P —. - . “U «\u .23 ”L a; .u... '- nnrt. . u ‘96» .tx u~t II v.9 O . c a .‘ol’. «.0 all .UJ 1.1L LIBRARY I 3'; _ v 800K BINDERY INC. LIBM ‘‘‘‘‘‘‘‘‘ S x "If :POI'T. HIGH“! ABSTRACT THE BIOLOGICAL CHARACTERIZATION OF CELLS OF A TRANSPLANTABLE TUMOR (JMV) DERIVED FROM A MAREK'S DISEASE LYMPHOMA By Elizabeth Ann Stephens This work was designed to resolve an important and contro- versial problem associated with JMV tumor cells, namely, the nature of their association with Marek's disease herpesvirus and whether the lesions associated with JMV were virus—induced or the result of trans- planted cells. JMV tumor cells were inoculated into day-old chicks in order to study the mean days to death, the number of chick lethal doses ne- cessary to kill 50 percent of the chicks and to observe enlarged leukotic livers and spleens. JMV tumor cells were also examined by co-cultivation in tissue culture, inoculation of susceptible chicks and inoculation of embryos for the presence of virus or viral antigens. JMV tumor cells were found to be totally free of replicating Marek's disease virus by the methods employed. Cultivation of JMV affected kidney cells and co-cultivation of JMV spleen tumor cells on duck and chick embryo fibroblasts failed to reveal any cytopathologic changes in the cultured monolayers. Chicks inoculated with JMV cell culture material failed to show any signs of disease. Four-day-old Elizabeth Ann Stephens embryos inoculated by the yolk-sac route were negative for virus lesions on the chorioallantoic membrane. The absence of Marek's di— sease herpesvirus particles in JMV tumor cells was confirmed by electron microscopic examination. Cell culture, indirect immunofluorescence and serum neutrali- zation assays for viruses other than herpesvirus were also performed. There appeared to be no evidence for reticuloendotheliosis virus or lymphoid leukosis viruses of subgroups A through D. An attempt to identify unknown replicating agents was unsuccessful by the methods used. JMV hyperimmune serum was assayed for Marek's disease virus- related antibodies by the indirect immunofluorescence, agar-gel pre- cipitin and serum neutralization techniques. There was no evidence of reactivity with cells producing Marek's disease virus or viral antigens. The existence of tumor antibodies was demonstrated on viable cell suspensions of the MSB-l Marek's disease lymphoblastoid cell line by the indirect membrane immunofluorescence technique. JMV Marek's disease tumor cells were tested by indirect membrane immunofluorescence with virus specific antiserum for the presence of Marek's disease virus-specific intra-cellular and membrane antigens and with JMV anti-tumor serum for the presence of tumor spe- cific antigens. There was no evidence of viral antigens. A distinct annular fluorescence was observed on the surface of the tumor cells stained with the anti-tumor serum. The tumor cell surface antigen was designated as a Marek's disease tumor-associated surface antigen (MATSA) . Elizabeth Ann Stephens JMV tumor cells were studied by the membrane immunofluores- cence technique to determine whether the proliferating lymphoid cells were of B or T cell origin. Since the tumor cells appeared to carry both antigenic determinants, this characteristic of JMV cells was not conclusive. In an attempt to determine the transplantable nature of JMV tumor cells, the B blood group alloantigenic markers on the lympho- blastoid cells were examined. The tumor cells appeared to be of non- host origin and to carry an alloantigenic marker identical with or at least clearly related to 82]. The presence of Marek's disease genome sequences was detected in the JMV tumor cells by nucleic acid hybridization technique. It was not possible to determine whether the cells contained a complete Marek's disease virus genome. THE BIOLOGICAL CHARACTERIZATION OF CELLS OF A TRANSPLANTABLE TUMOR (JMV) DERIVED FROM A MAREK'S DISEASE LYMPHOMA By Elizabeth Ann Stephens A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Poultry Science 1976 To my husband Sam ACKNOWLEDGMENTS I wish to express my sincere thanks to Dr. Richard L. Witter, Associate Professor of Pathology and Director of the United States Department of Agriculture Regional Poultry Research Laboratory and to Dr. Theo H. Coleman, Professor of Poultry Science, for their encourage- ment and guidance throughout the research and preparation of this thesis. I also wish to thank Dr. Ben R. Burmester, former Director of the Regional Poultry Research Laboratory, who encouraged me to con- tinue my education. To the other staff members of the Regional Poultry Research Laboratory: Dr. William Okazaki, Dr. Keyvan Nazerian, Dr. Lucy F. Lee and Dr. Jagdev M. Sharma, I wish to express my appreciation for their vital contributions necessary for the completion of this work. In addition I wish to acknowledge Dr. B. w. Longenecker, Department of Immunology, University of Alberta, Edmonton, Alberta, Canada, for his assistance in designing the B blood group experiments, and Dr. J. Lloyd Spencer, Animal Pathology Division, Health of Animals Branch, Agriculture Canada, Animal Disease Research Institute, Hull, Quebec, Canada, for his helpful criticism of the JMV work. I wish to thank Mr. Howard Stone and all the members of the genetics department at the Regional Poultry Research Laboratory for the extra effort extended in supplying me with the necessary experi- mental lines of chickens. I would also like to thank Miss Linda Offenbecker and Miss Francie Gulvas for their excellent technical assistance. For their co-operation and willingness to assist me with the handling of my experimental birds, I wish to express my thanks to our animal caretakers, Mr. Robert Lowe and Mr. Chester Holton. It is with the deepest gratitude that I acknowledge the love and understanding of my family. Through the efforts and encouragement of my husband Sam, our children: John, Jane, Daniel, Linda and Gary, and my parents, I was able to complete this work. iv TABLE OF CONTENTS Page LIST OF TABLES ........................ vi LIST OF FIGURES ....................... viii INTRODUCTION ...................... . . . 1 LITERATURE REVIEW ...................... 4 ETIOLOGY ............... . .......... 5 EPIDEMIOLOGY . . . . ................... . 7 METHODS FOR VIRUS ASSAY .................. 9 MAREK'S DISEASE TUMOR CELLS ................ ll JMV LYMPHOBLASTOID TUMOR CELLS ............... 2l MATERIALS AND METHODS .................... 27 EXPERIMENTAL DESIGN . . . .................. 36 RESULTS ...... . .................... 40 DISCUSSION .......................... 75 SUMMARY ........................... 87 RECOMMENDATIONS ....................... 88 LITERATURE CITED ........ . . ........... 89 LIST OF TABLES TABLES l. Titration of the JMV-S stock liver homogenate stored at -196°C . . . . ...... . ........ 2. Titration of the JMV-x462 stock liver homogenate stored at -196°C ................... 3. Titration of the JMV-x55 stock liver homogenate stored at -196°C ..... . . . . . ......... 4. Protection test with JMV—S hyperimmune sera . . . . . . . 5. Protection test with JMV—x462 hyperimmune sera . . . . . 6. Attempts to rescue an MD-like herpesvirus from JMV tumor cells . ....... . . . . ........ 7. Demonstration of the inability of JMV spleen cells to induce MD herpes-like lesions on the choriollantoic membrane of line 63 (MD antibody negative) embryos . . 8. Attempts to demonstrate MDV herpesvirus and antibody in chicks inoculated with JMV tumor cells . . ..... 9. Cell-associated nature of JMV spleen tumor cells and feather follicle extract . . . ............ l0. Summary of tests for detection of viral agents other than herpesvirus in JMV tumor cells .......... ll. Analysis of hyperimmune sera to detect antibodies induced in response to antigens associated with JMV tumor cells . . . . . . . .......... l2. Analysis of JMV cells for membrane antigen and tumor associated membrane antigen . . . . . . . . . . l3. Analysis of JMV lymphoblasts for B and T cell surface antigenic markers ............... l4. Attempts to demonstrate the transplantable nature of JMV-S tumor cells by differences in B blood alloantigenic markers on the surface of JMV _ tumor cells . . ............ . ....... Page 46 47 48 51 55 56 57 59 60 62 63 64 67 69 TABLES Page 15. Detection of MDV genome in JMV tumor cells by hybridization of JMV-DNA with H-labelled MDV'CRNA o o o o o o o o o o o o o o o o o o o o o 70 16. Description of JMV tumor cells analyzed by the electron microscope . . ..... . ....... . 73 vii FIGURE 1. LIST OF FIGURES Comparative titrations of three JMV sources demonstrating dose dependent latency effect ..... Enlarged tumorous spleen of a line Igchicken 8 days after inoculation with JMV Smear preparation of JMV-S spleen tumor cell suspension showing typical lymphoblastic cells . . . . Liver of chicken inoculated with JMV-S: a focus of lymphoblastic tumor cells is shown . . ...... Liver of chicken inoculated with JMV-S: a focus of lymphoblastic tumor cells infiltrated with small mononuclear cells is shown . . . . . . . . . . . Virus-induced lesions on the CAM of line 63 embryo inoculated at 4-days of age via the yolk-sak with Marek's disease virus .............. Three fields illustrating Marek's disease tumor- associated surface antigen (MATSA) on lymphoblasts from spleen of a chicken inoculated with JMV—S and stained with anti-JMV serum by the indirect membrane immunofluorescence technique ........ Electron micrograph of a JMV lymphoblast from the tumorous spleen of a chicken inoculated with JMV-S . . . . . ................... viii Page 41 43 43 45 45 53 66 75 INTRODUCTION Marek's disease (MD) is an infectious, neoplastic disease characterized by infiltration and proliferation of lymphoid cells in nerves and visceral organs, resulting in paralysis and death (Biggs and Payne, 1967). The etiologic agent is a highly cell-associated herpesvirus (Churchill and Biggs, 1967; Solomon gt_al,, 1968; Nazerian gngfl,, 1968), which can be transmitted to susceptible chickens either by direct contact with diseased chickens or by inoculation of infected material (Sevoian gt_al,, 1962; Biggs and Payne, 1963). Virus infection of cells may result in productive infection with antigen and virus pro— duction or abortive infection with the production of antigens and in- complete virus particles, both forms of which result in cell death; or non-productive infection, as exemplified by most lymphoid tumors or lymphob1astoid cells, which may result in transformation and prolifera- tion of the cells without expression of virus or viral antigens (Nazerian et_al,, 1975). MD lymphoma cells are non—productively infected and contain little or no infectious virus or viral antigens (Calnek §t_al,, 1970; Nazerian, 1971). MD viral genome has been demonstrated in lymphoid tumor cells and lymphoblastoid cell lines (Nazerian §t_al,, 1973; Nazerian and Lee, 1974; Lee §t_gl,, 1975). Tumor cells can transmit virus infection and reproduce the disease when inoculated into suscep- tible chickens (Calnek et al., 1970). Rapid serial passage of MD lymphoma cells in chickens has resulted in a reduced latent period and increased mortality (Jakowski §t_al., 1974; Sevoian §t_al,, 1964; R. Larose, personal communication; S. Schmittle, personal communication). One such virulent form, designated JMV, resulted from the serial pas- sage of lymphoma cells of the JM isolate of MD (Sevoian, 1964). The terms JM and JMV appear to be the source of some con- fusion. A “1ymphomatosis” (MD) isolate was given the code name JM by Sevoian gt_al, (1962). The designation, JMV, is not an acronym but is a code for a highly lethal tumor cell preparation which resulted from serial passage of cell suspensions from an ovarian lymphoma induced by the JM isolate (Sevoian gt_al,, 1964). JMV tumor cells may be considered a class of MD tumor cells because of their origin and because of their immunologic relationship to MD. Vaccination with herpesvirus of turkey (HVT) (Mason and Jensen, 1971; Sevoian and Weston, 1972) or attenuated MD virus (Spencer gt_al,, 1973) has been shown to protect chicks against JMV tumors. Likewise, JMV has been shown to protect against MD (Hong and Sevoian, 1974; Shieh and Sevoian, 1974; Kenyon gt_al., 1969). The controversial issue with JMV cells has been whether the tumor cells are host lymphocytes transformed by exogenous infection with a virulent herpesvirus, or whether they are of donor origin, i.e., a transplant. The findings of a herpesvirus (Yoon and Kenyon, 1975), cell-free virus (Hong and Sevoian, 1974; Shieh and Sevoian, 1974) and virus specific antibodies (Hamdy and Sevoian, 1973; Hong and Sevoian, 1974; Shieh and Sevoian, 1974, 1975) associated with JMV tumor cells are in contrast to reports that transmission of the tumor is dependent on viable intact cells (Jones et_al., 1969; Sevoian, 1967; Spencer and Calnek, 1967). JMV tumor cells have been widely used to challenge the im- munity of chickens vaccinated against MD (Mason and Jensen, 1971; Sevoian and Weston, 1972) and as a rapid test for genetic resistance to MD (Gavora gt_al., 1974); and yet, very little has been done to identify the nature of JMV tumor cells. In order to use these tumor cells as a tool in the study of tumor immunology and Marek's disease immunity it became important to characterize JMV tumor cells on the basis of their association with MDV, as well as other characteristics of biological importance. The objectives of this research were to resolve the nature of the association of Marek's disease herpesvirus with JMV tumor cells, to establish whether JMV tumors were virus-induced or transplants and to study certain biological characteristics of JMV tumor cells. LITERATURE REVIEW Marek's disease (MD) is a contagious neoplastic disease of domestic chickens caused by a herpesvirus (Churchill and Biggs, 1967; Solomon gt_al,, 1968; Nazerian gt_al,, 1968) and characterized by lymphoid infiltration in the peripheral nerves and development of lymphoid tumors in the visceral organs. Almost any organ or tissue can be involved, but the disease has a predilection for nervous tissue. Marek's disease was first described in Hungary by Joseph Marek (1907) as a polyneuritis, but has now been recognized throughout the world. During the first two decades of the century, both in Europe and the United States, the disease appeared to be confined to the nervous system (Kaupp, 1921; van der Walle and Winkler-Junius, 1924). Pappenheimer et_al, (1929) observed that about 10 percent of chickens with M0 had lymphoid tumors principally of the ovary. The disease became a serious problem to the commercial poultry industry in Europe and the United States during the late 1920's and throughout the 1930's. The increase in the incidence of the disease appeared to be associated with the increase in commercial poultry farms. A highly pathogenic form of the disease appeared around 1950 in the United States (Benton and Cover, 1957) and was characterized by an early on- set, high mortality and high incidence of visceral tumors. This acute form was epizootic in nature and appeared to be concentrated in geographic areas where poultry production was prevalent. Until the advent of vaccination (Churchill gt;313, 1969b; Okazaki gt_al,, 1970) the acute form was a serious cause of mortality to poultry in many countries. Clinically the earlier or mild form of the disease is char- acterized by progressive paralysis of one or more of the extremities. Morbidity and mortality are usually low. Lesions are predominantly in the nerves which become enlarged due to infiltration with lymphocytes of different sizes and plasma cells. In the acute form of the disease, which appeared around 1950, tumors are more prevalent and morbidity and mortality may be as high as 30 percent or more (Biggs, 1973). The lymphoma appears to be multifocal and affects the gonad, kidney, liver, spleen, lung, muscle, skin, bursa of Fabricius or thymus. Tumors are the result of infiltration of lymphocytes, plasma cells and large lymphoblastic cells referred to as"Marek's disease cells" (Biggs, and Payne, 1967). ETIOLOGY The selection of several lines of chickens with genetic resistance to MD (Hutt and Cole, 1948) and adequate isolation housing Sevoian.§t_al., 1962; Biggs and Payne, 1963) were of great importance in the study of Marek's disease. Sevoian gt_al, (1962) and Biggs and Payne (1963) successfully transmitted the disease serially from bird to bird with intact blood or tumor cells. The spread of the disease to uninfected chickens in contact with infected chickens conclusively established the contagious nature of MD (Biggs and Payne, 1963, 1967; Sevoian §t_al., 1963; Colwell and Schmittle, 1968). The discovery in cell culture of a highly cell-associated herpesvirus associated with M0 was a major breakthrough (Churchill and Biggs, 1967; Solompn §t_al., 1968; Nazerian §t_al., 1968). Additional evidence that a herpesvirus was the causative agent accumulated with investigations of natural and experimental cases of MD (Witter _e_t_a_1_., 1969a; Biggs §t_gl,, 1968; Ahmed and Schidlovsky, 1968; Bankowski gngfl,, 1969; Calnek and Madin, 1969; Eidson gt_al,, 1969). The feather follicle epithelium was found to be the maturation site of cell-free infectious virus. The transmission of the disease with cell- free feather follicle extracts provided further proof that MD herpes- virus was the etiological agent of MD. Enveloped virus was rarely seen by electron microscopy in tumors, nerves and other tissues of infected chickens (Schidlovsky §t_al., 1969; Calnek §t_gl,, 1970; Ubertini and Calnek, 1970) but was common in the nuclei and cytoplasmic inclusions of the feather follicle epithelium (Calnek gt_al,, 1970; Nazerian and Witter, 1970). The highly cell-associated nature of the virus both in cell culture and most tissues characterized the virus as a type B herpesvirus (Wilner, 1969; Churchill and Biggs, 1967; Nazerian 3311., 1968; Churchill, 1968; Lee $5311., 1969). The several isolates of MD, which have been obtained, varied considerably. Virulent or pathogenic isolates included JM (Sevoian §t_al., 1962), HPRS-16 (Biggs §t_al,, 1965), GA (Eidson and Schmittle, 1968a) and Cal-l (Bankowski §t_gl,, 1969). The apathogenic or mildly pathogenic isolates included HPRS-Bl4 (Biggs and Payne, 1963), WSU-GF (Kenzy et al., 1964), Conn-A (Chomiak et al., 1967) and HPRS-27 (Biggs and Milne, 1972). EPIDEMIOLOGY Chickens appear to be the principle animate reservoir of MD infection (Witter, 1972). Environmental factors such as exposure to contaminated air for less than 30 minutes have been sufficient to induce infection in newly hatched chicks (Chen and Witter, unpublished data). Infection has been accomplished by contaminated litter and droppings (Witter g3;§j,, 1968a), dust and dander (Beasley 23411., 1970) and feathers (Calnek et_al,, 1970). Vertical or egg transmission was proposed as a means of transmission (Sevoian, 1968); however, on the basis of ex- tensive further evidence it may be concluded that egg transmission does not occur or occurs only very rarely (Rispens gt_al., 1969; Solomon gt_al,, 1970). The portal through which the infectious material enters the host and initiates infection is not known, but the respiratory tract is a likely route (Calnek and Hitchner, 1969; Eidson and Schmittle, 1968b; Calnek gt_al., 1970). The portal of exit is the feather follicle epithelium (Calnek and Hitchner, 1969; Calnek gt_gl,, 1970; Nazerian and Witter, 1970). Kenzy and Biggs (1967) reported shedding of virus by infected chickens into the environment one or two weeks after in— fection. The disease spreads rapidly and the incidence of infection in a flock usually reaches 100 percent (Witter et al., 1970a); although, clinical signs do not necessarily follow infection (Chubb and Churchill, 1968; Witter gt_al., 1969a). Factors influencing the progression to clinical disease are the virulence of the infecting virus, dosage and route of exposure. Virulent isolates of MD cause a higher incidence of disease than the more apathogenic isolates (Biggs gg;j§L., 1968; Purchase and Biggs, 1967). Experimentally, the incidence of the clinical disease is dose-dependent (Calnek and Witter, 1972). The route of inoculation, such as intra-abdominal, results in higher incidence and shorter latent period as opposed to the nasal or oral routes or contact exposure (Witter and Burmester, 1967). Factors influencing the development and control of the disease include breeding and husbandry procedures. Genetic selection for re- sistance to MD can be accomplished in a few generations (Biggs gt_al.. 1968b; Cole, 1968). Resistance appears to be dominant and has no cor- relation with other production traits or resistance to lymphoid leuko- sis (Cole, 1970). Control by breeding for genetic resistance can be beneficial but costly and time consuming for the commercial industry. Conventional methods of sanitation and isolation used in prevention of infectious disease are of little use in the control of MD. Isolation type housing with filtered air positive pressure (FAPP) systems and sanitation procedures have shown success in reducing the incidence of MD under experimental conditions (Drury et_al,, 1969). Three types of vaccine have been developed for use in con- trolling MD. These are an attenuated Marek's disease virus (MDV) (Churchill §t_al,, 1969; Churchill gt_al., 1969; Biggs gt_al,, 1970), an apathogenic MDV (Rispens et al., 1972) and an apathogenic herpesvirus of turkeys (HVT) (Okazaki et_al., 1970; Purchase §t_al., 1971; Purchase gfl;j{L., 1972). MD appears to be the only naturally occurring lymphoma induced by a herpesvirus which is successfully controlled by vaccination. Since 1972 HVT has been in use as a commercial vaccine. Prior to its use the level of condemnations of broilers was almost 1.6 percent in the United States in 1970, the last year of statistics before the extensive use of the vaccine. By 1974 the level of condemnation was 0.29 percent, a reduction of 1.12 percent based on the assumption that without vac- cination losses would be at least 90 percent of 1970 level or 1.41 per- cent. Annual monetary losses due to MD have been reduced from $223 million to about $43 million (Agricultural Research Service, United States Department of Agriculture, 1975). METHODS FOR VIRUS ASSAY MDV can be propagated and assayed by several methods. The most sensitive method for detecting virus in chicken tissues is the inoculation of one-day-old susceptible chicks; this has been reported to be 10 to more than 1000-fold more sensitive than cell culture sys— tems (Witter gt_al., 1969b). Chicks are observed for any of several parameters of infection (viremia, antibodies, antigens, lesions) be- tween 2 and 10 weeks; the usual period being from 4 to 6 weeks (Witter gt_al., 1969b). Virus can be detected first in the lung and lymphoid tissues as early as 1 day after inoculation and, in most tissues, can reach a maximum titer of about 100 plaque forming units (PFU)/106 cells after about the 10th day and may persist at a lower level of 1-10 PFU/lO6 cells for the life of the chicken (Purchase, 1974). Fully 10 infectious enveloped virus can be found in the feather follicle epi- thelium around the 2nd to 4th week in titers of about 103PFU/106 cells (Calnek §t_al,, 1970). Assay of MDV has been done in duck embryo fibroblasts (DEF) (Solomon et_al., 1968; Witter gt_al,, l969a,b) or chick kidney cell cultures (Churchill and Biggs, 1967; Witter gt_al., 1969 a,b). Primary isolation is usually made from white blood cells, tumors or kidney cells (Solomon gt_al., 1971). The number of cells required to initiate a micro-plaque may vary considerable. Churchill and Biggs (1967) re- 2 5 ported that 10 to 10 tumor cells were needed to initiate a micro- plaque in tissue culture. Witter et al. (1969b) reported that at least 4 to 105 10 white blood cells were required to produce one plaque in tissue culture. The limited sensitivity of cell culture assay was presumably only detecting birds with high-titer viremias (Witter gt_al,, 1971). Therefore, the number of cells necessary to initiate a micro- plaque would undoubtedly depend on the level of viremia in the donor chicken. Inoculation via the yolk-sac of 4-day-old chick embryos with MDV chick kidney cell culture material or blood cells from infected chickens results in pock formation on the chorioallantoic membrane (CAM) (von BUlow, 1968 and 1971). The sensitivity of the assay has been shown to be similar to assay in cell culture, particularly when embryos from antibody-free dams are used (Biggs and Milne, 1971). The oncogenic herpesvirus of chickens is important as a model because MD tumors resemble certain tumors occurring in humans. Under- standing of the animal model may serve to help in further understanding of the etiology, epidemiology, control and recovery from human cancer. 11 MAREK'S DISEASE TUMOR CELLS There are several classes of tumor cells associated with M0 that are available for study. Tumor cells may be obtained directly from lymphomas induced by inoculation of chickens with M0 virus. In- fection may induce the development of progressive lymphoma with death or, in some instances, regression; however, it must be noted that only a low percentage of infected chickens develop lymphomas (Nazerian, 1973a). Lymphoma cells induced by inoculation of chickens with M0 virus are of host origin and have been shown to carry thymus (T) cell antigenic determinants on their surface (Payne and Roszkowski, 1972; .Hudson and Payne, 1973; Rouse §t_al,, 1973). The cells associated with the lymphoma are a mixture of different lymphocytes, blast cells and plasma cells (Biggs and Payne, 1967). The proportion of cells in MD lymphomas demonstrating a tumor-specific surface antigen (MATSA) ranged from 2.3 to 27.3 percent indicating that not all cells in the tumor are transformed (Witter gt_al,, 1975). Another class of tumor cells is represented by the lympho- b1astoid cell lines established in continuous culture from lymphomas of chickens inoculated with MDV. Two cell lines established by Akiyama gngfl, (1973a) and Akiyama and Kato (1974) from chickens inoculated with BC-l isolate of M0 were derived from an ovarian and splenic lymphoma and were designated as MOB-l and MSB-l, respectively. Two additional cell lines were established by Powell §t_al, (1974) from tumors of chickens inoculated with HPRS-16 isolate and designated as HPRS-l and 2. The MSB-l cell line was studied extensively (Akiyama and Kato, 1974; Nazerian and Witter, 1975) and found to be a homogeneous 12 population of transformed lymphoblastoid cells. The cell line appeared to express a high frequency (nearly 100 percent) of MD tumor-specific surface antigen (MATSA) (Witter gt_al., 1975) and T cell surface anti- genic markers (Nazerian and Sharma, 1975). ‘ A third class of tumor cells was produced as a result of a transplantable lymphoma from chickens inoculated with MDV (Jakowski gt_al,, 1974). The virus-induced tumor cells were passaged by inocu- lation into the pectoralis muscle resulting in tumors at the site of inoculation as early as 5 days from donor cell proliferation and virus- induced lymphomas of the visceral organs, which appeared after 21 days from host cell proliferation. The transplanted tumor line was shown to be a mixture of medium to large undifferentiated lymphoblastic cells with areas of necrosis. It has not been shown to date whether these tumors express MD tumor-specific surface antigen (MATSA) or T cell surface antigen. JMV tumor cells, developed by a similar rapid passage pro- cedure, have been classed as a transplant of donor origin (Spencer gt_al,, 1976). Cell suspensions produced from leukotic livers and spleens contained a predominance of lymphoblastoid cells (Spencer and Calnek, 1967); however, their association with M0 was not clearly es- tablished. Differences between virus-induced cells and tumor cells. MD lymphoma 'cells contain little or no infectious virus or viral antigens (Calnek and Hitchner, 1969; Spencer and Calnek, 1970; Schidlovsky et_al,, 1969; Nazerian and Witter, 1970; Purchase, 1970). Neither naked or enveloped virions could be demonstrated by electron microscopy; however, 13 infectivity of these cells could be demonstrated by jn_xitrg_cultivation or by inducing the disease when inoculated into susceptible chickens (Calnek gt_gl., 1970; Nazerian and Witter, 1970). The only cells known to produce complete enveloped virions are the cells of the feather follicle epithelium (Calnek gt_al,, 1970; Nazerian and Witter, 1970; Purchase, 1970). Virus particles are found in the nucleus and cytoplasm of these cells (Nazerian and Witter, 1970) and cell-free infectious virus is released from this site (Calnek gt_al,, 1970). Purchase (1974), Nazerian §t_al. (1975) and Nazerian (1976) described two different forms of infection caused by MDV. In the first form, i.e., productive infection, the cell produces viral deoxyribo- nucleic acid (DNA), virus-induced enzymes and antigens and Complete virus particles which acquire an envelope while maturing in the de- generating, stratified, squamous cells of the feather follicle epithelium. Complete enveloped virions are formed both in the nucleus and cytoplasmic inclusions. Another phase of the productive infection, i.e., abortive infection, has been found in the bursa of Fabricius, thymus, kidney and other organs without tumors in which nuclear and cytoplasmic antigens may be detected by immunofluorescence in cells undergoing degenerative changes. Naked viral particles are commonly found in these cells but enveloped virions are rare. The infectivity of these cells is cell- associated and is destroyed by killing the cells (Calnek gt_al., 1970; Nazerian and Witter, 1970; Purchase, 1970). Cells which demonstrate abortive infection contain viral genome and viral antigens but do not synthesize infectious virus (Purchase, 1974). Productive infection therefore, ultimately results in death of the cell. 14 A second form of infection, i.e., non-productive infection, is found in lymphoid cells of tumors and cell lines derived from tumors. Infectivity of these tumor cells is cell-associated. The virus DNA is present in multiple copies (Nazerian gt_al,, 1973) but is not expressed in the form of antigens or physical virious. This type of infection does not result in the death of the cell and thus provides a condition conducive to cell transformation and proliferation (Nazerian, 1976). For some reason however, in a low percentage (1-2 percent) of the lymphob1astoid cells the virus genome is spontaneously expressed and results in productive infection (Nazerian, 1976), which probably ex- plains the occasional fluorescing cells found in tumors (Calnek gt_al,, 1970). Intra-cellular viral antigens. Several intra-cellular virus-related antigens are detected in cells productively infected with MDV. These antigens have been detected by immunofluorescence and immunodiffusion techniques. Combined fluorescent antibody and electron microscopy studies have demonstrated that cells positive for antigens are positive for virus particles and, conversely, cells negative for antigens are negative for virus particles (Nazerian gt_al., 1969; Nazerian and Purchase, 1970). The immunofluorescence technique was also used to detect antigens induced by MDV in cell cultures and in tissues of infected birds (Spencer and Calnek, 1970; Calnek §t_al,, 1970). Antigens were found in the nucleus and cytoplasm of infected cells. Chubb and Churchill (1968) and Churchill §t_al, (l969a) using chick kidney cell cultures infected with MDV as the antigen, described 15 several immunodiffusion antigens associated with M0 infection. The A antigens were found in culture fluids and cell extracts. Two other antigens designated B and C were found in the extracts of infected cells. In contrast, tumor cells did not contain immunofluorescent viral antigens, precipitin antigens or infectious cell-free virus (Churchill and Biggs, 1968)and except only rarely there have been no virus particles observed (Schidlovsky §t_gl,, 1969; Calnek and Hitchner, 1969; Ubertini and Calnek, 1970; Nazerian, 1971; Akiyama et al., 1973b). Viral-membrane antigens. Cell surface viral membrane antigen has been reported on MD infected chicken kidney cell cultures (Chen and Purchase, 1970) and on Japanese quail embryo fibroblasts infected with either MDV or the cell-free HVT (Ishikawa gflngL., 1972). Chen and Purchase (1970) demonstrated a bright ring of fluorescence on the surface of rounded MDV infected cells of unfixed chick kidney cell monolayers and chick kidney cells in suspension. The specificity of the reaction was determined by the fact that only membranes of altered cells within virus plaques and not membranes of surrounding uninfected cells reacted with MDV specific antiserum. Early antigen. Nazerian (1975) reported the induction of an MDV antigen under conditions where DNA synthesis was inhibited. Lymphoblastoid cells of the MSB-l cell line were treated with thymidine DNA analogues, 5-iodo-2-deoxyuridine (IUDR) and 5-bromo-2-deoxyuridine (BUDR). After removal of the drugs, another antigen dependent on virus DNA synthesis was induced followed by virus production. The early antigen production could not be distinguished from the late antigen production by 16 serological methods because most sera from infected chickens has anti- bodies to both antigens. The results did suggest that MDV genome was associated with a large number of cultured lymphoma cells that did not ordinarily produce virus particles. A similarity can be drawn with the Raji cell, a human lympho- blastoid cell line derived from Burkitt lymphoma, which is negative for Epstein-Barr virus (EBV) and viral antigens but contains the EB viral genome (zur Hausen and Schulte-Holthausen, 1972). Treatment of the Raji cells with thymidine analogues has induced early antigen and virus particles (Hampar et al., 1972). Tumor antigens. An antigen was detected on the surface of MD tumor cells and lymphob1astoid cells of lines developed from MD tumors (Powell gt_al., 1974; Witter §t_gl,, 1975). Tumor cells in suspension were examined for membrane antigens with anti-tumor sera by a modifica- tion of the membrane immunofluorescence technique (Mfiller, 1961). The tumor-specific antigen did not appear related to virus structural proteins because cells positive for the tumor antigen did not react with anti-viral sera containing antibodies for membrane and intra- cellular antigens, furthermore, anti-tumor sera did not react with membrane and intra-cellular antigens of MDV infected cells (Powell gt_al,, 1974; Witter gt_al,, 1975). The surface antigen was not de- tected on normal chicken lymphocytes or on cells transformed by avian ribonucleic acid (RNA) tumor viruses. The antigen did not appear to be related to embryonic or histocompatibility antigens. The antigen was designated as a Marek's disease tumor-associated surface antigen (MATSA) (Witter et al., 1975). 17 Virus genome in tumor cells. As mentioned previously virus particles or virus specific antigens are found only rarely in tumors. The appear- ance of tumor-specific antigens on the surface of MD tumor cells ac- companied by the absence of virus particles supports the work of Nazerian et_al, (1973) that MDV-DNA is present in tumor cells. Marek's disease virus DNA was demonstrated in tumor cells from infected chickens (Nazerian gt_al., 1973) and in MD lymphoblastoid cell lines by nucleic acid hybridization (Nazerian and Lee, 1974; Lee gt_al,, 1975). Lee §t_al. (1975) demonstrated that all MD tumor cells, regardless of the MD isolate used, contained MDV-DNA. Transplantability of tumor cells. The transplantibility of MD tumor cells was demonstrated by Jakowski §t_al, (1974) and Theis EHLEgL. (1974). A transplantable lymphoma was developed in an inbred line of chickens by intramuscular injection of leukocytes from a Marek's disease virus infected chicken and was maintained by serial passage of virus-induced tumor cells in chickens of the same §_blood group histocompatibility genotype. Tumors developed at the site of inoculation and in the visceral organs. Karyotype studies, using sex chromosome markers, revealed the tumor at the site of inoculation to be of donor origin and the cells of the visceral organs to be of recipient origin (Jakowski §t_al., 1974). Detection of the §_alloantigens of the origi- nal tumor after repeated passage in chickens of a different §_genotype confirmed the perpetuation of the original §_phenotype in the cells of the transplantable lymphoma (Theis gt:al., 1974). .The pathogenicity of the transplantable lymphoma increased with each passage as indicated by an increase in the number of recipients developing tumors at the 18 site of inoculation and an increase in mortality with a shortened latent period. The progressive growth of the transplantable tumor cells suggested that the tumor represented a population of proliferating cells (Theis gt_al., 1974). Therefore, Theis gt_al, (1974) concluded that the recipient chickens did not contribute to the transplantable lymphoma. The occurrence of the transplant appeared to be an event of low inci- dence in that only 1 out of 15 attempts resulted in a continuously transplantable line. Even in histocompatible lines the successful transplantation of tumor cells may depend on optimal conditions for selection of a small population of transformed cells (Theis gt_a_1_., 1974). Because MDV was initially associated with the primary virus- induced lymphoma Theis §t_al. (1974) investigated the association of MDV with the transplantable lymphoma cell line. Both MD virus and antibodies were found in isolation reared recipients of the MD lymphoma cell line. Whether MDV was the only virus involved in the transformed transplantable lymphoma was not determined; however, attempts to control contamination by avian leukosis viruses were made by using COFAL nega— tive dams (dams free of the group-specific antigens of the avian leu- kosis viruses). 8 and T cell surface antigenic markers. Two populations of lymphocytes are recognized in the chicken; bursa-derived cells (B cells), which are responsible for humoral immunity, and thymus-derived cells (T cells) which are primarily concerned in cell-mediated immunity (Payne §t_al., 1974; Rouse gt_al,, 1973). Antisera directed specifically against chicken thymus or bursa cells have been used to study the origin of the lymphoid cells in Marek's disease lymphomas. 19 By means of the membrane immunofluorescence technique the majority of cells in MD tumors were found to be T lymphocytes (77 per- cent) with only a few (3-11 percent) B lymphocytes present (Hudson and Payne, 1973; Rouse §t_al., 1973). Powell gt_al,(l974) studied the MD lymphoblastoid cell lines HPRS-l and 2 for B and T cell antigenic markers and found that 100 percent of the cells stained for T cell de- terminants and 0 percent for B cell determinants. Additional work by Nazerian and Sharma (1975) with the MSB-l cell line also indicated about 99 percent T cells and 1—2 percent chicken B cells present in the cell suspension. The question of whether the T cells in the tumor were trans- formed by virus or were there as a result of a host immune response was not resolved. Hudson and Payne (1973) and Rouse §t_al, (1973) noted both T and B cells but did not prove whether the lymphoma cells were transformed or were normal invading cells. Hudson and Payne (1973) proposed that although T and B lymphocytes contribute to the tumor, the majority of cells were of thymus origin. Thus the lymphoid system may provide an initial target cell which, after infection, could lodge in the gonads and other sites where it could transform and express surface antigens. These altered T lymphocytes (now foreign) may pro- voke a host immune response and stimulate entry of antigen reactive lymphocytes into the tumor. This secondary antigen reactive response may be the result of both 8 and T lymphocytes in the tumor. Payne (1972) proposed two theories by which MD lymphopro- liferation could occur: (1) an "intrinsic mechanism" whereby virus within lymphoid cells causes altered cells to proliferate and form 20 tumors or (2) an "extrinsic mechanism" by which virus infected antigen bearing non-lymphoid cells illicit an immunological response by the host. It has not been clear whether MD lymphomas are caused by direct infection and transformation of lymphoid cells (intrinsic) or whether lymphomas are formed as a result of host reaction to virus antigens (extrinsic). Recent evidence has been presented that MD lymphoma cells (Nazerian et_al., 1973) and the MSB-l lymphob1astoid cells (Nazerian and Lee, 1974) favors the intrinsic hypothesis by showing that the cells contain a significant number of copies of MDV-DNA in the absence of virus and viral antigens. This has provided strong circumstantial evidence that true transformation may indeed occur in MD. The presence of T cell surface antigens and MDV genome associated with the tumor cells provides additional evidence that MD lymphomas are virus-induced transformed T cells. A direct jg_vitrg transformation of chicken T cells by MDV has not been done but would help to confirm the present data. B_blood group antigenic markers. The B_blood group locus of the chicken has been shown to be the major histocompatibility locus (Schierman and Nordskog, 1961). The production of transplantable MD lymphomas has depended on the use of this knowledge (Theis gt_gl,, 1974). Various reference alloantisera have been developed that are specific for the §_alloantigens on the surface of erythrocytes and lymphocytes and have been used to identify chicken populations selected for such parameters as histocompatibility and resistance to MD (Pazderka gt_al,, 1975). In addition to the technique of karyotype analysis, this technique based on the B_blood group alloantigenic markers has been used to 21 identify cell transplants. Specific agglutination reactions of lymphocytes in the presence of specific alloantisera can identify the alloantigens expressed on cells of MD lymphomas and thereby establish whether the MD lymphoma cells are the result of virus-induced recepient cells or whether they are transplanted donor lymphoid cells (Theis gt_al,, 1974). Theis gtgal. (1974) used specific alloantisera to agglutinate specific alloantigens (B1 and 82) on the surface of the transplanted tumor cells formed at the site of inoculation. It was concluded that the MD lymphoma was a replicating cell line and that passage of the lymphoma cells in birds of different B_genotypes did not alter the alloantigenic specificity of the cells. The visceral tumors which developed in some chickens after injection of the MD transplantable lymphoma cells were examined and the preliminary results suggested that these tumors did not develop as a result of metastasizing MD lymphoma cells. JMV LYMPHOBLASTOID TUMOR CELLS The JM isolate of MD, isolated by Sevoian gt_al, (1962) has produced a high rate of neural and visceral MD lesions in S-line chicks within 3 weeks after inoculation. Serial passage of affected tissues from experimentally infected chicks exhibiting a shorter latent period increased the pathogenicity not only in S-line chicks, but in other strains of chickens as well. Several other researchers have also noted that rapid serial passage of MD lymphoma cells in chickens resulted in a reduced latent period and increased morality (Jakowski et al., 1974 22 Theis gt_gl., 1974; R. Larose, personal communication; S. Schmittle, personal communication). Sevoian's virulent isolate, derived from multiple serial passage of JM-MDV lymphoma cells in MD resistant White- rock chickens (Sevoian, personal communication) was designated as JMV (Sevoian, 1967), because he interpreted this tumor to be induced by an especially virulent form of MD virus. This isolate has been used ex- tensively as a challenge virus for evaluating HVT-induced immunity (Mason and Jensen, 1971; Sevoian and Weston, 1972) and as a rapid test for genetic resistance to MD (Gavora §t_al., 1974). JMV has recently been proposed as a potential vaccine against MD (Sevoian, personal communication). JMV was highly lethal within 3 to 8 days after inoculation in all strains of chickens tested (Sevoian, 1967). The disease was characterized clinically by the sudden onset of generalized weakness and gasping a few hours prior to death. Enlarged livers and spleens, due to infiltration of lymphoblastic cells, occasional appearance of ruptured spleens, lymphoblastic leukemia in peripheral blood and tumors at the site of inoculation were noted on post-mortem examination (Spencer and Calnek, 1967). As previously mentioned most lymphomas are non-productively infected and the appearance of virus or viral antigens is rare. MD lymphoma cells can effectively transmit virus infection and induce disease when inoculated into susceptible chicks. However, the associ- ation of MDV with JMV tumor cells has not been clearly shown and has been a subject of controversy. The principle question to be answered 23 in this study is whether JMV-induced tumors resulted from viral trans- formation of recipient host lymphocytes or from the growth of trans- planted donor tumor cells. Detection of virus or viral antigens. Spencer et al. (1973a) reported a bi-phasic mortality in chickens inoculated with JMV tumor cells. Death and MD lesions occurring at the 5th week after inoculation in birds surviving the initial inoculation of JMV tumor cells, appeared to be caused, most likely, by MDV carried in the inoculum cells. Spencer however, did not infer that the initial JMV lesions were caused be a virus. Hamdy and Sevoian (1973) reported virus-specific complement fixing and serum neutralizing antibodies in JMV immunized chickens. Cell-free JMV obtained from JMV infected blood and an embryo attenuated cell-free JMV, designated JMV-A (Hong and Sevoian, 1974; Shieh and Sevoian, 1974, 1975), have been reported to stimulate chicks of lines N‘and P to produce high levels of virus-specific neutralizing and fluorescent antibodies against the JM isolate of MD. Yoon and Kenyon (1975) reported a JMV herpesvirus isolated from spleens of in- fected chickens which was distinct from MDV in virulence, tumor forma- tion and histopathology. Polykaryocytes were induced by the JMV virus in 3 days in chicken kidney cell cultures. Cytogenetic studies (Kim gt_al., 1972) indicated that donor genome, identified by sex chromosome markers, was not present in the cells of recipient birds, and therefore, the disease was a result of a virus associated with JMV tumor cells. In contrast to the reports that JMV lesions are induced by a virus associated with JMV tumor cells is the lack of contagion to cagemates of birds inoculated with JMV (Sevoian, 1967; Spencer and 24 Calnek, 1967). The lack of cell-free transmission of the disease with serum from JMV moribund birds was observed by Jones et_al, (1969). The dependency on viable tumor cells to reproduce the disease, the rapid onset of disease, tumors at the site of inoculation and the highly lethal nature of the inoculum cells were indicative of a cell trans- plant (Spencer and Calnek, 1967). Olmsted and Kenyon (1971) supported the transplant theory in their studies using whole-body irradiation to destroy the lymphoid element of recipient chickens. They concluded that the lymphoid proliferation must have been due to the proliferation of donor JMV lymphoblastoid tumor cells. Preliminary results by Witter SULJLL- (1975) demonstrated that JMV tumor cells were totally free of replicating MDV as indicated by the failure to isolate virus by co- cultivation of tumor cells with DEF jn_vjtrg_and by the absence of MDV specific antibodies in sera from JMV immunized chickens. Probably the best indirect evidence available to date that JMV tumor cells are antigenically related to MD lymphomas is the im- munologic response induced in chickens immunized with JMV, MDV or HVT. Mason and Jensen (1971), Sevoian and Weston (1972) and Shieh and Sevoian (1974) demonstrated that vaccination with HVT prior to challenge with JMV tumor cells protected chicks against the lethal effects of the tumor cells as measured by a reduction in mortality rate. Vaccination with HVT one week prior to inoculation of JMV tumor cells into the wing webof 5-week-old chickens significantly suppressed the develop- ment of wing web tumors (Spencer gt_al., 1973b). The mechanism by which HVT protects chicks against JMV is not understood. Mason and Jensen (1971) proposed that either protection was reflective of antigens 25 being shared between JMV or HVT, or that in some undefined manner HVT preempted cells critical to achieving JMV infection. Inoculation of low doses of JMV cells appeared to protect chicks against subsequent higher doses of JMV and MDV. As shown by Sevoian (1967) inoculation of mature dams with an initial sub-lethal dose of JMV tumor cells caused the chickens to be refractory to higher doses of JMV. Dayhold progeny from these dams were resistant to challenge with JMV and MDV up to 5 weeks of age. Kenyon §t_al, (1969) reported that sera from JMV inocu- lated survivors could passively immunize chicks against JMV. Adsorption of the antiserum with MDV infected cells significantly decreased the neutralizing capacity and was comparable to the results obtained by adsorption with JMV cells. Hong and Sevoian (1974) reported the existence of maternal antibody in progeny from adult chickens vaccinated with cell—free JMV and HVT. They concluded that progeny with JMV maternal antibody had more protection against JM-MDV infection and tumor induction than progeny with HVT or MDV maternal antibody. Tumor antigens on JMV tumor cells. As a result of the work begun in this research, which initiated further studies by Witter gt_gl, (1975), the unexpected finding that JMV antiserum was reactive with MSB—l cells, MD lymphoma cells and JMV cells suggested the possible existence of tumor-specific antigens related to MD. Other than that published by Witter gt_al. (1975) and the data presented in this thesis, there have been no other reports related to the existence of tumor antigens on JMV cells. There is no published information indicating the presence of MD viral genome associated with JMV tumor cells. There appears to 26 be no information at present as to the association of bursa-derived and thymus-derived lymphoid cells with JMV tumor cell transformation. The transplantable nature of JMV has been reported but additional evi- dence indicating a difference between the 8 blood group surface anti- genic markers of JMV tumor cells and those of the host lymphocytes has not been published. The controversial issues related to JMV tumor cells which should be resolved are first, the claims that infectious virus is responsible for JMV lesions and second, whether JMV can be considered a class of Marek's disease tumor. MATERIALS AND METHODS Chickens and embryos. The chickens used were Single—Comb White Leghorns from the MD susceptible lines 72, 100 and crosses 15 x 7 and 72 x 100, developed at the Regional Poultry Research Laboratory, East Lansing, Michigan (Stone, 1975), and the MD resistant Cornell N line (Cole, 1968). Four-day-old embryos of line 63 were used for yolk-sac inocula- tion. Line 72 is highly susceptible to MD (Stone, 1975) and is homozygous for the 83-blood group allele (Pazderka gt_al,, 1975). Line 100 is an inbred line developed from crossing line 6 males with line 7 females and then backcrossing to line 7 males for several gen- erations (Stone, 1975). Line 15 has been maintained since its inception in 1939 and is susceptible to MD. Line 6 is relatively resistant to 21 MD. The Cornell line N is homozygous for the blood group allele (Pazderka gt_al., 1975). All chicks were from dams negative for M0 antibodies except as otherwise specified. The chickens used in these experiments were maintained in Horsfall-Bauer isolators throughout their experimental period. Cell culture. Direct chicken kidney cell cultures (DCK) from JMV donor chicks were prepared and maintained in basal medium, Eagle (BME), with bovine fetal serum, 25 units/ml mycostatin, 100 units/ml penicillin- streptomycin, as previously described (Churchill and Biggs, 1967; Witter et al., 1968b). Cells were counted in a hemocytometer and plated at 27 28 5 x 106 cells per 60mm tissue culture dish. CEF and DEF cultures were prepared in tissue culture medium F 10-199 mixture with antibiotics as described according to the methods of Solomon et a1. (1971). Viruses. The JM isolate of MDV (Sevoian §t_al,, 1962) was cloned, by Dr. Richard L. Witter, Regional Poultry Research Laboratory, 3 times in DEF cultures from cell-free virus obtained from the feather tips of infected chickens and designated clone JM/102W. The material used had been passaged 43 times in chickens and 13 times in DEF cultures and consisted of infected DEF suspensions stored at -196°C. Uncloned preparations of the GA strain of MDV were also used. The reticuloen— dotheliosis virus (REV) strain T, was obtained from Dr. M. K. Cook» (Witter gt_al., 1970c). Stocks of the A, B, C, and 0 subgroups of Rous Sarcoma virus (RSV) were kindly supplied by Dr. W. Okazaki, Re- gional Poultry Research Laboratory. Virus and antibody assays. The phenotypic mixing test as previously described (Okazaki gt_al., 1974) was used to detect avian leukosis viruses of subgroups A, B, C, and D in stock suspensions of each JMV source, as well as in irradiated JMV-S cells. Line 100 C/0 (cells susceptible to all subgroups of avian leukosis viruses) CEF monolayers in cell culture medium containing 2 ug/ml DEAE-Dextran were inoculated with at least 103 focus-fOrming units of RSV-0. The JMV test samples were added 24 hours later and maintained at 37°C in a humidified chamber under CO2 for 7 days. Tissue culture fluids were harvested, frozen and thawed 3 times, centrifuged and inoculated at 0.5 ml super- natant fluid onto 24 hour cultures of C/E CEF monolayers. The mono- layers were overlaid with agar 24 hours later and examined approximately 29 7 days later for RSV foci. A neutralization technique as described by Ishizaki and Vogt (1966) was used to detect the presence of antibody to RSV subgroups A, B, C, and D in JMV antiserum. CEF monolayers infected with JMV spleen cells, maintained 7 to 21 days, were examined with REV positive antiserum by indirect im- munofluorescence for the presence of REV antigen (Witter gt_gl,, 1970c). Sera were assayed for REV antibodies by indirect immunofluorescence using REV infected CEF cell culture antigen (Witter gt_al., 1970c). A blind fluorescent antibody technique designed to detect unspecified replicating agents was based on indirect immunofluorescence staining with homologous hyperimmune JMV antiserum of cultures inocu- lated with JMV. The lack of positive staining reactions was considered evidence for the absence in test material of agents capable of repli- cating in the culture system and of inducing antibodies in chickens after inoculation. MDV was assayed by induction of herpesvirus cytopathologic changes in chick kidney, CEF and DEF cultures (Witter §t_al,, 1969b). Coverslips included at the first and third passage were fixed in ace- tone, and examined by indirect immunofluorescence for the presence of MD viral antigens. Four-day-old embryonated eggs of line 63 (negative for M0 antibody) were inoculated via the yolk-sac route and examined for pocks on the CAM at 14 days after inoculation (von BUlow, 1968 and 1971). Day-old susceptible chicks were inoculated, held in Horsfall- Bauer isolators for 4 to 8 weeks and observed for MDV-specific patho- logic or immunologic responses. Presumably these assay systems would also have detected the immunologically related herpesvirus of turkey (HVT) (Witter et al., 1970b). 3O Assay for MDV antibodies was done by several methods. De- tection of precipitin antibody was done by the agar-gel precipitin test(Chubb and Churchill, 1968; Churchill gt_§l,, l969a). Antibodies to virus-induced cellular antigen were detected by indirect immuno- fluorescence with acetone-fixed MDV infected chicken kidney cell cover- slip antigen (Purchase and Burgoyne, 1970; Spencer and Calnek, 1970). Neutralizing antibody was assayed by using one part serum diluted 1:5 or 1:10 and then mixed with an equal part of virus suspension such that the final dilution of serum was 1:10 or 1:20. The serum virus mixture was inoculated at 0.1 ml directly onto chick kidney monolayers, incu- bated at 37°C for 30 minutes at which time the media was replaced. JMV hyperimmune sera were examined for MATSA antibody according to procedures previously published (Witter §t_al,, 1975). JMV tumor cells. Three sources of JMV tumor cell preparations passaged 800, 250, and 60 times in chicks were designated as JMV-S, JMV-x462 and JMV-x55, respectively. The JMV-S was obtained from Dr. M. Sevoian, Amherst, Massachusetts. The JMV-x55 and JMV-x462 were obtained from Dr. B. W. Calnek, New York State Veterinary College, Ithaca, New York, who received them from Sevoian in 1965 and 1972, respectively. All stocks were received as frozen liver suspensions and represented different passage levels of the original JM-MDV lym- phoma from which JMV was derived. Stock tumor materials were prepared by inoculating day-old chicks of crosses 72 x 100 or 15 x 72 (MD antibody positive) and harvesting liver tumors from moribund chicks 7 to 12 days post-inoculation. The stock liver tumor materials were prepared as 10 percent tissue homogenates and stored with 10 percent dimethyl-sulfoxide at -l96°C (Spencer and Calnek, 1967). Single cell 31 suspensions of JMV spleen tumors for immunofluorescence and chick inocu- lation were prepared by removing the splenic capsule and connective tissue and gently teasing the cells with a scalpel. The crude spleen material was allowed to stand from 3 to 5 minutes before being filtered through gauze. The cells were then washed and centrifuged at 1500 rpm for 3 minutes (2 cycles) and resuspended in tissue culture medium or phosphate buffered saline (PBS). Spleen homogenates were prepared with a Ten Broeck tissue grinder in medium Flo-199 with 4 percent calf serum as 1:10 suspension on a weight per volume basis. Sonication was accomplished with a Bronwell Biosonik II cell disrupter (Bronwell Scientific, Rochester, N. Y.) for 3 one minute intervals at a setting of 70. The material was filtered through a 0.45 u filter (Nalgene Labware Division, Sybron Corporation, Rochester, N. Y.). Gamma irradi- ation of JMV-S spleen cells at 108 cells/ml was accomplished at 5000 r with a cobalt 60 source (Michigan State University, East Lansing, Michigan, Department of Food Science and Human Nutrition). Preparation of hyperimmune sera. JMV-S and JMV-x462 hyperimmune sera were prepared by inoculation of twelve-week-old 15 x 72 chickens with 69 and 178 CLDSO’ respectively, of liver homogenate, followed by two additional injections of 690 and 1780 CLD50 at the 15th and 18th weeks in some of the chickens. All chickens were bled at the let week. Membrane immunofluorescence technigue. Staining of unfixed lymphoid cells of the MSB-l cell line and JMV spleen tumor cells in suspension was done by the indirect membrane immunofluorescence technique as pre- viously described (Witter gt_al,, 1975). Approximately 2 x 106 lym- phoid cells/12 x 75 mm tube were suspended in PBS, washed and pelleted. 32 The cells were resuspended in 0.1 ml of 1:8 dilution of JMV hyperimmune serum and allowed to react for 30 minutes at 4°C. Cells were counter- stained with 0.1 ml of a 1:20 dilution of f1uoresceineisothiocyanate conjugated horse anti-chicken globulin (Roboz Surgical Instrument Co., Washington, D. C.) for 20 minutes at 4°C. After a final wash in PBS cells were suspended in 0.1 ml PBS/tube, a drop of cell suspension placed under a coverslip and examined immediately with a Leitz fluor- escence microscope with vertical illumination system for the presence of MATSA (Witter et al., 1975). MSB-lglymphoblastoid cell line. The lymphoblastoid cell line was ob- tained from Dr. S. Kato of Osaka University, Japan, and was derived from the spleen tumor of an MD infected chicken. The properties of the cell line have been previously described (Akiyama and Kato, 1974; Nazerian and Witter, 1975). JMV assay. One-day-old MD susceptible chicks were inoculated intra- abdominally with 0.1 ml of JMV tumor cells, placed in Horsfall-Bauer isolators, and observed through an experimental period of at least 21 days. Death from JMV tumor cells occurred between the 7th and 12th days after inoculation from enlarged leukotic livers and spleens. Chickens dead of non-specific causes, as confirmed by histopathology, were excluded from the data. Positive diagnosis was based on typical gross lesions or lymphoblastic infiltration of liver and spleen as confirmed by microscopic examination. Several lots of chicks were maintained in the same isolator since no horizontal transmission to control chicks within the isolators had previously been observed by 33 others (Spencer and Calnek, 1967) nor in any of the experiments conducted in this research. Quantitative assays were performed for each JMV source by inoculation of serial dilutions into separate lots of chicks. Titers were expressed as chick lethal doses (CLDSO) according to Reed and Muench (1938). JMV_protection test. A test was designed to measure the protective ability of JMV sera against jg_vivo tumor formation and death. Protec— tion was based on the ability of one ml of heat inactivated JMV serum to protect at least 75 percent of treated chicks against mortality re- sulting from inoculation of 50 CLD50 of JMV-S. Day-old chicks of line 100, five birds per lot, were each inoculated intra-abdominally with 1 m1 of the respective serum. The chicks in each lot were then challenged with 50 CLD50 of JMV-S one day later. Responses were measured as de- scribed for JMV assay. Assay for MDV virusespecific membrane antigen and MATSA. Unfixed coverslips of MDV infected DEF cell monolayer cultures and lymphoid cells in suspension were assayed for membrane antigen by indirect im- munofluorescence (Nazerian, 1973b) using anti-MDV and HVT sera. Unfixed, single cell suspensions of JMV and normal spleen cells were assayed for MATSA by indirect membrane immunofluorescence as has already been de- scribed. Cells in suspension were stained with 1:8 dilution of chicken JMV hyperimmune serum containing MATSA antibody and counter stained with conjugated anti-chicken gamma globulin as previously described. Detection of B alloantigenic markers. B2 and 82] antisera were kindly supplied by Dr. B. H. Longenecker, Edmonton, Alberta, Canada. This 34 experiment, designed by Dr. Longenecker, was done in an attempt to identify the alloantigens on the surface of JMV tumor cells. The in- direct membrane immunofluorescence technique was used to examine the cells. B and T cell surface antigenic markers. Unfixed, single cell suspen- sions of normal and JMV affected spleen cells were examined for B cell and T cell surface antigens by the indirect membrane immunofluorescence method. Anti-T and anti-B sera were kindly supplied by Dr. J. M. Sharma, Regional Poultry Research Laboratory, East Lansing, Michigan. Anti-T serum was produced in a turkey by three bi-weekly intravenous injections of 109 thymus cells from line 63 chickens and adsorbed 5 times with chicken erythrocytes (1:20 v/v) and repeatedly with bursa cells until all reactivity against 8 cells was removed. Anti-B serum was prepared by three intravenous injections of 109 bursa cells from line 63 chickens into ducks at 3 week intervals. The anti-B serum was adsorbed 6 times with chicken erythrocytes and repeatedly with thymus cells until reac- tivity against T cells was reduced or removed. DNA-cRNA hybridization. Normal spleen pools and JMV tumorous spleen 9 pools were processed to form cell suspensions of at least 10 cells per pool. These cells were given to Dr. Lucy F. Lee, Regional Poultry Research Laboratory, East Lansing, Michigan, for hybridization studies. DNA from normal CEF, GA-MDV infected CEF, and spleen from normal and JMV affected chickens were extracted according to published procedure (Lee §t_gl., 1971). Transcription of purified MDV-DNA into 3H-labelled complementary RNA and the technique of DNA-cRNA hyribidization have 35 been published elsewhere (Nazerian and Lee, 1974). Liquid scintilla- tion spectrometry was used to assay radioactivity on the membrane filters. Electron microscopy. Spleens were removed from each of 5 JMV-x55 and 5 JMV-S donors that were obviously moribund. Thin sections from each spleen were given to Dr. Keyvan Nazerian, Regional Poultry Research Laboratory, for examination by electron microscopy for virus particles (Nazerian and Burmester, 1968; Nazerian, 1970). EXPERIMENTAL DESIGN The primary objective of this research was to resolve the nature of the association of MD herpesvirus with JMV tumor cells and to establish whether these tumor cells were virus-induced or transplants. The implication of a virus associated with JMV lesions had been reported but there was no substantial evidence that would resolve the controversy. In order to determine whether replicating MDV was associated with JMV tumor cells the various methods presented were used in an at- tempt to resolve the question. 1. Cell culture techniques of the following type were used to assay for MDV. a. Direct kidney cultures were prepared from JMV moribund chicks and maintained and observed through a 7 day experi- mental period. b. JMV affect spleen tumor cells were co-cultivated with DEF and CEF cultures and maintained and observed through 3 passages or 21 days. c. The detection of MDV antigens in JMV inoculated cultures was done by the indirect immunofluorescence test. d. JMV inoculated cell culture material was assayed in day- old chicks which were then maintained for 21 days in isolators. 36 37 Four-day-old embryonated eggs were inoculated with JMV tumor material via the yolk-sac route and observed for typical MDV lesions on the CAM 14 days after inoculation. Day-old chicks were inoculated with sub—lethal doses of JMV tumor cells and observed through an 8 week experimental period for any signs of a low level of MDV in the inoculum cells that would ultimately result in MD infection. In order to determine whether cell-free herpesvirus was as- sociated with JMV tumor cells, intact JMV tumor cells and various preparations of disrupted JMV tumor cells were inocu- lated into day-old chicks which were held in isolators for 21 days. JMV tumor cells were subjected to gamma-irradiation in order to determine whether a latent herpesvirus was associated with the tumor cells. The cells were then co-cultivated on CEF and DEF cultures and observed for 21 days. After extensive efforts failed to reveal a herpesvirus as- sociated with JMV tumor cells the possibility of other avian tumor viruses was considered. The following methods were used to assay JMV tumor cells for other RNA tumor viruses. 1. 2. The indirect immunofluorescence technique was used to detect the presence of REV antigens in JMV inoculated CEF cultures. A blind fluorescent antibody technique was designed to detect unspecified replicating agents in cell culture and in the serum of chicks inoculated with JMV tumor cells. 3. 4. 38 A virus neutralization technique was used to detect the presence of RSV antibody in JMV sera. The very sensitive phenotypic mixing test was used to detect the presence of leukosis viruses in all three sources of JMV liver homogenate and JMV spleen tumor cells. Since all efforts to rescue a virus associated with JMV tumor cells had not produced positive results, the possibility of other anti- gens and viral genome was considered. The unexpected results of find- ing tumor specific antigen on the surface of MSB-l cells stained with JMV anti-tumor sera led to the following experiments. 1. The membrane immunofluorescence technique was used to detect the presence of antibody in JMV hyperimmune sera against viral and membrane antigen in MDV infected cells and tumor antigens on JMV cells. Day-old chicks were inoculated with JMV antisera to determine .whether the sera could protect chicks against a lethal dose of JMV tumor cells inoculated one day later. This test was done previous to the discovery of tumor-specific antigens; however the ability of the sera to protect chicks and the presence of tumor-specific antibody appeared to be related. In order to determine whether the tumor antigens observed on MSB-l cells and JMV tumor cells was specific for M0 non- productively infected cells, the technique of DNA-cRNA hybridi- zation was used to determine whether JMV tumor cells possessed MDV genome. The presence of MDV genome had been shown in MSB-l cells but no such evidence existed for JMV tumor cells. 39 4. JMV tumor cells were examined for the presence of any form of virus particle or viral antigens by the electron microscope. In an attempt to determine whether JMV cells were the result of a transplant the following assumption was made. Assuming that a transplanted cell would maintain the same B_blood group histocompati- bility antigen of the host in which it was produced, regardless of the number of times it was passaged in a host of another 8 genotype, an attempt was made to determine whether JMV tumor cells were a replicat- ing transplantable cell line. JMV tumor cells were examined for the presence of the particular 8 antigen of the host into which the cells had been inoculated. The absence of membrane staining with a specific alloantiserum homologous to the host genotype would imply that JMV was indeed a transplant and would probably express a different 8 alloantigen, namely that of the original host. As an additional biological characteristic JMV tumor cells were examined by membrane immunofluorescence for the presence of B and T cell surface antigenic markers. Since MD lymphoma cells had been shown to express T cell surface markers it was of interest to determine whether JMV cells expressed a similar marker. RESULTS Biological characteristics. Day-old chicks inoculated intra-abdominally with JMV tumor cells died between the 7th and 12th day depending on the dilution of the inoculum cells. The latency of the disease was in- creased approximately 1 day for each log dilution of JMV tumor cells (Figure 1). Prior to death the chicks were generally weak, emaciated and showed signs of respiratory difficulty. Affected livers and spleens were generally twice their normal size (Figure 2). In some cases spleens were ruptured and tumors were noted at the site of inoculation, especially with JMV-x55. The predominant cell in JMV lesions was a lymphoblast (Figures 3 and 4). The appearance of small lymphocytes in the lymphoblastic lesions (Figure 5) was not completely understood, but may be indicative of a host response to the transformed cells. There was no evidence of virus or viral antigens in any of the tumor cells examined by methods to be discussed. These characteristics were quite different from those associated with M0, such as longer latent period, the lack of tumors at the site of inoculation and a mixed popu- lation of lymphocytes, blast cells and plasma cells associated with the MD lesions (Calnek and Witter, 1972). The quantitative biological characterization of JMV tumor cells is presented in Tables 1 through 3 with the mean days to death for each dilution and the respective CLD50 for each JMV source presented. 40 Moan days to death 41 12 11 1O 1 2 3 4 5' 6 7 8 109,0 dllution °'———°Tltratlon of JMV-S x——-x Titration of JMV'X462 o———o Tltratlon of JMV-x 55 Figure 1. Comparative titrations of three JMV sources demonstrating dose dependent latency effect. The data were obtained from the results in Tables 1, 2 and 3. Figure 2. Figure 3. 42 Enlarged tumorous spleen of a line 7 after inoculation with JMV-S. 2 chicken 8 days Smear preparation of JMV-S spleen tumor cell suspension showing typical lymphoblastic cells. Wright X1580 43 uzqu&mm3g#gy Figure 2 O V. ' Cr.‘.. \.. ‘ 44 Figure 4. Liver of chicken inoculated with JMV-S; a focus of lymphoblastic tumor cells is shown. H & E X 390 Figure 5. Liver of chicken inoculated with JMV-S; a focus of lymphoblastic tumor cells (curved arror) infiltrated with small mononuclear cells (straight arrow) is shown. H & E X 630 45 Figure 5 Figure 4 46 FE F.O\omu_o emmm u mopwp=< Fe _.O\ms.m u omoso "Lap_h .mzmv Fm Low mm; vowgwa Fmpcms_gmaxu .8 .>oz 0“ xuoawpca soc m>wpmmmz u Jam o.P~ mxo mcoz o.- “\o m-o_ mo.pm N\o m-o_ o.P_ N\N eno— o_ u m N.m “\m m-o~ o— u m o.m “\N N-o_ a - a N.N “\N NE Lam _-o_ Amzmuv magma summw op mzmw cam: page» och pmoz cowpzpwo xuw_mugoz \.mom * .oomm_- pm umeopm mpmcamoEo; La>PP soopm m->:a any to cowumapwp ._ m_QML Fe F.O\ome_u ooo.map u "mo_wpc< Fe F.O\m~.m u omoso "Lapwp .mzmo Fm com mm: uo_gma Popcmswgmaxm .m .seonwpcm a: too a>wpomaz u Jam 47 mo.F~ m\o mcoz o.NF m\_ ouop Np - FF m._P m\m muop o_ - m m.m m\m ¢-o_ m u w N.w ¢\¢ m-o_ m 1 m m.m m\m N-op w - A N.“ ¢\e Na Jam _-o_ Amxmuv magma cumwu op mace cum: Page“ mcwp pmo: cowpapwo NwPFmpooz \.mom * .808m_- pm uagoum mpwcomoso; La>w_ xoopm Noex->zw ago to =o_umep_o .N apnea 48 _E _.O\omupu ooo.~mm u "mo_wp=< PE P.O\m~m.m u ugmuwh .mzmu Fm com mm: uo_gwa Foucwewgwqu .m .seonwpcm a: too m>LBammz - Jam Fm m\o mcoz MFN m\o o-o_ m_ o_ m.FP m\¢ muop __ o— N.o_ ¢\¢ ¢-o_ o.op ¢\¢ muop op m m.m m\m Nuo_ N m m.o m\m oo_ mam F-o_ Amxmuv magma spmmu op mxmv cam: Pmuop mcwp pmo: cowpspwo xuwpmugoz \.moa * .uommpn um umcoum mumcmmoso; Lm>w~ xuopm mmxu>zn ms“ Lo cowpmouwp .m mpnmp 49 Herpesvirus isolation. In order to determine whether JMV tumor cells were actively infected with a herpesvirus, extensive efforts were made to isolate an MD-type herpesvirus from the three sources of JMV tumor 6 cells. Direct kidney cell cultures of 5 x 10 cells per plate and 2 x 105 7 to 10 spleen tumor cells were co-cultivated on CEF and DEF, respectively, and were found negative for typical herpesvirus morpho- logical changes throughout their experimental period (Table 4). Since 102 5 to 10 MD tumor cells were shown to be sufficient to induce micro- plaques in cell culture (Churchill and Biggs, 1967), the number of JMV tumor cells presumably would have been enough to initiate cytopatho- logical changes. To demonstrate that the cultures were capable of expressing MDV plaques, control cultures of each type were super- infected with 1 x 103 PFU of JM/102W and were shown to develop charac- teristic foci. Coverslips included at the first and third passages were negative for MDV antigen when stained by indirect immunofluoresence with MDV or HVT antisera. Tissue cultures inoculated with JMV tumor cells failed to reproduce JMV or MDV lesions in day-old chicks, indi- cating that the infectious property associated with JMV tumor cells was not being propagated or maintained in cell culture after 7 or 21 days. There were no typical lesions of herpesvirus MD infection throughout the 21 day experimental period as determined by gross and histological examination. Gamma-irradiated tumor cells also failed to yield a latent herpesvirus based on the methods used in these experi- ments. The development of virus lesions on the CAM of chick embryos inoculated with MDV material (Figure 6) via the yolk-sac has been 50 Table 4. Footnotes. aDonor chicks were inoculated at l-day of age and tissues harvested at 7 days after inoculation from moribund chicks. bKidneys were removed from moribund chicks 7 days after inoculation, trypsinized and plated in 5 dishes at 5 x 106 cells per dish and maintained for 7 days. Spleens were removed from the same donors, single cell suspensions prepared by gently teasing the tissue with a scalpel. The cells were inogulated on to CEF and DEF 24 hour mono- layers at 2 x 106 and 1 x 10 spleen cells, respectively, 5 repli- gat: plates per donor spleen and maintained through 3 passages or ays. cCoverslips were included in each culture and examined at the first and last passage for evidence of MD antigen. dSpleen cell suspensions were prepared from 9 moribund donors at 7 days after inoculation, pooled and adjusted to l x 108 cells/ml and irradiated at 5000r for 13.1 minutes at 1 meter from a cobalt 60 source. The irradiated cells werg then inoculated 09 to 5 repli- cate dishes each of CEF at 2 x 10 and DEF at l x 10 cells per plate. eClone JM/102W was stored as DEF suspension at -l96°C. DEF 24 hogr monolayer cultures containing coverslips were inoculated with 10 PFU per dish, 5 dishes per vial tested. Abbreviations: CKC = Chick kidney cell culture CEF = Chick embryo fibroblasts DEF = Duck embryo fibroblasts PFU = Plaque forming units FA = Fluorescent antibody test MDV = Marek's disease virus HVT = Herpesvirus of turkey CPE = Cytopathologic effect Symbol: = Gamma-irradiation 51 az + + + mum mun mzmop\zo oz 1 1 1 mum oz 1 - - H5 62% $12: > mxo 1 1 1 mum m3 - - - “.8 62% m\o 1 1 1 gym awcuwx mmx1>zw m\o 1 1 1 mun m\o 1 1 1 mmu :mm_am m\o 1 1 1 oxo Aacuwx No¢x1>zo m\o 1 1 1 duo ma - 1 - to 52% m\o 1 1 1 uxu zmcvwx m1>2n mxuwsu cop mcwp upo mmU1F H>z >oz “no coax“ Fpmu umumm>gmg ucmm< cm megmpga mgzprmw uv> cH mammwu umpmm>omc zoom m=Lw>1mcwpumpmu Low mmamwcguwh .mFqu Loszo >zw soc» msgw>mmacmg mxwp1oz cm m:Ummc on mpaampu< .w mpnmp 52 Figure 6. Virus induced lesions on the CAM of line 63 embryo inoculated at 4 days of age via the yolk-sac with Marek's disease virus. 53 lllllllllllllllllllllllllllllllllllllllllIllllllllll MUNC 1 sun» 2 Figure 6 54 previously reported (von Bfilow, 1968, 1971; Biggs and Milne, 1971; Churchill, 1968), and has been shown to be nearly as sensitive as cell culture assay.(Biggs and Milne, 1971). Stock liver homogenates of JMV-S and JMV-x55 failed to produce lesions on the CAM (Table 5). An 8 week jn_viyg_experiment was designed to detect the presence of MDV in the JMV stocks (Table 6). Chicks surviving an ini- tial low dose at 1-day of age were given a second challenge with 104 viable JMV tumor cells at 4 weeks of age. There were no chickens diagnosed for M0 throughout the 8 week experimental period. Sera ob- tained at 8 weeks were negative for M0 specific precipitin and fluo- rescent antibodies. All lots were maintained in separate isolators. 3 Only lot 5, which had been inoculated with 10 PFU of JM/102W, was positive for M0 antibody. Lot 4 inoculated with 10 PFU JM/102W and 5.7 x 104 JMV tumor cells simultaneously was included to determine whether a low dose of MDV would have sufficient time to infect contact chicks in the same isolator before the high dose of JMV tumor cells killed the chicks. The results indicate that a low level of MDV would not be detected in birds dying from the JMV tumor cells during the first 7 days, but would possibly be detected in those birds surviving a sub-lethal dose of JMV tumor cells. Attempts to transmit the disease with cell-free filtrates of JMV-S, JMV-x462 and JMV-x55 spleen tumor cells and feather tip filtrates from JMV donors were unsuccessful (Table 7). The infectivity of JMV tumor cells appeared to be highly dependent upon intact cells. Simi- lar material from JM-MDV infected birds did demonstrate the cell-free infectious virus associated with the feather follicle epithelium. 55 a new Am use F mmpamhv mxuwco c? cowuwgpwa mzow>mga no woman mo: m~_mu Loss“ Lm>__.:u mmou mg» msow>mga no woman mm: voopn fiasco: was >o212n Low mmou ugh .AuwcmPpnsqcz .mcoumv mace .moxgnsm cw cowgmcu_u xgm:_e__mga a .A_Nmp .zopam co>v magw> com xmmmm ugmucmgm m we vow: coma mm; mumm umgmcozgaam upo1>mu1¢ mo cowumpsuocp umm1x~o>m N\o A _e _o.o noopm acoz “\L a _5 _o.o nooFm 3No_\2q o_\o o_ omosoe.mop am>ws mmx->2q o_\o o_ omosu¢.eo_ La>ws mmx->2q o_\o o, omosuw.mop La>ws m1>Za mxmu a. A nonm-x_o> mmcoammc .uwcwmem mozgnsm ammoo EarzoocH ucmm< ummu cowpm_:oocm co_mm4 mo omnszz meomuws LoE:H mo cowpmoso we mpzom .moagnEm Am>wummmc xuoampcm azv mm w:WF mo mcmcnsms uwoucmFFmowLosu mg» co mcowmwp mxw—1mmncm: oz moses? ob mppmo compam >za Lo xuwppnm:_ any we cowpmcpmcosmo .m m—nmp 56 Table 6. Attempts to demonstrate MDV herpesvirus and antibody in chicks inoculated with JMV tumor cells. Duration MDV antibody in of Inoculum a survivor serum Agent Lot test cells Dose AGP FA JMV-S 1 8 weeks Liver 1o4 - - JMV-x462 2 Liver 1o4 - - JMV-x55 3 Liver 1D4 - - JMV'S igd Liver 5.7x1o4 - - JM/102W 4 DEF 10 PFU JM/102W 5 DEF 1o3 PFU + + aJMV tumor cells were given at 4-weeks of age after an initial sub- lethal dose of 0-600 cells or about 0-50CLD50 for each JMV source. bJMV and JM/102W were inoculated simultaneously. 57 Table 7. Cell-associated nature of JMV spleen tumor cells and feather follicle extract. Spleen cells Feather Sonicate- tip Agent Age(days) Gross Intact Sonicate filtrate extract Donor bird JMV-S 7 + 4/5a 0/4 0/5 0/4 JMV-x462 7 + NDb 0/5 0/3 0/4 JMV-x55 7 + 4/5 0/5 0/5 0/5 JM/102W 22 + 1/1 1/5 0/5 4/5 aThe fraction indicates the number of line 100 (M0 antibody negative) chicks dead or killed with typical lesions of JMV tumor or MD per total chicks at risk. Chicks dead from nonspecific causes were ex- cluded from the data. The experimental period was for 28 days. bND means no data available because all inoculated chicks died of nonspecific causes. 58 Assays for other viruses. Tissue cultures inoculated with JMV tumor cells (described in Table 4) were examined for other avian tumor viruses. Attempts to detect REV antigen by indirect immunofluorescence with positive REV serum were unsuccessful. All three stocks of JMV tumor cells, as well as gamma-irradiated JMV-S cells were examined for avian leukosis viruses and found to be negative by the phenotypic mixing (PM) test. Sera obtained from hyperimmunization with JMV tumor cells were also negative for antibodies to RSV subgroups A through D as indicated by the inability of the serum to neutralize virus. The sera were also negative for REV antibodies by the indirect immunofluorescence test on positive acetone-fixed coverslip antigen. Hyperimmune anti—JMV serum did not react with acetone-fixed coverslip antigen from JMV inoculated tissue cultures as might have been expected if the JMV stocks contained an agent capable of replicating in both cultures and the host chicken (Table 8). Analysis of anti-JMV sera for herpesvirus antibodies. Sera from birds hyperimmunized with JMV tumor cells were analyzed by various methods (Table 9). The sera were negative for MDV specific antibody as indi- cated by the indirect immunofluorescence, agar-gel precipitin and serum neutralization tests. Viable cells of the MSB-l cell line stained with certain of the JMV sera by membrane immunofluorescence of unfixed cells in suspension exhibiting a distinct annular fluorescence. A small num- ber of the cells did not stain. Those sera expressing MATSA antibody with MSB-l cells also appeared to protect chicks inoculated at l-day of age with 50 CLD50 of the homologous JMV liver suspension (Table 9). 59 Table 8. Summary of tests for detection of viral agents other than herpesvirus in JMV tumor cells. Source material Tests Conducted CPEc tested PMa SNb REV 1Jnspec1fied REV Unspecifiéd’ JMV-S - - - y JMV-S - ND - JMV-x462 - - _ JMV-x55 - - - Positive controls + + + NA NA aThe phenotypic mixing test (PM) was done to detect the presence of lymphoid leukosis viruses. A positive test would be any plate con- taining foci as compared with the A, B, C, and 0 subgroups serving as positive controls. RSV-0 (negative control) on C/E cells gave no foci count. bA serum neutralization test was done to detect antibody to RSV of The sera did not. neutralize virus of subgroups A through D as compared with positive subgroups A through D in JMV hyperimmune sera. anti-A through D sera. CSpleen inoculated CEF cell cultures were examined through 3 passages (see Table 1) for any evidence of REV or an unspecified agent repli- cating in cell culture. No evidence of CPE was observed. d Acetone-fixed coverslips from the third passage were examined by in- direct fluorescent antibody technique with positive RE serum diluted 1:20 and unknown JMV antiserum diluted 1:20. stained with RE positive serum at 1:640, but not with negative serum diluted 1:20. There was no fluorescence observed with anti-JMV serum at 1:20. NA Not applicable ND Not done REV positive CEF antigen 60 Table 9. Analysis of hyperimmune sera to detect antibodies induced in response to antigens associated with JMV tumor cells. . a Serology Immunizat1on e MDV antibodyb MSBel Protection Agent Donor .Number of c d number 1nocu1at1ons FA AGP SN cells test JMV-S 1 3 <20 <1 <20 128 + 2 3 <20 <1 <20 8 - 3 3 <20 <1 <20 64 + 4 l <20 <1 <20 8 - 5 1 <20 <1 <20 8 - 6 l <20 <1 <20 4 - JMV-x462 7 3 <20 <1 <20 32 + 8 3 <20 <1 <20 128 + 9 3 <20 <1 <20 256 + 10 3 <20 <1 <20 16 + 11 1 <20 <1 <20 <4 - 12 1 <20 <1 <20 <4 - 13 l <20 <1 <20 <4 - aTwelve-week old 15 x 7 chickens maintained in Horsfall-Bauer units were inoculated with 69 and 178 CL050 of JMV-S and JMV-X462 liver homogenate, respectively. Two additional inoculations were given with 100-fold greater doses at the 15th and 18th weeks after the initial inoculation. All chicks were bled at the let week. bReciprocal of the highest dilution of serum staining acetone—fixed MDV infected monolayer chick kidney cultures by immunofluorescence (FA), reacting with MDV precipitin antigen (AGP) or neutralizing 50 percent or more of cell-free feather follicle derived MDV at a final dilution of 1:20 (SN). cThe data represent the reciprocal of the highest dilution of serum to give detectable membrane fluorescence on MSB-l cells in suspension MATSA . dProtection indicated by the ability of one m1 of undiluted hyperim- mune (MATSA positive) sera given at 1 day of age to prevent death in at least 75 percent of chicks challenged at the 2nd day with 50 CLD50 of the homologous JMV liver suspension. Death in control groups receiving normal serum before JMV challenge was 100 percent. 61 The actual results of the protection test are presented in Tables 10 and 11. The specificity of the sera for M0 tumor cells and the MSB-l cells has recently been reported (Witter §t_al,, 1975) and suggests the possible existence of tumor-specific antigens common to both types of tumor cells. Analysis of JMV tumor cells for herpesvirus associated antigens. JMV tumor cells were examined for membrane antigens (MA) and for MATSA by indirect membrane immunofluorescence (Table 12). JMV tumor cell prepa- rations were negative for virus specific MA but did express a distinct annular surface fluorescence when stained with MATSA positive JMV-S antiserum (Figure 7). Staining was confined to the large lymphoblastic cells of the JMV and MSB-l cell suspensions. Sera specific for MDV and HVT antigens did not stain the tumor cells but their specificity was shown by their reactivity with virus infected DEF cell culture antigen. Normal spleen cells in the preparation of JMV tumor cells did not stain with MATSA serum, nor did normal serum react with JMV or MSB-l cells, thus excluding the possibility that the reaction is due to immuno- globulin producing cells. B and T cell surface markets. JMV-S tumor cell suspensions were stained with antisera produced specifically against chicken B and T cells (Table 13). In three trials the JMV cells were stained strongly with anti-B serum and were equal in intensity to the staining observed with anti-B serum against bursa cells. Anti-T serum also stained JMV cells but to a lesser degree than the homologous system with anti-T serum and thymus cells. Although the intensity of staining seemed to .omo mo zoo1p “o Eooompooo o>wuooomog on» Go PE moo oo>vm moo: mxowgu .mozocm Ppo Low omega om mo: omo mo whoo1N no omcop_o;o >zoo .ooosm Loo mxowcu Lo osmosoc somoo— oopco>ogo Eagom powwowmmom Lo xooo .Eooom o>wuomoc ogu ope: oosoosoo mo mgo>w>szm ucoocoo mm co oomoo mo; cowpoouogoo .mv—owsu 00—. 95% m>wummwc Evonwucm OZ 5. vazmme mmz wmcoammg >26 mSHm 62 1 o o_ m\m oocoz 1 o op m\m o>wpomoz 1 o m_1F_ N.F_ o\o F momo_ 1 N Np m\m F oomop + m op o\P F momop + N N\o m come, 1 N N_ o\N m ammop + o o\o m mmmop ocowpoouogo meo>wosom omoom mxoo coo: oomcoomog mcowpon_coeew .oz Lo oz xuwpousoz >2o Lo .oz Eogom .oLom wooszsoQA; m1>2o cow: poop cowuoopogo .o— o_ooh .omo mo zoo1p uo Eosomwuoo o>wpooommc ozp No FE oco co>wm woo: mxowzo .mooogm Fpo Loy omozo om mo; omo Go ozoo1N po mmooppozo >2oo .ooosm goo moonszo somso_ oouco>oso Eogom Homeowwwom mo zoo; .Fogucoo Eooom o>wuomoc ozp sow: oogooeoo mo mgo>w>oom pcoogoo mm :o oomoo mo: cowpooposoo .mxowzo cop weep o>wuomoc zooompco a: ow oosomoos mo; omcoomog >zo ozho 63 1 o o.o_ m\m oocoz 1 o Pp1m N.m o\o o>wuomoz 1 o m~1pp o.op m\m F mmmo_ 1 o Np1m N.o_ m\m F ommo_ 1 _ NF1PF m._~ o\m P mmmop + m M\o m mNmoF + N N\o m mNmo— 1 o N_1m N.o_ o\o m mNmo_ + m m_ o\_ m mNmo_ + o mp m\p m ono— ooowpoouooo mgo>_>gom omooz mzoo coo: oomcoomog mcopuochossw .oz mo .oz znw_opgoz >2o 4o oz sosom .ooom moosewgoozz Nmox1>zo zpwz “mop :owpoopogo .F— oFQo» 64 .o>ppoooo u o ooo mo>pppmoo u + monogpm zgo> 1 ++ mo omgoom ocpcpopm .mogoppou oozopoooe omxpmoo :o moon mo: oocoomogoopmocoeep mocpm-opopmmoo poo oppoo Fooop>pocp co mpcoooo .oopoooo poo oppoo Looopooozo .ooppop>oo ogoooopm ozp w oppoo om pmomp po mo moposom mpooppoog m op oppoo o>pppmoo pcooooo :oozo .Lopoeopzoosm: o op oppou oppmopoozosxp mooop ocppoooo on oopoeppmo mppmo wo gonzozo ocoo poz u oz cooppoo ouopgom oopopoommo1oos=p omoompo m.xosoz n oz 1 <2 ”mooppop>ogoo< o o + o++ oz uumo 3No_\2o o o o o oz coopom posgoz o o.m w N.—o o o oo— p1omz o E .1. 93 o o 3 53% mmxéo o N.p w m.om o o um coopom Noox1>2o o oo.m w m.mw o o on oompom m1>2o Eooom A2o1ppco p>z1ppco >oz1wpco 1ozoezp ”opp: poospooop oopmo1mopomooo=pp oppoo po pcmogmo pcooooo .oomppco ooososos oopopoommo gosop oco cooppco ocogoEos Low oppoo >zo Lo mpmzpoc< .N_ opoop 65 Figure 7. A, B, and C: Three fields illustrating Marek's disease tumor-associated surface antigen (MATSA) on lymphoblasts from spleen of a chicken inoculated with JMV-S and stained with anti-JMV serum by the indirect membrane immunofluo- rescent technique. X1580 66 Figure 7 67 .ooppooooogo ppoo oooo op moo pcoogoo goo .mooxopzo No oopp posooo opo1xooz1m zoom woo: oppouo .m1>2o opp: oopopoooop o mxupzo cop x N mo No ocpp opo1ooo1op op m mo mcompom moogoszp Eoom oooooooo mcopmcoomom Losop >zoo . m oopooopmoo :oppoomo popow o+ o op o zoom opoom o :o oomoo oppmoopcp o>ppopom .popop goo oposom Loo oopcooo oppoo oop pmoop pzo oazmpmcopop m>ppopooo oopomoooopm oppoo pcoogoo .mooxooe upcooppco oooooom ppmo o oco o so» mpmopoozoszp >2o mo mpmopoc< .m_ opoop 68 indicate a B cell surface antigen, the staining with anti-T serum could not be ignored and therefore, the data appear to be inconclusive at this time. Alloantigenic markers on JMV tumor cells. In an attempt to determine whether JMV tumor cells were of donor or host origin, antisera directed against B2 and B21 §_blood group (major histocompatibility) antigens were used. The analysis of tumors produced in Bgl/Bgl-line N and §;-/ fig-line 72 hosts are presented in Table 14. Both 82 and 82] antisera reacted normally with their homologous small lymphocytes present in the JMV tumor preparations, as well as the homologous normal control chicks; however, the staining reaction of tumor cells in both lines differed from that of normal host lymphocytes. The transformed blast cells reacted to both 82 and 82] antisera, but the staining of tumor cells with anti-B2 serum was weak in comparison to the stronger stain- ing with B2] antiserum. The data presented indicate that 8 blood group alloantigenic markers on JMV lymphoblastic tumor cells differ from those on lymphocytes of the host in which the tumor was produced and that they carry an alloantigen closely related, if not identical, to the 821 antigen. DNA-cRNA hybridization. Table 15 represents preliminary results ob- tained by Dr. Lucy F. Lee. The data indicate that DNA extracted from 5 of 6 tumor cell suspensions having MATSA positive cells in excess of 40 percent hybridized to a degree with 3H-labelled MDV-cRNA, i.e., the cells were found to contain MDV-DNA sequences. The relative amount of DNA varied from tumor to tumor. 69 .mxopzo poopooo Foezo: soop oooooooo mcopmcoomom op pcmmogo mopzoozoszp oopoooooo1posooo .pposmo .m1>2o zpwz ompopzoocw mxopzo mo mooopom moooosop Eoow oogooooo mcopmooomzm co pcomooo mopzooooszp oopsooooo posgo: .pposmo .+ o op 0 mo opoom co oomoo oppmcopcp o>ppopom m1>2o opp; oopopooocw mxopzo wo mooopom moooosop Eogw oogooogo mcopmooomom op pcomooo mppoo ooop1pmopo .ooooo o .oopoopo>o moo; oposom Loo oppoo oop pmoop pzo ompmopoozoeoF1>2o oopo oozpocoo Eooomppo< oppoo poopcoo oppoo Logoulzzo coxopzo poopaooom ofioppmcopcp o>mpoFoNv ocpomoooopm mpooo w .mppoo Logop >2o No oooooom mop :o mooxgos upcooppcooppo ooopo m. op moooogomppo on oppoo gosop m1>2o No ooopo: opoopoopomooop ozp opogpmcoEoo op mpoEopp< .o_ opoop 7O 1 NpN omN p moezzp mpoo 1 oNp omp _ ooo poeooz + ooNN omNN _ oomo1>oz 1 com Npm o oopxp N 1 on Npm o oopxo _ omppeo ooopom poeooz + on ooo oN oopxp o + pom on NN ooFxN m + mom mom oN oopxp o + mom mom oo oopr m + on opo No oopxo.p N 1 moN ooN ON oopxop _ omppmo zoo—om m1>2o moocoooom N om; p om; ooz oooNpopoooz o>pppmoo mopoo N \mppoo * pooo oz ooppooop1: opp: zo mo coppoNpopoozz on oppoo Loosp >2o op osoooo >oz No coppoopoo .mp opoop m 71 .poopcoo o>pppmoo ozp mo tom: mo: >oz No mpopomp ppoopocp ooooopmooo moo: mooppooooooo ooo woos Lo poooooo oN mpcoou .oz ooppooop1zm opp; moopppm :o ooNNopooz; woo: zo o>pppmoo omoompo m.xoooz u >oz opoops Loo mpoooo 1 Zoo .oooppoo opepomom oosop oopopoommo omommpo m.xogoz u oooo< 72 Electron microscopy. The cell suspensions prepared from portions of the spleen sections given to Dr. Keyvan Nazerian are described in Table 16. Although each preparation contained MATSA positive cells, there was no evidence of herpesvirus or any other virus (Figure 8). 73 Table 16. Description of JMV tumor cells analyzed by the electron microscope. - % MATSA positive Agent Donor lymphoblasts EM JMV-S 83 - 50 - 77 - 81 - 4o — m-th-J JMV-x55 37 - 76 ' - 65 - 50 - 7O - 01-9de Abbreviation: EN = Electron microscope. 74 ooo.oNx .m1>2o opp: oopopoooop :oxopzo o No coo—om moooo23p mop Eogm poopoozoeop >zo o mo zooooooope ooopoo—N .m oozopo 75 N monopo DISCUSSION The major purpose of this research was to biologically char- acterize JMV and to determine whether lesions induced by JMV tumor cells were due to transplantation of the tumor cells or were induced by MDV released from the tumor cells. Confusion has arisen because MDV is a highly cell-associated virus and Sevoian (1964) developed JMV by repeatedly passaging in birds tumor cells that were originally induced by the JM isolate of MD. Yoon and Kenyon (1975) and Sevoian and others (Shieh and Sevoian, 1974, 1975; Hamdy and Sevoian, 1973; Kenyon gt_gl,, 1969; Kim gt_gl,, 1972) have considered that a highly virulent virus is released from JMV cells which induces the lesions observed. Spencer §t_al, (1973a) postulated that gross lesions prior to 14 days post inoculation were not virus induced but were due to cell transplantation and that survival was related to the host's ability to reject the transplanted cells. Lesions observed at a later stage (within 3 weeks post inoculation) were considered to be induced by MDV derived from inoculum cells. The results presented in this study demonstrated that JMV tumor cells were apparently lacking in infectious virus. This was based on the absence of (a) herpesvirus cytopathology or viral antigens in cell culture, (b) virus lesions in the CAM and (c) the production of lesions or immune response in sus- ceptible chickens. 76 77 Probably the most sensitive method for establishing that JMV was free of MDV was the repeated inoculation of chickens with JMV cells in increasing doses and the subsequent analysis of the resulting sera for evidence of herpesvirus infection. Since it has been previously 2 to 105 stated that 10 tumor cells are necessary to induce microplaques in cell culture (Churchill and Biggs, 1967), the number of JMV tumor cells assayed in either chickens or cell cultures certainly would seem sufficient to detect a herpesvirus. The data established that JMV cells must be intact in order to produce lesions. Furthermore no virus was isolated from feather follicle epithelium of JMV inoculated birds; whereas, virus was isolated from this site when birds were inoculated with the JM isolate of MDV. Jones §t_al, (1969) also re- ported that passage of JMV cell-free material was unsuccessful. An association between JMV and MDV has been indicated by immunization studies that have demonstrated an antigenic relationship between JMV, HVT, and MDV. Sevoian (1967) demonstrated that offspring of dams immunized with JMV tumor cells had a higher degree of resist- ance to JMV and MDV and that blood from MD infected chickens adsorbed protective antibodies from anti-JMV serum. Mason and Jensen (1971) were able to demonstrate that HVT protected chicks inoculated 7 to 9 days later with the lethal JMV agent and suggested that the protection was a reflection of antigens being shared between JMV and HVT. Sevoian and Weston (1972) also reported that HVT vaccination induced protection against JMV challenge. By means of complement fixation, serum neu- tralization and neutralization kinetics, Hamdy and Sevoian (1973) re— ported that JMV appeared closely related to JM virus, but that HVT was 78 less antigenically related to JM and JMV. Their results indicated that the complement fixation titer of JMV serum with JM virus-specific antigen was comparable with the homologous JM reaction and that the serum neutralization titer of JMV serum with JM virus exceeded the» homologous reaction. Spencer §£_El° (1973b) reported that vaccination with HVT 1 week prior to challenge with JMV significantly suppressed the development of wing web tumors. The inoculation of JMV tumor cells has also been shown to make chickens refractory to further chal— lenge with higher doses of JMV tumor cells or virulent MDV (Sevoian, 1967; Kenyon 35431., 1969; Hong and Sevoian, 1974; Shieh and Sevoian, 1974). The data presented here appears to corroborate these findings in that birds given an initial sub-lethal dose were able to survive increased doses of JMV cells. Analysis of the JMV sera indicated that there was no reaction with MDV infected cells by immunofluo- rescence or serum neutralization, nor with M0 specific antigen in the immunoprecipitin test. In addition hyperimmune sera, produced by a series of increasing doses of JMV cells, appeared to react by membrane immunofluorescence to cell surface antigens on MSB-l cells. The data also indicates that the titer of individual sera against MSB-l cells seemed to relate to the ability of the sera to protect chicks against 50 CLD50 of JMV tumor cells. Although the numbers of chickens included in the protection tests were not sufficient to establish statistical significance, there did seem to be a trend that would warrant addi- tional study. The limiting factor in the present study was the amount of immune sera available. The results on passive immunization against JMV challenge supports the work of Kenyon et a1. (1969), 79 Sevoian (1967) and Hong and Sevoian (1974) who observed a neutralizing effect in serum of birds which had survived JMV. The nature of the neutralizing effect was indicated by an increased latent period and reduced mortality. The bi-phasic mortality associated with JMV re— ported by Spencer gt_gl, (1973a) poses a question. In light of reports that inoculation of JMV cells appears to protect chicks against chal- lenge with MDV, it is interesting that chicks did not appear to be protected by the initial JMV cell inoculation and were diagnosed with M0 at the 5th week after inoculation. The possibility of adventitious infection of inoculum cells seems likely. The mechanim by which passive immunization protects against JMV has not been established. However, the results presented in this research make clear that the effect was not due to neutralization of infectious MDV as implied by Shieh and Sevoian (1974). It must be pointed out from their data that the JMV preparation used in their study was quite different from the JMV tumor preparations used in this study. More than likely the antibody reported on here is directed against tumor specific antigens or histocompatibility antigens on the tumor cells or both. Because there was no evidence that infectious MDV was associ- ated with JMV, the possibility that JMV cells were transformed by an avian RNA tumor virus was considered. JMV cells were examined for exogenous avian tumor viruses of the A through 0 subgroups and for REV. Tests for these viruses proved negative for any evidence of in- fection. The possibility of endogenous tumor viruses was considered but since nearly all avian cells contain or express the ubiquitous 80 endogenous avian tumor virus genome, the value of such tests in de- termining the course of the original transformation of JMV cells was questionable. Also negative results were obtained from tests for an unspecified replicating agent that would replicate in cell culture and induce an immune response in chickens. These findings added to the other evidence that JMV cells were totally free of replicating virus. The data tend to support the hypothesis of Spencer and Calnek (1967) that JMV tumors resulted from transplantation of inoculum cells. Their hypothesis was based on the sudden onset of the disease, tumors at the site of inoculation, and enlarged leukotic livers and spleens. These observations were confirmed in the present study. In order to lend further support to the hypothesis, the membrane immuno- fluorescence technique was used to demonstrate §_blood group allo- antigenic markers on JMV cells in an attempt to establish the trans- plantability of the cells. The §_gene is concerned with histocom- patibility (Schierman and Nordskog, 1961) and can be expressed through the function of the lymphocytes (Schierman and Nordskog, 1962). The antigens of the §_and §_system are common to lymphocytes and erythro- cytes, while A, D, and L antigens are erythrocyte specific (Schierman and Nordskog, 1962). B antisera have been shown to react specifically with their 8 erythrocyte antigen, but not with other known alloanti- genic markers (Pazderka gt_gl,, 1975). Based on the intensity of staining, JMV-S tumor cells appeared to carry antigens more closely related to 82]. The data suggest that JMV is a transplant and carries §_blood group surface antigen detectable with anti-B21 serum. The 81 .Egl allele has been associated with resistance to Marek's disease (Pazderka gt_al., 1975), a finding which makes the detection of the Bgl-allele on JMV cells important in tumor immunology and the study of regression. In this regard it is interesting that line N is homo- zygous for Bgl-and is resistant to MDV, but preliminary observations (unpublished data) indicate that line N is highly susceptible to JMV tumor induction. The observation that JMV-S hyperimmune serum reacted with JMV and MSB-l tumor cells, both derived from individual MD lymphomas, was considered evidence for the existence of a common tumor antigen since the antigen appeared unique to the tumor cell. This common antigen demonstrated on two classes of MD lymphomas in this study has now been called MATSA, an acronym for Marek's associated tumor specific antigen (Witter et_gl,, 1975). The possibility that the common antigen was merely a histocompatibility antigen was considered; however, the reaction was confined to the lymphoblastoid cells and was not found on normal lymphocytes in the same JMV preparation. In addition the observation that normal spleen cells did not fluoresce with JMV or MSB-l antiserum was considered evidence for the specificity of the reaction. Additional work has been done at the Regional Poultry Research Laboratory on the specificity of this tumor antigen (Witter et_al., 1975). As shown by the work presented here and by Witter §t_al. (1975) the lack of antibodies to viral antigens in MATSA sera may be related to the absence of MDV in JMV cells. It was also reported that MATSA 82 did not appear related to embryonic antigens contained in DEF and CEF since adsorption of JMV sera with normal DEF and CEF failed to reduce the MATSA activity of the sera. The specificity of the MATSA sera produced in chickens with JMV and rabbits with MSB-l cells was shown by reaction with M0 tumor cells of three classes; the MSB-l cell line, MD lymphoma cells induced by the GA isolate, and JMV tumor cells. The sera did not react with REV tumor cells, RNA avian tumor virus transplant RPL-16 or with the transplantable lymphoid tumor (TLT) cell line (Nazerian gt_al., 1976). There were antigenic differences noted between MATSA of JMV and MSB-l cells. From the titration of antisera on homologous and heterologous cells and by adsorption tests with homologous and heterologous antisera it was evident that a common antigenic determinant existed but that these antigens were not com- pletely identical. A similar type of cell surface antigen was reported by Powell §t_al, (1974) on cells from HPRS line 1 and MD lymphoma cells by using specific antisera raised in rabbits against suspensions of MD lymphoma cells. The high proportion of lymphoma cells and HRPS-l cells stain- ing with the anti-tumor serum also supports the finding of a tumor specific antigen. The MATSA reported in this study and by Witter §t_al, (1975) and the tumor specific antigen reported by Powell §t_al. (1974) may explain the protection against JMV by another MD related herpesvirus, HVT, and MDV protection by JMV. HVT is currently used as a vaccine against Marek's disease (Okazaki §t_gl,, 1970) and its effectiveness appears to involve activation of host cell-mediated immunity (Purchase 83 and Sharma, 1974). The appearance of a common tumor-specific antigen on both JMV and MD tumor cells suggests a possible cell mediated re- sponse to antigens on the tumor cells and may imply a possible mech- anism for HVT effectiveness. The immunologic response demonstrated in the protection tests and the ability of anti-JMV serum to detect tumor specific antigen is indicative that the chicken is able to recognize the tumor antigen and illicit an immune response. The importance of humoral immunity in tumor regression is not known but it was observed that immuno- logically competent birds (4 to 6 weeks of age) could withstand in- creasing doses of JMV cells. If humoral antibodies were induced by the first injection of cells, these may have blocked the tumor anti- genic sites on cells, thereby, preventing a cytotoxic or cell-mediated immune response from occurring. The occurrence of blocking antibodies has been previously reported (Hellstrfim and Hellstr6m, 1973). Attempts to classify JMV tumor cells on the basis of B or T cell markers were not conclusive. The predominance of T cell antigenic markers on MD tumors and cell lines has been well documented (Rouse gngfl;, 1973; Nazerian and Sharma, 1975; Powell gt_al., 1974; Powell and Rennie, 1974; Payne and Roskowski, 1972) and suggests that the transformed cells in MD tumors are T lymphocytes. In the three trials presented in this study, the intensity of staining on JMV cells with anti-B serum was consistently stronger than with anti-T serum on JMV cells, and compared with the homologous staining of anti-B serum and bursa lymphocytes. The weaker staining of JMV cells with anti-T serum could not be explained and differed from the homologous anti-T 84 serum with thymus cells. The variation from trial to trial in the percent of cells staining may have been due to differences in the conditions of the cells in the preparations. Staining of JMV cells was especially influenced by the stage of tumor development. For example there was a poor cell yield from necrotic spleens of birds near death. Because the data challenges the theory that only T cells are transformed in MD tumors, it will be necessary to conduct addi- tional experiments before a conclusion may be drawn. Development of a JMV cell line maintained jg_yjtrg_would provide a more homogeneous population of cells as opposed to the ig_viyg_system. The jg 3119 N system is confounded by many cell types in the lymphoma and both B and T cells may be present in tumor cells containing viral genome. Another explanation may be that the B cells are transformed and the T cells are present as part of the host's immune response. The specificity of the MATSA on JMV cells was further sub- stantiated by the contribution of Dr. Lucy F. Lee. The existence of MDV-DNA sequences in JMV cells was established by hybridization be- tween DNA extracted from those JMV-S tumor preparations having greater than 40 percent MATSA positive cells and 3H-labelled MDV-cRNA. The apparent lack of rescuable virus from JMV tumor cells may possibly indicate that JMV only has DNA sequences in common with MDV sequences that control induction of Cell transformation. The application of BUDR or IUDR to JMV tumor cells may induce virus production as in the Raji cell line or MSB-l cell line, in which case it could be assumed that the JMV tumor cells possessed complete genome. Although DNA hybridi- zation studies demonstrated that at least part of the MD genome is in 85 JMV tumor cells, the use of gamma irradiation at 5000 r, which is sufficient to inhibit cellular proliferation, was not able to induce rescuable virus from the tumor cells. The presence of MDV-like DNA sequences in JMV tumor cells and the lack of rescuable virus indicates a similarity with the non- producer Raji cell line, a human lymphoblastoid cell line derived from a Burkitt lymphoma (BL). The Raji cell line does not express Epstein- Barr virus (EBV) or viral antigens, viral capsid antigen or early antigen by the conventional methods of cell culture, egg inoculation or laboratory animal inoculation (Klein, 1973). Thin section of BL tumor biopsy examined by electron micros- copy is negative for virus (Klein, 1973). Similarly thin section of JMV spleen tumors expressing MATSA were also negative for virus particles. Complete viral genome was demonstrated by annealing Raji cell DNA with purified radioactive fragments of EBV-DNA (zur Hausen and Schulte-holthausen, 1972). Raji cells were made resistant to BUDR by incorporation of 100 ug of the drug into the culture medium for 6 months. After removal of the drug, repressed EBV particles were detected (Hampar gt_al,, 1972). Incorporation of IUDR and BUDR into MSB-l cells resulted in a higher number of cells actively pro- ducing virus, which indicated a close relationship between virus and host-cell DNA (Nazerian, 1976). Possibly the use of such drugs would induce JMV tumor cells to release complete virus. The importance of this research has been in the biological characterization of the JMV sources used in this study and has pointed out that not all preparations of JMV may be as described in this study. 86 Two controversial issues have been discussed and clarified. First the claims that infectious virus was responsible for JMV lesions have been refuted by the various techniques used to detect virus or viral anti- gens in the three preparations of JMV. Second, evidence that JMV is an MD tumor cell appears to be established because JMV and MD tumor cells have a common MATSA and have MDV-DNA sequences in common. No virus could be recovered from JMV cells by jg_yjyg_or in_xitrg_pro- cedures possibly because JMV tumor cells lack complete MDV genome; whereas, virus could be recovered from MD tumor cells. Work with B_ blood group alleles has provided support that JMV is a transplant. It appears from these data that JMV is a non-productively infected transplantable cell lacking any rescuable virus. The establishment of a JMV tumor cell line in continuous culture would be useful in further studies on neoplastic transformation and immunity to Marek's disease. SUMMARY This paper deals with the characterization of the lymphoblastic cells of JMV Marek's disease tumor and their inter-relationship with the host cell. An experimental model using B_blood group surface alloantigens to detect differences between tumor cells and host lym— phocytes suggests the probable non-host origin (transplantibility) of the tumor. JMV tumor cells appear to be devoid of replicating herpes- virus and viral antigens, but possess MDV-DNA sequences and carry Marek's disease tumor-associated surface antigen (MATSA). These trans- formed cells possess or appear to possess B cell surface antigenic markers, although conclusive evidence is lacking. The appearance of B cell surface markers is in contrast to other M0 transformed lympho- blastoid cell lines which have T cell surface antigenic markers. The importance of this work lies in three major areas. First, claims of infectious virus associated with JMV lesions have been refuted, but evidence for JMV as a class of MD tumor cell has been firmly estab- lished; these points have been highly controversial up to this time. Second, the finding of B cell markers on JMV cells may, if confirmed, constitute evidence contrary to a central theory of MDV-induced onco- genesis that only T cells can be transformed by MDV. Third, the characteristics established for this unique tumor cell now permit its use as a tool in further studies on NO: 87 RECOMMENDATIONS Further investigations into the transformation of bursa- derived cells would substantiate the findings of this study. If indeed JMV tumor cells are transformed B cells, they would provide another tool for further study of neoplastic transformation. The development of a JMV tumor cell line in continuous cul- ture would provide a homogeneous population of cells from which more conclusive data could be obtained concerning the B or T cell origin of the tumor cells. Treatment of such cells with BUDR or IUDR would possibly induce the expression of virus particles, which would further establish the association of MDV with JMV tumor cells. Further investigation into malignant transformation in the absence of complete virus should be done using JMV as a tool. 88 LITERATURE CITED LITERATURE CITED Agricultural Research Service, USDA; An Evaluation of Research on Lymphoid Leukosis and Marek's Disease, June. 1975. Ahmed, M. and G. Schidlovsky: Electron Microscopic Localization of Herpes Virus Type Particles in Marek's Disease. J. Virol. 2: 1443-1457, 1968. Akiyama, Y. and S. 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