PRODUCTEON, PUREFICATNXQ.P MD ENFECTED CELL “A” ANTIGEN mm for the Eegree of PhD.» MECHJGAN srmiumvmsm pamaxous — . ;_ 1973 f. ‘ ~ CHARACTEREATION 0F MAREK’S DISEASE 7 LIBRARY t”; Michigan State ' IJndveIekar This is to certify that the thesis entitled PRODUCTION, PURIFICATION AND CHARACTERIZATION OF MAREK' S DISEASE "A" ANTIGEN presented by Philip A. Long has been accepted towards fulfillment of the requirements for PhoDo degree in MOPOHO . // v '/ /\ / peéml/ %. [<2 15g; Major professor Date 3/5/73 0-7639 alumna av '5 HMS & SONS' 800K BINDERY IND. LIBRARY amps RS ”IIIGPDIT. mum; & ABSTRACT PRODUCTION, PURIFICATION, AND CHARACTERIZATION OF MAREK'S DISEASE INFECTED CELL "A" ANTIGEN By Philip A. Long Three immunodiffusion precipitin bands (A, B, and C) were formed between Marek's disease infected cell culture extracts and serum from naturally infected birds. Whether the loss of "A" antigen and patho- genicity during attenuation of the virus by passage in cell cultures is simultaneous or independent may be due to differences in antisers or differences in the strains of virus. This can be resolved only as the MDHV and attenuated MDHV antigen profiles are established by more rigorous chemical and immunologic methodology. Delayed hypersensitivity was detected in MDHV-infected chickens in which "A" antigen was present. The significance of "A" antigen and its relation to cellular immunity is unknown. The role of cellular immunity to the disease state or to the protection offered by the vaccine is yet unknown. The turkey herpesvirus which is nonrpathogenic for chickens has been used as the first vaccine successful for a virus induced neoplastic disease. Although the vaccine does not afford pro— tection in the same manner as other viral vaccines, it is effective against the manifestation of clinical disease. If the mode of action of the vaccine is determined, it may contribute to the understanding of human neoplastic disease and human oncogenic viruses. Philip A. Long The production, purification, and initial characterization of the "A" antigen of Marek's disease from GA-infected duck embryo fibroblast cell cultures were studied. The optimal antigen source was the serum free infected cell culture supernatant fluid harvested for six to eight times at 24 hr intervals. The antigen was purified ZOO—fold with 24% recovery by DEAE Sephadex A925 ion exchange column chromatography, iso- electric focusing, and preparative polyacrylamide disc gel electrophoresis. The antigen has an approximate molecular weight of 2 100,000 with 1 to 4 peptides (82,200; 56,890; 52,480; and 20650) as demonstrated by guanidine H01 agarose column chromatography and SDS-polyacrylamide disc gel electrophoresis. Immunocoprecipitation and periodic acid- Schiff analysis suggests the antigen is a glycoprotein. Trypsin degra— 1251 labelling suggests the antigenic determinant dation and in vitro is associated with the peptide(s) and tyrosine and/or histidine may be an essential component of the antigenic determinant. Immunoprecipitin activity of MDHV-A antigen was destroyed by 2-mercaptoethanol in the presence of 1M urea and 0.052 Brij 35 or 0.01M Dithiothreitol and 6M guanidine HCl indicating the importance of inter and/or intra chain disulfide bridge(s). The "A" has an approximate pI of 6.5 without dissociating agents and a pI of 6.68 i 0.03 in.lM urea and 0.052 Brij 35. PRODUCTION, PURIFICATION, AND CHARACTERIZATION OF MAREK'S DISEASE INFECTED CELL "A" ANTIGEN By M . '1 ‘4'" . aa‘ Philip A! Long A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health 1973 , '3 ACKNOWLEDGEMENTS The author wishes to express his sincere appreciation and thanks to Dr. Leland F. Velicer, Academic Advisor, and the members of my Academic Committee, Drs. D. H. Bing, V. H. Mallmann, and N. E. McCullough for their guidance, counsel and support throughout this study. A special appreciation goes to the author's wife, Mona, and our two children, Carol Ann and Christopher Allen, for their understanding in this long pursuit of intellectual enlightenment. I also wish to thank the numerous persons who have contributed to the research both directly and indirectly. This research has been made financially possible by the cooperative agreement with the USDA Regional Poultry Laboratory, East Lansing, Michigan, the National Institutes Post Doctorate Fellowship (Number F02 AIéSSSS-OB), and the Department of Microbiology and Public Health, School of Veterinary Medicine, Michigan State University. 11 TABLE OF CONTENTS INTRODUCTION . . . . . . . . . . . . . . . LITERATURE REVIEW. . . . . . . . . . . . . Herpesviruses. . . . . . . . . . . . Importance of Herpesviruses . Coding Potential. . . . . . . Structural Proteins . . . . . Cell Transformation and Membrane Antigens Shared Antigens in Herpesviruses. . . . . Marek's Disease. . . . . . . . . . . Etiologic Agent . . . . . . . . . Strains of Marek's Disease Viruses. Pathology . . . . . . . . . . . . . Serology. . . . . . . . . . . . . . Delayed Hypersensitivity and Cellular First Successful Vaccine for Virus Induced Neoplastic Disease . . . . . . . . smary O O O O O O O O O O O O O O 0 MATERIALS AND METHODS. . . . . . . . . . . Tissue Culture Reagents. . . . . . . Preparation of Primary (1°) Duck Embryo Fibroblasts (DEPs) Source of Marek's Disease Virus. . . MDHV-DEF Plate Cultivation and Infection MDHV-A Antigen PI‘OdUCtiOn. o e e o o e a storage Of MDHV-A. e e e e e o o o e e 0 Detection of the MDHV-A Antigen by Immunodiffusion Concentration of Large Volumes of MDHV-A Containing culture F1u1d O O C O C O C O O O Radioactive Labelling. . . . . . . . Immunity. Page UI UIb-L‘ww u \ONO‘GG 10 10 12 12 12 13 13 13 14 14 15 15 Radiactive Assay . . . . . . . . . . . . . . Sucrose Gradient Centrifugation. . . . . . . . DEAE Sephadex Ion Exchange Column Chromatography Isoelectric Focusing . . . . . . . . . . . . Immunocoprecipitation of MDHV-A. . . . . . . . Trypsinization of MDHV—A Antigen . . . . . . . Polyacrylamide Disc Gel Electrophoresis (PAGE) A. B. C. D. Acid-Neutral Protein Analytical PAGE. . . . Sodium Dodecyl Sulfate (SDS) Polyacrylamide Disc Gel Electrophoresis (SDS-PAGE) Analyses of Polyacrylamide Gels . Preparative Disc PAGE . . . . . . Purification of Chemicals. . . . . . . . . . A. B. C. Acrylamide. . . . . . . . . . . . BiS’fiCI’Ylamide e e e o a e o 0 Sodium Dodecyl Sulfate (SDS). . . Preparation of Antisera. . . . . . . . . . . A. B. RESULTS. . . . Pathogenicity and Virulence of GAeMDHV Chicken Antiserum . . . . . . . . Rabbit Antiserum. . . . . . . . . Optimal source Of MDHV-A o o o o o e o e o o o 0 Optimal Harvest Intervals. . . . . . . . . . . . Sucrose Gradient Centrifugation. . . . . . . DEAE Sephadex Ion Exchange Column Chromatography Isoelectric Focusing . . . . . . . . . . . . Analytical and Preparative Polyacrylamide Disc Gel ElectrophorESis O O O O O C O O C O O O 0 Sodium Dodecyl Sulfate Polyacrylamide Disc Gel Electrophoresis (SDS-PAGE). . . . . . . . . . . Trypsinization of MDHV-A Antigen . . . . . . . . . Immunocoprecipitation Analyses of MDHV-A Antigen . iv Infected DEFs Page 15 16 17 17 18 19 19 19 19 20 20 21 21 21 21 22 22 22 23 23 24 24 25 32 34 34 47 47 53 Guanidine Hydrochloride-Agarose Column Chromatography. . In vitro 125 Analyses of MDHV-A Antigen Double Labelling. . . . DISCUSSION 0 O O O O O O O O O 0 SUMMARY. . . . . . . . LIST 0? REFERENCES . . Purification by Differential I Labelling Of MDHv-A. e e o e e o o e e o e Page 55 56 56 59 66 67 Table 10 11 LIST OF TABLES Ih viva chick assay of 28th T.C. passage GAéMDHV DEFs for virulence and pathogenicity. . . . . . . . . . . . . . mHv-A AGN prOdUCtion. e e o a o e e e e o e o e o e o o o MDHV-A AGN production in roller bottle cultures with serum free media 0 O O O O O O O O O O O O O O O O O O O I O O 0 Effect of dissociating agents on MDHVeA AGN titer. . . . . Reproducibility of MDHV-A AGN with isoelectric focusing. . SDS—polyacrylamide gel electrophoresis of MDHV-A preparations . . . . . . . . . . . . . . . . . . . . . . . Effect of trypsin on.MDHV-A antigen immunodiffusion ac t 1Vi ty 0 O O O O O O O O O O O O O O O O O O I O O O O O Immunocoprecipitation analyses of MDHV-A antigen in mixed radioactively labelled infected and control cell culture fluids 0 O O O O I O O I O O O O O O I O O O O O O O O O O Immunocoprecipitation analysis of MDHV-A antigen in 14C- glucosamine labelled infected cell culture fluid . . . . . Analysis of MDHV-A antigen purification by differential double labelling O O O O O O O O O O I O O O O O O O C C 0 Analysis of MDHV-A antigen purification by differential dOUble labelling O I O I O O O I O O O O O O O O O O O O 0 vi Page 23 25 26 32 37 52 53 54 55 57 58 Figure 10 11 LIST OF FIGURES. Velocity sedimentation analysis of MDHV-A in a 5 to 202 sucrose gradient . . . . . . . . . . . . . . . . . Velocity sedimentation analysis of a stored MDHVsA positive preparation before (A) and after dissociation with urea and Brij 35 (B). e a e o o e e e o e o o o o DEAE Sephadex A-25 ion exchange column chromatographic analy818 0f mav-A I I I I I I I I I I I I I I I I I I I DEAE Sephadex A925 ion exchange column chromatographic analysis of MDHV-A by stepwise elution . . . . . . . . Isoelectric focusing of MDHV-A . . . . . . . . . . . . Analytical polyacrylamide disc gel electrophoresis of MDHv-A I I I I I I I I I I I I I I I I I I I I I I I I Analytical polyacrylamide disc gel electrophoresis and immunoelectrophoresis of MDHV-A positive preparations. Preparative polyacrylamide disc gel electrophoresis Of MDW’A. I I I I I I I ’ I I I I I I I I I I I I I I I Duplicate analytical polyacrylamide disc gel electro- phoresis of MDHV~A (Figures 6 and 7) stained with periOdic SCid-SChiff (PAS) a e e e e o o e e e e e o e SDS-polyacrylamide disc gel electrophoresis of MDHVQA and standard proteins. . . . . . . . . . . . . . . . . SDS-polyacrylamide disc gel electrophoresis of MDHVeA preparations I I I I I I I I I I I I I I I I I I I I I vii Page 28 31 33 35 36 39 41 44 46 49 51 INTRODUCTION Although many agents such as viruses, chemicals, and radiation induce neoplasia, common factors must be associated with the transformed neoplastic cells and the manifestation of clinical disease. The fundamental goal of cancer research, prevention of cancer in man, will be facilitated by the elucidation of the altered cellular mechanisms, the host defense systems, and their interrelationships in the neoplastic state. Since the isolation of the first oncogenic virus by Peyton Rous, 1911, researchers have sought to prove a viral etiology of cancer in man. Oncogenic viruses have been demonstrated in many animal species and it is logical to assume man also has virus-induced neoplasms. Animal tumor virus systems provide the models for understanding the interactions of the virus, cell, host, and resulting neoplasms. These models continue to yield new insights on potential control and prevention of neoplastic disease in animals and may provide the neces- sary understanding for human viral cancer research. None of the oncogenic herpesviruses provide a complete model system for studying oncogenesis or the virus-cell-host interrelationships. The limitations and difficulties represented in each system are distinct thus allowing comparative studies and subsequent extrapolation from one system to the other (52). The avian system is the most ideal model currently available based on these parameters: 1) the host species, 2) the availability of isogenic inbred chicken lines selected 1 2 for their susceptibility or resistance (19), 3) the availability of specific pathogen free birds and embryos for cell culture, 4) the ability to form chimeric chicks by embryonic parabiosis, 5) the experi- mental period for disease manifestation, 6) the compartmentalization of the immunologic system (thymus dependent cellular immunity and bursa Fabricius dependent humoral immunity), 7) the availability of cell free infectious virus (9), and 8) the first successful vaccine for virus-induced neoplasia (80). Even though numerous virus-specific antigens have been identified by immunofluorescence, immunoprecipitation, and virus neutralization the relationship of these antigens to the mature virion, to the disease syndrome, to each other, and their location in the infected cell is unknown. The first step in achieving an understanding of Marek's disease and the Marek's disease Herpesvirus (MDHV) will be the identi- fication, purification, and characterization of its induced structural and non-structural antigens. The specific aims of this research were to develop methods for the production and purification of MDHV "A" antigen and to determine its physical and chemical characteristics. The objective of this research was to provide new reagents, puri- fied MDHV-A antigen and a known monospecific MDHV-A antiserum, which could be used to study its role in this disease syndrome by various immunologic techniques. LITERATURE REVIEW HERPESVIRUSES Importance of Herpesviruses Marek's disease of the chicken has been established as a herpesvirus induced neoplastic syndrome (9,10,l4,lS,l6,18,27,75,76,94,99,102,108, 113). Recently other herpesviruses have been implicated as the etio- logic agents in the following cancers: 1) Lucké's renal adenocarcinoma of the frog (11,32,64), 2) Burkitt's lymphoma (21,25,26,43,53,54,60, 67,77,79,81,112) and other specific human lymphomas (44) by the Epstein-Barr virus (EBV), and 3) rapidly fatal lymphomas and reticulum cell sarcomas in a number of primates induced by a latent Squirrel monkey virus, Herpesvirus saimiri (68,69). Circumstantial evidence has been acquired which indicates possible roles for herpes simplex type 2 (RSV-2) in human cervical carcinoma (70) and EBV in human nasOpharyngeal carcinoma (44,95) and infectious mononucleosis (28,42,77). CodinggPotential Herpesvirus DNA (MW 6—10 x 107 daltons) (49,59,90,9l) has a poten- tial to code for a large number of structural and non-structural proteins. The non-structural proteins may serve only to supplement and augment the cell's enzyme complement. The extent to which the viral genome is expressed in infected cells and whether the expression is consistent in different cell types is yet unknown. These 4 virus-induced proteins may be antigenically distinct from those coded by cellular DNA and, therefore, may be assayed by immunologic methods. Structural Proteins Herpesvirus structural proteins are synthesized as immunologically unreactive polypeptides (37) in the cytoplasm (2,37,40,82,89,103) and are transported to the nucleus (2,37,81,102) where they are modified into immunologically reactive subunits (37). These subunits are incorporated into the nucleocapside within the nucleus (37) and the virion matures by acquiring an outer envelope as it passes through the nuclear membrane (96). Histones (6,38,88) and protamines (61) are synthesized in the cytoplasm in HeLa and Trout Testes cells, respectively, and are transported into the nucleus (6,38,61,88). This suggests that the protein transport may be a basic mechanism of eucaryotic cells. Control of cellular transcription has been attributed to both the acid and basic nuclear proteins. The inhibition of their synthesis or transport may play a role in carcinogenesis. Arginine rich proteins associated with herpesvirus DNA may affect cellular genome transcrip- tion evidenced by decreased cell histone synthesis following the appearance of the viral basic proteins in the infected cell nuclei (107). Cell Transformation and Membrane Antigens The cell transformation induced by other tumor viruses, i.e., Rous sarcoma, is associated with virus-specific cell membrane antigens (1,7,34,39,47,57,78,93,110). The abnormal growth of these transformed cells has been attributed to their loss of contact inhibition. Trans- plantation antigens have been isolated from cells infected with adenoviruses and from the membrane fraction of adenovirus-induced hamster tumor cells (45). Herpesvirus-infectec cell cytoplasmic 5 membranes have been shown to have contained virus-specific glycopro- teins identical to those associated with the viral envelope (3,48,50, 100,104). The specificity of the polypeptides was controlled by the viral genome and glycosylation was dependent on the specific cell type infected (105). Specific surface antigens on Burkitt’s tumor cells (21.53.54.60) and cells infected with Marek's disease herpesvirus (12,74) have been detected by immunofluorescence. A positive correla- tion existed between tumor regression and the antibody titer to the associated surface antigens in'sera of patients with Burkitt's lymphoma (21). Shared Antigens in Herpesviruses The first evidence of shared antigens was demonstrated by Mary Fink in 1968 (33) between the herpesvirus of Lucké's renal adenocarcinoma and the Epstein-Barr virus of Burkitt's lymphoma. Group-specific and type-specific antigens were found in the herpesviruses of Lucké's renal adenocarcinoma, Epstein-Barr virus, H. simplex, cytomegalovirus, and MDHV (51,71,83). More recently the results of immunodiffusion and immunoelectrophoresis suggest two antigens are shared in infectious bovine rhinotracheitis herpesvirus, MDHV, and Epstein-Barr virus (29). These findings support the concept of shared group-specific antigens in herpesviruses from diverse animal species. MAREK'S DISEASE Marek's disease (MD) was documented in 1907 as a clinical mani- festation of chickens by J. Marek (65) and has been referred to as range paralysis, neuralymphomatosis, and acute leukosis. It is an extremely contagious lymphoproliferative or lymphoinfiltrative disease characterized by lesions in the nerves, visceral organs, and skin. Etiologic Agent Van der Walle and Winkle-Junie (109) proposed in 1924 that MD was caused by a virus similar to that of herpes zoster of man. In vitro and in vivo experiments in the past five years provided the initial evidence that a cell associated herpesvirus was the etiologic agent (4,5,13.14.15.16,27,75,94,99,ll3). Koch's postulates were fulfilled only after low titers of cell-free infectious MD herpesvirus (MDHV) were obtained from infected cell culture (10.18.76) and infected bird feather follicle extracts (9). The MDHV is a group B herpesvirus (58) with the following characteristics: 1) the diameter of the enveloped virion is 200 to 400 mp, 2) the diameter of the nucleocapsid is 85 to 100 mm, and 3) the viral capsid has 162 hollow centered 6 x 9 mp cylindrical capso- meres arranged in icosahedral symmetry (9,27,75,76). The MDHV genome contains DNA with a buoyant density of 1.706 and a sedimentation coefficient of 568 which corresponds to a guanine/cytosine content of 462 and a molecular weight of l x 108 daltons, respectively (59). Strains of Marek's Disease Viruses Numerous strains of MDHV have been described and classified by the primary pathological manifestations as neurotropic. represented by the JM isolate (97,98), or viscerotropic, represented by the GA and RPL 39 isolate (22.85.87). Pathology The neural form of MD is characterized by paralysis and/or paresis of one or more of the extremities of chickens. The nerves, especially the vagus, brachial. sciatic, and the nerve of Remark, are grossly enlarged, and altered in color and general appearance. 7 In the visceral form the bird may or may not have signs of disease such as emaciation. ruffled feathers and lethargy but regularly have lymphoid tumors in the gonad. spleen. liver, lungs, heart. and proventriculus. The skin may have nodular enlargements associated with feather follicles. The nerve lesions of MD have been classified into two types based on histological differences (5): 1) Type A is characterized by the extensive infiltration of lymphocytes. plasma cells and dark staining "MD" cells, and 2) Type B is characterized by edema and the infiltra- tion of relatively few cells. The latter is thought to be a regressive stage. The visceral and skin lymphoid tumors are masses of pleomorphic lymphocytes either in a well defined area (nodular) or infiltrative throughout a given organ (diffuse). The intermediate and transitional layer cells of the feather follicle epithelium are vacuolated and many nuclei in the latter layer contain characteristic Cowdry type A inclusions. The lymphoid organ, bursa of Fabricius, usually exhibits both cortical and medullary atrophy. necrosis. and cyst formation. The clinical disease and pathology are influenced by the following parameters: the strain of MDHV (85.87.98), the age of the bird at exposure (5.97), the sex of the bird (19.85). and the genetic consti- tution of the host bird (5,19). Serology. Serologic investigations employing the Ouchterlony immunodiffusion agar-gel precipitin test (AGP) or the direct and indirect fluorescent antibody test (FA) have been used to assay antigens and antibodies in MDHV-infected bird tissues and sera. and antigens in MDHV-infected 8 cell cultures (l3,l4,55,56,7l,72,74.84,85,86,87,106,lll). The FA test was reported to be more sensitive than the AG? test (86). The data indicated no relationship existed between the two assay systems. The AGP test separates multiple antigen-antibody reactions and indicates the degree of identity among the antigens. Sera from naturally infected birds and sera from hyperimmunized birds usually forms one to three and six immunOprecipitin bands, respectively (14), with infected cell extracts. Three bands identified by both sera were designated A, B, and C. The "A" antigen was found in infected cell extracts and in concentrated infected cell culture supernatant fluid (14). Marek's disease herpesvirus "A" antigen (MDHV-A) synthesis in cell culture and the pathogenicity of the infected cells as monitored with the in viva chick assay were reported last simultaneously by the 33rd passage (17). Other results indicate either factor can be lost independently of the other (73,87,111). The conflicting results may be due to differences in the strains of virus. This can be resolved only as the MDHV and attenuated MDHV antigen profiles are established by more rigorous chemical and immunological methodology. Specific immunofluorescence (IF) was detected in cell cultures and bone marrow smears from infected birds by rabbit antisera induced by repeated injections of infected chicken blood preparations (55). Infected cell cultures exhibited both cytoplasmic and nuclear staining with the latter increasing during extended infection times (55). The same IF pattern was detected with hyperimmune chicken anti-JMAMDHV sera. The change in immunofluorescence from cytoplasm to the nucleus was correlated with the increased cytopathic effect (CPE) in the infected cell cultures (84.87). A FA study employing cloned MDHV isolates, their respective infected cells, and their induced antisera 9 in various combinations demonstrated no differences in the brightness of staining as seen by the naked eye (84). It was concluded that all the isolates were antigenically identical. This conclusion may be invalid because the sera were not cross absorbed to remove the anti- bodies specific for common or shared antigens. Immunofluorescence was not detected in lymphoid cells associated with lesions even though the buffy coat and tumor inoculums proved highly infectious for in vitra cell culture and in viva chick assay experiments (102). It is possible these cells contain a latent or integrated partially depressed viral genome and therefore fail to produce virus-specific antigens associated with productive infections. Delayed Hypersensitivity and Cellular Immupity Byerly at al. (8) skin tested MD—infected chickens for delayed sensitivity with three preparations, cell culture media and cell extracts from MD—infected cell cultures and feather follicle extract of MD-infected chickens. Chickens with and without gross lesions have positive reactions with both the feather follicle extract or the cell culture supernatant fluid; infected cell extract elicited positive reactions only in birds with gross lesions. Ninety-eight percent of the birds' sera had precipitins for l to 3 antigens when assayed by the agar gel precipitin test. Fauser at al. (31) demonstrated delayed hypersensitivity by both the classical in viva skin (wattle) test and the in vitra radial test for macrophage inhibition. A partially puri- fied "A" antigen preparation was used (donated by this author) and only infected birds responded in both test systems. Specific pathogen free chickens housed in isolators were negative by both tests. The "A" antigen was present but the significance and the relation to cellular immunity is unknown. 10 First Successful Vaccine for Virus Induced Neoplastic Disease The turkey herpesvirus (HVT) which is non-pathogenic for chickens (114) and is used as a vaccine (80) has an antigen which forms an immunodiffusion line of identity with MDHV-A antigen (114. and L. F. Velicer unpublished). The role of this related antigen in the oncogenic syndrome or the protection afforded by it is unknown. The vaccine does not inhibit either infection or subsequent replication of MDHV as vaccinated birds are viremic for both HVT and virulent MDHV. The vaccine only protects against the manifestation of clinical disease. The effects of cellular immunity or humoral immunity on the infection cycle, the neoplastic response, and/or action of the vaccine are also unknown. If the mode of action of the vaccine is determined, it may contribute to an understanding of human neoplastic disease and human oncogenic viruses. SUMMARY There is a vast amount of preliminary information which present! a complexity of significant questions concerning the synthesis and morphogenesis of the virus-specific antigens. their immunologic rela- tionships, their localization within the cell, their relation to the mature virion (structural vs non-structural), and their role in disease« or protection. The reagents previously used have lacked the speci- ficity required to make precise studies. Antigens need be identified and purified. and the monospecific antisera prepared and utilized. The experimental techniques. concepts. and knowledge developed from the previous work with herpes simplex virus and pseudorabies herpesvirus permits similar studies with the more complicated highly cell associated herpesvirus of MD. Results from oncogenic herpesvirus 11 research may provide information beyond that of virology; namely, the normal cell transport systems and regulatory controls. Understanding of the normal cell mechanisms and their alterations in the neoplastic state should provide the rationale on which effective therapeutic control or prevention of human cancer can be based. MATERIALS AND METHODS Tiggge Culture Reagents The reagents used were as cited by Solomon et al. (101) in the USDA Agriculture Handbook 4041 with the following modifications: 1) powdered Nutrient Mixture F-lO (NF-10) and Medium-199 (M-l99) (GIBCO, Grand Island. N.Y.) and tryptose phosphate broth (TPB) (DIFCO, Detroit, Mich.) was substituted for 10X concentrated liquid medium. 2) the volume of glass distilled water was adjusted. and 3) the medium was filtered with a 0.22 mu GS filter (Millipore Filter Corp.. Bedford, Mass.). Preparation of Primary (1°) Duck Embryo Fibroblasts (DEFg) The procedure used was as described previously in the Handbook 404 with the following exceptions: l) the embryo mash was trypsinized three to five times (20 min each), 2) the trypsinates after each harvest were kept on ice in 5% calf serum. 3) the combined trypsinates were filtered through three layers of cheesecloth prior to centrifugation and prior to seeding plates or roller bottles, and 4) the cells were kept on ice at a concentration of 2 x 107/m1 in NF-lO - M—l99 - 22 calf serum (growth medium) for up to ten days. 1Agriculture Handbook 404. Superintendent of Documents, U.S. Government Printing Office, washington. D.C. 20402. Price 20 cents. 12 13 Source of Marek's Disease Virus The CA strain of infected DEFs was obtained from K. Nazerian and codworkers (Regional Poultry Laboratory. East Lansing, Mich.) in the 17th passage and was passaged twice before being frozen for storage. The 19th passage served as our base stock. No greater than the 26th passage was used to infect cells for antigen production. Avian cells were stored in liquid nitrogen as described in Handbook 404. MDHV-DEF Plate Cultivation and Infection Approximately 4 x 107 1° DEFs in 20 m1 growth medium were seeded in 150 mm plastic tissue culture dishes (Falcon Plastics, Los Angeles. Calif.) and incubated at 37° in a 52 C02 atmosphere (Hotpack Corp.. Philadelphia. Pa.). The medium was changed after 16 to 20 hrs and a confluent monolayer was formed by 48 hrs. The monolayers were infected with a dilution of cells that induce maximum cytopathic effect (CPE) in 3 to 5 days. The infected DEPs were usually passaged l to 2 times on uninfected monolayers to obtain the number of plates required for roller bottle infection. See Handbook 404 for the procedure used in passaging avian cell cultures. The plastic dishes were reusable for up to 6 months. MDHV-A.An§igen Production MDHV-A was produced in MDHV infected DEF roller bottle cell cultures. Glass roller bottles (BELLCO. Vineland, N.J.) with a surface area of 1330 cm2 were equilibrated and stored in a 52 C02 atmosphere until needed. The bottles were rolled with 25 ml growth medium for 16 to 24 hrs. The rolled medium was replaced with 50 ml fresh growth 8 medium prior to seeding 4 x 10 I° DEFs. The cultures were incubated at 37° C and the medium was changed the following day. The cells were 14 monitored for the extent of growth and uniformity at 48 hrs, the pH was adjusted with isotonic NaHCOa, and stored I' DEFs were added if necessary. Routinely a monolayer was formed within 72 hrs. Each cell culture. When approaching confluency, was infected with 5 to 10 x 107 MDHV infected DEFs (2-4 150 mm plates/roller bottle) and incubated at 37° C. The growth medium was changed 16 hrs after infection. Each roller bottle was washed 3 times with 50 ml Hank's balanced salt solution (HESS) 72 hrs after infection and 25 ml of NF-lO - M-199 without calf serum (base medium) was added per roller bottle. The serum free culture fluid was collected and replaced with base medium every 24 hrs for six to eight days. Storage of MDHV-A The culture fluid was centrifuged at 10,000 rev/min for 30 min at 4° C in a Sorvall GSA rotor to remove cells and cellular debris and stored at -20° C. Twenty-five milliliter aliquots were concentrated 25- to 50-fold by negative pressure dialysis against 0.01M TRIS - 0.05 M NaCl - 0.01M EDTA pH 7.4 (TES) buffer. The concentrated culture fluid was titered for MDHV-A activity by immunodiffusion as described. Detection of the MDHV-A Antiggp by Immunodiffusion The general method was as described in Handbook 404 under agar gel precipitin test with these modifications: 1) the commercial agar (Noble Ion Agar) was purified by washing repeatedly with distilled water, once with 702 ethanol. once with acetone, and dried. 2) 0.01M barbital buffer pH 7.2 - 8% NaCl - 1.2% agar was used rather than 1.52 agar in phosphate buffered saline with 102 NaCl, and 3) 0.0012 sodium azide was used as the preservative. The antigen titers are expressed as the reciprocal of the last positive serial 2-fold dilution. 15 Concentration of Large Volumes of MDHV-A Containing Culture Plaid The culture fluid was filtered through an XM 300 membrane and concentrated on a PM 10 membrane by positive pressure filtration employing the thin channel Amicon concentrator TCF-lO (Amicon Corp.. Lexington. Mass.). The concentrate was dialyzed against 0.01M TRIS pH 7.4, centrifuged at 25,000 rev/min in an SW 27 rotor for 45 min and stored at -20' C. Radipactive Labelligg Infected and control DEF roller bottle cultures were labelled with 0.01 mCi 14C-amino acid mixture NBC-455 (New England Nuclear, Boston. Mass.) or 0.1 mCi 3H-Leu 30-50 Ci/mM (New England Nuclear, Boston, Mass.). Amino acid free MEM or Lou-free MEM were used as the respective medium without calf serum. Infected cultures were labelled after the first harvest of serum free culture fluid 120 hrs postinfec- tion. Normal cell cultures were labelled 96 hrs after seeding. Cell cultures were incubated with 50 ml Leu-free MEM or 47.5 ml amino acid free MEM for 8 hrs to deplete the respective amino acid pools. In the latter 2.5 m1 NF-lO - M+l99 base medium (1/20 normal amount of amino acids) was added with the 14C-amino acid label to supplement the amino acids that were absent in the label preparation. Control and infected cell culture supernatant fluids were harvested 48 hrs after labelling. Rgdioactive Assay One hundredth or one tenth milliliter aliquots from fractions obtained in analyzing radioactive labelled MDHV-A preparations were spotted on 2.3 cm dia Whatman 3MM filter discs. The discs were dried at 50' C, fixed with 52 trichloroacetic acid (TCA) at 4’ C for 20 min. 16 extracted with acetone twice for 10 min each, and dried at 50° C. The dried discs were added to vials containing 5 ml of phosphor scintilla- tion fluid and counted (Packard Instrument Co., Downers Grove. Ill.). Phpsphor Scintillation Fluid PPO 22.7 gms POPOP 1.9 gms Toluene 8 pints PPO - 2,5-Diphenyloxazole POPOP - 1,4 bis-[2-(46Methyl-S-Phenyloxazolyl)]-Benzene Sucrose Gradient Cepgrifugetion Linear 5 to 20% sucrose gradients were prepared in TBS buffer. centrifuged at 50,000 rev/min and 15° C for 11 to 12 hrs with an SW 50.1 rotor (Beckman Instruments, Inc.. Fullerton. Calif.). The 0.4 ml samples were layered on 4.8 m1 gradients. Separate gradients with bovine serum albumin (BSA) were used as a standard 4.48 marker (62) with each centrifugation. Thirty to forty fractions were collected from the bottom of the gradients by piercing the tube with a needle. Each undiluted fraction was assayed for MDHV-A activity by immunodif- fusion. When radioactive preparations were used ten lambda of the undiluted fractions were spotted on Whatman 3MM filters and processed as described. The fractions were serially diluted 2-fold to 1:8 and assayed for MDHV-A antigen at each dilution by the agar gel precipitin test. The 1:8 diluted fractions were monitored for optical density at 280 um. All MDHVeA positive fractions at the 1:8 dilution were further diluted (0.1 ml aliquots) 2-fold until the end point was determined. Positive fractions were pooled. dialyzed with 0.01M TRIS pH 7.4. concentrated, and stored at -20° C. l7 DEAE Sephadex Ion Exchange Colggg Chromatography DEAE Sephadex (A 25 or A 50) was charged as described by Pharmacia (Piscataway. N.J.) and equilibrated with 0.01M TRIS pH 7.4 (initial buffer). The concentrated MDHV-A positive culture fluid was dialyzed against initial buffer and clarified at 25,000 rev/min in an SW 27 rotor. The column size (1 x 50 to 4 x 500 cm) (Kontes Glass Co., Vineland, N.J.) depended on the quantity of protein to be processed. A concentration of 1 mg protein per 1 cc gel bed was found to be the maximum for complete absorption of MDHV—A. The DEAE Sephadex columns were formed and washed with 10 volumes of initial buffer before the application of the sample. The sample was applied in small aliquots at 30 min intervals and once the sample had been applied and absorbed for 2 hrs the columns were eluted with initial buffer until the eluate ) was free of protein as measured by optical density at 280 nm (0D280 (UA 4 UV Monitor, Instrumentation Specialties 00.. Lincoln, Neb.). The columns‘were eluted stepwise with 0.01M TRIS - 0.2M NaCl pH 7.4 and 0.01M TRIS - 2.0M NaCl pH 7.4. Each step was initiated only after the OD of the eluate returned to baseline readings. The baseline 280 reading was continued until at least one column volume had been applied before the next buffer was applied. The fractions were pooled, dialyzed with initial buffer. and concentrated, and stored at -20° C. Iggelectric Focueipg, The LEE-8100 isoelectric focusing column (LKB Inst., Rockville. Md.), 110 ml capacity, was used as described in the manufacturer's instructions for the anode at the bottom with the following exceptions: l) the sucrose gradient was formed with 1M urea (Schwarz/Mann. 18 Orangeburg. N.J.) and 0.052 Brij 35 (Polyoxyethylene Lauryl Ether) (Nutritional Biochemical Corp.. Cleveland, Ohio), 2) five grams of sucrose were added to the light solution. and 3) both the light and dense solutions were modified to contain only 50 ml each. Constant voltage (Instrumentation Specialties 00., Lincoln. Neb.) was applied at a level not to exceed 1.5 watts. The voltage was increased at 100 volt increments as the amperage allowed to a maximum of 800 volts and held until the amperage remained constant for at least six hours. Each 1.5 to 2.0 ml fraction (60 to 80) was assayed for pH, MDHV-A immunological activity, and optical density at 280 nm. When radioactive label was used 0.1 ml of each fraction was spotted on Whatman 3MM filters and processed as described. The positive fractions were pooled. dialyzed with 0.01M TRIS pH 7.4, concentrated. and stored at -20° C. Immugocoprecipitation of MDHV-A 14C-glucosamine and 14C-amino acid mix labelled MDHVvA prepara- tions containing 3H-Leu labelled control supernatant fluid were precipi- tated with 5-. lO-. 15-, and 20-fold quantities of MDHV-A positive or negative specific pathogen-free chicken serum for 2 hrs at room temperature. The samples were then reacted with rabbit anti-chicken IgG serum (Microbiological Associates, Bethesda. Md.) at 6 times the volume of chicken serum for 12 hrs. The precipitates were pelleted at 1800 G for 15 min and washed 4 times with 5 ml of cold HBSS. The precipitates were then dissolved in 0.3 ml of 0.1 N NaOH, spotted on Whatman 3MM filter discs, and processed as described. l9 Egypeinigation of MDHV-A.Antigeg The PM 10 concentrate containing antigen was mixed with trypsin at 2 mg/ml sample and incubated at 37° C for 8 hrs. A control sample diluted to equal volume with 0.01M TRIS pH 7.4 was handled in direct parallel. At 0, 2, 4. and 8 hrs 0.1 ml aliquots of both samples were removed, treated with an equal amount of soybean trypsin inhibitor (Worthington Biochemical Corp.. Freehold, N.J.). incubated for 10 min with intermittent stirring. diluted 2-fold with 0.01M TRIS pH 7.4, and analyzed for MDHV-A activity by the agar gel precipitin test. Polyacrylamide Disc Ge; Electrophoresis (PAGE) A. ‘Agid-Neutral Proteip Analytical PAGE. The resolution of acid-neutral proteins by PAGE was by the method of Davis (20) with these exceptions: l) a 32 polyacrylamide 1.0 cm spacer gel was formed from a 20:1.2 ratio of acrylamide to bis-acrylamide mixture, 2) both spacer and resolution gels were polymerized with N,N.N'.N'.-Tetra methylethylenediamine (TEMED) (Matheson, Coleman, and Bell, Rutherford. N.Y.) and ammonium persulfate, and 3) samples in 52 sucrose were layered on top of the spacer gel. B. Sodium Dodecyl;§ul§ate (SDS) Polyacrylamide Disc Gel Electro- phoresis (SDS-PAGE). A 1 cm 32 spacer and a 9 cm 102 resolution gel were used as above with the following exceptions: 1) the spacer gel buffer was 0.13M TRIS pH 6.8, 2) the resolution gel buffer was 0.24M TRIS at pH 8.8, 3) both gels contained 0.12 SDS. 4) the chamber buffer was 0.025M TRIS - 0.192M Glycine - 0.12 SDS at pH 8.0. 5) the voltage was kept constant. starting at 50 volts for 1 hr and then increased to 100 volts. and 6) the protein samples were adjusted to 12 SDS, 0.012 20 2-mercaptoethanol and 52 sucrose. heated at 90° C for 30 min and then clarified at 30,000 rev/min and 18° C for 30 min in an SW 50.1 rotor. Electrophoresis of the gels was performed with a Polyanalyst (Buchler Instruments, Fort Lee, N.J.) and a constant voltage/constant amperage power supply (Instrumentation Specialties Co.. Lincoln, Neb.). C. Analyses of Polyacrylamide Gels. Acid-neutral protein gels were analyzed by one of the four following methods. The SDS-PAGE gels were analyzed by only the first. 1) Stained with Amido-Swartz 108 as described by Davis (20) and scanned at 580 nm with a Gilford Spectrophotometer (Gilford Instruments Labs, Inc.. Oberlin. Ohio). 2) Stained with periodic acid-Schiff as described by Fairbanks et al. (30) and scanned at 560 nm (as above). 3) Sectioned lengthwise into 2 pieces (Canalco, Inc.. Rock- ville. Md.), overlayed with agar (0.852 or 8.52 NaCl) and assayed for precipitins by rabbit or chicken serum, respectively. 4) Cross sectioned into 1 mm sections (Diversified Scientific Instruments, Mountain View, Calif.) which were placed into agar wells cut by a #3 cork bore. The sections were assayed for MDHV-A with chicken serum by the agar gel precipitin test. D. Preparative Disc PAGE. A 3 cm long 32 spacer gel and a 1.5 cm long 102 resolution gel was used as described for the analytical gels of acid-neutral protein system. The acrylamide and bis-acrylamide were purified as described. The elution and electrode buffers were 0.005M TRIS - 0.038M Glycine pH 8.0 and 0.025M TRIS - 0.192M Glycine pH 8.0, respectively. The medium size gel column (Canalco, Inc.. Rock- ville. Md.) was used for a 1.5 ml sample (25 mg protein) which was 21 layered on top of the spacer gel as described earlier. Electrophoresis was initiated at 2 ms for 1 hr and then increased to 5 ma. As the dye band (phenol red) entered the resolution gel the ma was increased to 8 ma. The milli-amperage was kept constant. The column was eluted at 1 ml per min and l to 1.5 ml fractions were collected. Each fraction was assayed for MDHV-A activity and radioactivity when employed. The positive samples were pooled, concentrated. titered. recounted for total activity. and reanalyzed by analytical disc gel electrophoresis. Purification of Chemicals A. Acrylamide. One hundred grams of acrylamide (Eastman Organic Chem. Div., Eastman Kodak Co.. Rochester, N.J.) were dissolved in 1 liter of chloroform at 60° C (steam bath) with 1 tbsp of activated charcoal. The hot solution was filtered through a celite filter. The filtrate was cooled to -20° C for 24 hrs. The crystals of acrylamide were collected by filtration and dried under vacuum. The dried crystals were stored in brown bottles at 4° C under vacuum. B. Bis-acrylamide. Twenty-five grams of bis-acrylamide were dissolved in 1 liter of acetone by heating to 60° C (steam bath) with 1 tsp of activated charcoal. The hot solution was filtered, recrystal- lized and stored as described for acrylamide. C. Sodium Dodecyl Sulfate (SDS). Two hundred grams of SDS (Matheson, Coleman and Bell, East Rutherford. N.Y.) were dissolved in l L of glass distilled water by heating to 50° C. The solution was stirred for 12 hrs and l L of ethanol was added. The mixture was stirred for 12 hrs without heat. The solution was refrigerated for 24 hrs. The crystals were pelleted by centrifugation at 2000 rev/min 22 and 4° C for 1 hr with the GSA Sorvall rotor. The supernatant fluid was discarded and the pellet was resuspended in 952 ethanol at room temperature and then refrigerated for 12 hrs. The fluid was removed by vacuum, the crystals were dried at 37° C, bottled, and stored at room temperature. Preparation of Aptisera A. Chicken Antiserum. Blood was obtained by cardiac puncture from naturally infected chickens. The serum from each chicken was tested for immunological activity with a standard antigen preparation and reference serum. Those sera having only specific immunodiffusion activity for MDHV-A antigen.were pooled. dispensed into 2 ml aliquots, and stored at -20° C. B. Rabbit Angiserum. Rabbits were inoculated subcutaneously in each leg and in the neck region with one of the following antigen preparations in incomplete Freund's adjuvant (1.0 ml total containing 1 mg of protein): 1) DEAE Sephadex chromatography, 2) isoelectric focusing, or 3) infected cell extract. The emulsion was prepared by the dropwise addition of the sample to the adjuvant with constant mixing with a 1.0 ma syringe and 20 gauge needle. Subsequent injections of 1 mg of protein without adjuvant were injected subcutaneously at five sites 30 days after the initial immunization. This was repeated at two week intervals for 2 months. The rabbits were ear bled 3 days before inoculation and tested for precipitating antibody by agar gel precipitin test. Rabbits exhibiting precipitating antibodies were bled at 48 hr intervals (twice) and then inoculated. Each major bleed would yield approximately 15 to 20 m1 antiserum/rabbit. RESULTS Pathogeniciey and Virulence of GAHMDHV Infected DEFs The MDHV—infected DEFs at the 28th passage in cell culture which produced MDHV-A were assayed for pathogenicity and virulence. Clinical disease and lesions were noted in inoculated and uninoculated direct contact control chicks (Regional Poultry Laboratory Line 15 x 7 1 day old) held in modified Horsfall isolators. The infected cells and released virus from inoculated birds were virulent and pathogenic. During production of MDHV-A for this work the 26th cell passage was not exceeded. Table l. Ih viva chick assay of 28th T.C. passage GAFMDHV DEPs for virulence and pathogenicity Number Average Birds Treatment Gross Lesions Deaths Days8 A s Inoculatedb sled 4/8° 41 B 9 Direct contact 4/9 3/9 41 c 10 Inoculatedc 10/10 10/10 46.6 D 10 Direct contact l/lO 1/10 57 8Days to death for each bird divided by number of deaths. bEach chick was inoculated I.P. with 2 x 107 MDHV—infected DEFs 28th cell passage. cAs above with only 1 x 107 MDHV-infected DEFs. dNumber positive birds/number experimental birds. eNumber specific deaths/number experimental birds. 23 24 Optimal Source of MDHV-A Confluent I° DEF monolayers were inoculated with l x 107 infected DEFs/150 mm plate and the cells and fluid were harvested six to eight days later. These were centrifuged and the fluid was concentrated 50- fold/plate by negative pressure dialysis. The cell pellets were disrupted by homogenization (Dounce homogenizer) in 0.01M TRIS pH 7.4 and clari- fied at 50,000 rev/min in an SW 50.1 rotor for 30 min at 4° C. The clarified cell extracts were concentrated by negative pressure dialysis in TBS buffer to 0.5 ml representing a 400- or ZOO-fold concentration/ plate. The recovery of MDHVeA from the culture fluid was 32- to 128-fold greater than that from infected cell extracts (Table 2). The culture fluid was the optimal source of antigen; however, the presence of calf serum proteins would complicate subsequent purification of the antigen and a method for the production of MDHVqA in the absence of sera was sought. The use of serum free medium after infection and visible CPE had no significant effect on the overall MDHVQA production (Table 2). Optimal Harvest Intervals Antigen production may be continuous in the infection cycle as the spread of infection is cell-to-cell rather than by infectious cell free virus. A study was undertaken to determine the parameters for optimal production and recovery. The results (Table 3) based on 24, 48, 72 hr harvest intervals and an infected control held for 6 days (3 roller bottles/treatment) indicated that the daily harvest was optimal with a 1.2- to 7.8-fold greater recovery of MDHV-A. MDHVeA is not accumulated in the culture fluid for extended periods. Results of preliminary experiments indicated that 25 and 50 m1 of medium were required for 24 25 Table 2. MDHV-A AGN production Number Anti en Titer 4b Days Plates 2 Sera Culture Fluid Cells Postinfection 10 2 64 8 6 10 2 32 8 4 8 5 2 128 4 6 5 0 64 4 6 5 2 32 4 6 5 0 64 2 6 8The fluid from 5 and 10 plates (20 ml/plate) was concentrated by negative pressure dialysis to 2 and 4 ml, respectively. bCells were scraped from plates, washed with HESS 2X, and the final centrifuged pellet was suspended in 5 ml HESS. Cells were dis- ruptued by homogenization and centrifuged at 50,000 rev/min in an SW 50.1 rotor. The cell extracts were concentrated to 0.5 ml representing a 4- or 8-fold increased concentration to that of the culture fluids. and 48 hr harvest intervals. respectively, for optimal cell maintenance and prolonged MDHV-A production. Sucrose Gradient Centrifugation In order to obtain some knowledge about the size of the antigen a fresh PM 10 concentrate was centrifuged in a 5 to 20% sucrose gradient in TBS buffer pH 7.4. MDHV-A antigen was detected in over 652 of the sucrose gradient with the peak activity centered in the 3.5 to 3.83 region (Figure 1A). A leading shoulder of MDHV-A activity was noted. Recentrifugation of the peak fractions (Figure 1A - bracketed area) resulted in a sharpened MDHVHA antigen peak at the 3.53 region even though the leading shoulder appeared more pronounced. The antigen activity did not sediment as 26 Table 3. MDHV-A AGN production in roller bottle cultures with serum free media vaMDHV-A AntigenATiter at Harvesa Invervals Harvesta 24 hr 48 hr 72 hr Controle 1 4 2 8 16 3 32 64 4 128 128 5 64 6 16 54 8 32 Total AGN 252 208 72 32 aNF-10 - M—l99 (base medium) added 72 hrs postinfection and the first harvest was 24 hrs later or 96 hrs postinfection. b25 ml base medium/bottle concentrated to 8.5 mllbottle. c50 ml base medium/bottle concentrated to 0.5 ml/bottle. leO ml base medium/bottle concentrated to 0.5 m1/bottle. e100 ml base medium/bottle concentrated to 0.5 ml/bottle which was held with only pH adjusted for the entire 6 day cycle. far as the original sample and occupied a narrower percentage of the gradient (Figure 18). The 0D analysis of the fractions in Figure 280 1A demonstrated a complex heterogeneity of protein species throughout the gradient as compared to that of Figure 18. The BSAOD280 analysis (Figure 1A) demonstrated heterogeneity. This was reduced by the selection of the peak fractions (Figure 18). BSA standard markers used in all subsequent analyses were peak fractions obtained from sucrose gradients. The sucrose gradient centrifugation analyses suggest that MDHVeA antigen has an approximate 3.58 value 27 Figure 1. Velocity sedimentation analyses of MDHV-A in a 5 to 202 sucrose gradient. (A) A fresh preparation of antigen was con- centrated SO-fold by negative pressure dialysis (TES buffer). The sample was centrifuged for 12 hrs at 15’ C and 50,000 rev/min (SW 50.1 rotor). (B) A pool of the peak antigen and BSA fractions indicated in (A) by brackets was harvested, concentrated, and sedimented under identical conditions. A BSA 4.48 marker was centrifuged and processed in parallel gradients. Optical density at 280 nm of BSA ( 000 ) and MDHV-A sample (A»A A), and MDHV-A immunodiffusion activity (A A ). 28 U 31”. NOISflddIOONflWWI V‘AHOW ma ousuam SS m20_ho<¢u OmN.4<2¢Oz h 00 on On .1 «.r ‘1' .11. 0—11 “fill. 4‘ mmh on ltoe ”“082 9 AllSNSO TVDILdO 831“. nousnssnoomwm V—AHOW 4H ouowwm 1*. m20_bo(¢m DwN3<2¢OZ h 00 on on 8 O— m _ h — p _ — — - _ "“ "'.". "“.‘I —Jr 4 Q\ a... l <4 um \\ I. a. u. .s. a a 6.. so. u.- ... .‘oc . e“. g .00. e. , o.. co . 444 a o. «In 0 t. m I. .. ‘H ‘ N .‘ .o g .- —¢ 1 8. ( .. . . T 2 4 a a . ~~ ‘- cc. an? d .0 ‘ it .22. m ..on. O .2. 1... O L ..o~. (L _ 4 8.. _<< < A. a .4 “”083 9 AMSNSO 'IVOLLdO 29 (average of 5 determinations) when compared to BSA at 4.48 (Figure 1B and 28) (66). Antigen preparations that had been frozen and thawed repeatedly for removal of aliquots consistently had decreased antigen titers. Two possible explanations were considered: 1) proteolytic enzyme degradation with loss of immunological activity, or 2) aggregation of the antigen with itself or other proteins with a resultant drop in titer. To test the latter possibility a preparation stored at -20’ C which had decreased 4-fold in titer (64 to 16) was assayed by sucrose gradient centrifugation. The immunological profile (Figure 2A) was shifted to the bottom of the gradient and into the pellet. An experiment to dissociate the aggregated antigen by urea, Brij 35, and 2-mercaptoethanol was employed (46). The MDHV-A titer increased 4-fold after treatment for 5 hrs in IN urea - 0.052 Brij 35 at 37° C (Table 4). The addition of 2-mercaptoethanol decreased the immunologic activity within 1 hr and completely destroyed it by 3 hrs. Sucrose gradient centrifugation analyses of the control antigen preparation and dissociated preparation (Figures 2A and 2B) demonstrates the dissociation of MDHV-A and its shift in the gradient. Although MDHV-A shifts towards the top of the gradient the pellet contains approximately the same amount of 3H-Leu label indicating that most of the proteins were not dissociated by the treatment. Comparison of the antigen profile of Figures 28 and 1B suggests the leading shoulder may be a result of aggregation. 30 Figure 2. Velocity sedimentation analyses of a stored MDHV-A positive preparation before (A) and after dissociation with urea and Brij 35 (B). A 2 m1 aliquot of SO-fold concentrated MDHV-A culture fluid stored at -20' C for five months was concentrated to 1 ml. The sample was processed as follows: 1) 0.4 m1 of the concentrate was diluted with 0.4 ml 0.01M TRIS pH 7.4. 2) 0.4 ml of the concentrate was diluted with 0.4 ml 0.01M TRIS - 2.0M Urea - 0.1% Brij 35. 3) 0.2 ml of the concentrate was diluted with 0.2 ml 0.01M TRIS pH 7.4 held at 20' C (temperature control without centrifuga- tion analysis. The samples (1 and 2) were incubated for 5 hrs at 37' C. BSA samples were handled under identical conditions as described in 1 and 2. The non-dissociated samples (1 and BSA) were sedimented as in Figure 1 and the dissociated samples (2 and BSA) were sedimented at the same time in the presence of 1M urea. Endpoint titers of samples 1, 2, and 3 were 16, 64, and 16, respectively. Optical density at 280 nm of MDHV-A samples ( A.A A ) and MDHV-A immunodiffusion activity ( .A 15 ). 31 » UBlll NOISfldilOONnWNI V—AHOW 3 g 2 o v N - i l a a A a a I I I I I I '- ‘x - / s ..O N //a o o " 3 4 _o < 0 3 k?- of. ~52. . ~2 I J ‘l 1 l l l l l 1 1 l l I 1 v v v v r v I 7 ' v D s 2 N m (ll/2-01 ' mo ".1 H8 Halal NOISfldleONnWWI v-Auow qua: up! BSA 4.48 \ mum who; I! was an H; _ . . _____ _ _i .i__,i... _.. _._.,_ _ Figure 28 NORMALIZED FRACTIONS (36) Figure 2A NORMALIZED FRACTIONS m 32 Table 4. Effect of dissociating agents on MDHV-A AGN titer MDHV-A Antigen Titer Time--Hours after Initiation Treatmenta 0 1 2 3 4 5 6 Control 16 16 16 16 l6 l6 16 2M urea 16 32 32 32 64 64 64 1M.urea 16 32 32 32 32 64b 64 0.5M urea 16 32 32 32 32 32 32 1M ureac 16 8 4 0 0 0 0 a0.01MTRIS pH 7.4 with 0.051 Brij 35 urea concentrations variable. bImmunodiffusion - trace reaction. °0.01M4r21s pH 7.4 with 0.052 3:13 35 and 0.051 2-mercapto- ethanol. §§A§ Sephadex Ion Exchaggg Columg Chromatography A PM 10 concentrate was absorbed to DEAE Sephadex A 25 resin suspended in 0.01M TRIS pH 7.4. The resin was eluted with a linear 0.0 to 0.5M NaCl gradient in 0.01M TRIS pH 7.4. MDHV-A antigen was eluted by 0.05M to approximately 0.2M NaCl (Figure 3). A stepwise elution of MDHV-A from DEAE A 25 resin at 0.05M NaCl intervals extend- ing from 0.00 to 0.5M was performed to determine more accurately the exact NaCl concentration for the antigen release. The antigen was eluted at 0.05M, 0.1M, 0.15M and 0.2M NaCl. Preliminary results indi- cated a simple four step elution procedure could be used as a preparative step in purification as described. A dialyzed 60 m1 sample, which included 20 m1 of fresh 3H~Leu labelled MDHV-A positive sucrose gradient fractions and 40 ml PM 10 concentrated unlabelled MDHV-A preparation was applied to a l x 90 cm 33 (— 70 I MDHV—A I g .° . 50m— . I’J-o 5 - e r’ g E '0 . ’v” 3 ._ . ’’’’’ q I 3 . ,x g I- . ’I’ b 3 3°. 5. x’ . 0'3 x E o /”’o 4:- .o ”/ O.._- . -O 0... °. I/I’ ...e. .00. °. 0. J o4b’oe. 00.. ’I’ .. e... '0 e O -O.I I . . ”’l . n 1’ f0 1 30 ' 5'0 1 Figure 3. DEAE Sephadex A 25 ion exchange‘ column chromatographic analysis of MDHV-A. MDHV—A positive sucrose gradient fractions were concen- trated by negative pressure dialysis (0.01M TRIS pH 7.4) to 10 ml (18 mg protein - MDHV-A titer of 64). The sample was applied to a l x 8 cm gel bed equilibrated with 0.01M TRIS pH 7.4 buffer (10 m1) before elution with a 100 m1 linear NaCl gradient (0.00M to 0.5M NaCl in 0.01M TRIS pH 7.4). Two milliliter fractions were collected and assayed for MDHV-A antigens by the agar gel precipitin test (bracketed zone) and OD280 ( 000 ). 34 gel bed and eluted as described in Materials and Methods. The MDHV-A immunological activity was eluted by 0.2M NaCl and was found in two distinct peaks (Figure 4) which represented the fresh 3H—Leu and unlabelled stored antigen, respectively. The separation of these two distinct MDHV-A activities may be an inherent difference between aggregated and non-aggregated species or a molecular sieving effect of the resin-column and/or a combination of both. Isoelectric Focusing A MDHV-A positive DEAE preparation was isoelectric focused on a pH 3-10 gradient. The antigen was detected at pH 6.0 to 7.3 with the peak activity at pH 6.50. A protein precipitate was formed in the gradient which settled in the gradient and contained antigen activity. To eliminate protein precipitation a sucrose gradient with 1M urea and 0.052 Brij 35 (36,46) was used with the ampholine series pH 5 to 8. The MDHV-A antigen activity was found from pH 6.16 to 7.06 with the antigen peak at pH 6.69 (Figure 5). The pH gradient was linear from pH 4.5 to pH 7.4. The 3H-Leu content under the area of the antigen peak (titers 3 4) represented approximately 10% of the total protein applied to the gradient and represents 802 of the MDHV-A antigen. Isoelectric focusing of MDHV-A in the presence of urea and detergent has been reproducible with the antigen peak at pH 6.68 :_0.03 pH units (Table 5). Analytical and Preparative Polyacrylgmide Disc Gel Electrophogggig DEAE Sephadex chromatographed and isoelectric focused MDHV-A positive preparations were analyzed by polyacrylamide disc gel electro- phoresis as described. The respective stained gels revealed 16 and 10 OPTICAL DENSITV @ 280 nm 35 [I I c o I '1 7 _.5 v v v v M ,M. v ‘ t Iv V ~ 3 I V'. vvvvv v v v v v | V v v ‘7 vvvvvvvv v \\ fv ‘77 v VVV vv VJ '\ s .' $ '5. v v vv .0 "‘I“ I' v D. \‘. l.. .2322?!" L....I--.- . . .-. : . a A}... ' . . 20 40 60 .0 '00 ‘20 I‘D 160 FRACTION NUMBER (Bull/Fr) Figure 4. DEAE Sephadex A-25 ion exchange column chromatographic analysis of MDHV—A by step- wise elution. A 60 ml sample, which included 20 ml of fresh 3HrLeu labelled MDHV-A positive sucrose gradient fractions and 40 m1 PM 10 concentrated unlabelled MDHV-A preparation was dialyzed against 0.01M TRIS pH 7.4 buffer. The sample was applied to a 1 x 90 cm gel bed and eluted as follows: A) application, B) wash - 0.01M TRIS pH 7.4, C) elution 1 - 0.01M TRIS - 0.2M NaCl pH 7.4, and D) elution 2 - 0.01M TRIS - 2.0M NaCl pH 7.4. Six milliliter fractions were collected and assayed for 3H—Leu CHI/0.10 ml (. 0 0 ), Ongo ( V V ) and MDHV-A antigen by the agar gel precipitin test (bracketed areas). 3H Lou cm x 104/01 ml. 36 I3- v I -20 I?" I: “as: :I I. I' I' II- .I . {L .I ,— TT . —— IO-- II ,‘ _]o I: I Iv 7 9.. I I I I I i 6.69 .' g , _ I D- “ 7 5 : s 3" E . _ _ IY'I .' S 2 7" I" I IV'I ' E ”7 E- 'I I " . 8 53 :l I ‘I 0'. 2 ,( 6-- H I I ..' g z I}: I n‘" E s 5.. II I I 0' .. ( 5 a +' ; I 4 l ‘ I I " I I o n 4-- III 5 I6— II,‘ I 'II '. 3-- I I!“ I' h 2. L- 8— ...f I4 ll 4—-l I 1- I I' ' I 3 I0 T FRACTION NUMBER IIml/FII Figure 5. Isoelectric focusing of MDHV-A. A 5 ml fraction (25 mg protein) of pooled and concen- trated DEAE positive labelled peak (Figure 4) was extensively dialyzed against 0.001M TRIS pH 7.4. The sample was focused in a linear sucrose gradient with 1M urea, 0.052 Brij 35, and 1.22 Ampholines with pH range of 5 to 8. The sample was added to the light solution with the dissociating agents and incubated at 37° C for 4 hrs. The isoelectric focusing was performed as described in Materials and Methods for 72 hrs. 0ne milliliter fractions were collected and assayed for pH ( coo ), H—Leu ( A A A), and MDHV-A antigen by the agar gel precipitin test (AA). 37 Table 5. Reproducibility of MDHV-A AGN with isoelectric focusing8 pH Range Isoelectric AGN Titer Prot. Positive Point Peak Peak Exp. mg Fractions Fraction Fraction 1 25 6.16-7.06 6.69 32 2 25 6.08-7.15 6.70 64 3 20 5.75-7.19 6.65 64 4 35 6.00-7.12 6.68 128 5 75 5.54-7.46 6.67 64 Avg. 6.68 i 0.03 gAll experiments employed a sucrose gradient with the pH 5-8 ampholine, 1M urea, and 0.052 Brij 35. protein bands (Figures 6A and 6B). Immunodiffusion analyses of 1 mm segments from cross sectioned gels (bracketed zones) demonstrated that the antigen was in the region of proteins 3 through 8 (Figures 6A and 6B). The isoelectric focused preparation was analyzed by the modified immunoelectrphoresis of polyacrylamide gels as described. A chicken serum (Figure 7A) and the rabbit antisera induced by the DEAE Sephadex chromatographed preparation or infected cell extracts (Figure 78) demonstrated 1, 5 and l immunodiffusion precipitin bands, respectively. Electrophoresis of the isoelectric focusing preparation was per- formed on two sets of 12 gels (each) which were processed and immediately frozen at -76' C. One gel from each set was cross sectioned into 1 mm segments and analyzed for the location of the antigen as described. The remaining gels were sectioned and the appropriate sections were pooled and eluted with 0.01M TRIS pH 7.4. The eluted preparation (analytical gel preparation) was concentrated and reanalyzed by 38 Figure 6. Analytical polyacrylamide disc gel electrophoresis of MDHV-A. The broken line at 1 cm mark represents the interface of the 32 acrylamide spacer and the 7.52 resolving gel. The brackets repre- sent MDHV-A antigen on 1 mm segments from duplicate gels. The gels were stained with amido black and scanned with the Gilford spectrOpho- tometer gel scanner. The protein concentration applied per sample/gel were as follows: A) 300 ug DEAE Sephadex chromatographed product, B) 300 ug isoelectric focused product, C) 150 pg analytical gel elution product, and D) 100 us preparative disc gel product. Numbers equal visible protein bands and DB equals dye marker phenol red. 39 ‘7‘ 9 _i_ a... _p_ii_+- o 8.5:. mcmhmig-‘mo z. m02 ’!3 -o‘. ' .3 'i he 5,. iv g, A 13:. ._. I z 9. 9 .;. S. y : I .._. "z 6 J 3‘ G. I: ", 5 ~ ." a. a 5:. _ ., '1 4.. ., _ ‘ ‘ ‘ V * ' l. a V ‘ 5 V 'r Haw; ‘ $5}; ‘ ' ‘A . by '1 ‘7; Ia. ' 1 W» .4, K. ‘ I \ e I ... ! '9 I "L I. ' “ K ' .v " = ‘ .I-u. v a. 3..- ‘- V . “' n‘ , 47 chromatographed preparation had five stained bands corresponding with proteins #1, 3, 5, 9, and 11 (Figure 9B). The isoelectric focused preparation had four stained bands (#1, 3, 5, and 9) while the analy- tical gel preparation had three bands (#1, 3, and 5). Only two bands (#3 and 5) were associated with the purified preparative disc prepara- tion (Figure 9C). t Sodium DodecylgSulfate Polyagrylggide D;§g_ Gel Electrophoresis (SDS-PAGE) The four standard proteins, human transferrin, ovalalbumin, chymotrypsinogen, and cytochrome C, and the 4 antigen preparations from the DEAE Sephadex chromatography, isoelectric focusing, analytical and preparative disc electrophoresis were analyzed by SDS-PAGE. The relative mobilities to the dye band of the standard proteins when plotted against the logarithms of their respective molecular weight produced a straight line (Figure 11). The stained gels of the MDHV-A positive preparations exhibited from 4 to 16 peptides of which 6 exceeded the maximal range of the standards used (Table 6) (Figures 10 and 11). The peptides in the preparative disc preparation have approximate molecular weights of 82,200; 56,890; 52,480; and 20,650. Trypsinigation of MDHV-A Antigen The immunoprecipitin activity of the MDHV-A antigen in a DEAE Sephadex chromatographed preparation (titer of 64) lost 942 of its antigen activity within 2 hrs and was completely destroyed by trypsin within 8 hrs at 37' C (Table 7). In contrast the control sample's MDHV—A immunodiffusion activity remained constant at 64 and the addi- tion of soybean trypsin inhibitor had no adverse effects. 48 Figure 10. SDS-polyacrylamide disc gel electrophoresis of MDHV-A preparations. A 1 cm 31 acrylamide spacer and an 8.5 cm 102 resolution gel were employed (Materials and Methods). A) Standard proteins - Ht - human transferrin, 0v - ovalalbumin, Cn - chymotrypsinogen, Ce - cyto- chrome C, and DB - dye band phenol red; B) DEAE Sephadex chromatographed antigen preparation, and C) preparative disc gel antigen preparation. The gels were stained with amido black and monitored with the Gilford Gel Scanner. 49 AI? - s .‘. _,.'—-‘-—~.- - I6 A\“-— - “A’J l I 6 I‘ I5 ~J I3 I2 \ 0v IV IIIII.IIII JI3 /”“, 89IOII \ .I H II IIIII. .I III-.. II“|\III [I . I v. . N .. .I\ r t .I .. JI2 v 3456 7 .J 2 , I I i I 6 4 2 hiawl IL:I...rIL .WII+IITI#.I.ITII_ WIHIITIIILIIILF. DISTANCE IN CENTIME TE RS Figure 10 50 Figure 11. SDS-polyacrylamide disc gel electrophoresis of standard proteins (0) were plotted by their relative mobilities to the dye band vs the log of their molecular weight. The DEAF. Sephadex chromatographed preparation peptides ( 0 ) were plotted by their respective relative mobilities to the dye band and numbered for further identification. 51 \ \ \ d- 4-9 0\ —— HUMAN TRANSFERRIN (76,600) 8 e \ \ 9 e \ .T. .3 \ IO 0 II -n- .7 I2 o\ —— OVALALBUMIN (43.000) \ ' '6 \ 13 . \ \ \ \ - .5 \ \ \\ \ \ o -_ CHYMOTRYPSINOGEN (25,700) . .4 \ - \ I4 e \ 15 ‘- . \ - .3 \ \ \ \ \ \ r- -2 \ \ 16 ° \ \ \ \o _— CYTOCHROME 0 (12,400) ' 4" \ ‘\ \ \ I i I L 1 l I 1 l I j I I I I I l I I I .1 -3 5 -7 9 MOBILITY R, 'lM "IOW 901 ,. 52 Table 6. SDS-polyacrylamide gel electrophoresis of MDHV-A preparations Protein # M01. m. A B c I)“ be 1 c + - - - 0.025 2 c + - - - .074 3 ° + - - - .111 4 ° + + - - .136 5 ° + + -. - .161 6 ° ' + + + — .173 7 82,200° + + + + .210 8 72,450 + - - - .247 9 63,310 + + + - .272 10 56,890 + + + + .308 11 52,480 + + + + .333 12 44,160 + + + - , .383 13 36,730 + + - - .432 14 22,910 + + - - .568 15 20,650 + + + + .592 16 14,450 + + + - .691 Human transferrin 76,600 0.222 Ovalalbumin 43,000 0.383 Chymotrypsinogen 25.700 0.531 Cytochrome C 12,400 0.753 8Samples are preparations from the following steps in purification: A) DEAE Sephadex chromaotgraphy, B) isoelectric focusing, C) analytical disc gel electrophoresis, and D) preparative disc gel electrophoresis. be - distance protein migrated * distance dye migrated X length of gel before staining + length of gel after staining. cIndicates the molecular wt. of these peptides are beyond limits of the standard proteins for accurate extrapolation. 53 Table 7. Effect of trypsin on MDHV-A antigen immunodiffusion activity Antiggn Titer/Hrs Post Treatment Treatment8 0 2 4 8 b Trypsin-treated 64 4 Trace 0 Untreated 64 64 64 64 82 mg trypsin/1.0 ml of concentrated antigen from DEAE Sephadex column chromatography was incubated at 37° C. An aliquot was removed at the specified times and the trypsin was inactivated with an equal amount of soybean trypsin inhibitor. The untreated sample was diluted with buffer, incubated and subjected to soybean trypsin inhibitor. bThe aliquots were serially diluted 2-fold and assayed for MDHV-A immunodiffusion activity with monospecific chicken sera by the agar gel precipitin test. Immunocgprecipitation Analyses of MDHV-A Antigen Radioactively labelled antigen was mixed with.monospecific MDHV’A chicken antiserum or specific pathogen-free chicken serum and then the chicken IgG was precipitated by rabbit anti-chicken IgG. The coprecipi- tation of mixed label supernatant fluids from infected cells (14C- amino acids) and control cells (3H-Leu) provided an additional control over that of negative serum alone or an alternate precipitation system. The MDHV-A positive serum coprecipitate had a 14C-amino acid activity of 902 or 1.9-fold increase above background (Table 8). In contrast this serum precipitated only 5.4 and 5.8% 3Hr-Leu above that of the negative serum control. This difference may be accounted for by the slightly increased precipitate associated with the positive system and/or the limits of the procedure. Correction of the positive system for this difference would still yield a 1.82- and 1.86-fold increase of 14C- amino acid radioactivity above background. 54 Table 8. ImmunocOprecipitation analysis of MDHV-A antigen in mixed radioactively labelled infected and control cell culture fluids 14 ZbRecoveryin the Prgcipitgtea f C-amino acid c ’n-Leu" f (Infected) Net APB (Control) Net APB Sera Total (A—B) B Total (A-B) B A. MDHVQA Positived 10 6.40 3.10 90 3.9 .2 5.4 5 5.70 2.70 90 3.4 .2 5.8 B. Normale 10 3.3 3.7 5 3.0 3.2 EAverage of duplicate samples. bDEAE Sephadex chromatographed preparation of 1l'C-amino acid labelled infected cell supernatant fluid (titer of 8) with 6.8 x 103 cpm/20 ml and 3H-Leu labelled control cell supernatant fluid with 2 x 105 cpm/20 pl (20 ul/test sample). CZ 14C above nonrspecific background. dMonospecificMDHV—A positive chicken serum (titer of 4). The numbers represent the fold increase of chicken serum added (volume/ volume) to antigen preparation. Rabbit anti-Chicken IgG was added 6-fold in volume to chicken serum. eS pacific pathogenrfree chicken serum without.MDHVeA antibody (as in "d"). ' £1 BE above non-specific background. A 1l‘C--glucosamine labelled MDHV infected cell supernatant fluid was coprecipitated (as above) and the coprecipitate with positive serum had a radioactivity of 440 and 6822. or 4.4- and 6.82-fold above back- ground (Table 9). The specificity of the chicken serum was established as monospecific for MDHV-A by agar gel immunodiffusion and polyacrylamide disc gel immunoelectrophoresis (Figure 7 - A, C and D). 55 Table 9. Immunocoprecipitation analysis of MDHVeA antigen in 14C- glucosamine labelled infected cell culture fluid z 14c- lucosamine Recovered ig,the Precipitatea.b Net A.---Bc Sera Total (APB) B A. MDHVeA Positived 15 2.00 1.63 440 10 1.80 1.57 682 B. Normale 15 .37 10 .23 IAverage of duplicate samples. bSucrose gradient MDHV-A positive pool of 14C-glucosamine labelled infected DEF supernatant fluid (titer of 4) with 5.7 x 103 cpm/Z ul (2 ul/test). c: 1(‘C above non-specific background. dMonospecific MDHVeA positive serum (titer of 4). The numbers represent fold increase of chicken serum added (volume/volume) to antigen preparation. Rabbit anti-chicken IgG was added 6-fold in volume to chicken serum. eSpecific pathogen-free chicken serum without MDHV-A antibody (as in "d"). Guanidine Hydrochloridgfiégarose Column Chromatography A 3E-Leu labelled sample having 96 units of "A" antigen was chromato- graphed in the presence of 6M guanidine 8C1 (Gu-HcL) and 0.01M Dithio- threitol (ETT) by the method of Fish et a1. (35). The 1.2 x 980 cm 6% agarose gel bed was eluted with 6M Gu-Hcl and 0.01M.DTT at l m1/hr/ fraction. Radioactive peak fractions were pooled, dialyzed, and concen- trated before monitoring for MDHV-A immunodiffusion activity. The 56 antigen was present in the void volume (blue dextran marker) and less than 102 of the MDHV-A immunodiffusion activity was recovered. The peptide exclusion limit for 6! agarose is approximately 100,000 mol. wt. A treated sample not chromatographed but dialyzed and assayed for MDHVeA antigen also resulted in recovery of less than 151 of the anti- genic activity. 125 1h.v£tro I Eabelling of MDHVeA The analytical gel eluate preparation (titer of 32) was labelled with 1251 in vitro by the method of Eelmkamp (41). The method labels tyrosine and histidine molecules specifically in a reaction employing 125ICl. The labelled sample was devoid of all MDHV-A antigenic activity while the buffer control sample retained its antigenic activity*with no apparent decrease in titer. Analyses ongQHVeA Antigen Purification by Differential Double Labelligg A 3B-Leu labelled infected DEF supernatant culture fluid and a 1l‘C—amino acid mixture labelled control supernatant culture fluid were ‘ concentrated and assayed for the following: 1) MDHV~A immunodiffusion activity, 2) protein concentration by the method of Lowry (63), and 3) radioactivity as described. The two concentrated samples were pooled and processed by: l) ultracentrifugation, 2) sucrose gradient centrifugation, 3) DEAE Sephadex column chromatography, and 4) iso— electric focusing. All fractions at each purification step were assayed for MDBVeA activity and radioactivity. Antigen bearing fractions were pooled, concentrated, and assayed as described above. The final antigen preparation was purified 21.5-fold and possessed 43% of the original MDHVeA antigen (Table 10). The protein value may be too high since the 57 Table 10. Analysis of MDHV-A antigen purification by differential double labelling ,_7 X Recovegz Durigg Purification Infected Normal Method 'IEfi""'3i:E;E' 14C-AA Total/Proteina Concentration 100 100 100 100 Clarification 86b 53 71 57 Sucrose gradient 86 51 ' 51 42 DEAE Sephadex 62 22 15.8 19 Isoelectric focusing 43 I 1.9 1.37 2 aProtein determination by the Lowry procedure. bStored antigen preparation exhibiting aggregation. ampholines, urea, and Brij 35 reacted in the Lowry procedure to yield abnormally high readings. Thus the purification value may be low. A second experiment*was performed with the following alterations: 1) the labels were reversed, ll'C-amino acid mixture labelled infected DEFs and 3E-Leu labelled control DEFs, 2) absence of sucrose gradient centrifugation, 3) the MDHVeA positive concentrated preparation from isoelectric focusing was refocused, 4) the refocused antigen was further purified by preparative disc polyacrylamide gel electrophoresis, and 5) the protein values after the use of urea, ampholines, and Brij 35 were calculated from the initial cpm/mg protein ratios of the two separate samples. The final antigen positive preparation was purified ZOO—fold and 242 of the original MDHV-A antigen recovered (Table 11). The value for purification may be in error depending on the label distribution associated with the recovered protein. Calculation of net protein by the initial cpm/mg protein ratios would be correct only 58 Table 11. Analysis of MDHV-A antigen purification by differential double labelling X Rgcgyggy Durigg Purification Infected . Normal Total Method AGN IzC-AA 3E-Leu Protein Concentration 100 (400 units) 100 100 1008 100b Clarification 100°(400 units) 78 70 81.2 80 DEAE-Sephadex 60 (240 units) 56 36 44 43.9 Isoelectric focusingd 30 (120 units) 0.4 0.26 1.7 Prep disc electro- phoresis 24 ( 96 units) 0.112 0.012 0.12 aProtein determination by the Lowry procedure. bProtein determination by radioactivity/mg of original materials. cFresh antigen preparations exhibiting no aggregation. dPositive MDHVWA isoelectric focused antigen fractions were pooled, concentrated, and refocused. Refocused MDEVeA fractions were pooled, concentrated and purified further by preparative disc gel electrophoresis. if the label was uniformly distributed in the proteins. The differences in the protein values where both determinations were made are low indicating that the value may not be grossly in error. The poly- acrylamide disc gel, electrophoresis and immunoelectrophoresis data demonstrating the single protein and immunoprecipitin band suggest that the preparation was essentially pure. DISCUSSION The avian cell culture systems present numerous problems. First established cell lines are not available thus requiring the preparation of primary cells at weekly intervals. These cells are either used immediately, frozen in liquid nitrogen, and/or subcultured for later use. The storage of primary DEFs with 502 viability for up to ten days has provided greater flexibility in the culture system. It has reduced the number of primary cell preparations to once per week, eliminated the subculturing of cells, and permitted the seeding and reseeding of plates and/or roller bottles as needed during the week. MDHV-A antigen can be routinely produced in.MDBV-infected DEF roller bottle cell cultures. Two important findings, 1) the culture fluids being the optimal source of antigen, and 2) antigen production in the presence of serum free media, facilitate the purification and char- acterization by reducing the cellular protein and eliminating serum proteins from the crude MDHV-A product. The repeated harvests of culture fluid on a daily basis increased the total antigen recovery per production cycle and reduced the potential costs of producing MDHV-A. MDHV-A production in roller bottles has provided a system for large scale production with conservation of reagents, materials and labor. One roller bottle equivalent to ten 150 mm dishes requires l/8th the volume of culture medium for an equivalent yield of antigen per harvest. A reduced volume of culture fluid per unit surface area 59 60 for antigen production results in a greater initial concentration of MDHVFA and a decreased volume for further processing. The data also indicate that MDHVeA is not accumulated on an additive basis for extended time intervals in the culture fluid. This may be the result of released enzymes from degenerating cells associated with the MDHV CPE seen in infected cell cultures. This may account for the increased recovery of MDHVaA antigen on a 24 hr harvest interval. The extent of initial infection of DEF monolayers affects MDHVWA production. Infection by MDHV-infected EEFs to cause an observable CPE in three days permits optimal antigen production and recovery. Increased or decreased multiplicities of infection result in decreased antigen production and recovery. Extensive infections result in rapid degenera- tion of the monolayers. A suboptimal infection requires an increased lag period for cell-to-cell infection and optimal CPE formation. The cells become overcrowded, round, and detach from the glass surface. The entire monolayer has an increased tendency to slough from the roller bottle surface. When old cultures, six to eight days in culture, are optimally infected, they too fail to produce antigen of adequate titer or duration. Antigenically active MDHV-A was isolated and purified from crude serum free 3H-Leu labelled MDHV infected DEF cell culture fluid and a 14C-amino acid labelled control cell supernatant fluid. Initially the combined use of sucrose gradient centrifugation, ion exchange column chromatography, and isoelectric focusing on the differential double labelled preparation resulted in a purification of approximately 21.5- fold and the recovery of 431 of the immunodiffusion activity. A second differential double labelled purification experiment employing the preparative polyacrylamide disc gel electrophoresis (after isoelectric 61 focusing) purified the antigen approximately ZOO-fold with the recovery of 242 of the antigen activity. In contrast, Ross at al. (in press, J. Gen. Virol.) (92) reported an ammonium sulfate precipitation, DEAE Sephadex, and Sephadex 200 chromatography procedure resulted in a 20- fold purification and a 262 antigen recovery. The final preparative disc gel preparation contained 773 vs Protein (based on the initial cpm/ug protein - Lowry) and 96 units of antigen which represents 8.05 ug of protein per antigen unit. The original 14C-amino acid antigen material contained 420 mg protein and 400 units of antigen. If the product was pure and 8.05 us protein/unit of antigen is valid, than 3.22 mg of protein was antigen in the crude preparation or 0.762 of the total protein was antigen. The data of Ross et at. (92) that 800 ug of protein that has 12 units of antigen would give the purified antigen a value of 66.7 ug protein/unit of antigen and 5! of the total protein in the crude preparation was "A" antigen. Analysis of each purification product, DEAE Sephadex chroma- tography, isoelectric focusing, and preparative disc gel electrophoresis by analytical disc gel electrophoresis resulted in the detection of 16, 10, and l stain protein bands, respectively (Figure 6 - A, B and D). The single protein band from the preparative disc gel product was broad and corresponded to two protein bands, #3 and #5, of the DEAE Sephadex product. Ross et al. (92), whose antigen preparation was produced in 21 calf serum, reported the product (as described earlier) also produced a single band on analytical disc gel electrophoresis. It was further reported that elution of the antigen from disc gel had a single stained band when reanalyzed by disc gel electrophoresis even though the antigen was purified only 20-fold and the original material contained 22 calf serum. 62 The antigen was eluted from 22 disc gels with a 30 to 40% recovery. Analyses of 50, 75, and 150 ug protein of the eluted product resulted in l, 1, and 4 observable amido black stained bands, respectively, on both 7.52 and 10! polyacrylamide disc gels. The cross sectioning of duplicate gels into 1 mm sections and their direct analysis by immuno- diffusion indicated that the single protein (i7, Figure 6C) common to all gels was not immunologically active with the chicken serum. Two of the proteins (#3 and 5, Figure 60) observed at the higher application level were both in the range of the segments which had associated with them the single immunoprecipitin band. The two inactive proteins have been identified as protein '1 and #7 (Figure 6 - A and C) of the DEAE Sephadex chromatographed product. Secondly, the two MDHVeA immuno- diffusion reactive proteins also coincided with the two proteins having immunological activity in the ZOO-fold purified preparation (Figure 60). The two proteins were detectable by both amido black (Figure GD) and the periodic acid-Schiff stains, suggesting both proteins contained carbohydrate (Figure 90). Antiserum from rabbits immunized with the DEAE Sephadex Chromato- graphed preparation formed five immunoprecipitin bands with the iso- electric focused preparation on modified immunodiffusion polyacrylamide disc gel electrophoresis (Figure 731). In contrast 1 and 3.1mmuno- precipitin bands were seen with the preparative disc and analytical gel eluate preparations, respectively (Figure 7 - F and D). In all preparations PAGE-immunoelectrophoresis with chicken serum resulted in only one immunoprecipitin band (Figure 7 - A, C and E). Immunologically active MDHVeA antigen sedimented at approximately 3.58 suggesting a molecular weight of approximately 50,000 based on the BSA 4.48 marker. Although 6H guanidine HCl in the presence of 0.0MM 63 Eithiothreitol (Cleland's reagent - DTT) destroyed 902 of the immuno- logical activity, MDHVeA antigen was detected in the region of the void volume when chromatographed on 6% agarose eluted with 6M guanidine 801 and 0.01M.DTT (35). This suggests a molecular weight equal to or greater than 100,000. The purified MDHV-A preparation when treated with 12 SDS and 0.051 2-mercaptoethano1 at 90° C for 30 min and analyzed by 10% polyacrylamide disc gel electrophoresis with 0.1% SDS resulted in four amido black stained bands having approximate molecular weights of 82,200; 56,890; 52,480; and 20,650. The antigen preparation has not been shown to be pure by the most rigorous methods and the two immuno- logically reactive proteins have not been separated so the exact rela- tionship of these peptides to the antigen is unknown. Ross and co- workers (92) reported that the antigen has a molecular weight in the range of 70,000 to 90,000 based on G-200 gel filtration chromatography. Their purified antigen was analyzed on 7.5! SDS-polyacrylamide disc gels with two stained bands being observed with.molecular weights of approximately 50,000 and 90,000. The latter was the predominant species and may correspond to the major 82,200 peptide in our system. However, no correlation can be made concerning their single 50,000 mol. wt. peptide. Immunocoprecipitation analysis of lac-amino acid mixture (Table 8) or 14C-glucosamine labelled (Table 9) preparations suggests MDHVwA is a glycoprotein and this was supported by the carbohydrate profile of periodic acid-Schiff stained polyacrylamide disc gels (Figure 10 - B and C). Ross and co-workers (92) suggested the antigen is a glyco- protein based on autoradiography, immunoadsorption, and polyacrylamide disc gel electrophoresis PAS stain data. 64 Although Ross and codworkers (92) report their purified antigen has a broad pI ranging from 4.5 to 5.5 in the presence of 2M urea, our data suggest the antigen has a pI of 6.68 i 0.03 in the presence of 1M urea, 0.052 Brij 35, and a pH gradient of 5 to 8 (Figure 5 and Table 5). In the absence of urea, Brij 35, and in a pH gradient of 3 to 10 the antigen has an approximate pI of 6.5. The complete inactivation of MDEVeA.immunodiffusion activity by trypsin suggests the antigenic determinant is associated with the 1251 (35) destroyed the MDHV-A anti- peptide(s). In vitro labelling with genic activity which suggests that tyrosine and/or histidine may provide a functional group in the antigenic determinant. The antigenic activity was also destroyed by 2-mercaptoethanol in the presence of 1M urea and 0.052 Brij 35 (Table 4). The failure to regain immunoprecipitin activity after dialysis with 0.01M TRIS pH 7.2 or TES buffer suggests inter or intra cross linkage disulfide bridge(s) are essential in maintaining the antigenic integrity. Approximately 101 of the original antigenic activity was regained after 6M guanidine-801 with 0.01MIDTT treatment with and without chromatography. This could have represented the amount of antigen not completely dissociated. Epstein et al. (23,24) reported the results of refolding of disulfide bond containing proteins following reduction and unfolding in the presence of 8M urea and 2- mercaptoethanol. Upon controlled conditions most proteins regained 482 to greater than 951 of their functional activity. However, insoluble CM-cellulose trypsin, poly-DL-alanyl soluble trypsin and insulin had a recovery of native configuration of less than 101 as judged by enzymatic and biological activity. Byerly at al. (8) skin tested MD—infected chickens for delayed hypersensitivity with three antigen preparations which had been 65 processed by Bio-gel F—200 column chromatography containing MDHVeA antigen. Their data suggest there is a relationship between the development of delayed hypersensitivity reaction to some antigen and the development of lesions in the chickens. Fauser et a1. examined the production of Migration Inhibition Factor in vitro by lymphocytes from chickens with delayed hypersensitivity to Bacille Calmette Guerin (BOG) or with Marek's disease. Using Band 24, a purified antigen from Mycobaoterium bovis or 806, and the DEAE Sephadex column chromato- graphed MDEV-A antigen preparation (obtained from Long and Velicer) she demonstrated by the radial test migration inhibition in BCG and MD sensitized chickens and not in controls. Secondly, the in vitro results correlated with the in viva intra-dermal'wattle reactions. The question arises as to which protein or component of the DEAE Sephadex chromatographed preparation was responsible for the migra- tion inhibition or classical in viva delayed hypersensitivity response in HD-infected birds. Further preparation of the purified antigen is necessary for pro- ducing monospecific antibody for the biological assay of purity and other chemical and physical characterizations. Once a known mono- specific antiserum to a purified MDHV-A preparation is available serological studies concerning the antigen‘s location in infected cells, presence, absence, and/or alteration in avirulent strains, and the antigen's location within the virus may be undertaken. Secondly the purified antigen may be used to elucidate its role in cellular immunity and/or humoral immunity. Finally, the antigen may be used to determine if it has any role in the prevention or initiation of the clinical disease. SUMMARY MDHV-A antigen was produced in serum free media and the optimal source of the antigen was the infected-cell culture supernatant fluid. The antigen was purified 200-fold with 24% recovery by ion exchange column chromatography, isoelectric focusing, and preparative poly- acrylamide disc gel electrophoresis. The antigen has an approximate sedimentation coefficient of 3.58 and a molecular weight equal to or greater than 100,000 with l to 4 peptides ranging in molecular weight from 20,650 to 82,200. It may consist of two species having slightly different mobilities based on charge or molecular weight as demonstrated in 7.5 and 102 analytical polyacrylamide disc gel electrophoresis. Both stained bands (a single broad area) are associated with MDHV-A antigenic activity as determined with both chicken and rabbit antisera. The antigen has a pl of 6.68 i 0.03 in the presence of dissociating agents and an approximate pI of 6.5 in the absence of such agents. The MDHV-A antigen is a glycoprotein with the antigenic activity associated with a peptide. Tyrosine and/or histidine may be anv essential component of the antigenic determinant and either inter and/or intra chain disulfide bridge(s) are necessary for maintaining antigenic integrity of the molecule. 66 LIST OF REFERENCES 10. 11. 12. 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