In Vitro and In Vivo Studies with Epstein-Barr

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1 A n n a l s o f C l i n i c a l L a b o r a t o r y S c i e n c e, Vol. 3, No. 6 Copyright 1973, Institute for Clinical Science In Vitro and In Vivo Studies with Epstein-Barr Virus (EBV) A Review VIRGINIA C. DUNKEL, Ph.D. Immunochemistry Section, Biology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD ABSTRACT One of the most important findings in the area of oncogenic herpes viruses has been the demonstration of the Epstein-Barr virus (E B V ) in cells of cultures derived from Burkitt s lymphoma. In vitro studies indicated that, under the appropriate conditions, EBV could be cytopathogenic and result in cell death and, in this respect, act like other known herpesviruses. The more common response to the virus, however, appeared to be transformation and continuous growth of lymphoid cells. In vivo studies with non-human primates have resulted in no positive responses. Elucidation of the factors involved in determining whether or not the virus will produce a self-limiting lymphoproliferative disease or a neoplastic response requires further study. Introd u ction In 1956, Fawcett1 3 showed that virus particles similar to herpes simplex were present in certain tumor cells of the Lucke frog renal adenocarcinoma. The finding of such particles did not establish the etiological relationship between the tumor and the virus but did provide the first indication that a herpesvirus might have oncogenic potential. Since that time herpes viruses have been shown to be the etiologic agents of tumors in chickens, rabbits, 2 6 and monkeys; 3 3 to possibly be associated with tumors in sheep; 32 and most importantly, to be associated in some way with Burkitt s lymphoma, 21 nasopharyngeal carcinoma21 2 3,2 6 and cervical carcinoma1 in man (table I). H istorical B ackground The first of the human tumors to be associated with a herpesvirus was Burkitt s lymphoma. This tumor, occurring primarily in African children, was recognized and described as a clinical syndrome in the late 50 s by Burkitt. 3 By delineating the geographical distribution of the tumor, Burkitt also showed4 6 that its occurrence appeared to be related in some way to a mean minimum temperature above 60 F and an annual rainfall above 30 inches, i.e., tropical conditions. This information stimulated the possibility that an arthropod-vectored virus might be the etiologic agent for the lymphoma. Burkitt tumor biopsy material was, therefore, tested using classical virus isolation techniques8 and examined directly with the electron microscope. 12 All tests, however, failed to show that a virus was present in the tumor. It was not until cells from Burkitt s tumors had been grown in culture that Epstein et al9 observed particles with typical herpesvirus morphology. It had been assumed that growth of

2 V IRO LO G Y OF EBV-----A R E V IE W TABLE I H e r p e s V i r u s e s E t i o l o g i c A g e n t s o p T u m o r s Virus Host Disease Lucke virus Frog Kidney adenocarcinoma Marek s Disease Virus (MDV) Chicken Lymphoma Herpesvirus Saimiri Marmoset, owl, spider, and Lymphoma and leukemia (isolated from squirrel monkeys) Ringtail monkeys Herpesvirus Sylvilagus Cottontail rabbit Benign lymphoid hyperplasis and malignant lymphoma Herpesvirus Ateles Marmoset Lymphoma and leukemia (isolated from spider monkey) Owl monkey Lymphoreticular proliferation Ovine herpesvirus Sheep Pulmonary adenomatosis (Jaagsiekte) Epstein-Barr Virus (EBV) Man Burkitt lymphoma Nasopharyngeal carcinoma Infectious mononucleosis (generalized lymphoproliferative disorder) Herpesvirus Type 2 Man Cervical carcinoma tumor cells away from host defenses would allow replication of a virus. It was subsequently shown28 that, as far as neutralizing antibody is concerned, growth of viruscarrying cells continues in the presence of EB virus-neutralizing antibody without loss of viability of the cells or suppression of virus replication. After the detection of the virus by electron microscopy, a variety of preparations from the cell cultures were tested in both in vitro and in vivo systems. 11 Again, all attempts to show activity were negative. The virus appeared to be biologically inert and did not act like any of the known members of the herpesvirus group. The keystone to much of the subsequent work with Burkitt lymphoma was the demonstration by Levy and Henle, 3 1 using indirect immunofluorescence tests, that sera from all patients with Burkitt s lymphoma had high titers of antibody to the herpes virus, or Epstein-Barr virus (EBV ) as it is now called, found in cultured Burkitt lymphoma cells. Lower titers and fewer reactors were found among the controls. Henle and coworkers in a series of immunological studies determined that ( 1 ) antisera to known herpesviruses did not react in immunofluorescence tests with EBV-carrying cells; (2) certain EBV-positive human sera would not react with cells infected with known herpesviruses and (3) immunofluorescent positive sera could coat or agglutinate EB virus particles as demonstrated by electron microscopy. These findings in addition to subsequent immunological studies showed that EBV was a new and distinct herpesvirus. I n Vitro Studies Although the virus appeared to be biologically inert, Henle and co-workers22 were able to transmit it by cocultivation of normal human leukocytes with X-irradiated cultured Burkitt lymphoma cells. Viral antigen was detected in a small percentage of cells but, more importantly, they became blastoid and were capable of continuous growth. The first cell-free transmission of EBV was reported by Horoszewicz and Dunkel. 27 Using the non-producing human lymphoblastoid cell line and a multiplicity of 1, virus particles per cell, they were able to show that the events leading to the replication of EB virus were basically similar to those described for herpes simplex, but they occurred at a much slower rate. Virus particles attached rapidly to the surface of the cells and, after 8 to 1 2 hours, were observed to be in the

3 4 2 6 D U N KEL process of penetrating the cell membrane. The first signs of viral antigen synthesis were detected in the nuclei of infected cells between 14 and 18 hours. By 20 to 40 hours, virus particles in various stages of development were observed in the nuclei of infected cells. Envelopment of the virus occurred either during passage of the virus from the nucleus to the cytoplasm or by budding from the cell surface. Newly synthesized virus was released 40 hours postinfection and after this time enveloped, unenveloped and incomplete virus particles could be found. As with other herpesviruses, infection of the cells with this multiplicity of virus resulted in a steady decrease in the number of viable cells and death of the cultures by 120 hours. Under these conditions, EBV was cytopathogenic for the cells and, in this respect, acted like other herpesviruses. From other infectivity studies, it became apparent that EBV is cytotrophic for certain lymphoid cells. In addition to cells, other lymphoblastoid cells in culture could be infected. 2 8 In contrast to this, EB virus did not replicate in LM/DM cells, a lymphoblastoid line derived from a rhesus monkey with myelogenous leukemia. 6 With primary suspensions of cells from human, rhesus monkey and guinea pig embryo spleen and thymus, EBV was found to adsorb to spleen cells but not to thymus cells (table II). This was also found to occur with adult spleen and thymus cells from the same sources. 28 Although the virus adsorbed to the spleen cells, there was no evidence of replication during a 14 day period of observation. Transformation and continuous growth of lymphoid cells by EBV is probably the more common response with lower multiplicities of virus. A clear cut dose response to the virus has been demonstrated28 and there is correlation between virus concentration and a decline in the viability of infected cells. In addition, the virus carrier state can be established in human lymphoblastoid cells with low doses of virus. The initial cocultivation studies22 provided suggestive evidence of the growth stimulating effect of EBV. More definitive studies on the role of the virus in leukocyte transformation were provided by Gerber and coworkers. 15 They showed that infection of buffy-coat cells from a healthy adult who had no evidence of previous infection with EBV resulted in blastoid transformation of the lymphocytes and long-term growth. Cells from the same donor, when exposed to EB virus neutralized with positive serum, growth medium and culture fluid from a non-virus-producing cell line, failed to grow. Transformation of fetal human leukocytes with EBV was reported by Pope et al38 and Nilsson et al. 37 Other studies have shown37 that spontaneous transformation of leukocytes and the resulting establishment of lymphoblastoid cell lines occurred only with leukocytes from donors with serological evidence of previous EB virus infection. In addition, virus capsid antigen and other EBV associated antigens could be detected in these lymphoblastoid cells. This would seem to indicate that the virus exists in a latent state in man and is activated when the cells are placed in culture. Recently, Miller et al3 4 reported that peripheral leukocytes from the squirrel monkey and cotton-top marmoset also transformed following exposure to EBV. This is the first report of transformation of cells from New World monkeys by EBV. I n Vivo Studies Various studies have shown that the sera of certain non-human primates contain antibody to EBV and that lymphoblastoid cell lines established from the peripheral leukocytes of chimpanzees contain virus particles indistinguishable from the EB virus in human cells. This has suggested that non-human primates might be the animal of choice for in vivo studies with EBV. Newborn chimpanzees, rhesus and cynomolgus monkeys have been inoc-

4 V IRO LO G Y O F E B V -----A R E V IE W TABLE II I n f e c t i v i t y o f EBV f o b V a r i o u s C e l l s Established Cell Cultures Virus Attachment* Virus Replication** Human Lymphoblastoid + + NC RAJ1 + + LM/DM Rhesus Lymphoblastoid - - Primary Cell Cultures Human Embryo Spleen + Human Embryo Thymus Rhesus Monkey Spleen + Rhesus Monkey Thymus Guinea Pig Embryo Spleen + Guinea Pig Embryo Thymus * 37 C for 60 minutes. ** 14 day period of observation. ulated with fresh Burldtt lymphoma biopsy material, massive numbers of cells cultured from Burkitt tumors, and highly concentrated EB virus preparations. 30 To date, there have been no positive responses in these animals. Discussion Whether or not EB virus acts alone or in conjunction with a co-factor in the possible induction of Burkitt s lymphoma is still unresolved. The virus is intimately associated with lymphoid cells in culture and the viral genome has been detected by DNA hybridization in both tumor cells in culture17 and in cells from Burkitt tumor biopsy material. 18 The in vitro studies reveal that the virus can be responsible for both cytolysis and transformation. Elucidation of the factors involved in determining whether or not the virus will produce a self-limiting lymphoproliferative disease or a neoplastic response requires further study. References 1. A u r e l i a n, L.: Possible role of Herpesvirus hominis, type 2 in human cervical cancer. Fed. Proc. 32: , B i g g s, P. M., C h u r c h i l l, A. E., R o o t e s, D. G., a n d C h u b b, R. C. : The etiology of Marek s disease an oncogenic herpes-type virus. Perspect. Virol. 6: , BuKKrrr, D. P.: A sarcoma involving the jaws in African children. Brit. J. Surg. 46: , B u r k i t t, D. P. : A children s cancer dependent on climatic factors. Nature 194: , B u r k i t t, D. P.: Determining the climatic limitations of a children s cancer common in Africa. Brit. Med. J. 2: , D u n k e l, V. C. a n d M y e r s, S. L.: Continuous lymphoblastoid cell line from a rhesus monkey with myelogenous leukemia. J. Nat. Cancer Inst. 48: , D u n k e l, V. C., P r y, T. W., H e n l e, G., a n d H e n l e, W.: Immunofluorescence tests for antibodies to Epstein-Barr virus with sera of lower primates. J. Nat. Cancer Inst. 49: , E p s t e i n, M. A.: Virology and immunology of Epstein-Barr virus (EBV) in Burkitt s lymphoma a review. Oncogenesis and Herpesviruses. Biggs, P. M., de The, G., and Payne, L. N., eds. Lyon: IARC, pp , E p s t e i n, M. A., A c h o n g, B. G., a n d B a r r, Y. M.: Virus particles in cultured lymphoblasts from Burkitt s lymphoma. Lancet 1: , E p s t e i n, M. A. a n d B a r r, Y. M.: Cultivation in vitro of human lymphoblasts from Burkitt s malignant lymphoma. Lancet 1: , E p s t e i n, M. A., H e n l e, G., A c h o n g, B. G., a n d B a r r, Y. M.: Morphological and biological studies on a virus in cultured lymphoblasts from Burkitt s lymphoma. J. Exp. Med. 21: , E p s t e i n, M. A. a n d H e r d s o n, P. B.: Cellular degeneration associated with characteristic nuclear fine structural changes in the cells from two cases of Burkitt s malignant lymphoma syndrome. Brit. J. Cancer 17:56-58, F a w c e t t, D. W.: Electron microscope observation of intracellular virus-like particles associated with the cells of the Lucke renal adenocarcinoma. J. Biophys. Biochem. Cytol. 2: , G e r b e r, P. a n d B i r c h, S. M.: Complementfixing antibodies in sera of human and non

5 4 2 8 DÜNKEL human primates to viral antigens derived from Burkitt s lymphoma cells. Proc. Nat. Acad. Sci. USA 58: , G e r b e r, P., W h a n g - P e n g, J., a n d M o n r o e, J. H.: Transformation and chromosome changes induced by Epstein-Barr virus in normal human leukocyte cultures. Proc. Nat. Acad. Sci. USA 63: , G o l d m a n, M., L a n d o n, J. C., a n d R e i s h e r, J.: Fluorescent antibody and gel diffusion reactions of human and chimpanzee sera with cells cultured from Burkitt tumors and normal chimpanzee blood. Cancer Res. 28: , z u n H a u s e n, H. a n d S c h u l t e - H o l t h a u s e n, H.: Presence of EB virus nucleic acid homology in a virus-free line of Burkitt tumor cells. Nature 227: , z u r H a u s e n, H., S c h u l t e - H o l t i i a t j s e n, H., K l e i n, G., H e n l e, W., a n d H e n l e, G.: EBV DNA in biopsies of Burkitt tumors and anaplastic carcinomas of the nasopharynx. Nature 228: , H e n l e, G. a n d H e n l e, W.: Immunofluorescence in cells derived from Burkitt s lymphoma. J. Bact. 91: , H e n l e, G. a n d H e n l e, W.: Studies on cell lines derived from Burkitt s lymphoma. Trans. N. Y. Acad. Sci. 29:71-74, H e n l e, W. a n d H e n l e, G.: Evidence for a relation of Epstein-Barr virus to Burkitt s lymphoma and nasopharyngeal carcinoma. Comparative Leukemia Research. Dutcher, R. M., ed. Basel, Munich and Paris, Karger, pp , H e n l e, W., D i e h l, V., K o h n, G., z u r H a u s e n, H., a n d H e n l e, G.: Herpes-type virus and chromosome marker in normal leukocytes after growth with irradiated Burkitt cells. Science 157: , H e n l e, W., H e n l e, G., Ho, H. C., B u r t i n, P., C a c h i n, Y., C l i f f o r d, P., S c h y r y v e r, A., d e - T h e, G., D i e h l, V., a n d K l e i n, G.: Antibodies to Epstein-Barr virus in nasopharyngeal carcinoma, other head and neck neoplasms and control groups. J. Nat. Cancer Inst. 44: , H e n l e, W., H u m m e l e r, K., a n d H e n l e, G.: Antibody coating and agglutination of virus particles separated from the EB3 line of Burkitt lymphoma cells. J. Bact. 92: , H i n z e, H. C. a n d C h i p m a n, P. J.: Role of herpesviruses in malignant lymphoma in rabbits. Fed. Proc. 32: , It o, Y., T a k a h s h i, T., K a w a m u r a, A., Jr., a n d Tu, S. M.: High anti-eb virus titer in sera of patients with nasopharyngeal carcinoma: a small-scale seroepidemiological study. Gann 60: , H o r o s z e w i c z, J. S., D u n k e l, V. C., a n d G r a c e, J. T., Jr.: Biological properties of a herpes-like virus (HLV) from Burkitt lymphoma cell line. Fed. Proc. 27:262, H o r o s z e w i c z, J. S., D u n k e l, V. C., A v i l a, L., a n d G r a c e, J. T., Jr.: EB virus infection and propagation in human hematopoietic cells. Biblio. Haemal. 36: , L a n d o n, J. C. a n d M a l a n, L.: Seroepidemiologic studies of Epstein-Barr virus antibody in monkeys. J. Nat. Cancer Inst. 46: , L a n d o n, J.: Personal communication. 31. L e v y, J. A. a n d H e n l e, G.: Indirect immunofluorescence tests with sera from African children and cultured Burkitt lymphoma cells. J. Bact. 92: , M a c k a y, J. M. K. a n d N e s k e t, D. I.: Pathogenicity tests in lambs with an ovine herpesvirus. Oncogenesis and Herpesviruses. Biggs, P. M., de The, G., and Payne, L. N., eds. Lyon: IARC pp , M e l e n d e z, L. V., H u n t, R. D., D a n i e l, M. D., F r a s e r, C. E. O., B a r a h o n a, H. H., K i n g, N. W., a n d G a r c i a, F. G.: Herpesvirus saimiri and ateles their role in malignant lymphomas of monkeys. Fed. Proc. 32: , M i l l e r, G., S h o p e, T., L e s c o, H., S t i t t, D., a n d L y s m a n, M. : Epstein-Barr Virus: Transformation. Cytopathic changes, and viral antigens in squirrel monkey and marmoset leukocytes. Proc. Nat. Acad. Sci. USA 69: , M o r g a n, C., R o s e, H. M., a n d M e d n i s, B.: Electron microscopy of herpes simplex virus. I. Entry. J. Virol. 2: , Nn, S., M o r g a n, C., a n d R o s e, H. M. : Electron microscopy of herpes simplex virus. II. Sequence of development. J. Virol. 2: , N e l s s o n, K., K l e i n, G., H e n l e, G., a n d H e n l e, W.: The role of EBV in the establishment of lymphoblastoid cell lines from adult and foetal lymphoid tissue. Oncogenesis and Herpesvirus. Biggs, P. M., de The, G. and Payne, L. N., eds. Lyon: IARC, pp , P o p e, J. H., H o r n e, M. K., a n d S c o t t, W.: Transformation of foetal human leukocytes in vitro by filtrates of a human leukemic cell line containing herpes-like virus. Int. J. Cancer 3: , P u l v e r t a f t, R. J. V.: Cytology of Burkitt s tumor (African lymphoma). Lancet 2: , P u r c h a s e, H. G.: Role of herpesviruses in Marek s disease, a malignant lymphoma of chickens. Fed. Proc. 32: , W i t t e r, R. L., B u r g o y n e, G. H., a n d S o l o m o n, J. J.: Evidence for a herpesvirus as an etiologic agent of Marek s disease. Aircin Dis. 23: , 1969.