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.     

Transformation of tonsil lymphocytes by Epstein-Barr virus.

Tonsil lymphocytes obtained from children and adults at tonsillectomy were examined for susceptibility to infection with Epstein-Barr virus (EBV) in vitro. Of 37 specimens, 24 (65%) were transformed by EBV. Rate of transformation was higher in the cells from younger children than in those from older children and adults, but the susceptibility was not directly correlated to the titer of antibody against EBV in donor serum. All 22 transformed tonsil cell lines tested were carrying EBV-associated nuclear antigen, and 8 of them produced cytoplasmic IgA in 15-45% of cells. The results suggest that tonsil lymphocytes are targets of EBV.     

Epstein-Barr virus infection in humans: from harmless to life endangering virus-lymphocyte interactions

After the primary infection, that may or may not cause infectious mononucleosis, the ubiquitous Epstein-Barr virus (EBV) is carried for lifetime. The great majority of adult humans are virus carriers. EBV was discovered in a B-cell lymphoma (Burkitt lymphoma). EBV infection in humans is the example for the power of immune surveillance against virus transformed, potentially malignant cells. Although the virus can transform B lymphocytes in vitro into proliferating lines, it induces malignancy directly only in immunosuppressed hosts. EBV-induced growth transformation occurs only in B lymphocytes. It is the result of a complex interaction between virally encoded and cellular proteins. Different forms of the virus-cell and the cell-host interactions have evolved during a long period of coexistence between the virus and all Old World (but not New World) primates. The asymptomatic carrier state is based on a viral-strategy that downregulates the expression of the transforming proteins in the virus-carrying cell. In addition to the silent viral-gene carriers and the expressors of the nine virus-encoded genes that drive the growth program, virus carrying cells exist that show other patterns of gene expression, depending on the differentiated state of the host cell. Certain combinations contribute to malignant transformation, but only in conjunction with additional cellular changes. These are induced by direct or cytokine-mediated interactions with normal cells of the immune system.     

Growth in semisolid agar medium of human cord leukocytes freshly transformed by Epstein-Barr virus.

The semisolid agar method for the selective growth of transformed cells was applied to investigations of Epstein-Barr virus (EBV) transformation. When 1 X 10(5) HUMAn umbilical-cord blood leukocytes were seeded in agar medium immediately after EBV exposure, about 100 colonies developed in each dish. The occurrence of colonies correlated well with dilutions of EBV inoculum. These colonies were composed of lymphoblasts, were positive for EBV-determined nuclear antigen immunofluorescence, and consistently resulted in the establishment of cell lines. In cord leukocyte cultures exposed to EBV, cells capable of growing in semisolid agar medium appeared early and increased rapidly, though the total numbers of leukocytes and control cultures decreased during the first several days.     

Induction of Epstein-Barr virus-associated nuclear antigen during in vitro transformation of human lymphoid cells.

Human lymphoid cells isolated from the peripheral blood of adults, from cord blood, and from fetal liver, spleen, bone marrow, and thymus were cultivated with or without a cell-free preparation of Epstein-Barr virus (EBV) with demonstrated transforming activity. The cultures were examined for the EBV-associated nuclear antigen (EBNA) and for transfromation into permanent lymphoblastoid cell lines (LCL). EBNA, seen only in cultures that had received exogenous EBV, was detected between days 1 and 6 after addition of EBV, most frequently on day 3. EBNA-positive cells had a lymphoblastoid appearance. Transformation into established LCL became apparent between days 12 and 19. The addition of pokeweed mitogen to cultures containing EBV enhanced the development of EBNA, whereas phytohemagglutinin or concanavalin A had no such effect. Neither EBNA nor transfomration was observed in lymphoid cells from fetal thymus. In fetal spleen, bone marrow, and liver cells, EBV regularly induced EBNA and LCL transformation.     

Epstein-Barr virus-induced transformation of human leukocytes after cell fractionation.

The efficiency of transformation of human lymphocytes after infection with Epstein-Barr virus (EBV) was determined in fractionated and non-fractionated preparations derived from 16 human cord blood samples and two blood samples from adult donors. The transformation efficiency of macrophage-depleted leukocytes was consistently lower as compared to non-fractionated leukocytes. Additional depletion of B-cells resulted in a further decrease. Reduction of T-cells, however, did not influence significantly the transformation rate. In non-fractionated leukocyte cultures, as well as in macrophage-depleted and B-cell enriched cultures, colonies of transformed cells were regularly observed within the first week of cultivation. All cell lines established after EBV-infection revealed membrane-bound immunoglobulin. Reconstruction of macrophages-depleted, B-cell enriched or B-cell depleted cultures with autologous macrophages resulted in an increase of the transfromation efficiency up to the values of non-fractionated leukocyte preparations. Addition of heterologous human embryonic lung fibroblasts resulted in a similar increase. The results support the interpretation that EBV transforms only those cells of the hematopoetic system which are derived from the bone-marrow entity. The transformation efficiency is considerably increased by co-cultivation of lymphocytes with macrophages and heterologous human fibroblasts which seem to excert a feeder-layer effect by enhancing survival of lymphocytes in vitro.     

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.     

The transforming prototype of Epstein-Barr virus (B95-8) is also a lytic virus

The B95-8 isolate of the Epstein-Barr virus (EBV) has been described as a non-lytic transforming virus. We have performed experiments in order to determine if the B95-8 EBV is capable of super-infecting and replicating in EBV-genome-positive non-producer lymphoblastoid cells. Using concentrates of B95-8 EBV, prepared from 6 different B95-8 cell lines treated with 12-O-tetradecanoylphorbol-13-acetate (TPA), we demonstrated that virus concentrates could transform human or cotton-top tamarin B-lymphocytes and also lytically replicate in Raji cells, inducing EBV antigens and infectious virus. While the virus obtained from B95-8 super-infected Raji cells was able to transform cord-blood lymphocytes (CBLs) and super-infect Raji cells, transformation was abortive, with cell cultures only growing for up to 6 weeks. Transformation titers of the B95-8 virus concentrates ranged from 10(5) to greater than 10(8) transforming units/ml; early antigen (EA) induction ranged from 1% to 50% after superinfection of Raji cells, depending on the virus stock used, as determined by immunofluorescence. Southern blot analysis was carried out on the DNA prepared from B95-8 cells and virion DNA. The results were consistent with the published EcoRI restriction pattern for B95-8 EBV. The issue of whether the B95-8 cells produce virions with a dual biological phenotype or, rather, 2 biologically distinct viruses, is addressed.     

The interaction of non-transforming Epstein-Barr virus (EBV) with cell-associated EBV DNA in superinfected lymphoblastoid cell lines

Two human lymphoblastoid cell lines were established by the transformation of human cord-blood lymphocytes with transforming Epstein-Barr virus (EBV). One cell line (HLB-R1) was established with EBV obtained after the superinfection of Raji cells with HR-1 EBV and the other (HLB-Bl) was established from B95-8 EBV-infected human cord-blood lymphocytes. Both the HLB-R1 and HLB-B1 lines were susceptible to superinfection with HR-1 EBV. We found that EBV DNA was replicated in the superinfected cell lines and that transforming EBV was produced in both the HLB-B1 and HLB-R1 cells. The average titer of transforming EBV obtained in the HR-1 EBV superinfected HLB-B1 and HLB-R1 cell lines was 10(4) transforming units (TU)/ml, whereas the average titers of transforming EBV obtained by the superinfection of Raji cells was 10(1) TU/ml. Epstein-Barr virus capable of inducing early antigen (EA) in superinfected Raji cells (lytic virus) was not detected in any transforming virus preparation. Restriction enzyme digestion patterns of virus DNA isolated from HR-1 and B95-8 cells, as well as from superinfected cells, were compared. The EBV DNA that was replicated in the superinfected HLB-R1 and HLB-B1 cell lines showed a more complex pattern. Our data suggest that recombination between input HR-1 EBV DNA and latent cell-associated EBV DNA occurs. Presumably this recombination results in a change in the biological properties of the newly synthesized virus.     

Transformation of Madin-Darby canine kidney (MDCK) epithelial cells by Epstein-Barr virus latent membrane protein 1 (LMP1) induces expression of Ets1 and invasive growth

The Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) has a significant role in initiating EBV-associated lymphoproliferative disease and EBV-related malignancies. In view of clinical features related to the type of EBV latency, LMP1 may influence invasiveness of EBV associated tumors categorized as types II and III as represented on nasopharyngeal carcinoma (NPC). To screen for genes associated with invasion of epithelial cells transformed by LMP1, Madin-Darby canine kidney (MDCK) epithelial cells were transformed by LMP1. Stable transfection of a LMP1 gene into MDCK cells induced morphological change from cobblestone to a long spindle-shape, reduced cell-cell adhesion and caused high cell motility. Parental MDCK cells, which form spherical cysts in three-dimensional collagen gel matrix, form branching tubules following exposure to hepatocyte growth factor (HGF). MDCK cells transformed by LMP1 showed invasive growth to form branching tubules into collagen gel without HGF-treatment. mRNA differential display and Northern hybridization identified plasminogen activator inhibitor-1 (PAI-1), urokinase type plasminogen activator (uPA) and ets1 as genes upregulated during transformation by LMP1. Expression of a dominant negative type of Etsl in LMP1-transformed cells downregulated uPA expression and cell motility. Deletion of LMP1 cytoplasmic carboxy-terminal activating region 1 (CTAR1) domain abolished transformation, but a deletion mutant lacking CTAR2 domain still retained transforming and uPA-inducing ability. Expression of Ets1 was immunolocalized in tumor cells of NPC tissue which frequently express LMP1. Taken together, it is suggested that LMP1 induces expression of Ets1 which may contribute to invasion of NPC by stimulating cell motility and uPA expression.     

Re-evaluation of a transforming strain of epstein-barr virus from the burkitt lymphoma cell line,Jijoye

The biological properties of Epstein-Barr virus (EBV) from a Burkitt lymphoma cell line, Jijoye, were examined. The synthesis of virus capsid antigen (VGA) and early antigen (EA) in Jijoye cells was markedly enhanced by shift-down of the temperature of incubation from 37°C to 33°C. Cultures of Jijoye cells at 33°C released a large amount of transforming EBV (10^5.2 of 50% transforming doses/ml) into the culture fluid. However, no infectious virus was produced in all cultures at 37°C during the course of this study. The EBV (Jijoye EBV) from Jijoye line was found to possess only transforming activity, but not EA-inducing activity. Jijoye EBV lacks adsorbing capacity to Jijoye cells, in contrast to P3HR-l EBV which can adsorb to Jijoye cells. The Jijoye cells were highly susceptible to superinfection with P3HR-l EBV as demonstrated by the induction of EA, VGA and infectious EBV. The EBV induced by the P3HR-I EBV superinfection of Jijoye cells has also transforming activity but neither EA-inducing activity nor adsorbing capacity to Jijoye cells.     

Mechanisms of enhancement of Epstein-Barr virus-induced transformation of peripheral blood lymphocytes by a tumor promoter, TPA, with special reference to lowering of cytotoxicity of T cells

The mechanism of the previously observed enhancement of Epstein-Barr virus (EBV)-induced cell transformation of human lymphocytes by 12-0-tetradecanoyl-phorbol-13-acetate (TPA) was studied. A concentration of TPA (0.5 ng/ml) was used. In cultures of human cord blood lymphocytes infected with EBV in the presence of TPA, a larger number of EBV-associated nuclear antigen (EBNA)-positive and/or DNA synthesizing cells was observed than in the absence of TPA. In the virus-infected lymphocyte cultures of EBV-seropositive adult donors, in vitro regression of transformation, which is known to be caused by T lymphocytes, was suppressed by TPA, EBV-specific and -non-specific cytotoxicity of T cells generated in mixed cultures of peripheral blood lymphocytes (PBL) of seropositive adults and autologous lymphoblastoid cell line (LCL) cells was markedly lowered by the presence of TPA in the cultures, However, TPA had little effect on proliferation of T cells in stimulated cultures, and addition of TPA to the reaction mixture for the cytotoxicity test did not lower the cytotoxicity. These results suggest that TPA has an inhibitory effect on T cells which suppress EBV-transformation. THe effect on T cells, together with the direct promoting effect on proliferation of EBV-transformed cells, may explain the outcome of enhancement of EBV-induced LCL establishment from PBL of seropositive donors by TPA previously observed.     

In vitro transformation of human B-cell follicular lymphoma cells by Epstein-Barr virus

Human low-grade B-cell lymphoma cells cannot be readily maintained in long-term tissue culture. In an effort to obtain low-grade B-cell lymphoma cell lines for in vitro study, we used Epstein-Barr virus (EBV) as a transforming agent. T-cells were removed prior to EBV transformation by rosetting with sheep erythrocytes, followed by treatment with anti-T11 monoclonal antibody plus complement. The resulting cell population was cocultured with EBV prepared from tissue culture supernatants of marmoset cell line B95-8. Identical immunoglobulin gene rearrangements of tumor cells and EBV-transformed cells were the criteria used to determine that the transformed cells were of tumor origin. DNA was prepared from both biopsy tissue and EBV cell lines and digested with restriction endonucleases, and Southern blots were prepared by standard methods. B-cells isolated from biopsies of four low-grade B-cell lymphomas of follicular, small cleaved cell type and one of follicular, mixed cell type were transformed by EBV into rapidly growing in vitro tissue culture lines. Two of the five transformed cell lines had immunoglobulin heavy chain and light chain gene rearrangements which were present in cells from the original tumor biopsy, indicating that these EBV-transformed cell lines are of tumor origin.     

Epstein-Barr virus strain-specific differences in transformed cell lines demonstrated in growth characteristics, induction of viral antigens and ADCC susceptibility

Paired lymphoid cell lines were established by transformation with both B95-8 (B) and QIMR-WIL (Q) EBV strains, of cells either from cord blood or from peripheral blood or spleens of patients with leukemias or lymphomas. The morphologies of the transformed cell clumps differed consistently between B-transformed lines (B-lines) and Q-transformed lines (Q-lines) even after 1 year in culture. When the B- and Q-lines were compared for superinfection with P3HR-1 EBV, B-lines had a higher frequency of EA induction than did the Q-lines. The shape of dose-response curves for superinfection indicated that a much higher multiplicity of infection by P3HR-1 EBV was required for EA expression in Q-lines than in B-lines. This difference in superinfection was independent of the EBV receptor concentration on the two types of lines and reflected apparent control of the level of EA induction by the resident EBV genome of the transformed line. Transforming EBV could be rescued by P3HR-1 EBV superinfection of both B- and Q-lines originating from cells of patients with Hodgkin's disease and hairy cell leukemia but not from cord blood. The cord blood lines transformed with virus produced spontaneously from these B- or Q-lines, showed that the cell lines contained antigen-determining information of the resident genome in the original B- or Q-lines, respectively. Superinfected B-lines were more susceptible to ADCC with anti-EBV-positive sera than were superinfected Q-lines. These experiments demonstrate that distinct biology. differences exist in paired cell lines transformed by different EBV strains.     

Restrictions upon Epstein-Barr virus infection of the leukemic cell are demonstrated in patients with hairy cell leukemia

We tested the hypothesis that Epstein-Barr virus (EBV) might actually infect leukemic hairy cells in vivo by examining those cells for the EBV-receptor, EBV nuclear antigen (EBNA) and membrane antigen (MA), for spontaneous transformation and rescue of infectious virus and for presence of EBV genome. EBV-receptors were found on subpopulations of leukemic cells from each of 7 patients with hairy cell leukemia (HCL) tested. MA was present on low numbers (1-5 per cent) of fresh leukemic cells of 7 patients and in some instances occurred with a greater frequency after 3 to 5 days in culture, with or without 12-O-tetradecanoylphorbol-13-acetate. In 11 fresh leukemic cell preparations from 8 HCL patients, no EBNA was demonstrated. However, 2 samples after 4 days in culture expressed low frequencies of EBNA-positive cells. Spontaneous, EBV-positive cell lines were established with a high transformation efficiency from 3 HCL blood samples but not from 8 other specimens. Infectious EBV could be rescued from some hairy leukemic cell preparations by co-cultivation with cord blood lymphocytes. These results demonstrated that leukemic cell populations harbored infectious EBV, that the leukemic cells expressed virus receptors and suggested that a small subpopulation of leukemic cells might become infected in vivo at least transiently and possibly transformed in vitro by EBV. To test for the extent of occult in vivo infection of leukemic cells with EBV, Southern type hybridization studies were performed with a probe for EBV genome (Bam HI W). At a sensitivity level of 0.1 genome per cell, EBV genome was not detected in the leukemic cell populations of 7 patients. We conclude that host defence mechanisms protecting these individuals from EBV also prevent infections of the leukemic cell and/or most hairy leukemic cells are not suitable targets for both infection and transformation.     

Heterogeneity of Epstein-Barr virus derived from a nasopharyngeal carcinoma that has transforming and lytic properties

The biological activities of Epstein-Barr virus (EBV) from a human epithelial hybrid cell line derived from a nasopharyngeal carcinoma (NPC-KT) were studied. Low concentrations of virus from the NPC-KT cell line stimulated DNA synthesis of human cord blood lymphocytes (CBL) and induced CBL to form EBV nuclear antigen-positive continuous cell lines. The CBL exposed to higher doses of virus showed stimulated DNA synthesis 2 days after infection, followed by cell lysis. Virus from the NPC-KT cell line induced EBV-specific antigens in nonproducer Raji cells and reduced the colony-forming ability of the super-infected cells. The ratio of transforming activity to early antigen-inducing ability was not constant during cell passage. The data obtained suggest that NPC-KT clone 1 cells are producing virus with cytotoxic and transforming properties.     

In vitro transformation of hamster cells by herpes simplex virus type 2 from human prostatic cancer cells.

A cell-associated herpes simplex virus type 2 found in a human prostatic carcinoma induced in vitro transformation of hamster embryo cells. The transformed cells (YW-74) have been shown to be hamster cells by karyotype analysis. Their epithelial morphology and growth pattern, which are different from the parental cell, have remained stable through cell passages. The presence of herpesvirus antigens in the transformed cells was determined by specific immunofluorescence and colony inhibition tests. Immunofluorescence staining with specific anti-herpes simplex virus type 2 serum showed an intense and distinctive nuclear and perinuclear fluorescence in about 95% of the transformed cells. In addition, exposure of these transformed cells to herpes simplex virus type 2-sensitized lymphocytes resulted in inhibition of growth and colony formation, while no effect was seen with nonsensitized lymphocytes. Both observations are consistent with the involvement of herpesvirus type 2 in the transformation event. This virus, which does not produce a lytic infection and is not found either in extracellular spaces or supernatant fluid of the transformed cell cultures, is unique in the fact that it is cell associated, noncytopathogenic, and capable of transforming cells in vitro, and its antigens are clearly demonstrated in the transformed cells.     

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.     

Superinfection of epithelial hybrid cells (D98/HR-1, NPC-KT, and A2L/AH) with Epstein-Barr virus and the relationship to the C3d receptor

Three Epstein-Barr virus (EBV) genome-positive epithelial/hybrid cell lines (D98/HR-1, NPC-KT, and A2L/AH) were superinfected with EBV derived from P3HR-1 (HR-1), NPC-KT, and B95-8 cells. The HR-1 virus is lytic and induces early antigen in superinfected Raji cells; the virus is not capable of transforming B-lymphocytes. Virus preparations from NPC-KT cells have both transforming and early antigen-inducing properties, while B95-8 virus can only transform B-lymphocytes. It was possible to demonstrate EBV antigens after superinfection of D98/HR-1 cells with both HR-1 virus and NPC-EBV. The NPC-KT hybrid cells, which were originally prepared by fusing human adenoid epithelial cells (Ad-AH) and EBV genome-positive NPC explanted epithelial cells, were susceptible to superinfection with HR-1 virus but not to NPC-EBV. The A2L/AH hybrid cells, which were prepared by fusion between Ad-AH cells and lymphocytes transformed by B95-8 virus, could not be superinfected with any of the virus preparations. In order to further investigate the nature of the EBV receptor as it relates to other cell membrane components, we examined cell surface markers on Ad-AH, NPC-KT, A2L/AH, and D98/HR-1 cells using monoclonal antibodies and by rosette formation. We found that the NPC-KT, A2L/AH, and Ad-AH cell lines express the OKB2 antigen and that the common acute lymphoblastic leukemia antigen is expressed on the A2L/AH cells. We also found that NPC-KT parental cells and a clone of NPC-KT cells express erythrocyte antibody complement b and erythrocyte antibody complement d, as determined by rosette formation, but were negative for C3b and C3d when monoclonal antibodies against these two markers were used. The D98/HR-1 cells were also confirmed to be negative for C3b and C3d. The data suggest that the C3d receptor may be part of the EBV receptor but that the C3d receptor, by itself, is not the only receptor to which EBV can bind.     

Characteristics of cell lines derived from human leukocytes transformed by different strains of Epstein-Barr virus.

Variation of Epstein-Barr virus (EBV) in respect to its effect on the properties of transformed cells was probed. Human umbilical cord leukocytes from six different individuals were transformed in vitro by either B95-8 (B) or QIMR-WIL (Q) strains of EBV and subsequently 12 lymphoblastoid cell lines (six B-derived and six Q-derived lines) were established. The B lines and Q lines were different in the expression of EBV genome i.e. production of virus or viral antigens, and in other properties including growth pattern and immunoglobulin production. The most striking differences between the two groups lay in their capacity to produce infectious virus and in the shape of the cell-clumps. The results suggest that different strains of EBV may induce transformed cells with different characteristics.