Characterization of Virulent and Attenuated Strains ofPseudorabies Virus for Thymidine Kinase Activity,

Virulence and Restriction Patterns

M.D. McFarland, H.T. Hill and L.B. Tabatabai*

ABSTRACT

A pseudorabies virus mutant lack-ing thymidine kinase activity (TK-)was isolated and characterized. Themutant replicated as well in cell cultureas the parental TK+ strain, was nottemperature sensitive at 38.50 C, anddid not revert to TK+. Two pseudo-rabies virus field isolates and threecommercial modifled live virus vaccinestrains were compared for TK activityand virulence for the mouse; all strainsexpressed TK: Km values for thymi-dine of the viral TKs ranged from 2.9to 3.9,m; the commercial modifiedlive virus vaccine strains were reducedin virulence for the mouse two to ten-fold. The TK- mutant was avirulent forthe mouse. Restriction enzyme analy-sis of the pseudorabies virus DNAfrom the strains under study revealedthat two of the modified live virusvaccine strains are closely related andthat all three modified live virus vac-cine strains lack the largest PstI frag-ment characteristic ofthe other strainsincluded in the study.

Key words: Pseudorabies virus, thymi-dine kinase, virulence, restrictionendonuclease.

RESUME

Cette experience consistait a carac-teriser un isolat d'un mutant du virusde la pseudo-rage, depourvu de l'acti-vite de la thymidine kinase. II sedeveloppa aussi bien sur feuillet cellu-laire que sa souche parentale, pourvuede l'activite de la thymidine kinase,n'afficha aucune sensibilite a la tem-perature de 38,5° C et ne retourna pasen virus dote de l'activite de lathymidine kinase. La comparaison dedeux souches du virus de la pseudo-rage, isolees de cas cliniques, avec troissouches vaccinales commerciales at-tenuees, relativement a l'activite de lathymidine kinase et a la virulence pourla souris, revela qu'elles possedaienttoutes I'activite precitee, que les pro-prietes cinetiques de leur thymidinekinase variaient de 2,9 a 3,9,um et queles trois souches vaccinales etaient dedeux a dix fois moins virulentes pourla souris. Le mutant depourvu d'activitede la thymidine kinase se revela parailleurs tout a fait inoffensif pour lasouris. L'analyse de l'enzyme derestriction de I'acide desoxyribonu-cleique du virus de la pseudo-rage dessouches impliquees dans l'experience,revela une 'troite relation entre deuxdes trois souches vaccinales et l'absence,chez toutes les trois, du plus grosfragment de l'endonuclease de restric-tion PstI, caracteristique des autressouches incluses dans l'experience.Mots cles: virus de la pseudo-rage,thymidine kinase, virulence, endonu-clease de restriction.

INTRODUCTION

Pseudorabies is a disease of swinewhich is of major concern to the swineindustry in the United States and manyother countries of the world. Vaccina-tion is a commonly used method tocontrol losses due to pseudorabies. Anideal vaccine is one that is efficacious,safe and inexpensive to produce. Mod-ified live virus (MLV) vaccines are lessexpensive to produce than inactivatedvaccines and are thought to confergreater cell-mediated immunity (CMI).Modified live virus vaccines againstpseudorabies are available commer-cially in the US and elsewhere whichare considered safe for use in swine butmay cause mortality in other species(1,2,3).

It would be advantageous to char-acterize the genetic basis of pseudo-rabies virus (PRV) virulence so that,by selective mutagenesis, a MLV PRVvaccine could be created having max-imal safety and efficacy. Geck et al (4)have previously used restriction en-zyme (RE) analysis to distinguishattenuated European strains of PRVfrom virulent strains. The molecularbasis for viral attenuation associatedwith the physical changes in the PRVgenome noted by Geck et al remainsunknown.

Thymidine kinase (TK) is an enzymeencoded by many of the herpesviruses,including herpes simplex virus (HSV)and pseudorabies virus (PRV) (5).Viral TK has been shown to be a majordeterminant of neurovirulence forHSV (6,7) and PRV (8). The viral TK

*Immunobiology Program (McFarland), Veterinary Diagnostic Laboratory and Department of Veterinary Microbiology and Preventive Medicine(Hill), Iowa State University, Ames, Iowa 50011 and the National Animal Disease Center (Tabatabai), Ames, Iowa 50010.Part of a dissertation by the senior author in partial fulfillment of the requirements for the Ph.D. degree in Immunobiology, Iowa State University,Ames, Iowa.

Partially funded by USDA, National Veterinary Service Laboratories, Ames, Iowa and by State of Iowa ISU Fund #400-23-40.Submitted July 16, 1986.

Can J Vet Res 1987; 51: 334-339334

apparently plays a critical role in viralreplication in differentiated neuraltissue, which expresses very low levelsof cellular TK (9,10,1 1).

The present study was undertakento further characterize the biologicalproperties of attenuated strains ofPRV so that progress might be madetoward a MLV vaccine having optimalsafety and efficacy.

MATERIALS AND METHODS

VIRUSES AND CELLS

The Indiana-Funkhauser (IN-Fh)strain was obtained at the 16th cellculture passage (from Dr. E.C. Pirtle,National Animal Disease Center,Ames, Iowa); the NVSL 85-12 strainwas obtained at the third cell culturepassage (from Dr. R.L. Levings,National Veterinary Services Labora-tories, Ames, Iowa). Three commer-cially available modified live virus(MLV) vaccine strains (PR-Vac, Nor-den Laboratories, Lincoln, Nebraska;PseudoVax, Pitman-Moore, Washing-ton Crossing, New Jersey; Pseudo-rabies Vaccine, BioCeutic Laboratories,St. Joseph, Missouri) were rehydratedaccording to the manufacturer's in-structions before inoculation onto cellcultures. Viral stock cultures were pre-pared in MDBK cells (American TypeCulture Collection, ATCC-CCL-22)and titrated by plaque assay in six wellplates (Linbro, Flow Laboratories,Hamden, Connecticut). MDBK andLMTK- cells (12) were grown at 37°Cin Eagle's minimal essential medium(MEM) with Earle's salts and L-glu-tamine (GIBCO Laboratories, GrandIsland, New York) supplemented with10% heat-inactivated fetal bovine se-rum (FBS) and 50 ,g/mL gentamicinsulfate (GIBCO).

ISOLATION OF TK5AI PRV

The IN-Fh strain was cultivated forone passage in MDBK cells with me-dium containing 5,g/mL bromode-oxyuridine (BUdR) (Sigma ChemicalCo., St. Louis, Missouri) and a secondpassage in MDBK cells with mediumcontaining I0 ,g/mLBUdR,in MDBKcells. The resulting virus was plaquedunder 1% agarose overlay (SeaKemME, Marine Colloids Division, FMCCorp., Rockland, Maine) containing10 Atg/mL BUd R. Several large plaques

were picked and grown once in MDBKcells with medium containing 100 Ag/mLBUdR and twice more with mediumcontaining 100,ug/mL BUdR and200 ,ug/mL arabinofuranosylthymine(ara-T) (Sigma). Clones were plaque-purified twice at 38.50C, tested for TKactivity, and a stock culture of TK5A Iwas made by an additional passage inMDBK cells without BUdR or ara-T.

DETERMINATION OF REVERSION OF MUTANTTK5AI TO THE TK+ PHENOTYPE

To determine if mutant TK5A Ireverts to TK+ under nonselectiveconditions, a 75 cm2 confluent mono-layer of MDBK cells was inoculatedwith TK5AI PRV at a multiplicity ofinfection (MOI) of 0.01 to 0.1, andincubated at 37°C with MEM contain-ing 5% FBS, until 100% CPE wasobserved. Virus in the supernatant wassimilarly grown through ten passagesand tested for TK activity by thymidineplaque autoradiography. To select forTK+ PRV in the stock culture ofTK5A1, either 2.0 x 105 PFU or 1.2 x

107 PFU was inoculated onto a 75cm2monolayer of LMTK- cells and incu-bated in MEM containing 5 x 10-5 Mhypoxanthine, 5 x 10-7 M methotrexate(Lederle Parenterals Inc., Carolina,Puerto Rico), 1 x 10-6M thymidine andI x 10-6M glycine (HMTG) (13,14) forthree days at 370 C. The culture wasfrozen at -70°C, thawed, centrifugedto remove cell debris, and I mL ofsupernatant inoculated onto a secondLMTK- monolayer. The second and athird blind passage in LMTK- cellswith MEM containing HMTG wereconducted, and supernatant from thethird passage was tested for the pres-ence of PRV in MDBK cells withoutHMTG in the growth medium.

THYMIDINE KINASE ASSAYS

The TK phenotype of PRV strainswas determined by the thymidineplaque autoradiography method ofTenser et al (15). Confluent mono-layers of LMTK- cells grown in 60 mmculture dishes (LUX, Lab-Tek, Naper-ville, Illinois) were inoculated with 50to 150 PFU of PRV. Plates were over-layed with 0.5% carboxymethyl cellu-lose (Sigma) and incubated at 370 C for72 h. After removal of the overlay,monolayers were incubated for 6 hwith 2mL MEM containing 1.O,Ci

'4C-thymidine (55 mCi/ mmol, Amer-sham, Arlington Heights, Illinois),then fixed and stained with 0.1% crystalviolet in 10% formalin. After removalof the dish rims, monolayers were ex-posed to x-ray film (XAR-5, EastmanKodak, Rochester, New York) forthree to seven days.

Kinetic assays of PRV TK in cytosolextracts were performed as describedby Zavada et al (16), except thatLMTK- cells were used. For each ex-tract, one 75 cm2 confluent monolayerof LMTK- cells was inoculated withPRV at a MOI of 5 to 10, and incu-bated for 18 h at 370C. Infected cellsfrom each 75 cm2 culture were lysed inI mL lysis buffer. Substrate (14C-thymidine, 0.5 ,uCi/ mL, 55 mCi/ mmol,Amersham) concentrations rangedfrom 1.9 to 28.8 ,uM. Enzyme reactionswere incubated at several time inter-vals ranging from 0 to 20 minutes in a37°C water bath, stopped by immer-sion in boiling water for three minutes,and centrifuged to remove denaturedprotein. Fifty ,uL volumes of reactionsupernate were spotted onto 2 x 2cmsquares of Whatman DE-8 1 paper(VWR Scientific, San Francisco, Cali-fornia). Papers were air dried, washed,and counted in 10mL of scintillant(Ready-Solv HP, Beckman Instru-ments, Fullerton, California). Km andVmax values for viral TK were deter-mined from Lineweaver-Burk plots(17).

MOUSE VIRULENCE TEST

Tenfold dilutions of PRV stockswere made in MEM and 0.2mL wasinoculated intraperitoneally (IP) intoeach of five to six week old femaleCF-I mice (Sprague-Dawley, Indiana-polis, Indiana). Groups of five or tenmice were inoculated per dilution.Inoculated mice were observed dailyfor 14 days, and LD50 values determ-ined by the method of Reed andMuench (18).

RESTRICTION ENZYME ANALYSIS

Viral DNA was prepared fromPRV-infected MDBK cells by themethod of Van Alstine et al (3), withslight modifications (As modified byD. R. Jutting, National VeterinaryServices Laboratories, Ames, Iowa).Briefly, virus was pelleted through40% sucrose in 0.2 M phosphate

335

buffer, resusspended in LCM buffer(5% Na desoxycholate; 30mM Tris-HCI, pH 7.5; 125mM KCI; 0.5mMNa2EDTA; 3.6 mM CaCl2; 5mMMgCl2, 5% NP-40; 46,L mercapto-ethanol/ 100 mL) and extracted twice-with 1 mL Freon (1,1,2-trichloro-1,2,2-trifluoroethane). The viral pre-paration was layered over a 5 to 45%glycerol gradient (in LCM buffer) andcentrifuged at 100,000 x g for 1 h. Viralpellets were resuspended in TE bufferfor DNA extraction as described pre-viously (3). Following the phenol ex-tractions, 0.1 volume 3 M Na acetatewas added and the DNA precipitatedby the addition of 2.5 volumes of cold(-20° C) absolute ethanol and over-night storage at -20° C. Following cen-trifugation at 6000 x g for ten minutes,the ethanol was decanted, the pelletwas air dried, and was resuspended inI mL TE buffer. Viral DNA was di-gested with restriction endonucleasesBamHI (Bethesda Research Labora-tories, Gaithersburg, Maryland), KpnI,or PstI for 4 h, followed by electro-phoresis through 0.8% agarose for 24 hat 40 V. Bacteriophage lambda DNA(Bethesda Research Laboratories) di-gested with HindlIl was used as themolecular weight marker.

RESULTS

ISOLATION AND CHARACTERIZATIONOF MUTANT TK5AI

Mutant TK5AI was selected fromthe virulent IN-Fh strain of PRV forresistance to BUdR and Ara-T, andwas determined to have a TK-phenotype (Fig. 1). Mutant TK5AItitered equally in MDBK cells at 37°Cand 38.5°C and retained the TK-phenotype through ten passages inMDBK cells (Fig. 2). No virus wasrecovered from third passage super-natant containing HMTG, either fromthe initial inoculum of 2.0 x 105 PFU or1.2 x I07 PFU. In contrast, IN-Fh PRVreplicated well under selective condi-tions, and was present at high concen-tration after three passages in LMTK-cells and MEM containing HMTG.

DETERMINATION OF TK ACTIVITY OF VIRULENTAND MODIFIED LIVE VIRUS VACCINE STRAINSOF PSEUDORABIES VIRUS

The two virulent and three com-mercial MLV vaccine strains of PRVwere shown to have TK+ phenotypes

1 w

*..~~~~~~~~~~~W.. ...:..I.

..~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.. ......

A :BFig. 1. Thymidine plaque autoradiography results showing the TK+ phenotype ofPRV strain IN-Fh(3) and the TK- phenotype of mutant TK5AI (2). Dish 1: uninfected LMTK cells. A: crystal violet-stained cell monolayers. B: autoradiogram after exposure to cell monolayers incubated with '4(C)thymidine for six hours.

TABLE I. Kinetic Properties of Pseudorabies Virus Thymidine Kinase

Virus Strain

NVSL 85-12

IN-Fh

Norden

Pitman-Moore

BioCeutic

Km (jim)

3.53.33.7

4.24.03.3

3.02.92.7

3.74.04.0

3.22.72.8

aReciprocal of counts per minute, x 10-3bMean ± SD

336

Km Mean

3.5 ± 0.20b

3.8 ± 0.47

2.9 ± 0.15

3.9 ±0.17

2.9 ± 0.26

Vmax

3.3a4.52.5

2.83.03.4

5.05.22.9

5.55.53.8

6.76.71.7

Vmax Mean

3.4 ± 1.0

3.1 ±0.3

4.4 ± 1.3

4.9 ± 1.0

5.0 ± 2.9

A BFig. 2. Thymidine plaque autoradiography of tenth passage TK5A1 PRV (plates 1,2,3,5,6) andIN-Fh (plate 4). A: crystal violet-stained cell monolayers. B: autoradiogram after exposure to cellmonolayers exposed to medium containing 14(C) thymidine for six hours prior to fixation.

A B

Fig. 3. Thymidine plaque autoradiography results for the Bioceutic (1), Pitman-Moore (2), Norden(3), IN-Fh (4) and NVSL 85-12(6) PRV strains. PlateS5 uninfected LMTK- cells. A: cell monolayersstained with crystal violet. B: autoradiogram after exposure to cell monolayers exposed to 14(C)thymidine medium for six hours.

(Fig. 3). Lineweaver-Burk plots (Fig. 4)were drawn from initial velocity dataobtained from PRV-infected LMTK-cell lysates to determine the Km valuesand maximum velocities of viral TKfor the virulent and MLV PRV strains(Table I). Km values for thymidineranged from 2.9 to 3.9 MM.

MOUSE VIRULENCE

The six PRV strain included in thestudy were examined for virulence forthe mouse. Mortality data are shownin Table II. The MLV vaccine strainswere reduced in virulence for themouse approximately two to tenfoldrelative to the virulent strains. MutantTK5A I was avirulent for the mouse byintraperitoneal inoculation.

RESTRICTION ENZYME ANALYSIS

Viral DNAs from the six PRVstrains included in the study were di-gested by the restriction endonucleasesBamHI, KpnI and PstI (Fig. 5). Twoof the MLV vaccine strains (Nordenand Pitman-Moore) were shown to bevery similar in migration patternswhen examined with all three enzymes.The BioCeutic PRV strain differedconsiderably from all other strainswhen digested with BamHI. All threeMLV vaccine strains lacked the largestPstI fragment characteristic of theother three strains. Two BamHIfragments of mutant TK5A 1 increasedin molecular weight relative to theparental IN-Fh strain.

DISCUSSION

Previous studies have shown thatmodified live PRV vaccine strains cancause mortality in species other thanswine (1,2,3). Ericksen et al (19) haverecently reported mortality in cattlefollowing vaccination with a modifiedlive PRV vaccine. These reports indi-cate that the need exists to gain furtherknowledge of the genetic basis of PRVvirulence, so that an efficacious yetsafer vaccine might be producedthrough selective mutagenesis.

In the present study we have shownthat commercially available modifiedlive PRV vaccine strains retain con-siderable virulence for the mouse(Table II), a species commonly used insuch studies (20,21) and all possessTK+ phenotypes (Fig. 3). In contrast,the drug resistant mutant TK5AI was

337

* Rep 1* Rep 2

* Rep 3

-35 -30 -25 -20 -15

9.0 _ a

8.0

7.0 -

6.x

-10 -5 0 5 10 15 20 25 30 35

1 /(Thymidine), AM-' x 1 Q4

Fig. 4. Lineweaver-Burk plot (17) of initial velocity data from three replications of NVSL 85-12PRV-infected cell lysates.

Fig. 5. Restriction endonuclease analysis of the NVSL 85-12 (1), IN-Fh (2), TK5A1 (3), Norden (4),Pitman-Moore (5) and BioCeutic (6) strains of PRV. Digestion was performed with restrictionendonucleases BamHI, KpnI and PstI. Arrows indicate BamHl restriction endonuclease fragmentsthat varied in electrophoretic mobility between strain IN-Fh and mutant TK5A1.

shown to lack TK activity and wasavirulent for the mouse. This finding isin agreement with previous studiesassociating PRV TK activity withvirulence (8,22). Studies by Fraser andRamachandran (20) and Field and Hill(23) demonstrated that the nerveganglia and the CNS are the principlesites of PRV replication in the mouse,and McCracken et al (24) similarly

documented the neuronal spread ofPRV in calves. Studies with TKmutants of the related herpesvirus,HSV, suggested that viral TK wasessential for establishing a neuronalinfection (7,8,25). The findings pre-sented here support the role for PRVTK in determining virulence.

Herpesvirus mutants resistant todrugs which act specifically with viral

TK may express an enzyme withaltered kinetic behavior. McLaren et al(26) have characterized mutants resist-ant to acyclovir and have found themto express TKs having significantlyincreased Km values for thymidine.An increase in the Km value wouldalso probably be associated with adecrease in neurovirulence. In thepresent study we have shown that theKm values ofTK encoded by the MLVvaccine strains to be similar to thevalues of TK encoded by virulentstrains (Table I). The minor differ-ences noted are not likely to have anybiological significance.

Restriction enzyme (RE) analysisrevealed that all three MLV vaccinestrains lack the distinct PstI A frag-ment (highest molecular weight) char-acteristic of the NVSL 85-12, IN-Fhand TK5A1 strains (Fig. 5). Geck el al(4,27) observed that the absence of thePstI A fragment was a marker for theBartha K strain, and could be used todistinguish it from other avirulent andvirulent strains of PRV. Lomniczi et al(22) used marker rescue of wild-typeDNA sequences to restore the deletionin the PstI A fragment of the Barthastrain, which also restored wild-typevirulence. Although the gene withinthe PstI A fragment influences viru-lence, particulary for swine, the datapresented here indicate that PRV TKplays a much larger role in determiningvirulence for mice.

It was also observed that two BamHI fragments of mutant TK5A1 hadincreased in molecular weight relativeto the parental, TK+, strain (Fig. 5).Ben-Porat et al (28) also noted REpattern differences which existed be-tween a TK- PRV mutant and its paren-tal strain. Ben-Porat reported reitera-tions offragments below the BamHI Dfragment of the TK- mutant similar tothose observed in the Norden andPitman-Moore PRV patterns in thepresent study. Ben-Porat concludedthat the change in the RE pattern maybe related to a biological functionassociated with the loss ofTK activity.In the present study, changes in REpattern similar to those observed byBen-Porat did not occur. Wathen andPirtle (29) observed BamHI RE pat-tern changes after passage of a strain ofPRV in vivo similar to the changesobserved in the present study; theyobserved that the fragments which

338

TABLE II. Mortality in Mice after Inoculation with Virulent and Attenuated Strains of PRV

Dilution of Virus (Log,0)Virus strain Titera 0 -I -2 -3 -4 -5 -6 -7 PFU/LD50oc

NVSL85-12 1.4 x 108 29/30d 22/30 6/30 0/10 105IN-Fh 3.4 xIO7 19/20 24/30 3/30 1/30 270Norden 7.6 x 106 9/10 4.10 0/10 265Pitman-Moore 2.7 x 10' 9/10 4/10 0/10 941BioCeutic 1.1 x 107 9/10 5/10 0/10 274TKSAI 3.3 x I07 0/15 0/15 >107'PFU / mLbTwo-tenths mL inoculum/ mousecLD50calculated by the method of Reed and Muench (18)dNumber dead/ number inoculated

changed in electrophoretic mobilitymap to the inverted repeat sequences,which is a region associated with a highfrequency of molecular weight changesof restriction fragments. It is unlikelythat the RE pattern differences ob-served are associated with the changein TK phenotype.

In summary, the data presentedhere indicate that selection of a TK-mutant from a virulent strain of PRVmay be a route toward a vaccine ofincreased safety, but which wouldprobably retain the antigenicity of itswild-type parent. Studies on the effi-cacy of mutant TK5A1 as a vaccinewill be described in a separatemanuscript.

Interestingly, vaccinia virus TK hasalso been shown to be a determinant ofvirulence (30), and which also may beexploited as a selectable phenotype increating recombinant vaccines (30,31).

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