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

Zhu Ling, Yi Yue, Xu Zhiwen, Cheng Lu, Tang Shanhu, Guo, Guo Wanzhu

Description

PRV Fa is common in China and causes most of the pseudorabies in the pig industry. A PRV SA215 strain with deleted gE, gI, and TK genes was constructed to develop a commercial attenuated live vaccine. However, the physicochemical properties, growth pattern, penetration kinetics, and morphogenesis of the PRV SA215 and its parental PRV Fa strain are unclear. Results A series of experiments were conducted to characterize both strains and provide more information. PRV Fa and PRV SA215 were found to have similar penetration patterns, with about 5 min half-time of penetration. The SA215 strain exhibited a slight delay in entry compared with PRV Fa. In the one-step growth test, the titers of the SA215 strain were first detected at 8 h, rapidly increased, and peaked at 12 h. A plateau was formed between 12-36 h of culturing. PRV SA215 showed delayed replication and approximately 10-30-fold lower titers during 0-16 h of culturing compared with the PRV-Fa strain. After 16 h, the PRV Fa titers dramatically decreased, whereas those of PRV SA215 were prolonged to 36 h and reached a titer value equal to that of PRV Fa and then decreased. Both strains were sensitive to both heat and acid-alkali treatments; however, PRV Fa was relatively more stable to heat treatment than PRV SA215. Both strains could propagate in the cultures with pH values from 5.0 to 9.0. Cultures with pH below 3.0 or above 11.0 were fatal to both strains. Both strains had considerable resistance to freeze-thawing treatments. Morphogenetic investigations showed that typical phases in the maturation pathway were observed in the PRV Fa-infected PK15 cells, whereas secondary envelopment was not observed in the PRV SA215 strain. Instead, capsid aggregations with concomitants of electrodense materials were observed. Conclusions These results suggest that PRV SA215 is a promising strain for vaccine development

Sujets

• Pseudorabies
• MOrphogenesis

Informations

• Publié par: biomed
• Publié le: 01 janvier 2011
• Nombre de lectures: 686
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

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:[e-mail]
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
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VirologyJournal
2011,
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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
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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