17-31 Iva. - '-—-' wfi -'-s‘-a-<-.r ‘—-' f w—w—rvw ‘ SOME PROPERTIES OF ENFECTBOUS BRONCHITIS WRUS PROPAGATED IN ME ISOLATED CHOMOALLANTGEC MiMERANE Thesis for flu Dogma 05 pk. D. MlCHIGAN STATE UHEYEKSETY Yoshihiro Ozawa 1959 THESE c.3— This is to certify that the thesis entitled Some Properties of Infectious Bronchitis Virus Propagated in the Isolated Chorioallantoic Membrane presented by , Yoshihiro Ozawa ‘ has been accepted towards fulfillment of the requirements for Ph 0. degree in Microbiology and Public Health Major professor Date June 29, l959 0-169 LIBRARY Michigan State University &0 Jo. SO!!! PROHRTIIS Ol' INFECTIOUS BROHCHITlS mm: PROPAGlm II TH! 190nm CHORIOLHAN‘I‘OIC mm By YOSHIHIRO OZAIL A THESIS Submitted to the College of Veterinnry Medicine of Michigan State University of Agriculture and Applied. Science in partial fulfillment of the degree of DOCTOR O! PHI IOSOPH! Department of Microbiology and Public Health 1959 MHWMNTB The author ie grateful to Dr. Charlee H. Cunningham. Profeeeor of Microbiology and Public Health, for hie patient guidance, conetant stimulation, and thoughtful underetanding throughout the inveetigation and preparation of this nanuecript. The author in aloe indebted to Dr. L. C. Fergueon, Dean of the College of Science and Arte, and foraerly Head of the Department of Microbiology and Public Health, and Dr. H. J. Stafeeth, Acting Head of the Department of Microbiology and Public Health for their assistance in preparing thin nanuecript. The encouragement and cooperation of tire. II. P. Spring and Mrs. D. W. Church is greatly appreciated. ABSTMCT infectious bronchitis of chickens is an acute, contagious respiratory disease which is of economic importance to the poultry industry. The isolated chorioallantoic meabrane maintained in Hank's balanced salt solution has been found to be suitable for the pro- pagation of infectious bronchitis virus, Tangia M. The purpose of the present study was to investigate some properties of the Bendette embryo adapted strain of infectious bronchitis virus which was adapted to and propagated through 36 serial passages in isolated chorioallantoic membrane cultures. The growth curve or the virus exhibited five phases: a variable lag phase of 8 hours, a logarithmic phase during the next 52 hours, a primary decline phase over the following 60 hours, a stationary phase of 72 hours, and a secondary decline phase of 148 hours. The optimum incubation temperature was found to be 37°C at pH 7.0. The maximum yield of virus was obtained from cultures containing 200—600 mg of chorioallantoic membrane. Cultures incubated at 37°C for 215 to 81 hours prior to inoculation yielded the highest concent- ration of virus. Cultures inoculated immediately after preparation or 105 hours' incubation had a lower yield. The yield was lowest from cultures incubated for 1% and 216 hours. Viral infectivity decreased at the following retest log IDSO 0.113 per week at -25°C, and log IDSO 0.63 per hour at 37°C. During the first 30 minutes at #506, the rate was log 1950 0.15“ per minute followed by log ID50 0.0053 per minute for the subsequent 90 minutes. The activation energy for inactivation for the virus was l9.h6 X 103 calories per mole. Chemical fractionation failed to reveal association of in— fectivity with the ribonucleic acid portion of the virus. TABLE INTRODUCTION . . . . LITERATURE REVIEW. . MATERIALulflD METHODS RESULTS . . . . . . DISCUSSION . . . . . SUMMKRY . . . . . . BIBLIOGRAPHY . . . . OF COITINTS Page 15 19 53 55 Table II. III. IV. VII. VIII. IX. LIST Ol' TABLES Growth of lot and 6th Passages of ID? in CA)! Culture................... Growth of 16th Passage of ID' in 6th Culture . . foect of pH on Growth of 18th Passage-of 13' in CANCulture................. Growth of 23rd Passage of IBV in CA)! Culture at Various Temperatures of Incubation. . . . . . Growth of 23rd Passage of IBV in Different AacuntsofCAHinCulture. . . . . . . . . . Effect of Time 01' Incubation of CA)! Culture After Preparation and Prior to Inoculation WichBthPassageofIDV . . . . . . . . . . foect of Time of Incubation of 36th Passage of ID? in Hank's DSS Without Cut Culture. . . Thermostability of 25th Passage of CAM Cultured untu5°c................. Theraostability of 28th Passage of CAM Cultured twat-’ZSOGeeeeeeeeeeeeeaeee Page 20 27 29 30 31 31 32 32 LIST 01' PI GUESS Pigure 1. 5. b. 7. 9. 10. 11. Growth of 1st and 6th Passages of ID? in CM! Culture....................- Growth of l‘btn Passage of IN in CM! Culture. . . Growth of 16th Passage of ID? in CM! Culture. . . Effect of pH on Growth of 18th Passage of IDY in CAMCulture.................. Growth of 23rd Passage of IBY in OAK Culture at Various Temperatures of Incubation . . . . . . Growth of 23rd Passage of IBV in Different AmountsofCAMCulture. . . . . . . . . . . . Effect of Time of Incubation.“ CM Culture After Preparation and Prior to Inoculation Vith28thPassageofIBV. .. . . . . . . . . Effect of Time of Incubation of Both-Passage of 131‘ in Hank's BSS Without CAM Culture . . . Thermostability of 25th Passage of CM! Cultured twatu5°c.................. Thermostability of 28th Passage of CM! Cultured -IBVat-25°C................. Activation Dnerg for Inactivation of CAI! cm tmd 13v 0 O O O O O O O O O O O O O O O O 33 31+ 35 35 37 38 39 hi 1&2 “3 INTRODUCTIOI Tissue culture methods permit study of the host-parasite relationship and multiplication of viruses at the cellular level. The isolated chorioallantoic membrane maintained in a chemically defined medium has been found to be suitable for the propagation of several animal viruses. Cultivation of infectioue bronchitis virus in chorioallantoic membrane suspended in Hank's balanced salt solution offers the following advantages: (1) Base of preparation; (2) Large volume of culture may be obtained: (3) Serum. tissue extract, and other growth factors are not required: (n) The virus is free from other proteins. The objective was to study some properties of infectious bronchitis virus propagated in the isolated chorioallantoic meabrane suspended in Hank's balanced salt solution. LITERATURE REVIEW Infectious bronchitis is caused by the virus. 29.11% mill.“ It is an acute and contagious respiratory disease of chickens first described by Schalk and Bean“ in 1931. Symptomatically the disease is similar to lewcastle disease and laryngotracheitis. The mortality may be 70 to 80 per cent in chicks but death losses are negligible in older birds. The course of the disease is usually one to two weeks. ‘ but it may persist for several weeke in some instances. Broadfoot 12.13 t al and Sevoian 23; $.56 found that egg production and egg quality were markedly affected. user infection of adult birds several months may elapse before egg production returns to normal. Infectious bonchitis virus (13v) can be readily isolated from the lung and trachea of affected chickens. Pabricant and Levine” studied the persistence of the virus in eggs laid during and after an experimentally induced infection. The virus was first detected in the yolk two days after inoculation of the birds. The last successful virus isolation was from eggs laid #3 days after infection. The virus was also recovered from the trachea between one and four weeks after infection. Beaudette and Hudson9 first cultivated 137 in the cheris- allantoic membrane (CAM) of embryonating chicken eggs. They found that the virus did not produce gross lesions such as those produced by the viruses of laryngotracheitis, fowl pox, and pigeon pox. They also observed that with serial passage. the virus increased in virulence 2 and became lethal to the embryo. Delaplane and Stuartzu confirmed the observations of Beaudette and Hudson9 and also observed modification of the virus as the result of passage in the CL”. There was a decrease in virulence for chickens accompanied by a loss of immunogenicity. nelsiplnns23 reported that the allantoic cavity route of inocup lation was superior to the chorioallantoic membrane route for initial isolation of the virus. Dwarfing of embryos. which is a characteristic of 137 infection, was detected in the first passage via the allantoic cavity. According to Loomis‘gtHg;.hu gross lesions produced in chicken embryos after inoculation of IBV by the allantoic cavity route consist of the following: 1. embryos dwarfed as much as one-half normal size and having a firm ball-like shape characterised by curling with a.wry neck and feet deformed and compressed over the head, 2. congestion of the liver and deposition of uratee in the kidney. 3. living embryos are sluggish in their movements. h. the chorioallantoic membrane is thinner than normal and adherent to the inner shell membrane. 5. there are no visible lesions on the chorioallantoic mem- brane. 6. the amnionic membrane is thickened, dry, fibrotic, resists removal from the embryo, and restricts movements of the embryo. 7. feather development is immature and dry. According to a study by Cunningham and Jones21 on the effect of different routes of inoculation on the adaptation of the Van Roekel strain of IBV to the chicken embryo, chorioallantoic membrane inocula— tion produced mortality rates that did not show marked variation from passage to passage. The mortality rates following amnionic cavity inoculation were uniformly higher for the first five passages than those by other routes. Increasing adaptation of the virus by the allantoic cavity route produced higher mortality rates in the sixth and seventh passages. rho allantoic cavity route is the most desirable for primary‘ isolation of IBV due to the simplicity of operation and the appearance of pathological lesions in the first passage.21 Distribution of the Beaudette eggbadapted strain of 13V in the chicken embryos was studied by Cunningham and 31 Dardiry.2° who found the greatest concentration of the virus to be in the chorioallantoic membrane followed in decreasing order in the allantoic fluid, amnionic fluid, and liver. Yolk was innocuous. The maximum titer of the virus was obtained at the 36th hour after inoculation. The titer of the virus was greater in materials collected from living embryos than from embryos dead at the same postinoculation interval. Lllantoic fluid preparations of the virus stored at -35°C retained the initial titer for 30 days, but storage for 60 days resulted in a ten-fold decrease in titer.’ The virus was thermolable in eggs subJected to 99°! for 8 to 12 hours after the death of the embryo. The maximum embryo mortality rate occurred between the thh and 36th hour after inoculation. Hofstaduo found differences in the thermostability of 60 different isolates of 13' cultivated in eggs. Hhen virus infected allantoic fluid diluted 1 : 100 in nutrient broth containing 20 per cent horse serum with a final pH of 7.2 was subJected to 56°C, some strains were inactivated within 15 minutes while others were infective after #5 minutes. Page"8 reported that the Beaudette strain of ID? in the form of infected, undiluted allantoic fluid was inactivated within 10 minutes at 5o°c, 60 hours at 37°C, and 25 days at 22.25%. At the end of 20 weeks at WC, the virus was still infective. Sind157 studied the thermostability of 13 strains of m at various egg passage levels. Host strains of the virus in the form of undiluted. infected allantoic fluids were inactivated within 1&5 to 120 minutes at 56°C. Quantitative studies showed that thermal inactiva- tion of 13' followed a three halves order reaction rate which was indicative of a bimolecular reaction. It was assumed that in an early egg passage the virus existed in two phases: (1) 0 phase, in which some particles retained their original identity and were relatively thermostable. and (2) D phase, in which some particles were derived as a result of embryo culture and were thermostable. Bachrach 31 5.8 reported that thermal inactivation of type A, strain 119 of foot-and—nouth disease virus in bovine kidney tissue culture did not follow a first order reaction, and that the activation energy for inactivation of the virus culture was 27,200 calories per mole below 18°C. Bourdillonm reported that thermal inactivation of 8! strain of poliomyelitis virus did not obey a first order reaction. The maximum titer of ID? was obtained in eggs between 18 and 26 hours with Japanese strains52 and between 211, and 30 hours with the Connaught laboratory Vaccine Strain.38 There are only a few reports on the propagation of 13' in tissue culture. rahey and Crawley‘n cultivated the Connaught labora- tory Strain and the Beaudette embryo adapted strain9 in CM suspended in medium 597 (medium 199 minus purine and pyridine plus Hank's balanced salt solution). Maximum infectivity as determined by the SO ‘ per cent infectivity dose (11350) in embryonating chicken eggs was obtained between the Math to 513th hour with both strains. Vith the Connaught strain the 11350 was 105'1. The titer of the Beaudette strain ranged from 11350 105'28 to 108'28. both strains were grown in mon- key kidney and chicken-:embryo-heart cells but at a lower titer than with the CAN culture. He cytopathogsnic effects were observed in in.- fected cells. Buthala and Mathewsl’5 failed to observe cytopathogenic changes in monolayer cultures of chicken-embryo-kidney cells after inoculation with a lew York strain of IBV. Chomial: .Lt 5.16 observed a cytopathogenic effect in chicken- embryo-hidney cells in the second passage following the inoculation with the Beaudette strain. By the tenth culture passage the virus titered at 10-h” in cell cultures and 10-7 in embryos. In determining the growth curve in tissue culture it was found that the virus dis- appeared from the fluid in l} hours, reappeared in 16 hours, and reached maximum titer at 1&8 hours, where it remained for 2’4 hours and then declined. They also found that undiluted, infected allantoic fluid of both the Connecticut and Massachusetts strains of IBV passed serially five times in embryo kidney cells, failed to infect chicken embryos. The virus passed in embryo kidney cells caused cytopathogenic effects in chicken-embryo-fibroblast cultures. In the kidney cell cultures, the tissue culture propagated Beaudette strain of IBV was neutralised by both the Massachusetts and Connecticut type antiserum. but not by normal serum. However, the same virus. when tested in embryos. was neutralised only by Massachusetts type antiserum and not by normal serum or Connecticut type antiserum. Wright and Sagik6u reported plaque formation by the Beaudette strain of I37 in.monolayers of chicken-embryo-kidney cells. After a 3-day incubation period. the plaques were from 3.0 to 4.0 mm in diameter. Minced CAM culture was first used by teller and Indore63 for propagation of mumps and influensata viruses. The nutrient medium consisted of 3 parts of Bank's balanced salt solution (388) and 1 part of Simm's oxbloodpserum ultrafiltrate. In such media these viruses ‘ produced a measurable quantity of hemagglutinin. rulton and Armitageas, applying the same method.63 described a new technique of titrating the infectivity of egg-adapted influensa virus by adding virus dilutions to minced CAM suspensions. Robind and Inders51 prepared an extensive review of studies of animal viruses in tissue culture. It was emphasised.that many workers had investigated the factors influencing the growth of viruses such as compositien of the-medium, salt solution, type of tissue, and other V' '4 7“...- A K'TJ‘V'MV factors. Ackermann1 observed cellular respiration of the CAM following infection with the PR8 strain of influenza.virus. Sodium malonate,- 0.02 to 0.06M was found to have no inhibitory effect on the uptake of glucose by the membrane, but showed a partial inhibition of endogenous respiration of the tissue. Amounts of sodium malonate that reduced ougen uptake inhibited viral propagation, but were not virucidal. The action of sodium malonate indicates at least one reaction which is a metabolic step in the Kreb's cycle. Succinic dehydrogenase is affected by malonate and without a continuous supply of di-and tri— carboxylic acid, the entire cycle will be blocked. saton e_t_ 9;.29 found that butyl 3,5-diiodo-lI-hydroxy-bensoate, which uncouples phosphorylation from oxidation, had effects very similar to,dinitrophenol in inhibiting the growth of the P88 strain of influenza virus in CAM cultures and de-embryonated eggs. Ackermann and Johnson? concluded that the energy required for viral synthesis from 200mg of CAM in Simmonis solution was obtained from the oxidative phosphorylation activity of the host tissue. Ackermann and Maasaaba'u studied the growth characteristics of the P38 strain of influenza virus in CAM culture. The release or liberation of the virus from the CAM occurred without concurrent destruction of the membrane of the infected cell. There was no evidence of a general 'burst' phenomenon, and the destruction of the cell membrane did not seem to be essential to, or concomitant with, the release of virus. An early phase in the development of the virus, 2 hours post infection, was described to be sensitive to the action of CK -amino-p—methoxyphenylmethane-sulfonic acid. It was by virtue of this that virus multiplication was prevented. If this phase was allowed to go on to completion, replication of virus occurred even in the presence of the sulfonic acid, but the release of virus from the tissue was impaired. . . It was suggested that sulfonic acid might interfere with the adsorption or penetration of the virus, and that the initiation of infection and the liberation of new virus might be processed to share the same common character. It was also found that infection could be initiated in the presence of methoxinine (viral inhibitor) under which condition viral increase was disallowed. The function in viral development of one biochemical process which was inhibited by methoxinine was found to be completed at an interval after initiation of infection, and before the appearance of the infectious form of the virus. It was possible to define four stages of viral development in terms of sensitivity to methoxinine and 0(- amino-p—methoxyphenylmethane—sulfonic acid. Tenn and Tyrrell59 studied the kinetics of multiplication of the Lee strain of influenza virus in the chorioallantoic membrane in ‘ziggg employing the hemagglutination technic for measurement of virus concentration. A linear relationship was found between the logarithm of virus adsorbed and amount of membrane used. 0f the virus adsorbed, less than 10 per cent could be recovered from the membrane. 0! the recoverable virus 90 per cent, which in itself constituted only 2.5 per cent of the virus adsorbed, was neutralised by specific immune serum. Lee virus was adsorbed by the allantoic and chorionic layers of the membrane to a similar extent. Virus produced in membranes was 10 liberated rapidly and continually into the medium. Ackermann gt 93.5 found that under certain conditions, the P38 strain of influenza virus might bind to the chorioallantoic membrane and the infectious property was retained upon prolonged incubation of the complex. Apparently the bound active virus was not functioning in the initiation of viral increase. The bound infectious virus migit be partially. removed by extensive washing. The characteristics of the washing were suggestive of a reversible equilibrium type of binding. It was hypothesised that active virus was held by two types of binding at the same site: one type of binding being sensitive to the action of receptor destroying enzyme (RD!) of 11-31119. cholefl. the second type being sensitive to the blocking effect of o( -amino-p- methoxyphenylmethane sulfonic acid (”98). Virus could be held to the receptor site by either type of binding or both. It was found by Ackermann and’ Maassabb that p-fluorophenyl- alanine was phase-specific, did not interfere with the initiation of infection, but rather acted during the productive period of the infectious sequence. Baton gt 5.28 investigated the effect of thyroxin on CAM culture, and observed an increase in oxygen consumption of membrane suspended in 383 with 0.01 to l.0mg/m1 after a lag period of about 12 hours. A slight stimulation of growth of the P38 allantoic passage strain of influenza virus was observed in the presence of thyroxin. The reversible effect of hypotonic solutions on growth of influenza virus in CAM cultures has been studied by Eaton and Scale”. Growth of the virus was inhibited by exposure of the infected CAM 11 to Hank's ass diluted 1:2 or 1st. Sodium chloride added 18 or 2a hours after infection, although it stimulated growth of the virus, did not increase tissue proliferation. Loss of virus fnom heavily in.- fected tissue occurred on exposure to 50, 35, or 25 per cent 388. This was prevented by adjusting the solution to near normal osmolarity with glucose. Ishida and Ackermannul investigated the characteristics of the initial cell-virus complex using influenza A virus, and found that the complex was stable to dilution and to simple washing with buffered saline. It was insensitive to the action of m, but sensitive to the inhibitory effect of anti-viral serum. It was also found that the cell-virus complex could be formed at 3°C. Daniels _e_t_ 3.22 found minimal production of the P38 strain of influenza A virus in glucose deficient 358 when employed with the CAM adherent to the shell of ds-embryonated eggs. “hen glucose was added virus production was stimulated. According to Levine _e;_t_ _a_l_._.""3 potassium deficiency was found to suppress: (1) growth of chorioallantoic epithelium and fibroblasts, (2) multiplication of the P38 strain of influenza virus, (3) respira- tion of normal or virus-infected tissue by about 10 per cent. The results also indicated that potassium ion deficiency affected the virus-host interactions in two ways: (1) accessibility of virus attached to tissue to neutralization by immune serum, (2) intracellular synthesis of high energy bonds which was necessary for virus repro- duction. #7 According to Nagata _e_§ 9;. influenza virus and lswcastle disease virus could be subcultured in minced CAM suspended in medium containing only MI and 101, but viral infectivity diminished after three or four subcultures. Scott g§|§;,55 demonstrated that Newcastle disease virus (NDV) grew in isolated CAM suspended in Tyrode's solution. The Rankin-[J- 19% strain of ”V was propagated at 36°C and maintained through 30 serial passages inisolated CAM cultures. The factors influencing the growth of MDY in CAM culture have been studied by Zuschek gt 5.65'66'67‘ The maximum titer was obtained in cultures of CAM suspended in Tyrode's solution at pH 9.5. The smallest yield of virus was obtained at pH 2.7. The maximum titer was also obtained with Tyrode's solution that con- tained 0.2 gs of CaClz per liter. Greater concentrations of Ca012 appeared to deter growth of the virus. Potassium was also found tobs essential. Maximum yields of virus were obtained from the CAM sus- pended in Tyrode's solution that contained 0.14 gm of KCl per liter. Virus growth was inhibited in Tyrode's solution that was deficient in potassium. Magnesium ions did not overcome the inhibition of growth of the virus in Tyrode's solution deficient in potassium. An excess concentration of magnesium and glucose produced inhibitory effects on the virus. 'no significant inhibition of nnv was demonstrable in Tyrode's solution that contained 0.01 M of urethane, sodium malonate, or sodium fluoroacetate, but the growth decreased as the concentration of 2,1:— dinitrophenol was increased. Proflavin and sodium aside, at concentra- tions of 5 to no gm per milliliter were effective in inhibiting the production of IDV. Growth of the virus was limited to incubation be- tween 33°C and uz‘c but it grew best at u2°c. Production of the virus 13 followed a linear response after 36 hours of incubation. Brandtll cultivated twelve strains of IDY in roller cultures of minced CAM. Intracytoplasmic inclusion body formation in epithelial cells grown on cover slips was demonstrated with EDT and high—egg- passage mumps virus. ' lumerous attempts by Dunham and twinng to establish Type 2 poliovirus in the CAM membrane of chicken embryos were unsuccessful. Strains of cells derived from the CAM and growniin‘zitgg were readily infected after 5 transfers with not only Type 2 poliovirus but also Types 1 and 3. Multiplication of these viruses and cytopathogenic effects on the cultured cells were observed until the series were terminated after 10 or 20 passages. In general, all viruses appear to consist mainly, and some entirely, of proteins and nucleic acids. The nucleic acids of plant and animal viruses can.be either of the ribonucleic acid.(BNA) er desexyribonucleic acid (DEA) type, while bacterial viruses contain only DNA. Animal viruses appear to contain either DNA or REA but probably not both. The viruses which are known to contain RNA are those of foot—and-moutn disease?“ eastern and western equine encephalo- myelitis, poliomyelitis, influenza, mumps,50 fowl plague,53 and low- castle disease.11 Viruses which are known to contain.DNA are those of vaccinia, psittacosis, herpes, adeno, and rabbit papilloma.50 The infective RNA of Type 1 and 2 poliovirus was isolated by Alexander gt'g;.7 by the method of Gierer and Schramm who isolated infectious RNA from tobacco mosaic virus (TMV) with water saturated phenol in 1956. 11+ Colter and Bird” isolated an active m fraction of Meningo— encephalitis virus from infected Ehrlich ascites tumor cells. Infective RNA was also isolated from tissues infected with eastern equine encephalomyelitis virus by Hooker and Schlfer62 by treatment with phenol. Previous to this work, Schlfer53 found no in- fectivity of BEA obtained after phenol treatment of fowl plague virus. The reason for this was not clear. It was speculated that the EMA of fowl plague virus was relatively unstable or that, in contrast to tobacco mosaic virus, two units of RNA were needed for the formation of new infections particles; one for the production of’gyantigen and the other for the production of hemagglutinin. Infective EMA has been isolated from the viruses of mouse, oncephalomyelitisgu and foot-and-mouth diseaselu by treatment with phenol. furthermore, recent experiments by taper and Steerena have demonstrated the infectivity of tobacco ringspot virus nucleic acid prepared by a modification of the heat-denaturation.method, which uses hot RaCl for extraction. 61 extracted infectious EMA from tobacco mosaic virus Mecker with‘hot phenol. These facts suggest that the nucleic acid fractions of animal viruses might be the infectious units and the carriers of genetic information as in tobacco mosaic virus and also as in.DNA of bacterio- phage 'e “F57 MATERIAL AID METHODS Virus: The Beaudette strain of 13', lorth Central Infectious Bronchitis Virus Bepository,Code 1&2, was used throughout the experiment. This is an egg-adapted strain capable of killing lO-to 11-day-old chicken embryos within 36 hours after inoculation via the allantoic cavity. It has been through hundreds of serial passages in chicken embryos, but the exact number is unknown. Hank's Balanced Salt Solution: The balanced salt solution (BS!) was made with the following ingredients?“19 on a basis of grams per liter of double distilled water: la01, 8.0: m1,.0.b: lazEPOnJZEzO, 0.15: EZPO“, 0.06: wu,7320, 0.1: MgClZJflZO, 0.1: m0 , 0.35: Glucose, 1.0: and phenol red 0.02gn. Calcium chloride, 0.1hgm, nae prepared separately to avoid flocculation, and added to the other ingredients after sterilization in an autoclave for ten minutes at ten pounds pressure. Penicillin, 10,000 units, and streptomycin, 10 mg per 100 ml were added. The 383 was at approximately pH 7.5 Preparation of Ohorioallantoic tun-bran” and propagation of virus: Chorioallantoic membranes were collected from 10-to 12-day old asbryonating chicken eggs ”from a commercial, disease—free flock. The eggs had been in a Janesway Model 252 incubator at 99-99.5°r. The CAM was removed aseptically from the egg and washed in a sterile 15 lb petri dish containing about 20 m1 of sterile physiological saline. The membrane was then transferred through three succesive washings in 200 m1 of sterile saline contained in 500 ml Erlenmeyer flasks. The flasks were shaken vigorously by hand for thorough washing of the membranes. After the third washing, the CAM was removed to a sterile petri dish, and minced into small_fragments of 2 to 5 mm with scissors. The fragments were then placed in an 8 ounce prescription bottle containing 19 ml of 383. Unless specified otherwise, the cultures were incubated at 37°c. Minced CAM is the better medium for multiplication of 137 than whole CAM.33 It was necessary to adapt the embryo-cultivated virus to propagation in the isolated CAM in 383 by means of serial passage.33 for the first passage virus-infected allantoic fluid was used, but virus-infected 388 from the CAM cultures was used for subsequent transfers. Virus from each passage was harvested 60 hours after inoculation and stored at -25°C until used. for the maaority of the experiments virus from the 15th or’higher CAM passage was used. Infectivity Tests: A11 titrations of viral infectivity were performed with 10- to 11-day old embryonating chicken eggs. In some instances, titrations were performed immediately after harvest of the virus-infected fluid, but in others it was necessary to store the samples at -25°C until they could be used. Prior to titration, all samples of virus were centrifuged at 2,000 rpm for 5 minutes at h9C with an International Centrifuge (Model PBPl) to sediment fragments of tissue which were present. / h» J J Vt”, i‘v . I; ..q E I. \ 17 Serial ten-fold dilutions of the virus were made using 0.5 m1 rof sterile Difco nutrient broth and 0.5 ml of virus. Each of 5 embryos per dilution was inoculated with 0.1 m1 via the allantoic cavity. After inoculation the eggs were re-incubated and candied once a day. Embryo mortality during the first 24 hours after inoculation was considered to be due to non-specific causes, and these embryos were not included in calculations of mortality rates. 0n the 9th day after inoculation, all living embryos were examined for gross pathological changes associated with infection with 11”.“ Viral infectivity was calculated by the method of Reed and Muench“9 using embryo mortality and gross lesions as positive reponses. The results were expressed as the 50 per cent infectivity dose (1050) per 0.1 ml inoculum. Isolation of Nucleic Acid: The method described by Gierer and Schramm36 for isolation - of RNA from tobacco mosaic virus was used with a slight modification for attempts for isolation of RNA from IBV-infectod fluid. Mater saturated phenol was prepared and stored at 1+°c. To each of two sterile 16 x 125 mm scrswcap culture tubes in an ice, bath, 3 m1 of cold virus was added. An equal volume of water saturated phenol was added, the tube was closed tightly with a screw cap and the mixture was shaken vigorously by hand for 8 minutes at room temperature. After centrifugation for 3 minutes at 3,000 rpm at 14°C, the mixture had separated into the water and phenol phases. The water phase, 2.5 m1 from each tube, was transferred to other tubes in ice bath each containing 2.5 m1 of water saturated phenol. The mixture was again lb shaken vigorously for h minutes, and then centrifuged. The water phase was transferred as before and shaken with an equal volume of water saturated phenol for b minutes and centrifuged. The total water phase, about 2 ml, was transferred to another culture tube and the phenol was removed by five successive extractions with an equal volume of peroxide-free other. To remove the residual ether, dry nitrogen was bubbled through the solution at 0°C for about 10 minutes. The solution was adjusted to pH 6.8 to 7.0 with NaHC03 with phenol red as the indicator. This preparation will be referred as ”undiluted” nucleic acid even though there was a slight dilution from adjusting thO pHe new: unptation of 13" to cut Culture: It was necessary to confirm a previous report33 that the Demdette strain of 131 had to be adapted by serial passage to propa- gation in m culture. The result of the first and 6th passage of the virus is pre- sented in table I and Figure 1. These data served as the basis for further investigations of the characteristics of the virus propagated in CAI culture. and empha- sised that the aaxiaua yield of virus was present 60 hours after in- fection. for all experiments. virus from the 15th passage in GA! culture or higher was used. Growth of 131 in GA! Culture: In order to ascertain the pattern of viral aultiplication. twenty-seven cultures were prepared and inoculated with the 15th Passaga of flu virus .30. incubated 9% 37°C. At certain periods. the infected fluids were collected fronons culture and the virus was innediately titrated. the results an presented in Table II. he results for the first 60 hours after infection are shown in Figure 2. The general pattern was a decline or lag phase froa the initial titer of 106°3 to a ainiaua of 103-5 with a few fluctuations during the first 8 hours postinoculation. The lag phase was followed by a progressive increase or log phase to a aaxiaua titer of 106.5 at the 60th hour. 19 ”AL 20 The results from the 8th hour through the 10th day are shown in Figure 3. Free the minus titer of 106'5 at the 60th hour, there was a gradual decline during the next two and one half days to a titer of 105'3 at the 5th day. Following this. there was a stationary phase during the next 3 days. After the 8th day there was narked decrease to a titer of ioh'” at the 10th day. lffect of pH on Growth of 137: Ohorioallantoic nenbranes were suspended in 388 which previously had been adJusted to p! h, 5, 6. 7. 8, and 9 by the appropriate addition “ of either 0.1 I 301 or 0.1 l laOH. The pl of the 383 did not change appreciably after the addition of the GA! or the 17th passage of virus which was used as inoculum. At 8 and 60 hours after inoculation one nl of the infected fluid was collected and stored at -25°c prior to titration of viral infectivity and deternination of the pl! of the 60th hour sanples. (Table III and Figure h) At the 8th hour. there was a direct relationship between pH of the 388 and increase in viral infectivity. One exception was noted where at pH 9 the viral infectivity was 105.1: as compared to pH 8 where it was 10“”. At the 60th hour, naxinun infectivity of 106'8 occurred at p! 7.0 with lesser concentrations of virus at higher and lower pH values. The pH of the 383 collected at the 60th hour showed an increase of 1.5 and 0.5 units. respectively, for the initial values of p! h and 5. The 388 over the range of pH 6 to 9 showed a decrease of about 0.“ units for each initial value. 21 Fffect of Temperature on Growth of‘ 13': Chorioallantoic membrane cultures were incubated at 25. 30. 33. 35. 37. 39. “2. and “5°C for about two hours for thermal equilibrium prior to the inoculation with the 22nd passage of the virus. After 60 hours. the infected fluids were harvested and stored at -25°c until titrations were performed. As shown in Table IV and Figure 5. naximum multiplication of the virus occurred at 37°C where the titer was 106'2. Fron 25’0 to 37°C there we a direct relationship between infectivity and increased temperature of incubation. Growth of 13' in Different Amounts of CAI: After the final washing. the GAII was blotted dry between layers of filter paper. The nembrane was minced and 50. 100. 200. 300. 1500.. and 600 mg portions. respectively. were used for cultures. The 22nd passage of 13' was used as inoculum and the cultures were incubated at 37%. At the 8th and 60th hours. respectively. one a]. of 388 we re- moved froa each culture and stored at. -25“c until titrated. The results of the infectivity tests are presented in Table V and in Figure 6. At the 8th hour. there was‘a decrease in titer of all cultures. but the least amount of virus was present in those containing 50 and 600 mg of GAI. The greatest amount of virus was present in the cultures containing 200 and 300 ng of 0AM. At the 60th hour. there was an increase of virus in all cultures. but the greatest amount was present in those containing 200 mg or more of OAK. The least amount of virus was present in the cultures con- taining 50 and 100 mg of GAI. Using the extremes of the concentration 22 of virus. the titer of the culture containing M0 mg of CAI was about 102'° times higher than the culture containing only 50 mg of CAI. As shown in Table V. the pH of the 388 at the 60th hour de- creased in direct proportion to increasing amounts of CAM. The Effect of the Time of Incubation of CAI Culture After Preparation and Prior to Inoculation with 13': A series of CA! cultures was prepared. One culture was inocu- lated immediately after preparation and the others were incubated at 37°C for 2h. 57. 81. 105. 11415. and 216 hours before inoculation with the 27th passage of 137. The control. which contained 19 ml of 338 only. was also inoculated with the virus. After 60 hours' incubation. samples were removed from each culture and titrated immediately. As shown in Table '1 and Figure 7. the cultures incubated for 21; to 81 hours prior to inoculation had the highest concentration of virus. The cultures inoculated immediately after preparation or after 105 hours' incubation had a slightly lower yield. The yield was lowest from the cultures which had been incubated for 11414 and 216 hours. The control was innocuous after 60 hours' incubation. As a further test of the validity of the decrease of infectivity of the control. the 36th passage of the virus was used. The bottle containing only BBS and virus was shaken vigorously and one ml was removed and titrated immediately. The mixture was incubated at 37%. and samples were removed at h. 8. and 12 hours after inoculation. As shown in Table V11 and Figure 8. there was a rapid decline of viral infectivity. and the sample collected at the 12th hour was innocuous. Z3 Themstability: The thh passage of virus was dispensed in 1.5 ml portions in a series of 12 x 75 mm test tubes and incubated at 145°C in a “.th bath. After 2 minutes. the time required for the solution to reach thermal equilibrium. one sample was removed. This was considered to be at sero tiae. At subsequent intervals of 5. 10. 15. 25. 30. no. 50. 60. 70. 80. 90. 100. and 120 minutes. other samples were removed. Imme- diately after removal. each sample was frosen in an alcohol bath at ~30°c. and stored at -25°0 until titrated. The results of the titra— tions are presented in Table 7111 and in Figure 9. It is evident that inactivation was bimodal consisting of two first order reactions. During the first 30 minutes. inactivation proceeded according to the linear regression of T = 6.19 - 0.1515 I (minutes). Froa 30 nimtes throng: 120 minutes inactivation was I = 1.71 - 0.0053 1 (minutes). At 56°C. the virus. which had an initial titer of 106'2. was ‘ alnost completely inactivated within it minutes where only 2 of 5 embryos showed positive responses to the inoculum. The 28th passage of the virus was dispensed in 1.5 ml portions in 8 m1 screw cap vials and stored at.-25°c. Lftor 1, 2, 3, It. 6. 8. 10, 12. and 1h weeks the virus was titrated. The results are shown in Table I! and Figure 10. The virus decreased from 105'8 at the beginning of the experiment to 10!"2 at the 13th week. or T I 5.76 - 0.113 I ("Ck.)e lxtra‘ction of BIA from 1378 The Suth passage of 13' was adjusted to pH 7.0 with 1.15 per cent 2h lafiCOa. At the end of the extraction process. only about 1 ml of the fraction considered to contain m was obtained from 3 ml of the virus. The undilited BIA fraction and dilutions of lo‘r‘through 10"” were inoculated into two chicken embryos each. 0.1 ml per embryo. via the allantoic cavity. Two 7-week-old chickens received 0.2 ml of the fraction by the intranaeal. intramuscular and intra- tracheal routes. A CA! culture was also inoculated with 0.2 ml of the sample. The fluid from the CAN culture was harvested at the 60th hour and inoculated into eggs. No evidence of infection with IBV could be detected in any of the eggs or chickens. Activation Fnerg for Inactivation of Infectivity: The energy of activation. I. was calculated from the Arrhenius plotse. In calculating I. the following assumptions have to be made: the energy of activation for inactivation of the virus followed the rates of a mononolecular reaction: exponential inactivation was obtained at each temperature: slidit differences in pH of each sample did not affect the rate of inactivation of each sample: and difference in the passage of the virus did not cause marked differences in the rates of inactivation. The K values converted to day"1 were obtained from the slopes of the lines of the equations for thermostability of the virus at -25°c. 37°C. and h5°c. The slope. of the Arrhenius plot was -J-t.26 from which I was 25 le. mo per ries calo 103 .h6 x mately 19 roxi OPP be to ted cula cal 20 0.: m.m m.a a.o m.o m.m ~.m n.n ~.o omeaaea haw it: tit It: m.m w.m m.n n.~ 0.0 eweaaea and mmm on.” ma 8 on am 2 .3: m c eaaoaIeada E228 an a: .8 aged go a: can no maromc H nanmH ho Hedmwdfi nmnm ho masamo bu Quads comm 30 TABIIV (:30er OF 2311) PASSAGE Ol' 13' II DIM AITOUNTS OF CAN IR CUMURI Amount Log m 0/0. lml m./0 lml pH of out of CA1! 8th gour thmgour culture at he.) 60th hour-I 50 2.2 u.2 7.3 100 2.6 u.3 7.0 200 3.3 6.5 6.8 300 3.3 6.6 6.6 boo 2.6 6.8 6.5 600 2.0 6.2 6.3 ' CAM cultures at pH 7.5 at time of inoculation. 31 TABIEYI EFFECT 01' rm 0? IICUBATIOI 0F CAI! 0mm mm MARATIOI AND PRIOR TO IIOCULATIOI WITH 28TH ram 01' 13'. Time of incubation 0 2h» 57 81 105 1% 216 Log ID50/0.1m1. 5.7 6.2 6.0 6.2 5.7 5.5 5.3 TM '11 WT OF TI“! 01' INCUBATIOI OF 36TH PAS“ 01' 13' I! M's 388 VIM!!! CAM CUM. Time-ho urs O 1t 8 12 leg also/0mm. 5.3 2.8 0.5 o 32 run ml mosrimmrr or 2511! PASSAGE or out cum-um 113v AT “5%. 31:31:. log 1050/0. 11:1. “:3... Io 8 11,50 /0.1n1. O 6'3 “0 1.5 5 5'8 50 1.3 10 ”'3 60 1.3 15 3.5 70 1.5 20 2.8 80 1.5 25 2,5 90 1.3 30 1'8 100 1.0 120 1.1 will! WMILITI or 28!! master or on: CW 13v AT -25°0. log misc/0.1.1. Sea 5e5 505 5e3 5.5 503 he? use “02 “oz 933.659 ma co m: cm in €885 NH “.385an J} 4 a 1 * J; o 3 3 ems-can £8 m N t... ........ c \ L x "m \ 2 \ .u m _ xxx m” m 01:} ...Q. ._ _ .1.:... \\t.... x -67- "2. .. m. m t. c z I * 4 (:0 n O _ . I _ u m. 8m 0 .nflP—Buo 840 In an ho unogmdm mac and am.— .no E95 H gr! 3h d—a H,_-_____-__._—‘ oo manor-same m: cm an — ’I'I'O/OSGI 301 1 O go :40 In 5H ho naawdm mac." ho E05 N gawk 35 on absence—«n. m m x. o 1 1 ’tWI'O/OSGI 90': go :40 an ban ho Rwanda mama ho Enigma n gnu 36 FIGUBI 1} EFFECT 01' pH ON GROWTH OF 18TH PASfiAGI 01‘ ID? I! CAN 0mm 8 hours ' incubation 60 hours' incubation _ J l l l J 5 6 7 8 pH of initial CA1! culture noun 5 crown or 2331) PASSAGF or m In on: comm AT muons Wms or measures. 3? \ l J J I L ,5 ‘ I 0 F 25 . 3o 33 35 37 39 uz Temperature-0C. h5°0 38 noun 6 GROWTH OF 233]) PASSAQ OF 13' IN DIFFERENT AMOUNTS 01' CAR 1! CW 7 — mg. CA)! uoo 300 200 100 50 O 8 Time - hours 60 39 OHN 9327259 3: 6 m2 6 cm 3N n . . . l m 0 .bmn ho gawdm mBmN an: noHaaDogH on. «chum and nauagm as EH3 :40 .3 IoHaaDouH ho H23. .mo BOSE x. g: FIGURE 8 EFFECT OF TIME OF INCUBATIOI OF 36TH PASSAGI OF IN In m's 338 “11300! CAM comm Y = 5.32 - 0.613 x Time-hours eeavumn I same m ch as c3 cm 8 ca co cm 9. cm cm 2 q a 1 J .1 q d u _1 J u q 0 AW!“ \\I\\|®/ 1 H /®/@ll||6\ UI‘AU’ l N H “mooeo I Hhed I N L cm. . m g 0 N. a or m. \ u :36 .. 38 u » .co? 5 a: gagc :3 ac mocha 53 8 Sadficccxmmma m a: 42 n." muses I can on m m a . . j q 4 .1 N MHHeoI chem n h \n "(I I‘D/0961 30: L O .oomNI 94 SH go :40 no mean!" mamN ho Maneufiawoxmflme o." Enh it.3 F100” 11 ACTIVATIOI rmcr FOR INACTIYATIO! 0! CA! comm 13V . Q 2.5- 0 ”50° logmk/ l/T = .t.26 x 103 (I " 19.h6 x 103 calories I mol) 2.0r 1.5- 1.0 0. Log! -2.0' 12 L l l l l t 3-0 3.2 3.“ 3.6 3.8 “.0 (1/1 x 103) .‘IlIII I]. .‘ DISCUSSIOH A number of factors have been defined that profoundly influence the multiplication of viruses in tissue culture. Among these are the composition and pH of the medium. temperature of incubation. species and age of cells and the presence of naturally occurring inhibitors in the cells. Certain amino acids. carbohydrates. salt ions. vitamins or cofactors. and proteins are generally required for the growth of cells iglzitgg. Only glucose. glutamine. and salts26 are required for the prOpagation of poliovirus. Glutamine is probably required as is any other amino acid for the synthesis of virus protein. and glucose may be necessary for the source of energy. In addition. glucose may serve as the primary source for alanine. serine. glycine. ribose and desoxyribose. lipid and carbohydrates. and enters into the biosyn- thesis of purines and pyrimidines. The growth of m.” m§5end influenza virus“ are deleteriously affected by the absence of either>potassium. calcium. magnesium. or glucose in the extracellular medium in isolated CAN cultures. The natural susceptibility or resistance of cells of different kinds and origin is of primary importance.. The chorioallantoic membrane is an integral part of the developing embryo. and is composed of cells derived from the three primary germinal epithelial layers. When the CAM is grown in zitgg. the nature and metabolic environment of the CAN can be considered to be different from that which is part of the embryo. Some of the fragments of the CAM culture adhered to the surface of the uh “5 bottle. somewhat as explants. around which narrow zones of new cells were formed. Microscopic examination of these new cells revealed the presence of a predominance of epithelial cells. although a few fibro- blasts were present both under and at the periphery of the sons of epithelial cells. Serial passage was necessary to adapt the eggapropagated virus to cultivation in the isolated CAM. In the first passage. viral in- fectivity was relatively low. .The maximum titer was obtained at the thh hour after infection which agreed with the results obtained by Fergusongawho had previously adapted the same strain of virus to CAN culture. In embryo culture. the maximum titer was also obtained at the thh hour.38'52 but this was accomplished after the virus had been completely adapted to the egg. with the 6th passage. the titer at the thh hour was 100 times greater than for the same period with the first passage. The maximum titer occurred at the 60th hour. The interval to obtain the maximum titer for the 6th and subsequent passages in CA! culture was more than twice as long as in eggs. This is considered to be a reflection on the cultivation of the virus in the same basic cells but in different environment. I There was a marked difference between the viral infectivity in the first and 6th passages 5 minutes after contact of the virus and the CAM. With the first passage. the infectivity was 1000 times less than that of the 6th passage. This may be considered to be another indi- cation of a phase of the adaptation.procees of the virus to the CA! in a different environment. During the adaptation process in the new_ 1&6 cultural system. the molecular structure of IBV could have been modified so that the infectivity portion would become more stable after serial passage in the culture medium which had different osmotic pressure and hydrogen ion concentration. Fluctuations in infectivity of the 16th passage of virus within the first '4 hours post inoculation were similar to the results obtained by Scott _e_t_ 51.55 with Bewcastle disease virus in the isolated CA! in Tyrode's solution. The fluctuations with 137 may have been due to a temporary adsorption of some of the virus to non-susceptible host cells and release of the virus without completion of its multiplication cycle. Some of the virus may also have been inactivated by cellular debris and extracellular euteteccee39 which is similar to experiences with polio— virus.“6 The growth curve of the 16th passage of the virus during the first 60 hours may be divided into 2 phases; a lag phase of 8 hours and a log phase of the next 52 hours. The lag period indicated that about 8 hours were required for absorption of the virus to the cell. penetration into cell. and replication. Release of a new generation of the virus from the cell is unifested by the log phase. The 8 hour reproductive period for 13' corresponds to a 6- to 8-hour period for 1m. 55 This is in contrast to a 5- to 6-hour period for influensa vim-31 in the isolated CA! and 3 to 6 hours for poliov'irusu5 in monkey kidney cells. The log phase of II" was curvilinear as contrasted to the exponential log phase of IDV. The log phase of 137 was followed by a primary decline phase of 2 and one half days. a stationary phase of 3 days. and a secondary '47 decline phase of 2 days. . It is considered that during the log phase the ability of the original CAI cells to produce virus was exhausted as a result of in- fection by IBV'aad few functioning cells remained in the culture. The primary decline period may be a reflection of either inp activation of the virus or adsorption of the virus to the previously ' exhausted cells without replication. The virus may also have been absorbed into the newly formed cells of the can as the first phase of another multiplication cycle. The stationary phase probably represents an equilibrium between production of virus by the new cells and inactivation of virus that had already been released from all cells. At the end of the stationary phase. it is considered that all cells had lost the ability to reproé duos virus. and as a result the extracellular virus underwent inacti- vation as represented by the secondary decline phase. With 13V, the maximum yield of the virus was obtained from the chm culture at pH 7.0. whereas with the Roakdn.etrain.of lewoastle disease virus (IDV)65, the maximum titer was obtained in the isolated GAR in Tyrode's solution at pH 9.5. These facts suggest that 13' and ID? require different enzymatic systems for multiplication. and that the system necessary for the reproduction of 13V is active at pH 7.0 whereas that for nor requires a more basic environment. As a result of cellular metabolism. the pH of the cultures after 60 hours' incubation decreased 0.3 to O.h units per pH value when the initial pH of the 385 was 6.0 or'higher. Uhen the initial pH was 5.0 or lower, there was an increase in pH during the incubation period. This is considered to be a reflection of lac): of metabolic activity of the cell. Degeneration and necrosis might have occurred. Release of the cellular contents. which in themselves have a buffering action. may have caused the shift of pH of the extracellular medium towards neutrality. The titer. obtained from the cm cultures at pH 7.5 in the previous experiments were as high as 106'5 or approximately the same as that obtained at pH 7.0 in the present experiment. from these data it may be concluded that the optimum pH for 13' in CA)! culture is about 7.0. but that a pH range of 6.0 to 7.5 may be used for viral propagation. O The effect of temperature of incubation varies considerably with the virus-host system. The maxinl growth of some viruses seems to be correlated with the optimal temperatures for growth for the host cells while in other systems there is no such correlation. Colville 33 31,18 demonstrated that in cultures of avian embryonic lung. increasing the temperature of incubation from 3? to hl°0 did not prevent multiplication of influenza A virus. Farnham and lewton32 investigated the effect of variation of incubation temperature on the various stages in the growth cycle of herpes virus in Bela cells. While the adsorption of the virus to the cells was largely temperature independent. the rates of cell penetration by virus and of intracellular viral growth were considerably decreased at temperatures lower than 37°C. This has been correlated with a rate of viral inactivation which is higler at 37°C than at 32°o. “9 Zuschek e144,.” obtained the maximum growth rate of ID? in CM! suspended in Tyrode's solution and incubated at h2°C. The growth rate of EDT in the culture incubated at temperatures from 33 to h2°C followed a linear increasing response. The chicken. the natural host for 137. has a normal body temperature range from no to 141.6%. but this dose not necessarily mean that this is the optimum incubation temperature for cultivation of the virus in a tissue culture system. The effect of temperature on cultivation of 13' showed two linear responses: a rising phase between 25°C and 37°C. and a declining phase between 37°C and 155°C. from 25 to 37°C. there was a progressive increase in viral production which might indicate a parallel increase of metabolic ’activity and activation of cellular ensymatic systems that were responsible for reproduction of the virus. The decrease of viral activity above 37°C represents the opposite reaction. While there was no definite evidence that the virus multiplied in the culture at 25%. some of the infectivity was maintained for 60 hours. whereas the infectivity was completely IOUt ‘t tCSOCe The optimum temperature for the maximum yield of 13' is at 37°o. The relation between the amount of virus present and the number of cells available for adsorption may offer an explanation as to the low concentration cf virus present in the 50 and 600 mg our cultures at the 8th hour after inoculation. A theory is offered that with the 50 mg culture only a 50 small amount of the virus was adsorbed and the virus remaining in the medium was inactivated. With the 600 mg culture a greater amount of the virus was adsorbed due to more cells being available for adsorption of the virus. Although the 50 mg on culture was but a small portion of an entire CA“. average weight 200 mg. a sufficient number of cells was available for viral reproduction. Had there been no CL)! present. the virus would have been almost completely inactivated in the presence of 388 at the 8th hour sampling period as evidenced by this result in the experiment where only 338 and virus were present. As a further test of the validity of this theory as to the number of cells available for viral reproduction. samplings made at the 60th hour showed two extremes: (l) the maximum yield of virus was obtained from the cultures containing 200 to 600 mg of CHI. (2) the least amount of virus was obtained from the culture containing 50 or 100 mg of can. We significance is attached to the slight differences in the amount of virus obtained from the cultures containing 200 to 600 mg of CD! as only lo°'° infective doses separated the extremes. The low yields from the 50 and 100 mg of CAM are considered significant and support the theory that viral reproduction is dependent upon the relation of the number of cells available for infection and the mount of virus produced. The effect of the tins of incubation of the CHI culture prior to inoculation with 137 may perhaps be influenced by the multiplication 51 of the virus in the original cells as well as in the cells resulting from the explant zone of growth previously described. The latter might have been responsible for the slight increase of viral production in the cultures incubated from 2“ to 81 hours prior to infection as contrasted to the results obtained with the culture inoculated immediately after’preparation. It is considered that the progressive decrease of viral reproduction in the culture incubated for more than 105 hours represents a diminution due to exhaution of the viruseproducing capability of the cell. The virus in the form of infected allantoic fluid was inactivated in 5 minutes at 56°Cu8. The 2hth passage of the virus from CAN culture was inactivated in u minutes at 56°C. The slight difference in time of inactivation may be a reflection of the presence of less protective colloid in Hank's 388 than in allantoic fluid. When subjected to “5°C. inactivation of the virus proceeded according to two successive first order reactioni one for the first 30 minutes and the other over a subsequent period of 90 minutes. During the first 30 minutes. the fastest acting. or the most thermolabile component was inactivated. It is possible that the inactivated particles may have formed protective aggregates around the remaining active particles. thus accounting for the extremely low rate of inactivation during the subsequent 90 minutes. It is also possible that this bimodal type of overall reaction may be due to differential thermostability of original phase and derivative phase of viral particles in a.mixed population of the egg adapted strain of 137 as presented by Singh57. .53. Mi 52 Perhaps at 15%. with virus in the form of infected ass, selective separation occurred more readily than when the virus was Nbdected to 56°C in the form of infected allantoic fluid or 388. T1118 W I" OWN“! in part by the evidence that 2 of 5 embryo. inocula‘fl no. . 10-1 dilution of virus in ass and heated at 15% for 70 minutes. and 1 out of 5 embryos inoculated with a 10"2 dilution of the virus heated for 80 ninutes showed pathological alterations. that were typical of early egg passage or original phase of the virus“. The activation energy for inactivation of ID? in OD! culture was 19.1560 calories per nole below 155°C. I’revious work by as.“ with infected allantoic fluid showed an activation energy of 23.000 calories per nole. This difference in energy nay also be attributed to the protective action of colloidal substances in the allantoic fluid. All viruses on which chemical analysis have been made have been shown to contain at least nucleic acid in addition to proteins. The nucleic acids are considered to be the infective units. and are carriers of all the genetic properties of the viruses. Lack of infectivity of the fraction of 13' extracted with phenol may be due to the following: (1) the extracted nucleic acids are relatively unstable, (2) the infective unit of 13' is not con- tained in the fraction, and (3) other components of the virus are necessary for initiation of infection. further investigations are necessary to establish the reason for the negative results obtained from chemical fraction of 13'. 3. 5. 6. MY The Beaudette egg-adapted strain of infectious bronchitis virus was adapted to and propagated through 36 serial passages in minced chorioallantoic neabrane culture suspended in Hank's balanced salt solution. lach culture contained 1.0 n1 of virus and 19 ml of Hank's balanced salt solution. The growth curve of the 16th passage of infectious bronchitis virus exhibited five phases: a variable lag phase of 8 hours. a loan-- rithmic phase during the next 52 hours. a primary decline phase ’ over the following 60 hours. a stationary phase of 72 hours. and a secondary decline phase of #8 hours. The optinun incubation temperature ns 37°C at pH 7.0. The naxinun yield of virus was obtained fron cultures containing 200 - 600 ng of chorioallantoic neabrane. The chorioallantoic nenbrane cultures incubated at 37% for 21. to 81 hours prior to inoculation yielded the highest concentration of virus. The cultures inoculated immediately after preparation or after 105 hours' incubation had a lower yield. The yield was lowest from the culture inc‘ubated more than in» hours. Viral infectivity decreased at the following rates: Log 11350 0.113 per week at -25°0, and log 1050 0.63 per hour at 37%. At 15%, during the first 30 ninutes the rate was Log 11,50 0.15h- per ninutes followed by Log 1350 0.0053 per minutes for the subsequent 53 5h 90 ninutes. 7. The activation energ for inactivation of the virus was 19.!» x 103 calories per mole. 8. fractionation of 137 for isolation of ribonucleic acid failed to reveal the infectivity portion of the virus. 5. 6. 7. 8. 9. 10. ll. 12. 31311100355! Acker'nann. I. I. Concerning the relation of the Krebs cycle to virus propagation. J. Biol. Chem. £83. (1951): l$214128. Ackernann. I. I. and B. B. Johnson. Sons enery relations in a host-virus systu. J. lxptl. Med” 21. (1953): 315-322. Acke'rnann. I. I..and H. l‘. leased. Growth characteristics of influenza virus: The influence of a sulfonic acid. J. lxptl. 11.4.. 29.. (195“): 105-117. Lckernann. I. l.. and E. I. Haassab. Growth characteristics of influensa virus: Biochenieal differentiation of stages of develop-eat. J. lxptl. lied” m. (1951:): 329-339. notes-noon. w. w.. n. 1. Issues. and I. Ishida. Growth character-4 istics of influensa virus concerning the binding of virus by host cells. J. prtl. 11.0.. 19;. (195“): 595-55”. Ackerunn. I. I.. and B. 1'. Haaseab. Growth characteristics of influensa virus: Biochenical differentiation of stages of develop-eat 11..- J. lxptl. 11.0.. 193. (1955): 393—1102. Alexander. 1!. 3.. G. Koch. I. It. lountain. and O. Vanna-e. In- fectivity of ribonucleic acid fron polio virus in human cell aonolayers. J. sxpn. 11-0.. 129,. (1958): 183-506. Bachrach. B. I... 3. S. Breese. J. J. callis. I. 3. Hess. and R. I. Patty. Inactivation of foot and south disease virus by pH and temperature changes and fornaldehyde. troc. See. In. Biol. 11.4.. 21. (1957): its-2.152. Beaudette. r. 11.. .io 0. 19. Hudson. Cultivation of the virus of infectious bronchitis. J. h. Vet. lied. Assn" 29. (1937): 51.00. Bourdillon. J. Heat inactivation of nurine strain of SI polio- nyelitis virus. Arch. Biochea" 1. (1914»): 299—305. Brandt. 0. D. Inclusion body fornation with lewcastle disease and snaps virus in cultures of chicken enbryo cells. Virology. 5.. (1958): 177-191. Broadfoot D. 1.. B. 8 Poneroy. and I. I. Snith. lffects of infectious'bronchitis on egg production. J. her. Vet. Med. Ann... .121. (1950). 128-130. 55 i. iils )l’...’ ll. st'i' . .II : I 13s lb. 15. lb. 17. 18. 19. 20. 21. 22. 23. 2a. 50 Broadfoot. D. 1.. B. 8. Poneroy. and I. ll. Saith. lffects of infectious bronchitis in baby chicks. Poultry Sci... 15,. (1956): 757-762. Brown, 1.. R. 1'. Sellers, and D. 1.. Stewart. Infectivity of ribo- nucleic acid fron nice and tissue culture infected with the virus of l‘oot—end—mouth disease. Nature. _1_8_§. (1958): 535—536- Duthala. D. 1.. and J. lathews. Use of cellular cultures of chicken enbryo kidney tissue in virus studies. Cornell Vet.. 51. (195?): 118—159. Choniak, T. 11., R. I. Luginbuhl. and I. 1.. Jungherr. The propa- gation and cytopathcgenic effect of an egg—adapted strain of infectious bronchitis virus in tissue culture. Avian Disease; 2.. (1958): “56-“65. Colter. J. 3., and a. a. Bird. Infectivity of ribonucleic acid fron lhrlich ascites tuner cells infected with Henge encepha- litis. semi-s. 129. (1957): 859-800. Oolville. J. 11.. J. H. Dunbar. and H. R. Iorgan. Temperature and host cell factors in the growth of influensa virus (Pl 8 '81:“) in avian tissue in m. J. lmnunol.. 19. (1955): 2 269. Cunninshu. 0. H» A 2119.29.22.11 921.4: 13.11.3122- 3rd edition. Burgess Publishing 0a.. Minneapolis. Minnesota. (1957): 55. Cunninghan. 0. 8.. and 1. 8. ll Dardiry. Distribution of the virus of infectious bronchitis of chickens in enbryonated chicken eggs. Cornell Vet.. 3g. (19%): 381-388. Cunninghan. 0. 8.. and I. H. Jones. The effect of different routes of inoculation on the adaptation of infectious bronchitis virus to eabryonating chicken eggs. Proc. An. Vet. lied. Assoc" (1953): 337-3112. Daniels. J. 3.. II. D. Dates. and It. 1. Perry. lffect of glucose on the growth of influensa virus in deenbryonated eggs and tissue cultures. J. Innunol.. 99. (1952): 321-329. Delaplane. J. P. Technique for the isolation of infectious bronchitis or Newcastle virus including observations on the use of streptonycin in overconing bacterial contaninants. Proc. Nineteenth Ann. Pullorun Dis. 0onf.. (191:7). DCI‘pluC, Jo Pep “d He 0e Stuart. Eh. Md1f1abion Of infectious bronchitis virus of chickens as the result of propagation in embryonated chicken eggs. Rhode Island Agric. lxper. Sta. hill" £83. (19%): 1—20. 25. 26. 27. 28. 29. 30. 31. 32. 33. 31:. 35. 36. 37. 5‘? Dunhan. I. B.. and F. M. Dwing. Propagation of polio virus in chick embryo cell cultures. 1. cultivation of 3 virus types. Proc. Soc. pr. Biol. Died" 25. (1957): 637-639. lagle, 3.. A. l‘reenan. and I. Levy. Amino acid requirements of . monkey kidney cells first culture passage. J. Dxp. led" .122. (1958): baa-052. Baton. l. D.. and A. B. Scala. Reversible effect of hypotonic solutions on growth of influensa virus in tissue cultures. Proc. Soc. Exp. Biol. led" 93,. (1956): 289-297. Baton. 1:. D.. 1.. T. Adler. P. Bond. and A. R. Scala. The effect of thyroxin on respiration and virus growth in chick embryo tissue cultures. J. lnfect. 111... 2Q. (1950): 239.2%. Baton. M. D.. L. T. Adler. and I. 1. Perry. Virus growth and cellular. energ production: Effect of substances chenically related .to thyroxin on influensa virus. Proc. Soc. Exp. Biol. wsd.. .83.. (1953): 57-60. fabricant. J.. and P. P. Devine. The persistence of infectious bronchitis virus in eggs and tracheal exudates of infected chicks. Cornell Yet.. 3;. (1951): 2110-296. rahey. J. D.. and J. r. Crawley. Pr0pagation of infectious bronchitis virus in tissue culture. Canad. J. Micron _2_. (1956): 503-510. . Barnhnn. A. D.. and A. A. lewton. The effect of some environ- nental factors on herpes virus in Bela cells. Virology. 1. (1959): uu9.uoi. Perguson. G. 3. Growth of infectious bronchitis virus in sus- pended chorioallantoic nenbranss. ll. 8. Thesis. (1958). lichigan State University. Pranklin. I. l.. I. Vecker. and 0. Henry. Sons properties of an infectious ribonucleic acid from nouse encephaloayelitis virus. Virology. z. (1959): 220—235. Pulton. D.. and P. Arnitage. Surviving tissue s ension for influensa virus titration. J. 375. (Ga-bridge . g. (1951): 297-262. Gierer. A.. and G. Schrann. Die lnfektiositlt der lucleinslure aus Tabakonosaikvirus. 2. laturforsch” l}, 2,. (1956): 138-th. Banks. J. L. and B. I. Vallace. Relation of oqgen and teaperature in the preservation of tissues by refrigeration. Proc. Soc. Exp. Biol. led” 2;. (19(59): 196-200. 53 38. Hitchner. 8. B. and P. White. Growth-curve studies of chick embryo-propagated infectious bronchitis virus. Poultry Sci.. 21. (1955): 590-59u. 39. Holland. J. J.. and 1.. C. Is laren. The mamalian cell-virus relationship. 11. Adsorption. reception. and eclipse of poliovirus by Es1.a cells. J. In. Red" 1.22. (1959): 1487-5015. 1:0. Hofstad. I. 8. Stability of avian infectious bronchitis virus at 56° 0. Cornell Vet.. 29. (1950): 87-89. 1:1. lshida. D.. and I. I. Acksrnann. Growth characteristics ‘of in- fluensa virus: Properties of the initial cell-virus complex. J. lxptl. 11.1.. 112;. (1956): 501.515. 1:2. taper. J. l.. and B. 1.. Steers. Infectivity of tobacco ringspot virus nucleic acid preparations. Virology. 1. (1959): 127- 139. #3. Levine. A. 8.. P. a. Bond, 1. 1:. Scale. and 1:. n. Baton. 'Studiss on the relationship of potassium to influensa virus multipli- cation. J. 1anuno1.. 18, (1956): 386-392. M. Ioenis. 1.. D.. C. B. Cunninghan. I. 1.. Gray. and P. Thorp. Jr. Pathology of the chicken enbryo infected with infectious bronchitis virus. An. J. Vet. Bes.. 1;. (1950): 205-251. 1:5. Dwoff. A.. B. Dulbecce. I. Vogt. and I. Lweff. Kinetics ofithe release of polio-yelitis virus from cells. Virology. ;. (1955): 128.139. ho. charen. 1.. 0.. J. J. Holland. and J. T. Syverton. The nanmalian cell-virus relationship. 1. Attachment of poliovirus to cultivated cells of priests and non-priests origin. J. prtl. n.o.. 122. (1959): n75-hes. 1:7. lagata. 1., r. flatsumoto. x. Ohashi. 1. Iariya. and s. Istanabe. Studies on the mechanism sf influensa and Newcastle disease virus multiplication by tissue culture. lagoya J. lied. Science (Japan), 12. (195“): 67-73. 1:8. Page. 0. A. A study of the serun neutralisation test for infectious bronchitis sf chickens. Ph. D. Thesis. Hichigan State University. 195“. 1:9. Reed. 1.. J .. and H. fluench. A simple method of estinating fifty per cent endpeints. An. J. Hyg.. 22. (1938): ‘0934197. 50. Bivsrs. T. l.. and T. 1.. Borsfall. Jr. Viral £51 Bickettsial Infectigng g; 1‘13. 3rd edition; J. B. Lippincott Co.. Philadelphia. 1959). 51. 52. 53. 5“. 55- 56s 57. 53. 59. 60. 61. 62. 63 . 59 Robins. P. C.. and J. 1'. Indore. Tissue culture techniques in the study of animal viruses. An. J. wed. Sci.. 22.3. (1950): 316- 333- Sato. T.. T. Cugimori. S. Ishii. and I. flatsumoto. Infectious bronchitis of chickens in Japan.. 11. Identification of the causative agent as the virus of infectious bronchitis of chickens. Japan. J. lxptl. led.. _2_§. (1955): 1113-150. Schlfer. I. Sons aspects of aninal virus multiplication. “Perspectives in Virology“ by Pollard. John Wiley and Sons. Inc. 1:. r. (1959): pp. 21.2.2. Schalk. A. T.. and ll. 0. Haws. An apparently new respiratory disease of baby chicks. J. Am. Vet. lied. Assn.. 18. (1931): 1:13—1:22. Scott. 0. D.. K. P. Hansen. and 0. A. Brandly. A simplified procedure for the in m propagation of Kowcastlo disease virus. An. Vet. Med. Assn. Prec.. (1953): 312-315. 8ovoian. I.. and P. P. Levine. Effects of infectious bronchitis on the reproduction tracts. egg production and egg quality of luing chickens. Avian Dis.. 1.. (1957): 136-16“. Singh. 1. P. Thornal inactivation of infectious bronchitis virus infectivity at 56' c. n. s. Thesis. nichigen State University. 1957. Siser. 1. V. Teaperaturo activation and inactivation of the crystaline catalaso-hydregon peroxide systsa. J. Biol. Chen.. l2: (1%)! “bl-“73. Tenn. 1.. and D. A. J. Tyrrell. Influonsa virus nultiplication in the chorioallantoic nenbrano in vitre: Kinetic aspects of inhibition by 5. 6—Dichloro—l-B-D-Bibofuranosyl-bonsinidasole. J. 1:911. led” _1_0__9_. (195“): 591-562. van Booyen. C. I. A revision of Holmes's classification of aninal viruses. Subordor 111 (Zoophagineao). Canad. J. Micron _1_, (1951:): 227-2h8. Vecker. I. The extraction of infectious virus nucleic acid with hot-phenol. Virology. z. (1959): 21.1-21.3. Vscker. D.. and V. Schlfsr. lino infoktidso Kemponento von Bibonukleinsluro-Charoktor aus don Virus dor amorikanischen Pferde-lncephalonyolitis (type 0st). 2. Katurforsch.. 12; 2. (1957): “15-1117. Teller. T. 3.. and J. 1'. Indore. Production of henagglutinin by mumps and influensa A virus in suspended cell tissue cultures. Proc. Soc. pr. Bio1.. I. T.. 92. (19148): 1214-128. 65. 66. 67. 00 bright. B. 8.. and B. P. Sagik. Plaque fornation in monolayers of chicken sabryo kidney cells. Virology. 1. (1958): 573—57“. Zuschok. T.. K. P. Hanson. and C. A. Brandly. Factors influencing the growth of Kowcastle disease virus in isolated chorioallan- toic meabranos. An. J. Yet. no... 12. (1958): 191.195. Zucchek. T.. B. P. Hanson. and 0. A. Brandly. The influence of antimotabolites on the growth of lowcastlo disease virus in chorioallantoic membranes suspended in Tyrodo's solution. An. J. Vet. nee.. 12, (1958): 10014-1009. Zuschek. T.. B. P. Hanson. and 0. A. Brandly. Influence of tea- perature on the growth of lewcastle disease virus in chorio- allantoic monbranes suspended in Tyrodo's solution. An. J. vet. Bos.. go. (1959): 111-115. u. CT'._- _ n u