INFECnON AND IMMUNITY, March 1971, p. 373-377Copyright ( 1971 American Society for Microbiology

Vol. 3, No. 3Printed in U.S.A.

Immunodiffusion Studies of Purified EquineInfectious Anemia VirusHIDEO NAKAJIMA AND CHUZO USHIMI

Equine Inifectious Anemia Research Division, National Institute of Animal Health, Kodaira, Tokyo, Japan

Received for publication 20 October 1970

Antigenicity of purified equine infectious anemia (EIA) virus was examined byimmunodiffusion against sera obtained from horses experimentally infected withEIA virus. The purified virus reacted with the infected horse serum, and virus-specific precipitating antibody was demonstrated. Furthermore, it was found thatpurified EIA virus reacted against the serum of horses infected with all st;ains ofEIA virus which were antigenically different from one another. From the result,group-specific components of the virus rather than strain-specific ones were con-

sidered to be involved in the reaction. Serological reactivity was lost by adding anti-serum from the infected horse to the antigen. The precipitating antibody usuallyappeared in the serum 1 to 2 weeks after the first febrile attack of EIA and re-

mained for a longer period. Some characteristics of the purified antigen and speci-ficity of the reaction for EIA are described.

Equine infectious anemia (EIA) is worldwide indistribution and has been extensively investigatedfor many years. Many aspects of the disease,however, still require further investigation.Studies on the causative agent and its propaga-tion, immunology, pathogenesis, and diagnosisand on prevention of the disease are particularlyneeded.Many attempts have been made to elucidate the

physicochemical and morphological properties ofETA virus, and some characteristics of the virushave been previously reported (9, 10, 12-15).During these investigations a procedure for viruspurification was developed and highly purifiedand concentrated virus was successfully obtained(10).In the present investigation, the serological

reactivity of the purified virus, as measuredagainst the serum obtained from horses experi-mentally infected with ETA virus, was examinedby immunodiffusion. As a result, the purified virusreacted effectively, and viral specific antibody wasdemonstrated in the infected equine serum.The present report describes a standardization

of the immunodiffusion reaction and some char-acteristics and antigenicity of the purified antigen.Specificity of the test in EIA is also discussed.

MATERIALS AND METHODS

Cell culture and preparation of virus material. Hepa-rinized blood was obtained from horses, and leukocyteculture was carried out by a procedure previously de-

scribed (10) to obtain the starting virus material forpurification of the antigen. The horse leukocyte-adapted cloned strain (the P337 strain) of EIA viruswas used as the inoculum. Infectivity of virus materialswas titrated by using the horse leukocyte culture tech-nique as previously reported (3, 11).

Purification of viral antigen. The purification proce-dure for EIA virus has been previously described (10).Briefly, the virus was precipitated by ultracentrifuga-tion from approximately 1,000 ml of starting materialand suspended in 0.01 M phosphate buffer (pH 7.4;PB). This material was loaded onto a diethylamino-ethyl cellulose column, and the virus was eluted with1.0 M NaCl solution after contaminating protein wasremoved with 0.15 M NaCl solution. The eluate wasconcentrated and centrifuged in cesium chloride(CsCl) solution at 40,000 rev/min for 22 hr in an SW65 rotor of a Beckman model L2 ultracentrifuge. Aftercentrifugation, 1.5 ml of purified fraction whichshowed the highest infectivity was obtained and dia-lyzed against 0.01 M PB. This fraction was used as thepurified antigen.

Analytical ultracentrifugation. The purified anddialyzed virus fraction was analyzed by a Spinco modelE ultracentrifuge at an average speed of 25,000 and27,000 rev/min, and sedimentation coefficients of thevirus were measured by using ultraviolet adsorptionand schlieren optical systems.

Sera. Sera used in the present investigation wereobtained at various stages during the disease from ninehorses experimentally infected with EIA virus. Fourstrains of EIA virus, namely, the P337 strain, theWyoming strain, the German strain, and the Goshunstrain (2), were used as inoculum.

Agar gel diffusion. Gel diffusion plates were pre-pared on microscope slides (26 by 76 mm) by using

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4.5 ml of 0.8% purified agar (Eiken Comp., Japan),0.85% NaCi, and 0.01% thimerosal in 0.01 M PB.The wells were 3 mm in diameter, and six circumferen-tial wells were placed at a distance of 4 mm from thecentral well. The central well was usually filled with thepurified antigen, and the peripheral wells were filledwith sera. The plate was incubated in a moist chamberat 22 C for 4 days. When a precipitin line was formed,the plate was photographed unstained. It was nextwashed for 3 days in normal saline and rinsed for afew hours in distilled water. Finally, the slide wasdried, stained with amido black, destained, and photo-graphed again.

Neutralization test. Two strains of EIA virus whichwere antigenically different from each other were usedas indicator virus for the neutralization test. One wasthe P337 strain which had adapted to horse leukocyteculture and was used as the seed virus for preparationof the virus material. The other was the Wyomingstrain which had been supplied by the courtesy of C.D. Stein in 1955 and had been serially passaged onlythrough horses. To facilitate viral propagation inhorse leukocyte cultures, the latter was passagedthrough horse leukocytes four times by Y. Kono andonce by the senior author. The serological reactivitywas not altered during passages. A 0.1-ml amount ofthe mixture of indicator virus, which had approxi-mately 1050 median tissue culture infectious dose(TCID50)/0.l ml, and serially diluted serum was inocu-lated into 1-day cultures of horse leukocytes. The titerof neutralizing antibody was expressed in a mannerpreviously described (5).

Other measurements. Electron microscopy wascarried out as previously reported (10). The density ofviral fractions was determined by a gravimetricmethod by using 10 microliter capillaries (DrummondScientific Corp., Philadelphia, Pa.). The complementfixation test was performed by a method previouslydescribed (4). Complement-fixing (CF) antigenicitywas expressed as the reciprocal of dilution.

RESULTSStandardization of immunodiffusion procedure.

Purified EIA virus was tentatively examined byimmunodiffusion to determine whether it wouldhave serological reactivity with the serum ofexperimentally infected horses (no. 520 and 569)which had clinically shown typical symptoms ofEIA. As a result, a fairly clear and distinct pre-cipitin line was formed between the antigen andthe serum after 24 hr.With the purified virus and above-mentioned

sera, the concentration of agar gel, concentrationof NaCl in agar, incubation temperature, andincubation period were examined to determinethe best conditions for immunodiffusion. Conse-quently, it was found that 0.8% agar gel, 0.85%NaCl and incubation at 22 C were comparativelybetter among the conditions examined. A pre-cipitin line was developed within 24 hr in mostcases. It seemed, however, to become clearerwhen the slide was allowed to incubate for 3 more

days. Therefore, an incubation period of 4 dayswas used throughout the experiment.

Three kinds of antigen were prepared as fol-lows. One was purified and dialyzed virus with nofurther treatment (see above). The second was ob-tained by freezing, thawing, and shaking thepurified virus more than 10 times. The third wasprepared by treating the purified virus with anequal volume of ether at room temperature for5 min. Ether was immediately removed. Viralinfectivity was completely lost by this treatment.All of the antigen prepared above reacted withserum, and precipitin lines developed approxi-mate strength.The antigen and sera were diluted by a serial

twofold dilution and a reaction was carried out todetermine the serological reactivity and the anti-body titer. The strongest reaction was observedwhen undiluted antigen and sera were used. Pre-cipitin lines became almost invisible when theantigen and sera were diluted twofold.From the results obtained above, the virus

which had been purified and dialyzed to removeCsCl was used as antigen, and the reaction wasperformed without dilution of both the antigenand sera.Some characteristics of the purified antigen. The

viral antigen used in experiments was purifiedfrom 1,000 ml of infected horse leukocyte cul-tures, having a titer of approximately 106.5TCID0/0.5 ml, and concentrated to 1.5 ml.The virus ranging between 1.12 and 1.18 g/ml

in CsCl gradients was collected since the virusshowed broad distribution in the gradient. Infec-tivity and CF reactivity of the final materialincreased to approximately 108.0 TCID5o/0.5 mland 100, respectively. Its optical density at 280nm was mostly between 1.5 and 3.0, and thematerial was slightly white and turbid. The sedi-mentation coefficients measured by analyticalultracentrifugation were between 110 and 120S.Viral particles were observed by use of the elec-tron microscope, and most ofthem were disrupted(Fig. 1).

Reaction with sera obtained from horses infectedwith antigenically different strains of ELT virus.Recent evidence suggests that there are quite afew antigenically different strains of ETA virus.Actually, the P337 strain of the virus was neu-tralized with the serum of horse 520 which hadbeen infected with the same strain, but not withthe serum of horse 569 which had been infectedwith the Wyoming strain of the virus. In the pres-ent experiment, sera obtained from horses infectedwith the P337, the Wyoming, the German, and theGoshun strains of the virus which had beenproved to be antigenically different from oneanother (Y. Kono et al., Arch. Gesamte Virus-

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forsch., in press) were examined to determinewhether the precipitating antibody could bedemonstrated by using one strain (the P337 strain)of the virus as antigen. As a result, the antibodywas demonstrated against the antigen in all of theserum utilized.

In Fig. 2a, precipitin lines formed between theantigen (the central well) and sera (peripheralwells) which were obtained from three horses in-fected with the antigenically different P337 strainor the Wyoming strain, at various stages of the

disease, are presented. All precipitin lines formedwere connected with one another. This suggeststhat antibodies in the sera reacting with the anti-gen were all immunologically related to oneanother. Figure 2b shows the precipitin lines,stained with amido black, of the same specimenas demonstrated in Fig. 2a after completion of thereaction.

Serological reactivity of the antigen was lostwhen the antibody-positive horse serum wasadded to the antigen.

FIG. 1. Electron micrograph of purified and dialyzed equine infectious anemia viral antigen negatively stainedwith 2% phosphotungstic acid. Pleomorphic but predominantly spherical particles are seen, and most of them havebeen disrupted. An inserted photograph at the upper right-hand corner displays an intact virion. X 160,000.

b

...6......... 6::

1

AG.:P

:....... q3.6',

4FIG. 2. (a) Precipitin lines formed between the purified antigen (the central well; AG) and sera (the peripheral

wells) at various stages of the disease from horses infected with antigenically different strains of equine infectiousanemia virus. (1) Preinoculation serum; (2) serum which contained complement-fixing antibody, obtained from ahorse inoculated with the P337 strain; (3) serum which contained neutralizing antibody to the P337 strain, ob-tainedfrom the same horse as mentioned in (2); (4) serum which contained neutralizing antibody, obtained fromanother horse inoculated with the P337 strain; (5) serum which contained complement-fixing antibody, obtainedfrom a horse infected with the Wyoming strain of the virus; (6) serum which contained neutralizing antibody tothe Wyoming strain, obtained from the same horse as mentionied in (5). (b) Precipitin lines, stained with 0.1%amido black, of the same specimen as demonstrated in Fig. 2a.

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Horsa> 5690 5 D:s afteProculon 25 30 35 40Serum examined and its number

11-) ~~~~~2(-) 3(-) 4(+Inoculation

0 Wyoming straini)

0394

continued 45 50 55 60 65 70 75 80 855(+) 6t+)

FIG. 3. History of a horse experimentally infected with equine infectious anemia virus. Arrows indicate thetime ofserum sampling for immunodiffusion.

The purified antigen was therefore found toreact with the serum of horses infected with allstrains of ETA virus which are antigenicallydifferent from one another.

Reaction with sera obtained from infected horsesat various stages of the disease. CF and neutraliz-ing antibodies have thus far been demonstratedin EIA. However, the CF antibody is present foronly a short period after the first febrile attack ofthe disease (8). The neutralizing antibody, on theother hand, has been identified to persist for alonger period (5) although it reacts only typespecifically. In the present experiments, sera fromnine horses experimentally infected with EIAvirus were investigated at various stages duringthe disease to determine the time of appearanceof precipitating antibody and its duration. It wasfound that the antibody test became positive ineight of nine infected horses during the disease. Inmost cases, the antibody appeared 1 to 2 weeksafter the first febrile attack. In general, strongerreactions were seen when the serum obtainedmuch later was used.One of the typical cases is presented in Fig. 3

and 4. Horse 569 was inoculated with 1 ml of theWyoming strain of the virus. Body temperatureincreased on the 19th day after inoculation. Serawere obtained at the time of preinoculation, incu-bation, the peak of first pyrexia, and an appro-priate time subsequent to the fall in body tem-perature (Fig. 3). No positive reaction was ob-served by the time immediately after the fall inbody temperature. The precipitin line, however,became visible in 1 week after the fever had sub-sided (Fig. 4). Precipitin lines developed with theserum obtained at various stages of the diseasewere all connected with one another. The anti-body remained for at least 5 months after the on-set of the disease.

........

FIG. 4. Development of precipitating antibody dur-ing a course ofequine infectious anemia verus infection.The central well was filled with the purified antigen(AG), and the peripheral wells contained the sera.Serwn samples 1 to 6 are equivalent to those indicated inFig. 3. Serum 4 contained CF antibody, and sera S and 6contained neutralizing antibody.

DISCUSSIONThe specificity of the immunodiffusion reaction

for ETA that we have observed and described inthe present study is of great importance. Thefollowing evidence suggests that the reaction isspecific for ETA. (i) The antigen used in theexperiment was highly purified and concentratedETA virus. (ii) The antigen did not react with pre-inoculation sera but reacted only with sera ob-tained at some time after the first febrile reactionof ETA. (iii) The serological reactivity was lost

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when the antiserum was added to the antigen.(iv) The antigen did not react with the sera ofhorses infected with other viral diseases such asequine rhinopneumonitis virus and cytopatho-genic equine orphan virus (6, 7). (v) The anti-serum of infected horses did not react with suspen-sions of horse leukocytes, suspensions of culturedhorse leukocytes, bovine serum used for themedium, and the crude virus material used forpreparation of the antigen.

Serological reactivity of the purified viruspreparation decreased by twofold dilution, andthe precipitin line became almost invisible. It istherefore considered that a concentration of virusparticles equivalent to approximately 1080°TCIDw/0.5 ml is needed to show activity in thereaction.

Intact virions, however, are not considered tobe involved in the reaction for the followingreasons: the virion seemed to be easily disruptedas seen in Fig. 1; the antigen treated with ether orby freezing and thawing did not produce a re-markable change in its reactivity, and the pre-cipitin line was formed as early as within 24 hr inmost cases. Therefore, small components of thevirus must be involved. In addition, the antigenreacted with the serum of horses infected withantigenically different strains of EIA virus.Accordingly, the antigen involved in the reactionis not on the surface but is of the internal com-ponent of the virion. Therefore, it does not reacttype specifically but group specifically.

Precipitating antibody did not appear in one ofnine infected horses. This may indicate that anti-body was too weak to form a visible precipitinline in this horse. The antibody might have beendetected if the antibody fraction had been con-centrated.

Coggins and Norcross (1) recently reported onimmunodiffusion in EIA by using infected horsespleen as antigen and demonstrated the presenceof precipitating antibody. The relationship be-tween the two antibodies is unknown because ofthe difference in antigens employed.

ACKNOWLEDGMENTS

The authors express their thanks to M. Tajima for takingelectron micrographs of EIA virus, to K. Sekiguchi for the ana-

lytical centrifugation study of EIA virus, to Y. Kono for provid-

ing EIA immune serum and for determining the complement-fixing antibody titer in the horse serum, and to C. Kuniyasufor his valuable advice and help in performing the present study.They are also indebted to M. Ito for preparation of the photo-graphs.

LITERATURE CITED

1. Coggins, L., and N. L. Norcross. 1970. Immunodiffusionreaction in equine infectious anemia. Cornell Vet. 60:330-335.

2. Kobayashi, K., and Y. Kono. 1967. Serial passages of equineinfectious anemia virus in horse leukocyte culture. Nat.Inst. Anim. Health Quart. 7:1-7.

3. Kobayashi, K., and Y. Kono. 1967. Propagation and titra-tion of equine infectious anemia virus in horse leukocyteculture. Nat. Inst. Anim. Health Quart. 7:8-20.

4. Kono, Y. 1968. Characteristics of complement fixing antigenof equine infectious anemia virus. Nat. Inst. Anim. HealthQuart. 8:117-121.

5. Kono, Y. 1969. Viremia and immunological responses inhorses infected with equine infectious anemia virus. Nat.Inst. Anim. Health Quart. 9:1-9.

6. Kono, Y., and K. Kobayashi. 1964. Cytopathogenic equineorphan (CEO) virus in horse kidney cell culture. I. Isola-tion and properties. Nat. Inst. Anim. Health Quart. 4:10-20.

7. Kono, Y., and K. Kobayashi. 1964. Cytopathogenic equineorphan (CEO) virus in horse kidney cell culture. II. Im-munological studies of CEO virus. Nat. Inst. Anim. HealthQuart. 4:21-27.

8. Kono, Y., and K. Kobayashi. 1966. Complement fixationtest of equine infectious anemia. II. Relationship betweenCF antibody response and the disease. Nat. Inst. Anim.Health Quart. 6:204-207..

9. Nakajima, H., and J. Obara. 1964. Ether susceptibility ofequine infectious anemia virus. Nat. Inst. Anim. HealthQuart. 4:129-134.

10. Nakajima, H., M. Tajima, S. Tanaka, and C. Ushimi. 1969.Physicochemical studies of equine infectious anemia virus.Ill. Purification and electron microscopic observation ofthe virus. Arch. Gesamte Virusforsch. 28:348-360.

11. Nakajima, H., S. Tanaka, and C. Ushimi. 1968. Fractiona-tion of equine infectious anemia virus by diethylamino-ethyl cellulose chromatography and sucrose density gradi-ent centrifugation. Nat. Inst. Anim. Health Quart. 8:57-63.

12. Nakajima, H., S. Tanaka, and C. Ushimi. 1969. Physico-chemical studies of equine infectious anemia virus. I.Buoyant density of the virus. Arch. Gesamte Virusforsch.26:389-394.

13. Nakajima, H., S. Tanaka, and C. Ushimi. 1969. Physico-chemical studies of equine infectious anemia virus. II.Sensitivity of the virus to trypsin. Arch. Gesamte Virus-forsch. 26:395-397.

14. Nakajima, H., S. Tanaka, and C. Ushimi. 1970. Physico-chemical studies of equine infectious anemia virus. IV.Determination of the nucleic acid type in the virus. Arch.Gesamte Virusforsch. 31:273-280.

15. Tajima, M., H. Nakajima, and Y. Ito. 1969. Electron micros-copy of equine infectious anemia virus. J. Virol. 4:521-527.

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