Comparative Study of Three Methods for Detecting Avian Leukosis Viruses

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1 INFECTION ND IMMUNITY, My 1977, p Copyright C 1977 mericn Society for Microbiology Vol. 16, No. 2 Printed in U.S.. Comprtive Study of Three Methods for Detecting vin Leukosis Viruses EUGENE J. SMITH,* LYMN B. CRITTENDEN, ND JGODIN IGNJTOVIC United Sttes Deprtment ofgriculture, griculturl Reserch Service, Regionl Poultry Reserch Lbortory, Est Lnsing, Michign Received for publiction 13 December 1976 This investigtion s designed to compre detection limits for vin leukosis viruses fter infection of chicken fibroblsts ith deciml dilutions of Rousssocited virus type 1 (RV-1). t 5, 9, 14, nd 19 dys postinfection, cells ere exmined for group-specific (gs) ntigens by microtiter complement-fixtion (CF) tests for vin leukosis viruses nd by rdioimmunossys (RI) for the mjor gs ntigen hving moleculr eight of 27, (p27). Culture fluids, collected t the sme time periods, ere lso ssyed for reverse trnscriptse ctivities. We found tht minimlly infected cultures expressed virus proteins ithin 9 dys postinfection regrdless of method used. lthough p27 RI s consistently more sensitive thn CF or reverse trnscriptse ssys, sensitivity s only to- to fivefold greter hen concentrted suspensions of RV-, RV- 1, nd RV-2 ere compred. In terms of infectious units, the loest detectble virus titer s 6 x 13 infectious units s determined by RI end point dilutions. Hoever, our results led us to conclude tht hen concentrted cell extrcts re tested ith hmster ntiserum, CF is dequte for detecting infection. With pproprite ntiser, group-specific (gs) ntigens of vin leukosis-srcom viruses rect in complement fixtion (CF) tests (1) nd in specific competition rdioimmunossys (RI; 2, 13, 17). Previous reports indicted tht the RI for the gs ntigen hving moleculr eight of 27, (p27) ere 1 to 1, times more sensitive thn CF tests (13, 17), heres the loer limits for detecting relted type C ribonucleic cid viruses, s mesured by reverse trnscriptse ctivities, hve been reported to be 12 to 13 infectious units (7). Ech procedure requires specilied equipment nd regents; thus the criterion of choice my involve the limits of sensitivity. Becuse our experience ith hmster ntiser to Rous srcom virus-trnsformed hmster cells indicted tht CF s very sensitive, e compred detection limits s function of virus dose nd durtion of cultivtion of Rous-ssocited virus type 1 (RV-1)-infected chicken embryo fibroblsts (CEF). Using CF, p27 RI, nd reverse trnscriptse ctivities, e lso describe miniml detectble infectious units of three RNs: RV-, RV-1, nd RV-2. MTERILS ND METHODS Viruses nd cell extrcts. Secondry CEF from gs ntigen-negtive SPFS embryos (SPFS, Inc., Norich, Conn.) ere cultivted in medi previously described (5). Five sets of four 1-mm pltes, 5 ech contining 4 x 16 cells, ere seprtely infected ith.1 ml of decimlly diluted RV-1 rnging from 1-2 to 1-6. n dditionl set of uninfected cells represented controls. fter 5, 9, 14, nd 19 dys in culture, cells from to pltes of ech set ere shed in veronl buffer diluent (1), removed by scrping, nd centrifuged t 2, rpm for 1 min. Cell suspensions, circ 2% (vol/vol), ere freeethed t lest three times before they ere tested. t the time of ech collection, cells from compnion pltes ere removed by trypsinition nd reseeded t 4 x 16 cells per plte. Thirty-eight milliliters of culture fluids from ech set s clrified by centrifugtion t 1, rpm for 1 min, nd virus s sedimented fter centrifugtion for 1 h t 23, rpm in n SW27 rotor. Pellets ere resuspended in.38 ml of 1% Nonidet P-4 contining.2 M dithiothreitol nd stored in the vpor phse of liquid nitrogen before they ere tested for reverse trnscriptse ctivities. RV-, n endogenous leukosis virus, s collected from culture fluids of line 1 cells (4), nd RV-2 (18) s cultivted on infected secondry SPFS CEF. CF. Cell extrcts nd virus suspensions, diluted in microtiter pltes, ere incubted overnight t 4C ith 4 U (1:32 dilution) of hmster ntiserum nd five 5% hemolytic units of complement (Difco Lbortories, Detroit, Mich.). nticomplementry controls ere included in ll tests. ntiser ere prepred by injecting hmster cells trnsformed by the Schmidt-Ruppin strin of Rous srcom virus into hmsters (13). fter bout 8 eeks, nimls bering tumors ere periodiclly bled from the orbitl sinus. The ntigen end point titer for complete

2 VOL. 16, 1977 fixtion of complement by hmster ntiserum s pproximtely 1 ng of p27. Reverse trnscriptse ssy. Dilutions of Nonidet P-4-solubilied virus (.5 ml) ere incubted ith:.1 ml of [3H]thymidine triphosphte (specific ctivity, 76 cpm/pmol; Ne Englnd Nucler, Boston, Mss.);.1 ml ofpolyribodenylte:oligodeoxythymidylte (.2 units of bsorbncy t 26 nm; Collbortive Reserch, Wlthm, Mss.);.2 ml of mngnous cette (2.5 x 1-3 M); nd.1 of lox stock mixture contining.4 M Tris-hydrochloride (ph 8.1),.6 M KCl, nd.2 M dithioerythritol. fter 1-h incubtion t 37 C, duplicte smples nd controls lcking templte ere treted ith.1 ml of.1 M sodium pyrophosphte,.1 ml of yest ribonucleic cid (1 mg/ml), nd 2.5 ml of 1% trichlorocetic cid. Insoluble polymers ere filtered on nitrocellulose filters (.45-,um pore sie, HWP; Millipore Corp., Bedford, Mss.) nd shed ith bout 3 ml of 5% trichlorocetic cid. Filters ere counted in vils contining 5 ml of toluene-triton X-1 solution tht contined.4% Omnifluor (Ne Englnd Nucler). The verge number of picomoles of lbeled thymidine monophosphte incorported into n cid-insoluble product s corrected for counts in filters tht lcked templte. Competition RI. The mjor gs ntigens of n vin myeloblstosis virus hving moleculr eight of 27, (p27) ere purified by gunidine- HC1 gel chromtogrphy s described by Fleissner (6). 25-,ul portion contining 5 to 1 g of p27 s iodinted ith 1 mci of 125iodine nd 25,ug of chlormine-t (8). Lbeled ntigen s seprted from unrected iodine by elution from column (1 by 15 cm) of Bio-Gel P-1 ith.1 M phosphte buffer (ph 7.2). Double-ntibody immunossy mixtures consisted of the folloing:.1 ml of tofold seril dilutions of RV-1-infected cell extrcts or virl suspensions;.1 ml of 1:12,8-fold dilution of pig ntiserum to vin myeloblstosis virus;.1 ml of lbeled p27 (15, to 2, cpm/ng); nd.2 ml of buffer solution tht consisted of.1 M Tris (ph 7.9),.1 M NCl,.25 M ethylenediminetetrcetic cid,.2% bovine serum lbumin,.2% Triton X-1, nd norml sine serum (1:4). Duplicte smples ere incubted t 37 C for 3 to 5 h nd overnight t 4 C. 25-ml portion of undiluted got nti-pig immunoglobulin G, provided by the Resources nd Logistics Segment of the Ntionl Cncer Institute, s dded, nd solutions ere incubted t 37 C for 1 h nd finlly refrigerted for 3 h. Mixtures ere centrifuged t 2,5 rpm for 15 min, superntnts ere removed by spirtion, nd bound ntigen s counted in Beckmn model 3 gmm spectrometer. The verge percentge of binding s normlied to control mixtures tht lcked competing ntigen fter correcting for nonspecific binding (usully 5 to 1% of the totl input counts per minute). Becuse smll mount of competition s occsionlly observed t high protein concentrtions, greter thn 8% binding of the probe s considered insignificnt competition. Phenotypic mixing nd RV- ssys. RV-1 VIN LEUKOSIS VIRUS DETECTION 51 nd RV-2 ere titered by the phenotypic mixing ssy of Okki et l. (1). Briefly, C/O (susceptible to ll subgroups) cells ere co-infected ith Rous srcom virus-rv- nd the test smple. fter 7 dys, culture fluids ere collected nd ssyed on C/ E (resistnt to subgroup E only) cells. The ppernce of foci indicted the presence of non-subgroup E virus. RV- s mesured by dding the test smple to mixture of Rous-Jpnese quil (R-Q) cells (clone from Helen Murphy, Imperil Cncer Reserch Fund Lbortories, London) nd turkey embryo fibroblsts. fter 9 dys in culture, fluids ere ssyed on C/O nd C/E cells. ppernce of foci on C/O cells nd the bsence of foci on C/ E cells indicted tht subgroup E leukosis virus s present. RESULTS Chronologicl nd titrtion end points of RV-1-infected cells by CF, RI, nd reverse trnscriptse. CF ctivities in extrcts of chicken cells infected ith lo dilutions of RV-1 ere found 5 dys fter infection, nd the end point dilution s detectble by dy 9. Dt in Tble 1 lso indicte the percentge of binding of rdioiodinted p27 fter cell extrcts ere tested in competition immunossys. Under conditions of limiting ntibody, reduced binding of the lbeled probe, ttributed to specific competition by solubilied ntigen, s only mrkedly reduced in CF-positive smples. Reverse trnscriptse ctivities in pelleted nd concentrted smples equivlent to 5 ml of culture fluids from the sme RV-1-infected cells (Tble 1) prlleled tht found in cell extrcts by CF nd RI. In ll three methods, virus production s not observed beyond the 1-5 dilution of RV-1, even fter 19 dys of cultivtion. This greement in chronologicl nd virus titer end points indicted tht minimlly infected chicken cells required from 5 to 9 dys for RV-1 to be detected, regrdless of method used. Comprison of sensitivity limits of CF nd p27 RI. Extrcts of cells nd concentrted culture fluids collected 5 dys fter infection ith 1-2 dilution of RV-1 ere further exmined by dilution to respective end points in CF tests nd RI. Results indicted tht p27 RI of CEF extrcts nd culture fluids ere, respectively, 128 nd 32 times more sensitive thn direct CF (Tble 2). Note tht, despite the greter sensitivity of RI, the mount of RV- 1 produced 5 dys fter infection ith 1-5 dilution of RV-1 (Tble 1) s t lest 13 times less thn tht observed fter 9 dys of cultivtion; otherise, p27 ould hve been detected in cells collected 5 dys fter infection; i.e., percentge of binding ould hve been less thn 96% (Tble 1).

3 52 SMITH, CRITTENDEN, ND IGNJTOVIC TBLE 1. Detection ofrv-1 in serilly infected CEF by CF,p27 competition RI, nd reverse trnscriptse ctivities Dilution of RV-1 used Dys in culture fected ProcedutuerUin CF RIb Reverse trn scriptsec t 1:2 dilutions of 2% (vol/vol) CEF extrcts. Symbols: +, less thn 5% hemolysis of sheep erythrocytes; -, greter thn 5% hemolysis of sheep erythrocytes. b Numbers represent the percentge of binding of '25iodine-lbeled p27 by limiting mounts of ntiserum of vin myeloblstosis virus ith 1:4 dilutions of CEF extrcts. High vlues indicte less competition. d verge mounts in picomoles of [3H]thymidine monophosphte incorported into trichlorocetic cidinsoluble polymer fter 1-h incubtion t 37 C ith smples equivlent to 5 ml of culture fluid. TBLE 2. Comprisons beteen dilution end points of RV-1-infected CEF nd 1-fold concentrted culture fluids in CF tests nd p27 RI Smple Reciprocl of end points Procedure volume Supern- (ml) CEF' tnt' CF RI.1 32,768 16,384 Freee-thed extrcts (2%, vol/vol) ere used 5 dys fter infection. b Five dys fter infection, the culture fluid (38 ml) s centrifuged t 23, rpm for 1 h, nd the pellets ere solubilied in.38 ml of.1% Nonidet- P-4. Specificities nd sensitivities of p27 competition RI. Figure 1 shos dose response ith chromtogrphiclly purified, rdioiodinted vin myeloblstosis virus p27 s the probe nd three nonlbeled ntigens. s shon by others (3, 16), p19 nd p15 did not cross-rect ith p27 derived from vin myeloblstosis virus, nd the loer limit of detection (t 8% binding of the probe) s pproximtely.4 ng ith sine ntiserum. Immunossys ere lso conducted ith 1- fold concentrted suspensions of RV-, RV- 1, nd RV-2 (Fig. 2). Before concentrtion, smples of culture fluids from RV-1-, RV-2-, nd RV--infected cells ere stored t -7 C nd subsequently titered by the phenotypic mixing test (RV-1, RV-2), nd RV- ssys s described in Mterils nd Methods. In lo[ f- 7 6 L5C\ 5 cu 4.3 2F I *..2.1 O NNOGRM S FIG. 1. Dose response ith iodinted p27 s the probe nd competing ntigens. Symbols: p27 (), p19 (), nd p15 () in the presence of limiting mounts of sine ntiser to vin myeloblstosis virus (1:6,4). greement ith titers obtined by groth in tissue culture, competition for p27 by RV- nd RV-2 preprtions s similr, nd RV-1 competed bout 3-fold less efficiently * INFECT. : IMMUN..

4 VOL. 16, 1977 (Tble 3). This decresed efficiency reflects the loer virus titer of the ltter preprtion. Sensitivity limits for the detection of RVs. The pprent limits of sensitivity expressed in infectious units per milliliter ere bsed on end point dilutions of 1-fold concentrted suspen- 1.O \ Z 8- I' 6- C._. I/NTIGEN DILUTION FIG. 2. Double-ntibody p27 competition RI ith 1-fold concentrted suspensions of RV- (), RV-1 (), nd RV-2 () in the presence of sine ntiser to vin myeloblstosis virus (1:6,4). TBLE 3. VIN LEUKOSIS VIRUS DETECTION 53 sions of RV-, RV-1, nd RV-2 hen e used CF, reverse trnscriptse, p27 RI (Fig. 2), nd titers of undiluted fluids obtined by phenotypic mixing or RV- ssys. RI s only 2.5 times more sensitive thn either CF or reverse trnscriptse, nd the loest limit of detectble RV-2 s 6 x 13 infectious units. Note tht, lthough CF nd p27 RI end points ere not mrkedly different for RV- nd RV-2, RV-1 expressed only one-tenth the expected reverse trnscriptse ctivity compred to tht expressed by the other viruses on n infectious titer bsis. DISCUSSION Experiments designed to study the onset of detectble virus in RV-1-infected cultures by three independent methods indicted no significnt differences in chronologicl end points. RV-1 proteins ere redily detected in both cell extrcts nd culture fluids ithin 9 dys fter infection ith end point dilution, regrdless of the procedure used (Tble 1). The percentges of contribution to the totl protein content of RV-2 for three gs ntigens (p27, p19, p15) nd p27 lone ere pproximtely 75 nd 37%, respectively (3), but end point differences indicted tht p27 immunossys ere 32 to 128 times more sensitive thn CF, notithstnding the fct tht CF mesures ll five gs ntigens. Smple volumes ere.25 ml in CF microtiter pltes nd.1 ml in immunossy tubes; thus hen end points ere expressed on n equivlent smple volume bsis, p27 immunossys ere only 8 nd 32 times more sensitive thn CF hen superntnt fluids nd cell extrcts, respectively, ere used. Moreover, hen concentrted suspen- End point dilutions nd minimum number of infectious units of RVs detectble by CF, reverse trnscriptse, nd p27 RI End point dilutions detected ith: Minimum no. of IU detectble ith: Virus titer Culture (IU/mlud fluid Reverse Reverse tsec tse titer(ju/l) OF" trnscrip- RId CF trnscrip- RI RV ,192 5 x 14 5 x 14 1 X 14 RV x 14 5 x 15 4 x 14 RV , ,384 2 x 14 5 x 14 6 x 13 The number of infectious units (IU) per ml in undiluted fluids s bsed on virus titers obtined by phenotypic mixing nd RV- ssys. From these dt nd end point dilutions of preprtions ssyed by ech method, pprent limits of sensitivities ere clculted nd expressed s the miniml number of detectble IU. b Reciprocl dilution end points of gs ntigens in microtiter procedure, strting ith.25 ml of 1-fold concentrted culture fluids. c Minimum volumes (in milliliters) of unconcentrted culture fluids required for significnt incorportion of [3H]thymidine monophosphte into cid-precipitble polymer ith polyribodenylte:oligodeoxythymidylte s templte. d Reciprocl dilution end points in p27 competition titrtions, strting ith.1 ml of 1-fold concentrted culture fluids.

5 54 SMITH, CRITTENDEN, ND IGNJTOVIC sions of RV-, RV-1, nd RV-2 ere diluted nd tested by the three procedures, RI s clculted to be only to to five times more sensitive thn either CF or reverse trnscriptse (Tble 3). The loest limit of detectble virus corresponded to 6 x 13 infectious units. Becuse the methods discussed re pproximtely equl in sensitivities, selection of procedure for routine use in bsic or epidemiologicl reserch my be predicted on vilbility of regents (ntiser) or equipment. The convenience, high sensitivity, nd lo cost of CF testing ould be the preferred pproch for lrge-scle screening of flocks. The gs ntigens re redily detected by CF in egg lbumin from virus-shedding hens (15) nd fether pulps, heres liquid scintilltion or gmm rdition detectors re necessry for reverse trnscriptse ssys or RI. The CF test for vin leukosis viruses, s originlly described by Srm et l. (13), is bsed on the infection of leukosis virus-free CEF ith test smples. Thus, if tissue extrcts or ser contin vin leukosis-srcom viruses, susceptible, gs ntigen-negtive CEF could produce redily detectble mounts of virl ntigens. Hoever, in the bsence of virus infection, gs ntigen expression is controlled by dominnt host-cell gene (9, 11), nd extrcts from virus-free chickens my be positive hen tested directly by CF nd RI (15). Thus, more relible indictor of infection ould be reverse trnscriptse ssys if gs ntigen-negtive cells re not vilble. In our lbortory, reverse trnscriptse ssys nd p27 RI ere s sensitive s those exmined by others. Hoever, the use of high vidity hmster ntiserum nd recognition tht extremely smll volumes (.25 ml) of test mteril re used in microtiter ells reconcile, in prt, previous reports on mrked differences in sensitivities. CKNOWLEDGMENTS We thnk Pdmn Srm (Ntionl Cncer Institute, Bethesd, Md.) for providing the Rous srcom virustrnsformed hmster cell line used in prepring ntiser. pprecition is expressed to J. Gruber (Ntionl Cncer Institute, Bethesd, Md.) nd R. Wilsnck (Huntingdon Reserch Center, Bltimore, Md.) for providing got ntiser to sine immunoglobulin G. RV-1 nd RV-2 ere originlly obtined through the courtesy of P. K. Vogt (Los ngeles, Clif.). The cpble ssistnce of uror Whitson nd Frnces Gulvs is lso pprecited. This study s supported in prt by Intergency greement no. YOICP4214 ith the Virus Cncer Progrm of the Ntionl Cncer Institute. INFECT. IMMUN. LITERTURE CITED 1. Csey, H. L Stndrdied dignostic complement fixtion method, p In Public Helth Service Monogr. no. 74, Wshington, D.C. 2. Chen, J. H., nd H. Hnfus Detection of protein of vin leukoviruses in uninfected chick cells by rdioimmunossy. J. Virol. 13: Chen, J. H., W. S. Hyrd, nd H. Hnfus vin tumor virus proteins nd RN in uninfected chicken embryo cells. J. Virol. 14: Crittenden, L. B., E. J. Smith, R.. Weiss, nd P. S. Srm Host gene control of endogenous vin leukosis virus production. Virology 57: Crittenden, L. B., E. J. Wendel, Jr., nd D. Rtsch Genetic resistnce to the vin leukosis-srcom virus group: determining the phenotype of dult birds. vin Dis. 15: Fleissner, E Chromtogrphic seprtion nd ntigenic nlysis of proteins of the oncornviruses. I. vin leukemi-srcom viruses. J. Virol. 8: Gllo, R Reverse trnscriptse, the DN polymerse of oncogenic RN viruses. Nture (London) 234: Greenood, F. C., W. M. Hunter, nd J. S. Glover The preprtion of '31I-lbeled humn groth hormone of high specific ctivity. Biochem. J. 89: Hnfus, H., T. Hnfus, S. Ki, nd R. E. Luginbuhl Genetic control of expression of endogenous virus genes in chicken cells. Virology 58: Okki, W., H. G. Purchse, nd B. R. Burmester Phenotypic mixing test to detect nd ssy vin leukosis viruses. vin Dis. 19: Pyne, L. N., nd R. C. Chubb Studies on the nture nd genetic control of ntigen in norml chick embryos hich rects ith COFL test. J. Gen. Virol. 3: Sndelin, K., T. Estol, S. Ristemki, E. Ruoslhti, nd. Vheri Rdioimmunossy of the group-specific ntigen in detection of vin leukosis virus infection. J. Gen. Virol. 25: Srm, P. S., H. C. Turner, nd R. J. Huebner n vin leucosis group-specific complement fixtion rection. ppliction for the detection nd ssy of non-cytopthogenic leucosis viruses. Virology 23: Smith, E. J., J. R. Stephenson, L. B. Crittenden, nd S.. ronson vin leukosis-srcom virus gene expression: noncoordinte control of group-specific ntigens in virus negtive vin cells. Virology 7: Spencer, J. L., L. B. Crittenden, B. R. Burmester, C. Romero, nd R. L. Witter Lymphoid leukosis viruses nd gs ntigen in unincubted chicken eggs. vin Pthol. 5: Stephenson, J. R., E. J. Smith, L. B. Crittenden, nd S.. ronson nlysis of ntigenic determinnts of structurl polypeptides of vin type C tumor viruses. J. Virol. 16: Stephenson, J. R., R. E. Wilsnck, nd S.. ronson Rdioimmunossy for vin C-type virus group-specific ntigen: detection in norml nd virustrnsformed cells. J. Virol. 11: Vogt, P. K heterogeneity of Rous srcom virus reveled by selectively resistnt chick embryo cells. Virology 25: