AN ATTEMPT TO PROPAGATE THE MOUSE-ADAPTED LANSING STRAiN 0F POLIOMYELETIS VERUS IN THE DEVELOPING CHiCKEN EMBRYO Thesis fix {419 Degree 0*? M. 5. INCHEGAN STATE COLLEGE Lenore Jones 39:49 THESIS 0169 This is to cortitg that the thcsis entitled An Attempt to Propagafr the Mouse-Adapted Lansing Strain of Poliomyelitis Virus in the Develcping ChiCan Embryo l)l‘t.‘.§(.‘Illtfd In; Lenore Jones has been am‘vptml tmumls hlltillnwnt n] ”)0 requirements tnr ;&utcr's ‘hipvpin Bacteriofcgy 1/ ‘ ." I A / I k / Z” r l/ . 2‘}, [1“]: "I‘v-(Jj— (‘7 :x _/ A! - J a_ ,.\ “Aiilinl‘ lvrnfthM’f‘ Ihnn November 13, 1949 ‘ I“. l - I .Im.n.... Jul-.11"! .‘u'E-‘l 7".4- -"I- t'$4.q‘ ”a; 'l-'- ”TH-1.1 I ." I AN ATTEMPT TO PROPAGATL TEE MUUSb-ADAPTbD LANSING STRAIN UF PULIUMYSLITIS VIRUS IN ThE DLVLLUPING CnIChnN EMbRYO BY LENORE JONES A THLSIS 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 MASTER OF SCILNCE Department of BacteriOIOgy and Public Health 1949 ACKNOWLEDGMENT The author wishes to express sincere appreciation to the Michigan Department of Health, to Dr. G. D. Cummings, Director, Division of Laboratories, for the privilege of carry- ing out this study in the Virology Section; to Dr. Serge G. Lensen, Acting Chief, Section of Virology, for his example as a teacher and his valuable supervision and advice in this work; and to the personnel of the Section of Virology for their cooperation. The author also wishes to express appreciation to Dr. H. J. Stafseth, Professor and Head of the Department of Bacteriology, Michigan State College, for his helpful suggestions and criticisms of this thesis. QHQRHQ TABLE OF CONTENTS Page NO. Introduction I. General Considerations 2 A. Etiology of Poliomyelitis 2 B. Portal of Entry and Route of Invasion 2 C. Hist0pathology i D. Immunologic Types of Poliomyelitis Viruses 6 E. Definition of Lansing Type of Poliomyelitis Virus 7 II. The Use of Chicken Embryo Techniques in Virology 10 III. ‘Attempts to Propagate Poliomyelitis Virus in the Chicken Embryo 12 IV. The Propagation of Theiler Viruses, (TO and FA Strains), and the Columbia-SK, C(M) Lansing and MM Viruses in Chicken Embryos 14 ‘V. Tissue Culture Methods 16 VI. Experimental Methods used in the Attempt to Propagate the Mouse Adapted Lansing Strain of Poliomyelitis Virus in Developing Chicken Embryos 17 .A. Inoculum used for the First Egg Passage 17 B. Experiments in which the Amniotic Route was Used 18 - Techniques of Amniotic Sac Inoculation 19 - Material Harvested from the Eggs and Preparation of this Material for the Next Egg Passage 19 - Sterility Tests 20 - Selection of Embryonated Egg Material for Serial Egg Passage 20 - Results of Mouse Inoculation 21 - Description of the Experiments in which the Lansing Strain was Recovered from.Chicken Embryo Suspensions ' 21 - Results of Egg Inoculation by the Amniotic Route 23 C. Experiments in which the Intracerebral Route was Used 24 - Techniques of Intracerebral Inoculation 24 cit: Page No. - Material Harvested from the Eggs and Preparation of this Material for the Next Egg Passage 24 - Results of the Experiments Using the Intracerebral Route 24 D. An Attempt to Investigate the Possibility of the Virus Propagating in Chicken Embryos but Losing Infectivity for Nfice 26 E. Experiments in which a Latent Neurotropic MOuse Virus was Accidentally Propagated in the Chicken Embryo 27 - Description of Experiments 2? - Attempts to Differentiate between the Lansing Virus and the Virus which Produced Encephalitic Symptoms in Mice 29 VII. Discussion 30 IX. Bibliography 34 LVN-EH1. .1 IE INTRODUCTION Poliomyelitis is a common virus disease of man which presents, in clinically typical cases, flaccid paralysis of groups of voluntary mus- cles, resulting from the destruction of motor neurons in the brain and spinal cord. Most cases, however, run only a mild course characterized by fever, headache and upper respiratory and gastro-intestinal symptoms. Most poliomyelitis strains can be transmitted only to monkeys and chimpanzees. A few strains of poliomyelitis virus, the Lansing, the Yale -SK, MEFl, Phillips, WW and Wfd strains are pathogenic for mice, cotton rats and hamsters as well as primates. Several strains of polio- myelitis virus have been grown in tissue cultures but as yet there are no substantiated reports of the growth of strains pathogenic for monkeys in developing chicken embryos. At the present time, diagnostic tests are limited to the iso- lation of the virus and its identification'by the clinical manifestations and histopathologic findings of the disease reproduced in monkeys. The mouse neutralization test has been used, but this test is limited to the rodent-adapted strains. In the event that deve10ping chicken embryos could be used in the primary isolation of the poliomyelitis virus, or that rodent-adapted strains could be adapted to eggs, it would not only mean an increase in the host range but also a more practical, inexpensive method for experi- mental studies or diagnostic work. If the poliomyelitis virus could be propagated in the chicken embryo, the attempt might then be made to pre- pare a suitable complement-fixing antigen from egg material. It is the purpose of this paper to report the methods and re- sults of an attempt to propagate the mouse-adapted Lansing strain of poliomyelitis virus in developing chicken embryos. -1- I, GENERAL CONSIDLRATIONS A. Etiology 2£_Poliomye1itis The etiologic agent of human poliomyelitis is extremely small, its average diameter being estimated as 8-12 mu on the basis of filtra- tion experiments with gradacol membranes (Elford et a1, 1935)1. The virus is quite sensitive to oxidizing agents, ultraviolet light and heat, being inactivated by the latter within three minutes at a temperature of 50°C, (Shaughnessy, 1930)2. Inactivation of the virus has been shown to occur with 0.05 p.p.m. of free chlorine at a pH 6.85 - 7.40 in ten minutes, (Lensen et a1, 1947)3, and with 0.1 gm per cent mercuric chloride at 57°C within two hours, (Schultz and Robinson 1942)4. It is relatively stable even at extremes of the pH range and is resistant to ether, merthiolate, penicillin and streptomycin. Brodie (1935)5, found that it is inactivated in 3.5 days at icebox temperature by 0.3 per cent formalin, but resists 0.2 per cent formalin and 1 per cent phenol for 10 days. Preservation of the virus may be carried out by storing infected entire central nervous system (CNS) in 50 per cent glycerine in the refrigerator. According to Melnick (1946)6, the virus can be stored at -20°C as well as -70°C for twelve months without loss of titer. Virus suspensions, purified by ultracentrifugation and stored at a temperature of -25°C to -30°C will show a considerable dr0p in titer within a few days, (Lensen, unpublished observation). B. Portal and Route of Invasion Poliomyelitis virus produces histopathologic changes only in the CNS. It may be isolated, however, frmm the intestinal wall and the intestinal content as well as from.the CNS of fatal cases. The virus may also be found in the feces and nasopharyngeal washings of patients and -2- asymptmmatic carriers. It has never been found in the spinal fluid or lymphatics but has been recovered occasionally from the blood of patients, (ward, 1946)7 and (Koprowski, 1947)8, as well as from.the blood of monkeys infected by the intracerebral route, (Sabin, 1944)9. The portal and the route of invasion from the primary site of infection to the CNS has not been determined definitely. For a long time, it was believed that the virus entered, and passed from the nasopharynx by way of the olfactory nerves to the brain and spinal cord, but examina- tion of the olfactory mucosa and olfactory bulbs, at necropsy of patients, has only occasionally revealed virus or evidence of invasion (wae, 194s)1°. Regions of the alimentary canal from.which the virus has been isolated include the tongue, the walls of the oropharynx, the duodenum, i1eum.and colon (Sabin, 1947)11. Whether there is multiplication of the virus in the alimentary canal with the subsequent invasion and extension from.there to the CNS, or whether this is merely the pathway of excretion, has been the subject of considerable investigation and controversy. Chimpanzees have been infected with poliomyelitis by feeding them food which has been exposed to flies trapped in homes of patients ill with poliomyelitis, (Melnick, 1947)12. Experiments with the cynomol- gous monkeys indicate that the virus may enter through the pharyngeal or intestinal walls and after multiplying locally, reach the CNS by way of the autonomic nerves, (Burnet and Jackson, 1940)13. Faber, Silverberg and Dong (1948)14, also report infection of three of six monkeys (cynomol- gous, Masses irus)15, after ingestion of food mixed with poliomyelitis virus. Microscopic examination showed that the route of infection was not olfactory, but did show, by the distribution of lesions in the peripheral ganglia and CNS, extension from the oropharyngeal mucosa in two monkeys, via the trigeminal and cervical sympathetic nerves; and in -3- the third monkey, from.the intestinal mucosa via the intestinal sympathetic system to the CNS. It has been found in human cases of poliomyelitis occurring within 60 days after tonsillectomy that there was a marked incidence of the bulbar form (Aycock, 1942)16. This would suggest that tonsillectomy plays a part in the inoculation of the exposed fibers of the glossOpharyn- geal nerve which can then be followed by the extension of the virus along the axons of the glassopharyngeal nerve to the nuclei of the medulla and brain. Others claim that in human cases in which virus may be found in the intestine, it is merely an indication that this is the route of exre- tion of the virus. Helnick was able to isolate poliomyelitis virus from the stools of a monkey inoculated by the parenteral route, (Melnick, 1946)17. Howe (1948)18 states, "It is unlikely that the virus propagates in the intestinal contents or that its invasion extends as far as the CNS." He sites the work of Sabin and ward, (1941)19 and wae and Bodian, (1947)20, who have shown that neither virus nor hasions have been found in the coeliac ganglin, but he mentions the possibility of migration of the virus from.the gastro-intestinal tract along the vagus and visceral afferents to the CNS. C. Histopathologz Lesions of poliomyelitis are highly characteristic in their nature and distribution in the brain and spinal cord wherever neurons have been destroyed by the virus. Neuron damage involves chromatolysis , neuronal necrosis, neuronophagia and "outfall" of cells. This is accompanied by an inflammatory reaction in the surrounding mesodermal tissue in which the area becomes infiltrated with lymphocytes, plasma cells and macroPhages. Perivascular cuffing, (infiltration of adventitia by lymphocytes) is - 4 - nearly always present in the neighboring arterioles and venules. In the spinal cord and medulla, lesions are found primarily in the grey matter of the anterior horn, and to a lesser extent in the auto- nomic and sensory columns. In the brain, the precentral gyrus of the cerebral cortex, the vermis and the deep cerebellar nuclei, and occasion- ally the basis portis and inferior olives may show typical pathological changes. D. Immunologic Types 2f Poliomyelitis Viruses Two groups of experimental workers, Howe, Bodian and Morgan, (1949)21, and Kessel and Pait (1948)22, have shown that there are at least three immunologically different groups or types of poliomyelitis viruses. Three methods were used by these investigators in their immun- ological studies, namely: (1), injection of monkeys with one strain and reinjection with a hetero10gous strain; (2), cross immunity experi- ments (immunization of monkeys with.various strains, challenging them with the homologous strain and then rechallenging with a heterologous strain) and (3), neutralization tests in monkeys. The three types described by Howe, Bodian.and Morgan correspond with the three types described by Kessel and Pait wherever the same strains were used. Each found the Type I or brunhilde Type to include the followh ing strains, Brunhilde, Kotter, Minneapolis and Frederick. Howe et a1 added Per, Riley, Sudeck, Beich and MEFZ strains to this group, while Kessel and Pait included strains designated as BK, McK, Cu, Gu and 0p in their classification. Type II, or the Lansing Type, includes, according to Kessel and Pait, the Lansing and MV strains and according to Bodian (1949)23, the rodent-adapted strains, Lansing, Yale - SK, MEFI, Phillips, Wfd and‘WW strains and the non-rodent-adapted strains, the MV, McC and Aycock strains. Type III, or the Leon Type, was found by both groups of investigators to include only one strain, the Leon strain. This strain was shown to be unrelated to either the Lansing or Brunhilde Types. r. 431’”; L. the. w. E. Definition 2f the Lansing Type 2£_Poliomyelitis Viruses Poliomyelitis has been classified as a clinical entity since first described by Howe in 1840, but laboratory studies of the virus were not begun until 1909 when Lansteiner and Popper succeeded in trans- mitting the disease from a human cord suspension to the monkey. The next important step in the development of experimental work in poliomyelitis was the adaptation of the Lansing strain of poliomyelitis virus to the white mouse, (Armstrong, 1959)24. This was achieved by trans~ mitting the disease from a human cord suspension first to a monkey, then to cotton rats and subsequently to mice. Since then other strains of the Lansing Type have been shown to be pathogenic for mice, namely, the MEFI, WW,'Wfd, Yale - SK and Phillips strains. These strains meet the criteria of a poliomyelitis virus as outlined by the Committee on Nomenclature of the National Foundation for Infantile Paralysis. Criteria 2f & Poliomyelitis Virus (1) The strain must have been described as the cause of human poliomyelitis. (2) The rodent passage virus must be transmissible to primates. (3) The histopathological lesions in the spinal cord, medulla or brain of the paralyzed monkey must be characteristic for poliomyelitis. (4) The strain must be identified as a poliomyelitis virus by immunologic tests with.monkeys. (5) The host range of a poliomyelitis strain thus far is limited to primates with the exception of the Lansing Group which also are patho- genic for mice, cotton rats and hamsters. Any new virus which is typical with respect to host range should be classified only after complete consid- eration of its other properties. - 7 - is.“ All attempts to cultivate true poliomyelitis strains in the developing chicken embryo have been unsuccessful. (6) Another important preperty in identification of poliomyel- itis virus is its small particle size (ranging from 8-12 mu). Considerable confusion has arisen.from the association of the rodent-adapted Lansing Type of poliomyelitis viruses with the so-called "murine poliomyelitis" viruses. These viruses include the strains pro- ducing spontaneous mouse encephalitis, (e.g., Theiler (T0, FA and GDVII strains), as well as the Columbia-SK, the MM and the C(M) Lansing strain, (Schultz and bnright, 1948)25. They produce paralysis or encephalomyel- itic symptoms in mice but have not been shown to produce poliomyelitis in man. They have a wider host range than that of the Lansing Type and have been propagated in the embryonated chick egg. Because of their wide host range, the so-called "murine polio- myelitis" viruses are much easier to use for experimental studies. Some investigators believe that they may provide a.means of understanding better the true poliomyelitis viruses, especially if it could be shown that these viruses and the Lansing Type have a common evolutionary origin. 0n the other hand, the Committee on Nomenclature of the National Foundation for Infantile Paralysis believes that these murine neurotropic viruses have often been the source of confusion and error in work with poliomyelitis viruses in rodents. 'The Theiler viruses are of interest in any experimental work with the Lansing Type in mice because of the possibility of picking up this spontaneous mouse encephalomyelitis virus. The Theiler strains, the C(M) Lansing, Columbia-SK and MM strains are also of interest because they have been propagated in the -8- developing chicken embryo and many reports of the methods used in their adaptation to the embryonated egg have been published. Few reports have been published, however, on the methods used by the various investigators who have attempted to prOpagate true strains of poliomyelitis in the chick embryo. II. THE USE OF CHICKEN EMBRYO TECHNIQUES IN VIROLOGY Since viruses will grow only in living, susceptible cells, it has been a.major problem in experimental virology to find animal hosts which may be obtained in sufficiently large numbers for virus cultivation. Many viruses have been propagated in the developing chicken egg and the working out of chicken embryo techniques has promoted the develOpment of virology. Goodpasture and Buddingh, and Burnet and Beveridge were among the first to recognize the potentialities of virus proPagation in the chicken embryo. Their extensive research with vaccinia, fowlpox, Newcastle disease virus, etc., and their various publications on methods for virus propagation in the chicken embryo have been outstanding contri- butions to the development of diagnostic and experimental virology. Most animal viruses have been propagated in the chicken embryo but a few, e.g., poliomyelitis, herpes zoster, dengue and trachoma, have not been propagated in eggs in spite of the efforts of many investigators. Prepagation of viruses in the chicken embryo has three practical medical uses: (1) Identification of the etiologic agent by direct isolation of the virus concerned, e.g., in the differential diagnosis of smallpox and chickenpox. (2 Preparation of antigens for serodiagnostic work, c.g., the Frei test in lymphOgranulome infection; comphemant fixation in psittacosis, equine encephalomyelitis and influenza; and hemagglutination inhibition tests for influenza. (3) Preparation of vaccines for pro- phylactic use against yellow fever, influenza, 31d Eastern and Western equine encephalomyelitis. Different routes of inoculation are employed according to whether the chicken embryo is inoculated for primary isolation of a virus, for - 10- I‘ ,xflu 1. 58‘ r ‘- vaccine or antigen preparation, or whether it is to be used for a pock- counting method. Amniotic inocuh>tion exposes the inner epithelial lining of the amnion and the epidermal epithelium of the chicken embryo to the virus. This method utilizes the respiratory and gastro-intestinal tracts as portals of entry. Chicken embryos thirteen days of age are employed for the primary isolation of influenza A strains because at this stage in the development of the chicken embryo, the epithelial cells of the trachea are most susceptible to the virus. On the other hand, the less specialized cells of the eight—day embryo seem to be more susceptible to influenza B, 3" erpes simplex and vaccinia viruses. Allantoic inoculation is particularly useful in the propagation of the influenza and psittacosis group viruses for preparation of complement- fizing antigens. This route is also used in the production of influenza vaccine and in preparation of antigen for the hemagglutination test. Yolk sac inoculation is employed for the primary isolation of the viruses of lymphogranuloma venereum, psittacosis and mumps. The cells of the embryonic brain may be infected by intracerebral inoculation with the viruses of herpes, (Anderson, 1940)26, and rabies, (Dawson, 1941)27. Several viruses such as those of variola, vaccinia, herpes stnpk9x, fowlpox and influenza produce discrete Opaque lesions called "pocks" When inoculated on the chorioallantoic membrane. Burnet and his co-workers (1946)28? have employed pock-counting methods for the titration of virus suspensions and serum-virus mixtures, Chorioallantoic inocula- tion may also be used for primary isolation of some viruses, e.g., loup- ing ill and St. Louis encephalitis. These viruses when phaced on the chorioallantoic membrane will invade the body of the embryo. - 11 - III; PaiVluUs ATTLMPTS To PROPAGATE POLIOMYLLITIS VIRUS IN The CLICKLN LNBRYO Gard, in 1943, inoculated chicken embryos by the ohorioallantoic route with a monkey cord suspension of the L strain of poliomyelitis virus. Ea reported a single infection of a monkey which had been inoculated intra- cerebrally with 0.5 ml. of a chicken embryo suspension. Since the chicken )c. brain s-s ension was obtained from the first egg oaSSage only, there is >4 & '0‘ 'U the possibility that reproduction of the disease in the monkey was due to the passive transfer of the virus. All other workers were unable to prOpagate the virus of polio- myelitis in the devclOping embryo, deports have been published by Burnet . “ co , s r. (1955)28b, Stimpert (1909)“V; Kast and Kolmer (1948)”0; Mlordan and . ..,, :51 a . I 32 . Sa Fleitas (19ao) ; Schultz and nnrlght \10i8) ; and Jamieson and r '2 Powell (1948 5“b. Methods Described by_Burnet (1935)a8b Burnet attempted to prOpagate poliomyelitis viruses from five human cord suspensions and one monkey cord suspension (WV strain) in the chicken embryo. Ten per cent suSpensions of spinal cords were prepared by grinding and emulsifying the infected material in broth, centrifuging and removing the supernatant fluid for egg inoculation. Ten-day chicken embryos were inoculated with 0.1 ml. of this suspension by the chorioallan- toic route. Three to six days after the inoculation, chorioallantoic membranes and embryo brains were taken for the second ewe passage. n the chicken Ho No gross or microscopic lesions were observed embryo. Monkeys were inoculated with suspension from the first and second passages but none developed paralysis. I I Methods Described bv Riordan erd Sa Fleitas Riordan and Sa Fleitas (194€)31 reported that their efforts to erpagate in the chicken embryo the Lansing, Yale - SK, and Phi 11 ps strain, (mouse passe-3e virus), as well as two strains of poliomyelitis virus from a monkey cord, and a strain from a human spinal cord, were unsuccessful. Both the chorioallantoic and intracerebral routes were chosen for chicken embryo in culation. For the chorioallantoic route, six-day embryos were inoculated with 10 per cent and 20 per cent susoensions of Lansing, Yale - SK and Phillips strains and the human and monkey cord suspensions. For the in tracerebral route passages, ten-day chicken embryos were inoculated with 20 per cent susPensions. The eggs were he rvested daily or at regular intervals ranging from four to thirteen days after inoculation. Chorioallantoic membranes, brains, spinal cords and embryo minus the C.N.S. were he rvested. Suspensions from the first egg pass-gs were used for mouse and monkey infectivity tests. The mice and monkeys were observed for a period of thirty-five days and ell remained sell. . - . 53a and Jamlcson and Powell (19%8) \D V (X ) Schultz and Enright (10 have reno rted that they have attempted to nrooeete the Lansing strain in emtryonated c i cken eggs without success, but neither group has published description of the methods which they used. -13.. IV. T11: PROPAGATION or ‘l‘l‘sllz‘i 1111:1718 (170 M11) in slums), Tm- COLUNEIA SB”, C(16) LAT»? 1111c 111.1) m V"1‘:.:1_1s;.s 111 axioms 111111111103 Methods used 'n the Prooacetion of the Theiler Virus Several strains of mouse encephalomyelitis virus have been propagated in the chicken embryo by a numher of investigators, including Gard (1943); Riordan and Sa Fleitas (1946)31; Durhen and Parker (1943), and Schultz and finright (1948)32. Riordan and Sa Fleitas reported the cultivation of FA and TO strains of Theiler's mouse enceghelomyelitis viruses. The FA strain wes proPagsted through ten passages of the developing chicken embryo. It was found that emhryos from 6-10 days of age were satisfactory for inoculation. The chorioallentoio, yolk sac, allantoic, and intracerebral routes were used. The inoculum chosen was 0.1 ml. of e 20 per cent suspension of mouse cord and medulle evoejt for the intrecerebral route which was 0.03 ml. Ten oer cent susPensions of choricnllantoic memhrenes, yolk sacs or hest concen- whole embryos were used for subsequent e33 pssseges. The hi at \J Theiler‘s TO strein was passed through four generations in the chicken euhryo following the inoculation hy the chorioallantoic route. Virus was found to be present in the highest titer in the chorioallentoic membranes and chicken embryo after twelve days incubetion st 35°C follow- ing inoculation of six-day old eggs. The T0 and FA strains did not undergo any chenge after egg passage. The symptoms in mice inoculeted with infected egg suspensions were similar to those produced by mouse to mouse nassage. of Columbia-SK, MM and C(M) Lansing Strains Cultivation in the Chicken mmhryo Schultz and nnright (1947)32 reported the successful propagation - 14 - of the Columbia SK, the EM and the C(J) Lansing strains in the chicken embryo. They found that the hijhest cancentration of virus was obtained when chicken erbryos ranging from E-li days were inoculated. Th: incculum '— usod for the first egg paSngc was 0.1 ml. of a 10'” dilution of a mouse cnsion. The dilution 10-5 Wes cncsen because_it induced infection in three of three mice inoculated with 0.025 m]. intracerebrally. Dilutions 10-6 and 10"7 did not produce infection in all the mice inoculated. The routes of inoculation used were the chorioallantoic, the yolk sac and intraembryonic. heads, chorioallantoic membranes and ab- dominal viscera were harvested, ground to a 10 per cent suSpension, centrifuged at 1030 r.p.m. ”nd filtered through a handler candle. One- tenth ml. of this squension was used for the egg passage inoculum. Chicken entryos which died 2-5 days after inoculation vere stored in the refrigerttor. On the fifth day after inoculation, all the surviving chicken embryos were harvested, pooled with the deed embryos and suspen- sions w re orepared for the next egg passage. In all, thirty serial ego passages were carried through with the Columbia SK strain, ten passages with the MM strain and fifteen with the C(M) Lansing strain. The incidence of death among embryos in eggs incubated at 37°C was forty per cent lower than in eggs incubated at 35°C. Neutralization tests in mice showed that the Columbia-SK and the C(M) Lansing strains were neutralized by their homologous antiserum. Neutralization tests were not reported for the MM strain. -15- V. TISSUE CULTURE MbThODS Gildemeister (1933)34 claimed that poliomyelitis virus can be propagated through at least 18 tissue cultures using chicken embryo brain tissue, monkey serum and Tyrode suspensions. Carrel flasks were used. Subcultures were made twice weekly. Pauli (1934)55 was able to confirm.this but all other investigators, including Plotz (1938)36, Sabin and Olitsky (1939)37 and Kast and Kolmer (1957)38 were unable to repeat it. Sabin and Olitsky (1959)37 successfully propagated the MV strain of monkey passage virus using fragments of embryonic brain, obtained for young human fetuses. Enders, Wilber and Robbins (1949)39 report the cultivation of the mouse adapted Lansing strain of poliomyelitis virus in human embryonic tissue from the arm and leg. In this case, proliferation of the virus must have occurred in tissue which does not contain intact neurons, that is, either in the peripheral nerve processes or in cells of mesodermal origin. -16- VI. EXPERIMENTAL METHODS USED IN AN ATTEMPT TO PROPAGATEfTHE MOUSE ADAPTED LANSING STRAIN 0F POLIOMYELITIS VIRUS IN DEVELOPING CHICKEN EMBRIOS Attempts to propagate the mouse—adapted Lansing strain of poliomyelitis virus in embryonating chicken eggs were made using the method of serial egg passage. Three to five serial egg passages were carried out in each experiment. The routes of inoculation chosen included the amniotic and intracerebral. At least four chicken embryos were inocur lated in each passage. Fertile hens' eggs were inoculated after incuba- tion at 37°C for 7—11 days. They were then incubated at 35° or 37°C for 2-5 days before being harvested. To check for the presence of virus, material harvested from each egg passage was inoculated into at least five mice (0.03 ml. intracerebrally). See Table I. A. Inoculum Used for the First Egg Passage Mice were inoculated with the Lansing strain of poliomyelitis virus. Spinal cords and medullae were harvested from mice which became paralyzed 2-5 days after inoculation. In experiments 1 and 2, one per cent suspensions of Spinal cards and medullae were used, and in all the other experiments, ten per cent suspensions were used for the first egg passage inoculation. Whenever possible, eggs were inoculated the same day on which the mouse passage virus was harvested. Otherwise, the mouse cords 'and medullae were stored at 930°C. Mouse passage virus preparations AMlAKlMLl, AMlAKlMAb, AM14K1M47 and AMlAKlMAS were used. "A” designates the virus as it was received several years ago by the Section of Virology, Michigan Department of Health from Dr. Charles Armstrong. "M149 stands for the number of monkey passages carried out and “K" stands for one cotton rat passage. The second ”M" designates the number of mouse passages following the cotton rat pass- age. .17— Titrations were carried out to determine whether a pool of amniotic fluids from normal 10-day chicken embryos could inactivate the Lansing virus and also to determine whether in inoculating embryos by the amniotic route the virus would be diluted beyond a titer which would produce paralysis in mice. Virus suspensions were titrated using (1) physiological saline and (2), amniotic fluids from.normal 10-day chicken embryos as diluents. The dilutions were made and incubated for three days at 35°C in sealed test tubes to carry out as nearly as possible the conditions of incubation of the virus which prevail in the amniotic sac. A titration of the virus suspension diluted with penicillin solution (1000 units per ml. inoculum), and streptomycin (8000 units per ml. inoculum), was carried out to determine whether these antibiotics would inactivate the virus. In some experiments, 25000 units of strepto- mycin was used for egg passage inoculation, but 8000 units per ml. of inoculum was the largestéiose that mice could be given without developing convulsions. As before, dilutions were incubated three days at 35°C in sealed test tubes before the mice were inoculated. There was no appreciable difference in the titers. The LD5O titer of the virus suspension diluted with amniotic fluids was .02 per cent. A normal 10-day chicken embryo will yield approximately 3.0 ml. of amniotic fluid, and if 0.1 ml. of 10 per cent virus suspension is inoculated into the amniotic sac, the virus would be diluted to 0.3 per cent. Since this dilution is considerably lower than that of the LD50 obtained in the titration, recovery of the virus after the first egg passage would be due probably to passive transfer rather than prolifera- tion of the virus. B. Experiments in which the Amniotic Sac Inoculation was Used Nine experiments were carried out in which the amniotic sac was - 13 - chosen for the route of inoculation, (see Table I). The size of the inoculum ranged from.0.03 ml. in experiments 1 and 3; 0.1 ml. in experi- ments 4 - 8 to 0.25 ml. in experiment 9. Technigue 2f: Amniotic Sac Inoculation The eggs are candled and Opened over the air sac. A drop of saline is placed on the shell membrane, then the membrane is ruptured by placing the inoculating needle at an angle of 45° below the drop and applying pressue so that a small hole is made through which the drop of saline will pass to make a liquid wedge between the shell membrane and chorioallantoic membrane. ‘A portion of the shell membrane is then removed very carefully to give complete visibility of the route for amniotic sac inoculation. Gauge 22 or 23 needles, 5/5 inch in length, are used. The amniotic cavity is entered by introducing the needle through the allantoic sac as near the edge of the yolk sac as possible 'without rupturing it. The weight of the yolk will then serve to pull the amnion over the needle as it is pierced by a short, sharp thrust of the needle. The inoculated eggs are sealed with scotch tape and incubated at 350 or 37°C. The collection of the amniotic fluids is carried out by cutting away the scotch tape over the air sac, rupturing the allantoic sac and then very carefully dropping the entire contents of the egg into a sterile petri dish without breaking the amnion. The amniotic fluid may then be drawn off with a needle and syringe. Material Harvested from Eggs and the Preparation.2£ this Material for the Next Egg Passage Harvests consisted of (1), amniotic fluids pooled with brain and Spinal column; (2), amniotic fluids pooled with heads and torsos; (3), amniotic fluid; (4), brain and spinal column and (5), heads and torsos. In experiments 1 - 4, brains and spinal columns or heads and torsos were ground to a 10 per cent or a 20 per cent suspension with physiological saline. In experiments 5 - 8, amniotic fluids were pooled with the embryonic tissues and ground to a 10 per cent or 20 per cent suSpension with the physiological saline. These suspensions were centri- fuged at 3000 r.p.m. for 15 minutes and the supernatant fluids were used for the next egg inoculation and for mouse inocukition. Sterility Tests Blood plates and N.I.H. thioglycolate sterility broth were inoculated with each chicken embryo suspension and mouse passage virus suSpension before the suspensions were used as inocula for eggs or mice. Only sterile suspensions were used for the inoculation of the first and second egg passages. If bacterial contamination occurred in suspensions for egg passages 3 - 5, they were treated with antibiotics, (1000 units penicillin and 25000 units of streptomycin per ml. of inoculum, with a contact period of 30 minutes before the inoculation). When mice were inoculated, no more than 8000 units were used because a greater number of units cause convulsions. Selection 2£_nmbryonated Egg Material for Serial Egg_Passages Embryos dying within 24 hours after the inoculation were dis- carded. Suspensions prepared from the chicken embryos dying 2 - 5 days after inoculation in the first two egg passages were passed separately to the next egg passage and mice were inoculated. The surviving embryos were harvested after 5 days and pooled for the next egg passage and mouse inoculation. In passages 3 - 5, chicken embryos which died 2 - 5 days after inoculation were harvested and stored in the refrigerator. 0n the fifth - 20 - day after egg inoculation, all the surviving embryos were harvested, pooled with the dead embryos and suspensions were prepared for the next egg passage. Any suspensions showing bacterial contamination were treated with antibiotics as described under "Sterility Tests", before being used for egg or mouse inoculation. Results, 21', WW: Experimsnts l - 9 in which the Amniotic Route of Inoculation was Used Evidence of virus was obtained from chicken embryo suspensions which were harvested only from the first egg passages in experiments 3, 4, 5 and 8 when mice were inoculated. No mice became paralyzed after inoculation with suspensions from egg passage 2 - 5 in any of the experi- ments. In all cases in which paralysis of the mice occurred, the paralysis was typical of that produced by the Lansing strain. In experiment 9, however, where AM14K1M48 passage stock virus was used, atypical symptoms in mice were produced when they were injected with sterile chicken embryo suspensions frdm egg passage 1 - 5. These mice developed encephalitic symptoms, tremors, ruffled fur and weakness rather than paralysis of the legs and toes. This experiment will be described later in this report. Description.2£ the Experiments in_which the Lansing Strain Virus was Recovered from Chick Embryo Suspensions Experiment 3 The inoculum chosen for the first egg passage was 0.03 ml. of a 10 per cent suspension of mouse passage virus. Seven-day chicken embryos were inoculated by the amniotic sac route. Heads and torsos were harvested, ground to a 20 per cent suspension, centrifuged, and eggs and mice were inoculated on the same day with a portion of this supernatant fluid. The remainder of the suspension was concentrated seven times, approximately, by ultracentrifugation for the next egg passage and mouse inoculation. - 21 - One of ten mice inoculated with the 20 per cent suspension became paralyzed 18 days after the inoculation. One of ten mice inoculated with the con- centrated suspension of chicken embryo tissue became paralyzed after twenty days. None of the mice inoculated with embryo suspensions of the subsequent egg passage became paralyzed. Experiment 4 In experiment 4, a larger inoculum was chosen (0.1 ml.) and 9-day chicken embryos were inoculated. The harvest consisted of (1), amniotic fluids and (2), embryos brains and spinal columns. The amniotic fluids (undiluted), and brains and spinal columns were ground to a 20 per cent suspension in saline and were inoculated into mice and eggs on the same day as they were harvested to avoid storage of the egg passage material. Eight of ten mice inoculated with amniotic fluids became paralyzed with 7 - 17 dare. Only one mouse inoculated with the brain and spinal column suspension became paralyzed. In the subsequent egg passages, all the CNS suspensions were pooled, and all the amniotic fluids were pooled from each passage, and passed separately into eggs and mice. Mice were inoculated with suspen- sions of subsequent egg passages but none became paralyzed. ExPeriment 5 One ml. of a 10 per cent suspend.on of mouse passage virus was inoculated into 8-day chicken embryos. Heads, torsos and amniotic fluids were harvested, pooled and ground to a 10 per cent suspension. Again egg and mouse inocuhitions were made on the same day as the embryos were harvested. Four of ten mice became paralyzed 16-25 days after inoculation of the suspension from.the first egg passage. Suspension from.the second and the third egg passages did not produce paralysis in mice, however. Four embryos of the first egg passage died but only one of the suspensions from these eggs produced paralysis in mice. Experiment 7 In this experiment, only the chicken embryos dying 2 - 8 days after inoculation were harvested. From.each egg, serial egg passages were made. Ten-day chicken embryos were inoculated with 0.1 ml. of a 10 per cent suspension of mouse passage virus. Three of eight embryos died but only one proved to contain virus when mice were inoculated. Ten per cent suspensions of a pool of amniotic fluids and embryos were used for the second egg passage and mouse inoculation. All of the chicken embryos of the second egg passage survived. Results 2f Egg Inoculation by the Amniotic Route No specific gross changes were observed in chicken embryos which had died within 2 - 5 days after inoculation, or in embryos which survived this period, even when virus was recnvered from the egg qugnie usicnsicns. Virus was recovered from suspensions prepared from the U) .L. i“ c icken embryos which survived the five-day period after inoculation, as well as from a few embryos dying within this period. It was imvossible, however, to correlate death of the chicken embryo with presence of the virus. C. O v Inoculation was Used rout H acerehra the int_u PU O f P.) ") ‘b Intracerebrcl Experiments i__which the Four experiments were carried out u. 3 to 5 serial egg passr es. . of a U inoculation and each of those included Seven to eleven-day chicken embryos were inoculated with 0.03 a of 10 nor cent or 20 per cent suspension of mouse cords and medulla then incubated from 3 - 5 days at 35°C. The egg shell Intracerehral Method for the re candled and opened over the air sac. assod care- Tecnn_. membrane is removed carefully (as described under the amniotic route A.5/b inch, 27-gauge needle and a tuberculin syringe is used and is located, the needle is p- sac, then by a method). for inoculation. When the chorioallantoic sac and the amniotic into the craniul just posterior to fully through t.o sharp thrust, the needle is forced the eye. ‘Nith practice, the intracerebral inoculation may be made wi.h only occ+sional death of the embryo due to trauma. Materiel Harvested from the Eggs and Preparation 2:_this Materiel £3: the next Es? Pessare Material harvested from experiments 10 - 12 included (1), brains 2), amniotic fluids and brains and spinal columns, These pools were ground to a and Spinal columns, (3), amniotic fluids and heads and torsos. 10 per cent or 20 per cent susgension, centrifuged and the eip-rnatant and N.I.H. thioglycolate fluids were tested for sterility with blood plates strrility broth. lection of the em ryonated egg material for serial egg passage passages as for Route Se J was made in the same way for the intracerebral route chicken embryo harvested U passages in which the amniotic route was used. Results of the hxperiments usinc the Intracercbral KO specific changes were observed in the r),- ‘ - -25— 9 :‘mv of thee. ”d D. _A_r_1_ Attempt _t_g Investigate the Possibilig g: the Virus Propdgsting .in Chicken Embryos but Lgsing Infectivitz for Mice An attempt was made to investigate the possibility that there was prepagation of Lansing strain virus which became non—infective for mice after serial egg passage and therefore could not be detected. For this purpose, it was attempted to immunize mice by repeated intraperitoneal inoculation of various egg passage suspensions from experiments 4 - 8, passages 2 - 5, in which the amniotic route was used and from experiments 10 - 12, passages 2 - 5, in which the intracerebral route was used. The mice were challenged with 0.03 ml. of a suspension of Lansing strain mouse passage virus. The challenge inoculum.represented two LD50 doses. There was no protection. This would indicate that there was no virus in the egg material or that the egg suspensions were not suffi- ciently antigenic to produce, with the amounts inoculated, protection against an intracerebral challenge with infective Lansing strain virus. E. Experiment§_ig which E Latent Neurotrooic Mouse Virus gag Accidentally PrOpagated in the Chicken Embryo Evidence was found in experiments 9 (amniotic sac inoculation) and 13 (intracerebral inoculation) showing that a latent, neurotropic mouse virus had been picked up from the mouse passage (AM14K1M48) suspen- sion which was used for the first egg passage inoculum. When mice were inoculated with egg passage material, a few developed paralysis, but most of them showed encephalitic symptoms which were not characteristic for Lansing strain poliomyelitis. These mice showed tremors, ruffled fur, weak- ness of one or more legs and occasionally paralysis of the toes but not of the leg or foot. Death of the mice occurred within 24 hours of the onset of symptoms. A few mice did not show encephalitic symptoms but developed convulsions, with stiffening of the legs, which were followed immediately by death. In case these symptoms were caused by a bacterial infection, each sterility test was repeated before a suspension was used for egg or mouse inoculation. All suspensions were bacteriologically sterile when harvested except from one egg passage, (experiment 15, passage 5). This suspension was treated with 1000 units penicillin and 8000 units strepto- mycin per ml. inoculum. Description_g§ Experiments Experiment 2: Amniotic figggg Eight-day chicken embryos were inoculated with 0.25 ml. of a 10 percent suspension of mouse passage virus AM14K1M48. Amniotic fluids were harvested for egg and mouse inoculation. Three of eight mice became paralyzed ll - 13 days after inoculation, and one mouse presented encephal- itic rather than paralytic symptoms on the fourth day. Amniotic fluids were harvested from the subsequent egg passages (4 passages in all) for egg and mouse inoculation. All of the mice remained well (see'Table II). -27.. Experiment lg} Intracerebral Inoculation Eight-day chicken embryos were inoculated with 0.03 ml. of a 10 per cent suspension of mouse cords and medullae from passage AM14K1M48. Amniotic fluids and brains were harvested and pooled for the second egg passage. In subsequent egg passages, amniotic fluids and brains were harvested from.the living embryos and brains, viscera and amniotic fluid from.the dead embryos but remaining in good condition after death. In a few cases, the embryos were too small or disintegration of the tissues had begun and it was necessary to harvest chorioallantoic membranes and allantoic fluids as well. Results 2£_Mouse Inoculation with Egg Passage Material from Egperiment lg Egg Passage 1: When.mice were inoculated with suspend.ons from this passage, three of eight mice became paralyzed after 3 - 5 days. Three others died within the same period, without symptoms, and one mouse showed a questionable paralysis four days after the inoculation and died within 24 hours. Since the paralysis which did occur was the same as that produced by the Lansing strain, there was no definite indication that another virus was mixed with the Lansing strain from.the results of mouse inoculation with the material harvested from the first egg passage. Egg Passage 2: When.mice were inoculated with suspensions from both living and dead chicken embryos, they deve10ped encephalitic rather than paralytic symptoms, within a period of 3 - 5 days. These mice showed tremors, ruffled fur, spasticity and in two cases, paralysis of the toes of one foot but not of the foot or leg. Death of the mice occurred within 24 hours. Brains, medullae and cords were removed frmm these mice for a second mouse inoculation. Sterility tests with blood plates and N.I.H. thioglycolate broth showed that each suspension was sterile. These -28- suspensions were pooled and mice were inoculated. Two to four days after the inoculation, the mice from.the second mouse passage showed encephal- itic symptoms, tremors and spasticity, but none showed paralysis. Mice inoculated with egg passage suspensions from.passages 3 - 5 also showed encephalitic rather than paralytic symptoms with the excep- tion of one mouse which showed definite paralysis (see Table III - Mouse Inoculation after Egg Passage 5, experiment 13). Attempts to Differentiate between the Lansing Virus and the Virus which Produced Encephalitic §ymptoms in Mice In an attempt to differentiate between the Lansing virus and the virus which produced encephalitic symptoms in.mice, neutralization tests were carried out with CNS suspensions from.mice showing encephalitic symptoms and from a mouse which develOped paralysis. These mice had been inoculated with egg passage material from experiment 13, passage 4, (see Table II). The serum.chosen came from a pool of normal human serum.gamma globulin which had been used previously in immunological work to neutral- ize the Lansing strain. Normal horse serum.was used for the controls. There was no difference in the incidence of paralysis or of .encephalitic symptoms in the mice inoculated with either one of the CNS suspensions. The human serum.gamma globulin neutralized the virus in both suspensions. The LD50 of the virus with normal human serum gamma globulin was .003 per cent for CNS suspension from mice showing encephal- itic symptoms. The LD50 for the virus with normal horse serum was over .0001 per cent (end point was not reached). If neutralization of the virus had not occurred it would have been an indication that the virus was not related to the lensing strain. The fact that neutralization did occur was not considered to be a posit: ve f1nding, however because normal human serum,mav contain antibodies or a I to (L) I non-specific inhibitor for viruses other than the Lansing strain. Neu- tralization tests using a specific anti-Lansing serum were not carried out because this antiserum was not available at the time. A further attempt was made to differentiate'between the viruses by immunizing mice with the Lansing strain and challenging with the virus which produced encephalitic symptoms in mice.1 Ten mice were inoculated intraperitoneally with 0.2 ml. of a 10---»1 dilution of a Lansing strain. Two weeks after the last inoculation, they were challenged intracere- brally with 0.03 ml. of a 10--4 dilution of the virus which produced en- cephalitic symptoms. Ten.control mice were inoculated intracerebrally with 0.03 ml. of a 10-4 dilution of the same virus suspension. In both groups, 8 of the 10 mice inoculated developed encephal— itic symptoms 3 — 6 days after the inoculation. Thus, in this experiment, immunization with the Lansing strain virus gave no protection against the virus which produced the encephalitic symptoms in mice. After the results of mouse inoculation in experiments 9 - 13 were Observed, mice were inoculated with AM14K1M48 passage of Lansing strain again. The mice were checked very carefully and four of the mice inoculated showed encephalitic symptoms, tremors, ruffled fur, weakness of the legs, etc. Four of the mice showed symptoms typical for Lansing strain. Embryonated eggs were inoculated by the amniotic sac and intra- cerebral routes with.AMl4KlM48 passage of the Lansing strain and mice were inoculated with suspensions prepared from the first egg passage. Again some of the mice showed paralysis typical for Lansing strain and some showed encephalitic symptoms. 1. This procedure was based on data published by Kramer and Gear (1945)40 Mice were inoculated with spinal cord suspensions from passages AMlAKlMAé and AM14K1M47 and all mice showed paralysis typical for Lansing strain of poliomyelitis virus. It seemed evident, therefore, that another virus was present in the mouse passage (AMlLKlMAB) suspension. DISCUSSION The data presented show that when the amniotic sac method of inoculation was used, the Lansing strain of the poliomyelitis virus was recovered only after the first egg passage. Since the LD titer of the 50 virus was much higher than the virus dilution obtained when 0.1 ml. of a 10 per cent suspension of mouse passage virus was inoculated into the amniotic sac, it seemed probable that the virus was passively transferred in the first egg passage rather than that proliferation of the virus occurred. In two experiments in which the amniotic and intracerebral routes were used, some of the mice inoculated with suspensions from the first egg passage showed encephalitic symptoms and some showed paralysis. Most of the mice inoculated with suspensions from.subsequent egg passages in the experiment in which the intracerebral route was used deve10ped encephalitic symptoms. Since the same mouse passage suspension was used for the first egg passage in both experiments, this suspension was checked by mouse inoculation. Half of the mice showed paralysis and half showed encephalitic symptoms. Mice were inoculated with a suspension from the preceeding mouse passage and all showed paralysis typical for the Lansing strain. It seemed evident, therefore, that the virus which produced encephalitic symptoms in mice was picked up accidentally when mouse cords were collected for the AM14K1M48 Lansing virus passage. An attempt was made to differentiate between the Lansing virus and the virus which produced encephalitic symptoms by using neutraliza- tion tests with.normal human serum.gamma globulin. There was neutraliza- tion of the virus which produced encephalitic symptoms, but it was not considered as a positive finding because normal human serum.may contain antibodies or a-non-specific inhibitor for viruses other than the Lansing strain. When mice were immunized with Lansing strain and challenged with the encephalitic producing virus, the incidence of encephalitis was the same in this group as in the control group which were not immunized with the Lansing strain. In view of the different symptoms produced in the mice, and the fact that immunization with the Lansing strain did not protect mice when challenged with this virus, it seemed evident that it had been picked up accidentally from the mouse cord suspension used for the first egg passage. - 32 - SUMMARY An attempt to propagate the Lansing strain of poliomyelitis virus in developing chicken eggs has been made in this study. Both the amniotic and intracerebral routes were used. Three to five serial egg passages were carried out in each experiment. Whenever possible, storage of the mouse cords and egg passage material was avoided, and suspensions were prepared and inoculated the same day on which they were harvested. Lansing virus was recovered only from the first egg passage in the experiments in which the amniotic route was used. It seemed probable, therefore, that this was due to passive transfer rather than proliferation of the virus. Lansing strain of poliomyelitis virus was not recovered from any of the experiments in Which the intracerebral route was used. In one experiment in which the intracerebral route was used, a latent, neurotropic mouse virus was accidentally picked up and propa- gated through five serial egg passages. - 33 - 1. 2. 5. 4. 5. 6. 8. 9. 10. 13. 14. IX; Bibliography Elford, WlJ., Galloway, I.A. and Perdau, Jr., 1935, The size of the virus of poliomyelitis as determined by ultrafiltration analysis. J. Path. and Bact., 40:155-140. Shaughnessy, H.J., Harmon, P.H. and Gordon, F.B., 1930, The heat resistance of the virus of poliomyelitis. J. Prev. Med., 48149'1550 Lensen, S.G., Rhian, M., and Stebbins, M.R., 1947, Inactivation of partially purified poliomyelitis virus in water by chlorination. Am. J. Pub. Health, 57:869-874. Schultz, E.W., and Robinson, F., 1942, The invitro resistance of poliomyelitis virus to chemical agents. J. Infect. Dis.. 70:193-200. Brodie, M., 1935, Active immunization in monkeys against poliomyelitis with germicidally inactivated virus. J. Immunol., 28:1-8. Melnick, J.L., 1946, Storage of mouse-adapted strains of poliomyelitis virus and Japanese B encephalitis virus at subfreezing temper- atures. J. Infect. Dis., 79:27—52. hard, h., horstmann, D.M., Helnick, J.L., 1946, The isolation of poliomyelitis virus from human extra-neural sources in Search for virus in blood of patients. J. of Cl. Invest., 25:2 70-286. Foprowski, rZ., 1.0rton, T.W}, MCDermott, 1947, Isolation of polio- myelitis virus from human serum by direct inoculation into the laboratory mouse. U.S. Pub. Health 469.,“ V2. :1476-1478. Sabin A.B. 1944 Studies on the natu_ral history of poliomielitis. ’ ’_ ’ Jr J. J J. of hount Sinai heapital, 11:185-203. o-sv Vthe, HOE-0’ l 948, Viral and Rickehsial Infections of man, edited by Rivers, T. ‘fiv Ch. 10, 245-268. Sabin, A.B., 1947, Epidemiology of poliomyelitis. J. am. Ned. Assn., vol. 154, No. 9. Melnick, J.L., Von Magnus, H., 1948, Comparative susceptibility of cynomologous and other monkey species to poliomyelitis virus by the intracerebral and oral routes. Amer. Journ. Hyg. 48:107- 110. Burnet, F.M., and Jackson, A.V., 1940, Poliomyelitis; spread of polio- myelitis virus in cynomologous monkeys with particular reference to infection by pharyngeal-intestinal route. Australian J. Exp. Bio. and Med. Sci., 18:361-366. Faber, H.K., Silverberg, R.J., Dong, L., 1948, Poliomyelitis in Phillipine cynomologous monkeys after simple feeding. Am. J. Hyg., 48:94-98. 16. 17. 18. 19. 20. 22. 23. 24. 25. 26. 27. 28.8. 28.b 50. Aycock,’W.L., 1942, Tonsillectomy and poliomyelitis, epidemiologic considerations. Medicine, 21:65-69. Melnick, J.L., 1946, The recovery of poliomyelitis virus from the stools of experimentally infected monkeys and chimpanzees. J. 11711111111010, 53, 277-279. Howe, H.A., 1948, Viral and Rickettsial infections of man, edited by Rivers, T.M. Ch. 10, 245-268. Sabin, A.B., and Ward, R., 1941, The natural history of human polio- myelitis. I Distribution of virus in nervous and non-nervous tissue. J. EXP. Med., 73, 771-793. Howe, H.A., and Bodian, D., 1947, Isolation of poliomyelitis virus from throats of symptomless children. Am. J. hyg., 45, 219-222. Howe, H.A., Bodian, D., Morgan, 1.15., 1949, Differentiation of types of poliomyelitis virus. Am. J. Hyg., 49, 200-224. Kessel, J.F., Pait, 0.8., 1948, Resistaice of convalescent macaca mulatta to challenge with homologous and heterologous strains of poliomyelitis virus. Proc. Soc. Exp. Biol. and Med., 68:606-611. Bodian, D., 1949, wallingford poliomyelitis virus, another strain of the Lansing type infective in rodents. Proc. Soc. Exp. Biol. and Med., 70:1-5. Armstrong, 0., 1939, The experimental transmission of poliomyelitis to the eastern cotton rat. Pub. Health Rep.. 54, 1719-1721. Schultz, E;W., Enright, J.B., 1946, Cultivation of the murine SK strain of poliomyelitis virus in developing eggs. Soc. Exp. B101. and. Med... 6388-100 Anderson, K., 1940, Pathogenesis of herpes simplex virus infection in chick embryos. Amer. J. Path., 16:137-139. Dawson, J.R., 1941, Study of chick embryo-adapted rabies virus. Amer. J. Patho' 17' 177. Burnet, FKM., Beveridge, W.I.B., 1946, The cultivation of viruses and rickettsiae in the chick embryo. Spec. Rep. Series Med. Res. Council London, N0. 256:3-5. A Burnet, F.M., 1935, An attempt to propagate poliomyelitis virus in the deveIOping egg. Med. J. Australia, 1, 46-48. Stimpert, F.D., 1939, Cultivation of certain viruses using yolk of chick embryos as route of injection. Proc. Soc. Exp. Biol. and Med. , NeYo , 41 :483‘484. Kast, C., Kolmer, J.A., 1937, Unsuccessful attempts to cultivate the virus of epidemic poliomyelitis in various living tissue mediums. J. InfBCt. Dig.’ 61:60-64. Riordan, J.T., Sa Fleitas, M.J., 1946, Studies on the growth of murine encephalomyelitis viruses in fertile eggs. J. Immunol. 56:263-271. - 35 - 32. 33.9. 33.b 34. 35. 36. 37. 38. 39. 40. Schultz, E.W., Bnright, J.B., 1947, Further observations on the cultivation of strains of poliomyelitis virus in developing eggs. Proc. Soc. for Exp. Bio. and Med., 66:541-544. Powell, H.M., Jamieson, W.A., Culbertson, C.G., 1948, Injection of mouse adapted and egg adapted poliomyelitis-like virus in white rats. Proc. Soc. Exp. Biol. and Med., 68:80-81. Powell, H.M., Jamieson, W.A., 1948, On the immunology of certain mouse-adapted poliomyelitis virus strains ween cultivated in embryonated eggs. J. Infect. Dis., 83:238-242. Gildemeister, E., 1933, Uber die Zuchtung des poliomyelitis virus im.kunstlichen Nahrmedium. Deutsche Med. WChnschr., 59:877. (See reference 31, Riordan and Sa Fleitas) Pauli, P., 1934, Sulla sieroterapia della poliomielite anteriore acuta infettiva. Instituto sieroterapico milanese, 1:101. (See reference 31, Riordan and Sa Fleitas) Plotz, E., 1938, Les ultravirus des maladies humaines. Norbert Maloine, Paris, p. 1141. (See reference 31, Riordan and Sa Fleitas) Sabin, A.B. and Olifsky, P.K., 1936, Cultivation of poliomyelitis virus in vitro in human embryonic nervous tissue. Proc. Soc. Exp. Biol. & Med., 34:357-358. Kast, E. and Kohmer, J.A., 1937, Unsuccessful attempts to cultivate the virus of epidemic poliomyelitis in various living tissue mediums. J. Inf. Dis., 61:60-64. Enders, J.F., weller, T.H., and Robbins, F.G., 1949, Cultivation of the Lansing strain of poliomyelitis virus in cultures of various human embryonic tissues. Science 109:85-87. Kramer, S.D., Geer, H.A., 1945, The deveIOpment of active immunity in Swiss mice with infective and non-infective suspensions of poliomyelitis virus. J. Immunol. 50:275-281. - 35 - . .30 00:030.“ .msofimgoo mofipowam wcwmm 0%” wwuoe % . 0.889» 0.0 3050 sagas 033:9005 53:0 0 I n... nfimaaem 3000.3 mg» 0305 I m @000 I 9 phases 30.30 no 00:03 M55530 I m «0902 mmmmmmmmm 83090030000: 89m" 388.3 I 39 now .9988 n m m .m HHH aflomom mum E 330.9005” 030: .1. 0033. 03385 I HHH 0 need you 533003 um. o a lo. HH amused.“ mum _ 0 a. m m a .1 sowpddsoonH 00:0: 33am 03353 I HH swansm mom 53005 m m m m m m. m w m m m H oumawam I 830.9003“ mum 0.3.3003 05 00.30 cases no sodusommsm no." a no .Ha 3.0 :3...» 0303005” corpse x030 he: I H omdmoom mum memos 39825 n 2033086 so some? 35% mafia gm ofimfig as. E 9.1 Emma Human. r--————-———————| TABLE III EXPERIMENT #13 WITH THE LANSING STRAIN PASSAGE AMlhKthB METHOD OF INOCULATION - THE INTRACEREBRAL ROUTE Egg Passage I -8-day chick embryos were inoculated intracerebrally with 0.03 ml. of a 10% suspension of mouse cords and medulla from passage AMlhKlM EggpInoculation S S S S S S S Inocfilum.for egg passage II (1) 10% susp. brains, spinal columns, (living embryos) (2) 10% susp. brains, spinal columns, (dead embryos) J E Mouse Inoculation l PPP-l-DDDD _§gg Passage II S S S D D D Inoculum for egg passage III (1) 10% susp. brains, spinal columns, (living embryos) (2),10% susp. brains, abdominal viscera, extra embryonic fluids (dead embryos ! MOuse Inoculation(I) Manse Inoculation (2) i +3 +1) +1) +1) +13 +13 +13 +1) +13 +D +13 +13 +13 +13 EggpPassage III (DSSSSSD (27513131) Inoculum.for egg passage IV (1) 10% susp. brains, spinal columns, (living embryos) (2) 10% susp. brains, abdominal viscera, allontoic fluids (dead embryos) E Mouse Inoculation (l) MOuse Inoculation (2) . +D+DDDD sssss Egg Passage IV (1) SSSSDDDD (2) SDDD Inoculum.for egg passage V (l) 10% susp. brains, spinal columns, (living embryos) (2) 10% susp. viscera brains, amniotic fluids (dead embryos) - L r/ Mouse Inoculation (l) MOuse Inoculation (2) P+D+D+D+DSSSS \. EggLPassage V (l) S S S S S S S D D D D D (2) S S S S S S 8‘5 8 D D D Inoculum.for mouse inoculation (l) 10% susp. brains, amniotic fluids (living embryos) (treated with antibiotics because of bacterial contamination) (2) 10% susp. abdominal viscera, brains, amniotic flds. (dead embryos) (treated.with antibiotics because of contamination) “-mv T MOuse Inoculation (1) Mouse Inoculation (2) S S S S 5 4D 4D #D S D Note: S - surviving mouse or chick embryo D — dead P - mouse showing flaccid paralysis §D - mouse showing encephalitic sympotoms such as tremors, weakness of legs and sometimes convulsions, followed by dealh.within 2h hours. TABLE I EXPERIMENTS CONDUCTmD IN AN ATTEMPT TO PROPAGATA Thfl MOUSE ADAPTED LANSING STRAIN 0F POLIoMYhLlIIS VIRUS IN EMBRYONATMD CHICKEN EGGS (EXP. _§ 'H'Ehbryo Inocfi1a£iénl_n_lp .ig .. Embryonic Tissues Harvested E g m Results 3f Mpuse Inoculation after Passages in Eggs i No. 3 Mouse Age of Incubation E Tissues Harvested Disoosition of Egg Harvest ; Inoculum for the ; ngg Passage egg Passage Egg Passage ngg Passage Egg Passage . Passage Embryo i ‘- . neXt egg passage III IV V §Exp. #ii AE14K1M41 E 7-9 days 13-5 dags at gAmniotic fluids I (1) Amniotic fluids passed undiluted, f 0.05 ml. Q (1) 0/10+ 0/10 .2 0/10 i 0/10 not done Amniotic Sac Inoculation i E 3 L 37 C i E (2) tissues ground to a 20% susp. + in § S (2) 0/10 . i ' Inoculum for First Egg } ; . _“. v . g: p p g _ i . Saline - _-._- . . . , i ' '" Passage ' 0'0? ml. Of a iExp.d#2 AMldKlMél : 7-9 d%rs :5-5 dfiYS at lBrains, spinal columns, abdominal 5 Ground to a 20% suspension in saline 3 0.05 ml. - 0/10 0/l0 . 0/10 i not done - not done iEdTEEEp. of infected a f g o ? - . n -7 o , i ; s 1 . 37 C .v1scera (stored at 00 C) , , . mouse cords and medullae ; , 4 g i g _ (I .m_uwmiiw, , _1, ., E... . . g ; - . fExp. #6 AM14K1M41 ' 7 days :5-5 days at sheads and torsos (stored at ~5000) ? (1) Ground to a 2 % suspension in saline ‘ 0.05 ml. 3 (1; l/lO 0/l0 3 0/10 O/IO i not done ; ; 3700 g (2) 20% susp. concentrated 7 times 5 5 (2 1/10 E . ‘ §dxp. #4 AMIQKIMQB ' 7-9 days {4-5 days at {Amniotic fluids, brains and spinal ‘ (l) Amniotic fluids passed undiluted, g 0.1 ml. 3 (l) 8/l0 OVIO . O/lO 0/10 ' 0/10 2 ‘ 7 3500 icolumns, (passed on the same day) . (2) tissues ground to a 20% snap. in saline 5 § (2) 0/l0 0/l0)» ' 0/l0 1 O/lO 0/l0 (Exp. #5; AM14K1M4G 8-9 days 53-4 da 3 at (Amniotic fluids pooled with heads & ; Ground to a 20% susp. in saline i 0.1 ml. 3 4/10++ Q/lO i 0/10 ? 0/l0 O/lO f l ' ’ 35 C Storsos (Passed on the same day) A I ‘ 1/20 “ ' Amniotic Sac Inoculation . E , i f . 2 ' . 1 , Inoculum for First Egg fExp. #6! AM14K1M46 lst Pass. 53-4 dags at ,Amniotic fluids pooled with heads & § Ground to a 20% susp. in saline 1 0-1 ml. ‘ 0/10 0/10 f 0/10 j not done I not done Passage - 0.1 m1. of a Q E 11 days ' 35 C ’torsos (passed on the same day) 9 § . g E f / "‘*‘—“— ’ 0th P ' 'l ‘ = ‘ . ‘ 10% susp. of infected { 7-982a :88. 2 p _ _ mouse cords and medullae ; a . .- my ; E i ; .g g ; i 2 :Exp. #5; AM14K1M46 1 7-10 days 13-4 davs at §Amniotic fluids pooled with heads & f. Ground to a 20% susp. in saline 1 001 ml. 3' 0/l0 0/10 g O/lO 3 0/10 l 0/10 ? . 3560 gtorsos (passed on the same day) ‘ ‘ 1 ,,., i i P \ 3Exp. #8} AM14K1M46 1 10 dars :2-8 days at fOnly 5 dead embryos harvested ‘ Ground to a 20% susp. in saline ’ 0‘1 ml. f (1) 0/10 0/10 ? 3 1 g 3 3500 lAmniotic fluids pooled with heads & E .3 f (2) O 10 I 0/10 E i Q ’ ftorsos (passed on the same day) , __I ”up? _ 7 7 A_”H _Hj__w$§2_£_}9”ll ,i,_ gélgwml élMESt.QQQSMMIWhUpQEMdQnew~§~ not done :Exp.#10? AM14K1M46 3 let Pass. j5-4 dags at iBrains, spinal columns, (passed on the ; Ground to a 20% susp. in saline * 0.03 ml. f 0/l0 E 0/l0 g O/lO i 0/l0 é O/lO Z I ‘ 11 days 55 0 same day) } t f I § (1 , E , Other Pass. ; i 2 g i i , Intracerebral Inoculation; ‘ 7-9 days ' : ‘ A ”mwmw _ _ ,,_ - h._ _-.:E. . Vi_uf_ .wm~«-~~v~ «ww:“~~M-~-~-““é“"" “‘"“":" ' ' ””" E‘" Inoculum for First 5g 3 w_ - f . ”I _,_ _ ,wg _ . .1--. .mflwl.w,m ”Alli”- _MW-H-1 ”,_.V m - - I . p % I , Passa e _ 0.03 ml. SEEA éfixp.#ll ;AM14K1M46 : 7_10 days 5-4 dags at {Amniotic fluids, heads and torsos . Ground to a 20% susp. in saline 2 0.03 ml. 0/10 3 O/lO g O/lO i O/lO l Q/lO i6%fE§§éo of infected 2 : 55 C TPOOIed (passed on the same day) 3 g i _ 3 mouse cords and medullae % . g " . . ‘.' .' " D .. , , é " 3 ‘ * . iEXp.#12 AM14K1M46 7-10 days 55-4 da s at Amniotic fluids pooled With heads & 3 Ground to a 20% susp. 1n saline ; 0.05 ml. 0/10 f 0/10 J not done ! not done , not done 2 . i 55 C ltorsos or spinal column (passed on E E g i 3 ( 'the same day) i I - ‘ + 0/l0; numerator - number of mice paralyzed denominator - total number of mice inoculated ++ 1/20; suspensions from 4 dead embryos in the first passage were each inoculated into 5 mice - only one of the suspensions produced paralysis .nnf- .- .Icll II I curl I'll-Uh I I'.‘ .I .IJ' '| I I. iii, in 4M ”x d Jones MICHIGAN STAT II III! “II in] 31293 NIVERSITY LIBRARIES 3 015 8421 ““"fi