Epitope analysis of capsid and matrix proteins of North American ovine lentivirus field isolates

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0095-1137/91/071472-08$02.00/0

Epitope Analysis of Capsid and Matrix Proteins of North

American

Ovine Lentivirus Field Isolates

K. A. MARCOM,1 L. D.

PEARSON,'*

C. S.

CHUNG,'

J. M.

POULSON,'

ANDJ. C. DEMARTINI2 Departmentof

Microbiology'

and DepartmentofPathology,2 College ofVeterinary Medicine and

BiomedicalSciences, Colorado State University, Fort Collins, Colorado 80523 Received 29 January1991/Accepted22April 1991

Monoclonal antibodies(MAbs) directedagainst two phenotypically distinct ovine lentivirus(OvLV) strains were generated by fusion ofBALB/c SP2/0-Ag14 myeloma cellswithspleen cellsfrom mice immunized with

purffied OvLV. Hybridomas were selected by indirect enzyme-linked immunosorbent assay (ELISA) and analysis of reactivity on immunoblots. The majority (17 of 21) of the MAbs recognized the gag-encoded capsid protein, CA p27, of both strains. Four other MAbs recognized a smaller structural protein, presumably a matrix protein, MAp17. Three distinct epitopes on CA p27 and one on MA p17 were distinguished by the MAbs with competitionELISA. MAbs from each epitope group were able to recognize 17 North American field isolates of OvLV and the closely related caprine arthritis-encephalitis virus (CAEV). Analysis of the data indicated that theseepitopes were highly conserved among naturally occurring isolates. A representative MAb from each epitope group of anti-CA p27 MAbs reacted with four field strains of OvLV and CAEV on

immunoblots.Ananti-MAp17MAbrecognizedthe same OvLVstrainson immunoblots butfailedtorecognize

CAEV. MAbs whichrecognize conserved epitopes of gag-encoded lentivirus proteins (CA p27 and MAp17)are valuable tools. These MAbs can be used to develop sensitive diagnostic assays and to study the pathogenesis of lentivirus infections in sheep and goats.

Maedi-visna virus, progressive pneumonia virus (ovine lentiviruses [OvLV]) and caprine arthritis-encephalitis virus

(CAEV) are nononcogenic retroviruses belonging to the

subfamily Lentivirinae (4, 10, 34). These viruses areagents

of persistent, degenerative diseases in sheep and goats,

which are characterized by progressive pneumonia (4, 10), mastitis (10, 12), partialparalysis, erosivepolyarthritis, and

nonsuppurative encephalitis (10, 19, 30). Ovine andcaprine lentivirusesaretransmitted naturallyviaeither the gastroin-testinalroutefollowing ingestion of infectedcolostrum or the

respiratory route (19, 29). Infection with OvLV occurs

throughout manyofthe major sheep-producingareas ofthe

United States. In cull and range sheep from western and

midwestern states, seroprevalence of OvLV ranged from 1 to 90% (8, 16, 18, 23). In 1970, Gates and coworkers (15)

associated the disease inIdahowithreduced ewe

reproduc-tive efficiency and an increase in forced culling. Periodic

serological testingand slaughterorisolation of seropositive animalswill control lentivirus infection ofsheep and goats

(20-22, 32).

Ovine

progressive pneumonia

was first described

by

Marshin1923(33)and wassubsequently diagnosedin adult

sheep in nine counties ofMontana. In 1968,

Kennedy

etal. (27) isolated a virus from Montanasheepexhibiting clinical symptoms of progressive pneumonia. This virus closely

resembled anotherOvLV, themaedi-visnavirus, described 10years earlier bySigurdsson and Palsson (45). The CAEV wasisolated in 1980 from goats with chronic arthritis (7).

Previous studies show the close immunological

relation-ship of ovine and caprine lentiviruses (11, 44, 49, 51).

Immunoprecipitation of proteins from several isolates of

OvLVrevealahigh degree of relatedness, althoughdistinct

genetic differences are shown by hybridization studies (3,

44). Antigenic variationoccurs predominantly in the

enve-*Correspondingauthor.

lopeglycoproteins oftheruminant lentiviruses (13, 37, 48),

while the gag genes are highly conserved and share approx-imately 50% nucleotidehomology (38, 42, 43). The ovine and caprine lentiviruses have common antigenic determinants on the major capsid protein (4), although antigenic and

struc-tural variation of thisproteininsheepand goatlentivirusesis described(36).

Monoclonal antibodies (MAbs) which recognize con-servedepitopesongag-encoded proteins(CAp27,MAp17) are valuable tools in diagnostic assays and pathogenesis

studies with lentiviruses ofsheepand goats. In this report, we describe the production and characterization of MAbs which recognize structural proteins of OvLV. We

immu-nized mice with two phenotypically distinct field strains

(85/34and84/28)ofOvLVtogenerate 21 MAbsspecificfor

OvLV antigens. The in vitro

cytopathogenicity

of these strains ofOvLV rangesfrom highly lytic (85/34)to

persist-ent, slowly lytic (84/28), with varying degrees of syncytia produced in goat synovial membrane (GSM) cells (29). Theseviruses induced diseasein astrain-dependentmanner. The highly lytic strain rapidly induced clinical signs of

infectionandcaused more severelesions than didpersistent strains (29). When we use the

terminology

for retrovirus

proteins suggested byLeisetal. (31),themajority (17 of 21)

ofthe MAbs

recognized

the

capsid protein

CAp27 ofboth

isolates.Theremaining four MAbsrecognizedpresumablya matrixprotein,MAp17. Reactionwith MAbs from the four

epitopegroups inanindirectenzyme-linked immunosorbent

assay (iELISA) showed the conservation oftheseepitopes

amongnaturally occurringfieldisolates of OvLV.

MATERIALSANDMETHODS

Mice. BALB/c mice were used for all fusions and as a sourceofperitoneal macrophagesandthymocytesfor feeder cells in fusion andcloning

experiments.

Virus.Astrain of OvLV

(85/34)

thatishighly lytic in cell

1472

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culture was obtained from an animalexperimentally infected with lung exudate from an animal with anaturally occurring caseoflymphoid interstitial pneumonia. A persistent, slowly lytic OvLV (84/28) was isolated from an animal with a field case with lymphoid interstitial pneumonia and ovine pulmo-nary carcinoma (30). Both of these isolates were plaque cloned three times and subsequently propagated in GSM cells (provided by T. McGuire, Washington State Univer-sity) previously screened for latentretroviruses by molecu-larhybridization (42). Supernatants from infected cells were clarified (8,000 x g for 30min), and virus was concentrated byultracentrifugation (98,000 x g for 60min). The virus was then layered onto a 25 to 50% continuous-density sucrose gradient and centrifuged overnight in an SW40 rotor at 30,000 rpm. Fractions (0.5 ml) from the gradient were collected and analyzed for density and proteinconcentration and for reactivity with an OvLV CAp27-specific MAb. Peak fractions with a density of 1.15 to 1.16 g/cm3 were pooled and dialyzed against TNE (10 mM Tris [pH 7.2], 150 mM NaCl, and 1 mM EDTA). The protein concentration of the purified virus wasmeasured by utilizing bicinchoninic acid in a sensitive microtiter plate assay (47). Visna virus, CAEV, and H-24 were obtained from J. E. Dahlberg, National Cancer Institute, Bethesda, Md.

Fusion and selection ofhybridomas. Purified virus was used to inoculate mice in all fusionexperiments in an attempt to reduce thefrequency of hybridomas producing antibodies to cellular components. BALB/c mice were given three subcu-taneous inoculations (at 2-week intervals) of 20 to 60

jig

of sucrose-gradient-purified virus (85/34 or 84/28) disrupted with Triton X-100 and diluted 1:2 with purified adjuvant (RIBI Immunochem Research, Inc., Hamilton, Mont.). Three days prior to fusion of the mouse spleen cells to BALB/c SP2/0-Ag 14 myeloma cells, the mice received an intraperitoneal inoculation of 20 to 60 ,ug of virus. Spleen cells fromimmunized mice (5 x 107)andmyelomacells

(107)

were fused at 37°C in asolution of 1 g of polyethylene glycol 4000 (E. Merck, Darmstadt, Germany) in 1 ml of RPMI 1640-HM medium (Sigma Chemical Co., St. Louis, Mo.) containing 0.1 ml ofdimethyl sulfoxide (14). The fused cells were resuspended in complete medium (RPMI 1640-HM medium supplemented with 15% bovine calf serum [Hy-Clone Laboratories, Logan, Utah], 1x nonessential amino acids [Sigma Chemical Co.], 5 x 10-6M2-mercaptoethanol, 2 mM L-glutamine, and 50 ,ug of gentamicin per ml [Sigma Chemical Co.]) that alreadycontainedfeeder cellsconsisting

of peritoneal macrophages and thymocytes (28, 40) and dropped by pipet into 96-well, flat-bottomed Microtest III tissue culture plates (BectonDickinson, Lincoln Park, N.J.). To increase the cloningefficiency, 50 ,ug of STM mitogen per ml (RIBI Immunochem Research, Inc.) wasalso added. On days 2, 3, and 4 following the fusion, 100 ,ul of HAT (complete mediumcontaining 1 x

10'

Mhypoxanthine, 4x

10-7 M aminopterin, and 1.6 x

10'

M thymidine [Sigma Chemical Co.]) was added. The hybridoma clones were counted 9 to 12daysafter fusion and screenedforanti-OvLV

antibodies as detected by iELISA. If the hybridoma super-natants were also positive on an immunoblot, the cells were cloned at least twice by limiting dilution.

ELISA for selection of hybridomas producing antibodies with antiviral specificity. Hybridoma supernatants were screened by an iELISA. Purified OvLV oruninfected GSM cell lysates were dried into modified flat-bottomed 96-well ELISA plates (Corning Glass Works, Corning, N.Y.) over-night at 25°C. The optimal concentration of virus and GSM cell lysate was determined by titration against

OvLV-spe-cific MAb and cell-specific MAb. Endogenous peroxidase

present in the lysates was inactivated with 200 ,ul of0.6%

H202in methanol in each well for 20minatroom tempera-ture. After three rinses with phosphate-buffered saline (PBS)-Tween buffer (1.8 mM NaH2PO4 dihydrate, 8.4 mM Na2HPO4, 150 mM NaCl, and0.05% Tween-20), the plates

wereincubated at37°Cfor 1 h with BLOTTO block (1). The blocking solution wasflicked fromtheplates before 50 ,ul of hybridoma supernatants was added to both purified virus-coated wells anduninfected GSMlysate-coated wells. After incubation for 1 h at 37°C, the plates were then rinsed five

times with PBS-Tweenbuffer. Bound mouse

immunoglobu-lin was detected with 50 1±l of biotinylated rabbitanti-mouse immunoglobulin G (IgG) (heavy- and light-chain specific)

(Zymed Laboratories Inc., South San Francisco, Calif.)

diluted 1:2,000 in BLOTTO bufferfor 30minat37°C. After five PBS-Tween rinses, 50 ,u of streptavidin-conjugated horseradish peroxidase (Zymed Laboratories Inc.) diluted 1:2,000 in BLOTTO buffer was added to each well and incubated for 30min at37°C. After five PBS-Tween rinses, 100 ,ul of the chromagen-substrate solution was added to each well. The chromagen, 3,3',5,5'-tetramethylbenzidine (Sigma Chemical Co.), was prepared as a stocksolution (100 mg of 3,3',5,5'-tetramethylbenzidine in 10 ml of dimethyl

sulfoxide) and stored in the dark at room temperature. The chromagen-substrate solution consisted of 7.5 ,u of 30% H202, 50 mlof0.1 M citrate-acetate buffer(pH 6.0), and 0.5 mlof 3,3',5,5'-tetramethylbenzidine stock. The reactionwas stopped after 10 min with 50 ,ul of 1 N H2SO4. The optical density (OD) of the test wells was measured at 450 nm (MR600Dynatech PlateReader), and the data were collected on a personal computer. If the ratio of OD of virus/OD of uninfected cell lysate was >10, the supernatants were screened by immunoblot analysis to determine which viral antigens they recognized.

Polyacrylamide gel electrophoresis (PAGE). Samples (2 to 50 p.g of viral protein) were diluted 1:2 in a sample buffer containing2% sodium dodecyl sulfate (SDS) and 4% 2-mer-captoethanol and boiled for 5 min to denature the protein. The samples, along with prestained molecularweight mark-ers (Bethesda Research Laboratories Life Technologies, Inc., Gaithersburg, Md.) were thenapplied to a Tris-buffered discontinuous SDS-polyacrylamide gel (11% resolving gel with a 3% stacking gel). The proteins were separated elec-trophoretically (150 V for 45 to 60 min) andeithervisualized by staining with 0.25% Coomassie brilliant blue in 45% methanol and 10% acetic acid or electrophoretically trans-ferred with atransblotter (Bio-RadLaboratories, Richmond, Calif.) (11 V, overnight) onto nitrocellulose for immunoblot analysis.

Immunoblotting. Following transfer of the viral polypep-tides to a 0.45-,um-pore-size nitrocellulose membrane (Bio-Rad Laboratories), the membrane was blocked with 10%

skim milk in PBS for 1 h at 25°C. After rinsing with PBS containing 500 mMNaCl, 1 mM EDTA, and0.5% Tween-20,

the membrane was loaded into a Miniblotter 28 apparatus (Immunetics, Cambridge, Mass.). Then 60 p.l ofhybridoma supernatant or purified MAb was loaded into each channel for 1 h at 25°C. The blot was again rinsed andincubated for 1 h at 25°C with 60 p.l of alkaline phosphatase-conjugated goat anti-mouse immunoglobulin (heavy- and light-chain

specific) (Zymed Laboratories Inc.) diluted 1:250 in 10% skim milk in rinse buffer. The blot was rinsed before and after removal from theapparatus and thenbrieflyrinsed with distilled water. The chromagen(Nitro BlueTetrazolium) and substrate (5-bromo-4-chloro-3-indolyl phosphate) were used

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to detect positive reactions. When greater sensitivity was

required or more than one strain of virus was used, the

immunoblot was performed outside the apparatus.

MAb purification and biotinylation. Stable hybridomas were cultured in complete medium with 5% fetal bovine serum (HyClone Laboratories) for MAb production or

in-jectedintraperitoneally into pristane-primed mice for ascites production. The hybridoma supernatants or ascitic fluids were clarified (200 x g for 5 min), and MAbs were then

precipitated with 43% ammonium sulfateorpurified by using aGammaBind G affinity column (Genex Corporation,

Gaith-ersburg,Md.). The MAbswerethendialyzed against several

changesof TEN buffer (50 mM Tris, 1 mM EDTA, 150 mM NaCl [pH 7.4])or100mMNaHCO3 (pH 8). SelectedMAbs

ataconcentration of 1 mg/mlwerebiotinylated with

amino-hexanoyl-biotin N-hydroxysuccinimide (Zymed Laborato-ries Inc.) at a wt/wt ratio of 10:1 for 1 h, dialyzed against

TEN buffer, and then stored at -20°C.

Isotype determination. The isotypes of the MAbs were

determined by ELISA usingan isotyping kit (Bio-Rad

Lab-oratories).

Competition ELISA. Competitive inhibition assays were

performed to determine epitope specificity by adapting a

method used by Collins et al. (6). MAbs purified from GammaBind G columns were diluted in BLOTTO buffer,

and 50-,lI aliquots were incubated for 30 min at 37°C in modified flat-bottomed 96-well ELISA plates coated

over-night with 100 ng of purified OvLV (85/34) per well and blocked with BLOTTO buffer. Without removing the

com-peting antibody, 50

[lI

ofbiotinylated MAbwasaddedtothe wells and incubated for an additional 1 h. The plates were

rinsed five times with PBS-Tween buffer, and 50 ,ul of streptavidin-conjugated horseradish peroxidase diluted 1:2,000 in BLOTTO buffer was added for 30 min at 37°C. Plates were then rinsed five times with PBS-Tween buffer. The development of color and reading of the reactionswere

as described above for the iELISA. A reduction ofat least 45% in the binding of the biotinylated antibody in the presence of unlabeled antibody indicated that there was

competition for the sameepitope.

Detection of OvLV field isolates by using MAbs. Recent field isolates of OvLV were obtained froma flock ofrange

sheep by cocultivation of various tissues with ovinecornea

cells (apermissive cell line established by ourlaboratory).

Eleven of the recent field isolates and seven plaque cloned isolates (6 OvLV and CAEV)were passagedtwice in ovine

corneacells.Theinfected cultures werefreeze-thawed three

timesand usedtoinfect 75-cm2 culture flasks of ovinecornea

cells (80% confluent). Infected supernatants were collected

ondays 6, 10, and 14. Titers of each virus-infected superna-tant were determined on ovine cornea cells by using a

syncytium induction-dilution assay (29). The remaining

su-pernatant was concentrated 50-fold by ultracentrifugation

andstored frozenat-20°C. The virus-infectedsupernatants

were diluted 1:10 in 10 mM sodium borate buffer (pH 9.0) and coated in96-well plates overnight at37°C. An iELISA

wasperformed as described above, withsupernatants from each of the 21 MAbs reacted against each virus isolate. Two-way analysis of variance was performed on the data,

andtheTukeytestforhighly significant differenceswasused

to compare the mean ODs achieved with each MAb by

iELISA on each virus (46). The Spearman correlationtest

compared the ELISA OD and virus titerdatatodetermine whether a direct or indirect correlation existed between thesetwoparameters.

TABLE 1. Isotypesand antigen specificity of anti-OvLV MAbs

MAb Isotype _specificityAntigen

1B, 1D, 2F,2H, 3D, 3F, 4F,a Gl CAp27 5A,a 5C,a 5G, 7D, 8F, 9C,

9F,10C, 10G,a 12C

5F,9D Gl MAp17

2B G2b MAp17

6C M MAp17

aDerived against the 84/28 isolate; the remaining MAbs were derived

against the 85/34 isolate. All MAbs hadkappalight chains.

RESULTS

Production and selection of hybridomas. Fusion

experi-ments that produced the majority of our OvLV-specific

hybridomas involvedimmunizing mice with three sequential

doses of60

jig

of virus. The fusion efficiencyranged from 89 to 100% in the six fusions that were conducted. A total of 3,632hybridomaswas screenedby iELISA, and 440 hybrid-omas were further characterized by immunoblot analysis. Strong immunoblot reactivity against viral antigens was

observed with 144hybridomas thatwere dilution cloned at least twice following iELISA and immunoblot screening. Numerous hybridomas recognized antigens with Mrs be-tween90,000 to 150,000, but when these were screened by

immunoblotting against concentrated uninfected GSM cell supernatants,they allshowedreactivity with uninfected cell components. Fourof the 21clones werederived frommice immunized with thephenotypicallyslowlylytic isolate84/28

of OvLV.The 4clones from84/28-immunized mice and13of the 85/34-derived clones recognized the CA p27 protein of both virus strains. Four85/34-derivedclones recognized the structural protein MAp17 (Table 1). Thepredominant iso-type ofthe MAbs wasIgGl, withonly one IgG2b and one IgM isotype identified. All the MAbs demonstrated kappa light chains (Table 1).

Supernatants from 20 of these clones were then purified

over a GammaBind G affinity column (excluding the IgM

class MAb 6C). The average recovery ofpurified

immuno-globulin from200 mlofsupernatant was 1.7 mg.Titers ofthe

purified MAbs were determined against 100 ng ofpurified

virus per well, beginning with a concentration of 50 ,ug of MAb per ml. Themajority oftheMAbshadatiter of at least

400, withtheexception ofMAb 2F.ThisMAb hadatiterof 1,600,indicatingahigh degree of affinity fortheviralepitope

itrecognized.

Delineation of epitope groups by using competition ELISA. Three MAbs obtained from differentfusions were

biotiny-lated and used to identify epitope groups by competition

ELISA. Several patternsofcompetitionwere seenin these

experiments (Fig. 1). Percentagecompetitionwascalculated asfollows: (buffer- test/buffer) x 100, where the buffer = averagebuffer control- noantigenbuffer control andtest= test with antigen - test without antigen. The percentage

competitionwasdeterminedwith the

50-Rg/ml

valuesfor the

competing MAb. At least three distinct, partially overlap-ping epitopes on CA p27 were identified (Table 2). The MAbsrecognizingthe MA

p17

proteindidnotcompetewith any of the MAbs recognizing epitopes on the CA p27

protein.

Ammonium sulfate-purified ascitic fluids from several cloneswerereactedonimmunoblotsagainstfour OvLV field isolates and theclosely related virus, CAEV, to determine whether the viruses hadantigenic determinants in common

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1.00

0.80

0

C3 0.60 \

0.40

020

-0.00

.39 .78 1.56 3.12 6.25 12.5 25 50 100

COMPETING MAb (ug/ml protein)

FIG. 1. A representative competition ELISA experiment. Serial twofolddilutionsof unlabeled MAb (affinity purified andadjustedto

a standard protein content of 50 ,ug/ml) were preincubated with

purified OvLV (85/34) precoated ELISA plates. Without rinsing, biotinylated MAb 1B (1:100)was added, followedby

streptavidin-horseradish peroxidase and detected with thechromagen 3,3',5,5'-tetramethylbenzidine. Neither MAb 3DnorMAb SFdiminished the

signal; thus, they didnotcompeteforthesameepitopeasMAb1B.

MAb9Ccompeted with biotinylated MAb 1B toagreaterdegree thandid thehomologous unlabeled MAb 1B, indicating that MAb 9C hadahigheraffinity for the epitope recognized by thesetwoMAbs.

(Fig. 2). Equivalent concentrations of each purified virusas

determined by SDS-PAGE were usedon the immunoblots. The CAp27-specific MAbs recognized all five viruses, but the MA p17-specific MAb failed to recognize CAEV. Dif-ferent levels ofavidityfor CAEV CA p28 were seenin the

immunoblotstudy (Fig. 2). MAbs 3F and 5A demonstrateda

much weakerreaction with CAEV CAp28thandid MAb2F, while all threereacted withthe OvLV isolates with similar avidity. Thegag-encoded precursorp55was also identified byMAb 2F inthisblot;however, allfour MAbsrecognized the sameprecursorat lowerdilutions (datanot shown).

iELISA of field isolates of OvLV. We evaluated whether recentfield isolates and plaque-cloned isolates shared

con-servedepitopesontheir structuralproteinsbyperformingan

iELISA on several isolates of OvLV with each of the 21 MAbs. Infectious titers were obtained from the syncytium end-pointdilutionassaysofsupernatantfluidsfrom cultured

TABLE 2. Epitopegroupsdistinguished by anti-OvLV MAb competitionELISA

AtgnBiotinylated

Epitope MAgseiic MAbb

group CompetingMAb

Antificd

itya lB 3F 5A

I 5G CAp27 P -

-2H CAp27 P P

-1B, 1D, 2F, 7D, 9C, 9F, lOF, 12C CAp27 + +

-II 3D, 3F, 5C,1OGC CAp27 - +

-8F CAp27 - + P

III SAC CAp27 - P +

4FC CAp27 P P +

IV 2B,5F,6C,9D MAp17 - -

-aIsolate 85/34 propagated in GSM cells was purified and used at a concentration of100ngofproteinperwell in allcompetition experiments.

bPercent competition represents thepercentagethecompeting MAb

re-ducedthesignalfromabuffer controlsubstitutedfor thecompetingMAb.+, complete competition (65to 100%); P, partial competition (45to<65%);-,no

competition (<45%).

cDerivedagainstthe84/28 isolate.

TABLE 3. Meanopticaldensity, infectious titer, and homogeneousgroups of OvLVfield isolates

Titer Mean Homogeneous groupings Virus

(TCID_jml)a

ODb

byTukey'stest

(P<0.05)

H-24 180,000 0.726 A

Visna 10 0.428 B

85/14 6 0.331 C

85/34 56 0.322 C

85/62 100 0.308 D

102 10 0.236 E

148 10 0.229 E

126 6 0.228 E

116 18 0.224 F

100 10 0.218 F

125 18 0.217 F

101 18 0.212 F

84/28 6 0.205 F

CAEV 18 0.204 F

128 56 0.199 F

110 6 0.186 F

103 10 0.176 F

113 180 0.172 F

aTCID50,50otissue cultureinfective dose.

bObtained fromtriplicatedeterminationsof eachvirusagainstall 21MAbs.

cells infected with 11 recent field isolates and 6

plaque-cloned isolates of OvLV or CAEV(Table 3). The infectious titers of all viruses were low (6 to 180 50o tissue culture infective doses per ml), except that of H-24 (180,000 50% tissue culture infective doses per ml), ahigh-passage OvLV isolate which has lost its in vivo pathogenicity (29). Concen-tratedsupernatants from ovine cornea cellsinfectedwith the different virus isolates were tested against each of the 21 MAbs(Table 2). The mean ELISA OD values obtained from

triplicate determinations for all 21 MAbs weredifferentfor the viruses (two-way analysis of variance, P < 0.001). A posterioricomparison of the 18 viruses identified six homo-geneous reaction patterns, Athrough F (Tukey's test, P< 0.05; Table 3; Fig. 3). The OvLV isolates from range sheep (viruses 100 to 148, Fig. 3) demonstrated a homogeneous pattern of reactivity to the four MAb groups which was confirmedby statistical analysis. All but three of the isolates from the rangesheep belonged to group F, while the remain-ing three (102, 126, and 148) belonged to a closely related group, E(Table 3). The plaque-cloned isolates were classi-fied moreheterogeneously.The H-24 andvisnaisolates were separated intoreactionpatterns A andB,respectively(Table 3; Fig. 3). Isolates 85/14 and 85/34 were classifiedaspattern

B,with85/62representingaclosely related patternD(Table

3;Fig. 3). The CAEV and 84/28isolatessegregatedwith the

majority of the recent isolates from range sheep. The clas-sification of viral groups by ELISA OD was found to be

independent of virus infectioustiterbyusingthe Spearman correlation test.

Analysisof the combined meansforthefourMAbepitope

groups for each virus revealed significant differences (two-way analysisof variance,P < 0.001). Rankingthe mutually

exclusive MAbepitopegroups(Tukey'stest, P <0.05)from

highmeanELISA OD to low mean ELISA OD resulted in the orderIV, III, I,and II. This relative order for theepitope groupswas seen for all 18 virusesexceptH-24,102,and 103 (Fig. 3).

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MAb 2F

M

1

2

3

4

5 72>

46>

29>

a

mine_

18>

.a.. ,.-.

MAb 5A

M

1

2

3

4

5 72>

46>

29>

18>

MAb

3 F

M

1

2

3

4

5 29>

72>

46>

ES0

e

29>

18>

FIG. 2. Fourfieldisolatesof OvLV andone strain of CAEVwerereactedonimmunoblots witharepresentativeMAb from eachepitope

group. M, molecular mass markers in kilodaltons. Lanes: 1, 85/14 OvLV; 2, 84/28 OvLV; 3, 85/34 OvLV; 4, 85/62 OvLV; 5, CAEV.

Ammoniumsulfate-purified ascites ofMAbs 2F, 3F,and 5Adiluted 1:10,000 representedthree differentspecificities for CAp27 epitopes.

MAb 5Fdiluted1:200recognizeda MAp17-specific epitope. MAb 5F recognized onlythe OvLVisolates, unlike the three CAp27-specific MAbs, whichrecognizedall five viruses.

72>

MAb

S

F

M

1

2

3

4

5 46>

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GROL I _ GROtP 11 GROLP III GROLP IV

1.20 A

1.00

I

0.80 I

-~~~~~~~~~~~~~~~~~~~~

0.60

0.40 - T

020

C:0.00

-I

II

o H-24

LO

0.50

Q

0

0.40-0.30

0.20-0.10

0.00

B

l

C

I

D

B

I

VISNlA 85/ 14 85/34 85/62

E

I

102 148 126

VIRUS IDENTIFICATION

FIG. 3. Detection ofOvLV CA p27 (MAb epitope groupsI, II,andIII)and MAp17(MAbepitope group IV)insupernatantsfromovine corneacells infected with different isolates of OvLV. Twenty-one anti-OvLV MAbs were reacted against 11recent OvLV field isolates obtained fromaflock of range sheep andsevenplaque-cloned OvLV isolates by an iELISA. The mean OD value and standarddeviation of each MAbepitopegroup(Table 3)wereobtained for each virus. Themean+ standard deviationof the negative control (MAbs reactedagainst buffer)was0.056 +0.015. Tukey'stestordered the reactionpatternfor the fourMAbepitope groupsasIV, III, I,andII,witha P<0.05 againstthe 18different viruses. Six reactionpatternsfor theviruses (AtoF)wereidentified in which the meanswere notsignificantly different within each group by using Tukey'scomparisonofELISA OD andvirus (P<0.05).Allbut 3of the 11 recent field isolates were in group F, with the remaining 3 isolates (102, 126, and 148) placed into the next closely related group, E.

DISCUSSION

Wehave characterized21MAbsthatrecognized either the major capsid antigen (CA p27)orthestructuralprotein(MA

p17) of OvLV. These MAbs were initially selected for reactivity to OvLV antigens in an iELISA. Those hybrid-omas that also reacted positively in immunoblots were cloned at least twiceby limiting dilution. Although several

hybridomas produced MAbs that recognized antigens with

Mrs between 90,000 to 150,000, none ofthese antibodies

were specific for viral antigens. Treatment of the OvLV

inoculum with TritonX-100 mayhavesubstantiallyreduced the content of viral glycoproteins. Other researchers have also had difficulty generating MAbs specific for lentivirus glycoproteins (Mr of90,000 to 150,000) when they

immu-nized mice with complete virus preparations (2, 24, 48).

Glycoprotein-specificMAbswereobtained frommice

immu-nizedwithenvelope glycoproteins from visna virus purified by affinity chromatography (48). Antigenic variation among env-encodedglycoproteins of lentiviruses renders themless suitableastargetsfordiagnostictestsusinganti-glycoprotein MAbs (13, 37, 48). We chose to concentrate on CA p27-specific MAbs because thispolypeptide ishighly conserved among ruminant lentiviruses (11, 44, 51). Our MAbs also recognize thegag-encodedprecursorpolypeptide,p55 (Fig. 2,MAb2F) described by several researchers forlentiviruses of ruminant animals (2, 5, 24, 50).

The epitopes recognized by the 21 MAbs were

distin-guished by competition ELISA with three biotinylated MAbs. Analysis of our competition ELISA experiments identified four distinctepitopes. Some of the MAbspartially competed for two epitopes of the CA p27 antigen. Such MAbs may have recognized a site that overlapped two

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epitopes. Alternatively, attachment to one site by the com-peting MAb may have altered thetertiary conformation of a nearby epitope. The epitopes recognized by the 21 MAbs were consistently found in all 11 recent field isolates and laboratory-passaged plaque-cloned isolates of OvLV (Fig. 3). Moreover, aconsistentpatternof recognition by the four

epitope groups ofMAbsevolved when theywere tested on severalisolates ofOvLV. When the results were arranged

from highest ELISA OD to lowest ELISA OD, the sequence was MA p17-specific group IV followed by the CA

p27-specificgroupsIII, I, and II. Ourobservationsareconsistent

with the CA p27 and MA p17 proteins being conserved in OvLV and agree with those ofGogolewski etal. (17),who foundthat someantigenic determinants arecommonamong proteins of sheep and goat lentiviruses. The group IV MAb SF may have recognized a gag-encoded precursorprotein of

CAEVbyiELISA that was below adetectableconcentration

in an immunoblot.

Statistically different iELISA reaction patterns among the viruses may be attributed to factors such as the concentra-tion of viral antigen, diversity in epitopes expressed by a

particular isolate,strength ofantibodyaffinities,and

concen-tratioh differences in antibodies used in the iELISA. The

statistically categorized patterns of viral reactivity by

iELISA didnotcorrelatewiththeinfectious titersachieved by the various isolates. The quantity of intracellular and

extracellularCA p27 andMA p17wouldnotnecessarily be

expected to directly correlate with the infectious titer of

virus. There may have beendiversity among the isolates in theirability to regulate theproductionof proteins recognized

byourMAbs.Theconsistentpatternof relativereactivity by

the MAb epitopegroups argues againstdiversityin theviral antigens and in antibody affinity differences among the

isolates. Because the iELISAs were not normalized for epitope concentrationorMAbconcentration,wecannotrule out either ofthese as causes for the variations seen in the

iELISAs.

Theflock ofrangesheep from whichweobtainedourfield isolates may have been infected froma common source of OvLV,because theseisolateswereidentifiedasmembersof

two closely related reaction patterns of viruses by using statisticalanalysis. Queratetal. (44) classifiedOvLVonthe

basis ofprotein content, nucleic acid content, andlytic or

persistentnature in cell culture. Thehighlylyticviruses are

closelyrelatedtovisna virus, whereas thepersistentviruses are closely related to CAEV. In our study, the group F

(Table 3) viruses (CAEV, 84/28, and a majority

of

recent

isolates from range sheep)demonstrated aphenotype char-acteristicof theslowlylyticviruses describedby Queratand

coworkers (44). The viruses in groups B, C, and D

(visna

virus,85/34, 85/14,and85/62 [Table 3])weremore

typical

of thehighly lytic viruses.

The three CA p27 epitopes and one MA p17 epitope

defined in our study correspond with those found with

murine MAbs directed against human immunodeficiency

virus type 1 (HIV-1). Four different linear epitopes were identifiedby murine MAbs to CA p27 of HIV-1, with three of these MAbs recognizing conserved epitopes shared by HIV-1 and HIV-2 (25). Niedrig et al. (39) identified con-servedepitopes on HIV-1 by using murine MAbs directed

againstthe structural proteins MAp17 and CA p24. Three

different immunogenicregions were defined, one onMAp17

andtwoonCA p24.

The specificity of MAbs provides a distinct advantage over polyvalent serums that often have high background levels and false positives due to reactivity with other

anti-gens in several

diagnostic

assays. OurMAbs have

success-fully detected virus in whole

cells,

cell

lysates,

and cell culture supernatants in iELISA and immunoblotting.

Al-though our MAbs cannot

directly distinguish

qualitative

differences among isolates of OvLV, they can be used to

study

isolate-specific

differencesin the

production

of intra-cellular and extracellular levels of viral

antigen

and the

kinetics of virus

replication.

The MAbs described here

should be usefulforfurther

investigating

the

significance

of

distinct viral

phenotypes

in the

pathogenesis

of

OvLV-induced disease (26). These MAbs may also aid in

under-standing

the failure of

experimental

lentivirus vaccines to

preventdisease (9, 35, 41).

ACKNOWLEDGMENTS

This study was supported in part byNIH grant iROl A125770,

USDAspecialgrantCSRS 89-34116-4759,and the Colorado State UniversityExperimentStation1-56271.

WethankWalter J.Bruyninckxfor assistance withthestatistical analyses.

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