GROWTH OF INFECTIOUS BRONCHITIS VIRUS IN SUSPENDED CHORIOALLANTOIC MEMBRANES The:isforfhoDoonM.S. MICHIGAN STATE UNIVERSITY George Howarci Ferguson 1958 THESIS GROWTH OF INFECTIOUS BRONCHITIS VIRUS IN SUSPENDED CHORIOALLANTOIC PEI-{BEAMS By GEORGE HOWARD FERGUSON A THESIS Submitted to the College of Science and Arts of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Microbiology and Public Health 1958 This work is respectfully dedicated to MY FAMILY ACKNOWIEDGMEMS I wish to extend my appreciation to Dr. Charles H. Cunningham, Professor of Microbiology and Public Health, for his thoughtful understanding, patience, constant stimulation, instruction and guidance throughout the investigation and preparation of this manuscript. My appreciation is also extended.to Dr» L. C. Ferguson, Head of the Department of Microbiology and Public Health, for his constructive criticism of this manuscript. Sincere thanks are expressed to Mrs. M. P. Spring and Mrs. D. W. Church for their constant cooperation and consider- ation. TABLE OF CONTENTS PAGE INTRODUCTION......... ..... 1 LITERATUREREVIEW.................. 2 mmmmmmmonsunuu.........13 RESULTS.......................l6 DISCUSSION . . . . . . . . . . . . . . . . . . . . . . 27 3mm:.......................35 BIBLIOGRAPHY.....................37 LIST OF TABLES TABLE PAGE I. The Effect of Viral Growth on the pH of Hank's BSSwithCAM................... 20 II. First Passage Comparison of Growth of IBV in Whole and Minced CAM Suspended in Hank's 388 in Terms ofLogIDSO/O.lcc................ 21 III. The Effect of Hank's B83 and Certain Ion and Glucose Deficiencies in the Cultivation of IBV in Minced CAM in Terms of Log IDSO/OJ cc . . . . 22 LIST OF FIGURES FIGURE PAGE 1. Growth Curves of IBV in Whole and Minced CAM Suspended in Hank's B53 in the lat passage . . . . . 23 2. Growth Curves of the 6th Passage of IBV in CAM Suspended in Hank's 385 and Certain Ion.and Glucose Deficiencies . . . . . . . . . . . . . . . 2h 3._ Growth Curves of'the 9th Passage of IBV in CAM Suspended in Hank's 888 and Certain Ion and Glucose Deficiencies . . . . . . . . . . . ... . . 25 h. Growth Curves of the 12th Passage of IBV in CAM Suspended in Hank's BSS and Certain Ion and Glucose Deficiencies . . . . . . . . . . . . . . . 26 INTRODUCTION The refinement and expansion of tissue culture methods in virology have led to the discovery of heretofore unrecognizable viruses. The embryonating chicken egg affords an excellent medium for the cultivation of viruses and study of the host-parasite relation- ship, but the complexity of developing tissues, organs and systems does not permit a precise evaluation of a virus-cell interaction which my be accaplished better through cell culture. The cultivation of infectious bronchitis virus (IBV) on chorioallantoic membranes (CAM) suspended in a medium permits the study of: (l) the metabolism of tissue without the complication of the egg; (2) viral multiplication in a single tissue and (3) a medium free of embryonic urates and inhibitors. With the increasing knowledge of the action of different viruses in specific tissues, the compiling of information on definite viruses is becoming essential. h‘_ _‘.—_-—. i 4 LITERATURE REVIEW Infectious bronchitis (IB) is of particular economic importance in adult laying flocks, although chickens of all ages may be infected. The course of the disease is usually one to two weeks, but in some instances it may persist for as long as h to 6 weeks. Egg production will fall off along with softened and irregular egg shells.31 After infection of adult birds long periods of time are required before egg production returns to normal. In young chicks the most characteristic symptoms are nasal discharge, gasping, rales, and coughing.31 Tagpgia ‘pglliyb is the proposed name of the etiologic agent. The virus can be isolated from tissue or exudate from the re- spiratory tract throughout the respiratory phase of the disease, and is estimated to be from.65 to 135 mu in size.31’ 38 Domermuth and Edwards16 stated that by electron microscopy of infected CAM’the virus appeared to be 200 an in diameter, and was found separately, in pairs, in chains or in clumps of varying sizes. Cultivation of IBV has been done in embryonating chicken eggs 'where initially it is not lethal, but develops this property with subsequent passages according to Loomis gt al.36 Early embryo passage virus produces certain characteristic gross pathological lesions of the embryo consisting of: (l) CAM adherent to shell membrane and thinner than normal; (2) amnionic membrane thickened, dry, fibrotic, resisting removal from embryo,and restricted.movement of the embryo; (3) living infected embryos sluggish and weak; (h) embryos dwarfed as much as one-half size and having a firm ball-like shape characterized by curling with a wry neck and feet deformed and compressed over head and (5) feather development immature and dry. As embryo lethality increases with egg passages, host infectivity decreases.31’ 15 The allantoic cavity has been cited as the most de- sirable route of inoculation for primary isolation of IBV due to the appearance of pathological lesions in the first passage and.the more simple inoculation.36 This route is the easiest from a technical stand- point. Cunningham and 21 Dardiryn found that following the allantoic cavity route of inoculation the greatest concentration of the virus was in the CiM followed in decreasing order in the allantoic fluid, amnionic fluid and liver. Yolk material was innocuous. Hitchner and'White30 found the maximum titer of the virus in embryos to be at the 2hth to 30th hour after inoculation via the allantoic cavity. Cultivation of IBV in tissue culture has proved to be very rare. Only two articleslo’ 19 on tissue culture of this virus have been reported and both of them in the last two years. Buthala and Mathewslo studied the use of the chicken embryo kidney as a medium fer growth of various viruses. A strain of IBV which was in the 170th passage in chicken embryos was used. Trypsinized embryonic kidney cells, 1:200 dilution, were cultivated in a nutrient medium consisting of 0.5% lactalbumen enzyme hydrolysate, 2% calf serum and 97.5% Hank's balanced salt solution (BSS) at pH 7.8 failed to support growth of virus. Cytopathogenic effects were not observed. The Beaudette and Connaught strains of IBV were cultivated in whole CAM suspended in medium 597 (medium 199 minus purines and pyri- midines plus Hank's 383) by Fahey and Crawley.19 Maximum infectivity, infectious dose 50 (ID50) per'ml, as determined by chicken embryo inoculation was demonstrated between hB to 5b hours with both strains. In initial serial passages, harvests were made at hB hour intervals and high titers were observed after the third passage. The latent period for the Beaudette strain occurred over a period of from 12 to 18 hours post inoculation while the Connaught strain required as long as 20 to 26 hours. There were no changes in the pH of the control or infected cultures which indicated that cellular metabolism.did not occur. Both strains were grown in monkey kidney and chicken embryo heart cells. The Beaudette strain grew very little in whole embryo and chick liver cells, whereas, the Counaught strain could not be propagated in either. ‘An attempt was made to grow both strains inanrle's‘L strain cells, but growth could not be demonstrated in.either case. Ackermannl found that when lh-to 15-day old cm suspended in Simmls solution was infected cellular respiration remained approximate- ly the same. Sodium malonate 0.02 to 0.06 M exhibited.no inhibitory effect on the uptake of glucose by the membrane but showed a partial inhibition of’endogenous respiration of the tissue. Amounts of this compound that reduce oxygen uptake inhibit viral propagation,yet it is not virucidal. variations in oxygen tension influence the rate of virus propagation indicating aerobic oxidative processes taking place. The action of malonate indicates at least one reaction is a metabolic step in the Kreb's cycle. Succinic dehydrogenase is the enzyme 5 affected in this reaction and without the continuous supply of di- and tricarboxylic acids, the blocking of the whole cycle will result. Ackermann and Johnson2 concluded that the energy derived or required for viral synthesis from 200 mgm. of CAM in Simm's solution was obtained from the oxidative phosphorylation activity of the host tissue. Ackermann and Haassab3’ 1" 5 reported that sulfonic acid, p- fluorophenylalanine , methoxinine and alpha-amino-alpha -p-mthoxyphenyl— methanesulfonic acid inhibited the growth of influenza virus in whole CAM and Simm's solution. ’ Ackermann _e_t elf, and Ishida and Ackermann32 demonstrated the binding of influenza virus to the CAM suspended in a modified Sinun's solution and concluded that binding is of two types: ( 1) sensitivity to the receptor destroying enzyme action and (2) sensitivity to the blocking effect of alpha-p-methoxyphenylnethanesulfonic acid. Colville gt $.11 grew influenza virus on 11- and 19- day old CAM, lung, heart, liver, gut, skin and muscle. The best growth was demonstrated in lung and cm. The cm culture also grew equally well at 37 and 141 C. Bernkopfe cultivated influenza virus in 1h- to 15- day old on: of deembryonated eggs . The embryo was removed leaving the CAM intact along the wall of the shell. Tyrode's solution was placed in the shell, inoculated with virus and covered with sterile rubber caps. According to Daniels gt al.1h virus production was minimal in glucose deficient undefined balanced salt solution when employed with the CAM adherent to the shell of deembryonated eggs. When glucose was 6 added virus production was stimulated. Reduced oxygen consumption, 60 to 70 per cent of the control system, was characteristic of the glucose deficient medium. According to Levine gt al.33’3h adsorption of influenza virus to the can of 10- to 11- day old embryos in modified Hank's 1388 was not found to be affected by the deficiency of potassium ions in the medium. Respiration of the cells was decreased by 10 to 30 per cent. When the virus was grown in potassium deficient media there was a suppresion of : (1) growth of the CAM epithelium and fibroblasts, (2) multiplication of virus, and (3) respiration by 10 per cent. When potassium was added to the system the results were reversed. The lack of potassium also affected the accessibility of the virus attached to tissue to neutrali- zation by immme serum and also intracellular viral synthesis. Shulls and Rightsw" demonstrated the effect of influenza virus on the free amino acid pools of the CAM suspended in 1 part glucosol solution of Fulton and Armitage and 1 part phosphate buffer solution. Using membranes from lb- to 15- day old embryos it was shown after 17 hours of infection that slightly higher amounts of alanine , proline , glycine and glutamic acids were detected in the tissue. The Roakin strain of Newcastle disease virus was grown in iso- lated CAM membrane preparations maintained in Tyrode's solution by Scott 33 2;.” The strain was cultured through 30 serial passages and it was concluded that multiplication of the virus occurred because: (1) a significant increase in the quantity of virus inoculated occurred within a single culture and ( 2) the material from the last of a series of preparations contained the virus and represented a dilution far in excess of the original titer. 28’ 29 studied Pinter g_t_ 3;,” Liu ,5 3.1.95 and Henle gt g_l_., the deembryonating techniques prior to and after infection of CAH'with influenza virus using a mixture of modified glucosol and phosphate buffer solution. Phosphate buffered saline, Tyrode's solution, 10 per cent chicken serum in saline, normal allantoic fluid or even physio- logical salt solution gave similar results. Deembryonation after infection gave the better and more reproducible results. Release of the virus from the GAH’occurred.at constant rates for periods of 30 to 36 hours before yields decreased rapidly. In subsequent work, they hypothesized that hemagglutinins are produced in allantoic membranes. A greater concentration of non-infectious hemagglutinins were present in the GAMIthan in the suspending fluid and more infectious virus particles were in the fluid than in the tissue. In further work it was found non-infectious hemagglutinins were not released.from.the CAM following infection. .Addition of large amounts of seed virus after initial infection resulted in decreased infectious dose 50 (IDSO) per hemagglutinin ratios up to 2 to h hours. Virus added after this time had no influence. The distribution of external cell receptors played little, if any, role in non-infectious hemagglutinin production. A review of the multiplication of influenza virus in the ento- dermal cells of the allantois of the chicken embryo suspended in any 833 was conducted by Henle.27 The tissue remained viable and supported viral propagation for at least 2h hours. The medium was of no relative importance unless long incubation periods were used. In the case of long incubation periods glucose was essential for maximal titers. The virus could be controlled in such a manner that it would be permitted to undergo one cycle of multiplication and the time required for the first cycle extended over a period of 8 hours for influenza A and a period of 8 to 12 hours for influenza B virus. The conversion from incomplete virus to infectious virus was suggested as having been achieved by some limited material in the host cell. Multiplicity re- activation occurred when a cell adsorbed several inactivated virus particles and production and liberation of these particles by lysis occurred. Genetic recombination was suggested if host cells were infected with two different mutants . The offspring would contain not only the parent types but a combination of the two and the wild type, in which case all four types would retain their characteristics when infection of host tissue organ or cell with one virus prevented or partially inhibited simultaneous propagation of another viral agent. Zuschek _e_t 51.50" 51 studied factors that influenced the growth of Newcastle disease virus in CAM and Tyrode's solution. A deficiency of potassiun suppressed the growth of the virus. is the concentration of glucose. increased the amount of viral synthesis decreased. The mximml titer occurred when the medium was deficient in glucose. Haadsmm growth occurred in the medium with 0.1 g. of Mg Clz/liter, the usual amount present in Tyrode's solution. Concentrations of 0.2 g. calcium chloride per liter were necessary for maximal titers . Greater concentrations had an inhibitory effect on viral growth. Growth of the virus over a temperature range from 33 to 1:2 0 occurred best at 1:2 C. The growth of influenza virus was inhibited when cultured in CAM suspencbd in inorganic salts and glucose or pyruvate diluted 1 :2 or 1:1; according to Eaton and $0818.18 Restoration of the tenicity of the 888 with sodiun chloride as late as 1:2 hours post inoculation resulted in normal growth of the virus. A lzh dilution of 335 sustained normal multiplication with 0.11: M of glucose but the effect was not ob- served until 211 hours had elapsed. Gohd and Huang23 hypothesized that phosphate in excess in a CAM- BSS system led to depletion of intracellular calcium, thereby denying the cells a necessary substance for the synthesis of virus. This was demonstrated by allowing influenza virus to be adsorbed and penetration to occur before removing the tissue and placing it in phosphate buffer. Viral multiplication did not occur. The addition of calcium to the same culture permitted production of virus. Studies by Burr gt _a__1_.9 on the propagation of influenza virus in CAM in Hank's-Simm's serum ultrafiltrate, Earle's 388 and Medium 199 employed 9, 11, 13 and 16-day old membranes. As the age of the membranes increased the yield of virus decreased. The best results were obtained with membranes 9-days old but due to the small amom'lt of tissue it was necessary to use 11-day old membranes. The different media yielded approximately the same amount of virus . Virus propa- gation occurred mainly at the expense of the intracellular material and was independent of the nature of the extracellular medium. Influenza virus could be grown equally well in healthy surviving cells, in actively growing cells or in cells depleted of intracellular nutrients and undergoing degeneration. 0011th observed influenza virus synthesis in CAM suspended in a suitable medium at 25, 30, and 35 C. Some growth was detected at 10 22.5 c. No growth occurred at 20 c, but the tissue retained the ability to synthesize virus at 25 C or higher. The adsorption and penetration phases occurred at lower temperatures but no free virus was found within the cells, which led to the conclusion that after penetration the virus went into a latent period. The latent periods were from 2 to 3 hours at 37 C and 3 weeks at 20 C. The differences in the periods seemd to be related to the nutrition of the cell. Schlesinger and Karr, ’ 1:2 using CAM and Tyrode's solution, concluded that the maintainance or restoration of a normal supply of mucoprotein substrate is a function of the ability of cells of the allantois to maintain homostatic conditions under stress. In another report, they concluded that virus multiplied in cycles rather than continuously. In a study of the pyruvate metabolism of CAM infected with influenza virus, Wielgoszh8 used modified Simm's solution and found that pyruvate uptake was increased by infecting the CAM with the virus as compared with controls. Green and Beard26 conducted experiments to determine the enzyme properties associated with the dephosphorylation of adenosine tri- phosphate. They found that potassium, sodium, calcium and magnesium activated the enzyme at approximately the same rates, but sodiun proved slightly better than potassium. The enzyme was enhanced by these four ions acting alone or together. The divalent ions, separately or together, in the presence of either or both of the monovalent ions, strongly activated the reaction. The pH optimum was 7.16. Work conducted by AckermannT showed that the oxidative activity 11 of the mitochondria of the CAM was very low but the glycolytic activity was substantial. He reported that the endogenous supply of glucose and other essential nitrogenous requirements needed for influenza virus was completely adequate. He also fbund adenosine triphosphatase in minces of CAM. The CAM; which is highly vascular and constitutes the respiratory organ of the embryo, is formed from the fusion of the chorion and allantois. It reaches maximun growth at the 10th to 12th day of de- velopment and.a maximum weight of 2 gm.ho The CAthas proved to be an excellent medium for the abundant growth and culture of a large number of human and animal viruses.37 Inoculation of this membrane in embryonating chick eggs has been bene- ficial in the titration of viruses, isolation of viruses and for the production of vaccines. IBM has been found to multiply on the CAM but as yet has not been reported to produce specific lesions on it.10’ 19 Virus multiplication is said to be a cyclic phenomenon which consists of four phases: (1) the adsorption of viral particles to a susceptible host, (2) penetration of infecting particles into these cells, (3) synthesis of new viral particles, and (h) the release of new particles from benefactor cells. There are many factors that influence the growth of virus in embryonating chicken eggs. These are: (l) the age of the embryo; (2) the route of inoculation; (3) the volume and dilution of inoculm: (1:) the time and temperature of incubation and (S) the physiological and nutritional state. These same factors apparently would have the same degree of effect in tissue culture.22 .In the use of tissue culture there are many factors involving 12 homogeneity of the tissues used, to he considered. If the cells are heterogeneous one would expect that the virus eluted from these cells would not be eluted at the same time but over a period of time. If the cells are homogeneous, virus would be released at approximately the same time, over a period of time.7 Dulbeccol7 conducted work that showed in multicellular cultures the amount of virus released by single cells increased exponentially with time and that the number of cells releasing virus increased linear- ly with time. There are at least three different types of cells contained in the CAM'and it may be considered heterogeneous for two reasons: (1) because there are different types of cells and (2) cells of the same type may be in different physiological states.7 MATERIALS AND METHODS The Beaudette egg-adapted strain, North Central Infectious Bron- chitis Virus Repository, Code Number h2, was used. This strain is commonly used in serum neutralization tests and exhibits the ability to kill embryos within 36 hours post inoculation via the allantoic cavity. There are no records pertaining to the exact number of egg passages of this virus, but it has been through hundreds of passages. The passages for this study will be enumerated. The virus was passaged in eggs at the beginning of the experiment to establish a stock culture, and then used in the GAM'system where it was maintained in serial passage with Hank's BSS. Harvests of the infected fluid were collected and stored at ~30 C until used. Hanh's BSS contained the following ingredients on a grams per liter basis: 8.0 gm NaCl; 0.1. gm K01; 0.11; gm 01.012; 0.1 gm MgSOh-7H20; 0.1 gm.Mg012°6320; 0.12 gm NaZHP0h°12H20; 0.06 gm KHQPOh; 1.0 gm glucose; 0.02 gm phenol red; 0.35 gm‘NaHCO and 1 liter double dis- 3 tilled water. Modification of the amounts of the ingredients of this solution was made to enable the same amounts present in 20 ml to also/be present in 18.2 ml. This was accomplished by using the following formula: Example using NaCl 8.0 gm NaCl/liter 0.16 NaCl 1 NaCl 0.16" a 20ml 1787-3" "’ " fl” I I 0.17582 gm/20 m1 8.7910 gin/liter - the amount needed to enable 0.16 gm to be present in 18.2 ml. 11: Modification of the amounts of the ingredients of Hank's 383 on a grams per liter basis would then be: 8.7910 gm NaCl; 0.h395 gm K01; 0.1533 gm CaClz; 0.1099 gm HgSOho7H20; 0.1099 8m "8012-53203 0.1219 81'! NaZHPOh-IZH 0; 0.0659 on 10129014; 1.0990 glucose; 0.0220 gm phenol red; 0.3810 gm NaHC03 and 1 liter of double distilled water. Each ingredient of the 858 was prepared separately in a 101 concentration in 100 m1 of double distilled water. In this manner the complete 338 could be prepared or certain ingredients could be excluded for preparation of deficient media. No attempt was made to adjust the pH of the deficient media. The pH of the complete BSS was 7.3. The media were sterilized in an autoclave for 10 minutes at 10 lbs. pressure. Embryonating chicken eggs from a comercial, disease-free flock were incubated in a Jamesway model 252 incubator at 99-995 C. When the embryos were 10 to 11 days old they were candled. Tincture of metaphen was applied to the shell over the air cell. The shell over the air cell was cracked with forceps and removed. The shell membrane at the base of the air cell was peeled from the CAM. The CA)! was cut in a straight line with scissors from one side to the other and the endogenous mass, excluding the CAM, was emptied into a sterile petri dish. The CAM was then removed, washed in three 500 ml flasks each containing 200 m1 of physiological saline, minced into small fragments with scissors and placed in an 8 oz. prescription bottle containing 18.2 ml of Bank's BSS in which the total amount of the ingredients normally contained in 20 ml would be present in 18.2 ml. The membrane was then inoculated with 1 m1 of virus and 0.8 m1 of antibiotics which brought the total fluid in the bottle to 20 ml. The cultures were incubated at 15 37 C. At 12 hour intervals throughout a 60-hour period 1.5 m1 samples were removed and stored at -30 C until used for titrations. Infectivity titrations were performed using serial lO-fold dilution of virus and 5 eggs per dilution, 0.1 cc per egg, with inocu- 12 39 lation via the allantoic cavity. Calculation of infectivity was made by the method of Reed and Huench. When used for titration the samples were thawed and centrifuged lightly to sediment fragments of tissue which were present. Following inoculation the eggs were returned to the incubator and candled once a day. Embryo mortality during the first 21: hours was considered to be due to nonspecific causes and was not included in the final calculations. A post inoculation observation period of 6 days was used. Criteria for viral infectivity were: (1) embryo mortality and (2) gross pathological lesions . Embryo mortality occurred more frequently because this is an egg adapted strain which characteristically produces death. Gross pathological lesions consider- ed to be characteristic were balling, curling and dwarfing of the embryo to approximately one-half the size. These lesions were only observed at the limiting dilutions and, therefore, did not occur with regularity. The diluent used in all titrations was sterile nutrient broth. Bacterial sterility of all materials was determined by inoculating thioglycollate broth. RESULTS There was no special emphasis on pH during the period of the experiment, but some observations were made concerning the relationship between the control and infected cultures at 0 and 60 hours.21 The pH of Hank's solution was 7.68 but after the addition of antibiotics, membranes and virus the pH decreased approximately 0.3 units. The pH of the controls decreased about 0.2 units after 60 hours in contrast to the infected cultures in which there were slight fluctuations above or below the initial pH value (Table I). The hydrogen ion concentration of deficient media and.c0mp1ete media differed, but no attempt was made to adjust to any definite value. Previous work by'Springhs showed that IBV yielded higher titers when propagated in minced CAM than in whole CAM. The differences were most marked after several serial passages of the virus. To corroborate these findings growth curves of IBV in the lat CAM passage were con- ducted using whole CAM'and minced CAM with Hank's BSS. As shown in Table II and.Figure 1, virus was rapidly adsorbed during the lat 2h hours with both preparations but with the whole CAM there was a con- tinous decline of infectivity of the suspending medium for the next h8 hours. ‘Nith minced GAM,virus was released from the cells between the 12th.and 2hth hours. The titer remained fairly stationary from this time until the 60th hour but did not yield the original titer of 7.5. These results were similar to those obtained by SpringhE and indicated a slight advantage of using minced CAM as compared to whole CAM. 17 Through serial passage of the virus in minced CAM the pattern of growth demonstrated adsorption of virus to the cells within 2h hours as indicated by the precipitous decline of titer. Release of virus oc- curred as shown by the continuous increase in infectivity up to the 60th hour at which time the titer was as high as or higher than that of the seed.virus. This is evident when comparing the growth curves of the 6th, 9th and 12th passages where the periods of adsorption of virus and penetration into cells with subsequent liberation into the sus- pending medium are essentially the same for certain time periods (Table III, Figures 2, 3 and h). 'With 6th passage virus the typical cycle of infection occurred when Hank's BSS was used. At the 60th hour the titer of the virus was essentially the same as that of the seed virus used to initiate in- fection. The rate of adsorption of virus with calcium, magnesium and glucose deficient medium was the same as with the complete medium, but after the 12th hour release of virus from cells could not be detected by infectivity tests of the medium. Adsorption occurred with the potassium deficient medium but was not complete until 2h hours. This was followed by evidence of release of the virus from the 2hth through the 60th hour (Table III, Figure 2). Viral infectivity with the 9th passage virus of the deficient media followed essentially the same growth curve patterns, but at different numerical levels. There were lag, log, and decline phases with the exception of the potassium deficient medium. In this case, there was a stationary phase after the log phase rather than a definite decline phase. Viral infectivity of the glucose deficient medium 18 showed that the lag phase was at a slower rate than with the other media. iMaximum infectivity, 2.8, of calcium deficient medium occurred at h8 hours and was 0.2 units less than the seed virus. Glucose and .magnesium deficient media had a maximum infectivity at 36 hours. 'With glucose this was 0.h units higher than the seed virus, but that con- taining magnesium was the same as the seed virus. Viral infectivity of the potassium deficient medium showed an infectivity titer peak, 2.h, at 60 hours, but it was essentially at a stationary phase of 0.6 units below the initial titer of the seed virus. “With Hank's BSS lag and log phases only were detected. The rate of the log phase was most promi- nent from the 12th through the 36th hours followed by a slower rate to the termination of the experiment at the 60th hour. ‘At the 60th hour the titer of the virus was 2.1 units higher than the seed virus (Table III, Figure 3). [With 12th passage virus, infectivity of glucose and potassium deficient media followed essentially the same growth curves with the highest titer occurring at the h8th hour along with magnesium and calcium. There was a sharp decline for these media by the 60th hour. In.the log phase at the 2hth and 36th hours the numerical value of infectivities was the same for all deficient media. All deficient media exhibited a viral log growth phase extending over a period of 36 hours. Maximum infectivities of the deficient media were all above the value of the seed virus, potassium 0.2, glucose 0.3, calcium 0.7 and.magnesium 0.9 units. The maximum titer with Hank's BSS medium occurred at 60 hours but a stationary phase apparently began.at the 36th hour. Viral infectivity had a net increase of 2.1 units in Hank's BSS which was the same as with the 9th passage virus. 19 20 TABLE I THE EFFECT OF VIRAL GROWTH ON THE pH 0F HANK'S 883 WITH CAM Culture at Culture at Culture at Serial 0 time 60 hours 60 hours Control passage pH pH PH PH 8 7.35 7.27 7.37 - 9 7.35 7.30 70ho 7.17 10 7.35 7.35 7.h5 7.13 11 7.30 7.31 7.35 6.97 TABLE II lST PASSAGE COMPARISON or GROWTH OF IBV IN WHOLE AND MINCED CAM SUSPENDED IN HANK'S ass IN mus 0F 1.00 Ego/0.1 cc CAM A TIME IN HOIBS '61 12 2h 36 178‘ 60" “11019 705 205 200 107 008 103 Minced 7.5 1.0 202 200 107 105 21 22 TABLE III THE EFFECT OF HANK'S BSS AND CERTAIN ION AND GLUCOSE DEFICIENCIES IN THE CULTIVATION 0F IBV IN MINCED CAM IN TERMS OF LOG ID50/0.1 cc Time in Hours lst Passage Hank's 335 7.5 1.0 2.2 2.0 1.7 1.5 6th Passage Hank‘s $8 hell 0 205 305 308 he? Deficient of calcium h.h 0 O 0 0 0 glucose h.h 0 0 0 0 0 magnesium hell 0 0 0 0 0 potassium h.h 0.6 0 0.7 0.3 0.6 9th Passage Hank's BSS 3.0 0 1.8 17.0 h.6 5.1 Deficient of calcium 3.0 0.3 0.7 2.2 2.8 2.0 glucose 3.0 1.6 1.6 3.h 2.7 2.2 magnesium 3.0 0.h 1.0 3.0 2.h 2.0 potassium 3.0 0.6 10. 2.2 2.2 2.h 12th Passage Hank's 388 3.5 1.8 14.3 5.h 5.5 5.6 Deficient of calcium 3.5 l.h 2.5 3.5 h.2 2.0 glucose 3.5 0.5 2.5 3.5 3.8 3.0 magnesium 3.5 2.0 2.5 3.5 h.h 3.h potassium 3.5 0.5 2.5 3. 3.7 2.5 23 me on ma undo: 5“ 05.2. Ill-Ill 25 36:3 50 prong (1.8'80 a. In"... gl ’1.-. ‘ Bamfim 99.. 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