INFECTION AND IMMUNrrY, Dec. 1974, p. 1235-1240Copyright 0 1974 American Society for Microbiology

Vol. 10, No. 6Printed in U.S.A.

Some Properties of Precipitating Antigens Associated withInfectious Bursal Disease VirusK. HIRAI, E. KAWAMOTO,' AND S. SHIMAKURA

Department of Veterinary Microbiology, Faculty of Agriculture, Gifu University, Gifu, Japan

Received for publication 28 June 1974

Infection of the bursa of Fabricius and chicken embryo fibroblast cell cultureswith avian infectious bursal disease virus resulted in production of a number ofvirus-induced antigens. The antigens were specific, forming three precipitin linesby immunodiffusion with antiserum (designated PA-1, -2, and -3). To separateimmunoprecipitin from the remaining viral particles, two (PA-1 and PA-3) were

partially purified by subjection to two cycles of diethylaminoethyl-cellulosechromatography and filtration through a column of Sephadex G-150 gel. Theprecipitating antigen, PA-1, was found to migrate most slowly through the agar

gel, remaining serologically active after treatment with heating (56 C for 1 h),trypsin, lipolytic solvents, deoxyribonuclease, and ribonuclease. Its density was

1.27 g/ml. Morphologically the antigen displayed a doughnut-shaped structure 8to 12 nm in size. PA-3 migrated most rapidly through the agar gel. It was destroyedby treatment with heating and trypsin but not with lipolytic solvents, deoxyribo-nuclease, and ribonuclease. Density was about 1.25 g/ml. This suggests that theantigen is a part of viral structural components. PA-2 migrated through agar gelat a rate between that of PA-1 and PA-3. Because of its low concentration, PA-2was not further characterized.

Infectious bursal disease (IBD), also calledGumboro disease, is a viral disease of youngchickens (3). The disease specifically affects thebursa of Fabricius (BF) and sometimes otherlymphoid organs. Necrotic lesions of the bursalfollicles are the predominant changes. The dis-ease is of interest immunologically, since youngchickens affected by the disease are unable tocope satisfactorily with other infection, presum-ably because of the depression of bursa-derivedlymphocytes (1, 4, 9).

Propagation of IBD virus in embryos has beenreported by various investigators. However, in-formation on propagation of the virus in cellcultures has not been studied for a long time.Recently, Mandelli et al. (10) reported that anegg-adapted strain of IBD virus replicated inchicken embryo fibroblast (CEF) cells, produc-ing a defined cytopathic effect.

Previous work from this laboratory reportedthe development of an immunodiffusion reac-tion that has been established as useful for thediagnosis of IBD (7). The antigen used in thisreaction must be extracted from the typicallyaffected BF of chickens exhibiting the symp-toms of IBD. The specificity of this test for IBDhas been confirmed by several diagnostic labo-

' Present address: Department of Laboratory AnimalScience, Tokyo Medical College, Tokyo, Japan.

ratories. In the investigation of antigen proper-ties, it was also noted that precipitating anti-gens were produced in the medium from CEFcells infected with IBD virus.From the viewpoint of a comparative study of

the precipitating antigens, it will be of interestto clarify properties of the antigens that are notyet thoroughly studied. This paper deals withsome properties of the antigens.

MATERIALS AND METHODS

Virus and cell culture. Two strains of IBD viruswere used. The strain GBF-1 was described previously(6). It was prepared from the fifth and tenth passagesof the virus in susceptible chickens. An egg-adaptedstrain, 1/PV, was kindly supplied by G. Cervio of theInstituto Zooprofilattico Sperimentale della Lom-bardia E Dell' Emilia, Italy. It replicates in CEF cellsthat produce a cytopathic effect (10, 11). PrimaryCEF cell monolayers were prepared from 11-day-oldchicken embryos. The cells were grown in Eaglemedium supplemented with 5% calf serum and main-tained in Hanks balanced salt solution containing0.5% lactalbumin hydrolysate and 1% bovine fetalserum.

Antigen preparations. Typically affected BF ofchickens were harvested on the 4th day after inocula-tion. They were diced, diluted with an equal volumeof 0.01 M phosphate-buffered saline (PBS), andhomogenized in a Waring blender and Teflon homoge-nizer. The resulting homogenate was frozen and

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thawed 10 times. The materials were clarified bylow-speed centrifugation at 5,000 rpm for 30 min andhigh-speed centrifugation at 10,000 rpm. The super-natant was concentrated five times and used as crudeBF antigen (B-Ag).

Cell monolayers were inoculated with strain 1/PVof IBD virus. Cell culture medium, exhibiting 4+cytopathic effect 2 days after inoculation, was har-vested, and cellular debris was removed by low-speedcentrifugation at 5,000 rpm for 30 min. The superna-tant was added with ammonium sulfate (300 g/liter).After being stirred for 1 h, the suspension wascentrifuged at 10,000 rpm for 20 min. The precipitatewas dissolved in a minimal amount of PBS anddialyzed for 12 h against 500 volumes of the samebuffer to remove ammonium sulfate. The solution wascentrifuged at 10,000 rpm for 1 h and used as thecrude medium antigen (M-Ag).

DEAE-cellulose batch and column chromatog-raphy. The diethylaminoethyl (DEAE)-cellulose wasactivated with 0.5 N NaOH and then washed with dis-tilled water. It was equilibrated with PBS. One vol-ume of DEAE-cellulose was added to the crude B-Agor M-Ag, and after being stirred in the cold for 10 min,the gel was separated by centrifugation at 5,000 rpmfor 5 min. To remove the contaminating substances,the DEAE gel was washed with PBS. The antigenicactivity was eluted by 0.8 M NaCl in 0.0045 M phos-phate buffer (PB). The eluate was concentrated about12-fold by forced dialysis against polyethylene glycol(molecular weight, 6,000) and dialyzed for 12 h against100 volumes of PBS. The materials were applied to acolumn (30 by 2 cm) of DEAE-cellulose. Chromatogra-phy was carried out by the stepwise elution methodwith NaCl solution of various concentrations in PB.The flow rate was 0.5 ml/min, and the effluent frac-tions were continuously monitored at 280 nm with aToyo Uvicon (UV-540 M). Three-milliliter fractionswere collected. The maximal absorbancy and two frac-tions outside of the maximum were pooled and con-centrated for detection of the antigenic activity.Sephadex G-150 column chromatography. A

0.5-ml sample partially purified by DEAE-cellulosecolumn chromatography was applied to a column (100by 2.6 cm) of Sephadex G-150 (Pharmacia). The flowrate was adjusted to about 0.5 ml/min, and 3 ml ofeach fraction was collected in a tube.Heat stability test. Samples of antigens were

heated in a water bath at 56 and 78 C for 1 h. Theywere cooled immediately and assayed in an immuno-diffusion test for antigenic activity.Treatment of enzymes and lipolytic solvents.

Antigens were treated with 1% trypsin, deoxyribonu-clease (1 mg/ml), and ribonuclease (1 mg/ml) in PBSfor 12 h at 37 C. Antigens were added to an equalvolume of ether and chloroform. They were held at22 C for 1 h and centrifuged at 3,000 rpm for 10 min.The treated antigens were tested by immunodiffusionfor antigenic activity.

Density gradient. For density gradient determina-tions, about 4 ml of antigen was carefully layered over0.8 ml of saturated cesium chloride in PBS and thencentrifuged at 40,00 rpm for 24 h in a swinging-rotor

Hitachi RPS 40. Fractions were collected from thebottom through puncturing with a needle. Buoyantdensity was determined by measuring the refractiveindex of the samples in the refractometer.Membrane fiXltration. The partially purified anti-

gens were passed through a 10-nm membrane filter(Millipore Corp., Bedford, Mass.), checked for antige-nicity by immunodiffusion, and inoculated into thechickens as a test for separation of infectivity fromantigenicity.

Immunodiffusion. Agar gel precipitin slides wereprepared with diffusion medium containing 1% spe-cial Noble agar (Difco) in PBS and 0.01% merthiolate.Microscopic slides were flooded with 3 ml of meltedagar; the agar was allowed to solidify, and then wellsof 3 mm in diameter were cut at a distance of 4 mmfrom each other. The diffusion was allowed to proceedin a moist chamber at 22 C for 4 days.

Electron microscopy. Electron microscopy wascarried out as previously reported (5, 8).

RESULTSPrecipitating antigens in the infected BF

and cell culture medium. The crude antigens,B-Ag and M-Ag, produced three precipitin linesagainst antiserum. These antigens were desig-nated as precipitating antigens PA-1, -2, and -3,respectively. The designation for the precipitinlines is based on the position between theantigen and antibody wells (Fig. 1). PA-1formed the most intense line and was nearest tothe antigen well. PA-2 formed the weakest linebetween PA-1 and PA-3. PA-3 formed a sharpline and was located at the center positionbetween antigen and antibody well. Thus, thethree lines were common to B-Ag and M-Ag.The lines of PA-1 and PA-3 appeared as early as12 h, whereas PA-2 appeared at 36 h, afterdiffusion had been started. Precipitation didnot occur when concentrated normal BF andcell culture medium were examined by immu-nodiffusion against antiserum. After dilution ofantigens, the line of PA-1 was last to be ad-sorbed and was diminished with 32-fold dilu-tions. PA-2 was present in the lowest concentra-tion. PA-3 diminished with 16-fold dilutions.DEAE-cellulose chromatography. The ma-

terials eluted by 0.8 M NaCl of DEAE-cellulosebatch chromatography produced the three linesagainst antiserum as found in the crude B-Agand M-Ag. The samples were applied to theDEAE-cellulose column. Figure 2 shows theelution profile of the B-Ag removed from aDEAE-cellulose column in a stepwise fashionwith increasing concentrations of NaCl in PB.The absorbancy of ultraviolet light was recog-nized with the fractions eluted by each NaClconcentration. The antigenic activity was found

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FIG. 1. Immunodiffusion pattem of the crude anti-gens. The center bottom well (S) contained chickenantiserum to strain GBF-1 of IBD virus. The periph-eral wells contained the following antigens: (well 1)sample extracted from normal bursa of Fabricius;(well 2) crude antigen prepared from bursa of Fa-bricius of chickens inoculated with strain GBF-1 ofIBD virus; (well 3) crude antigen prepared from cellculture medium infected with strain 1/PV of IBDvirus; and (well 4) sample concentrated from normalcell culture medium.

The elution pattern of M-Ag was the same asthat of B-Ag described above.Gel filtration. The materials eluted by 0.2 M

NaCl of DEAE-cellulose chromatography wereconcentrated and partially purified further bySephadex G-150 chromatography (Fig. 3). Thetop fractions of two Sephadex peaks were con-centrated for examination in precipitin tests.PA-1 was found in the first peak but not in thesecond. PA-3 was not detectable in either peak.PA-1 eluted by gel filtration and PA-3 eluted

by 0.4 M NaCl from DEAE-cellulose chroma-tography proved to be immunologically distinct(Fig. 4).Some physicochemical properties of PA-1

and PA-3. Some properties of antigen weretested by the use of the partially purifiedmaterials with DEAE-cellulose column andSephadex G-150 chromatography (Table 1).PA-1 was stable to heating at 56 C for 1 h. Theprecipitin line of PA-3 disappeared at 56 C for 1h. PA-1 was resistant to trypsin, but PA-3 wassensitive. The antigens were detectable afterexposure to ether and chloroform. Deoxyribonu-clease and ribonuclease had no effect on either.PA-2 could not be tested because of its lowconcentration in the antigen preparations.Buoyant density. The activity of the PA-1

showed buoyant density of 1.26 to 1.28 g/ml in

FIG. 2. Chromatography of B-Ag on DEAE-cel-lulose. Elution pattern was obtained when 5 ml ofsamples was chromatographed on DEAE-celluloseequilibrated with initial PBS, pH 7.0; column size, 2.6by 30 cm; elution buffer, 0.01 to 0.6M NaCI in 0.0045M phosphate buffer; flow rate, 0.5 ml/min. Theantigenic activity was found in the peaks of 0.2 and0.4 M.

in those fractions eluted by 0.2 and 0.4 M(fractions 120 through 130 and 183 through 193).The antigen eluted by 0.2 M NaCl formed twoprecipitin lines, PA-1 and PA-3. PA-3 formedthe weak line. The antigen eluted by 0.4 MNaCl produced only a sharp precipitin line ofPA-3. The line of PA-2 was not observed inthose fractions eluted by 0.2 and 0.4 M NaCl.

FRACTION NMBER

FIG. 3. Gel filtration ofpartially purified PA-I on a

Sephadex G-150 column. The materials eluted by 0.2M NaCI of DEAE-cellulose chromatography wereconcentrated to 0.5 ml by forced dialysis againstpolyethylene glycol and chromatographed on a col-umn in phosphate buffer. Column size, 100 by 2.6 cm;flow rate, 0.5 ml/min. Antigenic activity (PA-1) was

found in the first peak.

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FIG. 4. Immunodiffusion pattern of the partiallypurified antigens (PA-1 and PA-3). The bottom well(S) contained chicken antiserum to strain GBF-1 ofIBD virus. The upper wells contained PA-1 partiallypurified from the DEAE-cellulose and SephadexG-150 column chromatography (well 1) and PA-3partially purified from the DEAE-cellulose columnchromatography (well 2).

TABLE 1. Some properties of IBD virus-associatedantigens

AntigenaTreatment

PA-1 PA-3

Temperature56 Cforlh +78 Cforlh _

Trypsin (1%) + +Ether (50%) + +Chloroform (50%) + +Deoxyribonuclease (1 mg/ml) + +Ribonuclease (1 mg/ml) + +

a Symbols: +, antigen forms a distinct precipitinline; -, antigen does not form precipitin line.

cesium chloride (Table 2). The buoyant densityof PA-3 varied from 1.24 to 1.26 g/ml. PA-2could not be determined due to its low concen-tration. The buoyant density of virions in ce-sium chloride was found to be 1.34 g/ml (5).

Filtration. The results of membrane filtra-tion studies indicated that the antigens passedthrough the 10-nm filter and were noninfectiousfor the chickens, whereas unfiltered rough B-Agwas infectious.

Electron microscopy. When the prepara-tions eluted by various NaCl concentrations ofDEAE-cellulose chromatography were exam-ined in the electron microscope, the intactvirions showing icosahedral configuration couldbe observed only in the 0.6 M NaCl eluates ofthe B-Ag and M-Ag (Fig. 5). PA-1 partiallypurified by Sephadex G-150 chromatography

regularly revealed a doughnut-shaped structureor its aggregates, which had a diameter of 8 to12 nm and a subunit arrangement (Fig. 6). Whenthe PA-3 preparations were examined in theelectron microscope, no distinct structure couldbe observed.

DISCUSSION

It is evident from the data presented here thatthere are three precipitating antigens specificfor IBD in the BF of infected chickens and ininfected cell culture medium. The antigens aresmaller in size than intact virions.The number of precipitin lines detected has

not been consistent (2, 7). Hirai et al. (7) founda precipitin line in the homogenate of theinfected BF of chickens; Almeida and Morris (2)presented evidence that two precipitin linesappear. These are possible explanations for theinability to demonstrate more than one precipi-tating antigen. The resulting 50% BF homoge-nate, further concentrated five times, is neces-sary to produce any reaction. It is possible thatother antigens were present in insufficientquantity to be detected by this method and thatthe test sera used in other experiments lackedsufficient antibody to any additional antigens.

TABLE 2. Cesium chloride density gradientcentrifugation of IBD virus-associated antigens

Antigen Buoyant density (g/ml)a

PA-1 1.26-1.28 g/mlPA-3 1.24-1.26Viral virion 1.34

aAntigenic activity determined by immunodiffu-sion reaction.

FIG. 5. Intact virions showing icosahedral configu-ration seen in the fraction eluted at 0.6 M NaCl ofDEAE-cellulose column chromatography of B-Ag.The micrograph was negatively stained with phos-photungstic acid. x 144,000. Bar represents 100 nm.

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FIG. 6. Doughnut-shaped structure observed in the PA-I preparation partially purified from DEAE-celluloseand Sephadex G-150 column chromatography of B-Ag. The sample was negatively stained with phosphotung-stic acid. x90,000. Bar represents 100 nm.

The three lines of the M-Ag were consistentlydetected in approximately a 500-fold concentra-tion of infective medium from CEF cells in-fected with IBD virus. A 100-fold concentrationof infective medium was sufficient to providePA-1 and PA-3 but not PA-2.The following evidence suggests that the

reaction of the B-Ag and M-Ag is specific forIBD virus: (i) the antigens did not react withpreinoculation sera but reacted only with seraobtained from natural infection and immuniza-tion of IBD virus; (ii) the serological reactivitywas lost when the antiserum was added to theantigen; (iii) the antigens did not react with thesera from chickens hyperimmunized againstinfectious bronchitis virus, Newcastle diseasevirus, infectious laryngotracheitis virus, andMarek's disease herpesvirus; and (iv) the anti-serum did not react with homogenates of nor-mal BF and cultured CEF cells, concentrationsof cell culture medium, or bovine serum used forthe medium.PA-1 migrated most slowly through the agar

gel. Its mean buoyant density of 1.27 g/ml wasslightly lower than of 1.34 g/ml for the virus (5).This may be due to the large size of the antigen.These facts are also suggested by the doughnut-shaped structure with a diameter of about 8 to12 nm found by electron microscopy in thesamples of PA-1. PA-1 remained serologicallyactive after treatment with lipolytic solvents,trypsin, and heating at 56 C for 1 h. Theproperties were identical with those of IBD

virus. Almeida and Morris (2) have observedthat the smaller particles (18 to 22 nm) alongwith hexagonal virus (60 nm) appear in the IBDvirus-infected bursal homogenated preparationby using immune electron microscopy. Accord-ing to them, although these particles weredifferent in features, they were antigenicallyrelated to each other. It is not known whetherthe smaller particles are related to PA-1. PA-1partially purified by Sephadex gel filtration wasnot infective. Although the significance andorigin of this antigen are not clearly known, it ishardly likely that the doughnut-shaped struc-ture (capsomere) consisting of intact virion (5)is associated with the antigenicity of PA-1.PA-3 migrated relatively rapidly through the

agar gel. The serological activity was destroyedby treatment with heating at 56 C for 1 h andtrypsin but not with lipolytic solvents. Themean buoyant density of 1.25 g/ml of PA-3 waslower than that of PA-1 and virus. PA-3 passedthrough the 10-nm filter and was noninfectiousfor the chickens. No infectious virus could bedetected in the plaque assay, nor were any virusparticles observed by electron microscope ex-amination of PA-3. In addition, when the par-tially purified virus was treated with sodiumdodecyl sulfate and submitted to agar gel, it wasdetected to yield precipitin lines identical withthe line formed against the PA-1 and PA-3(unpublished data). Accordingly, the antigeninvolved in the reaction might consist of a virusstructural component that is associated with

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the internal component of the virus' A numberof viral antigens may be detected by highlypurified and concentrated preparations of virus.More detailed information on this aspect will beprovided in a future report.The properties of PA-2 were not characterized

as were those of PA-1 and PA-3 because of thehigh concentration of B-Ag and M-Ag requiredfor its demonstration. This explains the largevolume of PA-2 available for study.

Intact virions are not considered to be in-volved in the reaction for the following reasons:the infective virus was eluted at a higher NaClconcentration than any of the antigens and alsoshowed a higher buoyant density than any ofthem; the particles were not observed by elec-tron microscopy in the antigen preparationspartially purified by DEAE-cellulose andSephadex G-150 chromatography; and the anti-gens passed through the 10-nm filter and werenoninfectious for the chickens.

Further studies are now in progress to charac-terize the precipitating antigens associated withIBD virus.

LITERATURE CITED

1. Allan, W. H., J. T. Faragher, and G. A. Cullen. 1972.Immunosuppression by the infectious bursal agent in

chickens immunized against Newcastle disease. Vet.Rec. 90:511-512.

2. Almeida, J. D., and R. Morris. 1973. Antigenically-related viruses associated with infectious bursal dis-ease. J. Gen. Virol. 20:369-375.

3. Faragher, J. T. 1972. Infectious bursal disease ofchickens. Vet. Bull. (London) 42:361-369.

4. Faragher, J. T., W. H. Allan, and G. A. Cullen. 1972.Immunosuppressive effects of the infectious bursalagent in the chicken. Nature (London) 237:118-119.

5. Hirai, K., and S. Shimakura. 1974. Structure of infec-tious bursal disease virus. J. Virol. 14:957-967.

6. Hirai, K., S. Shimakura, C. N. Chang, Y. Adachi, E.Kawamoto, N. Taguchi, Y. Suzuki, C. Itakura, F.Funahashi, Y. Tsushio, and M. Hirose. 1973. Isolationof infectious bursal disease virus and distribution ofprecipitating antibodies in chicken sera. Jap. J. Vet.Sci. 34:106-115.

7. Hirai, K., S. Shimakura, and M. Hirose. 1972. Immuno-diffusion reaction to avian infectious bursal diseasevirus. Avian Dis. 16:961-964.

8. Hirai, K., S. Shimakura, and E. Kawamoto. 1974.Electron microscope observation of infectious bursaldisease virus. Avian Dis. 18:467-471.

9. Hirai, K., S. Shimakura, E. Kawamoto, F. Taguchi, S. T.Kim, C. N. Chang, and K. Iritani. 1974. The im-munodepressive effect on infectious bursal diseasevirus in chickens. Avian Dis. 18:50-57.

10. Mandelli, G., E. Lodetti, A. Rinaldi, and G. Cervio. 1972.Charatteristiche colturali citologiche (IBA 1/PV). FoliaVet. Latina 2:399-425.

11. Petek, M., P. N. D'Aprile, and F. Cancellotti. 1973.Biological and physicochemical properties of the infec-tious bursal disease virus (IBDV). Avian Pathol.2:135-152.

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