Production of a Baculovirus-Derived gp5O Protein and Utilizationin a Competitive Enzyme-Linked Immunosorbent Assay for the

Serodiagnosis of Pseudorabies Virus

I. Prud'homme, E.-M. Zhou, M. Traykova, H. Trotter, M. Chan, A. Afshar, and M.J. Harding

ABSTRACT

The pseudorabies virus (PRV)gp5O envelope glycoprotein genewas cloned and expressed in arecombinant baculovirus. An anti-gp50 Mab (1842) recognized a pro-tein of approximately 40 kDain immunoblotting assays frominfected insect cell lysates, whilethis product was not present in cellsinfected with wild-type baculovirus.The recombinant protein was puri-fied by lectin affinity chromatogra-phy, utilizing lectins specific for0-linked oligosaccharides (Artocar-pus integrifolia and Glycine max).Competitive (c) ELISAs, usingeither crude or lectin-purified anti-gen, were devised for the detectionof antibodies to PRV in sera, andwere capable of monitoring sero-conversion by day 14 post-infection.Furthermore, a specificity of 100%and sensitivity of 98% (crude lysateantigen) or 96% (lectin-purifiedantigen) was found for a panel of80 swine sera, using the cELISA, ascompared to a serum neutralization(SN) test. These studies demon-strated that recombinant PRV gp5Oprotein shows promise as a cELISAantigen, for serodetection of PRV.

RESUME

Le gene de la glycoproteine gp5Ode l'enveloppe du virus de la pseu-dorage (VPR) fut clone et exprimedans un baculovirus recombinant.Lors d'etpreuves d'immunobu-vardage, un anticorps monoclonal

anti-gp5O reconnaissant une pro-teine d'environ 40 kDa a permis demettre en evidence cette proteinedans des lysats de cellules d'insectesinfectees avec le virus recombinantet non dans des cellules infecteesavec le virus de type sauvage. Laproteine recombinante fut purifrieepar chromatographie d'affinite uti-lisant des lectines specifiques (Arto-carpus integrifolia et Glycine max).Des epreuves ELISA de type com-pe'titif (ELISAc), utilisant soit del'antigene brut ou purifie par chro-matographie, furent mises au pointpour detecter des anticorps seriquesanti-VPR et furent en mesure desurveiller la seroconversion au jour14 post-infection. De plus, une spe-cificite de 100% et une sensibilite de98% (avec l'antigene brut) ou 96%(avec l'antigene purifie) fut obtenudans l'epreuve ELISAc avec unebatterie de 80 echantillons de se'rumde porc lorsque compare a l'epreuvede seroneutralisation. La proteinerecombinante gp5O du VPR demon-tre un certain potentiel commeantigene a utiliser dans une epreuveELISAc pour la se'rosurveillance dela pseudorage.

(Traduit par docteur Serge Messier)

INTRODUCTION

Pseudorabies virus (PRV) is analphaherpesvirus, causing severe neu-rotropic disease in a variety of domes-tic and wild animal species. Pigs arethe natural reservoir for PRV, and thevirus is maintained in swine herds byrepeated cycles of virus latency and

reactivation by general or local stim-uli. Typically, the virus causes severeencephalitis and often death in youngpigs, pneumonia in older swine, andalso abortions in sows, thus making itan important virus for both veterinarymedical and economical reasons.PRV is a reportable disease in manyregions which are attempting to eradi-cate or prevent virus entry into animalpopulations. England, Denmark andGermany have successfully elimi-nated the disease, and 13 additionalcountries have reported active eradi-cation schemes (1). Pseudorabies is anexotic disease in Canada, and limita-tions on animal entry and serologicalmonitoring programs are upheldto diminish the possibility of itsintroduction to the domestic pigpopulation.

Herpesvirus envelope glycoproteinsare involved with several initiationevents, including attachment to hostcells and virus envelope-cytoplasmicmembrane fusion (2). Viral glycopro-teins gll, gpSO and gH are indispensi-ble for virus penetration, howevergpSO-null PRV mutants are able tospread directly from cell to cell (3,4).In addition, envelope glycoproteins,notably gIll and gpSO, are majorimmunogens (5,6).The predicted molecular weight of

the gpSO amino acid chain is 45 kD(7), however, the gp5O precursorundergoes post-translational modifi-cations, to produce a mature formwith a molecular weight of 60 kDa.The protein sequence lacks consensussignals for N-linked glycosylation(Asn-X-Ser or Asn-X-Thr); rather 30-linked oligosaccharide structures

Animal Diseases Research Institute, P.O. Box 11300, Station H, Nepean, Ontario K2H 8P9.

Correspondence to Dr. M.J. Harding.Present address of Dr. I. Prud'homme: National Laboratory for Special Pathogens, Virus Laboratory, Building #10, Tunney's Pasture, Health Canada,Ottawa, Ontario KIA OL2.Present address of Dr. M. Traykova: Loeb Research Institute, 725 Parkdale Avenue, Ottawa Civic Hospital, Ottawa, Ontario K 1Y 4K9.

Present address of Dr. M.J. Harding: Pfizer Central Research, Eastern Point Road, Box 1195, Groton, Connecticut 06340, USA.

Received April 30, 1996.

Can J Vet Res 1997; 61: 286-291286

are found on mature gpSO, producedin either mammalian or insect cellcultures (8). In the latter system, the0-linked sugars are relatively smallnonsialylated moieties, the majorityof which is the monosaccharideN-acetylgalactosamine (GalNAc).ELISAs are widely used for sero-

logical detection of PRV (9,10). Morerecently, ELISAs, either as indirect orcompetitive formats, have beendescribed which discriminate betweennaturally infected animals and thosevaccinated with gene-deletion mutants(11,12). However, traditional screen-ing of swine sera for the presence ofPRV-specific antibodies is performedwith an indirect ELISA in which theantigen is derived from a wild-typevirus propagated in tissue-culture. Incountries where PRV is exotic, suchas Canada, the production of recombi-nant antigen for the PRV ELISAwould be advantageous because highsecurity facilities would not berequired for antigen production. Inaddition, protein produced by recom-binant means would elicit less batch-to-batch variation. This reportdescribes the construction, expres-sion, and preliminary assessment of arecombinant gpSO PRV antigen foruse in ELISAs.

MATERIALS AND METHODS

CELLS, SERA AND VIRUSES

Wild-type Autographica califor-nica nuclear polyhedrosis virus(AcNPV) and recombinants werepropagated in early to mid-exponentialphase Spodoptera frugiperda (Sf9)cells (American Type Tissue Collec-tion), in the presence of Sf900 serum-free medium (Gibco/BRL, Burlington,Ontario), including gentamycin(50 pg/mL), 2 mM L-glutamine and0.01% pluronic F-68. The anti-gpSOMab (1842; American Type TissueCollection, Rockville, Massachusetts,USA) has been shown to immunopre-cipitate PRV gpSO and neutralizePRV infection in the Mengeling-Vaughn porcine kidney cell line (13).Two swine (P34-76, P36-76) were

infected oronasally with the VR-135strain of PRV (American Type TissueCollection, Rockville, Maryland,USA) with a dose of 1000 TCID50. Anadditional pig (P2-77) was housed as

a sentinel animal. A 4th piglet (P49-84) was inoculated with a related her-pesvirus (bovine type 1; 14). Serumwas collected sequentially at varioustimes post-infection. Eighty sera orig-inating from both seropositive andseronegative PRV sera were providedby Dr. S. Goyal, University ofMinnesota, USA. The serostatus ofthe swine sera was determined by aserum neutralization (SN) test (15).PRV was grown in Vero cell mono-

layers and purified using potassiumtartrate gradients (16). Briefly, Verocells were infected with PRV accord-ing to standard protocols, and upondestruction of the monolayer, cellsand supernatants were frozen andthawed 3 times, then clarified by cen-trifugation at 3000 X g. The virus waspelleted by ultra-centrifugation at100 000 X g for 90 min, then resus-pended in 0.15 M NaCl, 0.06 M Tris-HCl, pH 7.4 and sonicated to disruptviral-cell membrane interactions.PRV was purified over a gradient of20-45% (w/w) potassium tartrate-TrisHCl, pH 7.4 at 79 000 X g for 18 h.Fractions were collected and proteinconcentration determined with anultraviolet light monitor. Protein-containing fractions were analysed byvirus titration on Vero cell monolay-ers in 96-well plates, and used asvirus controls in polyacrylamidegel electrophoresis (PAGE; 17), asdescribed below.

PRODUCTION OF BACULOVIRUSRECOMBINANT PRV GP50

A BamHI-NcoI fragment of theplasmid pMT 50-63 (3) encodinggpSO and a portion of gp63 of PRV(Ka strain), was subcloned into thepBlueBacIII transfer vector (Invitro-gen, San Diego, California, USA).Subcloning experiments were designedto result in a gpSO-baculovirus con-struct with the gp5O ATG start codonout-of-frame with the mutated ATTstart codon of the polyhedrin gene,present in the BlueBacIII transfer vec-tor. The presence of the PRV insert inthe recombinant plasmid was con-firmed by digestion with restrictionendonucleases BamHI and NcoI (dou-ble digest) and SalI.The gpS0-BlueBacIII recombinant

vector (5 jig) was co-transfected in thepresence of liposomes (20 jL) and1 pg linearized AcNPV DNA (Invitro-gen), according to the protocol pro-

vided by the manufacturer. Recombi-nant clones, identified using galac-tosidase colorimetric screening, weresubjected to 3 rounds of plaque purifi-cation. The presence of a gp50 insertwas confirmed in potential recombi-nants, in dot blot and Southernhybridization formats (18), utilizingbiotinylated BamHI-NcoI gpSO insertand additional reagents supplied in aPhotoGene kit (Gibco/BRL).

PROTEIN EXPRESSION ANDIMMUNOBLOTTING ANALYSES

Sf9 cells were infected with recom-binant gp5O-baculovirus at severaldifferent multiplicities of infection,ranging from 10-20 PFU/cell, ineither 6-well plates or spinner cul-tures. At various times post-infection,an aliquot of cells was collected andwashed twice in ice-cold PBS (pH6.8) and the cell pellet was resus-pended in phosphate buffered saline(PBS), pH 6.8, and stored at -70°Cprior to immunoblotting analyses.Pellets were diluted in loading buffer(final concentration of 62 mM TrisHCI, pH 6.7, 15% glycerol, 0.001%bromophenol blue), either with orwithout SDS at a final concentrationof 2%. Proteins were separated by12% PAGE (17) using buffer condi-tions in the presence or absence ofB-mercaptoethanol, to either allowintact or disrupted disulphide bonds.The gels were stained with Coomassieblue or electrophoretically transferredto a polyvinylidene difluoride (PVDF)solid support (19). Protein prepara-tions were also analyzed followingdirect dotting onto PVDF sheets.Unbound sites on the membrane wereblocked with 5% skim milk powder inphospate-buffered saline with 0.05%Tween-20 (PBST) for 30 min, thenincubated with PBST/1% skim milkpowder containing 0.1 ,ug/mL Mab1842 (ATCC, Rockville, Maryland,USA), an antibody specific for PRVgpSO (13). Following 3 washes withPBST, bound Mab was detected withhorseradish peroxidase (HRP)-goatanti-mouse IgG diluted 1/2000 in 1%skim milk powder for lh. Following 3final washes with PBST, the remain-ing colour reaction was developedwith diaminobenzidine as recom-mended by the manufacturer (Sigma,St. Louis, Missouri, USA). All stepsof the immunoblotting protocol wereperformed at room temperature.

287

PRV gp5O-63 insert

Polyhednnpromoter

lacZ gene Recombination sequence

gp5O-BIueBacilI12373 bp

Sall

Sall SaIl Amp resistance gene

Recombination sequence

Figure 1. Schematic representation of gp5O-BlueBacIII transfer vector. Locations of BamHI,NcoI and Sall restriction endonuclease sites, and PRV gp5O-63 insert, lacZ gene and recombi-nation sequences, are indicated.

.6 kbp=

Figure 2. Restriction endonuclease analysisof the recombinant transfer vector (gp5O-pBlueBaclll). Lane a: lambda/HindIII sizestandard; lanes b, c: gp5O-pBlueBacIII andpMT 50-63 recombinant plasmids followingBamHVINcoI restriction endonuclease doubledigestion; lanes d, e: gp50-pBlueBacIII andpMT 50-63 recombinant plasmids followingSaI restriction endonuclease digestion.

PURIFICATION OF RECOMBINANT GP50UTILIZING LECTIN AFFINITYCHROMATOGRAPHY

Methods for lectin affinity chro-matography were adapted from Hortinand Trimpe (1990). Preliminaryexperiments were performed to con-firm the optimal lysis buffer for main-taining protein antigenicity. Lysisbuffer solutions of 150 mM NaCl,10 mM or 50 mM Tris-HCl (pH 8),10 mM NaCl, 1.5 mM MgCl2 were

compared with detergent additives of0.05%, 0.1%, 0.2% and 1% octylphe-noxy polyethoxy ethanol (NP-40)or polyoxyethylene ether (TritonX-100). Cells were mixed with 1 mLlysis buffer per 3 X 106 cells. The sus-pension was incubated for 1 h at 4°C,then clarified by centrifugation at200 X g for 10 min. The supernatantwas applied to a 7 mm column, con-taining a 1:1 mixture of jacalin (Arto-carpus integrifolia)-agarose, knownto bind 0-linked oligosaccharidesand soybean (Glycine max)-agarose(Sigma), which is specific for trun-cated 0-linked oligosaccharides.Unbound material was washed fromthe column with 0.5 M NaCl, 100 mMTris-HCl, pH 7.5. Protein specificallybound by the lectin was eluted with20 mM methyl-ot-D-galactopyra-noside and 20 mM N-acetylgalac-tosamine (Sigma) in the same buffer.

COMPETITIVE ENZYME-LINKEDIMMUNOSORBENT ASSAYS

Crude lysates (representing variouscell concentrations) or lectin-purifiedgpSO (1:50 dilution of a pool of gpSO-positive fractions; 100 ,uL/well) werecoated onto ELISA plates (Nunc#2-69620, Gibco/BRL) by passiveadsorption at 4°C overnight in car-bonate buffer (0.06 M, pH 9.6). Simi-larly treated lysates from wild typeAcNPV-infected cells served as anti-

gen controls. After 5 PBST washes toremove excess antigen, the unboundsites were blocked with 3% gelatin inPBS for 30 min at room temperature.Pig sera (1:5 dilution in PBS), fromanimals with experimental or naturalPRV infections, were added (50 ,uL/well), along with Mab 1842 (50 ,uL/well) at a final concentration of0.2 ,ug/mL, and incubated for 1 h at37°C. The wells were then washed5 times with PBST and residual anti-bodies were detected with a 1:500dilution in PBS of HRP-conjugatedgoat anti-mouse antibody, followedby the substrate, 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid)diammonium; ABTS) in 0.1 M citratebuffer (pH 4.5) and H202. The plateswere shaken continuously for pre-cisely 10 min, then the optical density(OD) values of the enzymatic reactionproducts were measured at a wave-length of 414 nm, using a TitertekMultiskan MC microtiter plate reader(Flow Laboratories, MississaugaOntario). The OD values wereexpressed as a per-cent inhibitioncomparison according to the formula:% inhibition = [1 - (OD414 with com-petitor/OD414 without competitor)] X100. Samples were considered posi-tive if the OD reading was at least 2standard deviations above the mean of20 seronegative sera. Test sensitivitywas recorded as a percentage ofseropositives in the PRV gp5OcELISA/seropositives in the SN test.Specificity was measured as the per-cent testing seronegative in the PRVgp5O cELISA/seronegatives in the SNtest.

RESULTS

A schematic representation of thegpS0-BlueBacIII recombinant plas-mid is shown in Figure 1. Restrictionendonuclease digestion, using BamHIand NcoI, revealed the presence of agp5O insert of 2.1 kb (Figure 1;Figure 2, lane b). Incubation of gpSO-BlueBaclIl in the presence of Sallproduced 3 visible fragments, repre-sentative of the 5.2, 3.56/3.29,0.285 kb segments (Figure 2, lane d),which were predicted from thenucleotide sequence of BlueBaclll(Invitrogen) and the Rice strain ofPRV (7; Figure 1). After transfection,9 putative recombinants were

288

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B

80 kD+49.5kD32SkD -P

a bc d e f g h ijj

m~~~~o$a.

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_

Figure 3. SDS-PAGE (panel A) and immunoblot (panel B) analysis, using Mab 1842, of gp5O-AcNPV prepared in spinner cell cultures. Lane a: molecular weight marker; lanes b-f: gp-50-infected Sf-9 cell infected 0, 24, 48, 72 and 96 h post-infection; lane g: Vero cell control; lanesh, j: PRV-infected Vero cells; lane i: wild-type baculovirus control.

a b c d e f g h i j

49.5 kD -

32.5 kD--

Figure 4. Immunoblot analysis of lectin affinity-purified gp5O. Lane a: moleculmarker; lanes b-h: lectin affinity purification fractions 12/13, 15/16, 17/18, 19/23/24, 25/26; lane i: unprocessed wildtype baculovirus antigen; lane j: unprocesAcNPV antigen.

identified by colorimetric selection.Two baculovirus constructs, harbour-ing a full-length clone of the PRVinsert, were identified by hybridiza-tion to the biotinylated insert (data notshown). One construct (K-1) was

selected for further analysis.Spinner cultures infected with

10 PFU/cell produced similaramounts of the recombinant proteinbetween 48-96 h post-infection (Fig-

ure 3; panel B, lanes d-f). Thethe protein species variedapproximately 35-40 kDa. I

produced in mammalian tistures infected with wild-typossessed an approximate mweight of 57 kDa (Figure 3;lane j). No cross-reactingwere apparent in negativeVero cell or wildtype iinfected Sf9 cell prep

DPI

Figure 5. PRV cELISA, utilizing crude lysate(A) or lectin-purified (B) gp5O-AcNPV anti-gen to detect antibodies in two pigletsinfected oronasally with the VR135 strain ofPRV (P34-76; P36-76) and a sentinal piglet(P2-77).

(Figure 3, panel B, lanes g, i). Instatic flask cultures, recombinantgp50 protein was apparent by 48 hpost-infection, however relativeamounts began to decline by 72 hpost-infection (data not shown). At72 h following initial infection, a

smaller molecular weight species(approxiately 36 kDa) was also appar-

lar weight ent. Exponential cultures of Sf9 cells,120, 21/22, grown in 6-well plates and infectedssed gp5O- with 20 PFU/cell gpSO-AcNPV, pro-

duced several types of proteinmolecules, observed as a doublet at

sizes of approximately 40 kDa in immuno-between blots (data not shown). Lysis buffer'he gpSO containing 0.1-0.2% Triton-X in;sue cul- 150 mM NaCl, 50 mM Tris-HCl was

rpe PRV superior to other buffers studied, andiolecular these conditions were applied in sub-panel B, sequent protocols.proteins Following lectin affinity chro-control matography, dot and PAGE immuno-AcNPV- blotting analysis revealed that recom-arations binant gp5O was present in elution

289

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Figure 6. Correlation of SN and cELISAresults, utilization either crude lysate (A) orlectin-purified (B) antigen, for a panel of 80field sera, originating from American swine.

fractions #19 through 26 from thejacalin/soybean column (Figure 4lanes e-h). Subsequently, the serosta-tus of various swine sera was deter-mined, utilizing either a pool of frac-tions #19 through 26 from the affinitycolumn, or unpurified crude lysate asthe PRV antigen. The cELISAsdetected antibodies by 14 d post-infection, in both cELISA formats, in2 piglets experimentally infected withPRV (Figures SA and SB). Serocon-version of the sentinel pig probablyoccured at 30 d post-infection. Nocross-reacting antibodies weredemonstrated in either cELISA test insera from a piglet infected with arelated alphaherpesvirus, bovine her-pesvirus type 1 (data not shown).These results were similar to thatfound in a conventional indirectELISA (10), using antigen frominfected tissue cultures, and acELISA, with recombinant gIll anti-gen (Afshar A, unpublished results).

In addition, the cELISA tests werecompared to SN results generatedfrom a panel of 80 pig sera fromAmerican animals. The cELISA meanfor 20 SN seronegative samples(SN< 1:2) was 11% and 23% for crudeand lectin-purified antigen, respec-tively. Utilizing a negative cut-offvalue of 2 standard deviations abovethe mean for these same SN seronega-tive sera (ie. 23% and 43%, respec-tively), the sensitivity and specificityof 45 SN seropositive samples (SN >1:2) was 98% and 100%, respectively,using crude antigen (Figure 6A), and96% and 100%, respectively, for thelectin-purified antigen (Figure 6B).Fifteen additional sera with a border-line SN titer (1:2) demonstratedcELISA specificity of 47% and 33%,utilizing crude and lectin-purifiedantigen, respectively.

DISCUSSION

A major immunogen of PRV, gpSO,was expressed in the baculovirus,AcNPV, under control of the poly-hedrin promoter. A protein which wassmaller (ie. 35-40 kDa) as comparedto the authentic protein, was identi-fied in immunoblotting assays. Indi-rect ELISA tests using crude or lectin-purified antigen proved unsuitable,due to non-specific binding with neg-ative control antisera (data notshown). Typically, these cross-reactions can be decreased with theuse of purified antigens. Cross-reactions with negative antisera werealso observed with purified antigen ina indirect ELISA (data not shown),which may be due to antibodiesagainst swine cytomegalovirus (21),another herpesvirus, which is endemicin many Canadian swine herds. Theseearly results necessitated further stud-ies using a Mab-based cELISA for-mat, which was shown to provide ahigh level of sensitivity and speci-ficity. This study suggests that eithercrude or purified baculovirus recom-binant antigen is adequate as anELISA antigen for routine serologicalasays. However, crude baculoviruslysates may be cheaper to producethan lectin-purified antigens, depend-ing on the cost of the affinity chro-matography elution reagents.The gp5O protein species that were

detected in the gpSO-AcNPV lysate

varied between experimental proto-cols. This finding could be explainedby a variation in detection sensitivi-ties between experiments or effi-ciency of glycosylation betweenexperimental conditions (ie. PFU/cell,stage of the cell cycle). Variations inoligosaccharide content of a bac-ulovirus-gp50 construct has beenobserved depending upon the cell typeused for protein propagation (22).Glycosylation patterns also varyaccording to the stage in the infectioncycle in which the recombinant pro-tein is produced, ie. under control ofan earlier or later promoter (23).Recombinant gpSO degradationoccurred more rapidly in static cul-tures infected at a later stage of theinfection cycle, as compared to spin-ner cultures infected earlier. Otherinvestigations have revealed thatrecombinant protein degradation maydecrease as infection proceeds. Finalproduct titer is dependent on theinfection rate only if infection occursin late-exponential phase of the cellcycle (24). Many post-translationalmodifications accomplished in mam-malian cells also occur in insect celllines. The gpSO protein was morereadily recognized in immunoblottingassays by the gpSO-specific Mabwhen non-reducing conditions wereapplied, as compared to reducing con-ditions. Therefore, it appears thatdisulphide bond formation is occur-ring with the present gpSO-AcNPV-expressed protein.The immunoreactivity of the

recombinant gpSO protein in thecELISA warrants further investiga-tion, utilizing a more extensive panelof experimental and field origin pigsera, using either lectin-purified orcrude antigen. Limited sequencinganalysis of a number of PRV suggeststhat the PRV glycoprotein chosen forthese studies may be conservedamongst PRV strains (25). Moreover,all gene-deleted vaccine viruses thatare presently commercially availablein North America possess an unal-tered gpSO gene, thereby making agpSO-based ELISA an ideal screeningtest for detecting both wild-type andvaccinal PRV infections. Due to thefact that Canadian swine are not vac-cinated for PRV, an ELISA that dis-criminates between natural or vaccinetiters is not required for Canadianproducers and practitioners.

290

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ACKNOWLEDGMENTS

The authors wish to thank Dr. S.Goyal, University of Minnesota, forhis donation of pig sera, and Drs. MaxAppel and Gary Blissard, CornellUniversity, for review of themanuscript.

REFERENCES

1. KLUGE JP, BERAN GW, HILL HT,PLATT KB. Pseudorabies (Aujeszky'sdisease). In: AD Leman, BE Straw, WLMengeling, S D'Allaire, DJ Taylor, eds.Diseases of Swine. Ames: Iowa State Uni-versity Press, Ames: 1992: 312-323.

2. SPEAR PG. Entry of alphaherpesvirusesinto cells. Seminar Virol 1993; 4:167-180.

3. RAUH I, METTENLEITER TC. Pseu-dorabies virus glycoproteins gll and gp5Oare essential for virus penetration. J Virol1991; 65: 5348-5356.

4. PEETERS B, DE WIND N, HOOISMAM, WAGENAAR F, GIELKINS A,MOORMAN R. Pseudorabies virus enve-

lope glycoproteins gpSO and gll are essen-

tial for virus penetration, but only gll isinvolved in membrane fusion. J Virol1992; 66: 894-905.

5. BEN-PORAT T, DEMARCHI J, LOM-NICZI B, KAPLAN AS. Role of glyco-proteins of pseudorabies virus in elicitingneutralizing antibodies. Virol 1986; 154:325-334.

6. MARCHIOLI CC, YANCEY RJ JR,PETROVSKIS EA, TIMMINS JG,POST LE. Evaluation of pseudorabiesvirus glycoprotein gpSO as a vaccine forAujeszky's disease in mice and swine:Expression by vaccinia virus and Chinesehamster ovary cells. J Virol 1987; 61:3977-3982.

7. PETROVSKIS EA, TIMMINS JG,ARMENTROUT MA, MARCHIOLICC, YANCEY RJ Jr, POST LE. DNA

sequence of the gene for pseudorabiesvirus gpSO, a glycoprotein withoutN-linked glycosylation. J Virol 1986; 59:216-223.

8. THOMSEN DR, POST LE, ELHAM-MER AP. Structure of 0-glycosidicallylinked oligosaccharides synthesized by theinsect cell line Sf9. J Cell Biochem. 1990;43: 67-79.

9. MOUTU F, TOMA B, FORTIER B.Application of an enzyme-linked immuno-sorbent assay for diagnosis of Aujeszky'sdisease in swine. Vet Rec 1978; 102: 264.

10. AFSHAR A, WRIGHT PF, DULAC GC.Evaluation of an enzyme immunoassay fordetection of antibodies to pseudorabiesvirus in porcine field sera. Can J Vet Res1987(a); 51: 539-541.

11. VAN OIRSCHOT JT, HOUWERS,RZIHA HJ, MOONEN PJLM. Develop-ment of an ELISA for detection of antibod-ies to glycoprotein I of Aujeszky's diseasevirus: A method for the serological differ-entiation between infected and vaccinatedpigs. J Virol Methods 1988; 22: 191-206.

12. COOK D, HOWARD H, SNYDER M,MCMAHON P, KINKER D. The detec-tion of antibodies to the glycoprotein Xantigen of pseudorabies virus. J Vet DiagInvest 1990; 2: 24-28.

13. WATHEN LMK, PLATT KB, WATHENMW, VAN DEUSEN RA, WHETSTONECA, PIRTLE EC. Production and charac-terization of monoclonal antibodiesdirected against pseudorabies virus. VirusRes 1985; 4: 19-29.

14. AFSHAR A, WRIGHT PF, MYERS DJ,BOUFFARD A, DULAC GC. Specificityof the indirect enzyme-linked immunosor-bent assay for detection of pseudorabiesvirus antibodies in pigs exposed to bovineherpesvirus-1. Am J Vet Res 1987(b); 48:1461-1464.

15. PAPP-VIDD G, DULAC GC. Pseudora-bies. Adaptation of the counter-currentimmunoelectrophoresis for the detection ofantibodies in porcine serum. Can J CompMed 1979; 43: 119-124.

16. BUSH CE, PRITCHETT RF. Immuno-logic comparison of the proteins of pseu-

dorabies (Aujeszky's disease) virus andbovine herpesvirus-1. Am J Vet Res 1986;47: 1708-1712.

17. LAEMMLI UK. Cleavage of structuralproteins during the assembly of the head ofbacteriophage T4. Nature 1970; 227:680-685.

18. SOUTHERN E. Detection of specificsequences among DNA fragments sepa-rated by gel electrophoresis. J Mol Biol1975; 98: 503-517.

19. TOWBIN H, STAEHELIN T, GORDONJ. Electrophoretic transfer of proteins frompolyacrylamide gels to nitrocellulosesheets: Procedure and some applications.Proc Natl Acad Sci USA 1979; 76:4350-4354.

20. HORTIN GL, TRIMPE BL. Lectin affin-ity chromatography of proteins bearing0-linked oligosaccharides: Application ofjacalin-agarose. Anal Biochem 1990; 188:271-277.

21. DUNCAN JR, RAMSEY FK, SWITSERWP. Electron microscopy of cytomegalicinclusion disease of swine (inclusion bodyrhinitis). Am J Vet Res 1965; 26: 939-946.

22. HINK WF, THOMSEN DR, DAVID-SON DJ, MEYER AL, CASTELLINOFJ. Expression of three recombinant pro-teins using baculovirus vectors in 23 insectcell lines. Biotechnol Prog 1991; 7: 9-14.

23. SRIDHAR P, HASNAIN SE. Differentialsecretion and glycosylation of recombinanthuman chorionic gonadotropin (BhCG)synthesized using different promoters inthe baculovirus expression vector system.Gene 1993; 131: 261-264.

24. LICARI P, BAILEY JE. Factors influ-encing recombinant protein yields in aninsect cell-baculovirus expression system:Multiplicity of infection and intracellularprotein degradation. Biotechnol Bioengi-neer 1991; 37: 238-246.

25. HARDING MJ, PRUD'HOMME I,ROLA J. Specificity and nucleotypingstudies of a polymerase chain reactionassay for detection of pseudorabies virus.Can J Vet Res, 1997; 61: 157-160.

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