v'—.A. PARTiAL FRACTEONAYEON BY ULTRACENTREWfiATmN 0F NEWCASTLE DI$EASE VIRUS INTO COWLEMEEQT HXATION AND AN iw‘ECTEVE FRACT?QN Thesis for flu 9mm :9 My. D, MlCHfiGAN $13M? CGLEGE Rather Ewe-aims Johnwn H354. This is to certify that the thesis entitled PARTIAL FRACTIONATION BY ULTRACENTRIFUGATIW OF NEW CASTLE DISEASE VTRUS INTO COMPLLNE‘IT FIXATICN 1WD AN IY‘TFEC'IIVE FRACTICN presented by Bother Rodenious Johnson has been accepted towards fulfillment of the requirements for Ph.D. Bacteriology degree in \Jo ahkr’km Major professor Date July 7, 19511 0-169 PARTIAL FRACTICNATICA BI ULTHACENTMINUGATION OF NEWCASTLE DISEASE VIRUS INTO COMPLEMENT FIXATION AND AN INFECTIVE FRACTION BY Rother Rodenious gghnson A THESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Bacteriology and Public Health NEIL 7 gym”? 1m: H"7")’¢ ‘tr ACKNOWLEDGMENTS The author wishes to express his sincere thanks to Dr. Walter H. Mack, under whose inspiration, supervision, and interest this investigation was undertaken. He is also greatly indebted to Dr. H. J. Staf- seth, Head of the Department of Bacteriology and Public Health, for his kind guidance and valuable assistance. .0. ad ”I 4' PARTIAL FRACTIOHATION BY ULTRACENTRIFUGATION OF NEWCASTLE DISEASE VIRUS INTO COMPLEMENT FIXATION AND AN IR “CTIVE FRAC TION By I Bother Rodenious Johnson AN ABSTRACT Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DCETOB OF PHILOSOPHY Department of Bacteriology Year 1954 Approved AM FM hW< . T1 Soluble Hewcas1 'quu 4-!“- Complement fixation studies were done on an antigen consisting of Newcastle disease virus in hamster brains. In the first series of experiments, the complement fixation tests were carried out on the supernatant fluids and sediments of the first and second cycles of ultracentrifugation. It was found that the virus was removed from the medium at 114,000 time gravity. The sediments contained infective virus, complement fixing components and was capable of agglutinating erythrocytes. Both first and second cycle supernatant fluids were inactive for these three components. In the second experiment, a sample of the antigen was subjected to ultrasonic vibration prior to ultracentrifugation. As a result of this treatment, the supernatant fluids of both cycles of centrifugation contained complement fixing properties but were not infectious nor did it produce hemagglutination. Like the first experiment, the sediments from both cycles of centrifugation were infectious, fixed complement, and produced hemagglutination. The experiments, therefore, show that Newcastle disease virus contains a soluble complement fixation antigen. Besides other serological similarities, Newcastle disease virus compares to the related influenza viruses in this respect. INTR MATE BEE DI E SUId BIE TABLE OF CONTENTS INTRODUCTION . . . o o . . o o . MATERIALS AND METHODS . . . . . . . . . Propagation of Newcastle Disease Virus . . . . . Hemagglutination and Hemagglutination-inhibition teats o c o o o o o o o 0 Transmission of NDV to Syrian Hamsters . . Preparation of Complement Fixation Preparation of Erythrocytes . . . Titration of Hemolysin . . . . . . Preparation of Complement . . . . Titration of Complement. . . . . . Fractionation of Antigen . . . . . Titration of Antigen . . . . . . . Preparation of Antisera . . . . . Titration of Antisera . . . . . . RESULTS . . . . . . . . . . DISCUSSION . . . . . . . . . 0 SUMMARY . o o . . . . . . . BIBLIOGRAPHX . . o o . . . . . . Antigen 16 17 20 20 21 22 25 33 3h AB #9 68 72 73 LIST OF TABLES TABLE Page I. Titration of NDV (GB Strain) in 10 Day Old Embryon- 8.th Eggs 0 O O O O O O O O O 0 O O O O O O O 15 II. Procedure of Hemagglutination and Hemagglutination- Inhibition TGStS o o o o o o o o o o o o o o o o o 18 III. Test of Hemagglutination and Hemagglutination- Inhibition TGStS o o o o o o o o o e o o o o o o o 18 IV. Recordings of NDV (GB Strain) Transmitted to Syrian Hamflters o o o o o o o o o o o o o o o o o o 19 V. Titration Of HemOIYSin o o o o o o o o o o o o o o 23 VI. Complement Titration Using No Antigen . . . . . . . 26 VII. Complement Titration Using Hamster Brain Newcastle Disease Virus as the Antigen . . . . . . . . . . . 27 VIII. Complement Titration Using Normal Hamster Brain 3! the Antigen 0 o o o o o o o o o o o o o o o o o 28 IX. Complement Titration Using Normal Allantoic Fluid as the Antigen O O O O O O O O O C O O O O O O O O 29 X. Antigen Titration-Hamster Brain Adapted NDV (Not Ultracentrifuged) o o o o o o o o o o o o o o o o 35 XI. Hamster Brain NDV (Not Ultracentrifuged) With Rabbit Anti‘allantOic Fluid Serum o o o o o o o o o o o o 36 XII. Titration of First Supernatant Fluid of Hamster Adapted NDV After Ultracentrifugation . . . . . . 37 XIII. Titration of First Sediment of Hamster Adapted NDV After Ultracentrifugation . . . . . . . . . . . . 38 XIV. Titration of Second Supernatant Fluid of Hamster Adapted NDV o o o o o o o o o o o o o o o o o o 39 XV. Titration of Second Sediment of Hamster Adapted NDV ho XVI. Antigen Titration of Normal Hamster Brain . . . . . hl ii XVII. XVIII. XIX. XXI. XXII. XXIII. XXIV. XXV. XXVI. XXVII. XXVIII. XXIX. XXXI. LIST or TABLES (Cont.) Antigen Titration of Normal Allantoic Fluid . . . . Titration of First Supernatant Fluid of Hamster Adapted NDV After Ultrasonic Vibration and Ultra- Centrifugation o o o o o o o o o o o o o o o o o o Titration of First Sediment of Hamster Adapted NDV After Ultrasonic Vibration and Ultracentri- fugation o o o o o o o o o o o o o o o o o o Titration of Second Supernatant Fluid of Hamster Adapted NDV After Ultrasonic Vibration and Ultra- centrifugation 0 o o o o o o o o o o o o o o o o o Titration of Second Sediment of Hamster Adapted NDV After Ultrasonic Vibration and Ultracentrifugation Complement Fixation Reaction on Normal Hmmster Brain Tissue Suspension . . . . . . . . . . . . . Complement Fixation Reaction Using Normal Allantoic Fluid as the Antigen o o o o o o o o o o o o o o o Complement Fixation Reaction Using Hamster Brain Adapted NDV Antigen and Rabbit Anti-allantoic Fluid Serum. o o o o o o o o o o o o o o o o o o Complement Fixation of Antigen Prior to Ultra- Centrifugaticn o o o o o o o o o o o o o o o o o o Complement Fixation Reaction of the First Super- natant FIU1d o o o o o o o o o o o o o o o o o o Complement Fixation Reaction of the First Sediment. Complement Fixation Reaction of the Second Super- natant Fluid 0 o o o o o o o o o o e o o o o o o Complement Fixation Reaction of the Second Sediment Complement Fixation Reaction of the First Superna- tant Fluid After Ultrasonic Vibration and Ultra- centrifugation 0 o o o o o o o o o o o o o o o o o Complement Fixation Reaction of the First Sediment After Ultrasonic Vibration and Ultracentrifugation iii Page AZ #3 an MS as 53 5h 55 56 S7 58 59 60 61 62 XXXII. XXXIII. XXXIV. XXXV. XXXVI. LIST OF TABLES (Cont.) Complement Fixation Reaction of the Second Super- natant Fluid After Ultrasonic Vibration and Ultra- centrifugation 0 e e e e e e e e e e e e e e e e Complement Fixation Reaction of the Second Sediment After Ultrasonic Vibration and Ultracentrifugation Infectivity Test on Different Fractions in Embryo-~ Dated E885 e e e e e e e o e e e e e o e e e e Hemagglutination Test of the Hamster Adapted New- castle Disease Virus 0 o e e o e e e e e e e e e 0 Summary of Complement Fixation of Ultracentrifuged and Ultrasonic-ultracentrifuged Antigen Fractions and Control Reagents e e e e e e e o e e e e e e 0 iv Page 63 61L 65 66 67 INTRODUCTION On the basis of present knowledge, the tissue extract which contains virus is classified into two groups: 1) those containing a soluble antigen, and 2) those not containing a soluble antigen. Viruses in the first group are yellow fever, vaccinia, psittacosis, influenza, infectious myxomatosis and lymphocytic choriomeningitis. Only two viruses belonging in the second group have been studied, namely, Brown-Pearce car- cinoma of rabbits and papilloma of rabbits. Classification of the viruses into the above two groups has been done by differential ultracentrifugation and the re- sulting fractions are then’tested for infectivity and comple- ment fixing components. Hughes (1933) demonstrated that sera taken from menkeys recovering from severe yellow fever infections possessed a precipitin capable of reacting with a precipitinogen which occurred in the blood of monkeys during the period of acute illness. This precipitinogen was not the virus of yellow fever, but appeared to be associated with a protein of the albumin fraction of the virus. Its concentration reflected the sever- ity of illness. It disappeared with recovery, after stimulating the formation of a precipitating antibody. This precipitin was entirely independent of the protective antibody resulting from an infection. A similar precipitin occurred in the serum of humans who had recovered from.severe yellow fever infection. This precipitin reacted with the precipitinogen which occurred in the blood of monkeys during the acute phase of illness. Hughes and Theiler (193R) further demonstrated that two types of antibodies could result from yellow fever infection. The protective antibody resulted from.the presence of the virus itself, and was formed whenever the virus was present in ade- quate concentration, regardless of demonstrable evidence of infection. A precipitating antibody occurred subsequent to severe infections only, and probably reflected the response to products of cell destruction. Parker and Rivers (1936) found that the extracts of vac- cinal infected tissue, either dermal or testicular were freed from.virus by filtration through colloidion membranes which had an average pore diameter of 103.0 mu. These filtrates were tested for active virus in the testicles of rabbits and found to contain none. However, they did contain the soluble antigen as demonstrated by means of the precipitin reaction. Craigie and Wishart (1936) reported that the soluble substance which precipitated in presence of vaccinia immune serum was found in extracts of fresh dermal vaccine, and could be separated from the elementary bodies of vaccinia by means of centrifugation and filtration. Earlier (l93h) they found that the elementary bodies contained at least two agglutinogens (L and S) which differed markedly in their stability, parti- cularly to heat. The L agglutinogen was thermolabile at 56° C, while the S agglutinogen was stable at 95° C. In ad- dition to the soluble precipitable substance which was dis- tinguished by its occurrence in suspensions of fresh, dermal vaccine, precipitable substances have been recovered in sus- pensions of washed elementary bodies. In subsequent inves- tigations of L and S agglutinogens, it was observed that the S precipitable substance was present in solution in suspen- sions of elementary bodies which had been heated to inactivate the L antigen. However, control tests showed that heating was not entirely responsible for the liberation of S antigen, for most suspensions prior to heating were found to contain free precipitable substances which remained in the supernatant fluid when the elementary bodies were centrifuged out. Ele- ‘mentary body suspensions of varying age were therefore cen- trifuged and the supernatant fluids subjected to the precipitin test. Most of these supernatant fluids gave positive reactions in dilutions ranging from 1-2.5 to 1-80. The same results were obtained by Smadel and Wall (1938) and also Lavin and Dubos (19h0). Smadel and Rivers (l9h2) found that dermal filtrate pre- pared from the skin of rabbits infected with the virus of vaccinia contained the heat-labile ”L“ and heat-stable "S“ antigen of vaccinia which could be demonstrated by the preci- pitin technique. Shedlovsky and Smadel (19h2) showed that all of the serological activity associated with the heat-soluble and heat-labile soluble antigens of vaccinia were present in a single protein molecule. This differs somewhat from the con- cept of Craigie and Wishart (1936) who considered the two antigens to occur ordinarily in the form of a complex antigen which could be dissociated into two separate antigens under certain circumstances. The work on the complement fixation reaction by Bedson (1935) refers only to the use of dilute suspensions of psitta- cosis infected organs of mice. Lazaraus and Myer (1939) found that the large amounts of soluble protein in the crude virus suspensions made them of doubtful value for accurate serological work. However, they purified the virus by re- peated washings in the centrifuge and found that the infec- tive agent of psittacosis was the elementary body itself, a substance not separable from the elementary body. The elemen- tary body suspension contained at least two components, one destroyed at 70° to 75° C for one hour, and the other resisting the same treatment. Rake, McKee, and Schaffer (l9h0) found that lymphogranuloma venereum.virus propagated in the yolk-sac of embryonated eggs produced granules which were purified by differential centri- fugation. The sediment from.the second centrifugation repre- sented a preparation free from most yolkesac constituents and contained a rich suspension of the granules which were believed to be the elementary bodies of the infectious agent. Several tests of the sediment and supernatant fluid after centrifuga- tion showed nearly all of the mouse-infectious material to be in the sediment. The small amounts found in the supernatant fluid were believed to be due to insufficient centrifugation. However, they (I9hl) found that the G strain, after the same process, had a sediment complement-fixation titer of 1-100 and a supernatant fluid titer of 1-10. Bourdillon and Lennette (19h0) showed that the soluble antigen of influenza virus was closely related to the infec- tious moiety, which was suggested by preliminary experiments indicating that the infectious agent had about the same elec- trical mobility as the soluble antigen; however, this is at variance with Hoyle's and Fairbrother's conclusion (19370) that it was not possible to prepare a washed virus suspension entirely free from complement-fixing properties. Lennette and Horsfall (l9h0a)a1so demonstrated that the causAl agent of influenza virus is associated with a specific "soluble“ antigen. This antigen separable from the active virus, appeared to be responsible for the ig'zitgg immune reactions previously attributed to the virus itself. They showed further that the antigen remained in suspension even though the virus had been sedimented by centrifugal force. The antigen was consider- ably smaller than the virus and could be separated.from.it. Hoyle and Fairbrother (1937b)added evidence indicating that a soluble antigen was present in suspension of epidemic influenza virus and by relatively simple means (centrifugation) the virus could be entirely freed of this antigen. Friedewald (19h3) found that the complement-fixing anti— gen of influenza virus in allantoic fluid or mouse lung extract consisted of two distinct fractions. One was intimately asso- ciated with the virus particle and sedimented at the same rate as the hemagglutinin and infective particle in the centrifuge. This fraction, like the hemagglutinin and infective particle was repeatedly adsorbed by red blood cells and eluted from the cells on standing at room temperature or 37° C. It was not possible to dissociate this antigen from the virus particles by repeated washings in the centrifuge or by repeated adsorp- tion with red blood cells. A second fraction had a smaller size remaining in the supernatant fluid following centrifuga- tion sufficient to sediment most of the hemagglutinin and the infective particle. It was not adsorbed by fowl red blood cells. The two types of complement fixing antigens associated with the virus particle of the PB8, W.S., and swine viruses could be readily differentiated in cross complement fixation tests with ferret antisera in that the serum antibody titer was always much higher with the homologous antigen. The degree of specificity of the complement-fixation test with these anti- gens was comparable to that obtained with neutralization or agglutination tests. The soluble antigen from these virus strains, on the other hand, showed less specificity in cross complement-fixation tests, and the antisera reacted in rela- tively low titer. Henle, Henle, Groups and Chambers (l9hh) demonstrated that the two fractions from either influenza A or B virus differed in their optimal antibody relationship, in that the maximal serum titer of the sedimentable and adsorbable material was approximately four times higher than that of the non- sedhmentable and non-adsorbable fraction. Wiener gt_gl,(l9h6) discovered that two types of specific particles could be obtained from allantoic preparations of influenza A and B virus. The larger particles which possessed all the attributes of the virus and which showed a sedimentation constant of about 6003 was compared with the smaller components 303. In addition, some antigen was found to remain in the allantoic fluids after re- moval of the 6008 and 308. These components were calleddf3OS fraction. This material remained in the supernatant fluid upon repeated centrifugation. Analysis of the properties of 6008 and 308 components revealed marked differences. The 6003 fraction carried the infectivity, and it agglutinated chicken red blood cells (Friedewald, l9hh); it produced toxic lesions in.mice (Henle and Henle, l9hh); it possessed immunizing capa- city and acted as antigen in complement-fixation tests (Henle and Wiener, l9hS) and (Friedewald, l9h3). The 308 component, on the other hand, showed little infectivity, it did not agglutinate red cells, it was not toxic and was active only in the complement-fixation test. Only one common preperty existed between the two components, their ability to react as antigen in the complement-fixation test with specific immune sera against influenza. Moreover, by using centrifugation. Boyle and Fairbrother (1937a)found that the supernatant fluid of suspensions of mouse lungs infected with influenza "A" virus contained a soluble antigen, whereas the separated in- fective elementary bodies failed to react in the complement- fixation tests. Lennette and Horsfall (19h0b)confirmed the presence of a soluble antigen in mouse lungs but noted in ad- dition that the virus particles always contained some complement- fixing antigens. Henle and Wiener (l950)further showed that allantoic fluid infected with influenza virus A and B had three antigenic frac- tions as measured by the complement-fixation technique and that material sedimentable at 90,000 g in one hour contained both the 6008 and 308 components possessing two distinct antigens, while the supernatant f1uid<<308 contained only one which was similar to one of the sedimentable antigens. The 6008 unit of the PR8 strain had an antigenic titer of 1-32, the 303 unit of PR8 had an antigenic titer of 1-16, and the<:3OS unit in the supernatant fluid of PR8 strain had an antigenic titer of l-Bd Rivers and Ward (1937) demonstrated, as with vaccinia, that elementary bodies of myxomas could be obtained in a rela- tively pure state by centrifugation. Like vaccinial elementary bodies, they played a conspicuous role in that they either represented the etiological agent or were intimately associated with it. The bodies were specifically agglutinated by anti- myxoma serum.and agglutinated to a less extent by serum from rabbits convalescing from.fibroma, a disease closely related to myxoma. In virus-free filtrates of emulsions prepared from infected skin of rabbits, there was a soluble precipitinogen or precipitinogens specific for the malady. Moreover, a speci- fic precipitinogen or precipitinogens were demonstrable in virus-free serum of animals actually ill as a result of ex- tensive infection with myxoma virus. The supernatant fluid was tested for presence of virus by inoculation of animals (rabbits) and was found to be negative. Rivers, Ward and Smadel (1939) showed that a second soluble antigen of myxoma which was inactivated at a temperature of 50° C was present in filtrates made from tissues infected with live virus. This precipitinogen was usually inactivated by a temperature of 560 C for one hour; at times it may be inac- tivated in 15 minutes. No heat stable soluble antigen separ- able from.the virus was found in myxoma. The soluble antigen of myxoma was a heat labile protein which.had an isoelectric point near pH h.5 and was precipitated by a half saturated solution of ammonium sulfate. It could be partially purified by methods of differential precipitation based on variations in pH and electrolyte concentration. Rabbits receiving the labile soluble substance of myxoma developed homologous pre- cipitins and their sera agglutinated elementary bodies of myxoma, provided the dermal pulp from which the bodies were obtained contained the soluble substance; neutralizing anti- bodies did not appear, however, and the animals were not resistant to infection with the virus of myxoma. Smadel, Ward and Rivers (l9u0) discovered a second soluble antigen, separable from the virus, occurring in extracts of infected skin and in the serum of rabbits actually ill with infectious myxomatosis. Like the first antigen (A), the second (B) was heat labile and had certain characteristics of a globulin. The two antigens precipitated in different concen- trations of ammonium sulfate and could be separated by this method. Neither of the antigens, after being heated at 56° C, precipitated in the presence of specific antibodies. Smadel, Baird and Wall (1939) found that the lymphocytic choriomeningitis virus could be readily sedimented from sus- pensions of infected guinea pig spleen by ultracentrifugation at 30,000 r.p.m. for 20 minutes. The supernatant fluid freed of virus, fixed complement in presence of immune serum asttell as did the uncentrifuged material. The presence of soluble antigen was also demonstrated in extracts prepared from organs of infected mice. The same workers (19h0) showed that the soluble antigen of lymphocytic choriomeningitis which was readily separable from the virus was a relatively stable sub- stance and appeared to be of a protein nature. A specific precipitin reaction could be demonstrated when immune serum and non-infectious extracts of splenic tissue, obtained from 10 guinea pigs moribund with lymphocytic choriomeningitis, seemed to be manifestation of union of the same soluble antigen and its antibody. Smadel and Wall (l9h0) reported that the virus-free extracts of lymphocytic choriomeningitis containing consider- able amounts of soluble antigen failed to elicit anti-solubka substance antibodies and to induce immunity in normal guinea pigs. The same workers (l9h2) found that tissues of hamsters infected with the virus of choriomeningitis contained a speci- fic soluble antigen which was serologically identical with that found in tissues of other animals affected by the disease. The antigen was consistently demonstrable in complement-fixation titers of 1-8 to 1-32 in preparation of spleens taken from hamsters 5-8 days after injection of the virus. Kidd (1940) made clear through his filtration and centri- fugation experiments that serologically active substance of the Brown-Pearce tumor, like that of the virus papilloma, appeared to have a large particle size and weight, differing notably in this respect from the general soluble antigens. Furthermore, extract from the Brown-Pearce tumor containing the serologically active substance in quantity gave rise to no lesions upon injection into normal or tarred rabbits. Kidd (1938) found that the water clear supernatant fluid from the extract of rabbit papilloma which had been centrifuged from 6 hours at 18,000 r.p.m. was devoid of complement binding capacity and infectivity. However, the sediment contained 11 virus which bound complement in dilutions of 1-20 and l-hO and was pathogenic as demonstrated in rabbits. Friedewald and Kidd (19h0) showed that rabbit papilloma virus provided a notably favorable material for immunological investigatiOn.. The virus itself appeared to be antigenic, producing an anti- body that was capable both of neutralizing the virus and of fixing complement. Its immunological reactions were not complicated by any associated soluble antigen. The work of Reagan, Lillie, Poelma and Brueckner (19h?) showed that the California strain of Newcastle Disease Virus No. 11,9lh could be adapted to Syrian hamsters and was con- tinued through the 300th passage. Reagan gt 3; (l9h8) in adapting Newcastle disease virus to the Syrian hamster found that from the 70th through the 120th passage hamsters showed signs of central nervous system involvement which.were ob- served approximately 36 hours following inoculation; and most of the hamsters became moribund in h8 hours. From the 160th passage through the 190th passage, infected hamsters showed signs of central nervous system involvement in 2h hours and died within 36 hours of the inoculation. After the 190th passageinfected hamsters showed signs of central ner- vous system involvement in 12 hours, and 75 percent died within 2h hours. Reagan.g£ g; (1950) attempted to establish the hamster adapted Newcastle disease virus in ferrets and rabbits by using the hamster adapted virus after the 300th intercere- bral passage. It has been successfully passed, i. e. in 12 ferrets for 3 passages, and in rabbits through 5 passages after intracerebral inoculation. Ferrets, after the third passage and rabbits after the fifth passage, did not respond to the modified hamster virus and remained symptom free. "Modified virus“ referred to in this work is the New- castle disease virus after the fifteenth passage in hamsters. 13 MATERIALS AND METHODS Propagation of Newcastle DiSease Virus in Eggs In this work the GB strain of Newcastle disease virus was used. This strain was obtained from the Wisconsin Typing Station, University of Wisconsin, Madison, Wisconsin, where it is used for routine neutralization tests. The virus was passed five times in 10-day old embryonated eggs to increase the potency of the virus. Using an electric drill, two holes were bored into each egg to be inoculated without puncturing the inner shell membrane. One hole was bored above the air sac; another, 3 mm below the air sac (point of injection). Metaphene was applied to each egg to prevent contamination. Five hundred units of penicillin and 250 mg of streptomycin were used per ml of inoculum as a measure against bacterial contamination. Each egg was injected with 0.1 ml of Newcastle disease allantoic fluid using a 1 m1 sterile syringe with a 27 gauge needle, after which each hole was sealed with paraffin wax and the eggs placed in the incu- bator. The eggs were candled 12 hours after inoculation and any embryos dead at this time were considered to have died as a result of trauma. All embryos dying after the first candling were considered killed by the virus. In all cases the embryo died within 2h hours. The dead inoculated and uninoculated embryonated eggs (controls) were placed under refrigeration 11+ (Hi-8o C) for 12 hours to obtain a greater yield of allantoic fluid. After the eggs were chilled, the shell above the air use was painted with metaphene and this portion of the shell was removed; the inner shell membrane was removed also with a pair of sterile forceps. The allantoic fluids were har— vested by using sterile 10 ml syringes-and 20 gauge needles. The fluids were placed in sterile screw cap vials and kept under refrigeration (-200 C) until ready for use. The virus was prepared for titrations in ten-fold dilu- tions as follows: Ten tubes were placed in a rack and h.5 ml saline solution was placed in each tube from tube 2 through tube 10. Five-tenths ml of Newcastle disease virus in allan- toic fluid was placed in the second tube and mixed well; one- half ml of the material from tube 2 was transferred to tube 3. This procedure was continued through tube 10. The first tube contained undiluted Newcastle disease virus in allantoic fluid. The LDSO, was Calculated according to the method of Reed and Muench (1938). In all subsequent tests using eggs, the above procedure was followed. Table I illustrates the titration of the virus in eggs after five successive passages. TABLE I TITRATION or NDV* (cs STRAIN) IN 10 DAY OLD EMBBYONATED EGGS Virus Dilutions Virus -0 2 Dilution 10 10 10" 10'3 10'“ 110-5 10-6 10-7 10-8 10-9 10-10 u/u u/u u/u Mr M. 4/1; M. M. Ma out 0/u -l LDSO% - 10’8°5 *NDV - Newcastle disease virus Numerator of fraction denotes number of deaths. Denominator of fraction denotes number of embryonated eggs inoculated. 1S ’\ HemagglutinationngA) and Hemagglutination-inhibition (HIlgTests Chemically clean round bottom glass tubes (75 mm. by 12 mm. inside diameter) were used. Progressive two-fold dilutions of the virus from 1 in 5 through 1 in 5120 were prepared in 0.85 percent saline solution. To 0.25 ml. of each virus di- lution was added 0.25 ml. of serum to be tested except in the virus titration tubes where 0.25 ml. of saline solution was added. The tubes were shaken and 0.25 ml. of 0.5 percent chicken red cell suspension was added and the tubes were shaken again. The control tubes contained either saline solution and cells or serum and cells for the respective tests. The tests were incubated at room temperature 220 to 27° C. Readings of the virus titration tubes were made at 15, 30, and AS minute intervals; but with the tubes containing serum, readings were made at 15, 25, and 35 minute intervals. The degree of agglutination was read by viewing the tubes from the bottom of the rack through a mirror fixed at the preper angle. Distinct patterns of red blood cells were formed depending upon presence or absence of the hemagglutination. Where hemag- glutination was maximal, a uniform film of agglutinated red blood cells covered the entire bottom of the tube. Where there was no agglutination, the cells sedimented into a disc at the lowest point of the curvature of the bottom of the tube. Re- actions intermediate between complete agglutination or absence of agglutination, were seen as irregular clumps of cells 16 associated with a ring of finely aggregated or unagglutinated cells. Agglutination was recorded as + (complete agglutina- tion), - (no agglutination or complete inhibition of the ag- glutination by the serum), and : (partial or slight agglutina- tion or partial agglutination-inhibition). In the control tubes, the cells sedimented to the bottom of the tube and formed a central, sharply demarcated disc. The virus titer was the highest dilution of the virus in which complete hemagglutination was present. The serum titer was the lowest dilution of the virus in which complete hemagglutination-inhibition was present. Table II gives the procedure for the virus dilution for the HA and HI tests, and Table III gives the HA and HI titers of the Newcastle disease virus (GB Strain). Transmission of Newcastle Disease Virus to Syrian Hamsters One-tenth m1. of infected allantoic fluid from the fifth embryonated egg passage was injected intracerebrally into four h-week old hamsters. Table IV shows the hamster passages of the GB Strain, Newcastle disease virus. The hamsters from which brain material was used for pas- sage were sacrificed while moribund. A 10 percent brain sus- pension was prepared by grinding the brain tissues in a mortar with alundum as the abrasive and saline solution as the diluent. This suspension was centrifuged in a horizontal centrifuge for three minutes at 2,000 r.p.m. to throw down the coarse material. 17 TABLE II PROCEDURE 0F HEMAGGLUTINATION AND HEMAGGLUTINATION-INHIBITION TESTS Virus Dilution Material 1._l._l 1 l 1 1 Con- u.d. 3' 10 20 E5 0 160 320 one 1280 $2366 312'” trol gg Test: Virus dil. (ml. ) --------- 0.25 ml. per tube ---------- 0 o. 85% NaCl (ml. ) - -------- 0.25 ml. per tube --------- - 0.5 0.5% suspen- sion r.b.c. - -------- 0.25 ml. per tube - --------- 0.25 (1711.) HI Test: Virus dil. (ml.) - ----- ~-- 0.25 ml. per tube - ------ --- .0 Serum.dil. (ml. ) - -------- 0.25 ml. per tube - ------- -- 0.5 0.5% suspen- ' sion r.b. c. --- ------ 0.25 ml. per tube --------- - 0.25 (ml.) TABLE III TEST OF HEMAGGLUTINATION AND HEMAGGLUTINATION-INHIBITION TESTS Virus Dilution 1 .11.; 1 1 1 1 1 1 1 ' 1 Con- u.d. 5 10 20 H5 86 160 320 6110 12% 2560 3120 trol —_—: Material HA Test , + + + + + + + + + :p - - - GB NDV HI Test: GB NDV Rabbit , Anti-serum.+ + + + + + + + ‘1 - - - - Interpretation: HA titer of virus 6&0 HI titer of sera 320 HA - Hemagglutination HI - Hemagglutination-inhibition GB NDV - GB Strain Newcastle Disease Virus u.d. - undiluted 18 TABLE IV RECORDINGS OF NDV (GB STRAIN) TRANSMITTED T0 SYRIAN HAMSTERS Number of No. Animals Animals Paralyzed Number of days after Passages Inoculated* Moribund or dead Inoculation Paralysis Number Percent Occurred 1 h 2 SO 5-5 2 LL 2 50 6-6 3 u 1 25 S h A 2 50 5-5 5 h 3 75 5-6-6 6 1+ 3 75 ,S~S-5 7 u 2 So h-S 8 u 3 7S 3-3-h 9 h u 100 3-3-u-u 10 h h 100 3-3-h-5 11 u u 100 3-h-h—h 12 h h 100 3-3-u-u 13 u 1+ 100 3-3-3-3 1h 1+ it 100 3-3-3-13» 15 1L h 100 3-3-3-3 NDV - Newcastle disease virus * - Intracerebrally l9 Brain tissues were prepared in the above manner before each passage, and 0.1 m1. of the suspension was passed serially through 15 passages in hamsters before the adapted virus was used as an antigen in this work. Five hundred units of penicillin and 250 mg of streptomycin were added to each m1. of inoculum as a measure against contamination. Thio- glycolate and brain-heart infusion media were used for ster- ility tests at the time of passage. Preparation of Complement Fixation Antigen Antigen was prepared by injecting intracerebrally 0.1 m1. of hamster adapted Newcastle disease virus (after the fifteenth passage in hamsters) into h-week old Syrian hamsters. When the hamsters showed evidence of infection, they were sacrificed while moribund and the brain of each hamster was harvested aseptically and stored under refrigeration in screw cap vials at -200 C until ready for use. After the fifteenth passage of virus in hamsters it is considered to be modified virus. Preparation of Erythrocytes Erthrocytes were secured from sheep at the MiChigan State College sheep barn. Blood was drawn aseptically from the jugular vein of sheep in 20 m1. portions using a sterile 18 gauge needle and a 20 ml. syringe. The needle was removed from the syringe, and the blood was expelled in a sterile flask containing 20 ml. of Alsever's solution, and mixed 20 thoroughly to prevent the blood from clotting. This anti- coagulant was prepared in the following manner: Dextrose . . . . 10.25 grams Sodium Citrate . . . . . . . . . h.00 grams Sodium Chloride . . . . . . . . 2.10 grams Citric ACid e e e e e e e e e e 0.28 grams DIStilled Water 0 e e e e e e e 500 ml. The solution was sterilized in the autoclave for five minutes at lO-pound pressure. Just prior to the complement-fixation test the sheep cells were washed three times using four volumes of physio- logical saline solution. The cells were placed in a graduated test tube, physiological saline solution was then added and the suspension centrifuged for two lO-minute and one 20-minute period respectively at 2,000 r.p.m. No cells were used if after the 20-minute period the physiological saline solution showed any hemoglobin in the supernatant fluid. The final concentra- tion of the sheep cells in all tests was 2 percent packed cells in saline solution. Titration of Hmolysin Rabbit glycerinized anti-sheep hemolysin was obtained from Sharp‘ and Dohme Manufacturing Company, Philadelphia, Pennsyl- vania. A 1-100 stock solution of hemolysin was prepared by mixing 0.5 ml. of glycerinized hemolysin with 23.5 ml. of physiologi- cal saline solution, and 1 m1. of 5 percent phenol. This solu- tion was stable for months at refrigeration temperature (h-BO C). 21 The following dilutions of hemolysin were prepared from the (1-100 dilution) stock solution to determine the titer of the hemolysin: 0.5 ml. of 1-100 hemolysin + h.5 m1. of saline solution = l-l,000 0.6 ml. of 1-1000 hemolysin + 0.6 m1. of saline solution = l-2,000 0.2 m1. of 1—1000 hemolysin + 0.h ml. of saline solution = 1-3,000 0.1 m1. of 1-1000 hemolysin + O.h ml. of saline solution = l-h,000 0.1 ml. of 1-1000 hemolysin + 0.h ml. of saline solution = 1-5,000 0.1 ml. of 1-1000 hemolysin + 0.5 ml. of saline solution = l-6,000 0.1 ml. of 1-2000 hemolysin + 0.3 ml. of saline solution = 1-8,000 0.1 ml. of 1-2000 hemolysin + 0.h ml. of saline solution = 1-10,000 0.1 m1. of 1-2000 hemolysin + 0.5 ml. of saline solution = 1-12,000 0.1 ml. of 1-3000 hemolysin + 0.h m1. of saline solution = l-l6,000 Hemolysin was titrated once a week and two units of hemolysin were used in the complement, antigen and serum titrations. Two- tenths ml. of each dilution of hemolysin was transferred to clean tubes. To this was added 0.2 m1. of a 1-30 dilution of complement, 0.2 ml. of a 2 percent suspension of sheep cells, and O.h ml. of physiological saline solution. The constituents were thoroughly mixed in each tube and incubated for one hour at 37° C in a water bath. The hemolysin unit was the highest dilution that gave complete hemolysis. Usually a 1:6000 dilu- tion of the hemolysin produced complete hemolysis, therefore, 0.2 m1. of a 1:3000 dilution constituted the two units of hemolysin. Table V shows the results of the hemolysin titra- tion. Preparation of Complement All guinea pigs used were secured from the Michigan State Department of Health Laboratory. 22 TITRATION 0F HEMOLYSIN TABLE V Hemolysin Complement Sheep Saline Tube Dilution 1-30 Cells Solution Results 0.2 ml. Dilution 2% l 1-1000 002 ml. 0.2 m1. 001.]. ml. 145* 2 1-2000 0.2 m1. 002 ml. 00).], ml. )4." 3 1-3000 002 ml. 002 ml. 0.1.}. ml. )4.“ h. l-LLOOO 0.2 ml. 002 ml. 00L}. m1. 143* s 1-5000 0 o 2 ml 0 002 m}. o 0 all. ml 0 LL+ 6 1-6000 002 ml. 0.2 m1. 001‘. ml. 143'. 7 l-8000 0.2 ml. 0.2 ml. 0.h ml. - 8 l-l0,000 0.2 ml. 0.2 ml. 0.h ml. - 9 1-12,000 0.2 ml. 0.2 ml. 00’... ml. '- 10 1-16,000 002 m1. 002 ml. 00% ml. '- 11 None Non 0.2 ml. 0. ml. -* h+ Complete Hemolysis or)!- No Hemolysis Cell Control 23 Serum complement was obtained from guinea pigs which had been fasted 12 hours prior to bleeding. The pigs were bled from the heart in groups of ten, using a sterile 20 gauge needle with a 10 ml. syringe. After the pigs were bled, the needle was removed from the syringe and the blood expelled gently along the sides of the interior of the sterile tubes to prevent hemolysis. Each tube was stoppered with a cotton plug and slanted; the blood was allowed to clot at room tem- perature. After the clotting had occurred, each tube was rimmed with a sterile glass rod to break the clot. The tubes were centrifuged in a horizontal centrifuge at 2,000 r.p.m. for 15 minutes and the serum was removed from the clot by using a 10 ml. syringe with a 27 gauge needle and expelled into glass vials in one-half ml. portions. These vials were then hermetically sealed and stored under refrigeration at -200 C until ready for use. Aseptic technique was used in the above operation and complement prepared in this way was found to possess titers as high as 1-50 dilution after eight months storage. Titration of Complement Complement was titrated to find the complement unit or the minimum.amount of complement in presence of an excess of hemolysin that would bring about complete hemolysis. Two-tenths ml. of complement was placed into a series of 10 tubes including 1-10 through 1-60 dilution, two-tenths ml. of a 1-5 dilution 21+ of each antigen and two-tenths ml. of saline solution was added. The tubes were shaken to mix the constituents thor- oughly and incubated in a water bath for one hour at 37° C. The sensitized cell system consisted of a 2 percent sheep cell suspension and two units of hemolysin contained in 0.2 ml. each. Equal volumes of sheep cell suspension and hemolysin were mixed together and incubated at room temperature for 20 minutes prior to its use. Four-tenths ml. of the sensitized cells was added to each tube of complement dilution and in- cubated for an additional 30 minutes in a water bath at 370 C. The highest dilution of complement giving complete hemolysis after the additional 30 minutes incubation in the water bath was the complement unit. Two units of complement were used in the titration of antigens and complement fixation tests. If 0.2 ml. of the highest dilution of complement, for example l-hO, gave complete hemolysis, then two units of complement comprised 0.2 ml. of a 1—20 dilution. A control was used for red blood cells using physiological saline solution as a sub- stitute for complement and hemolysin. The titer of the com- plement was determined daily. Tables VI, VII, VIII, and IX show the degrees of hemolysis of the complement titrations in presence of the different antigens. Fractionation of Antigen The virus was used for antigen after the fifteenth passage in hamsters. 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The first tube contained the undiluted 10 percent suspension. The virus dilution ranged from 10'1 through 10'5. One-tenth ml. of each dilution of this material was injected into 8-day old embryonated eggs using 500 units of penicillin and 250 mg. of streptomycin as a measure against bacterial contamination. 31 Ultracentrifugation of Hamster Brain Adapted Newcastle Disease Virus 30 ml. of hamster adapted NDV ultra- centrifuged for 1 hour at llh,610 x gravity 2 ml. of supernatant fluid removed Sediment resuspended in 3 ml. of supernatant fluid Figure l 32 30 ml. of hamster adapted NDV ultra- centrifuged for 1 hour at llu,6lO x gravity Supernatant fluid discarded Sed ment resuspended in equal volume of serum saline solution l Recentrifuged for one hour at 11h,610 x gravity 2 ml. of supernatant fluid removed Sediment resuspended in 3 ml. of supernatant fluid Titration of Antigen Antigen was titrated using two-fold dilutions ranging from full strength through 1-128 of the centrifuged and un- centrifuged antigens. The centrifuged and the sonic centri- fuged antigens consisted of the supernatant fluids and the sediments after the first and second cycles of centrifugation in the ultracentrifuge. A sample of the control virus antigen and sediments of the first and second cycles of centrifugation was heated in a water bath for one hour at 56° C. This was done to deter- mine whether there was any difference in the complement fixa- tion reaction of the heated and unheated antigen. A sample of the uncentrifuged antigen was frozen in an alcohol and ice bath at ~60° C and thawed in a water bath at 37° C until a precipitate was formed, then centrifuged in a horizontal centrifuge for three minutes to sediment the pre- cipitate. This was done to determine whether more virus was liberated from the infected tissue when frozen and thawed than when ground in a mortar using alundum as the abrasive. Two-tenths ml. of each antigen dilution was added to 0.2 ml. of heated antiserum diluted 1-5 and two units of complement contained in 0.2 ml. Sheep cell control was prepared by adding 0.2 ml. of 2 percent washed sheep cell suspension to 0.6 ml. of physiological saline solution. Four complement controls were prepared by adding 0.2 ml. of two units of antigen to each of the h tubes, and 0.05 ml, 0.1 ml., 0.15 ml, and 33 0.2 ml. of two units of complement were added to each tube respectively. Physiological saline solution was added to bring the total volume to one ml. in each complement control tube. Hemolytic activity control of the serum was prepared by adding 0.2 ml. of antiserum and O.h ml. of saline solu- tion. Hemolytic activity control of the antigen was prepared by mixing 0.2 ml. of full strength antigen, 0.2 m1. of anti- serum.and 0.2 m1. of physiological saline solution substituted for complement. All tubes were incubated in a water bath at 37° C for one hour after mixing the components of each tube. The sensitized cell system consisted of equal volumes of a 2 percent washed cell suspension and two units of hemolysin contained in 0.2 ml. each. The sensitized cells were made up and incubated at room temperature 20 minutes before use. Four-tenths ml. of the sensitized cell system was added to each tube with the exception of the cell control tube. At the end of an additional 30 minute incubation period the tubes were read. The tubes with the highest dilution of an- tigen showing complement-fixation comprised the antigen unit. Two units of antigen were used in the complement-fixation test. Tables X through XXI show the antigen titrations. Preparation of Anti-Sera Anti-sera were prepared by injecting healthy adult rab— bits with normal allantoic fluid and Newcastle disease allan- toic fluid (GB Strain). 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