HEMAGGLUTINATION BY TRYPSIN- MODIFIED INFECTIOUS BRONCHITIS VIRUS By LEO JOHN CORBO A THESIS Submitted to the School for Advanced Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health 1957 This work is respectfully dedicated to MY FAMILY ABSTRACT Infectious bronchitis of chickens (IB) is an acute and highly contagious disease of economic significance to the poultry industry. The purpose of the present study was to determine if the serological techniques employed with the causative virus, Tarpeia _p_u_l_li, could be expanded to encompass hemagglutination. Infectious bronchitis virus (IBV) does not agglutinate red cells, and the only serological test which is applicable is the serum-neutralization test. Twelve strains of IBV, one of which was completely egg- adapted. were tested. Newcastle disease virus (NDV) and normal allantoic fluid (NAF) were used for controls. All virus was in the form of infected allantoic fluid of em- bryonating chicken eggs collected on the twelfth day of incubation. Newcastle disease virus and the egg—adapted strain of IBV were inoculated on the tenth day, whereas the other strains of IBV were inoculated on the ninth day. Normal allantoic fluid was collected on the twelfth day, also. Induction of a hemagglutinative agent in IBV was attempted through utilization of the proteolytic enzymes. trypsin, pepsin, papain, and rennin. Trypsin proved effective. ii . v—. M.- --—n ".n — A standard method of treatment (standard T process) was es— tablished. This consisted of mixing 1 ml of 1 per cent trypsin with 2 m1 of the sample. The system was incubated for three hours at 37°C. and then 1 ml of eggwhite trypsin inhibitor was added. The complete system was then tested for hemagglutinative activity at 23°C. All strains of IBV except the egg-adapted strain responded to treatment as evidenced by consistently high hemagglutination titers obtained when using chicken erythrocytes. Normal allantoic fluid and NDV were not affected by this treatment as NAF exhibited no hemagglutinative activity and that of NDV was unchanged. A 98 per cent decrease in viral infectivity accompanied tryptic modification of IBV. Adsorption of the hemagglutinative agent of modified IBV is rapid and complete at 37° and 23°C. but not complete at 4°C. Modified IBV has not been successfully employed in hemagglutination-inhibition tests. Normal chicken serum and anti-1B serum inhibit hemagglutination. The IBV hemagglutinin is distinguished from that of NDV and the nonspecific hemagglutinin of trypticase and tryptone on the basis of reactions with sera. iii Spontaneous elution of the hemagglutinative agent of modified IBV is slight at 37“ and 23°C. and more pronounced but not com- plete at 4°C. Hemagglutination by modified IBV occurs when chicken red cells are employed. No agglutination of the red cells of the dog, cat, rabbit, cow, sheep, horse, or human type 0 has been demon- strated. A direct correlation was observed between the optical den- sity of the modified IBV system, the hemagglutination titer, and the infectivity titer. It was demonstrated that the action of trypsin was directed to the virus, and not the chicken erythrocytes, for trypsin-modified red cells were not reactive. iv AC KNOWLEDG MENTS The author is grateful for the generous help, guidance, and instruction given to him by Dr. Charles H. Cunningham, Professor of Microbiology and Public Health. He is also indebted to Doctors Richard L. Bateman, Carl A. Hoppert, and Erwin J. Benne, and Professor Charles D. Ball for their guidance on the chemical in— terpretation of the many facets of the study; and to Doctor Lloyd C. Ferguson, the author is deeply appreciative for the kindness ex- tended in his administrative capacity. Grateful acknowledgment is also due to Mrs. Martha P. Spring. This study was supported by the Agricultural Experiment Station of Michigan State University. Leo John Corbo candidate for the degree of Doctor of Philosophy Final Examination: May 15, 1957, 8:00 A.M., Room 101, Giltner Hall. Dissertation: Hemagglutination by Trypsin- modified Infectious Bronchitis Virus. Outline of Studies: Major subjects: Virology and Bacteriology. Minor subject: Chemistry. Biographical Items: Born December 12, 1922, Elizabeth, New Jersey. Undergraduate Studies: University of Notre .Dame, 1940-1942, 1946-1947, 1950-1951. Graduate Studies: University of North Carolina, 1951-1952; Michigan State University, 1955-1957. Experience: Bacteriologist, Lobund Institute, Notre Dame, Indiana, 1952-1955. Society Affiliations: Society of American Bacteriologists, American Public Health Association, American As- sociation for the Advancement of Science, Society of Sigma Xi, The New York Academy of Sciences. vi TABLE OF CONTENTS INTRODUCTION ............................... HISTORICAL REVIEW ........................... Infectious Bronchitis .......................... Isolation of the virus ....................... Diagnosis of infectious bronchitis ............... Hemagglutination ............................ Viral hemagglutination ...................... Inhibition of viral hemagglutination ............. . Cell receptor sites ......................... Vaccinia and related hemagglutinins ............. Hemagglutination with arthrOpod-borne viruses ..... Action of enzymes in hemagglutinating systems ..... Enzymes and Enzyme-inhibitors .................. EXPERIMENTAL PROCEDURES .................... Materials and Preparations ..................... Methods .................................. EXPERIMENTAL RESULTS ....................... Hemagglutination ............................ vii 11 15 17 18 19 20 24 Z4 Z9 34 34 Adsorption Measured by Viral Infectivity pH of normal and viral—infected allantoic fluid . . . . Modification of infectious bronchitis virus by trypsin ..... ........................... Modification of infectious bronchitis virus by other proteolytic enzymes ................... Turbidimetric determination of hemagglutination Adsorption Measured by Hemagglutination ......... . . Rate of adsorption ........................ Maximum adsorption . . ................ . . . . . . Effect of trypsin on chicken embryos ........... Effect of trypsin on viral infectivity ............ Adsorption and inf ectivity ................... Relationship between infectivity titer and hemagglutination titer . ..................... Further Characterization of the Infectious Bronchitis Virus Hemagglutinin . . . . ..................... Elution ................................ Hemagglutination by different strains of infectious bronchitis virus ........ . ................. Hemagglutinative activity and initial recovery of infectious bronchitis virus in chicken embryos . . . . Effect of trypsin on red cells . . .- ..... . ....... ---------- Page 34 34 46 52 '64 64 64 67 67 68 69 69 71 71 71 71 74 Thermostability of the hemagglutinative activity of infectious bronchitis virus .................... Colloids ................................ Hemagglutination by peptones, polypeptides, and amino acids .............................. Agglutination of red cells of certain species ....... Hemagglutination- inhibition ..................... Hemagglutination—inhibition by serum ............ Effect of other inhibiting agents ................ DISCUSSION .................................. SUMMARY ................................... BIBLIOGRAPHY ............................... ix Page 77 77 81 83 84 84 90 92 104 106 TABLE 13. LIST OF TABLES pH of Normal and Viral—infected Allantoic Fluid from Twelve- day-old Chicken Embryos Hemagglutination by Trypsin-modified IBV ...... Hemagglutination by Trypsin-modified IBV ...... Hemagglutination by Trypsin-modified Normal Allantoic Fluid .......................... Hemagglutination by Trypsin-modified IBV and NAF (Standard T Process) .............. Effect of Incubation at 37°C. on Hemagglutination Titer .................... Effect of Time of Incubation at 37°C. on Hemagglutination in Standard T Process ........ Effect of Certain Enzymes on Hemagglutination Effect of Crystalline Trypsin on Hemagglutination Effect of Chymotrypsin on Hemagglutination ..... Effect of Chymotrypsinogen, Activated by Crystalline Trypsin, on Hemagglutination ....... Optical Density and HA Titer of IBV Strains in Standard T Process .................... Optical Density and HA Titer of IBV Strains in Standard T Process at Different Incubation Periods ............................... Page 35 36 38 39 44 45 46 47 49 50 51 53 54 T ABLE 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. Optical Density, HA Titer, and Infectivity of Dilutions of IBV 40-5 in Standard T Process Optical Density of Mixtures of T and EWTI ..... Rate of Adsorption of IBV 40-5 Hemagglutinin by Red Cells ........................... Total Adsorption of IBV 40-5 Hemagglutinin by Red Cells at 23°C. .................... Progression of Adsorption and Elution in Hemagglutination by IBV 40-5 ............... Hemagglutination by Strains of IBV after Modification in the Standard T Process ........ Concentration of IBV Hemagglutinin in Early Egg Passage ....................... Trypsin-modified Red Cells in Hemagglutination Tests ................................ Thermostability of IBV-infected and Normal Allantoic Fluids Prior to Standard T Process Thermostability at -30°C. of the Hemagglutinative Activity of IBV after Standard T Process ....... Colloidal Prussian Blue as an Indicator of the Charge on Reagents in the Standard T Process Hemagglutination by Peptones ............... Agglutination of Red Cells of Certain Species by T- modified IBV ....................... Hemagglutination-inhibition of IBV by Sera ...... xi Page 55 57 65 66 72 73 75 76 78 79 80 82 84 85 V O I ,7. Q 'l# ‘ ‘ ’7' TABLE Page 28. Hemagglutination—inhibition of IBV by Sera Inactivated at 62°C. for 20 Minutes and then Treated with T and EWTI .............. 87 29. Hemagglutination—inhibition Tests Using a Trypticase-Amino Acid Mixture .............. 89 30. Hemagglutination—inhibition (Alpha Procedure) . . . . 91 xii LIST OF FIGURES FIGURE 1. Hemagglutination Titer and Optical Density of IBV 40-5 after Standard T Process ............ II. Optical Density of Mixtures of T and EWTI ...... III. Schematic Representation of Reacting Products in the IBV-HA System ..................... xiii Page 56 58 61 of INTRODUCTION Infectious bronchitis is a disease economically important to the poultry industry. The serum neutralization test is the only serological procedure that may be employed for diagnosis of the disease. The object of the present study is to determine the potential of infectious bronchitis virus as a hemagglutinative agent. -‘ Il.‘-l— If r‘lll --—.~— _- r—-- L» .u-u—.-._ —'-W' «ms. .mm... *L‘mfl' F" HISTORICAL REVIEW Infectious Bronchitis Infectious bronchitis (IB), an acute and highly contagious reSpiratory disease of chickens, was first described by Schalk and Hawn (87) in 1931 in North Dakota. Since that time the disease has been recognized throughout the United States, in Canada, Great Britain, the Netherlands, Japan, and Brazil (54, 94). The causative agent is the virus Tarpeia pulli, which is spherical with filamentous projections and has a mean diameter of 70 mp as determined by electron microsc0py of the free elementary bodies (82, 83). Infectious bronchitis virus (IBV) within the cells of the chorio- allantoic membrane, has a mean diameter of 178 mu and is devoid of filaments. Some virus particles exhibit a discrete internal struc- ture which forms a pattern that suggests the possibility of a macro- molecular arrangement within the elementary bodies. Rows of clear spots of less electron density, about 20 mp in diameter, each of which contains a smaller, electron-dense body, make up this pat- tern (37). DE 863m. bill 51 The disease apparently is limited to chickens, and affects all ages, breeds, and sexes. Natural transmission is by direct contact or by aerosol. Experimentally, the disease may be transmitted readily following intratracheal, intranasal, and air sac inoculation (7, 35), but not by subcutaneous or intramuscular routes. Signs usually appear 24 hours after intratracheal inoculation. The most prominent signs are gasping, sneezing, coughing, and tracheal rales (5, 6, 34, 59). Isolation of the virus. IBV 'can readily be isolated from lung and tracheal material collected during the incubation period and throughout the respiratory phase of the disease. Chicken embryos are inoculated with these specimens and the outstanding gross alter— ations produced by the virus are a curling and dwarfing of the em- bryo (7, 35, 38, 69). These lesions are pathognomonic of infec- tion with IBV (69). Microscopic alterations include proliferation of mesodermal and ectodermal cells, edema of the amnionic membrane, necrosis and hemorrhage of the liver, interstitial nephritis, conges- tion of the spleen, and slight capillary congestion of the brain. Beaudette and Hudson (7) and Delaplane and Stuart (36) ob- served that following chorioallantoic membrane inoculation there is but slight embryo mortality in early passage of the virus. With pO HOT nom ‘0 e):l gluzf successive embryo passage the virulence of the virus increases as manifested by higher embryo mortality rates. Completely egg-adapted virus kills all embryos within 48 hours after inoculation (28). Com- plete adaptation varies with different strains. With some this has been observed at the sixty-fifth passage, and at the ninetieth and one hundred twentieth passages with others. Egg-adaptation is accompanied by loss of pathogenicity and antigenicity for chickens. Diagnosis of infectious bronchitis. Diagnosis of IE is based on the following: (1) clinical features; (2) isolation and identifica- tion of the virus; (3) neutralization tests; (4) cross-immunity tests (25). The ability of anti-IB serum to neutralize IBV was first re- ported by Beach and Schalm (5). Mixtures of virus and serum were not infectious for susceptible chickens. This method was later ap- plied with embryonating chicken eggs. Cunningham (24) reported the serum-neutralization indexes of normal birds, not previously exposed to IBV, would not be expected o ' 6 . . . l 517 :1: 100 037 or thirty-Six neutralizing doses. to exceed 10 IBV fails to agglutinate chicken erythrocytes, and the hemag- glutination-inhibition test cannot be used to identify either the virus Sut m0} or specific antibodies other than by an elimination process (4, 25, 32, 33, 38). Hemagglutination Many substances such as plant (80) and animal (90) tissue extracts, the higher fungi (40), bacteria (80), rickettsia (81), pleuro- pneumonia-like organisms (40), and viruses (55) may agglutinate red blood cells of various species. Some of the reactions are only an observable phenomenon without an understanding of the mechanism of reaction, but others such as those with bacteria and viruses are specific. Hemagglutination has been studied with many bacteria. Krauss and Ludwig (63) observed clumping of erythrocytes in the presence of staphylococci and vibrios. This was a direct bacterial hemagglutina- tion, the mechanism of which remains to be elucidated. Keogh, North, and Warburton (60) demonstrated adsorption of the polysaccharide antigen of Hemophilus influenzae on red cells and subsequent agglutination of the modified cells upon addition of ho- mologous antibacterial serum. The same reaction has been observed with other bacterial antigens and antibodies. In general, polysaccha- ride rather than protein antigens are adsorbed by the untreated erythrocyte. Assuming a two-step reaction in the modification of the red cell by polysaccharide antigen of various bacteria--(1) alteration of cell surface, and (2) attachment of antigen--Boyden (10) investigated a number of compounds to determine if they possessed the capacity to produce the first reaction and render red blood cells capable of adsorbing protein antigens. Certain inulin preparations were ef- fective, but tannic acid proved more effective. Either one in high concentration would agglutinate red cells. Modification of the cell must be carried out with great care for the reaction to occur. The treated cells are exposed to the protein antigen and thus acquire a new serologic specificity in that they are specifically agglutinated in the presence of homologous protein antibodies. Boyden demon- strated hemagglutination of tuberculo-protein modified and strepto- coccal-protein modified erythrocytes. Davidsohn and Toharsky (29, 30) reported that Comebac- terium H produced changes in human plasma and serum such that they possessed panagglutinating capacity. Panagglutination, the ag- glutination of erythrocytes by any normal human serum, was re- ported by Thomsen in 1927 (80). Viral hemagglutination. In 1941, Hirst (55) reported the in vitro agglutination of chicken erythrocytes by influenza virus-infected setti; ing hf (118638, allantoic fluid. Agglutination is characterized by a film of red cells covering the bottom of the tube. Normal allantoic fluid (NAF) in combination with red cells does not produce a pattern different from that of cells settling in physiological saline; i.e., a sharply demarcated disc of cells or "button" formation at the bottom of the tube. Hirst further demonstrated a strain specific inhibition of hemagglutinative activity by anti-influenza serum. Influenza virus hemagglutination was also discovered indepen- dently by McClelland and Hare in 1941 (72). They reported that human and guinea pig red cells, in addition to chicken erythrocytes, were agglutinable and that the virus was adsorbed by the cells. The first requirement for the exact study of the phenomenon was the establishment of specific criteria for the determination of hemagglutination titers. Hirst (56) used a method dependent on the optical density of the supernatant fluid. The absorbance of this fluid varies inversely with the rate and degree of agglutination and settling of cells. Salk (85) established the, pattern method for read- ing hemagglutination. Characteristics of hemagglutination permit separation of viruses into three groups: (1) influenza, mumps, and Newcastle disease; (2) vaccinia, variola, and ectromelia; and (3) a variety of ., .7 . 4 km ' v T w L_, "m for. Dan the ft] from ‘ #- other viruses--Japanese B, St. Louis and Russian encephalitis, mouse encephalomyelitis (Theiler's GD VII), West Nile fever, encephalomyo- carditis group, pneumonia virus of mice, foot-and-mouth disease, and fowl plague. The mumps (M), Newcastle disease (N), and influenza (1) group (MN'I) agglutinates red blood cells of a number of different animals. Chicken, guinea pig, and human cells are more commonly employed. Hemagglutination proceeds in two stages: (1) adsorption of the virus to specific receptors on the cell surface and subsequent agglutination; and (2) elution of the virus from the cells, leaving a modified cell surface (56, 57). The reaction is considered to be an enzymatic process associated with the virus particle. Separation of the enzymatic activity from the viral particles has not been accomplished, but soluble enzymes of essentially simi- lar quality have been obtained in culture filtrates of a few bacterial species. Electron microscopy indicates that agglutination is due to the formation of a lattice of red cells held together by multivalent virus particles (53). Definite evidence for the relationship of cell receptors for the MNI viruses is found in studies of the agglutinability of cells from which a virus has eluted. Elution of M leaves the cells _ "T- ’T‘bfi?‘x‘.-_" 7%" ‘ ‘ 111: m1"- - m.- .rara' agglutinable by N or I but not by M. Elution of N renders the red cells agglutinable only by I. Strain differences may be observed with influenza virus. Elution of influenza A leaves cells still agglutinable by some strains of influenza B virus. This provides the basis for the ”receptor gradient" theory (13). The principal investigations of the hemagglutination reaction with the MN'I group have utilized influenza virus. Adsorption of influenza virus to the surface of red cells occurs rapidly in physiological saline. Adsorption can be prevented in a solution of very low ionic concentration (18). In the absence of ions, none of the adsorptive or enzymatic interactions of influenza viruses with mucoid inhibitors or cell receptors occur. Visible agglutination by active virus may occur in solutions containing as little as 0.01 M monovalent cations. Indicator virus (virus heated at 55-56°C. for 30 minutes which shows no reduc- tion in hemagglutinative activity but which has lost its capacity to elute; i.e., lost its enzymatic activity) reacts in a lower concentra- tion of salts. Cations are the most important ions in the phenomenon. Cal- cium ions are more effective than sodium or potassium in allowing adsorption or enzymatic activity. The only anionic effect clearly 11-. Jifi‘ "d .‘ 10 demonstrable is that of sodium hexametaphOSphate, which inhibits hemagglutination and probably is due to an anticalcium action. Adsorption of virus by red cells at 0-4°C. is most effec- tive with 0.1 M salt concentration. Some adsorption occurs at 0.01 M concentration or less, and there is no sharp threshold of demar- cation. At 27°C. destruction of receptors occurs at salt concentra- tion below that required to produce visible agglutination. The rate of adsorption of influenza virus is related to tem- perature and numbers of cells (57). An increase of cells is accom- panied by an increase in the rate and degree of adsorption of hemagglutinin. The time for maximum adsorption is the same regardless of cell concentration. With the greater concentrations of cells the rate and degree of elution decreases. At 4°, 27°, and 37°C. adsorption proceeds rapidly during the first minute. Adsorption is less complete with rise in tempera- ture. At 4°C. the maximum adsorption (99.5 per cent of the virus) is not attained until 15 hours; at 27°C. (98.8 per cent), in 25 min- utes; and at 37°C. maximum adsorption (87.0 per cent) occurs in three to five minutes. The amount of elution observed at 4°C. at 18 hours is neg- ligible, but the degree of elution increases with temperature so m 2‘. V ~a- HM“! 11 that at 37°C. almost all of the adsorbed agglutinin is released in six hours. The infective agent of influenza virus suspensions is adsorbed by red cells simultaneously with the adsorption of hemagglutinins. Ninety-five per cent of the infective agent is removed in 15 minutes. Inhibition of viral hemagglutination. Three serum components are known to inhibit viral hemagglutination: (1) homologous antibody, presumably gamma globulin, Specific for the virus antigen; (2) "Fran- cis" inhibitor, active against indicator viruses; and (3) "nonspecific" inhibitor, characterized by its effect on unheated virus. Francis (44) observed that if indicator virus (LEE) is used to determine the hemagglutination-inhibition (HI) titer of Sera, very high values are obtained irrespective of the antibody content. Anderson (3) demonstrated that the ”Francis" inhibitor is destroyed by the receptor-destroying enzyme (RDE) of Vibrio cholerae as well as by active influenza virus. This allowed the development of the hypothesis that influenza virus contained an en- zyme essentially similar to the soluble RDE of Vibrio cholerae. Inhibitory action similar to that of the "Francis" inhibitor is elicited by a variety of physiological secretions and tissue ex- tracts containing mucoprotein. Some of these are human cervical 12 mucus, egg white, tears, human urine, and an extract from the sub- mandibular salivary gland of sheep. McCrea (75) identified the serum inhibitor of heated LEE virus as a component of the heat-stabile mucoprotein fraction of rabbit and human sera. Based on a dry-weight comparison, purified serum mucoprotein shows considerably higher activity than the serum from which it was derived. Serum mucoid inhibitor is relatively labile in that it is inactivated by heat at pH 4.6, by chloroform at pH 4.6, and by treatment with 90 per cent phenol. Inhibitory ac- tivity of normal serum is destroyed by trypsin, Chymotrypsin, pepsin, and cyanide-activated papain. All these reactions proceed more readily with increasing purity of the mucoid fraction. McCrea (73) reported destruction of nonspecific inhibitor in normal rabbit serum in 20 minutes at 62°C. Friedewald, Miller, and Whatley (45) demonstrated that saline extracts of human and chicken red cells contained inhibitor. These cells could also be agglutinated by influenza or mumps virus. When the virus receptor substance was removed from chicken erythrocytes by adsorption and elution with influenza virus, extracts of the cells no longer yielded the inhibitory substance. The inhibitory substance did not neutralize influenza virus in mice, and it failed to fix com- plement when mixed with influenza or mumps virus. Evidence was 13 obtained that some virus was released from the inhibitory substance after incubation for six hours at 22° or 37°C. Hirst (58) mixed equal portions of normal rabbit serum and crystalline trypsin (250 mg per cent), incubated at 37°C. for three hours, and noted a marked reduction in inhibitory titer using chicken red cells. Svedmyr (92) identified the inhibitor of influenza hemagglutina- tion which is present in normal allantoic fluid as a mucoid. Lanni and Beard (65), working with swine influenza virus, observed that egg white inhibitor is almost completely destroyed by treatment with dilute periodate. Murphy (78) reported optimal destruction of nonspecific in- hibitors of influenza hemagglutination present in serum by the use of RDE at pH 6.0. Wenners, Monley, and Jenson (95) utilized crystalline tryp- sin and sodium periodate, respectively, to treat serum and elimi- nate nonSpecific inhibitors of hemagglutination of chicken erythrocytes by mumps and Newcastle disease viruses. Davoli and Bartolomei-Corsi (31) reduced, and in some in- stances eliminated, the inhibitory activity of normal rabbit and human sera by adding a 3.81 per cent solution of sodium citrate to the serum. .-/ .." Ta J:- mrm _- 14 Sampaio and Isaacs (86) used crystalline trypsin (32 mg per ml of serum at 56°C. for 30 minutes) to destroy inhibitors in nor- mal ferret, chicken, rabbit, guinea pig, and mouse sera. Tamm and Tyrrell (93) reported that both normal allantoic fluid and human urine inhibit hemagglutination by the murine en- cephalomyelitis GD VII strain and influenza viruses. The inhibitors of GD VII are not affected by Vibrio cholerae filtrate or by active influenza viruses. The inhibitor contained in normal allantoic fluid combines with GD VII at 4°C. and dissociates from the virus at 22°C. By a combination of chemical purification and starch—zone electrophoresis, it has been shown that the GD VII inhibitor is chemically distinct from the influenza (LEE) virus inhibitor. Casals (21) reported that nonspecific inhibitors of influenza hemagglutination were removed from 1:5 dilutions of monkey and other animal sera by Seitz filtration. The removal of inhibitors was probably due to adsorption by the filter. An inhibitor of hemagglutination present in allantoic fluid suSpensions of certain strains of Newcastle disease virus, and in normal allantoic fluid of thirteen-day-old embryos, was largely re- moved by dialysis as reported by Williamson, Simonsen, and Blatt- ner (98). The inhibitor was heat-stabile and was not destroyed by RDE. Red blood cells suspended in allantoic fluids for varying 15 periods of time regained their agglutinability after washing. The in- hibitor retained complete activity. Cell receptor sites. The nature of red blood cell receptor sites has not been elucidated. After treatment with influenza virus or RDE there is a sharp decrease in the electrophoretic velocity of human erythrocytes, representing a diminution in the net negative charge (1, 50). Red cells treated with suitable amounts of periodate adsorb viruses of the influenza group, but neither spontaneous elution nor artificial removal by RDE occurs according to Fazekas de St. Groth (41). Erythrocytes treated with periodate—virus RDE (PVR cells) have active virus bound firmly to the modified receptors. PVR cells have the following properties: 1. They do not agglutinate spontaneously. 2. They are not agglutinated by the addition of influenza virus. 3. They agglutinate all cells susceptible to influenza virus a gglutinins . 4. They destroy receptors on normal cells and the virus inhibitor in mucoids. 5. They are agglutinated by specific antisera to the virus with which they are incorporated. l6 6. The agglutinating and enzymatic activities of these cells are neutralized by homologous antibody. The influenza virus receptors of chicken red cells resist destruction by a number of oxidizing agents (iodine, hydrogen per- oxide, potassium permanganate, and potassium dichromate), but are readily destroyed by sodium periodate at concentrations greater than that required for modification of cellular receptors (58). Burnet (14) states that the effect of periodate indicates the likelihood that the specific configuration of certain carbohydrate units is responsible for adsorption of virus and for susceptibility to en- zymatic action. Fazekas (42) reported that treatment of influenza virus with relatively large amounts of periodate destroyed infectivity, enzyme activity, antigenicity, and hemagglutinative activity. Cellular receptors in excised mouse lung and in the formalin- ized (0.1 ml of 4 per cent formalin) allantoic cavity can be modified so that they adsorb virus but neither spontaneous elution nor arti- ficial liberation by RDE occurs. Adsorbed virus is released from the untreated membranes upon introduction of RDE and also occurs spontaneously. Periodate treatment produces identical receptor modi— fication in vivo, both in mice and eggs (42). 17 Influenza virus infection may take place through periodate- modified receptors on which RDE cannot act. This may be deter— mined by: 1. Identity of infectivity end points in periodate-treated and in normal mice and eggs. 2. Reduction of the prophylactic effect of RDE in periodate- treated lungs. 3. Absence of interference in the allantoic cavity by heat- inactivated virus in the presence of RDE, if the active virus has been adsorbed to receptors modified by perio- date. Fazekas stated: "The assumption of enzymic receptor de- struction as the mechanism of infection (Hirst) is criticized, and a new hypothesis proposed: 'Preceded by specific adsorption of the virus to cellular receptors, infection is initiated by means of viropexis, the passive uptake of the infective particle by the host cell; enzymic destruction of receptors is not essential."' Vaccinia and related hemagglutinins. Vaccinia, variola, and ectromelia viruses possess a hemagglutinin that is distinct from the virus particle. Two hemagglutinating fractions have been found in vaccinia virus suspension of infected chorioallantoic membranes. 18 One is a soluble hemagglutinin and is completely separable from the infective virus particle whereas the other is closely associated with the elementary body (16, 46). Hemagglutinins of this group are inhibited by Specific immune sera but not by normal sera. Spontaneous elution of adsorbed hemag- glutinin does not occur, but the hemagglutinin can be removed by ad- dition of immune serum and the red cells are reagglutinable. Vaccinia hemagglutination is demonstrable only with chicken and pigeon cells (90). Hemagglutination with arthropod—borne viruses (21). The use of acetone and ether extracts of brain tissues of newborn mice in- fected with certain arthropod-borne viruses has made it possible to demonstrate hemagglutinins for chicken red cells associated with the following viruses: Dengue Type 1, Dengue Type 2, Eastern equine encephalomyelitis (EEE), Ilheus, Japanese B, Ntaya, St. Louis, Sindbis, Uganda S, Venezuelan equine encephalomyelitis (VEE), West Nile (Egypt 101 strain), Western equine encephalomyelitis (WEE), and yellow fever viruses. On the basis of temperature and pH requirements for hemag- glutination, the viruses may be divided into two groups: (1) those that require 37°C. and pH 6.4--EEE, VEE, WEE, and Sindbis viruses; l9 and (2) those that require either 4°C. or 22°C. and pH 7.0--Dengue Types 1 and 2, Ilheus, Japanese B, Ntaya, St. Louis, Uganda S, West Nile, and yellow fever viruses. Serological crossreactions may be found among viruses of each group, but antisera to one group do not react with antigens of the other group. Antibodies against certain viruses for which no hemagglutinin could be detected react with members of either one or the other group. Semliki Forest virus appears to belong to Group 1 and Russian far eastern and louping-ill viruses to Group 2. Action of enzymes in hemagglutinating systems. Rh-positive erythrocytes treated with trypsin and other proteolytic enzymes (pepsin, papain. erepsin; Chymotrypsin, bromelin, and Russel viper venom) are modified so that they show specific agglutination in saline dilutions of incomplete (albumin—active) anti-Rh sera (96). Agglutination of trypsin-modified cells by incomplete antibodies occurs in the absence of hydrophilic colloids which are required for the agglutination of unmodified cells. Trypsin, in low concentration, modifies red cells so that they are agglutinated by Saline dilutions of homologous antibody. When uSed in progressively higher concentrations, trypsin alters the red 20 cells so that they are agglutinated first by most normal sera and finally by saline. Agglutination of the more vigorously treated red cells by normal sera may be due to the T agglutinin found in many normal sera. The autoagglutination by saline of the most vigorously trypsin— treated erythrocytes, however, is a nonspecific effect (97). Morris (77) treated mouse brain suspensions of the GD VII strain of murine encephalomyelitis virus with trypsin which induced the viral agglutination of human group 0 cells at 20°C., whereas untreated virus agglutinated these cells only a 4°C. An effect on the red cell was excluded because washed, trypsin—treated cells failed to agglutinate at 20°C. in the presence of the untreated virus. Trypsin—treated virus, sedimented and washed by high—speed centrifugation, agglutinated human cells well at 20°C. Enzymes and Enzyme-inhibitors The major proteolytic enzymes of pancreatic juice are tryp- sin, Chymotrypsin, and a carboxypolypeptidase, each of which has been obtained in pure form. The combined activities of these en- zyrn es Was formerly believed to be due to trypsin alone. The en— zyme Combination is referred to as pancreatin (52). *————_ ,_.__..—_ ,4 ‘J g, ' ‘ our 2, 23;», , , ., A 21 Trypsin and Chymotrypsin may be classified as endopeptidases acting on peptide linkages either in the central or terminal portions of polypeptide chains. Bergmann and Fruton (8), through the use of synthetic peptide substrates, found that trypsin acts on peptide link- ages containing the carboxyl groups of either arginine or lysine. Chymotrypsin, on the other hand, was found to act on peptide link- ages involving the carboxyl group of tryosine and phenylalanine. The digestive action of trypsin and Chymotrypsin appears to involve the splitting of specific types of peptide linkages in the molecule, thereby producing either low molecular weight polypeptides or free amino acids. Trypsin has its greatest activity at pH 8 to 9. The optimum pH depends on ionic strength of the buffer, concentration of the sub- strate, and somewhat on the nature of the substrate. Carboxypolypeptidase activity is most likely due to a complex of many enzymes and is an example of an exopeptidase. In addition to the principal enzymes, pancreatic amylase and pancreatic lipase are found in the pancreatic juice. Activation of the major proteolytic enzymes of the pancreas is Presented in the following: 22 Precursor Activator Enzyme T rypsinog en Neutral pH or Trypsin Enterokinas e Chymot rypsinogen Trypsin Chymot rypsin Ca rboxypolypeptidas e Proca rboxypolypeptidas e Trypsin The peptide bonds are not the only groups attacked by the enzymes, since hydrolytic cleavage also occurs if the peptide bond is replaced by an ester bond. The mode and extent of cleavage are not completely deline- ated. Large amounts of free amino acids are formed when crude preparations of pancreatin act on casein or other proteins, but no amino acids are formed when casein is exposed to the action of crystalline trypsin (51). There are a number of naturally occurring trypsin inhibitors such as the soybean, colostrum, lima bean, ovomucoid, blood plasma, urine, Ascaris sp., and pancreatic inhibitors (66). Increased inhibi- tion of trypsin by serum is associated with disease processes that bring about cellular destruction. The diagnostic significance of in- creaS ed trypsin or Chymotrypsin inhibition is the same as that of increased fibrinogen concentration. It is a common, nonspecific r esPOIISe to a variety of pathological conditions, and has no value as a specific diagnostic test (33). 23 Gorini and Audrain (47) reported that after addition of one equivalent of ovomucoid to one equivalent of trypsin about 7 per cent of the tryptic activity remained. They ascribed this remaining activity to the enzyme-inhibitor complex itself. The addition of calcium to the system resulted in a gradual disappearance of ovo- mucoid. They concluded that the simultaneous presence of calcium and trypsin inactivated the ovomucoid and rendered it susceptible to proteolytic digestion. Inactivation did not occur in the absence of trypsin and occurred very slowly in calcium-free systems. Later (48), they concluded that the ovomucoid complex be- haved as an enzyme with a proteolytic activity corresponding to about 10 per cent of the activity of the trypsin present. Only traces of digestion of soybean inhibitor are noticed after prolonged incubation in the presence of trypsin (64). Lima bean inhibitor exerts its full action immediately upon contact with trypsin. The amount of trypsin inhibited is directly proportional to the amount of inhibitor present, indicating a stoichio— metric reaction (68). Green (49) states that the inhibition of trypsin by diisopropyl fluorolfl'iosphate, ovomucoid, soybean, and pancreatic inhibitors is of the competitive type, since it is overcome in the presence of the Substrat e , benzoyl- L- arginine ethyl ester. EXPERIMENTAL PROCEDURES The study was conducted in two parts: (1) the investigation of hemagglutinative activity associated with IBV; (2) the possible use of IBV hemagglutinative activity for diagnosis and characterization of IE. Materials and Preparations 1. Viruses: The strains of IBV used were from the North Central IBV Repository at Michigan State University, and are listed below: Reposi- tory Egg Code Pas- No. Code Isolated by sage 1 314 M. S. Hofstad, Iowa State College, Ames, 6 Iowa (1948) 2 33 M. S. Hofstad, Cornell University, Ithaca, 9 New York (1944) 3 104 M. S. Hofstad, Iowa State College, Ames, 4 Iowa (1947) 16 66 O. Hipolito, Universidade Rural de Estade 30 de Minas Gerais, Escola Superior de Ve- terinaria, Brazil (1955) 19 999 c. s. Roberts, Alabama Dept. Agr. (1956) 4 24 w, ._— w‘ i. Reposi- tory Code No. 20 21 23 24 40 41 42 Code 571 331 SIMS 586 25 Egg Pas- Isolated by sage C. S. Roberts, Alabama Dept. Agr. (1956) 9 C. S. Roberts, Alabama Dept. Agr. (1956) 6 C. S. Roberts, Alabama Dept. Agr. (1956) 13 C. S. Roberts, Alabama Dept. Agr. (1956) 9 M. P. Spring, Michigan State U. (1956) 0 H. Van Roekel, U. of Massachusetts 0 (1941) (291st bird passage) F. R. Beaudette, Rutgers U., New Un- Brunswick, New Jersey (1935) known (hun— dreds) The strains of IBV will be identified by the Repository code number and number of egg passages; e.g., 40—5 indicates repository code 40, fifth egg passage. IBV strain 42 is completely egg-adapted. This strain has been studied extensively and is often referred to in the literature as the Beaudette strain. A strain of Newcastle disease virus (NDV) (Accession 51-52- 308) which originally had been isolated at Michigan State University from lung and tracheal material of chickens infected from a natural ontbreak of the disease was used for certain comparative studies. 26 Normal allantoic fluid served as a control. All viruses were cultivated in embryonating chicken eggs via the allantoic cavity (0.1 ml inoculum). Nine-day embryos were used for all strains of IBV except the egg-adapted strain, which was inoc- ulated into ten—day embryos as was NDV. All normal and viral- infected allantoic fluids were collected from twelve-day embryos. At the time of harvest each allantoic fluid preparation was pooled, distributed in 30 ml screw-cap vials, and stored at -30°C. until used. At the time of use the virus suspension was thawed, centri— fuged at 3,000 r.p.m. for 20 minutes at 4°C., and the clear super— natant fluid was removed for the tests. Samples were frozen only once as repeated cycles of freezing and thawing have a deleterious effect on IBV infectivity. 2. Erythrocytes: Four adult Single Comb White Leghorns served as the source for erythrocytes which were obtained by cardiac puncture. Blood Was collected in chemically clean test tubes containing 1 ml of a 2 per- Cent sodium citrate solution for each 9 m1 of blood. The cells Were washed three times in ten or more parts of 0.85 per Cent saline, centrifuged twice at 1,600 r.p.m. for eight minutes. and 27 finally at 1,000 r.p.m. for 10 minutes. After the third washing the saline was removed and the packed cells were stored at 4°C. Cells were used in hemagglutination tests within one week of the time of collection. The author was the donor of human 0 cells. Cow, horse, sheep, dog, cat, and rabbit erythrocytes were collected from animals available in the College of Veterinary Medi- cine. 3. Diluents: 0.85 per cent sodium chloride. Bacto hemagglutination buffer (Difco), pH 7.2. 4. Sera: Normal serum was collected from chickens reared in isola- tion. Anti-IB serum was collected six weeks after intratracheal inoculation of adult chickens with 0.5 ml of IBV. All sera were Quantitatively assayed by serum-neutralization tests. 5. Enzymes: Trypsin, 1:250 (Difco). Trypsin (1 per cent) for hemagglutination (Difco). Trypsin, 2x crystalline, salt—free (Nutritional Biochemical Corporati on- - NBC). 28 Chyrnotrypsin, salt-free, from ethyl alcohol (NBC). Chymotrypsinogen, crystalline, salt-free (NBC). Papain, N.F. (Difco). Pepsin, 1:10,000 (Difco). Rennin, N.F. (Difco). 6. Enzyme inhibitors: Eggwhite trypsin inhibitor (NBC). Soybean trypsin inhibitor, 5X crystalline (NBC). 7. Amino acids and peptones: L-arginine, free base (NBC). Glycine, anhydride (NBC). L-lysine, monohydrochloride (NBC). L-methionine (NBC). L—phenylalanine (NBC). L-serine (NBC). L-tyrosine (NBC). Trypticase (Baltimore Biological Laboratories). Bacto-Tryptone (Difco). Ba cto- Tryptose (Difco) . Z9 3 . Bacterial filtrates: Clostridium welchii, types A, C, and D, were incubated at 37°C. for 18 hours in a medium of equal parts of pork infusion and thioglycollate broth. The cultures were centrifuged at 3,200 r.p.m. for 30 minutes at 4°C. and the supernatant fluid was collected. 9. Colloids: Arsenic trioxide. Red colloidal gold. Chromium hydroxide. Ferric hydroxide. Prussian blue. Silver. Methods 1. Hemagglutination (HA) test: Serial twofold dilutions of the virus samples were prepared With Bacto hemagglutination buffer through the range of 1:5 to 1:2560 or higher, as required. In a row of 12 x 75 mm tubes parallel to the tubes contain- ing the Virus dilutions, 0.25 ml each of the buffer, appropriate virus dilution, and 0.5 per cent suspension of chicken red cells were added 30 to each tube. In some tests cells from other species were used. The control tube contained 0.5 ml of buffer and 0.25 ml of cells. The tubes were shaken well and incubated for one hour at room temperature. In some instances they were incubated at differ- ent temperatures . The end point was the greatest dilution of virus which showed a pattern of complete hemagglutination. No attempt was made to read partial reactions. The titer, expressed as hemagglutinative units, was the re- ciprocal of the highest dilution of virus, before the addition of saline and cells, which produced complete hemagglutination. 2. Hemagglutination—inhibition (HI) test: a. Alpha procedure: This test was prepared in the same manner as the HA test except that 0.25 ml of serum, diluted 1:5, was substituted for 0.25 ml of buffer in each tube. The control tube contained 0.5 ml of serum and 0.25 ml of cells. The serum titer was the reciprocal of the lowest dilution of virus in which hemagglutination was completely inhibited. The HI titer of the serum was computed from the following formula : ‘-“"'W 1 italf - 31 Virus titer —-——_—— X dilution of serum = HI titer. Serum titer b. Beta procedure: Constant amounts of virus, expressed in HA units, were em- ployed with decreasing concentration of serum ranging from 1:5 through 1:2560. The serum titer is the reciprocal of the highest dilution of the serum in which hemagglutination is completely inhibited. The HI titer of the serum is computed for the beta procedure by multi- plying the serum titer by the number of HA units used in the test. The following formula is used: Serum titer X number of HA units = HI titer. 3. Hemagglutination test using trypsin—modified IBV: The HA test developed during the present study and used for investigation of characteristics of IBV employed the same procedure as the conventional HA test. The only difference was that IBV modi— fied by trypsin was used. The modified IBV was also used for the HI test. 4 - Viral infectivity: For quantitative determination of viral infectivity, serial ten- fOId dilutions of the virus were prepared in nutrient broth using I L __ 2‘ >’-— “fig-J 32 separate pipettes per dilution. Five eggs were employed per dilu— tion, and each was inoculated with 0.1 ml via the allantoic cavity. The eggs were incubated at 99-99.5°F. in an electric forced-draft incubator for five days following inoculation. Embryo mortality during the first 24 hours was considered to be due to nonspecific causes, and these were not included in the final results. Mortality and gross lesions were the criteria for infectivity of strains other than the egg-adapted strain 42, and were used in computing the titer, which was expressed as the 50 per cent infec- tivity end point, IDSO' according to the method of Reed and Muench (84). Lethality was the criterion for strain 42, and the titer was expressed as the LD50. 5. Serum-neutralization tests: Dilutions were prepared in the same manner as described for infectivity titration. Two rows of tubes were set up parallel to the dilution tubes. dilution of the test serum per tube. The second series contained 0.3 ml of nutrient broth per tube. To each was added 0.3 m1 of the corresponding virus dilution. Separate pipettes were used for preparing all mixtures. Inoculation was via the allantoic cavity, 0'1 ml per egg, five eggs per dilution, and incubation and One series of tubes contained 0.3 ml of a 1:5 -1 11w x; on ' 33 observations were made as described earlier. The series containing serum was inoculated prior to the controls to exclude the possibility of thermal environmental effects on the virus. The 50 per cent end point was used to evaluate all titrations. The LD50 neutralization index (LDSONI) was the difference between the reciprocal of the virus and the serum titers. The antilog was the number of neutralizing doses. _.__ W.—.'~ EX PE RIMENT AL RES ULTS Hemagglutination pH of normal and viral-infected allantoic fluid. Since the investigation would utilize trypsin and perhaps other proteolytic enzymes, it was necessary to determine the pH of normal and viral- F infected allantoic fluid to ascertain this environmental factor which influences enzyme activity. The results are presented in Table 1. The pH range of the samples tested was within the optimum, pH 8—9, for the activity of trypsin (79). Modification of infectious bronchitis virus by trypsin. The initial approach to modification of IBV by trypsin was concerned with the concentration of trypsin and the effects of time and temperature on the reaction. A l per cent aqueous suspension of trypsin (T) (1:250, Difco) was prepared and passed through a Seitz EK filter pad. Al- though T' is not completely soluble in water at this concentration, reference Will be made to the filtrate as l per cent T. Appropriate volumes of 1 per cent T were added to the allantoic fluid prepara- tions to give the desired final concentration of T; e.g., 1 ml 01' 34 35 TABLE 1 pH OF NORMAL AND VIRAL-INFECTED ALLANTOIC FLUID FROM TWELVE-DAY-OLD CHICKEN EMBRYOS Age of Embryo Sample When Inoculated pH NAFa 8.49 b IBV 40-5 9 days 8.02 IBV 42 10 days 8.09 NDv° 10 days 8.52 aNAF: Normal allantoic fluid. bIBV: Infectious bronchitis virus with respiratory code number. cNDV: Newcastle disease virus. 1 per cent T was added to 2 ml of allantoic fluid to give a final concentration of 0.33 per cent T. The 1 per cent T solution, alone or in combination with sa- line, served as the control. The results of these studies are presented in Tables 2, 3, and 4. Hemagglutination occurred with the controls as well as with the nor mal and viral-infected specimens. It was evident that higher 36 TABLE 2 HEMAGGLUTINATION BY TRYPSIN-MODIFIED IBV Strain Final Con- Tem- HA Titer (reposi- centration pera- Time HA after Ad- tory of Trypsin ture (min.) Titer dition of number) (Pct) (’C.) EWTI 40-6 0.5 4 150 320 40-6 0.5 23 120 1280 40-5 0.5 37 45 640 40-6 0.5 37 90 1280 2:11:21 0.25 23 o 1280 0 3:13:31 0.25 37 180 1280 0 40-6 0.25 4 225 640 40-6 0.25 4 270 320 40-6 0.25 23 120 640 40-8 0.25 23 120 640 40-6 0.25 23 180 1280 40-6 0.25 23 18o~ 1280 40-6 0.1 23 240 20 40-6 0.1 23 720 320 4 0-6 0.1 37 180 160 40- 6 0.1 37 375 640 40- 6 0.33 23 180 640 40- 6 0.33 23 240 640 40- 6 0.33 23 720 2560 40. 8 0.33 37 120 640 40- 6 0.33 37 375 2560 40- 8 0,33 37 180 2560 N __ L TABLE 2 (Continued) 37 Strain Final Con- Tem- HA Titer (reposi- centration pera- Time HA after Ad- tory of Trypsin ture (min.) Titer dition of number) (pct.) (° C.) EWTI 40-6 0.33 37 180 2560 40-5 0.33 37 180 5120 40-4 0.33 37 180 5120 40-5 0.33 37 180 640 320 40-5 0.33 37 180 5120 2560 40-5 0.33 37 180 1280 40-5 0.33 37 180 5120 40-5 0.33 37 180 5120 40-5 0.33 37 180 10240 40 5 0.33 37 180 10240 40-5 0.33 37 180 10240 40-6 0.33 37 180 2560 40-15 0.33 37 180 5120 2560 40-16 0.33 37 180 5120 2560 40-17 0.33 37 180 5120 2560 l -7 0.33 37 180 2560 2-10 0.33 37 180 5120 23—14 0.33 37' 180 2560 40-3 0.33 37 180 5120 40-25 0.33 37 180 1280 3:311:21 0.33 37 180 1280 o TABLE 3 HEMAGGLUTINATION BY TRYPSIN- MODIFIED IBV (egg-adapted strain 42; incubation at 37°C. for 180 minutes; final concentration of trypsin, 0.33 per cent) 38 HA Titer HA Titer after Addition of EWTI 640 640 640 640 1280 1280 2560 2560 2560 160 320 320 320 640 640 1280 1280 2560 2560 2560 2560 2560 2560 H . oooooooooooo, N CO 160 320 320 33!. I V fi_'_fi‘_fm we...“ TABLE 4 HEMAGGLUTINATION BY TRYPSIN- MODIFIED NORMAL ALLANTOIC FLUID 39 Final Con- Tem- HA Titer centration pera- Time HA after Ad— of Trypsin ture (min.) Titer dition of (pct.) (°C.) EWTI 0.5 4 150 320 0.5 23 120 160 0.5 37 45 320 0.5 37 90 320 0.25 4 225 40 0.25 4 270 20 0.25 23 120 40 0.25 23 180 20 0.25 23 180 80 0.1 23 240 40 0.1 23 720 160 0.1 37 180 10 0.1 37 375 160 0.33 23 240 640 0.33 23 720 160 0.33 37 120 40 0.33 37 240 640 0.33 37 375 320 0.33 37 180 40 0.33 37 180 160 0.33 37 180 160' 40 TABLE 4 (Continued) Final Con- Tem- HA Titer centration pera- Time HA after Ad— of Trypsin ture (min.) Titer dition of (pct.) (°C.) EWTI 0.33. 37 180 640 0.33 37 180 0 0.33 37 180 0 0.33 37 180 0 0.33 37 180 10 O 0.33 37 180 10 0 0.33 37 180 20 0 0.33 37 180 40 0 0.33 37 180 160 0 0.33 37 180 320 0 0.33 37 180 320 0 0.33 37 180 320 0 0.33 37 180 640 0 0.33 37 180 640 10 0.33 37 180 640 0 41 BA titers were obtained with IBV preparations than with the controls or NAF. Although the results were somewhat variable, the highest and most uniform HA titers were obtained with samples containing a final concentration of 0.33 per cent T and which had been incu- bated for three hours at 37°C. These conditions were selected for the basic procedure. To determine if the reaction was due to the virus or to the T alone, inhibition of T was attempted. Inhibition of T in the T-IBV mixtures at 100°C. for two minutes caused macroscopic precipitation of proteins which made the suspension valueless for the HA test. The supernatant fluid did not possess hemagglutinative activity. Resort was then made to naturally occurring T inhibitors. Eggwhite trypsin inhibitor (EWTI) was initially selected because of economy and solubility. Soybean trypsin inhibitor (SBTI) (5X crystalline) was used in the latter part of the study for comparison to EWTI. The T—SBTI complex exhibits no tryptic activity, and SBTI is resistant to tryp- tic digestion. EWTI is slowly digested by T and the T-EWTI com- plex may exhibit as much as 10 per cent of the digestive activity of T alone (47, 64). Following incubation of the samples with T, inhibitor was added and the HA test performed. A l per cent solution of EWTI 42 was used. The final concentration of EWTI in the mixture was equal to the T concentration. For example, 1 ml of l per cent T plus 2 m1 of sample resulted in a concentration of 0.33 per cent T. Ad- dition of 1 ml of l per cent EWTI to this mixture resulted in a final concentration of 0.25 per cent T and EWTI, respectively. This provides equal amounts of T and EWTI on the basis of weight by volume per cent, but not molality. Different volumes of T and EWTI may be used provided the final concentrations are equal. The molec- ular weight of EWTI is 28,000 as determined by osmotic pressure and 32,000 as determine by sedimentation diffusion. The average molecular weight of trypsin is 24,000 (66). Due to the relative insolubility of SBTI, a 0.25 per cent suSpension was used. To insure inhibition of T by an equal volume of SBTI, a 0.25 per cent solution of T was necessary. The results obtained with EWTI are presented in Tables , 3, and 4. The final mixture in the HA test was at pH 7.2, which is Within the range (pH 6 to pH 8) of stability of the influenza hemag- glutinin. Chicken red cells are spontaneously agglutinated or hem olyzed at greater extremes of pH (61). It is evident that hemagglutination is associated with the virus modified by treatment with trypsin. EWTI eliminated 43 hemagglutination by T in the saline controls and NAF. It is obvious that the hemagglutinative activity of the egg-adapted strain of IBV (IBV 42) was considerably less than that of other strains. SBTI, used in parallel with EWTI, provided identical titers with the strains tested. Based on these findings the viral modification procedure was standardized. The standard reaction system comprised 1 ml of 1 per cent T in combination with 2 ml of normal or viral-infected allantoic fluid. This mixture was incubated at 37°C. for three hours and 1 m1 of 1 per cent EWTI was then added; The final mixture was tested for hemagglutinative activity. This procedure was con- sidered to be the "Standard T Process." The system was maintained bacteria free until the addition of EWTI. Filtration resulted in diminished activity of EWTI. The diminished activity was a reflection of reduced concentration which likely resulted from loss of EWTI by adsorption to the filter. Repository strain IBV 40-5 was selected as the standard test strain. Further results, representative of the standard T process, are presented in Table 5. A temperature control was run in order to determine the effECt Of incubation at 37°C. on the virus and its role in the rm: '1. .‘ ' - mzurrw-vr TABLE 5 HEMAGGLUTINATION BY TRYPSIN- MODIFIED IBV AND NAF (STANDARD T PROCESS) 44 (35:13:21 IBV 40-5 IBV 42 NAF o 640 20 o o 1280 o o o 2560 20 o o 2560 o o o 2560 o o o 2560 o o o 2.560 0 o o 2560 o o o 2560 o o o 2560 o o o 2560 o o o 5120 320 o o 5120 o o o 5120 20 o 0 20480 0 o 0 2.0480 40 o 45 “\duction of the hemagglutinin. NDV was introduced as a positive control for hemagglutination. The results are presented in Table 6. TABLE 6 EFFECT OF INCUBATION AT 37°C. ON HEMAGGLUTINATION TITER Period of Incubation Strain None One Two Three Hour Hours Hours NDV 1280 1280 12.80 1280 IBV 40-5 0 0 0 0 IBV 42 0 0 0 0 NAF 0 0 0 0 Incubation for extended periods at 4°, 23°, or 37°C., in itself, confers no hemagglutinative activity to IBV (27). To exclude the possibility of a nonspecific reaction, further Studies on the tryptic activity were done, and NDV was included in the series. The effect of varied periods of incubation was concur- The results are expressed in Table 7. rently determined. The data further establish the optimum time of incubation as TABLE 7 EFFECT OF TIME OF INCUBATION AT 37°C. ON HEMAGGLUTINATION IN STANDARD T PROCESS 46 Time of Incubation IBV 40-5 IBV 40-5 IBV 40-5 NDV None - - . .......... 20 40 20 320 1 hour . . .......... 80 1280 640 320 2 hours . .......... 160 5120 2560 320 2'1/2' hOurs ........ 320 320 3 hours ........... 320 10240 5120 320 4 hours ........... 160 Modification of infectious bronchitis virus by other proteo- 1 acfifity in this system were: 1 . Pepsin, l per cent solution. 2. Rennin, l per cent solution. 3. Papain, 0.5 per cent solution. W. Other proteolytic enzymes tested to determine their The enzymes were prepared in distilled water, passed through Seitz filter pads, and employed in the following manner: One milliliter of enzyme solution was added to 2 ml of the normal and viral-infected allantoic fluids and incubated at 37°C. for A 47 three hours. No inhibitors were added. The enzyme-fluid mixtures were then used in HA tests. The results are presented in Table 8. TABLE 8 EFFECT OF CERTAIN ENZYMES ON HEMAGGLUTINATION . (incubation for three hours at 37°C.) E Enzyme IBV 40-5 IBV 42 NAF NDV —\ l H N°ne ........... o o o 320 TrYPSin ........... 5120 1280 160 320 Pepsi“ ........... o o o 320 Remain ........... o o o 320 Papain ........... o o o 320 Of the four enzymes employed, only trypsin was active on IBV at pH 8-9 as evidenced by HA. NDV was used as a control, and its hemagglutinative activity was in no way changed by the ac— tion of the enzymes. A further investigation of enzyme activity was undertaken through the use of crystalline trypsin (ZXT), Chymotrypsin, and c1"ymc’trypsinogen. Since no inhibitor for Chymotrypsin was avail- able. Chymotrypsinogen was used with ZXT, its activator, and 48 inhibition of Chymotrypsin in this system was controlled by inactivat— ing the ZXT with EWTI. Different volumes and concentrations of crystalline enzymes were employed in these tests. This was predicated on the factor of differential solubility of the crystalline enzymes as compared to T. Purity of crystalline enzymes as compared to T had also to be con- side re d . The activity of the crystalline enzymes is presented in Tables 9, 10, and 11. It is evident from the results that ZXT is more reactive than T in thuts system. One per cent ZXT cannot be substituted for 1 per cent T in the standard T process, whereas 0.25 per cent 2.xT may be used_ This discrepancy resulted from the differential solubilities Of T and ZXT. The ZXT is completely soluble in water at a concen- tration of 1 per cent, whereas the maximum solubility of T is ap- proXimately at a concentration of 0.25 per cent. Thus, 1 per cent ZXT Was more reactive and the resultant viral product had no ca- pacity for hemagglutination. At the optimum ZXT concentration, HA was evidenced in high titer with IBV, in reduced titer with the egg-adapted strain, IBV 42. and not at all with NAF. .- :1 may” TABLE 9 49 EFFECT OF CRYSTALLINE TRYPSIN ON HEMAGGLUTINATION —— Incubation EWTI T ° HA rypsln Sample at 37°C. 1 pct. 1 m1 1% 2 m1 40-5 1 hour 1 ml 0 1 ml 17.. 2 m1 40-5 2 hours 1 m1 0 1 m1 1°70 2 ml 40-5 3 hours 1 ml 0 lm1*0,1 6% 2 ml 40-5 1 hour 1 ml 0 1 m1 0 .1 6% 2 ml 40-5 2 hours 1 ml 0 1 ml 0.1 6% 2. m1 40-5 3 hours 1 m1 0 1 1101 0-33% 2 m1 40-5 1 hour 1 m1 0 1 ml 0.33% 2 ml 40-5 2 hours 1 m1 0 1 ml 0.33% 2 ml 40—5 3 hours 1 ml 0 1 ml 0.75% 2 m1 40-5 1 hour 1 m1 0 1 ml 0.75% 2 ml 40-5 2 hours 1 m1 0 1 ml 0.75% 2 m1 40-5 3 hours 1 ml 0 0-25 m1 1% 2 ml 40-5 3 hours 0.25 ml 1280 0.12.5 m1 1% 2 ml 40-5 3 hours 0.25 ml 2560 0.25 ml 1% 2 m1 40-5 3 hours 0.25 ml 5120 0.25 ml 1% 2 m1 40-5 3 hours 0.25 ml 5120 0-25 m1 1% 2 ml 42 3 hours 0.25 ml 160 °~25 m1 1% 2 m1 NAF 3 hours 0.25 ml 0 1 m1 1% ................. 2560 1 m1 1°70 . ........... 1 ml 0 \ l EFFECT OF CHYMOTRYPSIN ON HEMAGGLUTINATION (incubation for three hours at 37°C.) TABLE 10 50 C'31'1ymotrypsin Sample HA 1 ml 1% 2 m1 40-5 640 1 ml 1% 2 ml 42 80 1 ml 1% 2 ml NAF 0 0.25 ml 1% 2 ml 40-5 640 0.25 ml 1% 2 ml 42 40 0.25 ml 1% 2 ml NAF 0 1 m1 1% ....... 640 TABLE 11 EFFECT OF CHYMOTRYPSINOGEN, ACTIVATED BY CRYSTALLINE TRYPSIN, ON HEMAGGLUTINATION (incubation for three hours at 37°C.) 51 ChYm 0t rypsinogen Trypsin Sample ($32331 ) HA _\ 1 m1 1% 0.1 m1 1% 2 m140-5 0.1 m1 1230 1 m1 1% 0.1 ml 1% z m142 0.1 ml 160 1 m1 1% 0.1 m1 1% 2 m1 NAF 0.1 ml 0 0-2-5 ml 1% 0.1 ml 1% 2 m1 40-5 0.1 ml 1280 0-25 ml 1% 0.1 m1 1% 2 ml 42 0.1 ml 160 025 ml 1% 0.1 m1 1% 2 ml NAF 0.1 ml 0 1 m1 1% ................... o J‘— 52. It cannot be stated unequivocally that chymotrypsin produces a mOdification of IBV like that produced by T. Although inactivation was attempted, the possibility of residual chymotrypsin cannot be discounted since the enzyme, in itself, agglutinated red cells. Turbidimetric determination of hemagglutination. In the stand- ,1' ard T process a visible turbidity was produced upon addition of EWTI tC) the T-IBV mixture. In an attempt to correlate optical r density and HA, a Beckman Model B spectrophotometer was used and oI>tieal density was measured at a wave length of 500 mu. The results are presented in Tables 12, 13, and 14, and in Figure I. Hemagglutination varied directly with optical density and ID . 50 No turbidity is evidenced if ZXT and EWTI are mixed. The turbidity appears only upon mixing T and EWTI. Since T and not E” II is present during the initial incubation of the standard T proc- ess, a turbidity curve of T-EWTI was plotted maintaining EWTI constant. The results are presented in Table .15 and in Figure II. The production and nature of the precipitate in the T-IBV- EWTI 00mplex was considered to be the result of one of the follow— ing: (1) a reaction between free T and EWTI; or (2) a reaction between EWTI and some product or products of T digestion. 53 TABLE 1 2 OPTICAL DENSITY AND HA TITER OF IBV STRAINS IN STANDARD T PROCESS ‘ Strain HA Titer Op tical DenS1ty — F 1-7 640 0.24 3-6 1280 0.32 40-5 1280 0.42 40-5 2560 0.48 41-1 2560 0.50 2-11 5120 0.62 54 TABLE 13 OPTICAL DENSITY AND HA TITER OF IBV STRAINS IN STANDARD T PROCESS AT DIFFERENT INCUBATION PERIODS Strain 1.3.3212: HA Titer 352:1: 40—5 1 hour 1280 0.26 40-5 2 hours 5120 0.31 40—5 3 hours 10240 0.42 40-5 3 hours 10240 0.46 40-1 3 hours 0 0.055 40-25 3 hours 2560 0.28 TABLE 1 4 55 OPTICAL DENSITY, HA TITER, AND INFECTIVITY OF DILUTIONS OF IBV 40-5 IN STANDARD T PROCESS Embryo . Infectivity Dilution HA Titer Opt1ca1 Prior to Dens1ty Treatment (log 10/0.1 m1) None 2560 0.40 7.0 1 2 640 0 30 6.7 1:4 20 0.185 6.4 1 8 10 0 10 6.1 1:10 0 0.078 6.0 1 16 O 0 05 5.8 -w—u—w FIGURE I HEMAGGLUTINATION TITER AND OPTICAL DENSITY OF IBV 40-5 AFTER STANDARD T PROCESS 2 600 2400 2200 2000 .1800 1600 HA Titer 14001 1200 1000 800 600 _ 400 l l l i 1 1 —.2 -.3 -.4 -.5 ~.6 -.7 -.8 Log Dilution (Base 10) l -_9 I l -l.0 -l.l Optical Density ‘_.l--.._- .L TABLE 15 OPTICAL DENSITY OF MIXTURES OF T AND EWTI (wave length, 500 mu) 57 Tube 1% T Distilled 1% Optical N0 . (ml) Water EWTI Density (ml) (ml) 1 1.0 2.0 1.0 0.75 2 0.9 2.1 1.0 0.62 3 0.8 2.2 1.0 0.36 4 0.75 2.25 1.0 0.34 5 0.7 2.3 1.0 0.267 6 0.6 2.4 1.0 0.23 7 0.5 2.5 1.0 0.16 0.4 2.6 1.0 0.095 9 0.25 2.75 1.0 0.065 1 O 0.2 2.8 1.0 0.05 1 1 0.1 2.9 1.0 0.023 1 2. 3.0 1.0 0.00 1 3 1.0 3.0 0.00 58 FIGURE II OPTICAL DENSITY OF MIXTURES OF T AND EWTI 0 8 (1 m1; wave length, 500 mu) ' sit Opt1cal c>Den y o .u in C W 0.2. 0.1 ~10 .20 .30 .40 .50 .60 .70 .80 mlT .90 1.0 59 For further information on the nature of the turbidity, T was added to NAF, IBV 40-5, and IBV 42, followed immediately by addi- tion 0f EWTI. No turbidity was evident immediately in any of these mixtures, but after 15 to 20 minutes turbidity appeared with IBV 40-5 This is in keeping with the rate of appearance of the precipi- tate in the standard process. #9,]. M Later trials with a solution that contained 0.25 per cent T '4 prior- to filtration, and which produced HA titers comparable to a “1058 of 1 per cent T, produced no precipitate. It would appear that T has an affinity for some substrate or inhibi‘tOr present in NAF. It might be concluded that IBV 42, the egg‘adapted strain, does not act on allantoic fluid to provide alter- ation of the substrate. The metabolic processes or particulate na- ture of IBV 40-5 may produce a change in the substrate or act competitively to bind T. One explanation of the hemagglutination produced by T-treated NAF 01‘ IBV 42 in the absence of EWTI may be the direct adsorp— tion of the T-substrate complex on the red cell surface. A second poss1b1e explanation is an actual disruption of the T-substrate com- plex and the preferential adsorption of T onto the red cell. Thus, the T‘ red cell combination would provide the more favorable energy 60 State. The complexity of the red cell surface tends to lend credence to eith e r hypothesis. The term "substrate," which is generally used in discussion of enzyme activity, has been used to connote that agent in the allan- t01c fluid which combines with T. The term "inhibitor" may be ‘ t, more properly applied. Urine is one of the naturally occurring T i 1 ! inhibitors. The allantoic sac serves as a receptacle for kidney ex- cretions of the embryo. The concentration of urea in allantoic y fluid increases up to the fourteenth day of incubation. Uric acid Content increases throughout the entire incubation period. Creati— rune 13 also present in the allantoic fluid as are inorganic ions. Thus, the components of urine are present in NAF. Therefore, introduction of T into NAF would result in the combination of T and inhibitor to form an enzyme-inhibitor (EI) Complex which does not precipitate. EWTI, Addition of a second inhibitor, provides no stimulus for the dissociation of this initial EI complex which represents a more favorable energy state than that Of T‘ EWTI which precipitates. Reactive sites as presented in Figure III indicate the possible means of combination of T with urine trypsin inhibitor (UTI), EWTI, “ms particles, or red cell surface. 61 FIGURE III SCHEMATIC REPRESENTATION OF REACTING PRODUCTS IN THE IBV-HA SYSTEM UTI: EWTI: IBV: Red Cell Surface IBV Trypsin Urine Trypsin Inhibitor Eggwhite Trypsin Inhibitor Infectious Bronchitis Virus 62 The T-UTI union is postulated as occurring in only one man- ner, whereas two modes of reaction are postulated for T-EWTI. One mode Of reaction is postulated for the T-IBV union. Consideration must be given to a number of possible reactions between T and red cells, tWO of which are represented. The true nature of the components cannot be represented Since Only two planes are depicted, and, further, the true nature and Configuration of the reactive groups, other than the initial sites Of r“‘r‘aletion of T with substrate, are not known. The reagents as depicted serve as the basis for the follow- ing I'eatctions: 1 . The T— UTI union is possible and this combination could be adsorbed to the red cell due to the exposed group on the T molecule. 2. The T—EWTI union occurs and a precipitate is formed which is not adsorbed to the red cell. 3. UTI or EWTI is not adsorbed to the cell and does not cause agglutination. 4. UTI-T-EWTI represents a soluble complex which is not adsorbed on the cell surface. 5. T-IBV represents a true enzyme-substrate complex and the resultant modification of IBV provides a product w—. 63 which is adsorbed to the cell surface and causes aggluti- nation. T, therefore, has an affinity for both IBV and UTI present in the infected allantoic fluid. It preferentially reacts with the virus, is released, and reacts with more virus. The addition of EWTI serves to create an excess of inhibitor. The enzyme—sub- strate cycle is interrupted as enzyme, released from the enzyme- virus complex, is now bound in an E1 complex with EWTI as evi- denced by formation of the precipitate. The rate of formation of the precipitate indicates the plausibility of this explanation. The amount of precipitate, as determined spectrophoto- metrically, varies directly with the hemagglutination titer and would Freelude the possibility that hemagglutination was produced by T. It 8180 reflects increased viral concentration as determined by dilu- tion studies. It may be concluded that hemagglutination exhibited by T-IBV is either the product of adsorption of modified virus or of soluble degradation products derived from the virus through tryptic diges- t' 10“ Or a combination of both. 64 Adsorption Measured by Hemagglutination Rate of adsorption. Dilutions of IBV Were prepared to con- tain twenty or twenty-five HA units per 0.25 ml. One milliliter each of this preparation, HA buffer, and a 0.5 per cent red cell suspension were placed in tubes and incubated for varying periods at 4°. 23°, and 37°C. All reagents were at thermal equilibrium at the time of combination. At the termination of the contact period the suspensions were centrifuged at 3,000 r.p.m. for ten minutes at 4°C' The cell—free supernatant fluid was removed and tested for hemagglutinative activity. The results appear in Table 16. Adsorption was complete within five minutes at 23° and 37°C. At 4°C - adsorption was not complete within 25 minutes. Maximum adsorption. In an attempted determination of maxi- mum adsorption, constant amounts of cells were used with increas- ing HA units. A T-IBV preparation with an HA titer of 2560 was diluted 1:5 to contain 512 HA units per 0.25 ml. Multiple volumes of this con- centration were used. The results are presented in Table 17. There is but slight decrease in the ratio of adsorbed to nonadSOrbed hemagglutinin with increasing HA units within the range Studied A 65 TABLE 1 6 RATE OF ADSORPTION OF IBV 40—5 HEMAGGLU- TININ (20 and 25 units) BY RED CELLS (titers determined from supernatant fluid after deposition of cells) Tem— HA Titer after Adsorption pera— H 1 . . ture emagg “mm o 5 10 20 25 , ( C.) Min. Nlin. Min. Min. Nlin. J 37 Adsorbed with red cells 0 o o o o Nonadsorbed control 25 25 25 25 25 23 Adsorbed with red cells 0 o o o o Nonadsorbed control 25 25 25 25 25 Adsorbed with red cells 0 0 0 0 0 Nonadsorbed control 20 20 20 20 20 4 Adsorbed with red cells 5 5 5 5 5 Nonadsorbed control 25 25 25 25 25 X was} 66 TABLE 17 TOTAL ADSORPTION OF IBV 40-5 HEMAGGLUTININ BY RED CELLS AT 23°C. Reaction S stem HA Total Pct. 3' Titer Ad- Total HA °-5-P°t- s12; 8333' 21:33.; HA - ' Umts Buffer 5133131... natant (HA Ad- p Fluid units) sorbed 512 (0-25 ml) 0.75 ml 0.25 ml 10 512 98 1034 (0-5 ml) 0.5 ml 0.25 ml 20 1004 98 1536 (0.75 ml) 0.25 ml 0.25 ml 40 1496 97 2°48 (1 .0 ml) 0.00 ml 0.25 ml 80 1968 96 \\ ”if? 67 The adsorption is similar to that described by Magill (71) for influenza virus. The forces of attraction between hemagglutinin and chicken erythrocytes are governed by an orderly mechanism which effects a proportional distribution of hemagglutinin between erythro- cytes and suspending fluid. Adsorption Measured by Viral Infectivity Effect of trypsin on chicken embryos. For anticipated studies on the influence of T on viral infectivity, it was necessary to estab- liSh the effect of T and EWTI on embryos. Five embryos were inoculated via the allantoic cavity (0.1 ml per egg) with serial tenfold dilutions of T, EWTI, T followed by EWTI: and a mixture of equal parts of T and EWTI. Embryo mortality within 24 hours was produced by 0.3 mg of T- One milligram of EWTI produced no visible effect on the embryos as observed seven days after inoculation. The mixture of T and EWTI was innocuous. However, if 0.3 mg or more of T was inoculated and an equal amount of EWTI inoculated within 30 to 60 Seconds later, the T expressed its lethality and embryo mor- tality occurred. This lethal effect of T seemingly is not in keeping With the eelI‘lier hypothesis that T combines with an inhibitor present ¥_ Eh.“ _I'..:I 75 all.) IIJ. uni-In- —. 68 in NAF. This discrepancy may be resolved in any of three ways: 1 . There is an increase in urea in NAF up to the fourteenth day. Urate concentration and total nitrogen increase throughout in- CUbatiOn. The embryos were inoculated on the ninth day. It is possible that inhibitor may be present in lesser amounts than that contail'led in the twelve-day allantoic fluid used in the standard T I i PTOCeSs. The inhibitor can then inactivate only a portion of the T y introducted, thereby allowing the residual T to be adsorbed to cells 0f the embryo and cause death. 2. The T—UTI complex may be toxic. 3. The enzyme may selectively react with the embryonic C ells , Effect of trypsin on viral infectivity. For application in fu- t ure adsorption studies, the effect on viral infectivity of the T and E WTI Contained in the standard T process was determined. Strain IB V 40-5 with ID50 7.0 retained the same titer after incubation at 37° C- for three hours. The virus, as contained in the T—IBV-EWTI mi Xtu re, had an ID50 5.3. These data indicate a decrease of 1.7 log “hi ts 0r a fifty fold reduction of infectivity due to treatment of the V11» ~ us In the procedure. A \ A 69 Ldsorption and infectivity. The adsorption procedure consisted of combining 1 ml of T-IBV—EWTI with 1 ml of a 0.5 per cent red C911 suspension. This mixture was incubated at 23°C. for 10 min- utes and then centrifuged at 3,000 r.p.m. for 10 minutes at 4°C. The supernatant fluid was removed and its ID50 determined. The EWTI and red cell suspension used were not sterile, so antibiotics (10,000 units of penicillin and 10 mg of streptomycin per milliliter of preparation) were added. All dilutions have been con- Sidered in the expression of concentration in the following: 1 . Untreated virus ID50 6.2. 2. Virus after incubation at 37°C. for three hours ID50 6.2. 3. Virus after standard T process ID50 4.8. 4. Virus after standard T process and adsorption by red cells ID50 4.3. The reduction in viral concentration after combination with r ed cells implied that at least part of the hemagglutinin is inti— m ately bound to the virus particle or that the virus particle itself Se I‘Ves as the hemagglutinin. Relationship between infectivity titer and hemagglutination tite \r_ If it is assumed that one virus particle represents one infec- tiv e dose, values may be obtained such that HA units may be expressed a 1 ,5 70 in terms of infectivity units and a relationship between HA units and red cell concentration may be established. The ID50 of untreated virus was 6.2, or 1,581,000 infective £10883 per 0.1 ml. The HA titer was 2560 per 0.25 ml. By con- version, (2.5 X 1,581,000)/2560 = 1544 active particles per HA unit. This does not consider the inactive particles which may have been present and which may be modified by T to serve as hemag' vain». ‘ 5.4““ glutinative units . The 0.5 per cent red cell suspension contained approximately 7.500.000 cells per 0.25 ml as determined by direct count using a hemacy'tometer. If the total number of red cells are considered reSCtiVe, then one particle must serve as hemagglutinin for 4,858 cells_ It would be physically impossible for one virus particle the Size of IBV to Serve as the hemagglutinative bond for this number of cells. If the initial assumption, upon which the calculations are based, is accepted, then it must be concluded that something other than the viral particle enters into the phenomenon. Assuming the exposure of a specific hemagglutinin associated with IBV. the mOSt likely conclusion would be that part Of the hemagglutinative activity is intimately bound to the particle, since there is a diminution in infe - . QtlVity titer after adsorption, whereas the greater portion of the 7.3 71 activity is due to a hemagglutinin distinct from the particle per se. This Would parallel the hemagglutinative activity of vaccinia virus. Further Characterization of the Infectious Bronchitis Virus Hemagglutinin Elution. The progression of hemagglutination and subsequent elution of IBV in the HA test was studied at 4°, 23°, and 37°C. As ShOWn in Table 18, there is a direct correlation between the rate of adsot‘ption and an indirect relation between the rate of elution with respect to temperature. The adsorption of IBV appears to be that of chemisorption. Hemagglutination by different strains of infectious bronchitis ERE- IBV 40-5 was used almost exclusively for the development of the standard T process but it was also ascertained that other Strains could participate in the reaction. The hemagglutinative aetivity of certain other strains is presented in Table 19. A11 Strains except IBV 42, the egg-adapted strain, were modified and ylel‘i'ecl high HA titers. Hemagglutinative activity and initial recovery of infectious br witis virus in chicken embryos. When isolation of IBV from 1 “feet ed chickens is first attempted in chicken embryos, it is ~ Time TABLE 1 8 72 PROGRESSION OF ADSORPTION AND ELUTION IN HEMAGGLUTINATION BY IBV 40-5 30 min. 45 min. 60 min . l4 hrs~ t 4°C. 23°C. 37°C. Incomplete and indistinct 2560 2560 Most rapid set- tling of cells and clearest patterns Incomplete and indistinct 2560 2560 Still indistinct but read as 2560 1280 1280 640 2560 1280 Elution in dilutions greater than 1:20 Elution in dilutions greater than 1:320 No elution Some hemolysis 4 4| .‘. , 4k -53 HEMAGGLUTINATION BY STRAINS OF IBV AFTER MODIFICATION IN THE STANDARD T PROCESS TABLE 1 9 73 IBV Strain HA 40-5 2560 42 0 1-7 2560 2-10 5120 3-6 2560 16-34 1280 19-5 1280 20—10 2560 21-8 640 23-15 640 24—10 2560 41-1 5120 sometimes necessary to make more than one passage and rarely more than three passages before typical gross lesions of the em— bryo are manifest. To correlate hemagglutinative activity of T-IBV with egg Passage and positive reaction of the embryo, three different strains Were used, The results in Table 20 show that with strain 40 and 40'5'1C three egg passages were necessary before HA and viral activity Were demonstrated. Strain 41 was positive for both reac- tions on the first passage. These data indicate that hemagglutina— tive aetivity is associated with viral concentration. Egect of trypsin on red cells. To 2 m1 of a 25.0 per cent Saline Suspension of red cells, 1 ml of 1 per cent T was added and the rhiX‘ture was incubated at 37°C. for three hours. One milliliter of EWTI was added and the cells were washed in saline through three cycles of differential centrifugation. The cells were then used in the HA test with both modified and untreated IBV 40—5 and IBV 42 and NAF The data in Table 21 show that IBV does not agglutinate t . I’ypsl‘n‘treated red cells in contrast to agglutination of untreated Ce 113 as used in the HA test. This indicates that HA as observed in . the test is due to trypsin-modified IBV and not to trypsm- modified cells. \A 75 TABLE 20 CONCENTRATION OF IBV HEMAGGLUTININ IN EARLY EGG PASSAGE . iggggaggi Gifs‘éaiiliiiis Hr Tiier .___.________ 40-1 - . .. .................. 2/10 0 40-3 - . . ................... 2/10 ° 40‘3 - . ....... . ............ 7/10 5120 40-5-1(:_.1a .................. l/10 0 405~1(:—2a .................. 2/10 0 40'5-1C-3a .................. 8/10 256° 41.1 - ..................... 7/10 5120 a . Was IBV 40-5 was used to infect three-week-old chickens. IBV into reCovered from lung and tracheal suspensions and inoculated e gs. The last number indicates egg passage. \A 76 TABLE 21 TRYPSIN— MODIFIED RED CELLS IN HEMAGGLUTINATION TESTS HA Titer Virus Untreated Cells T—modified Cells T-IBV 40- 5 ........... 5120 0 T-IBV 42 .............. 0 0 T'NAF .............. 0 ° Untreated IBV 40-5 ....... 0 ° 77 Thermostability of the hemagglutinative activity of infectious bronchitis virus. Infected and normal allantoic fluids were heated for certain periods at 56°C. prior to use in the standard T process. The results are presented in Table 22. As meaSured by infectivity titration, IBV 40—5 resists 56°C. for 120 minutes (27). These data indicate that heat-inactivated virus remains susceptible to tryptic modification. Continued heating ex- tends Clenaturation and the resultant product does not evidence modification to the same degree as unheated IBV after T treatment. Virus, after T treatment, was stored at -30°C. for different periods after which its hemagglutinative activity was determined. The results are presented in Table 23. The results obtained showed such wide variation that no valid exPlana’tion can be offered. T-IBV could be stored at -30°C., but without any certainty of stability of hemagglutinative activity. Random sampling of T-IBV stored at 4°C. for similar pe- rio ds of time showed essentially the same results. Colloids. Precipitation reactions of arsenic trisulfide, Ch . romlum hydroxide, ferric hydroxide, red colloidal gold, and sil- Ve r e0lloids when combined with the reagents listed in Table 24 were 0f no Value as indicators of charge of IBV. 78 TABLE 22 THERMOSTABILITY OF IBV—INFECTED AND NORMAL ALLAN- TOIC FLUIDS PRIOR TO STANDARD T PROCESS Time I-IA Titer (min.) at 56°C. 40-5 42 NAF 0 640 0 0 15 640 0 0 30 640 0 0 60 640 0 0 90 640 0 0 120 640 0 0 150 640 0 O 180 160 0 0 THERMOSTABILITY AT -30°C. OF THE HEMAGGLUTINATIVE TABLE 23 ACTIVITY OF IBV AFTER STANDARD T PROCESS 79 . . Time , St rain 1:331 (days) ital 1 at -30°c. 1 er 40-5 640 1 10 40—5 1 10 40—5 640 1 5 40-6 2560 l 40 40—5 2560 2 20 2-11 640 Z 640 41-1 1280 Z 640 40-3 3 5120 2-10 3 5120 41-1 3 640 40-5 640 4 80 40-5 4 640 40 5 5120 8 160 40-5 1280 8 320 \ 80 TABLE 24 COLLOIDAL PRUSSIAN BLUE AS AN INDICATOR OF THE CHARGE ON REAGENTS IN THE STANDARD T PROCESS Reagent (0.2 ml added to 2.0 ml Reaction of Colloid of Prussian blue) T Precipitates in 15 minutes ' EWTI Stable at 1 hour T, EWTI Precipitates in 1 hour Distilled water Precipitates in 45 minutes NAF Precipitates in 30 minutes IBV 40-5 Precipitates in 30 minutes IBV 42 Precipitates in 30 minutes T, NAF, EWTI Stable at 14 hours T, IBV 40-5, EWTI Precipitates in 15 minutes T, IBV 42, EWTI Precipitates in 15 minutes None Precipitates in 1 hour 81 Colloidal Prussian blue that had been stored for nine months and which exhibited some spontaneous precipitation served to indi- cate charge differences. Combinations of colloid and reagents were Prepared as follows: 0.2 ml of reagent was added to 2.0 ml of the colloid and observed for precipitation. The results appear in Table 24. The stabilization of the colloid by EWTI and T—NAF-EWTI indicadies that each carries a net negative charge for the colloid . carries a negative charge also. This activity tends to support the earlier hypothesis that T is bound to an inhibitor upon introduction into NAF in vitro. If the T were free it would combine with the added EWTI and precipitate the colloid as did T-EWTI. Hemagglutination by peptones, polypeptides, and amino acids. Hemagglutination by the T-modified IBV was the stimulus for inves- tigation of HA by the products of T hydrolysis of other proteins. Serial twofold dilutions of 1 per cent aqueous solutions of trypticase, tryptone. and tryptose were tested for their hemagglutinative activity. The I'eslillts are presented in Table 25. Hemagglutination by trypticase and tryptone, but not by tryp- tose, SeI'Ves to emphasize the range of hemagglutinative agents and the question of the specificity of the T—induced IBV hemagglutinin. 82 TABLE 25 HEMAGGLUTINATION BY PEPTONES (titer expressed as reciprocal of g/ml) . 1 Per Cent t . Peptone T1 er EWTI Added Titer x Trypticase ................ 400 0.1 ml/ml 100 Tryptone . ...... . ......... 800 0.1 ml/ml 800 Trypt0se ................. o 0.1 ml/ml 0 Solutions of L-arginine, glycine, L—lysine, L-methionine, L‘Phlenylalanine, L-serine, and L—tyrosine, alone or in combination, exhi bited no hemagglutinative activity. An aqueous solution of trypticase, glycine, L-arginine, L- lysine. L-m-etionine, and L—serine, each at 1 per cent final concen— tration, had an HA titer of 1067 expressed as the reciprocal of grams per milliliter. Since the amino acids in themselves cannot cause agglutination, the titer, based on the weight of trypticase, would be 6400. Arginine and lysine represent the points of initial tryptic aetwlty . so a solution containing one gram each of trypticase, 1" arginine. and L-lysine in 100 ml of water was prepared. The titer 83 of this mixture was 133, but if based on the weight of trypticase alone it was 400. From the titers based on the weight of trypticase it is seen that the addition of the five amino acids enhances the hemagglutina— tive activity, whereas addition of arginine and lysine has no such effect. The approximate concentrations of metallic ions, amino acids, and Vitamins present in the peptone have been determined but the forms in which they exist is unknown so the mode of the reaction Of the added amino acids cannot be explained at this time. Agglutination of red cells of certain species. The hemag- glufiinative activity associated with most viruses is generally active on the red cells of at least a few species. The red cell spectrum of the IBV hemagglutinin is presented in Table 26. Hemagglutination by IBV is effective only with chicken erythro- Cytes. A 1 per cent solution of ZXT agglutinated human 0 cells. A trypticase-amino acid (arginine, glycine, lysine, methionine, phenylalanine, serine, and tyrosine) mixture had an HA titer of 1:100 (g/ ml) With both chicken and human 0 cells. wmm; 1.3 84 TABLE 26 AGGLUTINATION OF RED CELLS OF CERTAIN SPECIES BY T-MODIFIED IBV Species HA Titer Chicken 2560 Rabbit 0 Cat 0 Cog 0 Cow ‘ 0 Horse 0 Sheep 0 Human O 0 Hemagglutination-inhibition (HI) flemagglutination-inhibitionfl serum. Inhibition of the IBV hemeegglutinin was tested with known positive and negative sera. The reSults appear in Table 27. The inhibitory effect of the negative serum may be that of a nonspecific nature or it may indicate nonspecificity of the IBV he- magglutinin. HEMAGGLUTINATION-INHIBITION OF IBV BY SERA TABLE 27 85 . HA Titer IBV HA Alpha Procedure Beta Procedure Strain Titer Positive Negative Positive Negative Serum Serum Serum Serum \ 40- 5 2560 12800 12800 12800 12800 40‘ 1 5 1280 6400 6400 12800 12800 40‘ 1 6 10240 51200 51200 12800 12800 47- 320 1600 1600 12800 12800 Inactivation of nonspecific inhibitors in normal chicken serum was attempted by the following treatments which yielded negative 1. Inactivation at 56°C. for 30 minutes. 2. Inactivation at 62°C. for 20 minutes. 3. Addition of an equal volume of 3.81 per cent sodium ci— trate to the undiluted serum used in the beta test. 4. Filtration through Seitz EK pad. 5. Combination of equal volumes of serum and supernatant fluid from Clostridium welchii cultures and incubation at 86 room temperature for one hour. 6. Ether extraction of the serum at room temperature. Each of the three types of Clostridium welchii added to posi- tive IB serum produced a precipitate. No precipitate was formed When they were added to negative serum. These sera had been Stored at -30°C. from two to six months. The pH of the sera was 8-0» the Clostridium welchii supernatant fluid was 6.0, and the com- binatiOn was 6.7. A second trial with different cultures of the same Strains did not produce this effect. In this latter attempt the pH of the combination was 7.1. Differences in quantity and quality of pre- Cipitates of these positive and negative sera were observed upon ad- ditiol'l of concentrated hydrochloric acid. Serum containing anti- bOdi es against NDV but not IBV reacted in the same manner as did semm containing antibodies against IBV only. The precipitate is apparently an effect of pH and not a specific reaction between com- ponents in the system. Sera were inactivated at 62°C. for 20 minutes and then treated with T and EWTI as in the standard T process. The re- sults appear in Table 28. Identical results were obtained with positive and negative sera 1“ all tests except with IBV 40-23 and one test with IBV 40-5. TABLE 28 87 HEMAGGLUTINATION-INHIBITION OF IBV BY SERA INACTIVATED AT 62°C. FOR 20 MINUTES AND THEN TREATED WITH T AND EWTI HA Titer IBV HA Alpha Procedure Beta Procedure Strain Titer Positive Negative Positive Negative Serum Serum Serum Serum R 40 - 5 2560 12800 12800 6400 6400 40~ 5 2560 12800 12800 6400 6400 40 — 5 2560 ............ 1600 1600 40— 23 640 3200 3200 800 0 40.. 5 320 1600 1600 800 50 4 O— 5 2560 .......... 6400 6400 ably 10Wer than in the other tests which were performed at differ ent times. The HA titer of IBV used in these two tests was consider- It is possible that the reduced HA titers of the IBV Indicate an incomplete modification which may have resulted from a temperature fluctuation or reduced activity of the trypsin em— ployed . dEViatiOn from the standard would be reflected in the activity 0f bOth- — The IBV and sera were processed at the same time so any - h-fie-‘m ~v4“ - l - . i "t ‘ . 88 Crystalline trypsin was used later in treatment of sera, and either EWI‘I or SBTI was used as inhibitor. Positive and negative sera exhibited equal HI titers. As controls, the sera employed were tested for hemaggluti- native activity. Positive and negative sera treated in the following ways Showed no hemagglutinative ability: 1 . Untreated. 2. Inactivated at 56°C. for 30 minutes. 3. Inactivated at 62°C. for 20 minutes. 4. Standard T process with or without prior heat inactivation. Spontaneous agglutination by sera treated with 2XT was ob- served after incubation at 56°C. for 30 minutes even after the ad- dition of an equal amount of EWTI. The HI test was modified in that the IBV hemagglutinin and cells were combined prior to addition of serum. Positive and nega- tive Sera proved capable of disrupting the IBV hemagglutinin-red cell union as evidenced by lack of hemagglutination. The positive and negative sera were employed in HI tests With the previously used trypticase-amino acid mixture which had been Stored at -30°C. for three weeks. The results appear in Table 29 89 TABLE 29 HEMAGGLUTINATION-INHIBITION TESTS USING A TRYPTICASE—AMINO ACID MIXTURE HI Titer of Sera (alpha procedure) Sera Heated at 62°C. . t ' Titera Un reated Sera for 20 Minutes Positive Negative Positive Negative Serum Serum Serum Serum \ 1 0 6 7 0 0 o 0 2- 6 7 40 40 40 40 6 '7 0 0 0 0 Z 6 '7 2 0 20 2 0 2 0 \ a . No Virus present. There is a striking difference between the inhibition of the IBV and the polypeptide hemagglutinin by serum. Hemagglutination by IBV is completely inhibited by sera employed in the alpha pro- eedul‘e, whereas the highest HI titer obtained with the trypticase- amino acid mixture was 40. This indicates a difference in the na- \ure of these hemagglutinins. 7‘ 90 Effect of other inhibiting agents. To detect the effect of other agents that might inhibit the IBV hemagglutinin, untreated NAF, IBV 40-20 and IBV 42, and EWTI and SBTI were diluted 1:5 (1:4 in the case of SBTI) and used in place of serum in the alpha Procedure of HI tests. NDV was used as a positive control to show agglutination. The results are presented in Table 30. Normal and IBV-infected allantoic fluids contain inhibitor active against HA by NDV and IBV. Inhibitor is present in greater Concentration in the IBV-infected allantoic fluid as measured by NDV HA. Inhibition of the IBV hemagglutinin by EWTI but not by SBTI inClicates further that the hemagglutination is not a result of the activity of residual trypsin. EWTI, which is ovomucoid, represents One of the Francis-type inhibitors which are mucoid in nature. C>VoInticoid is not active against the NDV hemagglutinin. The supernatant fluid from C. welchii, type A, was diluted 1 :5 and tested for inhibition of hemagglutination with the IBV hemag- glutl'lnin. This preparation caused hemolysis but agglutination was apparent through the 1:80 dilution of virus. It may be concluded ‘hat the IBV hemagglutinin is dissimilar to that of vaccinia and eetromelia which is lipoidal in nature and is inhibited by lecithi- nase (89). A TABLE 30 HE MAGGLUTINATION- INHIBITION (ALPHA PROCEDURE) 91 HA Titer after H:r:;gigllu- HA Titer Inhibitor Combination with Inhibitor NDV 1280 T- IBV 2560 NDV 1280 NAF 160 T-IBV 2560 NAF 0 NDV 1280 IBV 42 80 T-IBV 2560 IBV 42 0 NDV 1280 IBV 40-20 80 T-IBV 2560 IBV 40-20 0 NDV 1280 EWTI 1280 T-IBV 2560 EWTI 0 NDV 1280 SBTI 1280 T-IBV 2560 SBTI 2560 DISC USS ION The action of trypsin on feline pneumonitis virus and the GD VII strain of murine encephalitis results in increased infectivity, Presumably by disaggregation of the virus. Trypsin has been em— p10yed for purification of psittacosis and other viruses. Trypsin aCtS to render particulate matter, other than virus, soluble, and Vims may be concentrated by centrifugation. Tryptic digestion reduces infectivity of potato virus X. A tenfOId reduction of infectivity of the LEE strain of influenza B Was reported following treatment with trypsin. This is the only report of reduction of infectivity of an animal virus by trypsin. IBV is an animal virus whose molecular structure can be CleteI‘mined in part by the action of trypsin as reflected in reduced infetztivity and induced hemagglutinative activity. Trypsin cleaves bonds formed by the carboxyl groups of the basic amino acids, arginine, and lysine. Thus, either arginine and/or lysine is pres- ent in the protein moiety of the virus. The spatial configuration of the protein molecule and its position in the virus particle permit herniation of the enzyme-substrate complex. 92 ‘4‘4_‘“— ‘ ficsfw 93 Specificity of the induced hemagglutinin has not been demon- strated by HI tests with anti-1B serum. The mode of inactivation 0f nonspecific inhibitor present in normal serum has not as yet been ascertained. Normal chicken serum may be rendered pan— agglu‘tinative through modification by trypsin. Although trypsin has been reported to inactivate nonspecific inhibitor of influenza virus in r1C>r-1'nal human, rabbit, ferret, mouse, guinea pig, and chicken Sera. it has not proved of value as employed in the present studies. In one trial with IBV a difference in modification of normal and im- mune sera was observed after T treatment. Further investigation 0f the concentration of T and time and temperature of the reaction may provide for proper modification. The RDE of Vibrio cholerae, thich was not available at the time that these studies were conducted, Should be utilized as well as sodium andpotassium periodate. The hemagglutinative activity of IBV induced by trypsin is distinguished from that of the peptones tested. Differential inhibi- tcry action of sera is so marked as to indicate a difference in the nature of these two agglutinins. A further distinction in the nature of these two hemaggluti- “ins can be derived from a comparison of the hemagglutination titers on a weight-per—volume basis. The end points of the poly— :peptide mixtures were determined on the basis of grams per 94 milliliter and the titer expressed as the reciprocal. A similar eval- uation can be made for the virus. The virus is a sphere of 70 mp average diameter with a 3 . .. 21 3 volume of 143,800 mp . One milliliter equals 10 mp . There— fore, if the infectivity and hemagglutination titers determined for a typical IBV strain are employed, the HA titer may be expressed in teI‘rns of mass per volume. The most common IBV HA titer was 2560/0.25 m1 and the 7 COI‘I‘esponding infectivity titer was‘ 10 /0.1 ml. If it is assumed that one virus particle represents one infectious unit, the total vol- hme of virus may be determined by multiplying the volume of one paI‘ticle by the total number of particles. Therefore, 143,800 mp3 8 l .1 or 105'1577, multiplied by 10 particles/ml equals 10 3 577 mp3. Th . . 3 . . 3 . . e volume of the particles in mp. d1v1ded by the mp. in one mil- liliter provides the volume of the virus in milliliters. Therefore: 1 3. 2 - 8 2 -8 10 1577/101: 10 7' 4 3 or 1.438 x 10 ml. If the density of IBV is assumed to be about 1.2, which is Within the range reported for other animal viruses of similar size and Shape, the weight of the virus in one milliliter of infected fluid is 1 '8 . . ~72 x 10 . In the HA test 0.25 ml of the IBV dilutions was 9 -3 .. used_ Therefore, 0.25 (1.72 x 10 ) equals 4.3 x 10 Expressed as the reciprocal of grams per milliliter, the HA‘titer of IBV is 95 6 x 1 0“, whereas the highest HA titer observed with the peptones was 1067. It was previously hypothesized that one infectious unit repre— sented 1,000 virus particles. Agglutination then could be more Plausibly attributed to the IBV particles. If this number of virus Particles is considered, the HA titer of IBV would only be reduced to 6 X 108 and there is still great distinction between the induced IBV hemagglutinin and that of the peptones. The true physical nature of the induced IBV hemagglutinative ELgent and its reaction with red cells possibly may be determined by differential ultrafiltration and electron microscopy. No enzymic quality of the virus itself is involved in the he- Inaggiutinative activity. This is evidenced by heating IBV. The HA titer remained constant after 150 minutes at 56°C. and a reduction in titer appeared only after 180 minutes. The virus is rendered noninfectious after 120 minutes. It may be concluded that denatura- tion of the virus protein proceeds at a rate such that after 180 minUt es there is modification which enhances or limits the activity of trypsin either by alteration of initial specific substrate or second- ary Sites of reaction. The carbohydrate portion of ovomucoid represents 20 per cent of its substance and consists of three molecules of mannose, \) ’ / / §Q * ”'7 \\\ I10 96 seven of acetylglucosamine, and one of galactose (71). The ovomu- coid may serve as a reducing substance through the action of the Sugars. That reduction alone is not the criterion for inhibition of hemagglutination was demonstrated when a preparation of EWTI, Which had been stored for two months and had lost some of its activity, was used in the alpha HI procedure. The initial HA titer Was 2560. With the ovomucoid the titer was 320, and not the ex- peCted zero. When a preparation representing a 1:5 dilution of 1 per cent each of EWTI and sodium thioglycollate was used as iul'3libitor the titer was 40. Sodium thioglycollate alone was not irlhibitory. The action of sodium thioglycollate was directed to the her13C>globin. The cells were characteristically agglutinated at a ViruS dilution of 2560 but had a green color which was probably due to the formation of sulfhemoglobin. A further consideration of the T-EWTI union and the inhibition of HA by EWTI in this test system may be predicated on the direct VaI‘iance of turbidity and HA titer. It was postulated that the tryp- Sin Which is free to combine with EWTI is that which was reactive With the virus since no turbidity is evidenced in the control fluid. The greater the trypsin reactive with virus, the greater the precipi- tation of the T~EWTI complex. The reduction in concentration of 97 EW'I'I represents a reduction in inhibitor of HA with a resulting higher titer. The IBV hemagglutinin differs from that of influenza virus as Seen in the rates of adsorption at different temperatures. Influ- enza virus is rapidly and most completely adsorbed to red cells at 4°C-. whereas the maximum rate and total adsorption of IBV is greater at 37°C. than at 4°C. Adsorption of influenza virus is characteristic of physical adsorption, whereas that of IBV is more representative of chemisorption. The HA reaction of IBV differs further from that of the MNI group in that spontaneous elution does not occur. It is generally considered that the prevailing forces involved in HA are electrostatic in nature. Ion-dipole or the lesser magni— tude Van der Waals' forces could account for this adsorption and the tenuous nature of such bonds, particularly amidst the complexity of Configuration and radicals in the protein moiety, could be reflected in elution. That elution occurs at 37°C. and not at 4°C. supports the existence of dipole forces in that thermal agitation would serve to disrupt these bonds. This type bond would also account for the more rapid and complete adsorption at lOWeI‘ temperatures. itifibfllj I ‘ trill" Ll llvl 98 This mode of reaction does not, in itself. provide for the ob- served reduction in net negative charge on the surface of the eryth- rocyte after adsorption and elution of the virus. Elution of influenza virus is considered to be the result of enzymatic destruction of cellular receptors by the virus. The net activity on the red cell is one of reduction as evidenced by the altered electrophoretic velocity and agglutinability. That the cells are not reagglutinable by the same strain of virus or those lower on the receptor gradient scale may indicate that the enzymatic mechanism is that of hydrolysis rather than of oxidoreduction which would be reversible in this system. It may be deduced from the over-all reaction of influenza virus HA that positively charged units, whether they be ionic or Polar, are necessary for adsorption. Spatial configuration is a de- termining factor since interionic attractive forces vary as r , . . . . - -7 Whereas interdipole attraction varies as r or r . The activity of cations in the adsorption phenomenon may be a Combination reaction with viral or blood cell protein. Proteins COInbine preferentially with calcium, magnesium, phosphate, and biQarbonate ions. The stability of the bond formed between pro- tein molecules and calcium or phosphate ions is due to the high e1ectrostatic action of the bivalent inorganic ions. Univalent ions at P05 99 such as those of sodium, potassium, or chlorine are less firmly bound. In the process of adsorbing counterions the ionized groups of the protein become neutralized and the over-all effect is a modi- fication of the electrochemical properties of the protein. If calcium combined with red cell protein there would be a reduction in net negative charge which would result in diminished forces of repulsion between the virus particle and the red cell since both virus and cell carry a net negative charge. Untreated IBV is incapable of agglutinating red cells. The T-modified IBV has hemagglutinative activity. The initial sites of tryptic activity are peptide bonds involving the carboxyl groups of arginine or lysine. Further enzymatic digestion proceeds but the sites of reaction are not known. From the initial reaction it is seen that the epsilon amino group of lysine and/or the guanido group of arginine may become eXposed. Substitution reactions with proteins indicate that at least a portion of the hydroxyl groups of hydroxyamino acids, the imida- zOle rings of histidine, and the guanido groups of arginine exist in the form of "free" reactive groups. The HA reaction is conducted at pH 7.2, and the epsilon amino group of lysine would carry a positive charge despite an assumed decrease in the pK of lysine 3 based on observation of the pK2 of glycine as a free amino acid and the peptide, hexaglycine. The positively charged groups could then form salt bridges with surface carbonyl groups of cellular protein. Relatively small numbers of these positively charged groups, as compared with di- poles, would have to be exposed to provide the stability which is manifest. Elution of the IBV hemagglutinin is probably not a true elution but is due to the tenuous nature of the lattice formation. This elu- tion, as reflected by compaction of cells, occurs only in the higher dilutions of IBV and stems from the nature of the proportional dis- tribution of the hemagglutinin among the erythrocytes. This is not the true compaction of nonagglutinated cells and does not exhibit ready streaming of cells when the tubes are tilted. Streaming is readily evident in untreated cells and those from which NDV has Gluted. This further tends to demonstrate a difference in the na- 1"ire of the bonding of the IBV hemagglutinative agent to the red cell. It has not been conclusively demonstrated that the egg-adapted StI'ain will react to the same extent in this System as other strains of IBV. In three tests in which the egg-adapted strain had an in- 7 . fectivity titer of 10 /0.1 ml only one had an HA t1ter of 320 after tJf‘eatment, whereas the other two had no hemagglutinative activity. 101 Consideration of one aberrant effect of the egg-adapted strain as expressed in high dilution upon embryo inoculation presents the possibility of the involvement of a factor other than that of concen- tration. This effect is the curling and dwarfing of the embryo rather than the lethal effect which is characteristic of egg-adapted strains. Foregoing any attempted explanation of its existence, cognizance must be given to the possible presence of an agent other than egg- adapted IBV or the presence of a modified form. Back—mutations of the strain have not been reported. If this agent is truly present, it may be responsible for the low HA titers sometimes exhibited by the egg-adapted strain. The reduced or absent hemagglutinative activity may be another expression of adaptation. Loss of pathogenicity and antigenicity for chickens is char- acteristic of egg-adaptation of IBV and may likely be the result of Structural changes in the virus protein. The lack of reSponse to tryptic activity by the egg-adapted strain may be a further reflec- tion of altered protein structure. The protein molecule of IBV may be elongate with exposed sites that are reactive with T and exposed groups that are essential for pathogenicity and antigenicity. The protein structure of the egg—adapted strain may be altered as a result of folding over of the polypeptide chains resulting in a lami- t1ate, globular molecule. The spatial configuration of this laminated 102. molecule may then result in the masking of these reactive sites and thereby account for altered activity. Another possible explanation for the differential hemaggluti- native activity of IBV and the egg-adapted strain may be based on the differential pathological effects of the two strains. The egg- adapted strain is more virulent and is lethal to embryos within 48 hours. From this greater virulence it may be deduced that a lesser number of particles represent an infective unit of egg-adapted virus than less—well-adapted strains of IBV. Therefore, identical infec- tivity titers of the two strains would not necessarily represent equality of total virus protein. If this is the case, the lesser hemagglutinative activity of the egg adapted strain would merely be a reflection of lesser substrate. Total nitrogen determinations (Kjeldahl) of NAF, IBV 40-5, and IBV 42 (egg-adapted) for distinction and correlation of virus Protein and hemagglutination were nonconclusive. The hemagglutinative activity of IBV is dependent on viral Concentration and may possibly be utilized as a diagnostic aid for the detection of the virus in embryos inoculated with tissue suspen- 8ions. The test is not presently adapted for assay of IBV anti- bOdies. The induced hemagglutinative activity and the concurrent reduction of infectivity are unique to this virus at this time. pa? I *4»; ! 103 So far as known, this is the first evidence presented on the participation of IBV in a hemagglutination system and possible ex- planation of chemical configuration of IBV protein. SUMMARY 1. Infectious bronchitis virus was modified by trypsin so 1' that it agglutinated chicken erythrocytes. Trypsin at a final con- {V centration of 0.33 per cent was added to the virus—infected allantoic fluid and incubated for three hours at 37°C. Eggwhite trypsin in- hibitor was added so that the final concentrations of trypsin and inhibitor were equal. This preparation contained the active hemag- glutinating agent. 2. A 98 per cent reduction in infectivity for embryos ac- companies trypsin modification. 3. The egg-adapted strain does not react to as great an extent in this system as do less-well-adapted strains. 4. The rate and degree of adsorption of the hemagglutinin Varies directly with temperature. Elution varies indirectly with temperature. 5. The hemagglutinin is present in two fractions: one in close association with the virus particle, and the second, which con- Stitutes the greater part, is distinct from the particle. 6. 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Blattner. Dialyzable factor in normal allantoic fluid inhibiting hemagglutina- tion patterns with Newcastle disease virus. Proc. Soc. Exp. Biol. and Med., §_9_ (1955), 206-209. [GUM USE ONLY M3133)! USE {tit-{L'I’ W "'Tittiijiii(it?))imuiritii“ 46 8007