INFECTION AND IMMUNITY, Apr. 1972, p. 467-473 Vol. 5, No. 4Copyright © 1972 American Society for Microbiology Printed in U.S.A.

Soluble Antigen of Blue-Tongue VirusC. S. WANG, D. C. LUEKER, AND T. L. CHOW

Departmenit of Microbiology, College of Veterinary Medicine and Biomedical Scienices,Colorado State University, Fort Collins, Colorado 80521

Received for publication 15 December 1971

A soluble viral antigen with possible diagnostic utility was obtained from blue-tongue virus-infected lamb kidney cells. It is a noninfectious proteinaceous substancewithout nucleic acid. The antigen is immunologically identical to one component ofthe viral particle, and in agar-gel diffusion tests it reacted with antiserum againstseveral blue-tongue virus strains.

Early studies of the antigenic plurality of theblue-tongue (BT) virus isolates demonstrated 10different BT virus strains (21). Later, 15 strainswere classified in South Africa (10, 11). In theUnited States, six BT virus isolates differing quan-titatively and qualitatively were differentiated invirus-serum neutralization tests in tissue culture(16). Klontz and co-workers (13) employed theagar-gel diffusion technique to detect precipitat-ing antibody in the serum of infected sheep andreported that their antigen was not the infectiousviral particle. In an immunodiffusion study of BTvirus, Jochim and Chow (12) noted that theantigen which reacted with antiserum to BT virusin agar-gel diffusion tests is soluble, is separablefrom the BT virus, and forms a precipitation linewith serum obtained from animals infected withdifferent strains of BT. Agar-gel diffusion testshave been used to detect precipitating antibodiesin the sera of infected cattle (20) and otherruminants (25).

This communication reports: (i) the isolationand purification of soluble antigen from BT virus-infected lamb kidney cells (LK), (ii) the identifica-tion of antigen origin by immunological means,and (iii) the suitability of the antigen as a diag-nostic agent to detect BT virus infection in ani-mals.

MATERIALS AND METHODS

Preparation of virus and cell cultures. Type 8 blue-tongue virus (BT-8), the American strain which hasbeen employed in commercial vaccines (17), was se-lected for this study. A stock preparation of BT-8 virusof the 28th serial passage in LK cells was obtainedfrom the Animal Disease Research Laboratory, Den-ver, Colorado. Virus was propagated in the secondpassage of LK cells grown in Earles' balanced saltsolution with lactalbumin hydrolysate (LE medium)plus 10%0 normal lamb serum. Primary LK cells wereprepared and stored by the method of Lindsay andChow (14).

Isolation of soluble antigen. The soluble antigen wasprepared as shown in Fig. 1. The confluent LK cellculture infected with BT-8 virus was harvested after96 hr of incubation. The growth media which con-tained both virus particles and soluble antigen wereclarified by low speed centrifugation (3,000 X g for30 min) and concentrated by dialyzing against poly-ethylene glycol (PEG) compound 20-M. The concen-trated supernatant fluid was again dialyzed against0.01 M phosphate buffer at pH 7.2 (1:20, v/v; 3changes) in the presence of toluene (Fig. 1, step 1).The nondialyzable materials were collected and freedfrom precipitate by centrifugation at 12,000 X g for30 min. The supernatant fluid was retained and desig-nated as BT-soluble-antigen-containing mixture (Fig.1, step 2). This material, after lyophilization, wasadjusted to 25% dry matter in 0.01 M phosphate buffer,pH 7.5, and then adsorbed on a diethylaminoethylcellulose (0.87 meq/g) column (2.5 by 18 cm) whichwas equilibrated with 0.01 M phosphate buffer, pH 7.5.Materials not adsorbed were the active substanceswhich were again concentrated by PEG and clarifiedby dialysis against the phosphate buffer and designatedas crude BT-soluble antigen (Fig. 1, step 3). To sepa-rate BT-soluble antigen from virus particles, ultra-centrifugation, at 78,000 X g for 90 min in 15 to 30%sucrose gradient in phosphate buffer, was used. Sol-uble antigen was recovered between fractions 2 to 5(1 ml per fraction) (Fig. 2). These fractions werepooled and designated as partially purified BT-solubleantigen preparation after removal of sucrose by di-alyzing against phosphate buffer (Fig. 1, step 4). Thismaterial was further purified by adsorption on apolymer of sheep anti-BT sera, an immunoadsorbentprepared by the method of Avrameas and Ternynck(4). The active material was eluted with 3 M MgCl2in tris(hydroxymethyl)aminomethane (Tris) buffer(0.01 M, pH 7.5) and then desalted by passing througha Sephadex G-200 column. BT-soluble antigen wasrecovered from the effluent between 5 and 16 ml. Theantigen was further concentrated by lyophilizationand designated as purified BT-soluble antigen (Fig. 1,step 5).

Agar-gel diffusion test. Double-diffusion tests wereperformed on slides by the method of Jochim andChow (12).

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1. BT VIRUS-INFECTED LK TISSUE CULTURECentrifuge at 3,000 X g to remove cell debrisDialysis against polyethylene glycol (PEG)Dialysis against phosphate-buffered saline (PBS)Centrifuge at 12,000 X g to remove precipitates

2. BT-SOLUBLE-ANTIGEN-CONTAINING MIX-TURE

Chromatography on diethylaminoethyl (DEAE)cellulose column (2.5 by 18 cm)

Dialysis against PEGDialysis against PBS

3. CRUDE BT-SOLUBLE ANTIGENSucrose gradient centrifugation (15 to 30%,

78,000 X g)Active materials between fractions 2 and 5 (1

ml/fraction)Dialysis against PBS

4. PARTIALLY PURIFIED BT-SOLUBLE ANTI-GEN

Adsorption with anti-BT serum polymerElution with MgCl2 in Tris bufferDesalting through Sephadex G-200Lyophilization

5. PURIFIED BT-SOLUBLE ANTIGENActivity 30 to 50 times that of 2

FIG. 1. Outline of BT-soluble antigen preparation.

Electrophoresis. Horizontal zone electrophoresiswas carried out on cellulose acetate atpH 8.6 in 0.05 Mbarbital and at pH 4.5 in 0.1 M acetate for 120 min at1.0 ma/1.0 cm. The sepraphore strips were activatedby soaking them in the appropriate buffer systemovernight; excess solution was removed with a buffer-dampened adsorbent pad, and 3 to 4 jliters of samplewas applied on the center of the strip. After elec-trophoresis, the strips were stained with 0.5% PonceauS in aqueous 5% trichloroacetic acid, and excess back-ground stain was rinsed off by swishing the stripssuccessively in three trays of 5% acetic acid. Dehydra-tion was accomplished by two rinses of absolutemethanol, and clearing was accomplished by dippingthe strips in a solution of 10% acetic acid in methanolfor 30 seconds prior to rolling onto a glass plate. Thestrips were dried at 60 C for 15 min, during whichtime they became transparent.

Determination of deoxyribonucleic acid, ribonucleicacid, and protein. The Burton modification (5) of thediphenylamine reaction for deoxyribose was employedfor the determination of deoxyribonucleic acid. Thy-mus DNA was used as a standard. Ribonucleic acidwas determined by the orcinol method (7), whichmeasures ribose. D-ribose was utilized as the standard.The method of Lowry et al. (15) was used to estimateprotein concentration with crystalline bovine albuminemployed as a standard.

Estimation of diffusion coefficient and molecularweight. Estimations of diffusion coefficient and mo-lecular weight of BT-soluble antigen by the agar-geldiffusion technique were performed by the method ofAllison and Humphrey (1, 2). Antisera prepared withchicken embryo origin (CEO) vaccine in both sheep

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7-0 .!

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X 4 6 8 10 12 14 16 is 20 22 24 26 28 so

FIG. 2. Sucrose gradient centrifugation ofBT-solubleantigen substance (78,000 X g, 90 min, BeckmanSW-25.1 rotor, 15 to 30% sucrose gradient). Symbols:(X) infectivity; (*) maximum dilutionforprecipitationline formation.

and cattle were employed, but only the diffusion co-efficient and the molecular weight of the gammaglobulin fraction of the bovine antiserum were usedfor calculation. They were 4.1 X 10-7 cm2/sec, and175,000, respectively (9). The angle between the pre-cipitation line and the antigen trough was measured.The diffusion coefficient of the antigen was calculated,and its molecular weight was estirhated by computingthe diffusion coefficient of the antigen relative to thatof the antibody.

For comparison, molecular weight of BT-solubleantigen was calculated from the elution volume fromSephadex gel filtration by means of the equationsderived by Squire (24).

Stability at different pH values and temperatures.Stability in different pH buffers was determined byincubating a 0.1% solution of BT-soluble antigen for1 hr at room temperature. The following buffers wereemployed: (i) Clark and Lub's HCl-KCl, pH 2.4;(ii) Soerensen's glycine-hydrochloric acid, pH 3.2,phosphate, pH 6.0 and 7.0, and glycine-sodium hy-droxide, pH 10.0; (iii) Limie's citrate, pH 4.0 and 5.0;(iv) Gomori's Tris, pH 8.0; and (v) Michaelis' bar-bital, pH 9.0. The compositions of the buffers weredescribed by Gomori (8), and the molarity of allbuffers was 0.01 M. After treatment of the antigen withthe aforementioned buffers, the samples were im-mediately neutralized by adding 0.1 N HCI or NaOH,and the antigenicity of each sample was tested againstsheep antiserum by agar-gel diffusion tests. Stability atdifferent temperatures was determined by incubating1 ml of a 0.1% solution of BT-soluble antigen in 0.01 Mphosphate buffer at pH 7.2 for 30 min at 25, 37, and56 C. Immediately after incubation, the antigen wastested against antiserum by the technique used above.

Extraction of antigens from BT-viral particles. Twomethods were employed in this study: trypsin diges-tion and glacial acetic acid dissociation.The trypsin digestion method is as follows. Trypsin

digestion of BT virus was performed by a modifiedmethod of Zwartouw et al. (27) and Marusyk (19).Viral antigens were obtained from BT viral suspen-sions (0.1%) in 0.01 M phosphate buffer at pH 8.0 byincubation with 0.4% (w/v) twice-crystallized trypsinfor 1 hr at 37 C in a water bath. Trypsin activity was

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stopped by the addition of an equal amount of soybean-trypsin inhibitor. Viral-antigen-containing solu-tion was obtained by centrifugation at 110,800 X g for2 hr and concentrated by lyophilization.The glacial acetic acid dissociation method is as

follows. Glacial acetic acid dissociation of BT viralparticles was conducted by the method of Mara-morosch and Koprowski (18). Two volumes of glacialacetic acid cooled to just above its freezing point wereadded to a cold virus solution (1 mg/ml). The result-ing solution was cooled to about 4 C and stirredoccasionally. After 1 hr, the solution was centrifugedat 110,800 X g for 2 hr, the supernatant solution wasdialyzed against 0.01 M phosphate buffer at pH 7.2and was then concentrated by lyophilization.

Preparation of antisera. The source of antigen usedfor preparation of antisera consisted of either com-mercial CEO vaccine or partially purified BT virus.Antisera to CEO vaccine and BT virus were preparedin two sheep each. Sheep were injected subcutaneouslywith one dose of 1 ml of CEO vaccine or 1 ml of BTvirus with 10'-3 tissue culture infective dose (TCIDw0)/ml by a method described previously (12). Cattleantisera to BT were obtained from A. Strating ofColorado State.University Virus Laboratory.

FIG. 3. Photograp,i of precipitation line formationby diffusion of BT-soluble antigen and antiseruim inagar-gel fronm wells at right angles to one anothler. BA,bovine antiserum; 01, 38 C; OA, ovine antiserum; 02,38 C; S, BT-soluble antigen.

BLUE-TONGUE VIRUS 469

RESULTSGeneral properties of BT-soluble antigen. Puri-

fied antigen was obtained as a white powder afterconstant weight was reached (48 hr) at roomtemperature in a vacuum desiccator over calciumchloride. At pH 7.2, the antigen dissolved readilygiving a clear, colorless solution in concentrationsup to 1% and behaved as a single component onhorizontal zone electrophoresis. The molecularweight of 208,000 daltons estimated from thediffusion coefficient agrees rather well with thevalue calculated from the elution volume ofSephadex gel filtration (Fig. 3 and 4). The antigenis a proteinaceous material and contains no de-tectable nucleic acid materials. It is heat-labile (56C, 30 min) and is inactivated below pH 6.0 orabove pH 9.0.

.20

.15

Y .10

.05 ,

0 2 4 6 8 10 12 14 16 18 20 22 40Fractions (0.5 .1/fr.)

FIG. 4. Calculation of molecular weight of BT-soluble antigen by equation derived from Sephadex gelfiltration (Sephadex G-200, 0.9 by 24 cm; effluent, 0.01M phosphate buffer at pH 7.3). Symbols: (*) bluedextran; (0) BT-soluble antigen. (VI VO)18 =(8.5/6.5)113 = 1.092; M113 = ClI3/g[l + g -(V/Vo)113] = 152(1.480 - 1.092) = 58.976; M =208,000 (reference 24).

* , -* X,~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~. * *~~~~~~~~

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..0

f.o

'E4-0

-2b

0 0 12 24 40 72 9 120HOURS

FIG. 5. Kinetics of BT-soluble antigen. Symbols:(*) maximum dilution for precipitation line formation;(0) infectivity.

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Kinetics of BT-soluble antigen production. Rouxflasks containing confluent monolayers of LKcells were washed once with warmed phosphate-buffered saline, inoculated with 5 ml of virusinoculum per flask (106 3 TCID5o/ml), and al-lowed to stand at room temperature for adsorp-tion for 1 hr prior to washing again with warmedphosphate-buffered saline. LE medium (90 ml)was added, and two flasks of BT-infected LKtissue culture were harvested at 0-, 6-, 12-, 24-,

FIG. 6. Precipitation line Jormation between BT-soluble antigent and antiserum. N-S, normal serum; A,antiserum; S, BT-soluble alntigenz; P, PBS; V, viralparticles; T, uninfected LK cell extracts; C, control.

48-, 72-, 96-, and 120-hr intervals, after which theprocedures described above were followed. Viraltitration was performed immediately after harvest.For production of BT-soluble antigen, the in-dividual harvests of culture material were con-centrated, dialyzed against phosphate buffer, cen-trifuged, and made up to 1 ml. Twofold dilutionsof the concentrated material were tested againstantiserum by agar-gel diffusion tests. The highestdilution of each sample which formed a pre-cipitation line was recorded (Fig. 5). The resultsshowed that virus titer and soluble antigen ap-peared after incubation periods of 6 and 24 hr,respectively. Both reached their maximum pro-duction after 4 days.

Immunological properties of BT-soluble antigen.Using the agar-gel diffusion test to determine theimmunological specificity of the purified BT-soluble antigen, it was found that 10 jig of theantigen in 0.01 ml of phosphate buffer formed aprecipitation line against sheep antiserum to BT-8virus; the controls (BT-soluble antigen againstnormal sheep serum, purified BT viral particlesagainst antiserum to BT-8 virus, and normal LKcell extracts against antiserum to BT-8 virus) werenegative (Fig. 6).Although antisera to different BT viral strains

(Spanish Fork and Morehouse) (A. Strating, M.S.thesis, Colorado State Univ., 1970) had no cross-reactivity with each other, all reacted with BT-soluble antigen isolated from BT-8 virus-infectedLK cells in agar-gel diffusion tests (Table 1),indicating that BT-soluble antigen behaves as acommon antigen among different BT viral strains.The origin of BT-soluble antigen. The origin of

BT-soluble antigen was studied by (i) direct com-parison with gel diffusion between BT-solubleantigen and viral antigens to determine whetherBT-soluble antigen is part of the virus and (ii) by

TABLE 1. Cross neutralizationz ofBT-8, SF, and MHviral isolates and agar-gel diffusion ofBT-8-soluble antigen against antiseraa

AntiserumbViral isolates or

BT-soluble antigenBT-8 SF MH

BT-8.+ - +SF...................... - + -MH.+ +BT-8-soluble antigen

(agar-gel diffusion) + + +

a BT-8, blue-tongue virus, strain 8; SF, blue-tongue virus, strain Spanish Fork; MH, blue-tongue virus, strain Morehouse.

b Symbols: +, positive reaction; -, negativereaction.

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blocking of the above precipitin reaction by firstabsorbing the antiserum with BT virus.

In the direct comparison method, antigens fromBT virus were extracted by (i) glacial acetic aciddissociation of the virus, and (ii) trypsin diges-tion. Although no result was obtained from theacetic acid procedure, gel diffusion test producedthree precipitation lines from the trypsin-digestedvirus. Both the BT-soluble preparation and thedigested virus shared a common antigen. Testswith BT-soluble antigen at a concentration of 2.5,ug per 0.01 ml gave no line formation but did givea line deviation at the same site (note the linedeviation of major band, Fig. 7) indicating a simi-larity between this antigen and that of viralantigen.In the blockingthe precipitin reaction method, no

precipitation line formation was found betweenBT-soluble antigen and the antiserum when theappropriate concentration of the antiserum wasfirst absorbed by BT virus (Fig. 8). The antiserum

FIG. 8. Agar-gel diffusion blocking test. A, anti-serum; S, BT-soluble antigen; P, phosphate-bufferedsaline; V-A, antiserum after adsorption with putrifiedvirus; T-A, anttiserum after adsorptioni wit/i normal LK

U."'..cell extracts; C, co/itrol.

used here was a 1:4 dilution, and the virus was108 3 TCID5o,/ml. The controls showed line forma-tion. This result substantiates the above observa-

FGi. 7. Compariso'i of BT-soluble atigen tn tion that BT-soluble antigen is immunologicallyparticle anltigenls. A, anltiserum; S, BT-soluble antigen;S-V, antigenis from BT viral particles; T-T, tryptic similar to one of the antigens extracted fromdigestion of niormal LK cells; T-I, trypsini antd its BT virus. It also indicates that BT-soluble antigeninhibitor. (1) BT-soluible anitigenz at a coiiceiitratioii of is related immunologically to a surface antigen of10 ug/0.01 ml; (2) at 2.5 ug/O.Ol ml. the virus.

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DISCUSSIONPurification of antigens synthesized during viral

infection is difficult (22) because viral antigens arepresent in small amounts and usually associatewith cellular materials. The same problems causedifficulty in purification of the virion (26). Whendialysis, chromatography, gradient centrifugation,and gel filtration were employed to isolate BT-soluble antigen, the final product contained cellu-lar materials.

Since the development of the immunoadsorbent(6) allowed purification of antibodies from anti-sera, the technique has been employed in the frac-tionation of antibodies and enzymes (23). A re-cently developed technique for preparing theimmunoadsorbent uses either ethyl-chloroformate(3) or glutaraldehyde (4) to convert the solubleprotein molecules into insoluble polymers whichare specific and stable immunoadsorbents. How-ever, little work has been done preparing anti-serum polymers for the isolation of virus-inducedantigens.

It is clear from the data collected in this studythat purification of BT-soluble antigen from BTvirus-infected LK tissue culture can be achievedby combining conventional protein fractionationmethods with the immunoadsorbent technique.Dialysis and chromatography removed 92 to 93%of the cellular material giving concentrated BT-soluble antigen detectable by the agar-gel diffu-sion test; the use of sucrose gradient centrifuga-tion allowed complete separation of BT-solubleantigen from virus, whereas the immunoadsorbenttechnique produced BT-soluble antigen in a highlypure state.

In this study, the antiserum used for antigendetection was prepared from a CEO vaccine com-pletely free of host cell (LK) materials. If free ofviral particles, an immunoadsorbent preparedwith this antiserum should absorb BT virus-spe-cific antigen from infected LK cells as our resultsconfirmed. The immunoadsorbent technique em-ployed has also shown a potential for the isolationof viral particles in a highly purified state suitablefor morphological study under the electron mi-croscope.

Kinetic study of BT-soluble antigen productionestablishing the optimal time for harvesting theBT virus-infected LK tissue culture agreed withthe assumption of previous workers (12). Theoptimal time for harvest occurred after 4 days ofincubation when the majority of the LK cells

showed cytopathic effects, and the viral titerreached its maximum.The work of Klontz and co-workers (13) show-

ing that the material which reacted with antiserumwas not composed of infectious particles has been

confirmed by our observation that the highlypurified, virus-free BT-soluble antigen containsno nucleic acid materials, is noninfectious, and isa proteinaceous material synthesized during BTvirus infection.A relationship was found between BT-soluble

antigen and BT virus. The immunological proper-ties of both the soluble antigen and virus weredetermined by antiserum prepared with CEOvaccine in sheep which was nonreactive with hostcell components, indicating that the soluble anti-gen generated during BT virus infection has noimmunological relationship to LK cell compo-nents but instead is viral related.Two methods were employed to determine if the

antigenic structure of the BT-soluble antigen isthe same as that of the viral particle: (i) directcomparison of the BT-soluble antigen with theantigens isolated from the virus and (ii) the use ofBT viral particles to indirectly block the pre-cipitation line formation between soluble antigenand antiserum. Direct comparison showed a con-nection between the precipitation lines producedby BT-soluble antigen and by trypsin-digested BTviral particles, suggesting that the soluble antigenis immunologically similar if not identical to oneof the components of the viral particles. Thissimilarity is further indicated by the deviation ofa precipitin line produced by viral componentsand antibody under the influence of BT-solubleantigen (Fig. 7).

Indirect evidence was obtained by the agar-geldiffusion block test, so named because the pres-ence of BT viral particles can block the formationof a precipitation line between BT-soluble antigenand antiserum. When the antiserum was firstabsorbed with BT virus (Fig. 8), no line formed,implying that the precipitin antibody present inthe antiserum is also specific to the component(s)of the virus, and is, therefore, adsorbed by thevirus.These observations indicate (i) the immuno-

logical identity between BT-soluble antigen andone of the BT viral antigens rather than with thoseantigens of theLK host cells and (ii) that the solu-ble antigen represents one of the componentspresent on the suiface ofBT viral particles, whichcould be isolated by trypsin digestion.From the above evidence and the correspond-

ence between the kinetics of the soluble antigenand viral titers (Fig. 5), it appears that the syn-thesis of this antigen is specifically associated withBT virus propagation as a precursor which wasproduced in excess.

Before a practical use of the BT-soluble antigencan be considered, the following question must beconsidered. Does this antigen represent a groupantigen among different BT viral strains that may

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be used as a diagnostic agent to detect BT virusinfection in animals as previously proposed (12)?In this and a previous study (12), BT-solubleantigen prepared from a BT viral strain was ableto react with antisera prepared with different BTviral strains in agar-gel diffusion tests, suggestingthat BT virus contains, produces, or both, across-reacting component in addition to thestrain-specific antigen. It appears that BT-solubleantigen represents this cross-reacting componentwhich is immunolcgically identical with a surfaceantigen of the virus, thus providing a basis for thepractical application of BT-soluble antigen fordiagnosis of BT viral infection in animals by use

of the agar-gel diffusion test.

ACKNOWLEDGMENTS

This investigation was supported by contract no. 12-14-140-1755-94 of the Veteriisary Biologic Division, Agricultural Re-search Service, U.S. Department of Agriculture.

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BLUE-TONGUE VIRUS 473

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