— — —— .— —— —— — —- .— —— —— —— —— — — — 145 876 THSF $TUDIES OF 7H5 DESSOCCATlON 0F ENFECTEOUS BRONCHETIS WRUS-ANTEBODY CGMPflEX Thaw: 5% HM DW 05 M. S. mcmm STATE WIVEESITY Mark F. Stinski 1966 LIBRARY Michigan State University STUDIES OF THE DISSOCIATION OF INFECTIOUS BRONCHITIS VIRUS-ANTIBODY COMPLEX By Mark F. Stinski A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Microbiology and Public Health 1966 ACKNOWLEDGEMENTS My sincere thanks to Dr. Charles H. Cunningham, Professor of Microbiology and Public Health, for his guidance throughout the investigation and the preparation of this manuscript. I wish to express my appreciation to Mrs. Martha Spring and Mr. Robert B. Lacey, Department of Microbiology and Public Health, for their inspiration and advice. I am greatly indebted to Dr. Philipp Gerhardt, Chairman of the Department of Microbiology and Public Health, for his excellent research and academic leadership. This study was supported by the National Institutes of Health Grant AI 055u9 and the Michigan Agricultural Experiment Station. 11 TABLE OF CONTENTS Page INTRODUCT I ON C O O O O O O O O O O O O 0 0 O 0 0 0 O O 1 LITERATURE REVIEW. 0 O O O O O O O O O O O 0 O O O O O 2 Infectious Bronchitis Virus . . . . . . . . . . . 2 Neutralization Reaction . . . . . . . . . . . . . 3 Dissociation. . . . . . . . . . . . . . . . . . . 5 MMEBIALS MD MEPHODS. 0 O O O O O O 0 O O 0 O 0 O 0 O 8 Vims O O O O O O 0 O 0 O O O 0 O 0 0 O O o 0 O 0 8 Anti sera. O O O O 0 O 0 O O O 0 O O 0 O O O O O O 8 Immunoglobulin. . . . . . . . . . . . . . . . . . 9 Diluent S O O O O 0 O O O O 0 0 O 0 O O O O O O O 9 Determination of Protein. . . . . . . . . . . . . 9 cell culture 0 O O O O O O 0 O O O O O 0 O 0 O O 0 10 Plaque Assay. . . . . . . . . . . . . 10 Effect of low and high pH on IBV infectivity. . . 11 Kinetics of IBV Neutralization by Anti- IBV- Chi Cken 86mm 0 O 0 O 0 O O O O O O 0 O O O O 0 ll IBV-antibody complex formation. . . . . . . . 12 Effect of pH on the stability of IBV-antibody complex 0 O O O O O O O O O O O O O O O O 12 Effect of pH on neutralization potential of antiserum prior to combination with IBV . . . . 12 IBV-antibody complex in an antibody-free SuspenSi on O 0 O O O O O O O O 0 O O O O O O 13 Dissociation of IBV-antibody complex at pH 1. 9 and reassociation . o o . . . 13 Effect of pH on dissociation of IBV-antibody complex in an antibody-free suspension. . . . . 13 Antibody dissociated at pH 1. 7 and 2. 7 from IBV-antibOdy complex 0 o o o c o o o o 1.3 Effect of dilution on IBV-antibody complex. . . o 14 Effect of dilution on IBV-antibody complex in an antibody-free suspension . . . . . . . . . 14 Effect of pH on cell- -IBV-antibody complex . . . o 15 RESULTS. . . . . . . . . . . . . . . . o . o o . . . 0 16 DISCUSSION . . . . . . . . . . . . . . . o . . . . . o 34 SUMMARY. . . . . . . . . . . . . o . . . . . . . o . . 38 LITERATURE CITED . . . . . . . . . o . . . o . . o . . #0 iii 10. 11. LIST OF TABLES Effect of low and high pH on IBV infectivity . Effect of pH on stability of IBV-antibody complex. 0 O O O I O O O O O O O O O O O O 0 0 Effect of pH on neutralization potential of antiserum prior to combination with virus. . . Dissociation of IBV-antibody complex at pH 1.9 and reassociation. . . . . . . . . . . . . . . Effect of pH on dissociation of IBV-antibody complex in an antibody-free suspension . . . . Antibody dissociated at pH 1.7 and 2.7 from IBV'antibOdy complex 0 c o c o o o o o o o o 0 Effect of dilution of IBV-antibody complex I. Inactivated antiserum. . . . . . . . . Effect of dilution on IBV-antibody complex II. Noninactivated antiserum . . . . . . . Effect of incubation at u C on IBV-antibody complex in an antibody-free suspension . . . . Effect of dilution on IBV-antibody complex in an antibOdy'free 81181381181011. 0 o c o o o o c 0 Effect of pH on dissociation of IBV-antibody complex after adsorption to cells . . . . . . iv Page 16 21 22 27 28 29 30 31 32 33 Figure 1. LIST OF FIGURES Page Kinetics of IBV neutralization by anti-IBV- chicken serum . . . . . . . . . . . . . . . . . 18 Dissociation of IBV-antibody complex. . . . . . 19 Percent dissociation of IBV-antibody complex as a function of H+ ion concentration . . . . . 2O INTRODUCTION The mechanism of neutralization of animal viral infec- tivity by antibody has been intensively studied. There has been no general agreement on the stability of the virus- antibody complex. The present investigation is concerned with the pH stability of infectious bronchitis virus-antibody complex and the interaction of the complex with chicken embryo kidney cells. LITERATURE REVIEW Infectious_§;9nchitis yigug Infectious bronchitis virus (IBV), the causative agent of a respiratory disease of chickens (59), is 830 to 1,000 A9 in diameter (4, 49), contains ribonucleic acid (RNA) (1, 48), and has a lipoprotein coat with pear-shaped surface spikes (4, 49). Some of the known physical properties of the virus are anisoelectric point at pH 4.05, anoptimal stability of infectivity at pH 7.8 (12),a density of 1.15 to 1.19 in sucrose (9, 15), and a sedimentation coefficient of 334 S (15). Viral protein contains sulfhydryl components as deter- mined with p-chloromercuribenzoate (53). Direct hemagglutination is not a characteristic of the virus. A noninfectious hemagglutinin can be isolated by anion exchange diethylaminoethylcellulose chromatography (5). It is 600 to 700 AP in diameter and contains 51% protein, 34% lipid and trace amounts of carbohydrate and RNA. Infectious bronchitis virus is sensitive to ether (1, 48, 66) and sodium deoxycholate (66). The virus can be propagated in embryonating chicken eggs (12) and cell culture (16, 10, 11). The mode of replication has not been definitely established. Viral particles adsorb to the membrane of chicken embryo kidney cells (CEKC) within 2 3 30 minutes,are detectable in the nucleus within 7 hours (48) and in the cytOplasmic matrix within 12 hours (49). After 48 hours viral particles accumulate in the interstitial areas and are present on the surface of the cell membrane. Virus is released from the cell by budding and obtains an envelope from the cell membrane (49). The frequency distribution of plaques formed in CEKC by the virus are according to the Poisson distribution. There is a linear relationship between the number of plaque forming units and the relative concentration (11). Heating the virus at 100 C or treatment with ether releases antigens 1, 2, and 3. The antigens are noninfective and nonhemagglutinative (66). Neutralization Reaction Neutralization of virus by antibody is dependent on the concentration of antibody, temperature, pH, and ionic strength of the suspending medium (13, 44, 45). Viral survival, as a function of time, is a linear decrease over 99.9% of the reaction. The decrease is according to -dc/dt = Kc where K is a proportionality constant, and -dc/dt is the rate of change of the concentration of virus (0) with time (t) (44, 26). The K value may be used as a quantitative index of the effect of one variable on the virus-antibody reaction (13, 44, 45, 22). A kinetic curve for anti-influenza-rabbit or fowl serum at 0 C had a lag phase and a phase of rapid exponential inactivation followed by a slower rate which was a reflection u of the heterogeneity of the antibody used to inactivate the virus (38) or was due to a resistant fraction of virus (13, 44). At higher temperatures the lag phase was not pronounced (13, 57, 43). Nonneutralized adenovirus was coated with antibody or trapped by aggregates to an extent sufficient to change the density of the particle (36). Three different types of combination between antibody and virus were detectable by electron microscopy: antibody in high concentration reacts with the virus through one of its active sites and radiates from the surface; in low concen- tration of antibody each antibody attaches to a single virus through sites at the ends of the molecule; at intermediate concentration of antibody, antibody molecules form bridges between adjacent viruses and cause aggregation (40). Electron microscopy provides no simple explanation for the interaction of neutralized virus with the host cell. Viral infectivity of a neutralization mixture is influenced by the host system employed (67, 34, 6, 38, 69). Lafferty (39) has hypothesized that neutralization of a virus by any single antibody molecule depends on the electrostatic nature of the host cell surface. Antibody molecules,XG, are cylindrical, 250 A0 X 30 to 40 A0 (23), and the active site is at the end (23, 2, 40). Antibody molecules to a single antigen may differ in molecular weight, electrophoretic mobility, and N-terminal amino acids (70). Dissociation Burnet et a1. (8) concluded that the reaction of animal viruses with antibody was freely reversible. Fazekas de St Groth and Webster (19) substantiated this using noninacti- vated serum with influenza. Dulbecco et a1. (13), Mandel (43), Kjellen (35). Lafferty (38), and Granoff (22) reported that neutralization was essentially irreversible at neutrality. If a state of equilibrium exist then dilution of the mixture should dissociate some of the combined reactants. Lafferty (38) reported recovery of infectivity by diluting after a brief period at 0 C. As the reaction time increased less infectivity was recovered on dilution. Dulbecco et a1. (13) and Mandel (44, 45) reported that poliovirus-antibody complex did not dissociate upon dilution. Granoff (22) substantiated this using Newcastle disease virus (NDV). However, Dulbecco et a1. (13) and Rubin and Franklin (57) reported a slight amount of dissociation (0.1 to 1.0%) of western equine encephalitis and NDV. Dilution of large amounts of poliovirus- antiserum mixtures resulted in 0.2% dissociation (44, 45). Andrewes (3) and Gard (21) observed that prolonged reac- tion time stabilized virus-antibody complexes. Lafferty (38) proposed that a freely reversible complex is formed during the first stage of the reaction. In the second stage, a stable complex is formed when the free site of an antibody molecule projecting from the virus combines with another antigenic site on the same virus. Divalent antibody reacted with influenza virus (38) and with adenovirus (35, 36) to form a stable com- plex. Monovalent antibody formed an unstable complex that was 6 dissociated by dilution (38, 36). Kjellen (36) substantiated that secondary reactions are responsible for efficient inactivation of adenovirus. The animal virus-antibody complex formed by the secondary reac- tion were dissociated by low pH. The dissociation resulted in quantitative recovery of viral infectivity (43, 35, 52, 22). As the pH increased the degree of recovery decreased (45, 22). Granoff (22) recovered infectious virus by dissociating NDV- antibody complex at pH 1.3 and pH 12.1. Poliovirus combined with minimal amounts of antibody and incubated for 120 days at 4 C can be recovered nearly quanti- tatively by dissociation at low pH. If large amounts of anti- body were combined with virus no infectious virus could be recovered by dissociation at low pH (44). Hummeler and Ketler (28), Mandel (44), Lafferty (38), and Granoff (22) concluded that the infectivity of the virus was not irreversibly altered by combination with antibody. Reactivated virus was reneutralized by the dissociated antibody (44) but at a slower rate than the initial neutraliza- tion (52). Kjellen (36) hypothesized that acidification caused new possible combinations between virus and antibody. Granoff (22) detected no difference in the rates of virus neutralization by untreated antiserum or antiserum acidified for 30 seconds, which indicated that antibody was not greatly altered. Isliker (29) concluded that at low pH influenza virus and antibody do not interact because they both have a net positive charge. At neutral pH the virus and antibody bear 7 Opposite net charges and interact. Virus-antibody complexes did not dissociate in an antibody-free suspension at neutral pH (45, 22). A nonavid antibody is one which is unable to form a stable complex with the virus surface. The complex formed by influenza virus and monovalent antibody dissociates when diluted at neutrality (38). Lafferty (38) interpreted the small amount of reversibility upon dilution at neutrality as indicative of monovalent, nonavid antibody which protected some viral particles from.neutralization by avid antibody. Westaway (69) found 0.5 to 14% dissociation of neutralized Murray Valley encephalitis and West Nile. He concluded that heat labile virus inhibitors reacted with viral particles and modified the attachment of avid antibody. Neutralized virus may be reactivated with fluorocarbon (32), ultrasonic vibration (39, 30), addition of inactivated virus (47, 13, 22), or by hypertonic saline solutions (63). A virus-antibody complex was adsorbed to susceptible cells (27, 44, 22). However, adsorption did not occur when the virus was effectively covered by antibody (27, 57). The role of antibody in neutralization of viral infectivity is thought to be prevention of penetration (57, 45) or uncoating (22). A buffer of low pH dissociates the antibody from the cell-virus-antibody complex without simultaneously inducing detachment of virus from the cell. The virus is then free to infect the cell (43, 44, 22). MATERIALS AND METHODS Virus The extracellular fluid of the 112th passage in chicken embryo kidney cells (CEKC) of the Beaudette culture of infectious bronchitis virus (IBV-42) was collected 60 hours post inoculation and stored at -60 C until used. The CEKC extracellular fluid was centrifuged at 10,000 x G for 30 minutes at 5 C (Sorvall RC-2B, rotor 33-34) to remove extraneous materials. The supernatant fluid was centrifuged at 74,000 x G for 60 minutes at 5 C (Beckman model L, rotor 50). The pellet was suSpended to original volume in phosphate buffered saline (PBS), pH 7.4 and stored at -60 C. This will be referred to as partially purified virus. Antisera Eight-month-old Single Comb White Leghorn cockerels were inoculated intranasally with 0.2 cc of IBV (16 x 105 chicken embryo infective doses 50). Six weeks later the chickens were reinoculated with 0.1 cc (8 x 105 chicken embryo infective doses 50) of virus. Three weeks after the second inoculation the chickens were fasted for 24 hours and exsanguinated. The sera were pooled, centrifuged, decanted, and stored at ~20 C. Before being used, the antisera were inactivated at 56 C in 8 9 a water bath for 30 minutes unless otherwise specified. Immunoglobulin Euglobulin was precipitated from the antisera by the method of Kendall (31). At 4 C 13 ml of saturated (NH4)2504 was added slowly to 26 ml of the pooled antiserum. The mix- ture was stored for 24 hours at 4 C, centrifuged, and the precipitate was resuSpended in PBS (0.1511NaClin.0.02 M phos- phate), pH 7.0, to a total volume of 26 ml. The procedure was repeated three times. Sulfate ions were removed by dialysis against PBS for four days at 4 C. The PBS was changed daily. Pseudoglobulin was separated from the euglobu- lin by dialysis against distilled water for three days at 4 C with daily changes of distilled water. The water insoluble euglobulin was resuSpended in PBS containing 0.3 M NaCl, pH 7.0, to a volume of 26 ml. Sedimentation coefficients of the immunoglobulin were not determined. Diluents The diluent for the virus and virus~antibody mixtures was phosphate buffered saline (PBS) with 2% new born calf serum, pH 7.3. The diluent for antibody was Hanks' Balanced Salt solution, pH 7.0. Determination of Protein The Folin-Ciocalteu phenol reagent as modified by Lowry et al., (42) was used to determined 100 ug or more protein per ml. The micromodification (42) was used for less than 10 100 ug of protein per ml. Standards were prepared with 10 to 300 ug of bovine serum albumin (National Biochemical Corp., Cleveland, Ohio). A Beckman DB spectrOphotometer was used. Cell Culture Primary chicken embryo kidney cell (CEKC) cultures, 107 cells per ml, were prepared from l7-to-18-day-old chicken embryos as described by Cunningham (10) and Cunningham and Spring (11). The cultural medium consisted of medium 199 in Hank's Balanced Salt solution. This was supplemented with vitamins, amino acids, and L-glutamine (General Biochemicals) all hav- ing a final concentration of 1% in Eagle's basal medium. In addition 5% newborn calf serum (Grand Island Biological Company), 100 units penicillin, 0.1 mg dihydrostreptomycin and 100 units Mycostatin per ml were added, respectively. The medium was adjusted to neutrality with 10% saturated NaHC03. Plastic (Falcon) petri dishes, 60 x 15 mm, were seeded with 4 ml of the cell suspension per dish. Incubation was at 37 C, 85% relative humidity, and 8% CO atmospheric 2 changes per hour. A satisfactory monolayer of cells was formed after 48 hours. Plaque Assay Four cell cultures were used for each virus sample unless indicated otherwise. The cultural medium was decanted from 48—hour-old CEKC monolayers. The monolayers were washed with ll balanced salt solution and each was inoculated with 0.5 ml of a particular suspension of virus. The inoculated and con- trol monolayers were incubated for 90 minutes. The fluid was decanted and the cells overlaid with 4 ml of the cultural medium containing 0.9% agar (Difco Noble). After the agar had solidified, the cultures were incubated 3 to 4 days. Neutral red, 0.1%, in PBS, pH 7.0, was added to the cultures. They were then incubated for 30 minutes at 37 C and 1 hour at 4 C. The number of plaques ‘was counted and the infectivity titer calculated using the enumerative dose response. The infectivity titer was expressed in plaque forming units (PFU) per ml. Effect of low and high pH on IBV infectivity Virus, 4 x 10# PFU per ml, was mixed with equal volumes of Sorensen's glycine-HCl, 0.1 N HCl acid, Sorensen's glycine~ NaOH, or 0.1 N NaOH. The final pH of the acidic and basic solutions was adjusted to pH 3.0 and 11.0, reSpectively, using 0.1 N HCl and 0.1 N NaOH. After incubation for 30 minutes at 4 C, the solution was diluted 10"2 and tested for virus. Kinetics of Neutralization The constant virus~decreasing serum neutralization test was based on the plaque reduction method (11). Equal volumes of virus, 5 x 104 PFU per ml, and anti~IBV-chicken serum were mixed and incubated at 37 C. At various time intervals, dilutions were made of each mixture and tested for virus. Virus control samples were processed in the same manner except that PBS was substituted for antiserum. 12 IBV - Antibody complex formation A concentration of antibody that would neutralize 99.9% of the virus was used. Virus and antibody were always mixed in equal volume and incubated in water baths for 1 hour at 37 C and then for 21 hours at 4 C unless otherwise Specified. Effect of pH on the stability of Egg-antibody complex Virus, 3 x 106 PFU per ml, was mixed with antiserum or immunoglobulin and incubated. Virusmantiserum mixtures were also incubated for 1 hour at 37 C and then for 14 days at 4, 23, 37 C. Test for dissociation of virus-antibody complex by high H+ ion concentration were made in ice-cold Sorensen's g1ycine~HCl containing 0.1% gelatin at pH 1.5 to 4.0. Based on preliminary examination it was necessary to dilute the virus dissociated at 1.5 to 2.7 10‘2. In the case of pH 2.8 or greater dissociation was not as complete and only a 10“1 dilution was required. After one minute, the pH of the reaction mixture was brought to pH 7 by a 10""2 dilution into neutral PBS. The mixture was tested for Virus immediately. Effect of pH on neutralization potential of antiserum prior to combination with IE! Antiserum diluted 1/80 was adjusted to pH 1.7 for 60 seconds at 4_C and then back to neutrality. Antisera pre- viously acidified or untreated were mixed with virus, 1 x 105 PFU per ml, and incubated at 37 C. At various inter- vals a portion of each was diluted and tested for virus. For the virus controls PBS was substituted for the antiserum. 13 IBV-antibody complex in an antibodyrfree suspension Virus and antibody were mixed and incubated as previously described. Virus-antibody complex was removed from the reac- tion mixture by centrifuging 24,000 x G for 1 hour at 5 C and then the sediment was resuspended to volume in PBS, pH 7.4. Dissociation of I§V~antibody complex at pH 1.9 and reassocia- tion Virus, 3 x 106 PFU per ml, was mixed with antiserum and incubated. Virus~antibody complex was removed from the reac- tion mixture and diluted 10'"1 in ice-cold Sorensen's glycine- HCl, pH 1.9. After 1 minute, two 0.5 ml samples were removed. One sample was added to 4.5 ml "PBS", pH 10.4. The final pH was 7.2. The other sample, the control, was diluted 10-3 to minimize the effect of antibody. Both mixtures were incu- bated at 37 C for 30 minutes. The control sample was tested without further dilution. The other sample was diluted 10-2 for compensatory dilution and then tested for virus. Effect of pH on dissociation of IBVmantibody complex in an antibodwaree suspension Virus, 9 x 105 PFU per ml, was mixed with antiserum and incubated. ViruSmantibody complex was removed from the reaction mixture and then acidified at pH 1.6, 1.7, or 1.9 as previously described. Antibody dissociated at pH 1.7 and 2.7 from IBV-antibody complex 5 Partially purified virus, 2 x 10 PFU per ml, was mixed with immunoglobulin, 164ng per ml, or antiserum both at a 14 concentration that would neutralize 99.99% of the virus. The mixtures were incubated and then virus-antibody complex was removed from the reaction mixture. The amount of_unneutralized virus was assayed and the sample was diluted 10'1 in Sorensen's glycinewHCl pH 1.7 or 2.7 without 0.1% gelatin. Test for dissociated virus was made as previously described. Antibody dissociated from virus was separated by centrifuging 74,000 x G for 30 minutes at 5 C. The pH of the supernatant fluid was adjusted to pH 7.0 and the amount of protein was determined. Undiluted samples were mixed with virus, 4 x 103 PFU per ml, and incubated at 37 C. At various intervals a portion of each mixture was diluted appropflately and tested for virus. Virus control samples were processed in the same manner except that PBS was substituted for the antiserum. Effect of dilution ontggv-antibody complex Virus, 4 x 105 PFU per ml, was mixed with non~inactivated antiserum, inactivated antiserum, or immunoglobulin and incu~ bated. Virus mixed with nonwinactivated antiserum was either incubated for 22 hours at 4 C or for 1 hour at 37 C and then 21 hours at 4 C. Threefold dilutions were made in PBS, pH 7.3. Undiluted and diluted samples were tested for dissociation immediately and after an additional 5 hours at 4 or 23 C. Effect of dilution on:;§V-antibody complex in an antibody-free suSpension Virus, 2 x 105 PFU per ml, was mixed with antiserum and incubated. Virus~antibody complex was removed from the reaction mixture. Undiluted samples and dilutions of 1/3 and l/9 were tested for dissociation. The undiluted sample was 15 also incubated at 4 C and tested for dissociation after 5 hours and 24 hours. To determine the amount of virus avail- able for dissociation and to establish that neutralized virus had been removed from the reaction mixture, tests were made for acidmdissociable virus as previously described. Effect of pH on cell-IBV-antibody complex Virus, 3 x 105 PFU per ml, was mixed with antiserum at a concentration that would neutralize 99.99% of the virus and incubated. Virus control samples were processed in the same manner except that PBS was substituted for the antiserum. Appropriately diluted neutralized virus suspension and virus control suspension were inoculated onto CEKC monolayers. After adsorption, the monolayers were washed with PBS, pH 7.3. Icewcold Sorensen9s glycine~HCl, pH 1.7 or 2.6, was added to the monolayer for 10 seconds and the cells were washed twice with PBS, pH 7.3. For control, monolayers were washed with only PBS, pH 7.3. The total time from the addition of Sorensengs glycineuHCl onto a monolayer to the final washing was 30 seconds. The cell layers were overlaid immediately with cultural medium containing 0.9% Difco noble agar. For cell controls, uninoculated monolayers were treated in the same manne I‘ o RESULTS Effect of low and high pH on IBV infectivity IBV was stabile for 30 minutes at pH 3.0 and labile at pH 11.0 (Table 1). TABLE 1. Effect of low and high pH on IBV infectivity Exposure Reagent Infectivity Per cent to pH titer 4 decrease (30 min) PFU/ml (X10 ) in infectivity 7.3 PBS (control) 4.08 O 3.0 Sorensen’s glycinewHCl 3.92 3°92 3.0 0.1 N HCl 3.75 8.09 11.0 Sorensen‘s glycinemHCl 0.00999 99.76 11.0 0.1 N NaOH 0.2560 93.73 Kinetics of neutralization The fir st part. of the curve is linear to 99.9% neutraliza- tion followed by a decrease in the slope (Figure l). The neutralization rate constant (K) may be calculated from: Co K = 2.3 D log 6; t where CO is the original concentration of virus, Ct is the 16 17 concentration at time (t), and D is the final dilution of the antiserum (44, 22). For antiserum dilutions 1/80, 1/160, and 1/320, K equals 8376 min.-l, 8364 min."l, and 1641 min._1 respectively. Immunoglobulin Immunoglobulin, 87 Pg per m1, neutralized l x 106 PFU of virus per ml, 99.99%. The immunoglobulin was tested by disc electrophoresis and only a single broad band in the gamma globulin region was found. Effect of pH on stability of IBV-antibody complex The complex formed with antibodies in anti-IBV-chicken serum or immunoglobulin was dissociated over the range of pH 1.5 to 4.0 (Figure 2). The maximum amount of dissociation was at pH 1.7 (Table 2, Figure 2). The amount of dissociation varied directly with the H+ ion concentration (Figure 3). At a pH lower than 1.7 the virus was inactivated by H+ ions and less infectious virus was recovered. There was approximately 5 to 20% inactivation of the virus controls. Dissociation was demonstrable after 14 days at 4 C but not after 14 days at 23 or 37 C (Table 2). Effect of pH on antiserum prior to combination with virus Acidified and nonacidified antiserum had similar K values. The data were used for the 5~to~30«minute test period for comparative purposes (Table 3). Per cent survival 100 10 0.1 0.01 r I I I I I I I I I‘. b‘ O O A O O . O C O O b- .- C .A A, 4A_ A (— .J l I J l i J l l J 10 20 30 4O 50 6O 7O 80 90 Time (minutes) Figure 1. Kinetics of IBV neutraliza- tion by anti-IBV-chicken serum. Antiserum diluted 1/80 (A). 1/160 (0). 1/320(0)o PFU/m1 107 105 10 I Ijl I IITr l l lllll lJlJl I 1 1 1111 l 1 l 1 1 1 1 1 Figure 2. M- b.) 45 67 8 pH Dissociation of IBV-antibody complex. Virus antiserum mixture (0), virus— immunoglobulin mixture (A). Per cent virus 70 60 50 4O 3O 20 10 20 IT I l i 1. _ .1 F O l l I l 1 2 3 4 pH Figure 3. Per cent dissociation of IBV-antibody complex as a function of H+ ion concen- tration. 21 TABLE 2. Effect of pH on stability of IBV-antibody complex Neutralization Dissociation PFU/ml after Per cent pH dissocigtion neutralized (X10 ) Virus dissociated 7.3 1.7 1.14 60.50 (99.9% neutralized 2.4 0.63 38.33 virus) 302 00018 13°95 Incubated 14 days at: 4 C 1.7 0.28 23.25 23 or 37 C 1.7 0 0 mmHH OH x oo.m om H moa x om.e m Homuosaoaaw m.somsoaom s.a mmma Hoa x 00.: om moa x 20.0 m mmm Afloaosoov m.s A 1.:HSV AHE\DmmV mm pm .SHE H pnmpmsoo opma pond» Azasv How Umpmmnp CoHpmNHHmedmz mpa>fip0mmsH nofipmnzosH pnmwmmm adhcmeQ< >mH Spas noapccaneoo on Hoahm achomapsm no Heapsopoc coapcmaamnpzcc no me go poommm .m mqm HE\Dmm HE\Dmm nmpoaoommao pcpmaoommap HE\Dmm ooNHHmedosss psmo pom msha> msha> m5ha> mm NchSoo scamnommdm ocamamponapsm no SH aoosaosousmH co soasoaoommao so me no sooccm .m mumsa 28 zaoumaeaxoamam n 2* some. mm.m moa x m.m moa x mom.m . m.m mmH mm.mm sea x Hmmm.m moa x o.H Hoe x m.o s.H adhcmapsm an chHHmHBSo: mana> o.m moa x om.m ow moa x om.: m 8.00? no.0 moa x owmm.m moa x m.m = s.m m.HH moH x ma.m so _., ._. . mos x oo.s m Adonpsoo maha>v 30H x 0:.N or mad mm.mm 30H x wmmm.m moa x o.H Hoa x m.m s.H m. w. mm w... w m \m) "mm. )m nsee awn de dA fieA T. 00 mq ou To .doI I an? d uwx xxa. Tse o.+ o o nuo Mm //1. w“ . m.» u.+ 7.1. Mste ./4.m //m m.am _ eLn . I. pea. saou mass was eLefls I I (o 10 14 IQ I I 71 a. u say a e .L A Pp“ p. P w D. oaxa emoaaao aoonaoss sdasnoamosdssa an pmwaamapscs maha> I'lllrnlIfl-r.rflvllirr I1 soaasoo aoopaosou>mH Sosa s.m one a.H we so oopsaoommao soopapss .o mamas 29 .pouaadhpdofi who; mSHH> on» mo Rm.mm 20:3 oHSPHHS Hmcawaho 03p Scam dobaaoo chm oped omega 0 .noapma>oo chopsmpm a 9 “mafia: wsashom madman go Hones: cwmambd m + so.o os.m om.m, (ww.a a m\H sH.a oo.m om.mH a m\H Qaaznoawondsaa u ma.H om.HN s oopsaaeso oats + msaa> so.o oo.a so.m om.m we m\H a... mo.m on.m. ea m\a ,. _ . 0 mm . mm.o om.eH ea oopsaaoss c .as m oo.o mo.m wo.m oo.a 6H m\a oo.o mm.m ma.ea oa m\a V - o a . mm.o om.mH ma assesses: o .as m o No.H sm.a om.m 0H axe mw.: :N.H Nu.m HH m\H adhomfipnm popdbapomsa . ma.m mo.ea HH oopsaaosa oaoN + msaa> oHsNDmm op onmHobcoo no women mmhdpaso .oommao mam m .Ihu co soaoaaso soaasaao psoo pom empocaxm n mbmm_m nonadz soapSHHQ Mocha .nm asymmapsm copmbapomsH .H .Hoagaoo aconapcwl>mH no soaPSHac mo 9009mm .5 mam¢a 30 .ooNaHmeSo: ohms mdhab on» no Rm.mm 20:3 ondpwaa Hondwaho 0:» Song po>anop who came moose a .codpmdbmo chmpnopm H n “mpdss wGHsHOL madman no senses cwmhmbd m HN.0 #3.: 0H.m Odwm. d m\H mm.mH Hm.m o.mm a m\H 0 MN I 3N.N 0.0: : UmdeaUGD @ .9: m os.o em.m No.0H o.on s m\a om.sa mm.o o.se s m\a - . O 3 I mm.m “.mm 3 UmpSHHUGD © .HS m Hm.o oo.m Na.m o.oe. e axe oo.mH do.s o.m¢ : m\H adnomfinsm A. ompm>apoosasoam I 350% 00H“ .3 Umpzflflfivgb OHmN AT mgfi> oasxsaa op - qoamnm>soo no woman mmHSpHSO .oomch ohm M Ilhu mo cmzoamsc Scandaap pzmo Ham Umpommxm D abmm.M HmDESZ SOHQSHHQ Hmpym ohm adhomapsm popcbapomsanoz .HH Moamaoo hponapsmi>mH no soapSHHU no poommm .w mqm¢9 .soapmaoommaoloaoc an dosaaacpon c .GOapmH>cc pudendum H n “nudes wzaahom tsunam mo Hones: owcachd m sea x om.m mm.w mm.sH m am a” moa x Ho.H as.m mm.mo m m . .. adhcwfipsm . popmbapomsH mod x :m.a ma.aa :H.om n oth + m5ha> oHs\saa soapmfioommap How .lkv mcndeSO canmaamam moha> 9 spam N no Honssz mhsom sonanmSm oohmnhponapnc an ad woaasoo successonsmH so soaoossosa co pooccm .a mamas 32 .coapmH>mp onmoscpm H n «mpazs wsHEHom madman no Hopes: owcac>¢ a o 05.5 sm.m ma.w m m\a mm.mm om.s am.mm m m\H adhomapsc popdbapomsa II mH.HH :H.om n popsadpsb + mshfl> Sodpmaoommac 3mm x .lhv monzpaso UoSOHQSc pace Mom pcpoomxm Q momm_m mo Honssz soapSHHQ SOHmGonSm omnpizuonapnm so ca Noamaoo hponapsml>mH no soapsaao mo poomgm .oa mqm4a 33 TABLE 11. Effect of pH on dissociation of IBV-antibody complex after adsorption to cells Inoculum pH Infectivi y Per cent PFU/m1 (X10) dissociation Virus + 7.3 33.0 - buffer 107 3500 "’ 206 3805 "' Virus + 7.3 0.009 - antiserum 1.7 7.860 22.47 2.6 .1.14 2.96 DISCUSSION The curve for the IBV-antibody interaction follows first— order kinetics with the reaction rate dependent upon time, antibody concentration, and temperature and is similar to the results reported by Dulbecco (l3), Mandel (44), and Granoff (22) with other virus-antibody systems. Acid stability (71) is the only criterion studied by which IBV differs from the general characteristics of myxo- viruses. At neutrality the IBV-antibody complex is stable. Dis- sociation occurs within 30 seconds at pH 4.0 or lower and confirms Granoff's (22) report that the amount of dissociation is dependent upon the H+ ion concentration. Mandel (44) and Granoff (22) both concluded that dis- sociation is not due to antibody denaturation based on the results, respectively: 1) dissociated virus and antibody could reassociate at neutrality, 2) acidification of anti- serum for 30 seconds has no effect upon the neutralization rate constant. It is unlikely, however, that a protein would not be denatured at low pH. It is possible that antibody changes configuration at low pH as does albumin (60) and returns to the original configuration at neutrality. Since acidification of virus and antibody below the isoelectric point would induce a net positive charge, the mechanism of 34 35 dissociation could be due to electrostatic repulsion between the chains of the antibody. This would alter the active sites with an accompanying reduction of van der Waals' attraction and the sites would no longer be complementary. A decrease of H+ ions results in less positive charge induction and as a result a smaller amount of dissociation. Since IBV was inactivated at a high pH, dissociation of virus-antiserum or immunoglobulin mixtures could only be proven in the low pH range where the virus is stabile. The inability to detect 100% dissociation may be due to reassocia- tion, lack of complete dissociation, or irreversible alteration of the virus. Dissociation of IBV-antibody complex after 14 days at 4 C with recovery of infectious virus emphasizes that anti- body does not permanently alter the infectious capacity of virus. This is in complete agreement with the reports of Mandel (44) and Granoff (22) using poliovirus and NDV, res- pectively, under similar conditions. It was not possible, however, to detect dissociation after 14 days at 23 or 37 C. The virus is inactivated at these temperatures (11). Antibody obtained from dissociated IBV-antibody complex will neutralize virus. The slow rate of neutralization could be due to some denaturation or simply the result of decreased protein concentration. These results favor the conclusion that antibody is denatured, butrwt irreversibly altered at low pH. Interaction between NDV and antibody has been reported by Burnet (8) to be completely reversible by dilution. This 36 report was refuted by Rubin and Franklin (57). Likewise, Mandel (44) and Granoff (22) concluded that the interaction is essentially irreversible by dilution. However, Mandel (45) reported 0.2% dissociation, when poliovirus and antibody were mixed in high concentration. When high concentration of IBV and antibody were mixed, dilution of the complex does not cause dissociation. If the complex is diluted and then incu— bated, dissociation, 0.06%, occurs. Dissociation is con- sidered a valid conclusion since there was no decrease in free virus in the undiluted mixtures during incubation. If, however, the antiserum is not inactivated and the reactants are incubated at 4 C, 0.3% dissociation will occur. Dissocia- tion was detected when the average number of plaques was not proportional to the dilution within the limits of the standard deviation. It is possible that the presence of heat labile non- Specific protein in noninactivated antiserum hampers the complete mechanism of neutralization and as a result causes a small amount dissociation by dilution. The IBV-antibody complex separated from the reaction mixture by centrifugation was stabile and no dissociation occurred after incubation at 4 C or by a 1/9 dilution. The complex dissociated only at low pH. These data confirm the previous findings that virus-antibody complex is essentially irreversible at neutrality. Anti-IBV-antisera were prepared in chickens. In contrast, Mandel (44) and Granoff (22) obtained antisera from animals which are not the normal host for the virus used. 37 The small amount of dissociation, 0.04%, of virus neutralized by immunoglobulin may have been the result of a slight amount of alteration of the active sites when the globu- lin was fractionated from antiserum. Lack of precise comple- mentarity could result in weak bonding between the virus and the antibody. The data presented confirm that a virus-antibody com- plex, and Specifically one with IBV, adsorbs to susceptible cells. Antibody can be dissociated from the complex without simultaneously inducing detachment of infectious virus from the cell. The mechanism of the effect of antibody at the cell membrane is not understood. Mandel (44) hypothesized that antibody blocks penetration of the cell by simple steric hindrance. Likewise, it may be possible that antibody affects cell membrane envagination. Granoff (22) proposed that anti- body prevents uncoating of viral protein. The experiments described in this investigation and the results obtained with other virus-host systems substantiate that virus-antibody complex dissociates at low pH, virus is not irretrievably deprived of infectiousness by neutralizing antibody, and virus-antibody complex is essentially irreversible at neutrality. SUMMARY Infectious bronchitis virus (IBV) is acid stabile and base labile. Virus neutralized by antibodies in either antiserum or immunoglobulin can be dissociated at pH 4.0 or lower and infectious virus recovered. A maximum of 60% dissociation of IBV-antibody complex occurred at pH 1.7. The amount of dissociation depends upon the H+ ion concentration. After 14 days at 4 C, the complex can be dissociated at pH 1.7 and infectious virus recovered. Antibody does not permanently alter the infectious capacity of the virus.- Antibody acidified for one minute at pH 1.7 was not irreversibly altered. Return to neutrality renatured the molecule. Acidified and nonacidified antiserum had similar neutralization rate constants. Infectious bronchitis virus and antibody dissociated at pH 1.9 reassociated at neutrality. The P8 of antibody dissociated from IBV was related to the 3* ion concentration. Dissociated antibody retained its capacity to neutralize virus. Dissociation did not occur when IBV-inactivated antiserum mixtures were diluted at neutrality. An extremely small amount of dissociation did occur when the complex was 38 39 diluted and then incubated at either 4 or 23 C for 5 hours. The largest amount of dissociation occurred when IBV-noninactivated antiserum mixtures were diluted at neutrality. The presence of heat labile nonSpecific protein in the serum may be related to the amount of dissociation by dilution. The IBV-antibody complex in an antibody-free suspension was stable and did not dissociate when incubated at 4 C or when diluted 1/9. 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