N.J. Young-BVDV荧光定量

1、JournalofVirologicalMethods138(2006)218222ShortcommunicationReal-timeRT-PCRdetectionofBovineViralDiarrhoeavirusinwholebloodusinganexternalRNAreferenceN.J.Young,C.J.Thomas,M.E.Collins,J.BrownlieDepartmentofPathologyandInfectiousDiseases,TheRoyalVeterinaryCollege,HawksheadLane,NorthMymms,Hateld,Hertfo

2、rdshireAL97TA,UKReceived21March2006;receivedinrevisedform3August2006;accepted29August2006Availableonline9October2006AbstractAnoveltwo-stepreal-timeRT-PCRassayusingSYBR®GreenIwasdevelopedforthedetectionofacuteBovineViralDiarrhoeavirus(BVDV)infectioninwholebloodfromcattle.Duringinfectionanimalsex

3、perienceacharacteristictransientleucopeniaandthenumberofcellspervolumeofbloodchangesovertime;soquantitationofviralloadbyreferencetoacellularhousekeepinggeneisnotidealasthismayhidesignicantanimaltoanimalvariation.Therefore,tofacilitatecomparisonofdifferentsamples,anexternalRNAreferencewasusedfornorma

4、lisationwherebyeachsamplewasspikedwiththeRNAvirus,CanineEntericCoronavirus(CECov),priortoRNAextraction,forcomparativepurposes.Real-timeRT-PCRwascarriedoutwithtwoprimersetsdesignedtoamplifyeithera156bpregionoftheBVDV5 -UTRora280bpregionoftheCECovnucleocapsidproteingene.Linearityandefciencyoftheassayw

5、asestablishedandthemethodassessedusingsamplesfromBVDV-challengedcalves.ViralRNAwasquantiedondays6and14post-challengebyreal-timeRT-PCR.Infectiousvirusisolationbytraditionalcellculturewasnegativeafterday7.Thisstudydemonstratesencouragingresultsforrapid,sensitiveandreliabledetectionofacuteBVDVinfection

6、andprovidesanalternativereal-timeRT-PCRmethodforuseonwholebloodsamplesorsampleswheresuitablehousekeepinggenesarenotavailable.©2006ElsevierB.V.Allrightsreserved.Keywords:Real-timeRT-PCR;BovineViralDiarrhoeavirus;Pestivirus;CanineEntericCoronavirusBovineViralDiarrhoeavirus(BVDV)isapathogenofworld

7、wideimportanceforcattle,causingsignicanteconomiclosses(Brownlie,1991).BVDVisclassiedwithinthePes-tivirusgenusoftheFlaviviridae(Heinzetal.,2000).Pestivirusesaresmall,envelopedvirusesthatcontainasingle-strandpositivesenseRNAmoleculeofapproximately12.5kb.Thegenomeistranscribedasasingleopenreadingframe,

8、ankedby5 -and3 -untranslatedregions(UTRs).TheBVDVpolyproteinisco-andpost-translationallycleavedtogiverisetoanumberofviralstructuralandnon-structuralproteins(Collettetal.,1988).AcuteBVDVinfectionisoftenclinicallyunapparentormild;clinicalsignsincludingtransientpyrexia,leucopenia,oculonasaldischargeand

9、depression.Althoughfrequentlyunnoticed,acuteinfectioncancontributetoorcauseavarietyofproblemsincludingdiseasesofthereproductive,respiratoryandentericsystems(Baker,1995).BVDVinfectionduringpreg-Correspondingauthor.Tel.:+441707666811;fax:+441707661464.E-mailaddress:nyoungrvc.ac.uk(N.J.Young).nancymayr

10、esultinfoetalinfection.Foetusesinfectedbeforetheonsetofimmunocompetencecanbecomepersistentlyinfected.Persistentlyinfected(PI)animalscontinuouslyshedvirusandactasamajorreservoirofinfectionforothercattle(Patonetal.,1999).Currentlaboratory-basedBVDVdetectionmethodsincludeserologicalassays,virusisolatio

11、n,antigendetectionanddetec-tionofviralRNAbyreversetranscription(RT)PCR(Sandvik,2005).Real-timePCRisnowbeingusedtoidentify,classifyandquantifymanyviralpathogensasitisahighlysensitiveandrapidmethodfordetectingviralnucleicacidsequencesinclinicalspecimens.Furthermore,real-timePCRisaquantita-tivetechniqu

12、eandassuchmaybeusedtoassessviralRNAlevels.Real-timeRT-PCRmethodsforgenotypingBVDVhavebeendescribedpreviously(BhudeviandWeinstock,2001,2003;LettellierandKerkhofs,2003).ThesepapershavefocusedonthedetectionandclassicationofBVDVintoeitherofthetworecognisedgenotypes(BVDV-1andBVDV-2)ratherthanmon-itoringt

13、hechangesinviralloadduringthecourseofanacuteorpersistentinfection.However,asreal-timePCRisaquantitative0166-0934/$seefrontmatter©2006ElsevierB.V.Allrightsreserved.doi:10.1016/j.jviromet.2006.08.008N.J.Youngetal./JournalofVirologicalMethods138(2006)218222219techniqueitmayalsobeusedtoassessviralR

14、NAlevelsduringthecourseofinfectionandrecovery.Usingreal-timePCRtoquantifytheviralRNAlevelsinBVDV-infectedanimalspresentsparticulartechnicalandbio-logicalchallenges.Collectionandprocessingofsamplesfromdifferentanimalsoveranumberofdifferentdaysinevitablyresultsinsomedegreeofsampletosamplevariationinth

15、etechnicaldetectionofthetargetnucleicacids.ThisarisesfromdifferencesinefciencyofRNAextraction,reversetranscrip-tionorPCRreactionswhichismadeparticularlysignicantbythesensitivityofthistechnique.Thesetechnicalvariationsareoften“controlled”forbynormalisationofthesignaltoaninter-nalstandard,typicallya“h

16、ousekeeping”gene,facilitatingthecomparisonofdifferentdatasets.Thispresentstwofurtherprob-lems:rstly,whilstthisiscommonpractice,suchhousekeepinggenesarerarelytrulystaticandmayalterunderexperimentalconditions(Thellinetal.,1999;Whelanetal.,2003).Totakeaccountofthis,bestpracticewouldrecommendassessingth

17、evalidityofanumberofdifferenthousekeepinggenetargetsandcomparingthemunderthetestconditionsused.ThislaborioustaskistheonlywaytoobjectivelyidentifymorestablemRNAsets.However,thisprocessisnotrelevanttotheBVDVsituationduringinfectionas,secondly,duringacuteinfectionwithBVDVanimalsexperienceameasurableleu

18、copeniaandthenumberofcellspervolumeofbloodchangesovertime.Traditionalmeth-odsofvirusisolationquantitatetheamountofvirusrecoveredfromaxedvolumeofbloodirrespectiveofthecellnumbersinthissample,whichduringacuteBVDVinfectionwilluc-tuate(PolakandZmudzinski,2000).BystandardisingtheviralRNAmeasuredbyreal-ti

19、mePCRtoahousekeepinggenesetthequantitationisessentiallydoneonapercellbasisratherthanapervolumebasisrenderingthecomparisonofsamplesfromleucopeniccalvesmoredifcultandprecludingasimplecomparisonwithmoretraditionalvirologicalmethods.Toovercomethesedifcultiesanoveltwo-stepreal-timeRT-PCRmethodusinganexte

20、rnalRNAreferencestandardwasdeveloped,whichhasbeenevaluatedonsamplesfromexperi-mentallyinfectedcattle.EachsamplewasspikedwithaknownamountofasecondRNAvirus,CanineEntericCoronavirus(CECov),priortoRNAextraction.Whiletheexternalstandardisnotanexactmimicofthetruetargetitissubjecttothesameexperimentalproce

21、duresandprovidesanexternalstandardforcorrectionofdifferencesintheefciencyofRNAextractionorRT-PCR.Furthermore,tofacilitateeaseofsamplehandlingthemethodwasbasedonquanticationofBVDVRNApervolumeoffrozenwholeblood.FiveHolsteinFriesiancalvesranginginagefrom2to4monthswereobtainedfromaBVDVfree-supplierandte

22、stedtoconrmBVDV-freestatusbyantigenandantibodyELISA.Thecalveswerehousedinapurpose-builtbarnwithappropriatebio-securitymeasuresinplacetoavoidexposuretoadventitiouspathogens.Afteraninitialacclimatisationperiodthecalveswerechallengedintranasallywith2×106TCID50ofvirus456497(earlypassageBVDV-1;kindl

23、ydonatedbyVLA,Weybridge,UK).EDTAbloodsamplesweretakenatappropriatedayspost-challengetoprocessforvirusisolationandreal-timePCR.AllEDTAbloodsamplestakenforvirologywerepreparedbylysisoftheerythrocytesusingammoniumchloridelysisbuffer.Thesamplesweresubsequentlypelletedandwashed,asdescribedinNobironetal.(

24、2003).TodetectBVDV,thesampleswerediluted1:5inmaintenancemediumandthenseeded,induplicate,ontofreshlypreparedfoetalbovinelung(FBL)cellsandincubatedfor5daysat37C,5%CO2.Afteroneroundoffreezethawanddilutionofmaterial,theinoculumwaspassedoncemoreontoFBLs,whichhadbeenpreviouslyseededontocoverslips,andincub

25、atedforafurther5days.Presenceofviruswasdetectedviaimmuno-uorescencestainingusingaBVDVhyperimmuneserumandaCy-3-conjugatedanti-bovineantibody.Scoringwasperformedusingauorescencemicroscopeandappropriatel-tersets.RNAwasextractedfromfrozenwholeblood(allowingsam-plesfromseveraldifferenttimepointstobeproce

26、ssedonthesameday)usingtheQIAampDNAbloodminikit(Qiagen).TheDNABloodMiniKitwasusedwithseveralmodications(madeindiscussionwiththesupplier)asitenabledpuricationofRNAfromfrozenwholebloodwhiletheViralRNAKitwasnotabletoprocessfrozenbloodsamples.First400lbloodwasthawedand50lCECov(1×105TCID50/ml)addedas

27、anexternalRNAreference.Thiswasaddedto400lproteasesolu-tionfollowedby400lbufferAL.Afterincubationat56Cfor10min,thesamplewascooledand460l100%ethanoladdedbeforeapplyingtothespincolumn.Aftertwowashes,RNAwaselutedwith50ltissueculturegradedH2O(Invitrogen).RNAwasreverse-transcribedusingSuperscriptII(Invitr

28、ogen)andrandom®primers(AmershamPharmaciaBiotech).SYBRGreenIdyewasusedinthisreal-timeRT-PCRmethod,whichbindstoanydoublestrandedDNAproducedinthereaction.Eachreactioncontained10lSYBR®GreenJumpStartTaqReadyMixforQPCR(Sigma),2.5lforwardprimer(10pmol/l),2.5lreverseprimer(10pmol/l)and5ltemplatecD

29、NA.Sampleswereanalysedintriplicate.ThePCRconditionswere94Cfor10minfollowedby35cyclesof94Cfor10s;53Cfor20s;72Cfor20s;80Cfor10safterwhichaplatereadwastaken.ThereactionswerecarriedoutonaDNAEngineOpticon2(MJResearch)anddataanalysedusingtheOpticonMonitorAnalysisSoftwareversion2.02.Theprimersweredesignedt

30、oamplifya156bpregionofBVDV5 -UTR(sense5 -TAGTCGTCAGTGGTTCACGCC;antisense5 -CCTCTGCAGCACCCTATCAG),ora280bpregionofCECovnucleocapsidgene(sense5 -CTCGTGGYCGGAAGAGTAAT;antisense5 -GCAACCCAGAMRACTCCATC)andweretestedtoconrmsensitivityandefciencyoverarangeofDNAcon-centrations.Primerefciencywasconrmedforeac

31、hexperi-mentusingtheformula:Primerefciency=101/slope(Pfaf,2001),wherebyavalueof2.0indicates100%efciency.ThisensuredPCRproductswereampliedatanefcientrateandexperimentswerecompa-rable.ThestandardsamplesusedwereplasmidDNAcontainingBVDVorCECovPCRproductsclonedintopGemT(Promega).AscDNAisapotentiallymorec

32、omplexsamplethanthepuriedplasmidDNAusedforthestandardcurveatestwascarriedouttoconrmthecomponentsofaviralcDNApreparationwouldnotbedetrimentaltothePCRreactionwherebystandardsampleswerespikedwithcDNAfromuninfectedbovinebloodandthe220N.J.Youngetal./JournalofVirologicalMethods138(2006)218222cyclethreshol

33、d(CT)valuescomparedtonon-spikedsamples.MeltingcurveanalysisofthePCRproductswasalsocarriedoutforeachexperimentandconrmedthatbothprimerdimersandnon-specicproductswereabsentanduorescencewasmeasuredattemperatureswhereonlyBVDVorCECovspe-cicampliconsweredetected(datanotshown).ThemeanCTvalueforCECovwasdete

34、rminedaftermultipleextractionsofCECovspikedblood(meanCT=12.457,S.D.=0.484;n=30).SamplesthatfellwithintherangeofmeanCECovCT±1S.D.werecorrectedasdescribedinNiesters(2001);theprocedurewasrepeatedforanysamplesthatwereoutsideofthisrange.AfterBVDVchallenge,leucopeniawasevidentinallcalves(Fig.1).Thede

35、creaseinwhitebloodcellcountsrangedfrom7%to52%comparedtothemeanpre-challengelevelsforallanimals3dayspost-challenge.Themeanreductionwasstatisti-callysignicantonday3(P=0.009),day5(P=0.025)andday6(P=0.017).ThisdemonstratesthecharacteristicleucopeniaobservedfollowingacuteBVDVinfectionandhighlightsthecomp

36、lexitiesfacedwhenundertakingreal-timePCRonacutelyinfectedcattleifnormalizingtocellularhousekeepinggenes.Primersweredesignedtoamplifya156bpregionofthehighlyconservedBVDV5 -UTR.Thelinearityofthe5 -UTRassaywasconrmedusingcDNAobtainedfromBVDVspikedblood(Fig.2A)withintherangeof7000ngto7ng,fromsam-Fig.1.W

37、hitebloodcellcountsfollowingchallenge.Countswerecalculatedasapercentageofthepre-challengebaselinevaluesforcalf1(),calf3( ),calf10( ),calf15(*)andcalf20( ).plescontaining2.1×104TCID50to2.1×101TCID50BVDV,indicatingthesensitivityofthetechnique.ThePCRefciencywas2.3andtheregressioncoefcientwas0

38、.993.Itwasnotpossibletoefcientlydetectvirusinsamplescontaininglessthan2.1×101TCID50BVDV.LinearityoftheprimerswasFig.2.OptimisationofBVDV5 -UTRreal-timePCRprimers.MeanCTvaluesof(A)seriallydilutedcDNAfromBVDVspikedblood,y=2.76x+30.06,r2=0.993,(B)plasmidDNAstandardcurve,y=3.42x+35.83,r2=0.995,and(

39、C)plasmidDNA( )andcDNAspikedplasmidDNA(×),y=3.55x+38.11,r2=0.997.Errorbarsshowstandarddeviation.N.J.Youngetal./JournalofVirologicalMethods138(2006)218222221Table1VirusisolationfollowingchallengeCalfDaypost-challenge356710141+3+10+15+20Virusisolationfrombuffycoatwasperformedbyimmunouorescenceass

40、ayaftertwopassagesinFBLcells.alsoconrmedusingplasmidDNAcontainingthe5 -UTRPCRproduct(Fig.2B)asthiswasusedforgenerationof5 -UTRstandardcurveineachexperiment.ThelinearrangeofthePCRwasfrom1×108to1×101DNAcopies/l,withanefciencyof2.02andregressioncoefcientof0.995.SpikingtheplasmidDNAstandardswi

41、thcDNAobtainedfromaBVDVnegativeanimalwasnotdetrimentaltotheefciencyofthereaction(Fig.2C)andtherewasnostatisticallysignicantdifferencebetweenCTvaluesobtainedfromplasmidcomparedtocDNAspikedplasmid(P=0.997).ThelinearrangeoftheCECovprimerswasalsoassessed2usingcDNAobtainedfromsamplescontaining1×10to

42、5×103TCID50CECovwithanefciencyof1.87andregressioncoefcientof0.990(datanotshown).ABVDVplasmidstandardcurverangingfrom1×108to1×101copies/lwasusedtoassess5 -UTRcopynumberineachsample.ThePCRefciencyforeachexperimentwasbetween1.96and2.07andtheregressioncoefcientwasgreaterthan0.992(datanots

43、hown).The5 -UTRCTvaluewasadjustedaccordinglybasedontheCTvalueoftheCECovreaction.Bystandardvirusisolationoneanimal,calf20,wasvirusnegativethroughoutthestudyperiod,whileviruscouldbeiso-latedfromtheremainingcalvesfor1,2or3daysbetweendays5and7post-challenge.Noviruswasisolatedfromanyanimalonday10or14post

44、-infection(Table1).Tovalidatethisnewmethodology,twotimepointswerecho-senforanalysis:day6post-infectionasthisrepresentsthepeakoftheviraemiaandalsoday14asthisrepresentsastagewhenvirusisolationindicatesallanimalstobevirusfree.BVDVviralRNAwasdetectedinwholebloodtakenatdays6and14post-challengeineverycalf

45、(Fig.3).Theestimatesforviralgenomecopy5numberrangedfrom1.95×105to5.88×105,and1.92×10to3.22×1055 -UTRcopies/mlbloodondays6and14,respectively.ViralRNAcopiespermlofbloodwerehigheronday6thanonday14inthreeofthevecalvesalthoughthisdifferencewasonlystatisticallysignicantincalf10(P=0.018

46、).TheremainingtwocalveshadverysimilarlevelsofviralRNAonbothdays6and14post-infection.Calf20whichhadbeenconsistentlynegativeforinfectiousvirusisolationwaspositiveforviralRNAatbothtimepointstested.ItwastobeexpectedthattherewouldbesomediscrepancybetweentheestimationoftheBVDV5 -UTRcopynumberoftheviralRNA

47、andtheinfectiousviralloadinthisstudyandresultsconrmthatRNAcopiesdonotcorrelatewiththenum-Fig.3.QuanticationofBVDVRNA.MeanBVDVRNAcopies/mlbloodfromcalvesonday6( )andday14( )post-challenge.Errorbarsshowstandarderrorofthemean“*”indicatessignicantvalues.berofinfectiousgenomespresentandthereforethepotent

48、ialforinfectivityandvirustransmission.LettellierandKerkhofs(2003)demonstratedusingaBVDVtype1virusstockthattherewere2400copiesofvirusgenomeperinfectiousparticle.Thispresumablyreectsaratioofinfectioustodefectiveviralgenomes.SimilarratioshavebeenobservedforYellowFeverVirus(Baeetal.,2003).Theresultsfrom

49、thecurrentstudywouldsupporttheseobservationsand,invivoatleast,itisproposedthatalthoughBVDVRNAisstillbeingdetected,infectiousvirushasbeencomplexedwithantibodiestherebyleadingtocessationofviraemia.Inthecaseofcalf20,virusneutralisingantibodytitresof1:564weremeasuredonday14(datanotshown),supportingthisp

50、remise.Inordertocomparesamplesitisnecessarytousesomeformofstandardisationandoftenhousekeepinggeneexpressionisusedtoachievethis.However,housekeepinggeneexpressioncanvaryandtheuseofthesegenesasinternalcontrolsshouldbecarefullyconsideredinrelationtocelltypeandthecellmetabolism(Thellinetal.,1999).Thelar

51、genumberofpub-licationsfocusingonthevalidationofinternalreferencegenesdemonstratesthedifcultyinselectingappropriatecandidates.Indeed,manyauthorsnowrecommendtheuseofatleasttwo(Dentetal.,1997)orthree(Bustin,2000;Vandesompeleetal.,2002)validatedhousekeepinggenestoensuretheaccuracyofdata.Inaddition,theu

52、seofhousekeepinggenestocontrolforthevariationinRNAextractionandcDNAsynthesisisdif-cultwhenaxedamountofbloodisanalysed(Johanssonetal.,2000).Thenormalrangeofleucocytecountsinbloodfromhealthycattleis412×109l1(Blood,1994),thusdemonstrat-ingapotentialthreefoldvariationincellnumbers.Furthermore,acute

53、BVDVinfectioncausestransientleucopenia,leadingtoareductioninnumbersofcirculatingleucocyteswhichcanbepronounced.Virusvirulenceaswellashostfactorsmayresultinthereductionofwhitebloodcellsrangingfrommildtosevere.Inthecurrentstudyallvecalveswerechallengedwiththesamedoseofvirusandthedegreeofleucopeniavari

54、edfromanimaltoanimal(Fig.1).Tofacilitatevirusquanticationperxedvolumeofbloodinthefaceofcharacteristicleucopeniaobservedincattledur-ingacuteBVDVinfection,anoveltwo-stepreal-timeRT-PCR222N.J.Youngetal./JournalofVirologicalMethods138(2006)218222methodwhichutilisedanexternalRNAreferencestandardwasdevelo

55、ped.AsecondRNAvirus,CanineEntericCoronavirus(CECov)wasusedasacontrolfortheeachstageofthemethod.ToenableeaseofsamplehandlingthemethodwasbasedonquanticationofBVDVRNApervolumeoffrozenwholeblood.EachbovinebloodsamplewasspikedwithaknownamountofthereferenceCECovpriortoRNAextraction.Thecoronaviruswasco-ext

56、ractedandsubsequentlyampliedinaquantitativemanner.ThisenabledvalidationofnucleicacidisolationandamplicationefciencyforeachsampleanddetectionofthosesamplesforwhichsamplepreparationoramplicationfailedbydeterminingthesignalgeneratedfromtheCECovstandard.Thissystemofspikingsampleswithaheterologousvirusha

57、sbeenusedpreviouslyforHepatitisCvirusquantication(Clelandetal.,1999;Castelainetal.,2004)andforthedetectionofEpsteinBarsvirusandCytomegalovirus(Niesters,2001).CurrentlythereismuchinterestincontrolstrategiesforBVDVinfectionofcattle.Tothisendrapidandreliablediagno-sisofbothpersistentlyandacutelyinfecte

58、dcattleisimperative.Moleculardiagnosticmethodsarebeingincreasinglyutilisedastoolsforthedetectionofnumerousviralpathogens.Theuseofreal-timeRT-PCRmethodstoestablishthepresenceorabsenceofBVDVRNAincattlemayhaveimportantimplicationsforfuturediagnosticscreeningandcontrolstrategies.AcknowledgementTheauthor

59、swishtothankDr.KerstinErles(RVC)forkindlydonatingtheCanineEntericCoronavirusandcorrespondingprimersequences.ReferencesBae,H.G.,Nitsche,A.,Teichmann,A.,Biel,S.S.,Niedrig,M.,2003.Detectionofyellowfevervirus:acomparisonofquantitativereal-timePCRandplaqueassay.J.Virol.Meth.110(2),185191.Baker,J.C.,1995.Theclinicalmanifestationsofbovineviraldiarrhoeainfection.Vet