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2024年7月26日发(作者:)
病毒颗粒计数的意义
我们知道噬斑法和TCID50法检测的是感染性病毒的浓度,并不体现病毒的总浓度(总颗粒数)。现在有越来越多的
研究显示,很多病毒在包装过程中由于缺失基因组,形成空心病毒。或者在合成过程中,基因的突变或者缺陷导致蛋白
的突变,造成病毒没有感染性。而这些非感染性病毒的数量在总病毒数量中所占的比例意义重大,能影响体内和体外的
研究结果。所以快速的病毒总浓度定量方法是非常必要的。
流感疫苗的生产中,从鸡胚培养来源的流感疫苗在使用专门的试剂裂解后,收获免疫原蛋白HA。非传染性的流感病毒
(被证实含有衣壳蛋白和部分的基因组)在裂解后同样能贡献免疫原蛋白HA。所以总病毒浓度的评估对流感疫苗的生
产意义重大。
此外,总病毒浓度的定量也有助于减毒疫苗的生产。减毒疫苗是复制缺陷的非感染性的但是能引发机体免疫反应的一种
疫苗。减毒疫苗既然不能复制,所以就不会引起细胞病变反应,而CCID50法则以细胞病变反应为判断基准。在某种意
义上来说,所有的减毒疫苗是由非感染的病毒颗粒构成,因此,采用总病毒浓度定量的方法是减毒疫苗唯一可靠的方法。
在动物疾病的预防和治疗中,采用感染性方法测得病毒滴度来决定注射的剂量,往往忽视了非感染性病毒颗粒在动物免
疫反应中的作用以及最终的药剂效果。比如噬斑法测得病毒滴度为1E6pfu,但是总病毒浓度是1E8vp/ml,在每一个感
染颗粒中,有100个颗粒没有被计数,最终可能影响动物治疗结果。
考虑到非感染性病毒颗粒的生物学作用以及在疫苗生产的作用,感染性病毒颗粒和总病毒颗粒的定量对于病毒疫苗的生
产和研究都至关重要,而病毒计数仪在10min内能快速获得病毒总浓度,所以能广泛应用于在病毒的研究和生产中。
WHYVIRALPARTICLEQUANTIFICATIONMATTERS
AswasdiscussedinourpreviousWhitePaper“AnOverviewofVirusQuantificationTechniques,”
lyonmeasuringinfectivityor
theamountofantigen,sgrowingevidence,however,
thatthenumberofnon-infectiousviralparticlesisofsignificantbiologicalimportanceandcan
untingdatasuggestsaneedforrapid
quantificationofthetotalnumberofviralparticlesinaviruscontainingsample,whichisnow
possibleusingtheinnovativeViroCyt®VirusCounter®2100.
Background:TheBiologyandBiologicalConsequencesofNon-InfectiveViralParticlesAlthough
viralparticlesmaybenon-infectiveforanumberofbiologicalreasons,defectiveviral
replicationisoftenthecause.
Forexample,viralcapsidswhichlackgenomes,maybeproducedduringthepackagingphase,
onsordefectsinviralgenomesalsoresultintheproductionof
vncluderelatively
minormutationsinkeygenescontrollingthevirallifecycleormuchlarger-scaledefects.
Inthecaseofso-called“defectiveinterferingparticles”(DIPs)discoveredininfluenza,verylarge
eplicativecycleispossibleonlyifthe
DIPparticlesareinthepresenceofreplication-competent,a
case,DIPsmay“piggyback”competentparticlereplicationoffsettingtheirdefects,andasa
result,DIPscompeteforresourcesagainstreplicative-competentparticlesandhaveevenbeen
tiontoprovidingpotentialcompetitionfor
criticalresources,ithasbeenmorerecentlydocumentedthatDIPsaffecttheseverityofinfection
ultofincreasingamountofresearchinto
non-infectiousinfluenzaparticles,otherclassesoftheseparticleshavebeendiscovered.
Noninfectiouscell-killingparticles(niCKP)foundininfluenzacultures7,interferon-inducing
particles(IFPs)8,andinterferoninduction-suppressingparticles(ISPs)9allplaysignificant
ervationthatthesenon-infectiousparticle
typesactuallymakeupthemajorityofparticlesinactiveinfluenzainfections9,raisesthe
vealsobeendocumentedinother
ueviralinfections,theyappeartoplayaroleinnaturalbiological
,genomereplicationerrorsduetothereversetranscriptionprocesscause
theformationofDIPswhichactivelycontributetoinfectionthrough“priming”ofCD4+T
tiontotheireffectonbiologicalsystems,monitoringnon-infectious
productionofthe
seasonalfluvaccine,ingpurification,
thevirusparticlesaresplitapartusingaspecializedreagentandtheimmunogenicHAproteins
-infectiousparticlesthatareknowntohaveaproteincapsid
andapartialgenomewillalso
ereforeessentialduringthis
processtoha
typesoated
vaccinesuseareplicationdeficientversionofavirustocauseanimmuneresponse,butwithlittle
heseattenuatedvirusesdonotreplicate,theywillnotcausethe
se,allattenuated
vaccinesconsistofnon-infectiousvirusparticles,andthus,theonlymethodstoreliablyquantify
heextensivebiologicalroleofthese
non-infectiveparticles,aswellastheirimpactonthedevelopmentandmanufactureofviral
vaccines,infectivetitersandtotalparticlenumbersarebothessentialforaccurateviral
characterization.
Theliteraturemakesitclearthatnon-infectiousviralparticlesareoffarmorebiologicalinterest
eenknownfordecadesthatthe
so-called“particle-to-PFU”ratiosformanytypesofviruscanbequitelargeandmayshow
considerablevariability12,suggestingthatparallelviralcultureswithdifferingparticle-to-PFU
rusesareknowntohaveextremelyhighparticleto
mple,varicellazostervirushasbeenshowntohavearatioof40,000:113,
whileothers–suchasbacteriophages–haveaparticletoPFUratioapproachingone,meaningall
icalregulatorsofviralinfectionandoftheimmunesystem,
non-infectiousviralparticlesareanaturalandnecessarycomponentofviralcultures,and
completecharacterizationofviralculturesrequirethatbothinfectiousandnon-infectious
gh,therearemultiplemethodswhichallowforinfectiousparticle
assessmentstobemade,untilrecently,optionsfornon-infectiousortotalparticlecountingwere
limitedprimarilytovisualizationviatransmissionelectronmicroscopy(TEM).However,dueto
thehighleveloftechnicalexpertiserequiredtoconductthesemeasurements,aswellasthe
needforsophisticatedandcostlyequipment,thistechniquehasprovenimpracticalformany.
Toaddresstheneedforviralresearcherstobeabletoaccurately,reliablyandeasilyquantifytotal
viralparticlecount,theVirusCounter®usCounterrelieson
fluorescentstainingofsurfaceproteinsandnucleicacidsfollowedbydetectionoffluorescent
aserexcitation,intactviralparticlesare
identifiedbycoincidentalproteinandnucleicacidsignals.
TheVirusCounter®2100,aToolfor“UniversalNormalization”Totrulynormalizeviralcultures,
thehe
complexinteractionsandpartially-understoodrelationshipsbetweeninfectiousand
non-infectiousparticlesinactiveviralcultures,accuratenormalizationrequiresthatboth
infectiousandnon-infectiousviralparticles(whichmaybededucedfromtotalparticlecounts)be
ast,determinationofparticle-to-PFUratioswasoftendifficultand
sometimesimpractical,sincethefewmethodsthatexistedforquantifyingtotalparticlecounts
werebothcostlyandtime-consuming,requiringsophisticated,expensiveandhighlytechnical
rast,implementationoftheVirusCounter2100reducesthetimerequiredto
roughly30minutesofsamplestainingand5-10minutesofinstrumentreadtime,limitingthe
cost,andlesseningthetechnicalexpertiserequiredtoobtainresults.
UseScenario:AnimalStudies–AccurateDeterminationofViralDosage
Viralchallengeintheappropriateanimalmodelisanimportanttoolinthedevelopmentof
r,theamount
ofvirususedisoftencalculatedsolelybasedoninfectivity-basedassaysand,ashasbeen
discussed,non-infectiveparticlescanoftenhaveeitherapositiveornegativeimpactinthe
mple,iftheinfectivetiteris
determinedbyplaqueassaytobe1E6pfu,butthetotalintactviralparticlecountisestablished
tobe1E8vp/ml,forevery1infectiveparticle,thereare100particlesthatarenotcountedas
infective,butmaybeinfluencingtheexperimentaloutcome,kingeachof
thesepropertiesfordifferentlotsofvirus,datesandothervariables,aclearandaccuratepicture
oftherelativecontributionofeachvariantispossible.
ComparingInfectiousandTotalParticleCounts
Tocompareinfectioustiterswithtotalparticlecount,samplesofinfluenzaH1N1,
Cytomegalovirus(CMV),RespiratorySyncytialVirus(RSV)andRubellaweremeasuredbyTCID50
assayorplaquetiter,n,total
particlecountsdeterminedbyeitherTEMortheVirusCounterwerestatisticallyidentical,while
titerbyTCID50measuredafractionofthetotalparticles,withcountsrangingfrom2-3.5orders
esultshighlighttherelative
abundanceofnon-infectiveparticlesasapercentageofthetotalpopulationacrossmultiplevirus
types.
UseScenario:VaccineProduction–TrackingandOptimizingYieldThroughoutthe
ManufacturingProcess
Although,therearemanypointsduringtheprocessofdeveloping,optimizingandproducing
vaccinesthatwouldbenefitfromrapidenumerationofviralparticles,oneofthemostsignificant
itenthannot,
thelongandcomplexstepsoftakingcrudematerialandtransformingitintoaproductreadyfor
litytotrackessentiallyinrealtimethe
quantityofvirusatbeginningandendofeachdistinctstagewillidentifywherelossesare
occurring,allgainsinefficiencyateachstepwould
leadtoconsiderablefinancialbenefits.
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