F THE STRUCTURAL PROTHNS“ ' DETERMINATlON 0 EASE ViRUS. ’ ‘ AND ANHGENIC ANALYSIS OF MAREK’S DIS Thesis for the Degree of Ph.rD. ' MICHIGAN STATE UNNERSITY Jl HSHlUNG CHEN 1972 rhp-giy. LIBRARY)‘ Michigan State University ' This is to certify that the thesis entitled Determination of the Structural Proteins and Antigenic Analysis of Marek's disease Virus presented by Ji flshiung Chen has been accepted towards fulfillment of the requirements for Ph. D. Microbiology degree in (Rikfgxcu z.L,¢.,.LLU Major professor B. R. Burmester, Director February 21+ 1972 Regional Poultry Research Lab Date 9 0-7639 ABSTRACT DETERMINATION OF THE STRUCTURAL PROTEINS AND ANTIGENIC ANALYSIS OF MAREK'S DISEASE VIRUS BY Ji Hshiung Chen Marek's disease virus (MDV) was propagated in roller bottle cultures of primary duck embryo fibroblasts and par- tially purified by sucrose density gradient centrifugation. Analysis of the proteins of MDV by electrophoresis in 7.5% polyacrylamide gel containing 0.1% sodium dodecyl sulfate (SDS) revealed that at least 8 proteins, designated VPI to VPVIII, were present in MDV. The VPI was the major protein. At least two proteins, VPII and VPIV, were found to be glycoproteins and therefore might be the envelope compo- nents. Comparative electropherograms of the proteins of herpes simplex virus and MDV and those of pseudorabies virus and MDV indicated that these herpesviruses had struc- tural proteins of similar size with only minor differences. The agar gel precipitin tests indicated that the rabbit anti-MDV serum reacted against SDS-disrupted MDV (the subunits of MDV) and had identities with rabbit Ji Hshiung Chen anti-SDS-disrupted-MDV serum. The MDV preparation also contained an antigen sharing partial identity with A- antigen of MDV and its antigenicity was not abolished by SDS treatment. The ferritin conjugation studies indicated that the rabbit anti-MDV serum contained antibodies reacted stronger against the subunits of MDV capsid proteins than antibodies against the whole capsid of MDV. The chicken anti-MD serum contained, at least, antibodies against viral capsid and envelope proteins. Both chicken and rabbit anti-MDV serums contained antibody against a sur- face antigen of MDV infected cells. Using the cell associated viral inoculum and fluorescent antibody (FA) technique, viral specific antigens were detected as early as 17 hours postinfection. As the infection proceeded, the FA antigen spread over the cytoplasm and nucleus of the infected cell. A mature, fluorescent plague of MDV was complete at about 60 hours postinfection. DETERMINATION OF THE STRUCTURAL PROTEINS AND ANTIGENIC ANALYSIS OF MAREK'S DISEASE VIRUS BY Ji Hshiung Chen 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 1972 ACKNOWLEDGMENTS I wish to express my sincere appreciation to Dr. Ben R. Burmester, Director of the U. S. Regional Poultry Research Laboratory and Professor of Microbiology and to Dr. Keyvan Nazerian of the U. 8. Regional Poultry Research Laboratory, for their encouragement and assistance in this study. I would also like to acknowledge the help and constructive criticism from Dr. Charles H. Cunningham, Professor of Microbiology and Dr. Leland F. Velicer, Assistant Professor of Microbiology. I also like to express my appreciation to Dr. Lucy F. Lee of the U. S. Regional Poultry Research Laboratory for her patience and generosity of using her laboratory facili- ties. I also like to acknowledge the financial support arranged by Dr. Delbert E. Schoenhard, Assistant Chairman of the Department of Microbiology and Public Health. My deep appreciation is also extended to all at the U. S. Regional Poultry Research Laboratory for their assist- ance and cooperation. ii TABLE OF CONTENTS Page ACKNOWLEDGMENTS O O O I O O O O O C O O O i i LIST OF TABLES O O O O O O O O O O O O O 0 LIST OF FIGURES O O O O O O O O O C O O . INTRODUCTION . . . . . . . . . . . . . . 1 LITERATURE REVIEW . . . . . . . . . . . . . 2 Etiologic Agent . . . . . . . . . . . . . 2 Strains of MD . . . . . . . . . . . . . 4 Pathology . . . . . . . . . . . . . . . 4 Gross Lesion . . . . . . . . . . . . . 4 Microscopic Lesion . . . . . . . . . . . 5 Serology . . . . . . . . . . . . . . . 5 The Replication of Herpesviruses . . . . . . . 7 Growth Cycle . . . . . . . . . . . . . 7 Viral Assembly . . . . . . . . . . . . 8 Structural Proteins of Herpesviruses . . . . . . 9 Synthesis of Structural Components of Herpesviruses in Infected Cells . . . . . . . . . . . 10 MATERIALS AND METHODS . . . . . . . . . . . 12 Virus . . . . . . . . . . . . . . . . 12 Preparation of Primary DEF . . . . . . . . . 12 Preparation of Chick Kidney Cells . . . . . . . 13 Preparation of Virus . , . . . . . 13 Preparation of Radioactively Labeled Virus . . . . l4 Purification of MDV . . . . . . . . . . . 15 a) The Supernatant Virus . . . . . . . . . 15 b) The Cytoplasmic Virus . . . . . . . . . 16 Determination of Protein Content . . . . . . . 16 Preparation of 3H Labeled DEF Proteins . . . . . 17 Determination of Contamination . . . . . . . . l7 Solubilization of Viral Proteins . . . . . . . 18 SDS-polyacrylamide Gel Electrophoresis . . 18 Estimation of the Molecular Weights of Viral Proteins 19 Preparation of Duck Embryo Cell Powder . . . . . 20 Preparation of Antisera . . . . . . . . . . 21 iii Adsorption of Serum with Duck Embryo Powder Agar Gel Precipitation (AGP) Test . Immunofluorescence Technique . Recrystallization and Concentration of Ferritin . . Preparation of Ferritin Conjugate . Ferritin Conjugation Technique for Electron Microsc0py . . . . . Preparation of Specimen for Electron Microscopy . . a) Thin Section . . . b) Negative Staining . Conditions for the Absorption of MDV on CK Cells . . a) Optimal Absorption Time . b) Absorption at 4°, 25° and 37° C . RESULTS 0 O O O O O 0 Purification of Virus . . Degree of Host Protein Contamination in Viral Preparations . . . Electrophoretic Pattern of the Viral Proteins Glycoprotein of MDV . . Of MDV Host Proteins in the Electrophoretic Profile of the Proteins of MDV . . . Comparison of Proteins of MDV and two Other Herpesviruses . . Estimation of the Molecular Weights of MDV Proteins . Conditions of the Absorption of MDV to CK Cells . . a) Optimal Absorption Time . b) Optimal Temperature . Analysis of MDV Antigen and Antibody by AGP Test . . The MDV Antigens in CK Cells Detected by FA Technique . . . . . Detection of MDV Antigens by Ferritin Conjugated Antibody . . . . . DISCUSSION . . . . . . SUMMARY....... LITERATURE CITED . . . . - APPENDICES . . . . . . 1. Reagents and Isotopes 2. Buffers, Solutions and iv Page 22 22 22 23 24 25 26 26 28 28 28 28 29 29 34 34 38 41 41 45 45 45 45 50 56 59 61 67 69 77 78 80 LIST OF TABLES Table Page 1. Protein contents of viral preparation . . . . 33 2. Purity of MDV preparations . . . . . . . . 35 3. Distribution of radioactive amino acids in the different structural proteins of MDV . . . . 39 4. Molecular weights of MDV proteins . . . . . 47 5. Effect of temperature on the adsorption of MDV to CK cells D O O O O O O O O O O O 51 LIST OF FIGURES Figure Page 1. Sucrose density gradient analysis of MDV from infected culture medium . . . . . . 30 2. Electron micrograph of MDV from the sharp band of the supernatant virus . . . . . . . 31 3. Sucrose density gradient analysis of MDV from the cytoplasmic extract of infected cells. . 32 4. Electropherogram of the structural proteins of MDV from the sharp band of the supernatant Virus. 0 O O O O O O O O O O O O 36 5. Electropherogram of the structural proteins of MDV from the cytoplasmic virus . . . . . 37 6. Electrophoretic analysis of MDV glycoproteins . 40 7. 'Determination of host protein contamination in supernatant virus. . . . . . . . . . 42 8. Determination of hosr protein contamination in cytOplasmic virus. . . . . . . . . . 43 9. Electropherogram of MDV and HSV proteins. . . 44 10. ElectrOpherogram of MDV and PrV proteins. . . 46 11. Molecular weight determination of the structural proteins of MDV. . . . . . . 48 12. The adsorption curve of MDV to CK cells . . . 49 13. The AGP test of rabbit and chicken anti-MDV serums with MDV, SDS—disrupted MDV and A-antigen o o o o o o o o o o o o 52 14. The AGP test of rabbit antiserums with SDS- disrupted-MDV and MDV o o o o o o o o 54 15. The FA antigen of MDV in CK cells . . . . . 57 16. The FA antigens of MDV in CK cells. . . . . 58 17.' The MDV antigen in infected cells detected by ferritin conjugated with chicken anti-MD serum, . . . . . . . . . . . . . 60 vi INTRODUCTION Marek's disease (MD) is the most common of the lymphOproliferative disease of chickens. The etiological agent is highly cell associated and belongs to the herpes- virus group. The MD virus (MDV) can be prOpagated in chick kidney (CK) cell, duck embryo fibroblast (DEF) and chicken embryo fibroblast (CEF) cultures. A relatively low yield of virus is usually obtained from these cultures. The work so far reported showed that antigens produCed from cells infected with MDV can be detected by agar gel precipitin (AGP) and fluorescent antibody (FA) tests. In order to understand the properties of MD anti- gens, the author prOposed to study the structural proteins of MDV and to use the AGP, FA and ferritin antibody conju- gate techniques to detect the location of the antigens in the infected cells. The structural proteins of MDV were analyzed by SDS-polyacrylamide gel electrophoresis and its electrophoretic pattern was compared with those of two well known herpesvirus--herpes simplex virus (HSV) and pseudorabies virus (PrV). LITERATURE REVIEW Marek's disease (MD) is a neoplasia of lymphoid tissue in chicken. Due to the mononuclear infiltration of the peripheral nerves and the inflammatory character of some of peripheral nerve lesions, Marek (36) identified this disease as polyneuritis. Recently Payne and Biggs (48) showed that MD is characterized by the neoplastic- like proliferation of lymphoid cells in the nerves and the visceral organs, particularly the gonad. Etiologic Agent In 1924 Van der Walle and Winkle-Junius proposed that MD was caused by a virus similar to that of herpes Zoster in man (68). The attempts to prove the viral etiology of MD were unsuccessful until 4 years ago when circumstantial evidence was provided by the discovery, in England (13) and in U.S.A. (42,63), of a herpesvirus associated with MD. The subsequent discovery that the feather follicle epithelium of MD infected chickens is the virus maturation site and the successful transmission of the disease with the cell-free virus prepared from these follicles (8,41) firmly proved that the herpes virus is the etiological agent of MD. The MD virus (MDV) belongs to the group B herpes- virus (32). Enveloped virions obtained from the epithelial cells of feather follicles have a diameter of 200 to 400 mu (8,41). The nucleocapsid has a diameter of 85 to 100 mu with 162 cylindrical capsomeres arranged in icosahedral symmetry (2,8,19,42). The capsomeres are hollow centered and measured 6 X 9 mu. In tissue culture, viral particles appear as naked nucleocapsids and most of them are in the cell nucleus (19,42). Enveloped virions can be found in the cytoplasmic inclusions of CK cell culture derived from kidney tumor of MD infected chickens (2). Virions are rarely observed in tumors (8,19,42,57); however, naked nucleocapsids can be found occasionally in the nuclei of cells in the bursa of Fabricius (9) and in Schwann cells (67). Enveloped virions are frequently present in the nuclei and in the cytoplasmic inclusions of the epithelial cells of the feather follicles (8,42). The MDV contains DNA (32) and its replication is inhibited by 5-iododeoxyuridine (13,61) and S-bromodeox- yuridine (39). Centrifugation in a cesium chloride gradient in a preparative ultracentrifuge indicates that viral DNA contains 56 to 57% guanine and cytosine (32). However, upon analytical ultracentrifugation, this DNA has a buoyant density of 1.706 corresponding to guanine and cytosine content of 46% (33). When centrifuged in an analytical ultracentrifuge, the DNA of MDV has a sedimentation constant of 56 S corresponding to a molecular weight of 100,000,000 daltons. This DNA hybridizes with RNA isolated from infected cells and not with RNA from uninfected cells. Strains of MD Various strains of MD, such as JM (59), GA (16), RPL39 (49), HPRS 814 (5,6), and HPRS Bl6-20 (50,51) have been described. No difference in serology has been shown among these strains. However, they differ in pathological manifestations. Certain strains, such as GA and RPL 39 (16,51), are more viscerotropic than neurotropic, whereas the strain JM is more neuropropic than viscerotropic (59). Pathology Gross Lesion The MD is characterized by paralysis or paresis of one or more of the extremities of chickens. Such chickens usually have grossly enlarged nerves. In addition, infected chickens often have lymphoid tumors which occur most com— monly in the gonad, liver and lungs. The gross pathology is subject to influence by the strain of MD (50,59), the age at the time of exposure (6,58), genetic constitution of the host (6,7,15) and sex (7.50). Microscopic Lesion Based on differences in histopathology, the nerve lesion of MD has been classified into 2 types. The A type lesion (6) consists of infiltration of small, medium and large lymphocytes, a few plasma cells and large dark staining cells which are referred to as "MD cells." The B type lesion (6) is characterized by edema and infiltration of only a few lymphocytes and plasma cells. The lymphoid tumors of the visceral organs consist of a mass of pleomorphic lymphocytes. Lesions in the bursa of Fabricius are cortical and medullary atrophy, necrosis and cyst formation. The lesions in the epithelium of feather follicles are in the intermediate and transitional layer. The cells are filled with clear vacuole. Many of the nuclei in the transitional layer contain characteristic Cowdry type A inclusions. Serology Several serological techniques have been described for the detection of antigens induced by MDV infection in tissue culture and in 2129. Sera for the detection of MDV antigens may be obtained either from survivors of infection or from hyperimmunized chickens. The agar gel precipitation (AGP) test was first developed by Chubb and Churchill (11). They employed CK cell cultures infected with MDV as antigen against sera from MD infected chickens. Using this test Churchill et a1. (14) detected 3 lines of precipitation which they designated as A, B, and C. The A antigen was detected in the supernatant fluid of infected cultures. The B and C antigens were detected in the cell extract. Depending on the sera used, sometimes as many as 6 different lines of precipitation could be detected. The nature of these antigens is still unknown. Using the fluorescent antibody (FA) test, 3 morphologically different antigens have been described. They are diffuse nuclear antigen, diffuse cytoplasmic antigen, and cytoplasmic granular antigen (40,49). A surface antigen has also been detected on the infected cells by the FA test (10). Whether or not these antigens are related to the viral structural proteins is not known. Combining the technique of FA and electron microscopy, Nazerian and Purchase (40) found that the cytoplasmic granular antigen never contained recognizable virions. The nuclear antigen often contained viral particles but they were not the sole content of this antigen. The diffuse cytoplasmic antigen, on rare occasions, contained some virions. According to Purchase, no antigenic differences could be detected among the strains of MDV by the FA test (49). Using the AGP test, Churchill, et a1. (14) found . . ._.’ 4%.... ‘ I l L '-._‘_" that the cell culture attenuated MDV lacked the A antigen whereas the virulent strains possessed this antigen. However, some of the virulent strains also lacked A anti- gen (51). Except for one herpesvirus of turkeys (HVT), no common antigenicity between MDV and several other herpes viruses were detected by the FA test (49). The Replication of Herpesviruses The studies of the replication cycle, viral assembly and the structural proteins of MDV have been hindered by the strict cell association of this virus. The replication of herpesviruses will refer to 2 herpesviruses-—herpes simplex virus (HSV) and pseudorabies virus (PrV). Growth Cycle Once the herpes virus adsorbs to the cell, pene- tration is relatively rapid and the virus is taken into the cell in a pinocytotic vesicle (23). The duration of the eclipse phase varies among herpes viruses: 3 hours for PrV in rabbit kidney cells (26) and 5 hours for HSV in human epidermoid carcinoma No. 2 (HEp-2) cells (54). The reproductive cycle of HSV in HEp-2 cells lasts from .13 to 19 hours (54), whereas the cycle for PrV replication :in.rabbit kidney cells is about 10 to 12 hours (26). At t:he end of the reproductive cycle, the yield of HSV is lC),000 to 100,000 virions per HEp-2 cell (69) and about idle same amount of PrV per rabbit kidney cell (26). Viral Assembly In HSV infected BHK 21 cells, the viral DNA is first detected about 5 hours after infection and the rate of viral DNA synthesis increases until 7 hours and then re- mains constant (56). The PrV DNA is first detected about 2 hours after infection and its rate of synthesis increases until 9 to 10 hours after infection (27,29). Viral protein synthesis in the HSV and in the PrV infected cells precedes the formation of infectious virus and viral proteins are made in excess (22,56). The viral proteins are not associated with viral particles immediately after their synthesis. The assembly takes place at the later stages of the replication cycle (22). The site of the herpesvirus assembly is the nucleus of infected cell (21,37). The first observed change in the nucleus of the infected cell is the margination of the chromatin. The electron dense particles (about 30 to 40 mu in diameter) are formed near these chromatin areas (37). As the infection cycle proceeds the particles appear to increase in diameter to about 50 mu and become less electron dense. Then the particles become surrounded by a membrane and the entire structure has a diameter of 70 to 100 mu (37). In certain strains of HSV the Virions form intranuclear crystals (18,38). However, the virions in the cytoplasm possess a double membrane with a diameter of 100 to 130 mu. Therefore, Morgan, et a1. (37) concluded that the site of viral development of herpesvirus is in the nucleus where the initial particles are formed and are enclosed by a single membrane. The second membrane seems to be acquired as the virions are released into the cytoplasm. Structural Proteins of Herpesviruses Although the application of acrylamide gel electro- phoresis to the analysis of viral proteins was developed in 1965 (66), analysis of the structural proteins of different herpesviruses is still in a primitive stage. This probably accounts for discrepancies in results so far reported. Using the macroplaque strain of HSV propagated in HEp-2 cells, Spear and Roizman (64) found that HSV contained at least 8 to 9 proteins with molecular weights ranging from 24,000 to 125,000 daltons. Olshevsky and Becker (47), using the HF strain of HSV propagated in the BSC line of monkey kidney cells, reported that at least 9 proteinS' existed in HSV with molecular weights ranging from 20,000 to 140,000 daltons. The latter group also reported that proteins II and VIII were present in capsid and proteins 'VI and VII were present in the viral core, whereas proteins III, IV, and V were associated with the viral envelOpe. The PrV, propagated in rabbit kidney cells possessed at least 8 proteins. The major component of these proteins fund a molecular weight of 120,000 daltons (62). When the 10 detergent treated PrV was analyzed on polyacrylamide gel electrophoresis, 6 distinct peaks were observed and the viral envelope was found to contain 4 distinct glycoproteins (28). Weinberg and Becker (71) compared the electrophoretic patterns of the structural proteins of HSV and of EB virus, the latter being a herpesvirus isolated from cultures of human lymphoblasts derived from Burkitt's lymphoma. They ' found that the 8 proteins of both viruses are similar. Synthesis of Structural Components of Herpesviruses In Infected’Cells Electron microsc0pic studies Showed that the core and nucleocapsid of HSV were assembled in the nucleus (44) and the nucleocapsids were enveloped from the inner layer of the nuclear membrane (44). For both HSV and PrV the struc- .tural proteins were found to be synthesized in the cytoplasm of infected cell early in the infection cycle and then tranSfered into the nucleus (3,20,64). The synthesis of viral structural proteins was not inhibited by cytosine arabinoside (3), a DNA inhibitor. Not all the proteins synthesized in the cytOplasm were transported into the nucleus. This may contribute to the compartmental segre- gation of herpesvirus antigens as detected by FA test (43,53). Although it is believed that the envelope of the 11erpesvirus is derived from the cell nuclear membrane (44), 11 the study using the ferritin-conjugated antibody indicated that both nuclear and cytoplasmic membranes of infected cells contributed to the viral antigens (45). However, the glycoproteins associated with the envelope of PrV are present only in a small amount in the cytoplasm of infected cells (4). Therefore, the antigenically altered membrane in the cytoplasm of PrV infected cells are not identical with the envelope proteins of PrV (4). In the case of HSV it was reported that the glycoproteins of the viral envelope and membranes from infected cells share common features and that the minor differences depended only on the different strains of virus and host cell used (30). The envelope proteins of HSV are synthesized concurrently with viral structural proteins and immediately after they are synthe- sized they bind to the smooth membrane in the cytoplasm and become glycosylated in situ (65). MATERIALS AND METHODS Virus The GA strain (16) of Marek's disease virus (MDV), cloned and propagated for 19 passages in duck embryo fibroblast (DEF) cultures, was used in all the experiments. Stocks of virus-infected cells were stored in liquid nitrogen at 1 x 107 or 2 x 107 cells/ml in a 2 ml vial. The MP strain of herpes simplex virus (HSV) (17) was obtained from Dr. B. Roizman, University of Chicago. Pseudorabiesvirus (PrV) (labeled NADL, T[-]) was obtained from Dr. H. G. Purchase, Regional Poultry Research Labora- tory. Both HSV and PrV were propagated twice in primary DEF and then stored at -700 C. Preparation of Primary DEF Embryonated duck eggs are obtained from Truslow Farm Inc. Chestertown, Maryland and Ridgeway Hatcheries, La Rue, Ohio. All cell culture procedures employed sterile technique. After 14 days incubation, embryos were removed :from the eggs, washed 3 times with phosphate buffer saline :solution (PBS), minced into small fragments by vigorously stirring in a trypsinization flask and rinsed 3 times with IPBS. The fragments of embryo tissue were treated with 0.02% trypsin in PBS for 45 minutes at 370 C. The 12 13 trypsinized cells were then filtered through cheesecloth, centrifuged at 400 X G for 5 minutes, resuspended in growth medium and the cell density adjusted to 2 X 107 cells/ml. For plating, 2 X 107 cells in 20 m1 of growth medium were added to each 150 mm petri dish. Roller bottle cultures with between 30 and 40 X 107 cells in 100 ml of growth medium added to each bottle were incubated at 370 C and rotated at.2 revolutions per minute. A monolayer had usually formed by 36 hours. Preparation of Chick Kidney Cells Chicken kidney (CK) cell cultures were prepared as described by Churchill (58). The pool of kidney tissue from 5-day-old or older chickens hatched and reared in isolation was washed twice with PBS and then stirred in 0.025% trypsin in PBS at 370 C. The trypsinized cells were collected at 10 minute intervals, passed through cheesecloth, and centrifuged at 400 X G for 5 minutes. The cell pellets containing a minimal amount of red blood cells were pooled and resuspended in culture medium. The 6 cells were counted and 8 X 10 cells were plated in each 60 mm plate and 2 X 107 cells in each 100 mm plate. Preparation of Virus The growth medium from roller bottles with a confluent cell monolayer was replaced with maintenance Inedium and GA-MDV infected cells were added at a ratio 14 of 1:5 of infected cells to normal cells. After 12 to 24 hours, the inoculum was drained and fresh maintenance medium was added. When the cells had undergone extensive .cytopathic changes, usually at 72 hours post infection, the monolayer was scraped into the medium, transfered to centrifuge tubes and centrifuged at 10,000 X G for 5 minutes. The virus in the supernatant culture medium will be referred as "supernatant virus," whereas the virus in the cell pellet will be referred as "cytoplasmic virus." Preparation of Radioactively Labeled Virus For the labelling of MD viral DNA, the medium was replaced at 36 hours postinfection (pi), with medium 199 without thymidine to which had been added 0.2 uCi/ml of 14C-thymidine or 4 uCi/ml of 3H-thymidine. The proteins of MDV were radioactively labeled by replacing the medium with medium 199 with 1/10 of the normal concentration of amino acids te which had been added 1 uCi/ml of 14c- reconstituted protein hydrolysate (RPH) or 5 uCi/ml of 3H-RPH. Glucosamine labeled MDV was prepared by growing the infected DEF culture in medium 199 containing 5 uCi/ml of D-glucosamine-6-3H. . For the preparation of HSV or PrV with radio- actively labeled protein, DEF monolayers were inoculated at a multiplicity of infection of 2 or 5, respectively, and 16 or 4 hours later the medium was replaced with medium 15 199 containing 1/10 of the normal concentration of amino acids and 0.2 uCi/ml of l4C-RPH. Viruses were harvested at 40 to 48 hours, or 24 hours pi, respectively, according to the original multiplicity of infection. Purification of MDV a) The Supernatant Virus The culture medium was brought to 50% saturation with ammonium sulfate and the precipitate allowed to form for 1 hour at 4° C. The precipitate was sedimented by centrifugation at 10,000 X G for 5 minutes, dissolved in virus buffer, and dialyzed overnight at 4° C against virus buffer. This virus concentrate was placed in centrifuge tubes and underlayered first with 10 ml of 20% sucrose and then with 3 m1 of 65% sucrose solution in virus buffer. It was then centrifuged for 90 minutes in a SW 27 rotor of a Beckman L2-6SB ultracentrifuge at 21,000 rpm. The virus band at the interface between 20% and 65% sucrose was collected, diluted with virus buffer, and layered on top of a 12 to 52% continuous sucrose gradient in virus buffer and then centrifuged at 25,000 rpm for 45 minutes in a SW 27 rotor of a Beckman L2-65B ultracentrifuge. One ml fractions of the gradient were collected from the bottom of the tubes and 0.1 ml portion of each was precipitated with cold 10% trichloroacetic acid (TCA) and the precipitate collected on a Millipore filter (Millipore Corp., Bedford, 16 Massachusetts). The filters were placed in scintillation vials, dried, 5 ml of toluene based scintillation fluid was added to each vial and the radioactivityivas counted in a Beckman LS—lOO scintillation counter. b) The Cytoplasmic Virus The infected cells were allowed to swell in reticulocyte standard buffer (RSB) for 30 minutes at 4° C and then disrupted with 4 to 10 strokes in a Dounce homogenizer. When examined under a phase microscope, about 90% of cells had been disrupted while most nuclei were still intact. The homogenate was centrifuged twice at 700 X G for 7 minutes at 4° C to remove the nuclei. .The detergent nonidet P40 (NP40) was added to the supernatant fluid until a final concentration of 0.5% was reached. The mixture was incubated at 4° C for 30 minutes and then centrifuged at 10,000 X G for 10 minutes. This supernatant fluid was layered on top of a 12 to 52% sucrose gradient and centrifuged as previously described, section (a). Determination of Protein Content f The protein of the viral preparations, horse spleen ferritin, and gamma globulin were determined by the method of Lowry, et al. (34) using crystalline bovine serum albumin as the standard. 17 Preparation of 3H Labeled DEF Proteins The DEF cultures were grown for 48 hours in medium 199 with 1/10 of the normal concentration of amino acids and 10 uCi/ml of 3H-RPH. The culture medium was separated from the cells by centrifugation, and an equal volume of saturated ammonium sulfate was added. The resulting pre- cipitate was dissolved in virus buffer and cushioned by centrifugation at the interface between 20% and 65% sucrose in virus buffer. This protein was referred to as host proteins from culture medium. The 3H-RPH labeled DEF cells were treated as for the preparation of the virus from the cytoplasmic extract of infected DEF with the exception that the sucrose gradient step was omitted. Determination of Contamination The homogenate of 3H-RPH labeled DEF was mixed either with the ammonium sulfate precipitate of the un- labeled medium from infected DEF cultures or with the homogenate of the unlabeled infected cells. The radio- activity was counted and the protein content was determined. The specific activity was expressed as counts per minute (cpm)/ug of protein. The initial mixture usually had a specific activity of over 500 Cpm/ug of protein. The virus was then purified from the mixture as described above. The specific activity of the end product was again determined. The percentage of contamination was expressed 18 as the ratio of the specific activity of the end product to that of the initial mixture. Solubilization of Viral Proteins ‘The purified viral preparations were solubilized in a solution of 1% sodium dodecyl sulfate (SDS) and 1% 2—mercaptoethanol (2—ME) in 0.1 M sodium phosphate buffer pH 7.0 by incubation at 37° C for 3 hours. The solubilized proteins were then dialized for 24 hours against 0.01 M phosphate buffer pH 7.0 containing 0.1% SDS, 0.1% 2-ME, 7% sucrose and 0.01% bromphenol blue. SDS-polyacrylamide Gel Electrophoresis This technique was similar to that described by Maizel (35). The following components were used: Stock A: acrylamide 4.125 gm bisacrylamide 0.110 gm make up to 25 ml with distilled water Stock B: .10% SDS 0.500 m1 1 M sodium phosphate buffer pH 7.0 5.000 ml distilled water 19.500 ml Stock C: 1.5% ammonium persulfate in water. For a 7.5% gel, 22.7 ml of stock A was mixed with 25 m1 of stock B and deaereated for 5 to 10 minutes under vacuum. Then 0.015 ml of N,N,N',N'—tetramethylethylene diamine (TEMED) was added to 9.54 ml to yield approximately .10 ml of gel mixture which was used to prepare three l9 6 X 100 mm gels. After the gel solution had been poured into the gel tube, water was carefully layered on and polymerization continued to completion in 40 minutes at 25° C. The electrophoresis buffer contained 0.1% SDS in 0.1 M sodium phosphate buffer, pH 7.0. The gels were "pre-electrophoresed" at 5 mA/gel for 45 minutes at 25° C. The solubilized proteins in 0.01 M sodium phosphate buffer pH 7.0 containing 0.1% SDS, 0.1% 2-ME, 7% sucrose, and 0.01% bromphenol blue were layered on top of the gels. About 70,000 to 100,000 cpm of radioactive viral proteins were used. Electrophoresis was performed at 7 mA/gel for 11 hours. The gel was then sliced into 2 mm fractions and each slice was immersed in 0.5 m1 of concentrated H202 and incubated at 50° C for 12 hours. Five ml of scintillation fluid containing 7 parts of Omnifluor in toluene and 6 parts of Triton X 100 were added to each vial. Radioactivity was measured in Beckman LS-100 scintillation counter. Estimation of the Molecular Weights of Viral Proteins The molecular weight estimation was performed on 7.5% polyacrylamide gel containing 0.1% SDS similar to that described by Shapiro, et al. (60). The following electro- phoretically purified proteins, provided by Dr. J. A. Boezi, Department of Biochemistry, Michigan State University, were 20 used as markers: trypsin (m.w. 23,300), pepsin (m.w. 35,000), aldolase (m.w. 40,000), fumarase (m.w. 49,000), catalase (m.w. 60,000), bovine serum albumin (m.w. 68,000). and phosphorylase (m.w. 94,000). These marker proteins were solubilized under the same conditions as those used for the viral proteins. The mixture of marker proteins and viral proteins were electrophoresed in parallel gels. After electrophoresis, the gels were fixed in 10% TCA at room temperature and stained with a solution containing 0.4% Coomassie blue, 10% TCA and 33% methanol for 12 hours. They were then destained in a solution containing 10% TCA and 33% methanol. The distance which each marker protein had migrated was plotted against the logarithm of the respective molecular weight to construct a standard curve. The molecular weights of the viral proteins were deter— mined by extrapolating from the standard curve. Preparation of Duck Embryo Cell Powder The DEF cell suspension was centrifuged at 2,000 X G for 30 minutes and the pellet was resuspended and washed 3 times with PBS. The pellet from the final washing was resuspended in PBS, filtered through cheesecloth to remove the course particles, and then resedimented by centri— fugation at 2,000 X G. The supernatant fluid was discarded. TThe pellet was suspended in acetone and then transfered to £1 Buchner funnel containing Whatman No. 1 filter paper. 21 The precipitate was washed with generous quantities of acetone to remove as much water as possible. The resulting filter cake, after being dried at 37° C, was a fine, light- colored powder. Preparation of Antisera The chicken anti-MDV sera were prepared by inocu— lation of one-day-old line 151 (15) chicks intra-abdominally with 2 X 106 GA-l9 infected DEF. Chicks were reared in a Horsfall-Bauer isolator for 10 weeks. The chickens with symptoms of MD were removed for collection of serum. At the end of 10 weeks, sera were collected from all surviving chickens. The titers of antibody to MDV antigens were determined by the AGP test. For the preparation of rabbit anti-MDV sera, 1 mg of partially purified MDV or SDS disrupted MDV preparations in 0.5 ml were mixed with an equal quantity of complete Freund's adjuvant and 0.25 ml was injected into each foot pad. Four weeks after the primary injection, the rabbit was injected intramuscularly with similar material. This was repeated at 2 week intervals for 2 months. Blood for serum was withdrawn periodically by heart puncture. The serum was absorbed twice with duck embryo powder and tested for antibody activity by the AGP and immunofluorescent antibody (FA) tests. 22 Adsorption of Serum with Duck Embryo Powder The serum was mixed with duck embryo powder at the ratio of 1 ml to 50 mg. After incubation at 37° C for 1 hour and 4° C overnight, the mixture was centrifuged at 40,000 X G for 30 minutes. The serum was carefully removed without disturbing the sediment and was then stored at -200 C. Agar Gel Precipitation (AGP) Test For the quantitative and qualitative determinations of antibody in chicken serum, a 0.8% agar in phosphate buffer containing 8% sodium chloride was used. The antigen was prepared from MDV infecced CK cell cultures as described by Chubb and Churchill (11) or from DEF culture infected with MDV by a similar procedure. For the detection of MDV antibody produced in rabbit serum, a 0.8% agar gel in virus buffer was used. The antigens used were partially purified and SDS—disrupted viral preparations. Immunofluorescence Technique For the detection of antigens in MDV infected cells, cell monolayers on coverslips were infected with the appropriate amounts of GA-l9 so that discrete plaques could be identified. At a proper time interval the in- fected cells on the coverslips were rinsed once with PBS, fixed in acetone at 4° C for 1 minute, and than air dried. 23 The coverslips were attached horizontally to the top of rubber stoppers with adhesive tape, moistened with PBS and then the antiserum was applied. They were then incu- bated in a humid plastic chamber for 30 minutes at 25° C. Thereafter, the coverslips were submerged in PBS and rinsed by gentle stirring for 15 minutes. The PBS was removed from the plastic chamber by an aspiration device and the coverslips were then flooded with the appropriate dilution of fluorescein conjugated anti-chick or anti- rabbit gamma globulin for 30 minutes. The coverslips were then removed from the stoppers, rinsed once in distilled water and mounted on glass slides in 90% glycerol and 10% PBS or in Elvanol (52). Coverslips were examined under fluorescence microscope with HB 200W lamp, BG12 4mm excitor filter at 510 mu. Recrystallization and Concentration ofiFerritin Ferritin was recrystallyzed by the method of Howe, et al. (24). One gm of ferritin was diluted with 100 ml of 2% ammonium sulfate at pH 5.85. Then 33 m1 of 20% cadmium sulfate was added to precipitate the ferritin. The suspension was stored at 4° C overnight and then centrifuged at 1,500 X G for 2 hours at 4° C. The crystal- line pellet was redissolved in 100 ml of 2% ammonium sul- fate andrecrystallized 5 times as described above. After the final recrystallization, the ferritin crystals were 24 dissolved in 75 ml of 2% ammonium sulfate, then an equal volume of saturated ammonium sulfate was added with constant stirring. The precipitated ferritin recovered by centri- fugation at 1,500 X G for 20 minutes at 4° C, was dissolved in distilled water and precipitated two more times in the same manner at 50% saturation with ammonium sulfate. Sufficient water was added to the final precipitate to allow transfer to dialysis tubing. The preparation was dialyzed against running cold tap water for 24 hours and then against cold 0.05 M phosphate buffer pH 7.5 for another 24 hours. The dialyzed ferritin was concentrated by centrifugation at 100,000 X G for 2 hours. Three- fourths of the colorless supernatant fluid was removed and the pellet was rediSsolved in the remaining one-fourth of the supernatant fluid after storage at 4° C overnight. It was sterilized by passing through a 0.45 u Millipore filter and then stored at 4° C. Preparation of Ferritin Conjugate Ferritin in 0.05 M phosphate buffer pH 7.5 was mixed with 0.3 M borate buffer pH 9.5 to give a final concentration of 20 to 25 mg/ml of ferritin in 0.1 M .borate buffer. Xylylene diisocyanate (XC) was then added in the proportion of 0.1 ml per 100 mg of ferritin. The Imixture was stirred vigorously for 45 minutes and then Cenatrifuged for 30 minutes at 1,500 X G. The brown 25 supernatant fluid (ferritin intermediate) was used im- mediately for conjugation. Purified gamma globulin was added to the ferritin intermediate at a ratio of 1:4 w/w. Fresh 0.3 M borate buffer was then added to maintain the molarity of 0.1 and pH of 9.5. This reaction mixture was stirred gently at 4° C for 48 hours and dialyzed against 0.1 M ammonium carbonate overnight at 4° C. This step was to inactivate the residual isocyanate group which could interfere with the specific tagging of the ferritin. The conjugate was then dialyzed for 4 to 5 hours against 0.05 M phosphate buffer pH 7.5 at 4° C. Unconjugated globulin was removed by 3 cycles of centrifugation of the mixture at 225,000 X G for 5 hours. The pellet of ferritin conjugated globulin was resuspended each time in 0.05 M phosphate buffer pH 7.5. The final solution was sterilized by filtration through a 0.45 u Millipore filter. Ferritin Conjugation Technique for Electron Microscopy Cell monolayers on the coverslips were fixed for 30 minutes in 5% formaldehyde in 0.1 M phosphate buffer pH 7.2 (24). They were then treated with 10% DMSO in 0.1 P4 phosphate buffer pH 7.2 for 10 minutes and placed in a 100 mm Petri dish which was submerged in a mixture of dry 26 ice and ethanol. After thawing at room temperature, the monolayers on the coverslips were rinsed once with PBS. For the direct staining technique, the conjugate was applied to cells on the coverslips which were placed in a humid chamber at 37° C for 1 hour. The coverslips were then washed 4 times with PBS, scraped off and centri- fuged. Thin section of the pellet was prepared as described below. For the indirect staining technique, the anti-serum was first applied to cells on coverslips and incubated and washed as above. The conjugated anti-globulin was then applied on the monolayers and incubated, washed, scraped off and pelleted as described above. Preparation of Specimen for Electron Microscopy a) Thin Section The pellet of infected cells which had been stained with the ferritin conjugate, as previously described, was fixed in 1% glutaryl-aldehyde in 0.1 M phosphate buffer, pH 7.2, for 1 hour at 4° C. It was then rinsed twice with 0.1 M phosphate buffer pH 7.2 and postfixed with 1% osmium tetroxide for 45 minutes at 4° C. The Specimen was washed once with 0.1 M phosphate buffer pH 7.2 and dehydrated in the following steps: 27 Dehydrating mixture Dehydrating time (minute) 30% ethyl alcohol, cold 15 50% ethyl alcohol, cold 15 75% ethyl alcohol, cold 15 95% ethyl alcohol, room temperature 30 100% ethyl alcohol, room temperature 30 100% ethyl alcohol, room temperature 30 100% ethyl alcohol, room temperature 30 Propylene oxide, room temperature 15 Propylene oxide, room temperature 30 Propylene oxide, room temperature 30 The specimen was immersed in 50%-50% propylene oxide-epon mixture overnight to allow the epon to penetrate. It was then embedded in epon. Polymerization was completed by incubation at 60° C for more than 72 hours. The gelatin of each block was removed by submerging the block in warm water. A small truncated pyramid with an area less than 1 mm2 was formed by trimming the top of each block with a razor blade. Sections of about 60 mu thick were cut with a LKB ultramicrotome using a glass knife. The sections formed a ribbon which was picked up with a copper grid, stained with lead citrate and uranyl acetate, and coated with carbon. The sections were examined in Siemens Elmiskop 1A electron microscope. 28 b) Negative Staining About 20 ul of 2% potassium tungstate was mixed with approximately 10 ul of viral preparation and 5 ul of 0.05% sucrose. A portion of the mixture was picked up with a carbon coated copper grid and about 1 minute later the excess fluid was removed by blotting the edge of the grid. The grid was air dried and examined. Conditions for the Absorption of MDV on CK Cells a) Optimal Absorption Time The CK monolayers were infected with an appropriate amount of GA-MDV. At 0, 1, 2, 4, 6, 8, 10, 12 hours after infection, duplicate plates were rinsed twice with culture medium, fresh culture medium was added, and they were incubated at 37° C. The plaques were counted 4 to 5 days later. The number of plaques was plotted against the adsorption time. Thus, the optimal absorption time was the time when the plaque number reached a maximum. b) Absorption at 4°, 25° and 37° C Three sets of CK monolayers were infected with MDV and incubated at 37° C, 25° C and 4° C respectively for 3 hours. The monolayers were rinsed twice with culture medium and fresh medium was added. After incubation at 37° C for 4 days, the plaques were counted. The Optimal temperature was the one which had the greatest number of plaques. RESULTS Purification of Virus The culture medium from 3H-thymidine labeled MDV infected DEF, when examined on 12 to 52% sucrose gradient centrifugation after it was precipitated in 50% saturated ammonium sulfate and redissolved in Virus buffer, had 2 bands. There was a sharp band near the bottom of the tube and below this was a rather diffuse band (Figure 1). The negatively stained sharp band contained predominantly small aggregates of naked nucleocapsids and a few enveloped virions (Figure 2). The diffuse band contained naked nucleocapsids. The viral preparations from NP40-treated cytoplasmic extract of 3H-thymidine labeled GA-MDV infected DEF, when analyzed on 12 to 52% sucrose gradient centrifugation, had a single sharp band (Figure 3). This band consisted of mostly naked nucleocapsids and very few enveloped virions. The yield of MDV protein per roller bottle was only 50 ug from the sharp band of the supernatant virus, 60 ug from the diffuse band of the supernatant virus, and 150 to 200 ug from the cytoplasmic virus (Table 1). 29 3O 30 W 3H CPM x 10'2 ’ up 0 I 5 l l l 1 IO 20 3O FRACTION NUMBER P¢ellei Figure 1.--Sucrose density gradient analysis of MDV from infected culture medium. 31 Figure 2.--Electron micrograph of MDV from the sharp band of the supernatant virus. X 80,000. 32 -3 H CPM X IO 1° 3 FRACTION NUMBER Figure 3.-—Sucrose density gradient analysis of MDV from the cytoplasmic extract of infected cells. 33 moufl> m.mna mom ovo.e mma oem.m UHEmmadoumu m.oe oe oom.a He OHH.N cane snowmen monfl> m.Hm me owe mm one.e acne ncehcceodcm Quocm cannon Amsvmaupom Mom Amsvaouoe Amsvmaueom Hem Aosvamuoa Hem mmmHm>< Ammaupoc omvm .pdxm Ammauuon vmva .umxm .mcoflumuomoum HmHH> wo mucopcoo chDOHmII.H mqmde 34 Degree of Host Protein Contamination in VIfal Preparations The contamination of the host protein in MDV prepa- ration was determined by mixing 3H—RPH labeled uninfected DEF cell homogenate with either ammonium sulfate precipitate of the unlabeled GA-MDV infected culture medium or unlabeled GA-MDV infected DEF cell homogenate. The results (Table 2) indicated that the supernatant virus from the sharp band had the least host protein contamination, yet the host protein still consisted of 15 to 16% of the viral prepa- ration. The supernatant virus from the diffuse band con- tained 19 to 20% host protein whereas the viral preparation of the cytoplasmic extract of the infected cells contained about 22 to 23% host proteins. Electrophoretic Pattern of the Viral Proteins of’MDV When the rebanded viral preparations from the infected culture medium and cytoplasmic extract were solu- bilized and analyzed by SDS-polyacrylamide gel electro- phoresis, at least 8 proteins were distinguishable in each preparations (Figures 4 and 5). The first 2 MDV proteins from the supernatant virus designated as VPI and VPII, contained about 35% of total labeled amino acids whereas VPV, VPVI, and VPVIII consisted of 33% of the labeled amino acids. The VPIII, VPIV, and VPVII were minor proteins each containing less than 10% of 35 TABLE 2.--Purity of MDV preparation.a Specific Activity(cpm/ug protein) Expt. 3 b . Per Cent H-RPH-labeled normal Contamination DEF cell homogenate Purified plus unlabeled infected MDV material 1 630b 101d 16 120e 19 . 800c 176 22 ; t;“‘ b d :1 2 600 89 15 Q 122e 20 850° 196 23 aHomogenate of normal DEF cells, grown in the presence of 3H-RPH, were mixed with either 50% ammonium sulfate precipitate of unlabeled culture medium from in- fected cells or homogenate of unlabeled infected cells. The virus was then purified as described in the text. Specific activities of purified viral preparations and the original homogenates were determined, and their ratios were calculated to determine the per cent of contamination. b3H-RPH labeled normal DEF cell homogenate mixed with 50% ammonium sulfate precipitate of unlabeled culture medium from infected cells. C3H-RPH labeled normal DEF cell homogenate mixed twith unlabeled infected cell homogenate. dTop viral band from infected culture medium. eBottom viral band from infected culture medium. 36 s— vs" ”i" W vi" “5“” WV" 3,... 2N l 1 IO 20 3O 4O 5O FRACTION NUMBER Figure 4.--E1ectropherogramof the structural proteins of MDV from the sharp band of the supernatant virus. 37 s— i" “‘i'V‘i'Vi’” “3“?” “i“ “i" ‘1’ 94— , X 23— O. O 12*— . 3 FRACTION NUMBER Figure 5.--Electr0pherogram of the structural proteins of MDV from the cytoplasmic virus. 50 "I r: 38 the labeled amino acids. Nearly 13% of the labeled amino acids stayed near the origin of the gel (Table 3). The amino acid in VPII of cytoplasmic virus was considerably lower than that from the supernatant virus. This indicated that VPII may be a viral glycoprotein since cytoplasmic viral preparation contained less enveloped virions and had been treated with NP40 which lysed the envelope. .0 L7 Glycoprotein of MDV In order to study the viral glycoproteins which are primarily present in the viral envelope, infected DEF was labeled with 3H-glucosamine and the virus was purified from the culture medium. Then 3H-glucosamine labeled and l4C-RPH labeled viruses were mixed, solubilized and analyzed by SDS-polyacrylamide gel electrophoresis. The 3H- glucosamine was associated with VPII and VPIV (Figure 6a). When the viral preparation was treated with NP40, both 3H-glucosamine peaks were drastically reduced (Figure 6b). The 3H-glucosamine was not detected either in the diffuse band of the supernatant virus or the sharp band of the cytoplasmic virus which was treated with NP40. These results indicated that VPII and VPIV were 2 glycoproteins of MDV. 39 ‘ In... 1.] flu, o.oH e.m m.n m.oa o.oa HHH> o.m m.v e.m m.v m.m HH> o.ma m.ma v.mH o.eH m.ma H> v.0a m.m m.ma m.m m.m > o.m N.h m.m e.o m.m >H H.m o.m v.m v.0 m.m HHH m.mH H.mH m.eH m.oa m.mH HH «.ma m.mH m.>H v.om e.ma H e.ma o.ma e.ma m.ma N.HH caaaho mmmuo>< v.pdxm m.umxm m.umxm H.umxm .oz cflmuonm vaceououd Hmnfl> ca mceom OCHEMimm .msue> mmoomflc m.xmumz CH Desmond mcflmwoum Housuosupm #COHTWMHU mflu. CH WUHUM OCHEM m>HMUMOHmuMH MO COHfiSQHHNWHQIlom Maw—”mama“. 40 Tie N..o_ x 2% In 5 4 « _ ‘0' -'- " ' Frocrion Number 1.--: 4 N9 x ado o: (b) NP40 treated. Figure 6.--E1ectrophoretic analysis of MDV glycoproteins. (a) untreated; 41 Host Proteins in the Electrophoretic Profile of the Proteins of MDV It is possible that the host proteins which consti- tute 15 to 20% of the proteins in viral preparations may be responsible for certain protein peaks in Figures 4 and 5. In order to test this, the l4C-RPH labeled MDV was mixed with 3H-RPH labeled normal DEF cell homogenate. The mixture was then solubilized and electrophoresed on SDS- polyacrylamide gel. The results indicate that normal host proteins do not migrate identically to viral proteins in polyacrylamide gel (Figure 7). The same is true for the host protein in the culture medium of uninfected culture (Figure 8). Furthermore, the electrOphoretic profile indicated that host proteins contained more of the medium and small size proteins. Therefore, the proteins shown in the electrOpherograms of MDV preparations are viral proteins. Comparison of Proteins of MDV and two Other Herpesviruses To compare the protein profiles of MDV and 2 other herpesviruses, 3H-RPH labeled MDV was mixed either with l4C-RPH labeled HSV or PrV. Each mixture was purified solubilized and analyzed by electrophoresis in 7.5% polyacrylamide gel containing 0.1% SDS. Similar electropherograms were observed when 3H-MDV and l4C-HSV proteins were coelectrophoresed except for the following minor differences (Figure 9): (a) No MDV protein ‘Ih. 42 Ti N.o_ x 2% I» 5432 44 _ J “I 50 1.--; Nb. x 2% 0: Fraction Number Figure 7.--Determination of host protein contamination in supernatant virus. 43 .msufl> owfimmamoumo aw cowumcfiemucoo swwuoum umo: mo cowumcflsumumcil.m enamwm .mnEaz cozooe .1 -) 20: x was H9 @ ( 1.----.1 me x 2% o: 44 _ 5 ale N.o. x 28 In FRACTION NUMBER Figure 9.--Electropherogram of MDV and HSV proteins. 45 corresponded to HSV protein VI, but the shoulder of MDV VPV seemed to resolve into a minor peak, (b) No HSV protein corresponded MDV VPVIII. The protein profiles of MDV and PrV were also similar except for the following minor differences (Figure 10): (a) No PrV protein corresponded to MDV VPIII, (b) No PrV protein corresponded to MDV VPVI; instead, PrV protein 6 migrated behind MDV VPVI. Estimation of the Molecular Weights of'MDV Proteins The average molecular weights of MDV proteins ranged from 33,000 to 101,000 daltons (Table 4) as derived from the standard curve (Figure 11). Conditions of the Adsorption of MDV to CK Cells a) Optimal Adsorption Time Adsorption of MDV to CK monolayer reached a plateau after 8 hours pi (Figure 12). The rate of absorption after 8 hours decreased and the plaque count was about 80 to 90% of that of control of which was absorbed for 24 hours. b) Optimal Temperature Since CK monolayer tended to peel off the plate at 37° C after the MDV was absorbed at 4° C for more than 5 hours, the absorption time of 3 hours was chosen to determine 46 1.----.1 Nb. x 2% o: 5 4 3 2 I _ L _ _ _ a a a 2 .. ”a 40 ele N.o_ x 2% In 0 5 3O 20 FRACTION NUMBER Figure 10.--Electropherogram of MDV and PrV proteins. 47 mm mm em mm HHH> mm mm oe mm HH> we we mm me H> mm mm mm mm > am pm on om >H me He mm ms HHH mm om mm mm HH HOH hm boa mm H moonw>¢ m.umxm m.umxm H.umxm .oz Asepamcv OH x “roam; Hmaoomaoz aflopoum m .mCHwDoum mSHH> mmmmmflc m.xmumz mo mucmflmz Hofisomaozll.v.mqm¢9 48 5 If: I X '09 Phosphorylase A c9 8 '— 3, 7 — VP I BSA 3 5 —— Catalase tr 5 *- Fumarase j 4 e T Aldolase I) P ' 8 3 r VP VI ”3'" —I . O Trypsun 2 2 — VPVI 4 |x|() 1 I IL I i 20 40 60 80 origin Figure ll.--Molecular weight determination of the MIGRATION DISTANCE (mm) structural proteins of MDV. IOO 49 '1. I00— 0 ° 0 O O O O 75... 5 g 6 2:» 8 25— LL 1 l I l l l O I 2 4 6 O 0 I2 I8 Figure 12.--The adsorption curve of MDV to CK cells. 50 the optimal temperature for the adsorption of MDV to CK monolayer. Adsorption at 37° C was more favorable than 25° C or 4° C (Table 5). Analysis of MDV Antigen and Antibody by AGP Test The following results were obtained with the AGP Test: A. The rabbit anti-MDV serum formed 2 precipitation lines when reacted with homologous antigen (Figure 13a) and 3 lines when reacted with SDS disrupted MDV preparation (Figure 14). This serum also formed 2 lines against A- antigen; one of which shared partial identity with the line formed between chicken anti-MD serum and A—antigen (Figure 13b). B. The rabbit anti-SDS—disrupted MDV serum formed 3 lines against homologous antigen. These 3 lines had identity with the 3 lines formed between SDS-disrupted MDV and rabbit antiserum produced against MDV (Figure 14a). The rabbit anti—SDS-disrupted-MDV serum formed 1 line against MDV (Figure 14b). This serum also reacted with A-antigen and had partial identity with the line produced between chicken anti-MD serum and A-antigen (Figure 13b). C. There was no reaction between chicken anti-MD serum and partially purified MDV or SDS-disrupted MDV (Figure 13c). 51 .mmsomam mo HmQESZQ .Houecoum m.m mm Hm mm He mm muses m U co H.©H mm ow eh mm mm mason m U omm «.mm com vow boa mom mad muooc m U ohm OOH mom Hmv nae mmm vam mmusoc NH 0 ohm mEHB w mmmnm>¢ v.pmxm m.umxm m.pmxm H.9mxm COHDQHOmnd musumquEmB .maamo MU Ou >Q2 mo COHDQHOmpm ecu co musumummfime mo pomwmmli.m mqmde 52 Figure l3a.--Reaction of rabbit antibodies with MDV. a: rabbit anti-MDV serum b: rabbit anti-SDS-disrupted—MDV serum c and d: normal DEF homogenate e: MDV In 0.8% agar in virus buffer. Figure 13b.--Reaction of chicken and riabbit antibodies with A-antigen. a: rabbit anti-MDV serum b: MD positive chicken serum c: MD negative chicken serum d rabbit anti-SDS-disrupted—MDV serum e A-antigen In 0.8% agar in phosphate buffer with 8% sodium chloride. Figure 13c.——Relation between MDV, SDS-disrupted MDV, and A-antigen. a: A-antigen b: SDS-disrupted—MDV c and d: MDV e: chicken MD serum In 0.8% agar in phosphate buffer with 3% sodium chloride. 54 Figure l4a.——Reaction of rabbit antibodies with SDS-disrupted MDV. a: rabbit anti-MDV serum b: rabbit anti-SDS—disrupted-MDV serum c and d: Normal DEF homogenate e: SDS-MDV Figure l4b.--Reaction of rabbit antibodies with MDV and SDS-disrupted MDV. a: rabbit anti-MDV serum b: SDS-MDV c: rabbit anti-SDS—disrupted—MDV serum d: MDV 55 56 The MDV Antigens in CK Cells Detected by FA Technique The rabbit anti-MDV serum with an FA titer of 1:160 was diluted with PBS and used through out the experi- ment. Between 0 to 17 hours p.i. bright fluorescent stain was only found in scattered round cells originating from the inoculum. The newly formed antigen detectable in CK cells was first found at 17 hours p.i. Two CK cells adjacent to a round DEF cell had bright stain (Figure 15a). The FA plaques became more recognizable at about 19 to 20 hours p.i. (Figure 15b). In certain occasions exclusive cyto- plasmic stain could be observed at 19 hours p.i. (Figure 15c). Fine granular stain was first noticed at 22 hours p.i. (Figure 16a). After 24 hours p.i., as the infected cells appeared rounded up, the fine granular stain lost its distinction and the entire cell was brightly stained (Figure 16b). The late large FA plaque consisting of round cells started to appear as early as 24 hours p.i. and was complete at about 60 hours p.i. (Figure 16c). Since the infection process was not synchronous, different stages of FA stain development-—from diffuse stain in morphologically normal cells to the formation of round cells of a mature FA plaque--could be observed from 24 to 48 hours p.i. 57 Figure 15.—-The FA antigen of MDV in CK cells. (a) 17 hours postinfection. (b) and (c) 19 hours postinfec— tion. Approx. X230. 58 Figure l6.—-The FA antigens of MDV in CK cells. (a) 22 hours postinfection.(b) 24 hours postinfection. (c) 60 hours postinfection. Approx. X230. 59 Detection of MDV Antigens by Ferritin Conjugated Antibody With chicken serum, the ferritin particles were found to tag cytoplasmic membrane of partially disrupted infected cells. In fully disrupted cells specific tagging of the nucleus of infected cell and the viral nucleocapsids was also observed. More ferritin particles could be found in areas with nucleocapsids compared with areas lacking them (Figure 17a and 17b). Tagging was not observed on the viral core or the inner side of the capsid. The envelope of the virion was also tagged with ferritin (Figure 17c). The normal cells treated with antiserum and the infected cells treated with normal serum did not react specifically with ferritin conjugate. Both cytoplasmic and nuclear matrices of infected cell were tagged with ferritin when rabbit anti-MDV serum was used. The viral nucleocapsids had some ferritin parti— cles tagged but to a lesser extent than those which appeared on the cytoplasmic and nuclear matrices of the infected cell. Again, normal cells treated with antiserum and infected cell treated with normal serum did not have specific tagging of ferritin particles. 60 a. ‘n_ y .13 M Figure l7.--The MDV antigen in infected cells detected by ferritin conjugated with chicken anti-MD serum. (a) Portion of the nucleus of a dis- rupted DEF infected with MDV. (b) Higher magni- fication of a). (c) An enveloped virion taggened with ferritin particles. DISCUSSION Purification of enveloped virions is often compli- cated by host protein contaminants as documented for arbovirus (1) and herpesvirus (55). The same difficulty was encountered in the purification of MDV. The cell association nature of MDV and its slow growth caused a poor yield of virus (Table 1) and high rate of contamination (Table 2). Nevertheless, MDV prepared from the medium of infected cell cultures had less host protein contamination than those from infected cells themselves. The virions from the medium of infected culture may be excreted from the infected cells, but most likely they are from infected cells which were detached from monolayer and floated in the medium. These cells have viral specific inclusions and enveloped virions and are easy to disrupt by simple shaking. Analysis of purified MDV on SDS-polyacrylamide gel electrophoresis revealed 8 distinct proteins. An additional protein with a molecular weight larger than that of VPI might be present since about 13% of labeled amino acids stayed near the origin of the gel. However, the evidence for its existence based on the results from coelectrophoresis of the proteins of MDV with those of HSV and PrV was not 61 62 conclusive. Of these 8 proteins the first 2, VPI and VPII, constituted about 35% of the total labeled amino acids. About 33% was present in VPV, VPVI, and VPVIII, whereas VPIII, VPIV and VPVII were minor proteins. The VPI was the major component of the MDV proteins with a molecular weight close to the protein II of HSV (47) and protein 2 of PrV (62). This conclusion was drawn from the compara- tive study of the protein patterns of HSV and MDV, and PrV and MDV (Figures 9 and 10). Some proteins of MDV, HSV and PrV have same size as indicated in the similarity of the migration rate in the gel, but their amino acid compositions may be quite different. A similar observation was also reported for proteins of Epstein-Barr virus (EBV) of Burkitt's lymphoma and HSV (71). Because of the low yield and impurity of the MDV preparations, it was not possible to establish a direct relationship between these proteins and the structural components, i.e., envelope, capsid and core, of MD virion. Such a relationship had been established in HSV (47) and PrV (28,62). The glucosamine labeling into VPII and VPIV and their sensitity to NP40 indicated that they might be the components of the viral envelope. The NP40 dissolves the cytoplasmic membrane (46) and virion-associated envelope (47), but the possibility that they might be associated 63 with virus modified cellular membrane could not be excluded. There may be more than 2 glycoproteins present in the MDV envelope as is in HSV (47,62) and PrV (28), but such was not detected for MDV in these experiments. This is a discrepancy in the molecular weight of the major proteins among herpesviruses. Although the proteins of MDV, HSV and PrV are similar in size, the molecular weight of VPI of MDV is about 10% less than the molecular weight of the protein II of HSV and 20% less than the molecular weight of the protein 2 of PrV. This dis- crepency might be due to the method used. The molecular weight of VPI of MDV was determined in parallel gels with marker proteins and making corrections as suggested by Weber and Osborn (70). The value obtained might be subject to more error than the one obtained by using radioactive marker proteins. The partially purified MDV contained protein with an antigenicity similar to that of A—antigen. This may indicate that A-antigen is a structural component of MDV. However, A—antigen may be present in the MDV preparation as a contaminant; thus, it is difficult to conclude that A-antigen is part of the structural proteins of MDV. The SDS-treated MDV induced antibody against A-antigen-like protein in rabbits. This indicated that A—antigen-like activity present in the MDV preparation could not be abolished when treated with SDS. 64 The reactions in the AGP test between rabbit anti- MDV serum and SDS disrupted MDV indicate that MDV injected into rabbit disintegrated into structural subunits. This may be due to the action of certain unknown enzyme(s) on MDV. The 3 lines resulting from the reaction between rabbit anti-MDV and SDS disrupted MDV did not share antigenic identity with those obtained from the reaction of rabbit anti—MDV serum and MDV. These reactions indicate that viral capsid protein subunits of MDV do not have the same antigenic character as the complete viral capsid. Similar observation has also been reported for PrV (20,25). Since CK cells were not uniformly infected and cell associated virus was used, the FA antigens were not detected as early as those of HSV and PrV. Nevertheless, FA antigen of MDV was detected as early as 17 hours p.i. when the cells were morphologically normal. This was about 7 to 8 hours earlier than that reported by Purchase (49). The FA antigens were more prominent in the cytoplasm than those reported by Purchase (49) who observed that nuclear FA staining was more prominent. The FA antigens described in the present report are similar to that of Kottaridis and Luginbuhl (31). The differences of the appearance of FA antigens between the present report and that of Purchase (49) may be due to the time of replication cycle when the observation was made and the serum used. In 65 the present report, the observation was made at early stage of replication whereas Purchase observed most of the FA antigens at the time when CPE had already appeared. The antisera used may also contribute to the difference. In the present report, the rabbit anti-MDV serum was used whereas in Purchase's experiment chicken anti-MD sera were used. The partial identity of A-antigen and purified MDV preparation in the AGP test indicated that besides A-antigen, rabbit anti—MDV detected other antigens in infected cells. This may partially explain the discrepancy in the appearance of FA antigens observed in the present report and that of Purchase. The finding that certain cells had only cytoplasmic FA antigen at 19 hours p.i. may indicate that viral antigen is first synthesized in the cytOplasm of the infected cell and is then transfered to the nucleus, since at the later stage of infection most constituent cells in an FA plaque are extensively stained in both cytOplasm and nucleus. This may also indicate that infection of MDV in CK cells is a slow process quite different from that of HSV (54) and PrV (26). However, it is possible that the earlier appearance of FA antigens would be detected if large quantities of cell-free virus was employed to infect the CK cells. 66 The lack of differences in the staining of FA antigens when rabbit anti—MDV or rabbit anti-SDS-disrupted— MDV serums were used reflected that rabbit anti-MDV serum contained antibody against the protein subunits of MDV. The various stages of FA plaque development between 24 to 48 hours p.i. might be due to the asynchronous stages of the inoculum. The ferritin conjugate experiment indicated that chicken anti-MD serum contained antibodies against MD viral envelope and capsid proteins. Chicken antiserum may contain antibodies against various non-structural viral prOteins produced during the process of infection. The results with the ferritin conjugated with rabbit anti-MDV serum were in agreement with those ob— tained with the AGP test. The rabbit antiserum reacted stronger against the protein subunits than against the whole viral capsid proteins. This may explain why more ferritin particles tag the cell matrix than viral parti— cles. The tagging of cytOplasmic membrane with ferritin particles confirmed the existence of surface antigen on MDV infected CK cells which has been detected by the FA technique (10). SUMMARY 1. Virus was partially purified from the culture medium and from cells of MDV infected DEF culture by sucrose density gradient centrifugation. 2. The proteins in MDV were analyzed by electro- phoresis on 7.5% polyacrylamide gel containing 0.1% SDS. 3. At least 8 proteins were detected by SDS- polyacrylamide gel electrophoresis and 2 of them were glycoproteins. The molecular weights of these 8 proteins ranged from 33,000 to 101,000 daltons. 4. Comparative electropherogram of the proteins of HSV and MDV, and those of PrV and MDV were similar with minor differences. 5. The MDV and SDS~disrupted MDV preparations contained an antigen which shared partial identity with the A-antigen of MDV. The antigenicity of this antigen was not abolished by SDS. 6. Three antigens were detected in SDS disrupted MDV when it was reacted with rabbit anti-MDV or anti-SDS- disrupted-MDV serums. The rabbit anti—MDV serum shared identity with rabbit anti-SDS-disrupted MDV serum when reacted against SDS disrupted MDV. 67 68 7. The MDV preparation seemed to disintegrate into the subunits when injected into rabbit. 8. Subunits of MDV capsid were antigenically dif— ferent from those of whole capsid. 9. The FA antigen was first detected at 17 hours postinfection and before 20 hours postinfection the FA stain was predominantly in the cytoplasm. 10. Fine granular cytoplasmic FA antigen was detected at 20 to 22 hours postinfection. After 22 hours, the FA antigen spread over the entire cell and the FA staining plaque was observed 60 hours postinfection. 11. Chicken anti-MD serum contains antibodies against the viral capsid and envelope proteins of MDV. Rabbit anti-MDV and anti—SDS-disrupted MDV reacted stronger against cytoplasmic and nuclear matrix of infected cell than against viral particles. 12. Surface antigen was also detected by ferritin conjugates when both chicken and rabbit anti-MDV sera were used. LITERATURE CITED 69 10. LITERATURE CITED Acheson, N. H., and I. Tamm. 1970. Purification and properties of Semliki Forest Virus nucleocapsids. Virol. 41:306-320. Ahmed, M., and G. Schidlovsky. 1968. Electron microscopic localization of herpesvirus type particles in Marek's disease. J. Virol. 2:1443—1457. Ben-Porat, T.; H. Shimono; and A. S. Kaplan. 1969. Synthesis of proteins in cells infected with herpesvirus. II. Flow of structural viral proteins from cytoplasm to nucleus. Virol. 37:56-61. Ben-Porat, T., and A. S, Kaplan. 1970. Synthesis of proteins in cells infected with herpesvirus. V. Viral glycoproteins. Virol. 41:265-273. 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Weber, K., and M. Osborn. 1969. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. Biol. Chem. 244:4406-4412. Weinberg, A., and Y. Becker. 1969. Studies on EB virus of Burkitt's lymphoblast. Virol. 39: 799-806. APPENDICES 77 APPENDIX 1 REAGENTS AND ISOTOPES Reagents: 1. 2-mercaptoethanol (2-ME) and sodium dodecyl sulfate (SDS) are fromMatheson Co. Inc. Norwood, Ohio. Acrylamide, bisacrylamide, and N,N,N',N'-tetramethy- lethylene diamine (TEMED) are from Eastman Kodak Co. Rochester, New York. Ammonium persulfate, ammonium sulfate and dimethyl sulfoxide (DMSO) are from Fisher Scientific Co. Fair Lawn, New Jersey. Bromphenol blue is from Allied Chemical Inc. Morristown, New Jersey. Trichloroacetic acid (TCA) is from Merck and Co. Rahway, New Jersey. Xylylene diisocyanate (XC) is from Polyscience, Inc. Paul Valley Industrial Park. Warington, Pennsylvania. Coomassie blue is from Colab laboratories Inc. Chicago Heights, Illinois. Nonidet—P40 (NP40) is from Shell Oil Co. Chicago, Illinois. 78 B. 79 Isotopes: l. D—glucosamine—l-14C hydrochloride and D-glucosamine— 6—3H hydrochloride are from New England Nuclear Corp. Boston, Massachusetts. Methy-3H-thymidine, methyl-14C-thymidine, 3H— reconstituted protein hydrolysate (RPH) and l4C-RPH are from Schwarz Bioresearch Inc. Orangeburg, New York. APPENDIX 2 BUFFERS, SOLUTIONS AND MEDIA Buffers: l. Phosphate buffer saline (PBS). 2. Reticulocyte standard buffer (RSB): 0.01 M Tris- HCl pH 7.4, 0.005 M MgCl2 and 0.005 M NaCl. 3. Virus buffer: 0.02 M Tris—HCl pH 7.4 and 0.15 M NaCl. 4. Sodium phosphate buffer (0.1 M and 0.01 M) pH 7.0. 5. Phosphate buffer (0.05 M) pH 7.5. 6. Phosphate buffer (0.1 M) pH 7.2. 7. Borate buffer (0.1 M and 0.3 M) pH 9.5. Solutions: 1. 2% ammonium sulfate pH 5.85 and saturated ammonium sulfate. 2. 20% cadmium sulfate. 3. 0.1 M ammonium carbonate. 4. 5% formaldehyde in 0,1 M phosphate buffer pH 7.2. 5. 10% DMSO in 0.1 M phosphate buffer pH 7.2. 6. 1% glutarylaldehyde in 0.1 M phosphate buffer pH 7.2. 7. 1% osmium tetroxide. 80 .J‘ 81 8. 2% phosphOtungstic acid (PTA). 9. Uranyl acetate and lead citrate. C. Media: 1. For DEF culture: Growth medium: Medium 199 and F10 at the ratio of 4:5, 5% tryptose phosphate broth, 0.084% sodium bicarbonate, 100 units/ml of penicillin, 100 ug/ml of streptomycin, 25 units/ml of mycostatin and 4% heat-inactivated calf serum. Maintenance medium: The same as growth medium except that the serum concentration is reduced to 1%. 2. For CK cell culture: Growth medium: 80 parts of Eagle's basal medium, 10 parts of tryptose phosphate broth, 3 parts of 2.8% sodium bicarbonate, 5 parts of bovine fetal serum, 100 units/ml of penicillin, 0.01 mg/ml of streptomycin, and 25 units/m1 of mycostatin. Maintenance medium: The same as growth medium except that the serum concentration is reduced to 1%. fl ”1 ”I II” H