病毒颗粒计数的意义

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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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本文标签： 病毒颗粒感染性浓度蛋白