Growth, physicochemical properties, and morphogenesis of Chinese wild-type PRV Fa and its gene-deleted mutant strain PRV SA215

Zhu
etal
.
VirologyJournal
2011,
8
:272
http://www.virologyj.com/content/8/1/272

RESEARCH

OpenAccess

Growth,physicochemicalproperties,and
morphogenesisofChinesewild-typePRVFa
anditsgene-deletedmutantstrainPRVSA215
LingZhu
1
,YueYi
1
,ZhiwenXu
1
,LuCheng
1
,ShanhuTang
2
andWanzhuGuo
1*

Abstract
Background:
PRVFaiscommoninChinaandcausesmostofthepseudorabiesinthepigindustry.APRVSA215
strainwithdeletedgE,gI,andTKgeneswasconstructedtodevelopacommercialattenuatedlivevaccine.
However,thephysicochemicalproperties,growthpattern,penetrationkinetics,andmorphogenesisofthePRV
SA215anditsparentalPRVFastrainareunclear.
Results:
Aseriesofexperimentswereconductedtocharacterizebothstrainsandprovidemoreinformation.PRV
FaandPRVSA215werefoundtohavesimilarpenetrationpatterns,withabout5minhalf-timeofpenetration.The
SA215strainexhibitedaslightdelayinentrycomparedwithPRVFa.Intheone-stepgrowthtest,thetitersofthe
SA215strainwerefirstdetectedat8h,rapidlyincreased,andpeakedat12h.Aplateauwasformedbetween12-
36hofculturing.PRVSA215showeddelayedreplicationandapproximately10-30-foldlowertitersduring0-16h
ofculturingcomparedwiththePRV-Fastrain.After16h,thePRVFatitersdramaticallydecreased,whereasthoseof
PRVSA215wereprolongedto36handreachedatitervalueequaltothatofPRVFaandthendecreased.Both
strainsweresensitivetobothheatandacid-alkalitreatments;however,PRVFawasrelativelymorestabletoheat
treatmentthanPRVSA215.BothstrainscouldpropagateinthecultureswithpHvaluesfrom5.0to9.0.Cultures
withpHbelow3.0orabove11.0werefataltobothstrains.Bothstrainshadconsiderableresistancetofreeze-
thawingtreatments.Morphogeneticinvestigationsshowedthattypicalphasesinthematurationpathwaywere
observedinthePRVFa-infectedPK15cells,whereassecondaryenvelopmentwasnotobservedinthePRVSA215
strain.Instead,capsidaggregationswithconcomitantsofelectrodensematerialswereobserved.
Conclusions:
TheseresultssuggestthatPRVSA215isapromisingstrainforvaccinedevelopment
Keywords:
pseudorabiesvirus,gene-deletedmutantstrain,growthandphysicochemicalproperties,
morphogenesis

Background
infectionisoftenfatal,andanimalsdiefromcentralner-
The
pseudorabiesvirus
(PRV)isamemberofthevoussystemdisorders,whereasolderinfectedpigs
Alphaherpesvirinae
subfamilyinthefamily
Herpesviri-
usuallyprimarilydeveloprespiratorysymptoms.Similar
dae
.ItisthecausativeagentofAujeszky
’
sdisease[1].tootheralphaherpesviruses,PRVinfectionisalife-long
ThediseasecausedbyPRVwasfirstobservedincattlelatentinfectionoftheperipheralnervoussystem.These
anddescribedas
“
maditch
”
[2].Swineistheprimarylatentlyinfectedpigscanserveassourcesofrenewed
hostandreservoirofthisvirus.PRVisabletoinfectinfectionwhenthelatentvirusgenomespontaneously
mostmammalsandsomeavianspecies.Inyoungpig-reactivatesandinfectiousvirusesareproduced.Inpreg-
lets,aswellasintheothersusceptiblespecies,PRVnantsows,PRVinfectionsmaycausethedeathof
fetusesand/orabortion.Thus,PRVisapathogenwith
majoragriculturaleffectsandeconomicimportance[3].
*
1
Correspondence:wzguo@126.com
Pigsarecommonlyvaccinatedwithattenuatedlive
AnimalBiotechnologyCenterofSichuanAgriculturalUniversity,Yaan,
PRVvaccines[4]tocontrolthedisease.Thesevaccine
Sichuan(625014),PRChina
Fulllistofauthorinformationisavailableattheendofthearticle
©2011Zhuetal;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommons
AttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,andreproductionin
anymedium,providedtheoriginalworkisproperlycited.

Zhu
etal
.
VirologyJournal
2011,
8
:272
http://www.virologyj.com/content/8/1/272

strainsareattenuatedbyinactivationofoneormore
genesthatencodenonessentialproteins.Studiesfound
thatglycoproteingEisdispensableforviralgrowthin
animalsorinthecultures[5,6];however,itisnecessary
forvirulenceandvirionreplication[7,8],andplaysan
importantroleinvirionspreadfromcelltocell.Avirus
withonlyadeletionofthegEgeneiscapableofrepli-
cating
invivo
and
invitro
withconsiderablevirulence.
Thus,mostofthecommercializedattenuatedlivevac-
cinesareconstructedbasedongEdeletioncombined
withoneormoreofothergenedeletionssuchasTK
[9].However,thiskindofvaccinehasanattenuated
virulenceandrestrictedinfectivity.Inactivationofviral
geneswasreportedtooftenresultinreducedreplication
capacity.ThehighlyvirulentstrainNIA-3,theinterme-
diatelyvirulentstrain2.4N3A,andthenon-virulent
strainBarthawerecomparedinastudyofthepatho-
genicityofdifferentPRVstrains.Theresultsshowed
thatthedegreeofvirulenceisdirectlyrelatedtothe
abilityofthesevirusestoreplicateinthenasalepithe-
lium[10].Reducedreplicationlowerstheviralantigen
supplyandmayimpairtheimmunogenicityofthe
vaccine.
AgenedeficientstrainSA215(gE-/gI-/TK-)wascon-
structedinourlaboratorybasedonaChinesePRVFa
strain[11].ThePRVFastrainistheearliestisolated
typicalstrainthatcausedtheprevalenceofpseudorabies
inChinaandcausedlargeeconomiclossesinthepig
industry.Thebiologicalandphysicochemicalproperties
andmorphogenesisofbothstrainsarestillunclear.Pre-
liminarystudieshaveshownthatthePRVSA215strain
hasbiologicallysecurepropertieswithhighimmuno-
genicityandlong-termimmunity,andcouldbedevel-
opedasacommercialattenuatedlivevaccine.However,
itsgrowth,physicochemicalproperties,andmorphogen-
esisduringmultiplicationhavenotbeeninvestigated.In
thecurrentpaper,theresultsofrecentstudiesonthe
physicochemicalpropertiesandgrowthpattern,penetra-
tionkineticsandmorphogenesisarepresentedto
providemoreinformationabouttheChinesetypical
wild-typePRVFastrainanditsprogeny,thePRV
SA215strain.
Materialsandmethods
Virusesandcells
ThePRVSA215strainwasconstructedbyourcenter
(AnimalBiotechnologyCenterofSichuanAgricultural
University)basedontheChinesePRVFastrain.PRV
SA215carriesgE,gI,andTKgenedeletions.The
stronglyvirulentwild-typePRVFastrainwasobtained
fromtheInstituteofChinaVeterinaryMedicineInspec-
tion.PK15cellsandVerocellswerepurchasedfrom
CCTCC.DMEM,trypsin,andfetalcalfserumwerepur-
chasedfromGIBCOInc.Methylcellulosewasobtained

Page2of9

fromSigmaInc.Verocellswereusedinthevaccine
strainpropagation,plaqueformationtest,isolationand
titrationofthePRVvirusinnasaldischarges,andsera
neutralizationassays.
PRVPassaging
VerocellswereculturedinDMEMat37°Cand5%
CO
2
.Thenutrientsolutioncontained10%fetalcalf
serumand100U/mlofpenicillinandstreptomycin.
Thegrowthliquidswerepouredoutwhenadensecell
monolayerwasformedinthecultureflasks.Thecell
monolayerswerewashedtwicewithcalcium-andmag-
nesium-freewater.1-2dropstrypsinwereaddedinto
eachflasktotrypsinizethecells.Thecellsweresplit1:2
forsubsequentcultures.
Plaqueformationtest
Thetestwascarriedouttomeasurevirustitrationfol-
lowingourregularlaboratoryprotocol.Briefly,theVero
cellsweregrownon6-wellplatesuntiltheappearance
ofcellmonolayers.Thevirussampleswerediluted10
timesandthesampleswithappropriateviraltiterswere
selectedforinoculation,and0.1mlofthissamplewas
addedintoeachwell.Thesubjectculturesamplesinthe
platesweregentlyshakentomixthesampleswelland
allowedtoadsorbfor1hat37°C.Asolutionof1%
methylcellulosewasaddedtothewellstocoverthecul-
turemedium.Afterthetestedsampleswereculturedfor
3-5daysat37°CinaCO
2
-controlledincubator,the
methylcellulosewasaspiratedand1mlofformalincrys-
talpurplestainingsolutionwasaddedtofixandstain
thesamplesfor20min.Thestainingsolutionwas
removedbywashingwithtapwater.Theplaqueswere
countedandthePFUwerecalculatedbasedonthe
volumesoftheoriginalsamples.
Penetrationkinetics
Thepenetrationkineticsofbothstrainswasassayed
usinglow-pHinactivationoftheextracellularvirus
basedonthemethoddescribedbyMettenleiter[12].
Briefly,PRVFaandPRVSA215wereinoculatedonthe
PK15monolayercells.Theinputvirusamountwasca.
200PFUperwellinasix-welltissuecultureplate.
Afteradsorptionfor1hat4°C,theinoculantswere
removedandthecellswerecoveredwithnutrientsolu-
tionat37°Ctofacilitateviruspenetration.AlowerpH
solution(pH3.0,40mMcitricacid-10mMKCl-135
mMNaCl)wasaddedtothewellsfor2mintoinacti-
vatetheextracellularvirusesafter0,5,10,20,and40
minofpenetration.ThelowerpH-treatedcellmono-
layerswerewashedtwiceandcoveredwithmethylcel-
lulosesolution.Afterculturingfor2-3days,thecells
werefixedandstained.Theplaqueswerecountedand
thepenetrationpercentageswerecalculatedby

Zhu
etal
.
VirologyJournal
2011,
8
:272
http://www.virologyj.com/content/8/1/272

comparingwiththenumberofplaquesformedinthe
PBS-treatedcontrol.
One-stepgrowthanalysis
One-stepgrowthanalysiswasperformedaccordingto
themethoddescribedbyKluppetal.[13].ThePK15
monolayercellswereinfectedwithPRVFaandPRV
SA215at0.005multiplicityofinfection(MOI)ineach
wellofasix-welltissuecultureplate.Theviruseswere
allowedtoadsorbfor1hat4°C.Theextraviruseswere
removedandtheinfectedcellswerecoveredwithnutri-
entsolutionat37°C.After90minofcultureandpene-
tration,theextracellularviruswasinactivatedwithalow
pHsolution.Themaintenancegrowthsolutionforthe
cellswasthenswitched.Thecellsupernatesandt