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Antigenic variation of neutralization-sensitive epitopes of caprine arthritis-encephalitis lentivirus during persistent arthritis.

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Copyright© 1988, American Society for Microbiology

Antigenic Variation of

Neutralization-Sensitive

Epitopes of Caprine

Arthritis-Encephalitis Lentivirus during Persistent Arthritis

TRAVIS C. McGUIRE,* LINDA K. NORTON, KATHERINE I. O'ROURKE,AND WILLIAM P. CHEEVERS

DepartmentofVeterinaryMicrobiology and Pathology, Washington State University,Pullman, Washington 99164-7040

Received 14March 1988/Accepted2 June 1988

Caprine arthritis-encephalitis virus (CAEV), a naturally occurring lentivirus of goats, causes disease

characterized by virus persistence andrecurrentarthritis. Thesestudies demonstrate in vitro neutralization of CAEV infectivity byserumfrom goats infected with CAEV. Serum neutralizing activitywas notdetectable until 10to36 monthspostinfection, and titerswererelatively low(.1:8). Serum neutralizationwascaused by

antibody andwasvirusspecific. Antigenic variants of CAEVwereisolatedfrom cell-free joint fluid ofarthritic goats 9 to 18 months postinfection. The delayed appearance of neutralizing antibody and the subsequent

development of antigenic variantsmaypromoteCAEV persistence in vivo and provideastimulusforrecurrent

arthritis.

Animportant characteristic of lentiviruses is persistence ininfected hosts resulting inprogressiveorrecurrentdisease (24, 31, 32; W. P. Cheevers and T. C. McGuire, Adv. Virus Res., in press). In hosts withcompetent immune systems,

one mechanism of persistence is restricted lentivirus gene

expression in infected cells (3, 11, 27). Although these infected cells contain the virusgenome, theypresumably do notmake either enough viralproteinsto be recognized and destroyed by the immune system or enough virusto infect other cells. However, such latently infected cells do not

provide an explanation for recurrence of clinical disease

noted with infections by equine infectious anemia virus (EIAV) (19) and caprinearthritis-encephalitis virus (CAEV) (6, 17). The emergence of virus mutants from latently infectedcells whichare notrecognized by existing neutral-izing immune responses could exacerbate disease and

en-hance persistence by infection of other cells. Theoccurrence

of antigenic variants during infection is documented for EIAV(19, 22) and visna virus(25) andmaybeapropertyof all lentiviruses (22, 33), including human immunodeficiency virus(HIV), which exhibitsgenetic variationovertime(12). Initial demonstration ofantigenic variation of neutraliza-tion-sensitiveepitopes ofalentivirusduring persistent

infec-tionrequires that several criteria bemet(19, 25). (i) Infection is initiated withahomogeneous virus population, whichmay

be obtained by a series ofterminal dilutions. (ii) The host

makesa serumneutralizingantibody responsetothe infect-ing virus which is detected in vitro. (iii) Subsequently, virus which isnotneutralizedby theserumantibody that

neutral-izes the infecting virus is isolated from the host. Antigenic variation ofCAEV, alentivirusgenetically relatedtoEIAV (4), visna virus (29), andHIV(4), has been difficulttostudy. Previously, neutralizing antibodywasnotdetectedinseraof

CAEV-infected goats (18, 28), preventing evaluation of antigenic variation. Hyperimmunization of two CAEV-in-fected goats with CAEV andMycobacterium tuberculosis induced serum neutralizing antibody, providing the impetus

for further studies (28). In this report, which confirms the results ofa recent report(8), we demonstrate that

neutral-izing antibodyappearsin the serumofgoatsexperimentally infected withCAEV, although theappearanceisdelayed and

doesnotoccurinallgoats.Further,antigenic variants of the

*Correspondingauthor.

infecting viruswereisolated from the cell-free synovial fluid

offourgoatswith activearthritis 9to18 months postinfec-tion (p.i.). This later observation, taken together with the demonstration ofan antigenic variant of a CAEV isolate

from Australia (8), demonstrates that antigenic variation of neutralization-sensitive epitopes of CAEV occurs during

persistent infection. It is hypothesized that the delayed

appearance of neutralizing antibody and the subsequent

development ofantigenic variants provide stimulus for re-currentarthritis and promoteCAEV persistence in vivo.

MATERIALSANDMETHODS

Virus sources. In vitro propagation of CAEV was in

primary goat synovial membrane cells. Appearance of syncytial cellswasusedastheindicator of virus infection(6, 17). The CAEV 63 isolate described inprevious studies (18)

was used eitheras anuncloned stock (CAEV 63U) orafter

cloning (CAEV 63C) by three serial terminal dilutions. The origins of the other CAEV isolates (designated Co, 89, and 52) and thetwosheep lentiviruses, visnavirusand

progres-sive pneumonia virus, have been detailed previously (21). Animals and virus infection. All goats were purebred

Saanens. The CAEV-infectedgoats weremaintained

sepa-rately from the CAEV-freegoats. Tengoats (group 1)were

obtainedbycesarean section and infectedat10daysofage

with 1.0 ml of 106.2 50% tissue culture infective doses

(TCID50s) of CAEV 63C (0.5 ml intravenously and 0.5 ml intra-articularly) (17). Obtaininggoatkidsby cesarean

sec-tion, maintaining those kids isolated from the mother, and feedingthem CAEV-free milk prevent most natural

trans-missionfrom mothers withCAEV (6). Fifteengoats(group 2) were from CAEV-free mothers and were infected by

feeding10 mlperdayofcolostrumcontaining1060TCID50s

ofCAEV 63C at1through5daysofage. Fivegoats(group 3) were obtained by cesarean section and infected in the samewayasgroup 1 exceptthat CAEV63U wasused (1). Each CAEV-infected goat group had three to five control goats that were sham-inoculated with noninfected tissue

culture medium and maintained separately throughout the experiments. Infected and control goat groups were

man-aged similarly withregardtoration, vaccinations, and

par-asitetreatments.

For virus isolation, synovialfluidsampleswere collected

from the inoculated radiocarpal joints of group 1 goats. 3488

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Synovial fluid was diluted 1:10 with Dulbecco minimal

essential medium and centrifuged to pellet cells, and the

upper two-thirds of the supernatant was removed for virus

isolation (17). Virus isolates from group 1goats 18, 23, 24, and 51 made at 18, 12, 18, and 9 months p.i., respectively, were used for antigenic variation studies. These virus

iso-latesweremade when thecarpus was significantly enlarged,

indicating active arthritisaspreviously described (17). Virus

isolateswere expanded in synovial membrane cell cultures,

and infectivity titers were determined and neutralization

assays were done by the virus reduction method described below.

Virus neutralization assays. Two methods were used to

determine in vitroserumneutralization of CAEV. The initial

detection of serum neutralizing antibodies and all studies

exceptthedetermination of neutralizingantibody titerswere

done by a virus reduction method (9). To determine virus

reduction, 0.1 ml ofgoatserumheatedat56°C for 30minwas

added to 0.1 ml of Dulbecco minimal essential medium containing 102.8to

104-4

TCID50s

ofCAEV 63C. The mixture

was incubated for 1 h at 37°C and then for 18 h at 4°C. Threefold dilutions of the mixturewere assayed for virus in

4wellsperdilution ofa96-well plate. The platewasseeded

with 6 x

103

goatsynovial membrane cells in 5% fetal bovine

serum in Dulbecco minimal essential medium. Plates were

incubated 13 to14daysat37°C, Giemsa stained, and scored for syncytial cells. The

TCID50

was calculated by the

method of Reedand Muench (30). To calculate the percent

neutralization (reduction), the virus titer determined after incubation withatest serumwas subtracted from themean

virus titer (determined after incubation with control serum

samples) and the result wasdividedby the mean virus titer

(determined after incubation with control serum samples)

andmultiplied by 100.

To measure serum neutralization titers, a constant virus

andserumdilutionmethodwasused(18). Serum titerswere

determined twiceby using fourfoldserumdilutions with four

wellsperdilution. Input virus of

101-4

to101.7wasincubated

with serum dilutions for 1 h at 37°C. The volumes of

reactantsand virusdetection methodswere asdescribed for

thevirus reduction method.

Todemonstrate that theserumneutralizing activitywasin

theimmunoglobulin fraction, immunoglobulin Gl (IgGl) and IgG2 wereisolated fromtwo serumsampleswhich

neutral-ized CAEV andfromtwoserum samplesfrom age-matched

CAEV-freegoats. TheIgGl and IgG2, whichcomposemost

of thegoatserumimmunoglobulin, wereisolatedby

ammo-nium sulfateprecipitation and separatedonDEAE-cellulose

asdescribedpreviously (14). Purity of the isolated

immuno-globulin fractions was assayed by polyacrylamide gel

elec-trophoresis in sodium dodecyl sulfate, followed by Coomas-sie brilliant blue staining.

Immunofluorescence assaysfor p28 were compared with

syncytial cell formationtomeasureCAEV neutralization. A

monoclonal antibody to CAEV p28 was used as the first

antibody in indirect immunofluorescence assays to detect CAEV infection (21). The F(ab')2 fragment of rabbit anti-bodiesto mouseimmunoglobulins conjugatedtofluorescein isothiocyanate (Organon Teknika, Malvern, Pa.)wasusedas a secondantibody.

To determine whether in vitro CAEV neutralization was

caused by serum binding to noninfected cells, undiluted

serumsamples (0.05 ml)wereincubated with synovial

mem-branecells inmicrodilution wellsfor 60 minat37°C. Serum

[image:2.612.316.558.95.162.2]

was washed away, and CAEV was titrated on the washed cells asdescribed for the titer reductionassay.

TABLE 1. Neutralization of CAEV63C by serum samplesfrom CAEV-infectedgoats

Time p.i. of No.neutralizing/

serumsample no.

testeda

1 CAEV 63C 1 yr 9/10

2 CAEV 63C 2 yr 7/15

3 CAEV63U 9mo 0/5

3 CAEV 63U 5 yr 3/5

aNeutralizationbyaserum was defined asavirus titerreduction of>3

standarddeviations from the meanlog1ovirus titer determined after incuba-tion with serum samples from 3 or4age-matched,CAEV-free goats run in the sameassay.

RESULTS

Table 1 presentsresults of virus neutralization by serum

samples fromthree groupsofgoatsexperimentallyinfected with CAEV. Ofgroup 1 serum samples, taken 1 year after

infection, and group 2 serum samples, taken 2 years after

infection,90 and46%, respectively, neutralized CAEV 63C

used for initialinfection (Table 1). In apreviousreport(18),

we failed to demonstrate neutralizing activity in serum

samples fromgroup 3 goats collected 2 to 39 weeksp.i.with

CAEV63U. Here (Table 1), serum samples from group 3 goats, taken 36 weeks p.i. failed toneutralize CAEV 63C,

confirming our initial observation. However, three of five

samples taken 5 years after infection of group 3 goats

neutralizedCAEV63C (Table 1).

Serumneutralizationof CAEV blocked bothsyncytialcell

formation and the appearance of viral p28, determined by

immunofluorescence assay with monoclonal antibody to p28, indicating that neutralization blocked virus protein production (Table 2). IgGl and IgG2 isolated from two

neutralizing serum samples contained neutralizing activity, demonstrating that serum neutralization was caused by antibody (Table 2). Pretreatment of indicator cells with

neutralizingserumbefore virus titration failedtoaffectvirus

titers, rulingoutnonspecific blocking of CAEV infection by thebindingofantibody to the cell surface (Table 2).

Measurement ofneutralizing antibody in six goats from group 2 at intervals after infection illustrated its delayed appearance and relatively low titer (Table 3). Neutralizing antibodies were absent in all six goat serum samples at 3

months, in50% ofthe samplesat10months, andin33% of

thesamplesat 15months p.i. All six goats hadneutralizing antibodiesat36months p.i., with titers ranging from2 to8.

Serumsamples fromage-matched noninfectedgoats had no

neutralizingactivity. Therelatively low neutralization titers

showninTable3,determinedby reaction with

101.4

and

101'7

TCID50s by the constant virus and serum dilution method,

are similar to titers observed in some serumneutralization assays ofHIV (5, 35). However, when

104-3

TCID50s of

CAEVwereincubatedwithundilutedserum as

described

for thevirusreductionmethod(9), four low-titerserumsamples

(.1:2)

from group 1 goats reduced the infectivity by as much as 99.9% (Table 4). Therefore, subsequent serum

neutrali-zationexperimentsweredonebythe virus reduction method with undiluted serum.

Theungulate lentiviruses are genetically related(29) and have antigenic cross-reactivity of the structural and

enve-lope proteins (10). To determine whether epitopes recog-nizedby neutralizing antibody areconserved aswell, three

distinct CAEV isolates and two sheep lentivirus isolates

were assayed by using two serum samples from group 2 goats that neutralized CAEV 63C. One serum, G26,clearly

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TABLE 2. Characterizationof in vitroneutralization of CAEV 63C bytwo serum samplesfromgroup2goats infectedwith CAEV 63C for 1year

Expt. no. Treatment Virus assay Serum source Virusneutralization(%)a

1 None Syncytialcellformation G14 99.9

G26 99.9

2 None Immunofluorescence(antibodytoCAEV p28) G14 99.1

G26 99.8

3 Purificationof

IgG,

andIgG2 Syncytial cell formation G14 97.0

G26 87.7

4 Incubation' Syncytialcellformation G14 0.0

G26 36.0

a The virus reductionmethod was used, and the mean virus titer from tests with serum samples from two age-matched, CAEV-free goats was used as100%

forcalculation of percent virus neutralization by an infected goat serum for experiments 1 and 2. For experiment 3, IgGl and IgG2 isolated from serum samples from two age-matched, CAEV-free goats were used as100%. The isolatedIgG1 and IgG2 from each CAEV-free and CAEV-infected goat serum was adjusted to

15mg/ml.

bIndicator cells were incubated with either test or control serum before addition of virus dilutions for titration.

failed to neutralizethree CAEV isolates and the two sheep

lentivirus isolates (Table 5), demonstrating that these

iso-latesdo not shareneutralizing epitopes recognized by

anti-bodiesinG26serum. The other serum, G14, neutralizedthe

infecting viruses CAEV 63C and CAEV 89 but failed to neutralize theother isolates tested.

Variation in epitopes recognized by neutralizingantibody was shown in subsequent joint isolates from four goats (Table 6). These antigenic variants were isolated from cell-free synovial fluidaspirated fromarthriticcarpaljoints 9to 18monthsp.i.In each case,serumcollectedatthesame time as orbefore thejoint isolate neutralized the infecting virus

(CAEV 63C) but failed to neutralize the subsequentjoint isolate. Significant neutralizing antibody against two of the

joint

isolates (G24 and G51) appeared 3 to 12months later (Table 6).In ourprevious work(2), theCAEV isolatesfrom

G18and G24 (Table 6) were neutralized by a

complement-dependent reactionwithrabbitantiseratoCAEV 63C. These

rabbitantisera (2) apparently recognize one or more com-monneutralization-sensitive epitopesshared by CAEV 63C

and the isolates from G18 and G24. In contrast, serum

samples from goats infected with CAEV 63C recognize neutralization-sensitive epitopes on CAEV 63C which are notpresent on G18 or G24isolates.

DISCUSSION

Thisstudy hasdefined several characteristics ofthe

neu-tralizing-antibody response in goats experimentallyinfected with CAEV. First, infected goats made neutralizing

anti-TABLE 3. Serumneutralizationtitersof group 1goatsagainst infectingvirus(CAEV 63C)

Titeratmopi.a:

Goat

10 15 36

16 2 <2 8

18 2 2 2

24 <2 <2 2

22 <2 2 8

23 <2 2 2

51 2 8 2

aThe neutralization titeris the reciprocal ofthehighest serum dilution preventing infectionin50% ofthewells.Theinputvirus was10i7TCID50s for goats 16, 18, and 24and 101-4TCID50s forgoats 22, 23, and 51. For each

interval, four control serum samples fromage-matched, CAEV-free goats

wereevaluatedconcurrently with both input virusamounts; neutralizing titers were <2for all control serumsamples, as well as for all test serum samples at

3monthsp.i.

body,althoughthere was individualvariationin the response and not all goats responded even 5 years after infection. Second,theinitial appearanceof neutralizing antibodywas

delayed for months or years, while an antibody to viral

proteinsappears within weeks (1). The early appearance of antibodiesto CAEV proteins (1) and the lackof functional immunosuppression caused by CAEV infection (7) do not support an immunosuppressive effect of the virus as an

explanation for the delay in antibody appearance. Also,

peripheral blood mononuclear cell proliferation reactionsto

CAEV antigen are detectable by 14 days p.i. and increase thereafter (1). Third, neutralizing antibody to CAEV 63C

failed to neutralizetwo ovinelentivirus isolates and

gener-allyfailedtoneutralize CAEVisolates from othergoats. The latter result is similar to previous observations made with hyperimmune serum (28) and infected goat serum (8).

Fourth, neutralizing titers against the infecting virus were

relativelylow and did not alwaysrise progressively during persistent infection (Table 3) (8). Even though the

neutral-izing titers were usually low, the reaction is considered specific because it was absent in serum samples from

age-matched, sham-inoculated, CAEV-free goats and because

CAEV-neutralizing serum failed to neutralize other related lentiviruses.

Fifth,

antigenic variants arose in the

synovial

fluid of arthriticgoats asearlyas9monthsp.i.In twocases,

antigenic variation wasfollowed bytheappearance of

neu-tralizing antibody to the new variant. The delayed appear-ance ofneutralizing antibody to infecting virus and to new

[image:3.612.54.553.95.189.2]

antigenic variants indicatesthattherate-limiting step in the

TABLE 4. Neutralization ofrelativelylargeamountsof CAEV 63Cby undilutedserumsamples from group1goats

infected with CAEV 63C for1 year

Serumsource Survivingvirusa Virusneutralization(%)b

G18 407 97.6

G22 871 95.0

G23 224 98.7

G51 <15 >99.9

Control sera(mean) 17,173 NAC

a Undiluted sera were incubated with virus and the surviving virus (TCID50s per0.1ml)wasdeterminedby titrationasdescribedfor the virus

reduction method.

bThe virusreductionmethod wasused, andthe mean virustiter fromtests

with serumsamplesfrom twoage-matched,CAEV-freegoatsand one fetal

bovineserumsample(controlserumsamples)wasusedas100%tocalculate percent virusneutralization.

INA,Notapplicable.

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TABLE 5. Neutralization of goat and sheep lentivirus isolates by serumsamples from group 2 goats infected with CAEV 63C

for 2 years

% Virus neutralization of lentivirusisolatesa

Serum source

CAEV Co CAEV 89 CAEV 52 PPVb Visna virus

G14 40.9

83.1c

63.0 57.9 0

G26 0 48.2 50.2 0 10.2

a The virus reduction method was used, and the mean virus titer from tests with serum samples from two age-matched, CAEV-free goats and one fetal bovine serum sample was used as100%otocalculatepercent virus neutraliza-tion for each isolate. Neutralizaneutraliza-tion of CAEV 63C was99.9%owith both G14 and G26.

bPPV,Progressive pneumonia virus.

cNeutralization was significant when the

log1o

virus titer determined after incubation with test serum was >3 standard deviations below themeanlog1o

virustiterdetermined after incubation with three controlsera.

cell-free appearance ofantigenic variants of CAEV is the

slow development of selective pressure by antibody rather than a slowly mutatingvirus.

Although antigenic variation occurs in other lentivirus

diseases, there are differences in the appearance of the variants during the disease course. Antigenic variants of

EIAV aregeneratedasrapidlyas15days afterinfection, and

at least six variants can appear during the first year after infection (19). Episodes of clinical disease are associated with the appearance ofcell-free antigenic variants in the plasma. In studies with visna virus,antigenic variants were

isolated1to 3yearsafterinfection and theirappearance was

usedtoexplain the irregularcourseof disease inthecentral

nervous system (23, 25, 26). In twolong-term studies, 25% (34) and 16% (20) of the visna virus isolates were

antigenic

variants, the remainder being similar to infecting virus. Thesefindingswereusedto argueagainsttheimportance of visna virus antigenic variants in the pathogenesis ofbrain

lesions. These observations may result from the method used to isolate the viruses for study; visna virus variants were isolated from peripheral blood cells orcerebrospinal fluid cellsbycocultivationwithsusceptible cells (20, 25, 34).

Invitrococultivationmayactivatereplication of virusthat is

restricted in vivo(13). In contrast, EIAVantigenic variants

were isolated from cell-free plasma (19), and CAEV

anti-genic

variantsreported herewereisolated fromcell-free fluid fromjoints with active arthritis. Suchcell-free virus variants have the potential to infect new pools ofcells in vivo and initiateinflammatory processes. WithregardtoHIV,genetic variation of isolates is extensive (12), but it is not clear whether HIVantigenic variantswilloccur andresembleone

ofthe animallentivirus intheirpatterns ofappearance. Thecharacteristics ofthe neutralizing-antibody response and the appearanceofantigenic variantsmay contribute to

the recurrence and

progression

ofarthritis seen in a large

number of CAEV-infected goats (6, 17). Joint lesions are

characterizedby massive accumulations oflymphocytes and macrophages inthesynovium (6). High titers of antibodyin

synovial

fluidtoviral

antigens,

especially

gp135

(15), and a

progressiveincrease in the in vitroproliferative response of

peripheral blood lymphocytestoCAEVantigens (1) demon-strate avigorous immune response toCAEVantigens. The

failure of neutralizing antibody to limit infection of

[image:4.612.314.556.102.274.2]

suscep-tiblecells duringthe first few months afterinfection ofthe goat could generate alarge poolofpersistentlyinfected cells. Even if the eventual appearance of neutralizing antibody limits thespread of infecting virus to othercells, the emer-genceofantigenicvariants could keeptheinfected-cellpool

TABLE 6. Comparison by neutralization of infecting virus CAEV63C and virus isolates from thejointsofgroup1 goats

9to 18 monthsp.i.

Serumsource % Virus neutralizationofa: (mop.i.) Jointisolateb Inoculum virus

G18(11) 0 99.6c

G18(24) 0 84.0c

G18(36) 51.1 91.8c

G23 (12) 37.2 97.9C

G23 (18) 65.4 92.8c

G23 (24) 72.4 91.5c

G24(12) 10.9 93.6c

G24(24) 84.6c 78.0

G24(36) 87.5c 95.5c

G51(9) 16.7 92.0c

G51(12) 81.7c 999c

G51(18) 99.1c 99.9c

a For each virusisolate,threeserumsamplesfrom the goat in whichthe

isolatewas made and four serum samplesfrom noninfected,age-matched

controlsfor each infected goatserum wereconcurrentlyevaluated,each in

duplicate.The meanvirus titer fromtestswith four controlserumsamples

wasusedas100%oforcalculation of percent virus neutralizationbyaninfected goatserum.

bJoint isolates fromG18,G23,G24,andG51wereobtained18, 12, 18,and

9monthsp.i.,respectively.

cNeutralizationwassignificantwhen thelog1ovirustiter determined after incubation withaninfected goatserum was>3standard deviations below the

mean log1ovirus titer determined afterincubation with four controlserum samples.

replenished. Virus which

spreads by

cell-to-cell contact

without neutralization

by antibodies,

as

suggested

for visna

virus(16), could also

replenish

theinfected-cell

pool.

How-ever, a

delayed

neutralizing

antibody

response and the

development

ofa numberof

antigenic

variants

during

per-sistent disease wouldbe sufficientto

provide

thecontinued

inflammatory

stimuli for

progressive

arthritis. An alternate

possibility

isthat

antigenic

variationisan

epiphenomenon

of lentivirus

persistence

andhas norolein

replenishing

persis-tently infected cells.The

ability

toisolate

antigenic

variants of CAEV from cell-free

synovial

fluid

provides

alentivirus

systemto

clearly

dissecttherole of viral

antigenic

variantsin the recurrence and

progression

of arthritis and in the

main-tenanceof

persistently

infected cells.

ACKNOWLEDGMENTS

We thank Janice Carlson and Alberta Brassfield for technical assistance.

This work was supported by Public Health Service grant AM 27680fromtheNational Institute ofArthritis,Musculoskeletal and Skin Diseasesand U.S.DepartmentofAgriculturegrants 84-CRSR-2-2442,85-CRSR-1-1731, 58-AHZ-2-679, and BARD US-937-85.

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on November 10, 2019 by guest

http://jvi.asm.org/

Figure

TABLE 1. Neutralization of CAEV 63C by serum samples fromCAEV-infected goats
TABLE 4.CAEV Neutralization of relatively large amounts of 63C by undiluted serum samples from group 1 goatsinfected with CAEV 63C for 1 year
TABLE 6.CAEV Comparison by neutralization of infecting virus 63C and virus isolates from the joints of group 1 goats9 to 18 months p.i.

References

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