ELECTROKME’TPC STUDEES 0N ERYTHROCYTES TREATED WITH MODEHED ENFEC‘CEOUS BRONCHWIS VIRUS Them {at {he Degree :2? M. S. MEI-MEAN STATE UMV’ERSETY Niiambar Biswai 19633 OVERDUE FINES ARE 25¢ pER DAY PER II‘EM Return to book drop to remove this checkout from your record. ‘ u’\ , ‘ N) be, - 7 JV " {nag-i.” ELECTROKINETIC STUDIES ON ERYTHROCYTES TREATED WITH MODIFIED INFECTIOUS BRONCHITIS VIRUS By Nilambar Biswal A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Microbiology and Public Health 1963 ACKNOWLEDGMENTS I wish to express my gratitude to Dr. C. H. Cunningham, Professor of Microbiology and Public Health, for his most valuable guidance throughout this investigation. I am greatly indebted to Dr. J. J. Stockton, Chairman of the Department of Microbiology and Public Health, for his kind advice and encouragement. My sincere thanks are also due to Mrs. M. P. Spring, Department of Microbiology and Public Health, for the help rendered and interest shown by her in this investigation. Finally, I wish to take this opportunity to express my deep appreciation to all my friends for their helpful criticisms during this investigation. 11 TABLE OF CONTENTS INTRODUCTION . LITERATURE REVIEW. Electrophoresis Erythrocyte . Direct Hemagglutination Receptor Destroying Enzyme Inhibitors. Indirect Hemagglutination. Infectious Bronchitis Virus MATERIALS AND METHODS Viruses. . Normal Allantoic Fluid. Erythrocytes Diluents Trypsin. . Eggwhite Trypsin. Inhibitor Receptor Destroying Enzyme Electrophoresis Apparatus. Hemagglutination. Treatment of Erythrocytes with Virus Treatment of Erythrocytes with Trypsin Treatment of Erythrocytes with RDE. RESULTS DISCUSSION . SUMMARY BIBLIOGRAPHY iii 0 O O C O LIST OF TABLES Table l. ElectrOphoretic mobility of erythroctyes in O. O67M phOSphate buffer at pH 7. 35 before and after reaction with trypsin- modified IBV . . . . . . . . . . . . 2. Electrophoretic mobility of erythrocytes in O. O67M phosphate buffer at pH 7. 35 treated with different reagents. . . . . 3. Electrophoretic mobility of erythrocytes in O. O67M phosphate buffer at pH 7. 35 treated with different reagents. . . . A. Electrophoretic mobility of normal and trypsin- treated chicken erythrocytes in O. O67M phosphate buffer at pH 7. 35 treated with different reagents. . . . . 5. Electrophoretic mobility of RDE-treated— chicken erythrocytes in O. O67M phOSphate buffer at pH 7. 35 treated with different reagents. . iv Page 24 26 27 28 29 INTRODUCTION Infectious bronchitis virus (IBV) normally does not cause hemagglutination. When modified with trypsin, the virus agglutinates erythrocytes of chickens and turkeys only. The hemagglutination is attributed to a modification of the virus to permit adsorption to the surface of the erythrocytes. The surface of the erythrocyte is a macropolyanion that imparts a net negative electric charge. When treated with virus, some of the ionizable groups of the erythrocyte surface preferentially adsorb Opposite ions supposed to be present on the surface of the virus particle, thereby reducing the surface charge density of the erythrocyte and hence its electrophoretic mobility. Electrophoresis offers a means by which precise determination of this physical entity on a biological surface can be made. This study was undertaken to ascertain the electro- phoretic mobility of erythrocytes before and after treat- ment with IBV and trypsin-modified IBV. LITERATURE REVIEW Electrophoresis Electrophoresis is the movement of charged particles suspended in a liquid medium, under the influence of an applied electric field (Overbreek and LiJklema, 1959). The electrophoretic mobility, v, is calculated from the electro- phoretic velocity (V) per unit field strength (X) (Abramson _g§_§l., 1942) as follows: v = V/X, expressed in_p per sec. per volt per cm. The field strength X is determined according to Ohm's law and is dependent upon the specific conductance of the solution (Ks), the cross sectional area of the cell (q) in cm2, and the current (I) in amperes as follows: X = I/q Ks. The microscopic method of electrophoresis deals with the direct observation of microscopically Visible particles as they migrate in the electric field. Four types of electrophoresis cells are available and each of them claim some advantage over the others: (1) Northrop-Kunitz wide, flat, rectangular, horizontal type (Northrop—Kunitz, 1925; Abramson, 1929a); (2) Vertical flat cells (Abramson et al., — 2 3 1942; Ponder and Ponder, 1955; Angers and Rottino, 1961); (3) Lateral flat cell (Hartman §t_§l,, 1952); (4) Cylin- drical cell (Bangham 33 al., 1958b, Seaman and Heard, 1961). In all the flat type of cells, when the particles are suspended in a suitable medium and~the current is applied there is an electroosmotic flow of the liquid along the surfaces of the cell wall and the liquid returns through the center. Therefore, it is only at the two stationary levels, which are intermediate to the center and the interior surfaces of the cell, that the liquid is motionless. For accurate measurement of electrophoretic mobility, the location of the stationary level with respect to the depth of the cell must be determined. Smoluchowski's equation (O.2llX or 0.789X), when X is the depth of the cell, may be used only when the cell width is great com— pared to the depth. Otherwise, Komagata's correction (Stationary level a 0.5 + 0.2887‘Y/ l + -l4%2i) where A is the ratio of the cell width to the depth must be used (Bull, 1951). The use of the micro-electrophoresis cell has been of considerable help for investigation of the surface phenomena related to biological substances. Besides the knowledge of the surface charge density on the erythrocytes in normal (Abramson, 1929b; Furchgott and Ponder, 1941; Bateman and Zeliner, l956; Angers and Rottino, 1961) and disease conditions (Rottino and Angers, 1961), bacteria, Spermatozoa and submicroscopic substances adsorbed on to 4 the surface of the carrier particles can be studied (Brinton and Lauffer, 1959). Submicroscopic substances such as pro- teins, lipoproteins, antibodies, detergents, and viruses can be adsorbed on to the surface of microscopically visible particles such as glass, quartz, collodion, carbon, mineral oil, ferric oxide, ion exchange resins, silica gel, aluminum oxide and even air bubbles. The electrOphoretic mobility of a particle covered with adsorbate is characteristic of the adsorbed material and not of the particle. Uniformity of results depends upon complete covering of the particle with the adsorbate (Miller_et_al., 1944). Erythrocyte The surface of the erythrocyte offers a unique tool for observation of many biological phenomena. The cell stroma has an inner fibrous layer and an outer layer of plaques (Moskowitz and Calvin, l952) consisting of long, thin rods composed of a lipid-protein-carbohydrate complex (Hiller and Hoffman, 1953) which serves as the receptor to which certain viruses may become attached. The lipid-rich protein fraction is also a potent inhibitor of PR—S influ- enza indicator virus (Howe, 1951). Depending on the pH and ionic strength of the sus- pending medium, the erythrocyte has a specific electrOphore— tic mobility characteristic of the species (Abramson and Moyer, 1930; Abramson et_al;, 1942). Furchgott and Ponder (1941) found that the electrokinetic behavior of human 5 erythrocytes is mainly associated with the lipid fraction of the cell stroma. The phosphoric acid group of cephalin may be mainly responsible for such behavior. Protein does not have any significant role in the electrophoretic mobility of erythrocytes. According to Bangham et_al. (1958a) phos- phate groups on lipids are responsible for the negative electric charge on sheep erythrocytes. Heard and Seaman (1960) and Seaman and Heard (1960) consider the surface structure of the human erythrocyte to be a macropolyanion imparting a definite electric charge which can be modified by opposite ion association and possibly by adsorption of hemolysate. The authors attribute the negative charge on the erythrocyte to be due principally to a carboxyl or sulfate group or a mixture of both. At a physiological pH and ionic strength there is no positive grouping and evidence for the phOSphate groups being mainly responsible for the net negative charge is circumstantial. Madoff and Eyler (1961) reported that the negative charge on the erythrocyte is mainly due to sialic acid of varying degree in different species of mammals. N— glyco- lylneuraminic acid is present in the stroma of the erythrocytes of the sheep, pig, horse, and ox (Klenk and Uhlenbruck, 1958) and N-acetylneuraminic acid is in the stroma of erythrocytes of man and chicken (Klenk and Lemp— frid, 1957; Klenk and Uhlenbruck, 1958). There is a linear relationship between the percentage of total N-acetylneuraminic 6 acid released and the per cent reduction in the electro— phoretic mobility or total surface charge on human erythrocyte (Eyler et_al,, 1961). The erythrocyte behaves unusually and differently to a variety of substances of animal and plant origin. Gela- tin, egg albumin, casein, hemoglobin, and fibrinogen do not adsorb to the surface of the erythrocyte and its electro- phoretic mobility is not affected (Abramson §t_al,, 1942). According to Abramson and Moyer (1930), hemolysis caused by chloroform, freezing and thawing, and water does not significantly alter the mobility of the erythrocytes of rabbits. Rottino and Angers (1961) however, reported that hemolysis due to ruptured cells, chronic inflamatory diseases and malignant ne0p1astic diseases alters the electrophoretic mobility of human erythrocytes. Trypsin reduces the electrophoretic mobility of human erythrocyte by 25% (Ponder, 1951), and a glycopeptide containing sialic acid is released from the human erythrocyte (Cook et_§l,, 1960). Agglutination of erythrocytes by extracts from plant (Netter, 1956) and animal tissues (Stone, 19A6), higher fungi (Fahey, 195A), bacteria (Netter, 1956), rickettsia and pleuro—pneumonia—like organisms (Fahey, 1954) has long been established. Endowed with an antigen and receptor on its stroma, the erythrocyte is agglutinable by complementary structures in human sera and some viruses, respectively. 7 That influenza virus agglutinates chicken erythrocytes was a significant step forward in virology (Hirst, 1941; McClelland and Hare, 1941). Hirst (1942a, 1942b) demon— strated that influenza virus adsorbed to the chicken erythr- ocyte and formed bridges between adjacent erythrocytes with resulting agglutination. After a reaction period dependent upon time and temperature, the virus eluted from the surface of the erythrocyte. It was soon discovered that hemagglu- tination was not a characteristic of all viruses and that not all erythrocytes from mammals or birds were agglutinable. Different mechanisms of hemagglutination are involved and viruses can be classified under several groups (Hirst, 1959). Direct Hemagglutination l. Arthropod-borne viruses The virus particle itself is possibly the hemagglutinin (Sabin and Buescher, 1950) and very Specific conditions are necessary for hemagglutination. Japanese B, Western equine, yellow fever, and dengue viruses are some known agents of the group that have a wide difference in antigenicity. They are not known to contain any enzymatic activity (Anderson, 1959). 2. Lipoportein hemagglutinins The hemagglutinin is distinctly separable from the virus particle. There are two sub-groups: (a) Pox viruses that include vaccinia, variola, and ectromelia; (b) Psittaco- sis-lymphogranuloma venerum group including meningo- 8 encephalitis virus. Lecithinase inactivates the hemagglu— tinin which may be a 1ip0protein. 3. Echo viruses and Coxsackie B3 The hemagglutinin is associated with the virus particle which elutes from the surface of the erythrocyte. The receptors of this group of viruses are different from those of the myxoviruses. Hemagglutinin is active against human type 0 erythrocytes. A. Adenoviruses More than 18 different types of viruses of this group Specifically agglutinate erythrocytes from different species of animals. 5. Miscellaneous group Encephalomyocarditis viruses agglutinate only sheep erythrocytes. Pneumonia Virus of mice agglutinates mouse and hamster erythrocytes. The hemagglutinin appears to be a part of the virus particle. The murine poliomyelitis (GDVII strain) virus has a hemagglutinin which agglutinates human erythrocytes at low temperature. The polyoma virus of mice, an oncogenic virus, agglu- tinates erythrocytes from many mammalian species. 6. Myxoviruses Mumps, Newcastle disease virus, fowl plague, and strains of influenza virus are included in this group. The 9 hemagglutinin is associated with the virus particle con- taining the enzyme, neuraminidase. The interaction of influenza virus and the erythrocyte is similar to the formation of an enzyme-substrate complex (Hirst, 1942a). The initial attachment is inoic in nature and the hydroxyl group of a polysaccharide chain present on the substrate is responsible (Buzzell and Hanig, 1958). The virus finally elutes from the erythrocyte due to the action of neuraminidase on the receptor of the erythrocyte. According to Hanig (1948), there is a decrease of the net surface charge of the human erythrocyte and the electro- phoretic mobility which may be attributed to any of the following: (1) loss of charged substances from the cell surface, (2) change in the spatial configuration of charged groupings on the cell surface, or (3) adsorption by the erythrocyte of charged elements of the solvent. Hanig also concluded that the reduced electrophoretic mobility was a result of the destruction of the virus receptors on the erythrocyte surface. Bateman §t_al, (1956) have attributed the reduced electrophoretic mobility to the formation of additional free positively charged groups on the surface of erythrocytes. Ada and Stone (1950), Stone and Ada (1952) reported that the reduction in electrophoretic mobility after treat— ment of human erythrocytes with myxoviruses and receptor destroying enzyme (RDE) was in close approximation to the "receptor gradient" proposed by Burnet et a1. (1946). 10 However, Newcastle disease and swine influenza viruses in allantoic fluid deviated from their normal position in the receptor gradient, indicating that there may be some inhibitors in the allantoic fluid for these viruses, and that the loss of agglutinability was n0t a direct function of the total residual electric charge on the erythrocyte surface. Receptor Destroying Enzyme Receptor destroying enzyme (RDE) (Burnet 22 a1., 1946; Burnet and Stone, 1947) is an induced (adaptive) exo-enzyme of Vibrio cholerae that has played an important role for a better understanding of some of the biological concepts underlying the virus—erythrocyte receptor reaction, inhibi— tion of hemagglutination, and inactivation of both receptor and inhibitor by virus. This enzyme of V, cholerae is now known as neuraminidase (Gottschalk, 1957). It is defined as the specific «d — glycosidase cleaving the ad — ketosidic linkage joining the potential keto group of a terminal N-acylated neuraminic acid to an adjacent sugar residue in a disaccharide, trisaccharide, or polysaccharide (Gottschalk, 1958). Neuraminidase can be prepared in crystalline form (Schramm and Mohr, 1959; Ada and French, 1959). The enzyme can also be prepared from a variety of microorganisms such as Clostridium welchii, Clostridium tertium, Pseudomonas fluorescens, Pseudomonas pyocyanus, Pseudomonas stuzeri, Lactobacillus bifidus, and Diplococcus pneumoniae. 11 The myxoviruses possess 9C - neuraminidase and the enzyme, assumed to be present in patches on the virus sur- face, seems to be associated with the virus hemagglutinin (Gottschalk, 1960). Howe gt_§1. (1961) suggested that myxovirus hemagglutinin and neuraminidase have separate identities. Burnet (1942) pr0posed a ”receptor gradient" or the order in which the myxoviruses may be graded according to their receptors. According to Hirst (1959), there are two hypotheses to explain the receptor gradient: (1) several different kinds of receptors are on the erythrocyte surface and different kinds of viral enzymes may be involved although there is yet no evidence for such multiplicities, and (2) accessibility of similar receptors on the erythrocyte surface for viral enzymatic action. This does not explain the situation where the hemagglutination titer is not a direct function of the total residual charge on the erythro- cyte surface. Inhibitors (Ada and Stone, 1950), or muco- protein receptor substances, having different configurations (Howe et_al,, 1961) may be responsible. Inhibitors ;_-ctFrancis-(l947) reported that influenza virus heated at 56 C for 30 minutes agglutinates erythrocytes but does not elute from them. Francis identified this as ”indicator virus." Normal serum strongly inhibits hemagglutination by indicator virus but not non—heated virus. The inhibitors 12 identified thus far are mucoproteins (Hirst, 1959) and some mucolipids (Rosenberg gt_§1., 1956; Rosenberg and Chargaff, 1958). Mucoproteins are defined as conjugated proteins with multiple hexosamine containing oligosaccharides or small polysaccharides as the prosthetic groups. The pros- thetic groups are covalently linked to the protein core (Gottschalk, 1952, 1954). Inhibitory muscoproteins are present in a variety of sources such as human and rabbit serum, ovarian cysts, sheep salivary gland (McCrea, 1948, 1952a), normal human urine (Tamm and Horsfall, 1952), tissue extract (Hirst, 1959), sputum mucoid and brain mucoid (Howe §t_§1,, 1961), meconium from infants (Curtain §£_§l,, 1953; Pye, 1955; Zilliken, et_§13, 1957), and erythrocyte stroma (Howe, 1951; Howe gt_a1., 1957; McCrea, 1953b). All these known inhibitors, however, lose their activity when treated with intact myxoviruses, RDE (neur- aminidase), trypsin, or periodate (0.001M) (Gottschalk, 1960). Sialic acid, a group name for acylated neuraminic acids, is released from the inhibitors through the action of neuraminidase. Neuraminic acid is the basic unsubstituted structure C9H1708N, common to all the inhibitors. Not all sialo-mucoproteins are inhibitory. To qualify as a virus hemagglutinin inhibitor, the mucoprotein must have a substrate for neuraminidase, and compete successfully with the receptor on the cell surface for the virus. The successful competitor, either the erythrocyte receptor or 13 the mucoprotein, exerts the more attractive force and has the relatively greater number of functional groups for the virus particle (Gottschalk, 1960). Mucolipids from ox brain (FOlCh.EE.§l°: 1951) and human brain have a low degree of inhibitory capacity towards PR-8 (Rosenberg et_al., 1956; Rosenberg and Chargaff, 1958) and PR 301 (Howe et_§1., 1961) indicator virus. Indirect Hemagglutination Indirect hemagglutination (Lennette, 1959) is due to the action of specific immune serum on erythrocytes treated withtannic acid and then coated with viral or rickettsial antigen. Tanned sheep erythrocytes to which herpes simplex virus has been adsorbed agglutinate with specific immune serum (SCOtt.EE.§l-: 1957). Brown_et'a1. (1962) have reported that the horse erythrocyte can be modified with tannic acid to give an in- direct hemagglutination test for two strains of IBV. Infectious Bronchitis Virus Infectious bronchitis virus, Tarpeia pulli (Merchant and Packer, 1961; von Rooyen, 1954), is the etiological agent of infectious bronchitis of chickens. The virus is a sphere with a diameter of 65 mp to 135 mp (Reagan E£.§l°) 1950); Reagan and Brueckner, 1952; Nazerian, 1960). It exists in two phases, the thermolabile D phase and the thermostable 0 phase (Singh, 1960). The optimum pH for stability is 7.8. The isolectric point is about pH 4.05 14 (Cunningham and Stuart, 1947). The approximate density of the virus is 1.15 (Buthala, 1956). The virus can readily be cultivated in the chicken embryo. The virulence of the virus for chicken embryo is increased through serial passage (Cunningham, 1957). The Beudette strain or egg—adapted strain of IBV can be cultivated in chicken embryo kidney cells, chicken embryo fibroblasts (Spring, 1960), chicken liver and heart cells (Fahey and Crawley, 1956), and in the isolated chori— oallantoic membrane (Ferguson, 1958; Ozawa, 1959). Infectious bronchitis virus in allantoic fluid does not cause hemagglutination. Modification of the virus with trypsin induces agglutination (Corbo and Cunningham, 1959) of erythrocytes from turkeys and from chickens older than three weeks. The same receptors on the chicken erythrocyte may be involved with both influenza (PR-8) and infectious bronchitis virus (Muldoon, 1960). The hemag- glutinin is associated with the virus particle (Nazerian, 1960). Specific inhibition of hemagglutination of anti—IBV serum has not been accomplished. Inhibition also occurs with normal chicken serum (Corbo and Cunningham, 1959; Muldoon, 1960). Trypsin, sodium or potassium periodate (0.9 to 0.01 M), zymosan, and RDE do not remove inhibitors of the virus hemagglutinin present in normal and immune sera (Muldoon, 1960). Trypsinanodified IBchfly'adsorbs to and agglutinates chicken erythrocytes according to studies with the indirect 15 fluorescent antibody technic (Stultz, 1962), and electron microscopy (Nazerian, 1960). TrypsinemodifiedlflMfin allantoic fluid does not pre- cipitate with its antibody in agar-gel-medium whereas non— trypsinized infectious bronchitis virus precipitates with its antibody (Tevethia, 1962). Brown et a1. (1962) have reported that horse erythro— cytes can be modified with tannic acid to give an indirect hemagglutination test for two strains of IBV. MATERIALS AND METHODS Viruses Infectious bronchitis virus, 41 (IBV-41), the repository code for the Massachusettes strain, Newcastle disease virus (NDV) and PR—8 strain of influenza virus were used. The viruses were cultivated in ten—day—old embry- onating chicken eggs, inoculum 0.1 ml per egg. The allantoic fluid from the IBV-infected embryo was harvested 72 hours postinoculation whereas NDV and PR-8 influenza virus were harvested 48 hours postinoculation. The pooled viruses were stored at -70 C in screw cap vials. At the time of use they were thawed at room temperature, centri— fuged at 1400 g for ten minutes, and the supernatant fluid was removed and used for experimental purposes. All cul- tures were tested for bacteriological sterility in Brewer thioglycolate medium (Difco). Normal Allantoic Fluid Normal allantoic fluid (NAF) was collected from thirteen-day—old embryonating chicken eggs to serve as the control for the viruses contained in allantoic fluid. 16 l7 Erythrocytes Blood was obtained from a Single Comb White Leghorn cockerel and from a turkey by cardiac puncture. Human type 0 blood and blood from a cow and horse were obtained by venipuncture. The blood was collected in tubes containing 1 ml of a 2% (w/v) sodium citrate solution for each 6 m1 of blood. The blood was centrifuged immediately and the plasma was removed. One volume of the packed erythrocytes was then washed three times by centrifugation for ten minutes per wash, using about thirty volumes of saline solution for each wash. After the last wash, the saline was removed from the packed erythrocytes which were stored at 4 C for as long as four days. At the time of use, the erythrocytes were diluted to an apprOpriate concentration with buffer for a particular experiment. Diluents Saline, sodium chloride 0.85% (w/v) in double distilled deionized water, and Bacto (Difco) hemagglutination buffer (pH 7.35 :_0.05) were used. Sorensen's 0.067M phosphate buffer (Clark, 1925) at pH 7.35 was also used. Dextrose (dehydrated) 1% (w/v), was added to prevent hemolysis during electrophoresis. Trypsin Bacto-trypsin (Difco 1:250) 1% in double distilled deionized water was incubated for 15 minutes at room 18 temperature, passed through a Seitz EK filter and the fil- trate was stored at -30 C until used. Eggwhite Trypsin Inhibitor A one per cent solution (w/v) of egg white trypsin inhibitor (ETI) (California Corporation for Biochemical Research) in double distilled deionized water was stored at ~30 C until used. Receptor Destroying Enzyme A 1y0philized sample (Behringwerke, Marburg-Lahn, Germany) was reconstituted to volume in double distilled deionized water and serial two-fold dilutions were prepared with calcium borate buffered saline solution (Burnet and Stone, 1947). Electrophoresis Apparatus A Northrop-Kunitz flat, horizontal type electr0phore— sis cell apparatus (Arthur H. Thomas Co.) was used. The electrical circuit consisted of a VOKAM power pack (Shandon Scientific Co., Ltd., London) that can convert AC to DC with a variable potential from O to 400 volts and a current strength varying from 0 to 80 milliamperes. In the experiments, 100 volts with a current strength of 5 milli- amperes were used. The optical system consisted of a monocular microscope with a 4 mm 0.66 NA high dry objective and 10X eye piece with a micrometer disc. A Reichert Viewscope was mounted 19 over the eye piece for ease of viewing the specimen. The micrometer disc lines projected at the level of the elec- trophoresis cell were 33°33.P apart. This measurement was made by superimposing the image of the disc upon the rulings of a hemocytometer placed at the level of the electrophoresis cell. The zinc electrodes were cleaned with water to remove the deposition formed on them after being used four to five times. The electrodes were used with a saturated zinc sulfate solution prepared in deionized water. The electrophoresis cell was thoroughly washed with acid cleaning solution, rinsed with double distilled water, and coated with gelatin solution and then with the buffer to be employed before each individual test. The two station- ary levels of the electrophoresis cell were determined after the interior of the cell was coated with one per cent gelatin in water (Abramson_et_al., 1942). Measurements made at the two levels were similar. After several prelim— inary electrophoretic tests in which the measurements were essentially the same, it was more convenient to make readings at only one of the two levels. Usually ten electrophoretic tests were made of each sample and the results were averaged. There was little difference in the measurements for each sample when the direction of the current was reversed. Tests were performed first with normal erythrocytes. 20 The electrophoretic mobility, v, of the erythrocyte is expressed as the velocity per electrical field strength (X) in volts per cm. velocity/ X : distance traveled / X time < ll >< II I/ 9 KS , where I = current in amperes; q = cross sectional area of the electrophoresis cell in cm2; and KS = specific conduc- tivity of the buffer. The following were the constants for the experiments: I = 0.005 amperes q = 0.12 cm2 KS = 0.0045 ohms"l cm’l. x = O 005 amps = 9.26 volts/cm. 0.12 cm2 x 0.0045 ohm‘l cm’l A typical example for v = velocity/X = 10.85 p sec‘1 9.26 volts cm‘l = 1.17 P sec'l volt'l cm‘l Hemagglutination Modification of IBV by trypsin was based on the pro— cedure described by Muldoon (1960). Allantoic fluid containing the virus was thawed at room temperature, centrifuged at 1400 g for 10 minutes and the supernatant fluid was collected. To two volumes of the supernatant fluid, one volume of 1% trypsin was added. After the mixture 21 was incubated in a water bath at 56 C for 30 minutes, one volume of 1% ETI was added and the mixture was incubated at room temperature for at least 15 minutes. The procedure for the hemagglutination test was the same for all viruses (Cunningham, 1960). Serial two—fold dilutions of the virus were prepared in hemagglutination buffer. To each of a series of 12 x 75 mm tubes was added 0.25 ml each of diluted virus, saline solution, and 0.5% erythrocytes. The tubes were shaken for about 10 seconds and then incubated for 1 hour at room temperature. The hemagglutination titer expressed as HA units was the recip— rocal of the highest dilution of the virus in which hemagglutination was complete. Treatment of Erythrocytes with Virus All the erythrocytes were used with trypsin-modified IBV, but only chicken erythrocytes were used for NDV and PR-8 influenza virus. The ratio of virus to erythrocytes and the optimum time for the establiShment of equilibrium were based on the electrophoretic mobility of the treated erythrocytes. The following was established as the standard proce- dure. To 0.25 ml of packed erythrocytes was added 5 m1 of virus containing 100 HA units per 0.25 ml and the mixture was incubated for four hours at room temperature. The erythrocytes were then washed with buffer for a particular experiment and a final suspension of 0.1% erythrocytes was made. 22 Control samples of erythrocytes were prepared in the same manner except that buffer was used in place of the virus suspensions or other reagents. Chicken and turkey erythrocytes were treated with non-trypsinized IBV, NAF, and NAF treated with trypsin and egg white trypsin-inhibitor (NAF + T + ETI) in the same manner as for trypsin—modified IBV. The treatment of the erythrocytes with these reagents also served as a control as they did not cause hemagglutin- ation. Treatment of Erythrocytes with Trypsin Erythrocytes were treated with trypsin in the same manner as they were treated with the viruses. When four VOlumes of trypsin were added to one volume of packed erythrocytes and incubated for 30 minutes at 37 C the same result was obtained as at room temperature for four hours. Chicken and turkey erythrocytes were treated with trypsin, washed, and then treated with trypsin—modified IBV, NDV, PR-8 influenza virus, RDE, non—typsinized IBV, NAF, and NAF treated with trypsin and ETI. The erythrocytes were washed finally and resuspended in the appropriate buffer. Treatment of Erythrocytes with RDE To 0.25 ml of packed erythrocytes was added 5 ml of RDE diluted in calcium borate buffered saline to contain 100 RDE units per ml. The mixture was incubated at 37 C for four hours. The erythrocytes were then washed and some 23 of these erythrocytes were further treated with PR-8 influ- enza virus and trypsin-modified IBV. Titration of RDE was done according to the procedure described by Burnet and Stone (1947). To two-fold serial dilutions of the enzyme, an equal volume (0.25 ml) of 1% chicken erythrocytes was added. The mixture was incubated at 37 c for 30 minutes. 0ne drop (0.04 ml.) PR—8 influenza virus, containing 10 HA units, was added to each tube con— taining the RDE treated chicken erythrocytes. The tubes were shaken for a few seconds and then incubated for 30 minutes at 37 C. The end point was the highest dilution of the enzyme in which partial agglutination occurred. The' units were expressed in RDE units. Standard error of the electrophoretic mobility was calculated after Waugh (1943). Formula: {m = %;= where 07H is the estimate of the standard deviation of the means, 6; is the standard deviation of the samples and N is the number of observations in each experi- ment. RESULTS Erythrocytes from the horse, cow, and human did not agglutinate with trypsin—modified IBV and there was no reduction of their electrophoretic mobility. Erythrocytes from the chicken and turkey agglutinated with trypsin- modified IBV. Electrophoretic mobility was reduced from 1.17 to 0.92, a 21.4% reduction, for the chicken erythro- cytes and from 1.27 to 1.05, a 17.4% reduction, for the turkey erythrocytes (Table 1). TABLE l.—-E1ectrophoretic mobility of erythrocytes in 0.067M phosphate buffer at pH 7.35 before and after reaction with trypsin- modified IBV. Electro horetic Agglutination mobi ity Per Cent Source by typsin- Al sec’lvolt‘lcm’l reduction of modified trypsin-mEH} of erythrocyte IBV Normal IBV mobility Chicken + 1.17 0.92 21.4 Turkey + 1.27 1.05 17.4 Horse — 0.91 0.91 0 Cow - 0.93 0.93 0 Human — 1.31 1.31 0 24 25 Agglutination of chicken and turkey erythrocytes and reduction of electrophoretic mobility (Table 1) occurred only when trypsin-modified IBV was used. Non—trypsinized IBV, NAF, or NAF treated with trypsin and ETI did not cause hemagglutination or reduction of the electrophoretic mobility (Table 2). Trypsin reduced the electr0phoretic mobility of the chicken and turkey erythrocytes from 1.17 to 1.04, an 11.1% reduction, and from 1.27 to 1.22, a 3.9% reduction, respectively. There was no reduction of the electr0phoretic mobility of these erythrocytes after treatment with a mix- ture of trypsin and ETI. Erythrocytes treated with trypsin and then with NAF, NAF treated with trypsin and ETI or IBV did not have any further reduction of electr0phoretic mobility. When trypsin-treated erythrocytes were treated with trypsin—modified IBV the electrophoretic mobility was further reduced (Table 3). The electrophoretic mobility of chicken erythrocytes, 1.17 was reduced to 0.92/p.sec'lvolt‘lcm‘l, a 21.4% reduc- tion, after treatment with trypsin-modified IBV; to 0.68, a 41.9% reduction, after treatment with PR-8 influenza virus; to 0.53, a 54.7% reduction, after treatment with NDV and to 0.42, a 64.1% reduction, after treatment with RDE (Table 4). 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