Hemagglutination by Equine Infectious Anemia Virus

Transcription

1 INFECTION AND IMMUNImTY, Aug. 1976, p Copyright X 1976 Americn Society for Microbiology Vol. 14, No. 2 Printed in U.S.A. Hemgglutintion by Equine Infectious Anemi Virus HIROSHI SENTSUI* AND YUJI KONO Equine Infectious Anemi Reserch Division, Ntionl Institute of Animl Helth, Kodir, Tokyo, 187 Jpn Received for publiction 24 My 1976 Equine infectious nemi (EIA) virus which ws propgted on n equine derml cell line gglutinted guine pig erythrocytes. Virl fluids contining bout men tissue culture infective doses/ml showed hemgglutinting (HA) titers rnging from 16 to 32 units/0.05 ml. Results of cesium chloride equilibrium density grdient centrifugtion reveled tht the hemgglutinin ws inseprble from the virus prticles. The hemgglutintion rection persisted over wide rnge of temperture nd ph, nd the bsence of divlent ctions did not decrese its ctivity. The HA ctivity ws stble t 4 C but not t The ctivity ws destroyed by virus-disrupting lipid solvents nd modertely sensitive to proteolytic enzyme. Neurminidse enhnced HA ctivity slightly. Phospholipse C hd no effect on HA titer, lthough it completely inctivted infectivity. It ws reltively stble to ultrviolet irrdition. Thus, the hemgglutinin ppers to be closely ssocited with virus prticles, nd its ctivity is dependent on the presence of its lipids nd proteins. Hemgglutintion ws inhibited by ser from horses infected with EIA virus. Hemgglutinin receptors on the erythrocytes were inctivted by proteolytic enzyme nd formldehyde but were not influenced by neurminidse, sodium deoxycholte, or KIO4. Equine infectious nemi (EIA) is n cute or chronic virl disese of the horse nd other equide. The chrcteristics of this disese re intermittent fever with viremi (5, 10), nemi (3), progressive wekness (3), nd persistent virl infection for life (22). Some properties ofeia virus re similr with those of ribonucleic cid (RNA) tumor viruses (20). It is n enveloped ribonucleic cid virus (18) tht is sensitive to ether (16). It hs deoxyribonucleic cid (DNA)-dependent stge in its growth cycle (11) nd contins n enzyme tht produces DNA from virl RNA templte (RNA directed DNA polymerse) (H. Nkjim et l., Annu. Meet. Jpn. Soc. Vet. Sci., 80th, 1975). It is lso reltively resistnt to ultrviolet irrdition (15). Electron microgrphs hve shown tht EIA virus mtures by the budding process on the cell surfce (11, 23), nd virl prticles pper pleomorphic, lthough most re nerly sphericl with dimeters between 90 nd 140 nm (17, 23) Ṡerologiclly, ELA viruses possess t lest two different ntigens, i.e., one ssocited with the envelope nd the other ssocited with the nucleocpsid. The former is strin-specific ntigen nd is detectble by neutrliztion (9, 12). The ltter is common ntigen mong 325 vrious strins of EIA virus nd is detectble by complement fixtion (CF) (6, 7) nd immunodiffusion tests (1, 19). There hs been no previous report on hemgglutintion by EIA virus, probbly due to the inbility to obtin high concentrtions of highly purified virus. Recently, however, these difficulties hve been eliminted by the estblishment of EIA virus-infected cell line tht produces high concentrtions of virus nd cn be further concentrted nd prtilly purified (14). This development prompted us to reinvestigte the hemgglutinting (HA) ctivity of EIA virus, nd it ws found tht EIA virus gglutintes guine pig erythrocytes. This pper describes the properties of the hemgglutinin nd the inhibition of hemgglutintion by ser of the horses infected with EIA virus. MATERIALS AND METHODS HA ntigen. The source of HA ntigen consisted of 7-dy culture fluids of n equine derml cell line which propgtes EIA virus persistently. The infected cell line, primrily estblished by W. A. Mlmquist (14), ws obtined by us through the courtesy of H. Nkjim of our institute. The cell line ws cultivted in medium contining 10% fetl clf serum nd 90% Egle miniml essentil medium. The infected culture fluid ws centrifuged for 20 min t 1,000 x g, nd the resulting supern-

2 326 SENTSUI AND KONO INFECT. IMMUN. tnt fluid ws further centrifuged for 120 min t 100,000 x g. The pellet ws resuspended in phosphte-buffered sline solution (PBS) t ph 7.2 to 1/ 25 of the originl volume. Infectivity titrtion. These were crried out by the end point method in tube cultures of norml horse leukocytes (8). After inocultion with 0.1 ml of seril 10-fold dilutions ofvirl mteril, the cultures were kept t 370C until cells were gretly dmged. Culture fluids were hrvested nd subinoculted two times onto horse leukocyte cultures, nd the infective end point ws determined by testing the CF ctivity of ech culture fluid. The infectivity ws expressed s the men tissue culture infective dose per milliliter. CF tests. Ech smple to be tested ws mixed with n equl volume of ethyl ether t room temperture for 10 min. After removing the ethyl ether by centrifugtion nd evportion, it ws redy for use in the CF test. The CF tests were crried out by the method described previously (7, 8). Hemgglutintion nd HI test. The microtiter method ws used. To ech seril twofold HA ntigen dilution in 0.05 ml, n equl volume of 0.5% erythrocytes (RBC) in PBS (ph 7.2) ws dded. The mixture ws incubted t room temperture for t lest 3 h. The reciprocl of the highest virus dilution showing complete hemgglutintion ws considered s the HA titer (HA units/0.05 ml). In hemgglutintion inhibition (HI) tests, 4 HA units of ntigen in ml ws dded to ech seril twofold dilution of serum in ml of PBS. After incubtion for 60 min t 37 C, 0.05 ml of 0.5% guine pig RBC suspension in PBS ws dded. The tests were llowed to stnd overnight t room temperture nd red the following morning. The reciprocl of the highest serum dilution showing complete inhibition of hemgglutintion ws considered s the HI titer. Preprtion of ser for HI titers. To remove nonspecific HA inhibitors, ech 0.25-ml serum smp!e ws diluted with n equl volume of PBS, heted t 56 C for 30 min, nd mixed with 0.5 ml of 25% kolin solution of PBS. The mixture ws held t room temperture for 20 min with occsionl shking nd then centrifuged t 1,000 x g for 20 min. To the superntnt fluid, 0.1 ml of pcked wshed guine pig RBC ws dded to bsorb isohemgglutinins which occsionlly ppered fter feverish condition. Adsorption took plce t room temperture for 20 min with occsionl shking, fter which the smple ws centrifuged for 10 min t 1,000 x g. The superntnt fluid ws then redy for use in the HI test s 1:4 dilution. Tretment of EIA virus hemgglutinin with chemicls nd enzymes. One volume of the vrious test regents ws mixed with 10 volumes of HA ntigen. The finl concentrtion of the regents used, temperture, ph, nd time periods of tretment re shown in Tble 1. Trypsin (bovine pncres, type 2, 2x crystllized) nd neurminidse (Clostridium perfringens type V, purified) were obtined from Sigm Chemicl Co., St. Louis, Mo., nd phospholipse C (Bcillus cereus) ws from Boehringer Mnnheim Corp., New York, N.Y. Control hemgglutinin smples, preserved or hndled under identicl conditions without tretment, were titrted for HA ctivity in prllel with treted smples. Regent lone ws lso used s control to test its direct effect on RBC. Ultrviolet irrdition on the EIA virus hemgglutinin. Portions (0.5 ml) of four identicl HA ntigen smples contining 16 HA units were plced in petri dishes (14-mm dimeter) nd irrdited by 15-W germicidl lmp t distnce of 34 cm with continuous shking. One smple ws tken 5, 10, 20, nd 30 min fter inititing irrdition to determine the rtes of inctivtion of HA ctivity nd infectivity (Tble 2). Therml stbility of EIA virus hemgglutinin. HA ntigen ws held t 40, 370, nd 56 C for given periods of time s shown in Tble 3 nd immeditely preserved t -80 C. After incubtion of the finl ntigen t ech temperture, HA ctivities of ll smples were titrted t one time. Equilibrium density grdient centrifugtion of EIA virus hemgglutinin. Four milliliters of the ntigen ws crefully overlid onto 1 ml of sturted cesium chloride (CsCl) solution. The mteril ws centrifuged without mixing t 40,000 x g for 22 h in n SW50.1 rotor of Beckmn L2-65B ultrcentrifuge. The frctiontion of the solution into ml portions ws crried out using n ISCO model D density grdient frctiontor. The density of frctions ws determined by the grvimetric method using 10-,ul cpillries (Drumond Scientific Co., Broomll, P.). Selected frctions were tested for virus infectivity nd concentrtion of CF nd HA ntigens. Adsorption of EIA virus onto guine pig RBC. One milliliter of virus fluid nd 0.5 ml of wshed pcked guine pig, horse, or goose RBC were mixed nd held overnight t 4 C. The mixture ws then centrifuged t 1,000 x g for 20 min. The resultnt superntnt fluid ws tested for virus infectivity. Tretment of RBC with vrious regents. A 0.5% suspension of guine pig RBC in PBS ws mixed with n equl volume of ech regent in PBS. The incubtion times, tempertures, nd regent concentrtions re shown in Tble 5. The RBC were then wshed three times with PBS nd resuspended in the originl volume of PBS. HA titrtions were mde in prllel with treted nd untreted RBC. RESULTS Hemgglutintion with RBC from vrious species. RBC from horses, cows, sheep, rbbits, guine pigs, mice, geese, nd chickens were subjected to HA tests. Only RBC from guine pigs were gglutinted consistently, nd of 20 smples tested ll were eqully susceptible to gglutintion. The RBC concentrtion tht showed high nd reproducible titer ws 0.5% suspension (Fig. 1). Effect of vrious tempertures nd ph vlues on the HA rection. No difference in HA titers ws observed t 40C, room temperture, nd 37 C. The hemgglutintion pttern ws

3 VOL. V.,9HEMAGGLUTINATION 14, 1976 BY EIA VIRUS 327 TABLE 1. Effect of vrious regents on hemgglutinting ctivity nd infectivity of EIA virus Agent Condition of tretment t Finl concn HA units/ 37C TCID5o/ml (log) 0.05 ml Ether 5 min, ph % 0 <1.50 Chloroform 5 min, ph % 0 <1.50 Control Sodium deoxycholte 60 min, ph % 0 <1.50 Control Formldehyde 60 min, ph % 0 < % Control KI04 60 min, ph % Control Trypsinb 60 min, ph % Control Neurminidse 30 min, ph units/ml,c Phospholipse C 30 min, ph % 16 <1.50 Neurminidse nd 30 min, ph units/ml,c 64 <1.50 phospholipse C 0.02% Control TCID5O, Men tissue culture infective dose. b Trypsin ctivity ws stopped by dding trypsin inhibitor (0.2%) to the mixture of trypsin nd virus. c Determintion mde using NAN-lctose. TABLE 2. Unit Effect of ultrviolet irrdition on HA ctivity nd infectivity Irrdition time (min) HA units/0.05 ml TCID./ml (log _ 1.50 : " I TCID5., Men tissue culture infective dose. not ltered even fter holding t ech temperture for 18 h. In ddition, there ws no distinct difference in HA titers when the rection ws performed t ph rnge between 5.5 nd 7.5 nd in the presence or bsence of clcium or mgnesium ions. Effects of vrious gents on HA ctivity. To elucidte the chemicl nture of the hemgglutinin, HA ntigen ws treted with vrious gents s shown in Tble 1. Ether tretment completely inctivted hemgglutinin nd infectivity; no HA ctivity ws recovered from either the wter or ether phse. Chloroform nd sodium deoxycholte lso completely destroyed HA ctivity s well s infectivity. Formldehyde, depending on concentrtion, lso inctivted HA ctivity nd infectivity. Trypsin reduced HA ctivity modertely but hd no effect on infectivity. In contrst, KIO4 hd no definite effect on HA ctivity but inctivted infectivity significntly. HA ctivity ws incresed fourfold by neurminidse tretment but not ffected by phospholipse C. However, phospholipse C destroyed infectivity completely. Cotretment with both enzymes showed no dditionl effect beyond the neurminidse effect on HA titer. Effect of ultrviolet irrdition on HA ctivity. Inctivtion of HA ctivity nd infectivity by ultrviolet irrdition is summrized in Tble 2. HA ctivity ws more stble thn ws infectivity. The former ws reduced by fctor of min fter irrdition, though the ltter's titer ws decresed to 1/106 of its originl infectivity. Effects of vrious tempertures on HA ctivity. The results re summrized in Tble 3. HA ctivity ws reduced fourfold in 30 min t 560C or 8 dys t 37 C. It ws reltively stble t 40C, t which temperture it ws decresed only twofold in 1 month. Reltionship of hemgglutinin to the virus prticle nd CF ntigen. The distribution of HA nd CF ntigens nd infectivity fter CsCl grdient centrifugtion re illustrted in Fig. 2. They were ll found in the density rnge between 1.10 nd 1.21 g/ml. The peks of ctivity of both ntigens nd infectivity were t density of 1.16 g/ml. The CF ntigen could not be detected unless ech frction ws treted with ether.

4 328 SENTSUI AND KONO TABLE 3. Stbility of hemgglutinting ctivity t 560, 370, nd 4 C Stbility of HA ctivity t: HA units/0.05 ml 56 C (min) C (dys) C (dys) Incubted t indicted temperture for designed period. Adsorption of infective EIA virus onto guine pig RBC. Infectivity of the virl fluid which ws mixed with guine pig RBC ws decresed bout 10-fold, but other fluids which were mixed with horse nd goose RBC showed INFECT. IMMUN. no definite decrese in infectivity, indicting tht the virus ws dsorbed only onto guine pig RBC (Tble 4). Attempts to chrcterize the receptors of hemgglutinin by tretment of RBC with vrious compounds. Trypsin nd formldehyde hd detrimentl effects on the gglutinbility of RBC. Neurminidse, KIO4, nd sodium deoxycholte, on the other hnd, hd no effect (Tble 5). HI ntibody in the infected horse serum. HA ctivity ws inhibited by ntiser collected from horses infected with EIA virus (Tble 6). HI ntibody ws first detected 2 to 3 months fter infection nd persisted t lest until 270 dys. The HI titers observed were in rnge of 1:16 to 1:256 (Fig. 3). Nonspecific HA inhibitors or isohemgglutinins were found in some ser. However, they were lwys removed by tretment with kolin nd dsorption with guine pig RBC, without influencing HI ntibody titers. DISCUSSION Since HA ctivity of EIA virus could not be seprted from infectivity or CF ntigen, which is component of the virus prticle (4), or by CsCl grdient centrifugtion, nd since the infectivity of virl mteril mixed with guine Downloded from I1'A e * * *b 0 S on September 26, 2018 by guest.%-.- A, 0 i...z.;... IX0 * 0 0. * * FIG. 1. Reltionship between RBC concentrtion nd HA ntigen titer. The line indictes positive limit.

5 VOL. 14, ho c: 8 40._ 'U s 1U1 _ I15 : 't 1.31 *1: 1. +.OL < (Top) Frction Number (Bottom) FIG. 2. Distribution of infectivity (A), HA (C), nd CF (0) ntigens ofconcentrted EIA virus fter cesium chloride equilibrium density grdient centrifugtion. TABLE 4. Adsorption of EIA virus onto erythrocytes Erythrocyte TCID50/ml (log) Guine pig 4.75 Goose 5.75 Horse 5.50 Control 5.75 TCID50, Men tissue culture infective dose. b Mixture of virus fluid nd RBC ws kept t 4 C overnight. After centrifugtion, the superntnt fluid ws titrted for infectivity. TABLE 5. Agglutinbility ofguine pig erythrocytes by EIA virus fter tretment with vrious gents Tret- HA Agent Finl concn ment units/ t 37"C 0.05 ml (min) Trypsin 0.2% 60 0 Formldehyde 0.25% 60 4 Sodium deoxy % cholte-ki x 10-5% Neurminidse 0.18 units/mp' Control j Determintion mde using NAN-lctose. HEMAGGLUTINATION BY EIA VIRUS 329 pig RBC ws reduced bout 10-fold, it would pper tht the EIA virus hemgglutinin is closely ssocited with the virus prticle. Stbility of HA ctivity, however, did not lwys correlte with tht of infectivity. Lipid solvents destroyed not only HA ctivity but lso infectivity, but KIO4 nd phospholipse C tretments decresed infectivity without influencing HA ctivity. On the contrry, trypsin reduced HA ctivity without decresing infectivity. In ddition, HA ctivity ws more resistnt thn infectivity to ultrviolet irrdition nd formldehyde tretment. These results indicte tht lipoprotein or some other ether-sensitive substnce my ply n importnt role in the ttchment of EIA virus to the erythrocytes. These findings lso show tht the hemgglutinin hs different qulittive properties from infectivity nd my be due to loction differences from the determinnts of infectivity. A chrcteristic property of EIA virus hemgglutinin is its ether sensitivity. A number of viruses hve been shown to gglutinte erythrocytes of vrious species (2). Almost ll virl hemgglutinins, from not only nked viruses but lso enveloped viruses such s myxoviruses nd rboviruses, re stble or enhnced by ether tretment (2). The only exception, to our knowledge, is the rbies virus, the hemgglutinin of which is ether sensitive. In compring EIA virus hemgglutinin with tht of rbies virus, we noticed tht they differed in bsic properties. Rbies virus hemgglutinin ctivity is completely destroyed by trypsin nd phospholipse C nd is not influenced by ultrviolet irrdition (13). The hemgglutinting property of EIA virus ws enhnced by tretment with neurminidse, wheres murine leukemi virus treted with the sme enzyme reveled hemgglutinting properties not shown by untreted virus (21). However, phospholipse C tht enhnces TABLE 6. HI ntibody response in horse ser fter inocultion with EIA virus Horse Preinocuno. ltion Postinocultion (dys) <4 <4 < <4 <4 < <4 <4 < <4 <4 < <4 <4 8 _b <4 < Reciprocl of serum dilution tht showed definite inhibition of hemgglutintion. b -, Not tested.

6 330 SENTSUI AND KONO INFECT. IMMUN. J.;H J: rt.. : if.\ t 4 V ). ih KK- ;.]qsi )o J 8-4->!...I,...1-, 9 9 Downloded from * m FIG. 3. Development ofhi ntibody in horse (no. 594) infected with the EIA virus. A line indictes zone ofpositive HI rection. the HA ctivity of murine leukemi virus in coopertion with neurminidse (21) hd no effect on EIA virus hemgglutinin. These results show tht ETA virus hemgglutinin hs unique properties. Since the gglutinbility of RBC ws eliminted by trypsin nd formldehyde, nd since KI04, sodium deoxycholte, nd neurminidse tretment filed to inctivte the rectivity, the results suggest tht the ctivity is ssocited primrily with the proteins of the erythrocyte surfce, most probbly the receptors, rther thn with crbohydrte or lipid residues, nd these receptors for EIA virus hemgglutinin re different from those for myxoviruses (2). 1.A -e-- HI Antibodies ginst EIA virus hve been demonstrted by CF (6), immunodiffusion (1, 19), nd neutrliztion (5) tests. In the present studies, ser from infected horses showed high HI titers, with the highest being greter thn 1:256. The HI ntibody, which ppered reltively lte (60 to 150 dys) fter inocultion nd which persisted for long period, similr to neutrlizing ntibody (5, 12), is distinguishble from CF nd immunodiffusion ntibodies, which cn be detected soon fter the onset of infection (1, 6, 19). Studies on the reltionship between HI nd neutrlizing ntibodies still remin for future investigtions. It hs been reported tht there re mny immunologiclly distinct types of EIA virus (9, on September 26, 2018 by guest

7 VOL. 14, ). It remins to be determined whether ll possess HA ctivity nd whether the hemgglutinins re common or type specific. LITERATURE CITED HEMAGGLUTINATION BY EIA VIRUS Coggins, L., N. L. Norcross, nd S. R. Nusbum Dignosis of equine infectious nemi by immunodiffusion test. Am. J. Vet. Res. 33: Howe, C., nd T. Lee Virus-erythrocyte interctions. Adv. Virus Res. 17: Ishii, S Equine infectious nemi or Swmp fever. Adv. Vet. Sci. 8: Kono, Y Chrcteristics of complement fixing ntigen of equine infectious nemi virus. Ntl. Inst. Anim. Helth Q. 8: Kono, Y Viremi nd immunologicl responses in horses infected with equine infectious nemi virus. Ntl. Inst. Anim. Helth Q. 9: Kono, Y., nd K. Kobyshi Complement fixtion test of equine infectious nemi. fl. Reltionship between CF ntibody response nd the disese. Ntl. Inst. Anim. Helth Q. 6: Kono, Y., nd K. Kobyshi Complement fixtion test of equine infectious nemi. I. Specificity of the test. Ntl. Inst. Anim. Helth Q. 6: Kono, Y., nd K. Kobyshi Specificity of ssy of equine infectious nemi virus in horse leukocyte culture. Ntl. Inst. Anim. Helth Q. 7: Kono, Y., K. Kobyshi, nd Y. Fukung Antigenic drift of equine infectious nemi virus in chroniclly infected horses. Arch. Gesmte Virusforsch. 41: Kono, Y., K. Kobyshi, nd Y. Fukung Distribution of equine infectious nemi virus in horses infected with the virus. Ntl. Inst. Anim. Helth Q. 11: Kono, Y., T. Yoshino, nd Y. Fukung Growth chrcteristics of equine infectious nemi virus in horse leukocyte cultures. Arch. Gesmte Virusforsch. 30: Kono, Y., K. Kobyshi, nd Y. Fukung Serologicl comprison mong vrious strins of equine infectious nemi virus. Arch. Gesmte Virusforsch. 34: Kuwert, E., T. J. Wiktor, F. Sokol, nd H. Koprowski Hemgglutintion by rbies virus. J. Virol. 2: Mlmquist, W. A., D. Brnett, nd C. S. Becvr Production of equine infectious nemi ntigen in persistently infected cell line. Arch. Gesmte Virusforsch. 42: Nkjim, H., Y. Mizuno, K. Ysud, nd C. Ushimi Physicochemicl studies of equine infectious nemi virus. V. Effect of ultrviolet irrdition on virus infectivity. Arch. Gesmte Virusforsch. 41: Nkjim, H., nd J. Obr Ether susceptibility of equine infectious nemi virus. Ntl. Inst. Anim. Helth Q. 4: Nkjim, H., M. Tjim, S. Tnk, nd C. Ushimi Physicochemicl studies of equine infectious nemi virus. III. Purifiction nd electron microscopic observtion of the virus. Arch. Gesmte Virusforsch. 28: Nkjim, H., S. Tnk, nd C. Ushimi Physicochemicl studies of equine infectious nemi virus. IV. Determintion of the nucleic cid type in the virus. Arch. Gesmte Virusforsch. 31: Nkjim, H., nd C. Ushimi Immunodiffusion studies of purified equine infectious nemi virus. Infect. Immun. 3: Nowinski, R. C., L. J. Old, N. H. Srkr, nd D. H. Moore Common properties of the oncogenic RNA viruses (Oncornviruses). Virology 42: Schifer, W., nd J. Sznto Studies on mouse leukemi virus. II. Nchweis eines virusspezifischen Himgglutinins. Z. Nturforsch. 245: Stein, C. D., L. 0. Mott, nd D. W. Gtes Some observtions on crriers of equine infectious nemi. J. Am. Vet. Med. Assoc. 126: Tjim, M., H. Nkjim, nd Y. Ito Electron microscopy of equine infectious nemi virus. J. Virol. 4: