocytes (PBL) from an untreated chronic lymphocytic leukemia (CLL) patient produced progressively growing lethal tumors in 4 of 11 whole body irradiated (440 rads) nude mice. In one tumor bearing mouse there was splenomegaly and generalized enlargement of lymph nodes. Chromosomal analysis and membrane immunofluorescence revealed that cells in all the 4 s.c. tumors and a proportion of cells in the enlarged spleen and lymph nodes had human chromosomes and contained human kappa or lambda chains demonstrating that these were polyclonal human B-cells. Epstein-Barr virus associated nuclear antigen could be detected in 100% of cells in all the 4 EBV transformed B-cell lines in vitro and aliquots of cells from several s.c. tumors and metastatic lesions examined. Successful serial transplantation into irradiated nude mice was possible for at least 3 generations with one of the 4 s.c. tumors. During serial transplantation, spread of tumor cells to the spleen and lymph nodes could be detected in all the 3 passage mice investigated; however, there was no evidence in any mouse of dissemination of tumor cells into the bloodstream or into any organ other than lymph nodes and spleen. s.c. tumors also developed in a proportion of irradiated nude mice after inoculation of cells from two other s.c. tumors and the metastatic spleen and lymph nodes, but all these tumors regressed during the first or second transplant passage. Two % of PBL from the untreated patient and 4% of EBV transformed PBL maintained in vitro were found to have trisomy of chromosome 12 which is the most frequently reported anomaly associated with human CLL B-cells. It is highly probable that the cells with trisomy were derived from the leukemic clone of this patient. Cells with this trisomy predominated in most metastatic sites compared to the parent s.c. tumors. Inoculation of irradiated nude mice with EBV transformed PBL from this patient after chlorambucil therapy (100% metaphase plates with 46,XY,11q+ karyotype) or with EBV transformed PBL from 2 normal adults failed to produce any progressively growing tumor in a total of 12 irradiated animals observed greater than 300 days. Although there are several reports of EBV induced immortalization of CLL B-cells in vitro, we have not seen any previous report on the successful serial transplantation and dissemination of EBV transformed CLL B-cells in nude mice.     

Lymphoblastoid transformation and kinetics of appearance of viral nuclear antigen (EBNA) in cord-blood lymphocytes infected by Epstein-Barr Virus (EBV).

Human cord-blood lymphocytes were infected with B95.8 Epstein-Barr virus (EBV) before and after separation into B- and T-cell populations. Lymphoblastoid cells exhibiting B-cell characteristics appeared after 2 to 3 days of culture in the total population and in the separated B-cell subpopulation but not in the T-cell subpopulation. EBV nuclear antigen (EBNA) was detected concurrently with the appearance of lymphoblastoid cells. The proportion of EBNA-positive cells corresponded to that of lymphoblastoid cells, and reached 50% after 4 days. EBNA was present only in cells with B-cell markers. These observations indicate that only B-cells are susceptible to EBV infection, that the transformation occurs within a few days and that EBNA is a valid early marker for susceptibility to EBV transformation.     

Epstein-barr-virus hypersensitivity of lymphocytes from patients with ataxia-telangiectasia

Ataxia telangiectasia (AT), an autosomal recessive disorder with a high incidence of lymphoreticular malignancies including Epstein-Barr virus (EBV)-induced lymphoproliferative disorders (LPD), was investigated to assess the susceptibility to EBV infection and oncogenesis. When the patients' lymphocytes were infected with B95-8 EBV, there was a tendency toward an enhanced growth in semisolid agar, as compared with the healthy donor counterparts. Among the preparations tested, from 14 patients, 2 cell lines showed extremely high colony forming efficiency. The lymphocytes from patients with AT did not contain a large number of EBV target cells, as determined by the maximum frequency of EBV-determined nuclear antigen (EBNA) induction prior to cellular DNA synthesis. Fourteen different lymphoblastoid cell lines derived from the 14 patients with AT were then examined for their EBV inducibility and superinfectibility. By treatment with 12-O-tetradecanoyl-phorbol-13-acetate TPA) and culturing at a lower temperature of 33-degrees-C, early antigen (EA) induction occurred approximately 6-fold and 5-fold higher, respectively, as compared with the lymphoblastoid cell lines derived from healthy controls. Viral capsid antigen (VCA) was also induced significantly by TPA or culturing at lower temperature in the lines from patients with AT, but only slightly in the control counterparts. When the lymphoblastoid cells from patients with AT were exposed to P3HR-1 EBV, EA and VCA syntheses were approximately 6- and 12-fold higher, respectively, than those in the cells derived from the healthy controls. This evidence suggested B lymphocytes of patients with AT were highly susceptible to EBV infection and possibly linked to the development of EBV-induced LPD.     

Enhancement of outgrowth of EB virus-transformed cells from normal human peripheral blood by a tumor promoter, TPA

Effect of 12-0-tetradecanoyl-phorbol-13-acetate (TPA) on the establishment of lymphoblastoid cell lines (LCL) following spontaneous tranformation of B cells by Epstein-Barr virus (EBV) from peripheral lymphocytes of healthy EBV-seropositive adults was examined. In the lymphocyte culture with TPA (0.5 ng/ml), the frequency of establishment of LCL increased and the time required for appearance of transformation decreased. A larger number of EBV-positive cells in the peripheral blood were detected in the assay by co-cultures with cord blood lymphocytes in the presence of TPA than in the absence of it. The enhancement of LCL establishment by TPA was largely abolished in the culture with EBV neutralizing antibody-containing human umbilical cord serum. This suggests that the enhancement by TPA may be caused mostly by transformation of coresident B lymphocytes infected with active virus induced from the EBV genome-positive cells by TPA.     

Persistence of transforming and nontransforming Epstein-Barr virus in high passages of P3HR-1 cell lines

At least three laboratories have reported that the P3HR-1 line, which had originally produced transforming Epstein-Barr virus (EBV), now produces only the nontransforming variant. Studies to determine whether these findings were universal or a consequence of specific cell lines or culture conditions were undertaken in P3HR-1 cultures of identical HLA types from five sources. All of the EBV preparations derived from cell lines cultured at 32, 34, and 35 degrees C transformed cord blood lymphocytes, whereas virus propagated at 37 degrees C did not usually transform. Furthermore, indirect immunofluorescence revealed that a monoclonal antibody directed against transforming EBV membrane glycoprotein bound to 10-12% of the P3HR-1 cells that had been continuously propagated at 34 degrees C, but the antibody did not bind to the same cells cultured at 37 degrees C. Although virus expression was completely repressed in transformed cord blood cells, transforming virus could be rescued by superinfection with nontransforming P3HR-1 EBV. Cells transformed with P3HR-1 virus induced poorly differentiated lymphomas in athymic nude mice after seven or eight passages. Whether all P3HR-1 cells have the potential to produce detectable quantities of transforming virus remains to be determined.     

Cellular localization of an Epstein-Barr virus (EBV)-associated complement-fixing antigen in producer and non-producer lymphoblastoid cell lines

Anti-complement immunofluorescence (ACIF) was used to study the complementfixing antigens of human lymphoblastoid cell lines. These cell lines carry the Epstein-Barr virus (EBV) genome although only producer cultures synthetize EBV-specific antigens (virus capsid antigen, VCA and early antigen, EA) detectable by direct and indirect immunofluorescence, usually in less than 5% of the cells. The ACIF test revealed an antigen localized in the nucleus of the lymphoblastoid cells. In contrast to EA and VCA, this antigen was present in over 90% of the cells of both producer and non-producer cultures. The antigen was shown to be specific for EBV by comparing the reactions of 52 sera in the ACIF test. Sera giving the nuclear reaction contained antibodies to VCA, EA or antigens detectable by complement fixation tests on cell extracts, but sera without EBV antibodies failed to give the reaction. Weak, equivocal or discordant reactions occurred with six sera with low titres in VCA, EA or complement fixation tests. Cell lines derived by transformation of human and primate lymphocytes by EBV gave the nuclear reaction. Control cells with no known association with EBV were non-reactive. These included foetal lymphocytes transformed by phytohaemagglutinin, cell lines derived from breast cancer, glioma, normal glia, pleuritis maligna and myeloma, and two marmoset lymphoid lines carrying Herpesvirus saimiri (HVS). In preliminary experiments, the ACIF test was used as a tool to trace the EBV genome at the cellular level. Cells from two Burkitt lymphoma biopsies, one tested after biopsy and one after passaging in nude mice, contained an EBV-specific antigen. Three clones of cells derived from hybrids of mouse somatic cells and a human lymphoblastoid cell line also contained such an antigen, but the number of reactive cells varied from clone to clone. A fourth clone was non-reactive.     

Epstein-Barr virus (EBV) in Chinese pediatric Hodgkin disease: Hodgkin disease in young children is an EBV-related lymphoma.

BACKGROUND: The Epstein-Barr virus (EBV) is thought to be involved in the pathogenesis of some Hodgkin disease (HD) cases. EBV may be associated particularly with childhood HD, a disease rare in the West compared with developing countries. In this study, a large series of Chinese pediatric HD cases has been examined to determine the age-specific prevalence of EBV. METHODS: Paraffin sections from 104 pediatric and 52 adult Chinese HD cases were examined for EBV-RNA (EBERs) and EBV latent membrane protein-1. RESULTS: Most pediatric cases arose in boys and showed an histology of mixed cellularity. Prominent interfollicular involvement was seen frequently in the childhood cases. EBV was identified in tumor cells in 113 of 156 (72%) HD cases but was more frequent in pediatric cases (93 of 104; 89%) compared with adult cases (20 of 52; 38%) (P < 0.01; chi-square test). EBV was found in 86 out of 91 (95%) cases in children aged 3-10 years and in 7 out of 13 (54%) cases in children aged 11-14 years (P < 0.01; chi-square test). The virus was less frequent in cases in young adults than in old adults, although this trend was not significant (P > 0.05; chi-square test). Pediatric HD was associated with EBV irrespective of histologic subtype. In adults, EBV was associated more frequently with mixed cellularity than with other subtypes. CONCLUSION: To the authors' knowledge, this is to date the largest series of pediatric HD cases studied for EBV. Study findings provided further evidence that HD is etiologically heterogeneous. The authors believe that pediatric HD now should be regarded as a distinctive EBV-related lymphoma.     

Properties of a baboon lymphotropic herpesvirus related to Epstein-Barr virus.

Three lymphoblastoid cell lines were established from splenic lymphocytes of a lymphomatous baboon (Papio hamadryas) by co-cultivation of the lymphocytes with X-irradiated cells of marmoset or baboon lymphoblastoid cell cultures; the baboon splenic lymphocytes failed to grow when cultured alone. A herpesvirus, associated with each cell line, was identified by immunofluorescence, molecular hybridization and electron microscopy. Antigenic comparison with Epstein-Barr virus (EBV) showed that the baboon herpesvirus and EBV shared cross-reacting viral capsid antigens (VCA): 20 of 20 (100%) anti-VCA (EBV)-positive human sera and 55 of 62 (89%) baboon sera reacted with the baboon lymphoblastoid cells and baboon sera stained EBV VCA in P3HR-1 and EB-3 cells. No nuclear antigen, as assayed by anti-complement immunofluorescence tests, was detected in baboon lymphoblastoid cells when human or baboon anti-VCA positive sera were used. Baboon anti-VCA-positive sera also failed to stain EBV nuclear antigens (EBNA) in Raji or P3HR-1 cells. Preliminary molecular hybridization studies showed only approximately 40% homology between viral DNA of baboon cell lines and DNA of EBV derived from P3HR-1 cells.     

Calcium concentration defines two stages in transformation of lymphocytes by Epstein-Barr virus

Lymphocytes from Epstein-Barr virus (EBV) seronegative donors were either stimulated with Protein A or infected with EBV and cultured in growth medium containing a range of calcium levels (700 microM to less than 2 microM available calcium). Three calcium end-points for the proliferation of B lymphocytes were defined: 18 microM for mitogenically-stimulated B cells; 8 microM for a pre-12 h event in EBV transformation and less than 2 microM for a post-12 h event in EBV transformation resembling the end-point for the emergent lymphoblastoid cell line. EBV-infected lymphocytes cultured in calcium-depleted medium expressed the EB virus nuclear antigen and proliferated after addition of calcium chloride. The present study should be useful both in defining the sequence of events in EBV transformation and in studying properties specific to the transformed phenotype.