Porcine parvovirus: virus purification and structural and antigenic properties of virion polypeptides.

0022-538X/83/020842-13$02.00/0

CopyrightC) 1983, American Society for Microbiology

Porcine Parvovirus: Virus Purification and Structural and

Antigenic Properties of Virion Polypeptides

THOMASW. MOLITOR,1 H.S.

JOO,1

ANDMARC S.COLLETT2*

DepartmentofLargeAnimal Clinical Sciences,College of Veterinary Medicine, University of Minnesota, St.

Paul, Minnesota55108,1 and Departmentof Microbiology, University of Minnesota Medical School, Minneapolis, Minnesota554552

Received18October1982/Accepted10 November 1982

Porcine parvovirus

(PPV) was extensively

purified from infected

swine fetal

homogenates by CaC12 precipitation

followed by

CsCl

density centrifugation. Two

species of particles possessing PPV-specific

hemagglutinating activity were

observed banding

at

densities of

1.39

and

1.30

g/ml, representing

full and empty

20-nm

virion particles,

respectively. Both classes of particles contained three

major polypeptides,

A, B, and C, with respective molecular weights of 83,000,

64,000,

and

60,000. The amount

of

polypeptide

A was similar in both

species

(approximately

10%); however, the

B

protein

wasmostabundant in the

1.30-g/ml

particles,

whereas the

C

protein

wasthe

major

polypeptide found

in the

1.39-g/ml

particles. Antisera generated

toeach

sodium

dodecyl

sulfate-polyacrylamide

gel-purified virion

structural

protein had reactivities

qualitatively

similar to those of

conventional antisera raised against intact PPV in avariety of standard serological assays. The antisera generated

against

the individual sodium dodecyl

sulfate-denatured

PPV

polypeptides

were

able

to react with

native, intact

PPV

virions

and

were

capable of neutralizing virus

infectivity.

Porcine parvovirus

(PPV) is a

ubiquitous

in-fectiousagent of pigs. The etiological role ofthis

virus

in

reproductive failure

in

swine

is well

established

(13, 20, 21). Most

often, disease

caused

by

PPV

is manifested

asfetal death and

mummification, although infertility,

abortion,

stillbirth, and neonatal death

may

also

be

conse-quences

of

in utero PPV

infection

(13,

21).

Humoral

immunity, either

as aconsequence

of

naturalexposure or

from

vaccination,

will

often

prevent

viremia in

pregnant sows and thus

pre-vent

transplacental

virus

infection

and

subse-quent

fetal

death

(15, 20).

PPV, like other

parvoviruses,

isa

small,

non-enveloped virus

containing single-stranded

DNA

(23).

PPVappears to bea

nondefective

or

autonomously

replicating parvovirus,

similar in

this respect to the more

extensively

studied

parvoviruses

such as the minute virus

of

mice

and the Kilham rat

virus,

and in contrast to the defective

adeno-associated

viruses

(35).

Very little is known of the molecular features of PPV. We

began

our

study of

PPV

by

develop-ing

a scheme for

obtaining

the virus in

highly

purified form

from

infected swine

fetal tissue

and then

proceeded

with studies on the virion

protein

composition.

We report here our work

on the

isolation

and

purification

of PPV from

infected fetuses

and the initial

characterization

of various

features

of

the three

major

virion

polypeptides.

Weprepared

antisera

to each

puri-fied

viral

polypeptide and

compared the reactivi-ties of these antisera in a variety of tests. We

found

that the three

virion

proteins

of PPV were

both

structurally and antigenically quite similar.

Finally,

the denatured, gel-purified virion

pro-teins

were

able

to

elicit

virus-neutralizing

anti-body.

MATERIALS ANDMETHODS

Virus propagation in infected animals. Pregnant sows, at approximately 35 to 40 days of gestation,

were purchased from a local swine producer. The

serological status of these sows was not important because no transplacental immunity has been

ob-served in swine (30). At 40 to 50 days, sows were

laparotimized, and fetuseswereinfected via amniotic fluid inoculation with PPV (0.2ml;1,024 hemaggluti-nating unitsper 50,ul;107 50%tissue cultureinfective doses)aspreviously described (25).Thevirusused for inoculation was PPV strain NADL-8, originally

ob-tainedfrom the National Animal DiseaseLaboratory,

Ames, Iowa. Ten to 15dayslater, the sows were taken to slaughter; the entire uterus of each was collected and transported backtothelaboratory.Pooled

inter-naltissues fromeachfetus(lung, kidneys, liver,heart,

spleen,andintestines)werecollected andmincedin 50 mMTris-hydrochloride-25 mM EDTAbuffer, pH 8.7

(TE buffer). After overnight freezing, minced tissue fluids fromeachfetusweretestedby hemagglutination (HA)ofguinea pig erythrocytes forpresenceof virus (seebelow). Tissues fromfetuses that werehighlyHA 842

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PPV: PURIFICATION AND ANTIGENIC PROPERTIES 843 Infectedfetuses(viscera)

20%homogenate/sonic extractin TE buffer HAassayindividual fetuses

HApositive HAnegative: discard

-12,000 x g, 30min

Supernatant Discard pellet

Adjustto 25 mMCaCl,,4°C, 30min

12,000 x g, 20 min

Discard supernatant

Resuspend in TEbuffer

Clear

Supernatant

- Discontinuous CsClgradient

120,000x g, 24 h

Fractions

K Determine fraction density

Assayfor viralantigen by HA

Poolvirus peak (1.39 g/ml),dialyze againstTEbuffer Repeatdiscontinuous CsCl gradient

Poolvirus peak(1.39g/ml),dialyze againstTEbuffer PurifiedPPV

FIG. 1. Scheme forpurificationofPPV.

positive (>1:i,024)werepooled,dilutedtoa20%(wt/ vol) suspension in additional TE buffer, and further homogenizedinaWaringblender. Thesuspensionwas

frozen and thawed twice andwassonicatedat4°Cfor three 1-min intervals beforeviruspurification.

Virus purification. The procedure for the purifica-tion ofPPVfrommummified fetuseswasadaptedfrom theprocedure of Tattersalletal. (33) used for minute virus of mice(Fig. 1).All manipulations werecarried

out at 4°C. Sonicated homogenates of HA-positive fetalviscerawerecleared of cellular debris by

centrif-ugation at 12,000 x g for 30 min. To the resultant supernatant was added CaCl2 dropwise to a final concentration of 25mM, and the viruswasallowedto precipitate for 30 min. The precipitate was collected

by centrifugation at 12,000 x gfor 20 min and was

suspended by gentle sonictreatmentin TEbuffer. Any insoluble material was removed by brief

centrifuga-tion, and the cleared supernatant was adjusted to a final CsCl density of 1.32 g/ml in TE buffer. This solution waslayeredonto anequal volume ofa CsCI solution in TE buffer having adensity of1.40 g/ml. Centrifugationwasperformedat120,000 x gfor18 h at 4°C (Beckman SW41 rotor). Gradients were frac-tionated(approximately25to30fractions),the

refrac-tive index of each fraction was determined, and the presence ofviral antigenwas assayed by HA.

Frac-tionscontaining hemagglutinating activitywerepooled

andsubjectedto asecondcycle ofdiscontinuousCsCl centrifugationsasoutlinedinthelegendtoFig.2.The

pooled HA-positive fractions from the second CsCl gradientswerethendialyzed againstTEbuffer.

Radioiodination of viralproteins. Viralproteinswere labeled with

1251,

using the chloramine T method of Hunter(8). Purified virus (50 to100 ,ug) inTEbuffer (50,il)wasdisrupted by boilingin1% sodium dodecyl

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sulfate (SDS) for3 min. After coolingto room tem-perature, 100 ,Ci of '25I (carrier free; New England

Nuclear Corp.) was added, followed by 50 ,ug of

chloramineT. After 2min atroom temperature, the

reaction mixture was applied to a Sephadex G-50 column (1 by 12 cm)equilibrated with0.01 Msodium

phosphate (pH 6.8). The excluded peak of

radioactiv-itywas collected and pooled.Thespecific activity of the radiolabeled proteins was approximately 5 x 106 cpm/,ug.

Viralpolypeptide purification. Viral structural

pro-teins, either '25l radiolabeled or unlabeled, were

boiled inSDS-containing sample buffer for2 minand

then were subjected to electrophoresis in SDS-con-taining 10% polyacrylamide gels (17). Proteins were

localized either byautoradiography orby Coomassie

brilliant blue staining ofend sections of thegels. The individual protein bands were excised, and the

pro-tein-containing gel pieces were subjected to another cycleofelectrophoresis on asecond SDS-containing

polyacrylamide gel. After protein localization in the second gel, the excised gel pieces were either used

directlyforone-dimensional partial proteolytic peptide mapping(seebelow)orusedfor theextraction of the protein contained within. Isolation ofviral proteins

frompolyacrylamide gel pieceswaseitherbyrepeated

elution in 0.05 MNH4HCO3-0.1%SDSat37°Corby isotachophoresis inSephadex G-50columns (1). The

leadingbufferwas50 mMTris-hydrochloride (pH 6.8),

andtheterminating bufferwas50 mMTris-glycine (pH

8.8). Bromophenolbluewasusedas atracking dyefor

theleading edgeofprotein.Thedye-containing

Sepha-dexwasremoved from the columns and washed with

watertorecovertheprotein.Bothproceduresresulted

inrecoveries ofproteinof65to90%. Topreparethe

individualvirionpolypeptidesforuseinanimal

immu-nizations, proteinsweretwicegel purified andeluted from the polyacrylamide gel pieces in 0.05 M

NH4HCO3-0.1% SDSasdescribed above. The eluates

werelyophilized, suspendedin1to2ml ofTEbuffer,

andextensively dialyzedover aperiodofseveraldays

againstbuffercontaining10mMpotassium phosphate (pH 6.8), 40 mM NaCl, 1 mM EDTA, 1 mM

2-mercaptoethanol, and 50%glycerol. The method of Lowryetal. (18)wasemployed todetermine protein

concentrations, using bovineserumalbumin (BSA)as astandard.

Peptidemapping.The one-dimensionalpartial prote-olysis mapping procedure of Cleveland et al. was

employedasoriginallydescribed(4). '25I-labeledPPV proteins, purified by two cycles of

SDS-polyacryl-amide gel electrophoresis, were subjectedto

electro-phoresis in SDS-containing 15% polyacrylamide gels

in the presenceof either Staphylococcus aureus V8

protease (Miles Laboratories, Inc.), elastase

(Wor-thington Diagnostics), or chymotrypsin

(Worthing-ton).

Preparation ofantisera. Normal porcine sera were

collected from adult pigs that were free of

PPV-specificantibodiesasdeterminedbyhemagglutination

inhibition(HAI;seebelow) andby

immunoprecipita-tionof radiolabeledPPV-infectedcultured celllysates (datanotshown).Adultpigseracontaining antibodies toPPV, as determined by HAI, werecollected from sows orgilts naturally exposedtoPPV.Fetalpigsera

containing antibodies to PPV were prepared by the

amnioticinjectionof 70-to85-day-oldfetuseswith 0.2 ml ofPPV NADL-8(1,024hemagglutinationunits per

50,u1). Fetuseswerecollected21 dayslaterandbled. Rabbit antisera against intact PPV and gel-purified

viral polypeptides were prepared in the following

manner. Five-pound (2,268-g) New Zealand white rabbitswerebledbytheearveinstoobtainpreimmune (normal)rabbitsera.Thesesera weredemonstratedto

lack antibodies specific forPPVantigensby HAI and

by immunoprecipitation of radiolabeled PPV-infected

cultured cell lysates; they were therefore used for

subsequentimmunizations. Rabbitswereinjected

sub-cutaneously at multiple sites along the back with either 50,ug ofintact,CsCl-purified,fullvirus particles

or50,ug ofgel-purified polypeptidedissolvedin1mlof

TEbufferthathad been emulsifiedinanequalvolume

ofcomplete Freund adjuvant. One rabbitwasused for

each antigen preparation. Rabbits receiving purified viral polypeptides were injected at 3 weeks and 7

weeksaftertheinitial immunizationwith 50jigof the

respective gel-purified protein in incomplete Freund

adjuvant. The rabbit that received intact virus was

boosted once at 3 weeks with 50 jig of virus. All rabbits were maintained in individualcages. Rabbits

injected with live virus did not show demonstrable

virusshedding, and other normalrabbitsmaintainedin the same animal room remained negative for PPV

antibodies throughout thecourseof this experiment.

Table 1 summarizes the various antisera used inthese studies.

HA and HAI. PPV antigen was detected by the

hemagglutination ofguinea pig erythrocytesas

previ-ously described (14). Antibody titers to PPV were

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measuredby anHAltest(14). Briefly, heat-inactivat-edsera werefirstabsorbed with 25% kaolin in borate-saline solution (pH 9.0)for 20 min and centrifuged.

TABLE 1. Various antiseraused in thepresentstudy

Antiserum

Abbrevia-

Methodofproduction/source

tion

Normalpigserum NPS HAI-negativeadult animal

AdultpigaPPV PaPPV Naturallyexposedfield animal

FetalpigotPPV FoxPPV Experimentalvirusinfectionbyamnioticinjectionof pregnantsow Normal rabbit serum NRS Pre-immunizationseraof rabbitsused below

Rabbit aintactPPV RaPPV Immunization withCsCl-purifiedintactvirionparticles

RabbitotApolypeptide RaA Immunizationwithgel-purifiedApolypeptide; 10-week post-immu-nizationserum

Rabbit aB polypeptide Ra B Asabove withB polypeptide Rabbit a Cpolypeptide Ra C Asabove withC polypeptide

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PPV: PURIFICATION AND ANTIGENIC PROPERTIES 845 This wasfollowed byincubation for 60 min with50%

guinea pig erythrocytesand centrifugation (500 x gfor 10min). This procedure removed natural nonspecific

hemagglutinins. Sera were then serially diluted with

phosphate-bufferedsaline (PBS) in microtiter plates. A standard amount of cell culture-propagated PPV (8

hemagglutination units per 50 .d) was added to all wells and allowed to incubate for 3 h at 22°C or

overnightat4°C.Then a0.6%suspension of guinea pig

erythrocytes was added to all wells andincubated at 22°C for 2 h. The endpoint is expressed as the recipro-calofthehighest dilution resulting in 50% inhibition of HA.

Other serological assays. An indirect

fluorescent-antibody(IFA) test,previouslydescribed byBommeli

et al. (3), was used to compare the reactivities of various antisera with infected cells in culture. Briefly. PPV-infected ST (swine testicle)cells grown on

Lab-Tek slides were treated with acetone and air dried. Various antisera, diluted 1:8 in PBS, were incubated with the monolayers at 37°C for 60 min, after which

theywerewashedandfurther incubatedwith 0.1 mlof

a 1:50 dilution of a 1-mg/ml solution of protein

A-fluorescein isothiocyanate conjugate (Pharmacia Fine

Chemicals. Inc.). Controls consisted ofknown

PPV-positive and-negative sera, uninfected ST cells, and

pseudorabies virus-infectedcells. Agar gel

immunodif-fusion (AGID)wasperformedaspreviously described (16) to testforthe presenceofprecipitating antibodies.

Protein transfer to nitrocellulose and reaction with antisera. PPVproteins wereelectrophoretically trans-ferredfromSDS-polyacrylamide gels to nitrocellulose paper(Schleicher&SchuellCo.)by the procedureof Bittner et al.(2). After transfer, gelswerestained with Coomassie blue toestimate the efficiency of transfer. Routinely, 70 to95% ofthe proteins weretransferred

tothenitrocellulosesheets. Theprocedureof

Syming-ton etal. (31) wasemployedtoimmunologicallydetect proteins. Briefly, the sheetscontainingthetransferred

proteins were placed in asolution of4% BSA-0.01% sodium azide for 18 h at 22'C. Purified immunoglob-ulinG(100 to 200

p.g;

obtained by ammoniumsulfate precipitation) from various antisera was added. and

incubationwascontinuedwithgentle shaking for16to 24 h at22°C.Sheets were then washed five or six times (5 min per wash) with BSA-sodium azide solution.

Protein A (Pharmacia), radioiodinated bythe proce-dureofHunter(8)in BSA-sodiumazide solution(107

cpm; 8x 106

cpm/[.g).

wasadded, andthe sheetswere gently shaken at 22°C for 3 to 4 h. The sheets were again washed five or six times(10 min perwash)with

BSA-sodium azide solution, air dried, andexposed to X-rayfilm.

Electron microscopy. CsCl-purified virions,

exten-sively dialyzed against TE buffer, were analyzed by

negativestaining with4% phosphotungsticacid, using standard procedures (7). Grids were examined in a Zeiss 10transmission electron microscope at a plate

magnification of40,000 withan80-kV beam. The ability of the various antisera to form lattices with intactvirion particles was tested by an immuno-electron microscopy procedure(7). Various sera

(di-luted 1:10 in TE buffer) were mixed with equal vol-umes of purified PPV in TE buffer and incubated

overnightat4°C.Themixtures were then diluted 1:100 with water and centrifuged at 30,000x g for 30 min to

pellettheimmunecomplexes.Thepellets were

resus-pended in a small volume ofTE buffer, negatively stained, and analyzed in the electron microscope as described above.

Virusinfectivity neutralization. Standardvirus infec-tivity neutralization assays were performed in

tripli-cate. Sera(1:2or 1:10dilutionsin PBS,total volume 0.3ml)were mixed andincubated with0.3 mlofvirus

(8 HA units per 50

[LI)

for 60 min at 22°C. A 200-,ul

volume of this mixture was then added to Leighton

tubes containing recently trypsinized ST cells

previ-ously washed with PBS. Controls included known positive sera. known negative sera. no serum, andno virus. Theinocula were allowed to absorb for 90 to 120

min. after which the cultureswere washed with PBS andfresh growth mediumwasadded. Fourdaysafter

inoculation,the Leightonslipswereremoved, treated with acetone, and air dried. The slips were then stained directly with fluorescent-antibody conjugate.

washed, and observed under a fluorescent

micro-scope. The fluorescent-antibody conjugate consisted of fluorescent isothiocyanate conjugated to porcine

anti-PPVantibodies producedingnotobioticpigs(21).

Virusinfection of cells wasconsidered positive when

multiple foci offluorescentlylabeled cellswere appar-entonany given Leightonslip.

RESULTS

Purificationof PPV from infected fetuses. The

purification

procedure for PPV from

infected

porcine

fetuses isoutlinedin

Fig.

1.Asfound for

minute virus of mice (33) and H-1 virus (24),

PPV is

significantly

purified

from cellular

mate-rial at

high

pH and low

ionic

strength

in the presenceof EDTA andis

greatly

enriched

for by

CaCl.

precipitation.

CaCl

precipitation

of the

virus was found to be essential for obtaining

highly

purified

preparations

and wasfar superior

to other methods

of

virus

concentration

(e.g.,

12%

polyethylene

glycol

or

30%

ammonium

sulfate).

The

CaCl-precipitated

virus was

sus-pended

in the high-pH buffer in thepresence of

25 mM EDTA, and the soluble

virus-containing

material was then subjected to discontinuous CsCldensity centrifugation (Fig. 2). Asdetected by HA, viral antigen appeared in two major, well-resolved peaksatbuoyantdensities of 1.39 and 1.30

g/ml (Fig.

2A). These two

peaks

were

routinely

seenin different fetal preparations and

usually

contained

equal

amounts of

hemaggluti-nating activity. To further enrich for these two viral antigen-containing species, we subjected

the

pooled

fractions of each

density

peak

to a

second cycle of discontinuous CsCl

centrifuga-tion.The

1.39-g/ml

density

particles

were run on

a

density

gradient

identical to the first

gradient

(Fig. 2B), and the lighter

1.30-g/ml

particles werecentrifugedin aslightlylessdensegradient (Fig.

2C).

Ineach case, a

single

HApeak of viral antigen wasobserved,

banding

atthe same den-sity as found in the first density

gradient

run.

The

HA-positive

fractions of both the 1.39- and

the

1.30-g/ml particles

were individually pooled

and

dialyzed

against

TE buffer.

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The

purity

of these two viral

antigen-contain-ing fractions was assessed by negative-contrast electron microscopy (Fig. 3A) of samples and

Coomassie brilliant

blue

staining

in

SDS-con-taining 10% polyacrylamide gels electropho-resed(Fig. 3C). Particles banding at a density of 1.39 g/ml had a diameter of approximately 20 nm. They excluded the phosphotungstic acid stain (Fig. 3A) and therefore appeared as charac-teristic "full" (DNA-containing) parvovirus viri-ons. Particles of similar size were found in the 1.30-g/ml density band together with varying amounts

of

debris (Fig. 3B). However, these particles appeared to largely take up the nega-tive stain; therefore, they most likely represent "empty" (DNA-lacking) virion capsids. These results appear to be similar to those obtained

from

other, more thoroughly studied parvovirus systems (35). Wehave not attempted to identify viral particles that may be present at

intermedi-ate

densities

or that may represent various

im-mature or partial genome-containing virions.

The polypeptide

composition

of the

material

present in the 1.39- and

1.30-g/ml

density bands

was analyzed by

SDS-polyacrylamide

gel

elec-trophoresis (Fig.

3C). Both

preparations

ap-peared

to

contain three

predominant

protein

bands with

molecular

weights

of

83,000, 64,000,

and 60,000.

The three

proteins

of

the "full"

particles appeared

to

be

highly enriched,

where-as

minor, presumably

contaminating protein

species

were present in the

"empty"

particle

preparations.

The presence

of

three

polypep-tides

of

the

observed molecular

weights

in

highly

purified virus

preparations

is very

characteristic

of

parvoviruses (5, 10, 11, 26-28, 33),

and we

therefore conclude

that these three

polypeptides

are

the structural

proteins

of

PPV. The

structur-al

proteins of parvoviruses

have

previously

been

termed A, B,

and C in

descending order of

molecular

weight

(32),

although VP1, VP2,

and

VP3

have also been

used

(19, 24).

The

relative

distribution of the three

PPV

structural

proteins should be noted.

In

the

1.39-g/ml

particles,

the

60-kilodalton

(kd) protein (C

orVP3) appears to be most

abundant,

whereas in the

1.30-g/ml particles,

the64-kd

protein

(Bor VP2) is present in the greatest amounts

(Fig.

3C).

Although this difference

in the B and C

proteins

betweenfull and empty

virion

particles

exists,

the amount of the 83-kd

protein

(A or

VP1) appearsto

remain

constant

(approximately

10%

of the

total).

This observation has been made with several

independent

PPV prepara-tions and has also been observed in other

parvo-virus

systems(5, 24).

Structural relatedness of PPV

polypeptides.

Previous studies of

parvovirus structural

pro-teins have indicated that the

A, B,

and C

poly-peptides

are very

closely

related as

determined

4

(0

z

I

E

-p

(0) z 0a

FRACTION NUMBER

FIG. 2. CsCl density centrifugation of PPV. PPV

was precipitated with CaC12 from virus-containing homogenates ofinfected swinefetuses, suspended in

TEbuffer, and prepared for discontinuous CsCl densi-ty centrifugation as described in the text. Gradient fractions were assayed for PPV-specific hemaggluti-nating activity (HA) and density (A). The two peak regions ofHAactivitywereindividually pooled. The1.39-g/mldensity peak wassubjected to discontin-uousCsCl centrifugation asecond time in agradient identical tothat used the first time (see the text) and assayedsimilarly (B).The1.30-g/mldensity peakwas

centrifuged in a second gradient of slightly lower density than the first. The pooled fractions from the first CsClgradient wereadjusted to afinalCsCl

density of1.25g/ml and layeredonto anequal volume

ofCsCl solutionhavingadensity of1.30g/ml. Centrif-ugation and subsequent gradient fraction assay (C)

wereperformedasdescribed inthe text.

by

various

peptide

mapping procedures (12, 19,

22,

34). In these systems,

it

appears that the

peptides

of the C

protein

are contained within

the

peptides

of the B

protein,

whichareinturn a

subset of those of the A

polypeptide.

To deter-mine whetherasimilar

situation existed

with the

PPV

structural

proteins,

we

performed

one-dimensional

partial proteolysis mapping

studies

onthe three PPV

polypeptides.

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[image:5.496.267.444.68.385.2]

PPV: PURIFICATION AND ANTIGENIC PROPERTIES

i,30g/ml

A.

B

E '

0 oL

C. i I

92- ,

-A 68- -_

_- C

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45- _

FIG. 3. Analysis of CsCldensity gradientfractionscontainingPPV-specifichemagglutination activity. The 1.39-and1.30-g/mlpeaks of HAactivity(Fig.2BandC,respectively)wereindividually pooledandextensively dialyzedagainst TE buffer. Portions of thedialyzedmaterialswereanalyzed bynegative

staining

inthe electron microscope(see thetext).(A)

1.30-g/ml

densitymaterial. (B)1.39-g/ml

density

material.Bar,40nm.Another portionof thedialyzedmaterials wassubjectedtoelectrophoresisinan

SDS-l0o

polyacrylamide gel.Thegel wasthenstained with Coomassie brilliant blue and destained(C).Molecularweightstandardswereincluded in

anadjacenttrack in thegel(numbersrepresentkilodaltons).

The

proteins

of

highly purified

PPV

(1.39-g/ml

particles)

were

radiolabeled in vitro with 1251 and

separated

by SDS-polyacrylamide

gel

electro-phoresis. To

ensure

the

purity of each

polypep-tide, the three proteins

were

individually

sub-jected

to a

second cycle of polyacrylamide gel

electrophoresis before

any

analyses

were

per-formed. A

portion

of each

of the

twice-gel-purified proteins

was

electrophoresed

on a

third

polyacrylamide

gel (Fig.

4a).

Using

the

partial

proteolytic

mapping

proce-dure

originally

described

by

Cleveland

et

al.

(4),

we

compared the

partial digests of the

1251_

labeled PPV A, B, and C

proteins by using three

different

proteases:

S.

aureus

V8

protease,

elas-tase, and

chymotrypsin

(Fig. 4). The V8

prote-ase

patterns

were

nearly identical for

the

three

polypeptides.

The

elastase and

chymotrypsin

patterns of

each

of the

three

proteins

werealso

very

similar, with

manypeptides from the three

proteins

migrating identically.

Itappeared,

how-ever, that

several of the

peptide bands derived

from protein

A

migrated

more

slowly than the

similar

peptides in the B protein digest.

Similar-ly,

certain peptides in the B

protein pattern

migrated

more

slowly than the

corresponding

C

protein

peptides (Fig. 4).

This suggests

that

these

fragments

are

terminal

portions of the

respective

proteins, which

are

otherwise

very

similar in

primary

structure.

Use of individual PPV structural

proteins

to generate

PPV-specific

antisera.

The data

report-ed

above

suggest

close structural similarities

among the

three virion

polypeptides A, B, and C

of PPV. To further compare

these

polypeptide,

we

felt it would be useful

toprepare

antisera

to

each individual SDS-denatured

protein. To

this

end, highly purified

PPV

preparations

were

dis-rupted and

subjected

to

preparative

SDS-poly-acrylamide gel

electrophoresis

to separate the

three viral

proteins.

After

localization

of the

protein bands,

each was excised and then was

subjected

to a

second

cycle of gel

electrophore-sis

as was

done

for

the

above-described

125I1

labeled

proteins. The

protein

bands

of

the

sec-ond

polyacrylamide

gel

were

localized

and

extracted from the

gel pieces

as

described

above.

Figure

5

shows

a

Coomassie

blue-stained

polyacrylamide gel

of

aliquots

of each of the

gel-purified

proteins

and

a

sample

of the

starting

virus preparation. After these

procedures,

we

routinely

were

unable

to

detect

contamination

of

VOL. 45,1983 847

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848 MOLITOR, JOO, AND COLLETT

(a)

r. C:

.; A

0. *

i.-(b)

II

.

0

43

*

i

1**

(c)

S

.

*9.

O..

_*

.0

9.

I;

_

(d)

sg

-U

F~rA. '¼-;;..; F

FIG. 4. Partialproteolysis mapping of

125I-labeled

PPVstructural proteins. Purified virions (1.39 g/ml) were disrupted and radiolabeled with 1251 as described in the text. The three individual polypeptides, A, B, and C, were gel purified twice (see the text), and a portion of each was re-electrophoresed in a third SDS-10% polyacrylamidegel to assess theirpurity (panel a). Portions of each polypeptide were then subjected to partial proteolytic hydrolysis during electrophoresis in SDS-15%polyacrylamide gels as described previously (4), using (b) V8 protease, 0.1 ,ug per track; (c) elastase, 1 ,g per track; and (d) chymotrypsin, 5 ,ug per track. After electrophoresis, gels were dried and exposed to X-ray film. The numbers in the margins represent approximate molecular weights (in kilodaltons).

one protein

with

the

others,

even when

1251

labeled

proteins

were

similarly

purified

(Fig. 4).

However,

we

do

feel that

contamination below

the

sensitivity of

our tests

could have existed.

Each

of these

gel-purified proteins

was

used

to

immunize

rabbits.

In

addition,

intact PPV

was

used as an

immunogen.

At3 and 7

weeks

post-immunization,

the

rabbits

receiving

the

gel-purified

proteins

were

boosted with additional

protein.

At

various times after

the

primary

im-munization, small samples of

serawere

obtained

from each

rabbit and assayed for PPV-specific

antibodies

by

an

HAI

assay. The immune

re-sponses of each rabbit are shown in

Fig.

6. All

of

the

immunogens elicited

a

PPV-specific

re-sponse;

however, it

appeared that intact virus

was

able

to generate

the strongest and

most

rapid

response. It must be

noted

here, however,

that

only

one

rabbit

was used

for

each

immuni-zation.

We

feel

that

this

precludes

any

conclu-sions

concerning

the

antigenic

strength of

the

various immunogen

preparations.

A summary

of

the

various antisera used in this

study

is

provid-ed

in Table

1.

Serological

tests

comparing

various

PPV-specif-ic antisera. A

variety of standard

serological

tests was

performed, using

conventional

anti-sera toPPV and

antisera

generated by

the

SDS-denatured,

gel-purified

viral

proteins.

The

re-sults of

thesetests are

summarized

in

Table

2.

Although

the

antisera

generated against

the

gel-purified proteins

exhibited lower HAI titers

than

antisera

produced

from intact virus

(Fig.

6;

Table

2), these antisera did

react

with viral

antigens

in

infected

cells in an IFA assay

and did

contain

precipitating

antibodies

as

determined

in

an

AGID

assay.

Ability of antisera to

recognize

denatured and native PPV

antigens.

Wewent ontocompare the

reactivities of

the

various

PPV

antisera

to

both

denatured

viral

proteins

and

native,

intact

virus

particles.

Because

antisera

were

generated

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PPV: PURIFICATION AND ANTIGENIC PROPERTIES 849

C B A ST

L..L. .-..II. i

200- 116-

92-

68-IN~

_

45-FIG. 5. Purity of PPV polypeptides A, B, and C used forrabbit immunizations. Purified virions (1.39g/ ml) were disrupted by boiling in SDS-sample buffer and electrophoresed in preparative SDS-101% poly-acrylamide gels. After localization (see the text), the threepolypeptides A, B, and C were excised and then wereindividually subjected to a second cycle of elec-trophoresis. A portion of the three protein prepara-tions obtained after gel elution anddialysis (see the text) was thenelectrophoresedin anothergel together with a sample of the starting viruspreparation(ST). Thegel was stainedwith Coomassie brilliant blue and destained.

2 4 6 8 10 12 14

POST-IMMUNIZATION TIME (WEEKS)

FIG. 6. Immuneresponse of rabbits injected with

intact virions and isolated PPV structural proteins. Individual rabbits were immunized with 50 ,ug of

intact, CsCl-purified virus (1.39-g/ml particles), gel-purified polypeptide /A, gel-purified polypeptide B,

andgel-purifiedpolypeptideCasdescribed in thetext. Rabbits receiving gel-purified polypeptides were

boostedat3and 7weeksafter the initialinjection,and

the rabbit receiving intact virus was boosted at 3

weeksonly.Atvarious times aftertheprimary

immu-nization, blood samples werecollected, and the sera were assayed for PPV-specific antibodies by

hemag-glutination inhibition (see the text).

TABLE 2. Serological tests comparing the immune responseof rabbits and swine immunized with either

intact PPV or gel-purified structuralproteins

Antiserum Serologicaltest

resulta

HAI IFA AGID

Normal pig <4 -

-Adultpiga PPV 8,096 + +

Fetalpig a PPV 8,096 + +

Normalrabbit serum <4

Rabbit aintact PPV 8,096 + +

Rabbit a A polypeptide 1,024 + +

Rabbit a Bpolypeptide 32 + +

RabbitaCpolypeptide 2,048 + +

a The serological assays, HAI (hemagglutination inhibition), IFA (indirect fluorescent-antibody), and AGID(agargelimmunodiffusion),aredescribed inthe text. -, Nodetectable reactivity; +,clearly positive reactivity. As the IFA and AGID are difficult to evaluate quantitatively, no attempt is made here to distinguish antisera reactivities.

against the

SDS-gel-purified proteins,

we

might

expect

these

sera tocontain

antibodies directed

more toward linear determinants on the

pro-teins, whereas antisera produced by

immuniza-tion

with

intact

virions

might contain antibodies

to

higher-order

structural

determinants. PPV

a-(f) 0- Go u

z cr : c

I2 4 5

FIG. 7.

Reactivity

of variousantiseratodenatured

PPV

polypeptides.

Punified PPV was

disrupted by

boiling

in

SDS-sample

buffer, and the viral

polypep-tideswereresolvedon anSDS-10%

polyacrylamide gel.

The

proteins

werethen

electrophoretically

transferred

tonitrocellulosepaper(2),

strips

werecut, and

individ-ual

strips

were incubated with various antisera (see

Table1), followedbyreaction with

"N-I-abeled

protein A(seethetext).The washed

strips

werethenexposed

to

X-ray

film. The exposure time for all

strips

shown

was thesame(10 min). Upon

longer

exposure(5to6

h), a

qualitatively

similar

reactivity

of R aPPV with

the PPV

proteins

wasrevealed; however,no

reactivity

wasobserved with NRS.

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particles

were disrupted by boiling in SDS and

2-mercaptoethanol,

resolved by electrophoresis in

an

SDS-polyacrylamide

gel, and transferred to

nitrocellulose

paper.

Identical

strips of

the

pro-tein-containing nitrocellulose

paper were then

incubated with

the

various antisera, followed by

incubation

with

125I-labeled

protein A to

local-ized bound immunoglobulin.

The results of this

"Western

blot" analysis

are shown

in Fig.

7.

Normal

rabbit

serum

showed

no

reactivity

to

the

PPV proteins (Fig. 7, track 1). The individual antisera generated against each of the viral pro-teinsshowed very strong reactivity with all three PPV structural proteins (Fig. 7, tracks 3 to 5).

Antisera obtained by immunization with intact

virus reacted

very

poorly

with

the denatured

antigens (Fig. 7, track

2). However,

longer

auto-radiographic

exposures (10

times) did

reveal

some

reactivity

to the three

viral proteins.

Simi-larly, porcine

antisera obtained from

naturally

infected

adult

pigs

or

infected fetuses

reacted

poorly

with the

denatured

PPV

polypeptides

(data

not

shown). Thus,

as expected,

antisera

raised against

the

SDS-gel-purified proteins

re-acted

very

well with

the

denatured polypeptides.

It is

of

interest here that all antisera

reacted to all

three viral structural proteins.

Even

though

anti-serumwas

generated by

immunization with

each

individual viral

protein, each antiserum,

in

addi-tion

to

recognizing

the

immunizing antigen,

rec-ognized equally

well

the

other two

viral

pro-teins. This result is consistent with

the

close

structural

similarities

among the three

proteins

(Fig.

4).

However,

an

alternate

interpretation

of

these

results

is

that our

immunogen preparations

of

the

gel-purified proteins

were

sufficiently

con-taminated with

the

other

polypeptides

that an

immune

response was

also

mounted

against

these contaminant

proteins.

To

determine whether the various antisera

were

able

to

recognize

native, intact virus

parti-cles,

we

tested their

ability

to causethe

immuno-specific aggregation (lattice formation) of

PPV

virions

in an

immunoelectron microscopy

assay

(7). Antisera

were

mixed with

purified virus,

and

any aggregates formed were collected by

centrif-ugation.

The

pelleted

material was then

ana-lyzed after negative staining in the electron

microscope. Immunospecific

lattice

formation

occurred with antisera

generated against

native

virus

(Fig. 8, panel 2) and with the antisera

generated

against

the

individual gel-purified,

de-natured proteins

(Fig.

8, panels 3 to 5), but not withnormal, preimmune serum (Fig. 8, panel 1). Ability of antisera to neutralize virus infectiv-ity. The results

described

above indicate that

antisera produced

by

immunization

with

SDS-gel-purified

PPV

proteins

are able to recognize

native, intact virus particles

as well as linear

determinants on the denatured viral

polypep-tides.

We

finally

asked whether these antisera

contained antibodies

capable

of

neutralizing

vi-rus infectivity. Using a direct immunofluores-cenceassay,wefound that antiserum

generated

by each of the

gel-purified

proteins did indeed contain

neutralizing

antibodies (Fig.

9; Table

3).

It appears,however, that the titer of the neutral-izing antibodies in each of the antiersa

produced

against the individual proteins was lower than that found in antisera generated against the intact virus. The neutralizing titers of the sera paralleled the

HAI

titers described above

(Table

2).

DISCUSSION

Swine

fetuses, either naturally

exposed to or experimentally inoculated with PPV, actively support virus

replication,

which ultimately re-sults in fetal death. As PPV is

often difficult

to grow tohigh titers in cells cultured

in vitro,

we initially concentrated on characterizing the

mo-lecular

features of

PPV aspropagated

in infected

animals. Recently, a brief report on certain aspects of PPVreplication in culturedfetal

por-cine

kidney cells has appeared

(29). The

avail-ability

of both

an animal model

and

an in vitro cell culture system for virus

propagation

pro-vides an excellent opportunity for the detailed and controlled study of the molecular, immuno-logical, and pathogenic

characterization

of PPV.

Propagation of

PPVin

swine fetuses

provides

anabundant source

of

virus material.

Employing

procedures used

for

the

purification of

previous-ly

studied parvoviruses

(33), we have been able

to obtain

highly enriched

virion

preparations.

These

preparations contained

two

predominant

forms

of

the 20-nm

virus

particle:

"full"

(DNA-containing) particles with

a

density

in

CsCl

of

1.39

g-ml, and "empty"

(DNA-deficient)

parti-cles with the

lighter density

of1.30

g/ml.

Three

major

polypeptides, designated

A,

B,

and

C,

were

consistently found

in both of these virus

species, having

molecular

weights of 83,000,

64,000,

60,000,

respectively.

Other

parvoviruses

have been shown to consist of three structural proteins with molecular

weights

in the

following

ranges: A, 93,000 to 73,000; B, 80,000 to64,000;

C,

67,000 to56,000

(32).

Wetherefore conclude that theseproteins are the structural

proteins

of

PPV.

Polypeptide

A

routinely represented

ap-proximately 10%

of the total viral

protein

in

both the full and the empty viral

particles.

Polypeptide

C appeared most abundant in the

"full"

particles,

whereas the B

protein

was present in the greatest relative amounts in the

"empty" particles (Fig.

3). All of these results

are

consistent

with

previous observations

in

other

studied

parvovirus

systems

(35).

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PPV: PURIFICATION AND ANTIGENIC PROPERTIES 851

4

-FIG. 8. Immunoelectron microscopy of

purified

PPV reacted with various antisera. Various antisera (see Table 1)wereincubated withpurified, intact virusparticlesand tested for theirabilitytoform immune

specific

virus-antibody latticesasdescribed in thetext.

ruses(34), the defective adeno-associated virus-es(12, 19), and,morerecently,witha

densonu-cleosisvirus(22) have shownthat theA, B,and C viral structural proteins in their respective systems have extensive sequence homology withoneanother. Itnow appearstobeaccepted that the lower-molecular-weight polypeptides

aresubsets of thelarger proteins; however, the meansof their derivationand the mechanism of

putative RNAorprotein processingremain

ob-scure. Inour limited structural analyses of the

PPV proteins, we found that the three viral

proteins were also very closely related to each other inprimary sequence.

Avariety of antiseraweregeneratedto inves-tigate certain antigenic properties of PPV and its constituent proteins. Ourgreatest interest

cen-tered

on

the

antisera

that were raised against

the

SDS-denatured, gel-purified

viral structural

proteins. Antibodies elicited by

the

individual

gel-purified polypeptides

were

qualitatively

sim-ilar

to

antibodies in

sera

from naturally infected

adult

pigs

or

rabbits

experimentally immunized

with intact virus particles in several standard

serological

assays,

including

HAI, an IFA test,

and an

AGID

assay

(Table

2).

However,

the

antisera

generated

against

the

denatured,

gel-purified

proteins

reacted more avidly with the

denatured

antigens

than

did

the

conventional

antisera

(Fig. 7). Still,

these

antisera

were all

able

to

recognize

and

immunospecifically

aggre-gate

native

intact virion particles (Fig. 8).

Final-ly,

all

antisera

directed against PPV,

whether

produced by

natural

infection, immunization

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852

FIG. 9. Virusinfectivityneutralizationbyvarious antisera detected by direct immunofluorescence. Various

sera

(diluted

1:10inPBS)weremixed andincubated with PPV and then were placed on cultures of ST cells, as

described in thetext.After4days, PPVcellularantigensweredetected with afluorescent-antibody conjugate andviewed under afluorescentmicroscope(see the text). The seraused were (1) NRS; (2) R a PPV; (3) R a A; (4) R a B; (5) R a C (see Table 1 forabbreviations).

with intact virus

particles,

or

immunization

with

the

individual, SDS-gel-purified

viral structural

proteins,

were

able

to

neutralize virus

infectivity

(Fig. 9; Table 3). Antisera prepared

against

individual

parvovirus structural

proteins

in other

systems have

failed

to

elicit

a

virus-neutralizing

response

(6,

9). Several

explanations

may ac-count

for this

apparent

discrepancy.

We

feel

that

the method of

antigen preparation

may be an

important factor.

We

find it

interesting

that each

of the antisera

generated

against

the

individually gel-purified

viral

proteins reacted with all

three structural

proteins and that each viral

polypeptide

was

capable of

eliciting

virus-neutralizing

antibody.

These results

are

consistent

with the

sequence

homology

among the

three

proteins

and suggest

that

the

virus-neutralizing

determinant(s)

is

present on

all

three

viral

structural

proteins.

However,

we

feel that due

to

the

possible

minor

contamination of each

immunogen preparation

with the

other structural

proteins, and

to

the

fact

that the

neutralizing

antibody

response

could

have been

a

result of

such

contamination,

any

conclusions

concerning

the

nature

of

PPV-neu-tralizing

antigenic

determinants is

unwarranted

at

this time. The

generation

of monoclonal

anti-bodies

to these

proteins should

provide

more

conclusive

information

concerning

the number

and

distribution of

virus-neutralizing antigenic

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PPV: PURIFICATION AND ANTIGENIC PROPERTIES TABLE 3. Viral infectivity neutralization of various

antisera

Culture fluid Fluorescent

antigen

Antiseruma (HA)b cells (%)C

1:2 1:10 1:2 1:10

Noserum 256 256 >80 >80

Novirus <4 <4 0 0

Normal pig serum 256 256 >80 >80

Fetal

pig

aPPV <4 <4 0 0

Normal rabbit serum 256 256 >80 >80 Rabbit a intact PPV <4 <4 0 0

Rabbit a A <4 <4 0 0-5

RabbitaB <4 32 0 30-40

Rabbit aC <4 16 0 5-10

aFor standardization, rabbit a A, a B, and a C

antisera were normalized to the same HAI titer and thenwere diluted either 1:2 or 1:10 in PBS.

bCellculture fluids were tested at 3 days postinfec-tion for the presence of extracellular (culture fluid) virus by hemagglutination of guinea pig erythrocytes (seethe text).

cAcetone-fixed cells were stained at 3 days postin-fection with PPV-fluorescent antibody conjugate for thepresence of intracellular virus as described in the text.The percentageof fluorescent cells was estimated fromatleastfiveindependent fields of view.

determinants on the PPV structural polypep-tides.

ACKNOWLEDGMENTS

We thank Dennis Anderson, Shirley Halling, and Allen Lemanforassistance andnumerousenlighteningdiscussions, SusanWells forcontinuing support, andCindy Kosman for manuscriptpreparation.

Portionsofthis work weresupported bygrant MIN-26-074 from theMinnesotaAgricultureExperiment Station.

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