Higher titers of some H5N1 and recent human H1N1 and H3N2 influenza viruses in Mv1 Lu vs. MDCK cells

MISSING IMAGE

Material Information

Title:
Higher titers of some H5N1 and recent human H1N1 and H3N2 influenza viruses in Mv1 Lu vs. MDCK cells
Physical Description:
Mixed Material
Language:
English
Creator:
Hamilton, Sara B.
Wyatt, Diane E.
Wahlgren, Brett T.
O'Dowd, Maureen K.
Morrissey, Jane M.
Daniels, Deirdre E.
Lednicky, John A.
Publisher:
BioMed Central (Virology Journal)
Publication Date:

Notes

Abstract:
Background: The infectivity of influenza A viruses can differ among the various primary cells and continuous cell lines used for such measurements. Over many years, we observed that all things equal, the cytopathic effects caused by influenza A subtype H1N1, H3N2, and H5N1 viruses were often detected earlier in a mink lung epithelial cell line (Mv1 Lu) than in MDCK cells. We asked whether virus yields as measured by the 50% tissue culture infectious dose and plaque forming titer also differed in MDCK and Mv1 Lu cells infected by the same influenza virus subtypes. Results: The 50% tissue culture infectious dose and plaque forming titer of many influenza A subtype H1N1, H3N2, and H5N1 viruses was higher in Mv1 Lu than in MDCK cells. Conclusions: The yields of influenza subtype H1N1, H3N2, and H5N1 viruses can be higher in Mv1 Lu cells than in MDCK cells.
General Note:
Hamilton et al. Virology Journal 2011, 8:66 http://www.virologyj.com/content/8/1/66; Pages 1-9
General Note:
doi:10.1186/1743-422X-8-66 Cite this article as: Hamilton et al.: Higher titers of some H5N1 and recent human H1N1 and H3N2 influenza viruses in Mv1 Lu vs. MDCK cells. Virology Journal 2011 8:66.

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
© 2011 Hamilton et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
System ID:
AA00024410:00001

Full Text

PAGE 1

METHODOLOGY OpenAccessHighertitersofsomeH5N1andrecenthuman H1N1andH3N2influenzavirusesinMv1Luvs. MDCKcellsSaraBHamilton1,DianeEWyatt2,BrettTWahlgren3,MaureenKO Dowd1,JaneMMorrissey1,DeirdreEDaniels1, JohnALednicky4*AbstractBackground: TheinfectivityofinfluenzaAvirusescandifferamongthevariousprimarycellsandcontinuouscell linesusedforsuchmeasurements.Overmanyyears,weobservedthatallthingsequal,thecytopathiceffects causedbyinfluenzaAsubtypeH1N1,H3N2,andH5N1viruseswereoftendetectedearlierinaminklungepithelial cellline(Mv1Lu)thaninMDCKcells.Weaskedwhethervirusyieldsasmeasuredbythe50%tissueculture infectiousdoseandplaqueformingtiteralsodifferedinMDCKandMv1Lucellsinfectedbythesameinfluenza virussubtypes. Results: The50%tissuecultureinfectiousdoseandplaqueformingtiterofmanyinfluenzaAsubtypeH1N1,H3N2, andH5N1viruseswashigherinMv1LuthaninMDCKcells. Conclusions: TheyieldsofinfluenzasubtypeH1N1,H3N2,andH5N1virusescanbehigherinMv1Lucellsthanin MDCKcells.BackgroundTheinfectivityofinfluenzavirusescandifferamongthe variousprimarycellsandcontinuouscelllinesusedfor suchmeasurements[1,2].Astheterm infectivity has manymeaningsinvirology,inthismanuscript,infectivityisbroadlydefinedastheabilityofavirusparticleto enterahostcellandformviableprogenyvirions.Measuresofinfectivitydependnotonlyontheinherentsusceptibilityofaparticulartypeofcellforagiven influenzavirus,butalsoonthemethodologyusedfor infectingthecells[suchasthelengthoftimethevirus isleftincontactwiththecells,astheaffinity/avidityof avirusforitsreceptor(s)mayvaryaccordingtocell type],thequasispeciesdistributionwithinaparticular influenzavirusstock,andothervariables. Accurateviableviruscount sareessentialforinhalationexposurestudieswithaerosolizedviruses[3],for correlationofviablecount togenomeequivalencein levelofdetectionstudies,andotherrelevantworkwith influenzaviruses.QuantitativeRT-PCRmethodsarenot suitable,astheydonotdistinguishbetweenviableand non-viablevirusparticles.Indeed,infectiousinfluenza virusparticlescompriseaminorsubpopulationofbiologicallyactiveparticles(BAP)withinaviralpopulation [4].TheotherBAPincludeinterferonsuppressingparticles[4,5],defectiveinterferingparticles[4,6],andnoninfectiouscell-killingparticles[4,7]. Madin-Darbycaninekidney(MDCK)epithelialcells arewidelyusedfortheisolationofhumaninfluenzaA andBvirusesandthedeterminationofinfluenzaA virustiters[1,8-11].However,we(S.HamiltonandJ. Lednicky,unpublished)andothers[2,12]haveobserved thatallthingsequal,thecytopathiceffects(CPE)of manyinfluenzaAvirusesaredetectedearlierinamink lungepithelialcellline(Mv1Lu)(AmericanTypeCultureCollection[ATCC]CCL-64)thaninMDCKcells. TheuseofMv1Lucellsforthedetectionofinfluenza virusesisnotnovel;forexa mple,thecellsaresupplied byacommercialsource(DiagnosticHybrids,Inc., Athens,OH)toclinicallaboratoriesforthatpurpose.In MDCKandMv1Lucellsgrownasamonolayer,CPE *Correspondence:jlednicky@PHHP.UFL.edu4EnvironmentalandGlobalHealthDepartment,CollegeofPublicHealthand HealthProfessions,UniversityofFloridaatGainesville,Box100188, Gainesville,FL32610,USA FulllistofauthorinformationisavailableattheendofthearticleHamilton etal VirologyJournal 2011, 8 :66 http://www.virologyj.com/content/8/1/66 2011Hamiltonetal;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and reproductioninanymedium,providedtheoriginalworkisproperlycited.

PAGE 2

duetoinfluenzavirusesgen erallyconsistsofvisible changesintheappearanceofnucleiininfectedcells, andtheformationoffocalenlargedgranularcells ornon-specificcelldeterioration,followedbydetachmentoftheswollencellsfromthegrowingsurface. Occasionally,influenzavirus-infectedMv1Lucellsform spindle-shapedgranularcellsthatdonotdetachfrom thegrowingsurface.AbasiccomparisonofMDCKand Mv1LucellsisgiveninTable1. TheacronymMv1Lustemsfrom Mustelavison (Americanmink)lung (nowreclassifiedas Neovison vison ).Minkarehighlyrelatedtoferretsandaresusceptibletoinfluenzaviruses[13].Weareperformingvariousstudiesofinfluenzavirusesindomesticatedferrets ( Mustelaputoriusfuro ),andaskedwhetherMv1Lu cellsmightbeadvantageousfortheisolationand/or enumerationofH5N1andotherinfluenzavirusesinferrettissuespecimensorsecretions.Anunderlying assumptionofourswasthatinf luenzavirusesinspecimensderivedfromferretswi thactiveinfluenzainfectionswouldeffectivelyattach,replicateandefficiently produceprogenyvirionsinMv1Lucells.Moreover,we wishedtoknowwhethervirusyieldsmightdifferin MDCKvsMv1Lucells.Welearnedthatthevirusyields ofmanylow-passageinfluenzaAvirusstrainswas higherinMv1LucellsthaninMDCKcells,evenwhen thevirushadnotbeenadaptedforgrowthinferrets.Results1.ValidationofcelllinesWhereasvalidatedlow-passageMDCKcellsareusedin somelong-establishedinfluen zaresearchlaboratories, suchcellsarenolongereasytoobtain.Togaininsights applicabletocurrentrealities,MDCKandMv1Lucells obtainedfromvariouscommercialoruniversitysources wereevaluatedforthiswork(theidentityofmostofthe supplierscannotberevealedduetolegallybindingclient confidentialityagreements).ThemorphologicalcharacteristicsoftheMDCKandMv1Lucellsvariedamong thebatchestested,andtheyalsovariedinsensitivityto influenzaviruses,celllongevity,andcellgrowthkinetics/ properties.Furthermore,e speciallysincethecelllines wereestablishedlongago,theyhadbeenpropagatedby othersincellculturemediasupplementedwithfetal bovineserumthathadnotbeengamma-irradiatedprior tocryopreservation/archivi ng.Notsurprisingly,many batchesofbothcelllinescontainednumerousmultinucleatedlargesyncytia,cytoplasmicinclusionbodies,perinuclearorcytoplasmicvacuoles,andothersignsofviral contamination,eveninthepresenceofgamma-irradiated serum(someexamplesareshowninFigure1). Varioustypesofcommerciallypreparedcellculture mediaandserumsampleswereextensivelyevaluated (datanotshown).AbatcheachofMDCKandMv1 Lucellsthatlackedovertsignsofviral(orother)contamination,andhadminimalanomaliesdetectableby microscopyusingphase-contrastobjectives(nosignsof non-specificcelldeterioration,rarevacuolesandabnormalnuclei,nogranulation,andlessthan1syncytium per6fieldsat200magnification),andthatsupported high-titeryields( 408PFU/ml)of Influenzavirus A/Wisconsin/67/2005(H3N2)(usedasanindicator strain)inaseriesofpilotstudies,werechosenforthis work.Duringpassage,thece llsweresub-culturedata minimumratioof1:4toamaximumof1:20whenthey werenearlyconfluent.Thed imensionsoftrypsinized MDCKandMv1LucellsweremeasuredusingaCASY 1resistancemeasuringmulti-channelcellanalyzersystem(RocheInnovatisAG,Reutlingen,Germany).Those measurementsindicatedthattheMDCKcellswere largerthantheMv1Lucells.Forexample,oneday post-seedingandcultivatio nunderoptimalconditions (MaterialsandMethods),MDCKhadameandiameter of18.4 m(Figure2),whereasthemeandiameterof Mv1Lucellswasabout12.3 m(Figure3).Asshown inFigure2and3,MDCKcellsconsistentlyproduced moredebristhanMv1Lucells(compareareasofleft peaks[levelofdebris]inbothFigures).Celldimension andamountofdebriscorrelatedwiththesourceofboth serumandgrowthmedia.Forexample,MDCKcells growninoptimalgrowthmediumbutsub-optimal serumhadadifferentprofileat1daypost-seeding:the amountofdebriswascomparablyhigherandthecell dimensionmorevariable(Figure4).2.PandemicH1N12009virusescanbepropagatedin Mv1LucellsWedeterminedthatvariouspandemicH1N12009viruses includingstrainsA/California/04/2009andA/California/ 07/2009canbereadilypropagatedinMv1Lucells.As showninFigure5,A/California/04/2009,obtainedforthis workasanMDCK-passagedvirus,isabletocompleteits replicationcycleinMv1Lucells.InFigure5,MDCKand Mv1Lucellswereinfectedatthesamehighmultiplicity ofinfection(MOI)(approx10);whereassomeenlarged nucleiandcytoplasmicgranulation(evidenceofinfection withinfluenzavirus)areevidentintheMDCKcells,most Table1CharacteristicsofMDCKandMv1LucellsCharacteristicMDCKMv1Lu MorphologyEpithelialEpithelial GrowthpropertiesAdherentAdherent SourceKidneyLung GenderFemaleadultMaleandfemalefromfetuses Doublingtime129hr20hr1Doublingtime(thiswork):TimeforcelltoduplicateinDMEMunderoptimal conditions.Hamilton etal VirologyJournal 2011, 8 :66 http://www.virologyj.com/content/8/1/66 Page2of9

PAGE 3

oftheMv1Lucellsweredestroyed48hrspost-infection. AtaMOIof0.1orless,nearlycompletedestructionofthe cellswasdelayed,requiringmorethan48hrs,butstillfollowingthepreviouspatternobservedwithhighMOI: nearlycompletedestructionoccurredfirstinmonolayerof Mv1Lucells.Sincetheinfectedcellsformedcytopathic effects(CPE)typicalofinfluenzavirusesatlowandhigh MOI,theobservedeffectswerepresumedtobeindependentofeffectsattributabletothosecausedbynon-infectiouscell-killingvirusparticles.ThevirusintheMv1Lu cells(Figure5)wasputativelyshowntobeaninfluenzaA virususingacommercialsolidphaseELISAtest A B C D E F Figure1 MicroscopicanalysesofculturedMDCKandMv1Lucells .A.MDCKcells,normal(200magnification);B.MDCKcells,aberrant; largeinclusionbodyandvacuolation(arrow);C.MDCKcells,aberrant;Giantcellwithinclusions(arrow);D.MDCKcells,aberrant;perinuclear vacuoles(arrow);E.Mv1Lucells,normal(200);F.Mv1Lucells,aberrant;syncytium(arrow). Hamilton etal VirologyJournal 2011, 8 :66 http://www.virologyj.com/content/8/1/66 Page3of9

PAGE 4

(QuickVueInfluenzaAkit,QuidelCorp.,SanDiego,CA) kit,andconfirmedasA/CA/04/2009(H1N1)byRT-PCR andsequencingoftheviralhemagglutinin,neuraminidase, andmatrixgenes(datanotshown).3.Mv1LucellscanbeusedforinfluenzaAvirusplaque assaysToourknowledge,Mv1Lucellshavenotbeenusedfor influenzavirusplaqueassa ysbyotherlaboratories (supportingliteraturewasnotfound).Wenowdisclose thatwehaveusedMv1Lucellsforplaqueassaysofa varietyofinfluenzaAviruses.Examplesareshownfor A/Mongolia/244/2005(H5N1)[Figure6]andforA/ California/04/2009(H1N1)[Figure7].4.TitrationofinfluenzaH5N1virusesFollowingthegeneraloutlinedepictedinFigure8,the plaqueand50%tissuecultureinfectiousdose(TCID50) Figure2 MDCKcellprofileunderoptimalconditionsandserum,onedaypost-seed .Ordinate,cellcount(CNT);abscissa,diameterofcell orotherparticles( m). Figure3 Mv1Lucellprofileunderoptimalconditionsandserum,onedaypost-seed .Ordinate,cellcount(CNT);abscissa,diameterofcell orotherparticles( m). Hamilton etal VirologyJournal 2011, 8 :66 http://www.virologyj.com/content/8/1/66 Page4of9

PAGE 5

titersof4differentH5N1influenzavirusesgrownin embryonatedchickeneggsweredeterminedinMDCK andMv1Lucells.ThetitersoftheH5N1viruseswere higherinMv1LuthanMDCKcells(Table2).Forthese H5N1viruses,CPEwereusuallyevidentupto6hrearlierinMv1LucellsthaninMDCKcells(datanot shown).Similarresultswereobtainedwithvirusesfirst propagatedinMDCKorMv1Lucellsinsteadofeggsas showninFigure8:highertitersresultedwhenMv1Lu cellswereusedforplaqueorTCID50determinations (datanotshown).5.TitrationofseasonalandpandemicH1N12009 influenzavirusesAgainfollowingtheproceduredepictedinFigure8,marginallytosignificantlyhigherviraltiterswerealsocalculatedinMv1LucellsthanMDCKcellswithvarious egg-grownseasonalandpandemicH1N12009influenza viruses(Table3).AsforH5N1viruses,similarresults wereobtainedwithviruses firstpropagatedinMDCK andMv1LucellsinsteadofeggsasshowninFigure8: highertitersresultedwhenMv1Lucellswereusedfor plaqueorTCID50determinations(datanotshown). Figure4 MDCKcellprofileunderoptimalconditionsbutw ithlower-qualityserum,onedaypost-seed .Ordinate,cellcount(CNT); abscissa,diameterofcellorotherparticles( m). A B Figure5 AppearanceofMDCKandMv1Lucells48hrafterinfectionofthecellsatahighMOIwithapandemicH1N12009virus EnlargednucleiandcellulargranulationbutnocellulardetachmentareevidentintheMDCKcells(panelA),whereasmostoftheMv1Lucells havedetachedorareshowingadvancedCPE(panelB). Hamilton etal VirologyJournal 2011, 8 :66 http://www.virologyj.com/content/8/1/66 Page5of9

PAGE 6

DiscussionAsforMDCKcells,Mv1Lucellscanbeusedforboth influenzaAvirusTCID50andplaqueassays.Andin establishingparametersfo rtheenumerationofvarious influenzaAstrains,weobservedthathighervirustiters wereattainedformanyinMv1Lucellscomparedto thetiterobtainedinthemorecommonlyusedMDCK cellline.Takentogether,itmaybeadvantageoustouse Mv1Lucellsforcertainapplications,suchasforobtainingestimatesofadelivereddoseofaerosolizedinfluenza virusininhalationexposurestudies[3],andforthe detectionofrelativelysmallamountsofinfectiousinfluenzavirusparticlesintissueorsecretionspecimens. Thoughnotspecificallymentionedinthebodyofthis manuscript,wenotedthatMv1Lucellsmustbe handledasdescribed(MaterialsandMethods).When Mv1Lucellsaremaintainedconfluentformore than1weekwithoutsplittingthemforvirustitration, variabilityintheCPEcausedbyinfluenzavirusesoccurs, Mv1 Lu MDCK Figure7 ComparisonofplaquesformedbyA/California/04/2009(H1N1)inMv1LuandMDCKcells -1 -2 -3 N o n-inf ected -4 5 Figure6 PlaqueassayofA/Mongolia/244/2005(H5N1)inMv1 Lucells Hamilton etal VirologyJournal 2011, 8 :66 http://www.virologyj.com/content/8/1/66 Page6of9

PAGE 7

asreportedbySchultz-Cherry etal [2].IntheimproperlymaintainedMv1Lucells,theappearanceofCPE istypicallydelayedcomparedtoMDCKcells,andthese Mv1Lucellscannotbeusedforplaqueassays.Materialsandmethods1.CellsMDCKandMv1LufromDiagnosticHybrids,Inc.were selectedforthiswork.TheMDCKcellswereobtainedat passage68andusedthroughpassage78,Mv1Lucellsat passage71andusedthroughpassage88.Bothcelllines wereculturedasmonolayersat37Cand5%CO2.Both celllineswereroutinelypropagatedineitherDulbecco s ModifedEagle sMedium(DMEM)(Mediatech,Inc., Manassas,VA)orEarle sMinimalEssentialMedium (EMEM)(InvitrogenCorp.,Carlsbad,CA).Bothmedium formulationsweresupplementedwith2mML-Alanyl-LGlutamine(GlutaMAX ,InvitrogenCorp.),antibiotics [PSN;50 g/mlpenicillin,50 g/mlstreptomycin, 100 g/mlneomycin(InvitrogenCorp.)],and10%(v/v) heat-inactivatedgamma-irradiatedfetalbovineserum (HyClone,Logan,Utah).Sodiumpyruvate(Invitrogen Corp.)andnon-essentialaminoacids(Hyclone)were addedtoEMEM(eachatamanufacturer srecommended finalconcentrationof1).Priortovirologytests,thefollowingprecautionarystepsweretakentoreducepossible mycoplasmaandothercontam ination:thepre-banked cellswerepropagatedinplasmocin(Invivogen,San Diego,CA)containinggrowthmediafortwoweeks, followedbytwoweeksintheabsenceofantibioticsto determinewhetherfast-growingmicrobialcontaminants werepresentorabnormalmorphologicalchangeswould occur.ThecellswereconfirmednegativebyPCRforthe presenceofmycoplasmaDNAusingaTakaraPCR MycoplasmaDetectionkit(TakaraBio,USA,Thermo Fisher),andbymycoplasmatests(cultureandDNA staining)performedbyacommercialtestinglaboratory (BioniqueTestingLaboratories,SaranacLake,NY).The celllineswereobserveddailytomonitorconfluencyand checkedfornormalmorphology.Theyweresplitwhen theyreached~90%confluency.Cellcountswereperformedonharvestedculturesusingeithertrypanblue dye-exclusionhemacytometermethodologyorbyautomatedcellsizeanalysis(CASY1counter). ActivelygrowingMDCKandMv1Lucellswere plantedat1-3103viablecells/wellin96wellmicrotiterplatesaminimumofthreedayspriortoassay.Likewise,6-wellmicrotiterplateswereplantedatleastthree dayspriortoperformingaplaqueassayataseeding densityof3-6105viablecell/well.Cellbankswere preparedbyfreezingactive lygrowingcellsinstandard growthmediumcontaini ng5%DMSO.Afterrapid thawingoffrozenvialsof cells,eachvialwascentrifuged,thefreezemediumwasdiscarded,andthecell pelletwasresuspendedingrowthmediumforplanting culturevessels.2.InfluenzavirusesInfluenzavirusH5N1strainsA/Vietnam/1203/2004and A/Mongolia/244/2005werefromarchivesoftheSoutheastPoultryResearchLaboratory,andA/Iraq/207NAMRU3/2006wasfromtheNationalBiodefenseAnalysisandCountermeasuresCenter[NBACC],which obtainedthevirusfromNavalMedicalResearchUnit No.3[NAMRU-3],Cairo, Egypt(37)(Table1).The H5N1viruseswerereceivedaslow-passagestocks,and theiridentityverifiedusingviralgenomicsequencing. OtherinfluenzaviruseswereobtainedfromtheCenters forDiseaseControlandPreventionorfromtheATCC.3.ViruspropagationLow-passagestocksoftheH5N1viruseswerepropagatedintheallantoiccavitiesof10-day-oldembryonated Table2Titersofegg-grownH5N1virusesinMDCKandMv1LUcellsH5N1Virus MDCKcells Mv1Lucells Log10TCID50/ml Log10PFU/ml Log10TCID50/ml Log10PFU/ml A/Vietnam/1203/20047.26.8.28.27.9.2 A/Mongolia/244/2005 6.4.1 6.2 8.2 7.8.2 A/Iraq/207-NAMRU3/2006 8.8.2 8.3 10.2 9.9aTCID50datafromthreeseparateexperiments,plaqueassaydatafrom1(A/Iraq/207-NAMRU3/2006)or3(A/Vietnam/1203/2004andA/Mongolia/244/2005) experiments. V irus Egg Incubate at C Refrigerate egg Collect allantoic fluid MD C KM v 1 L u Figure8 Outlineofprocessusedforcomparingtitersof egg-grownvirusinMDCKandMv1Lucells Hamilton etal VirologyJournal 2011, 8 :66 http://www.virologyj.com/content/8/1/66 Page7of9

PAGE 8

chickeneggs(ECE)for24hat37C;theallantoicfluid washarvested,centrifugedforclarification,andstoredat -80Cforuptooneyearorinthevaporphaseofliquid nitrogenforlongerstorage[3,11,14,15].Allworkwith H5N1viruseswasconductedinaBiosafetyLevel3 enhancedlaboratory.SubtypeH1andH3influenza viruseswerepropagatedinECEorinMDCKcells (usingthesamesubstratetheywerepropagatedinat theATCCorCDC).4.CalculationofTCID50valuesTCID50valuesweredeterminedbyinfectingMDCKand Mv1Lucellsin96-wellmicrotiterplateswithserial dilutionsofvirusandcalculationoftheTCID50four dayslaterbythemethodofReedandMuench[16]. CellsforTCID50determinationswereinserum-free completeEMEMcontainingL-1-tosylamide-2-phenylethylchloromethylketone(TPCK)-treatedtrypsin,and re-fedwiththesamemedium.Asthespecificactivityof theTPCK-trypsinwasrelativelyhigh,thefinalconcentrationsofTPCK-trypsinwere1.0 g/mlforMDCK cellsand0.1 g/mlforMv1Lucells.5.PlaqueassaysForplaqueassays,newlyconfluentMDCKorMv1Lu cellsinsix-welltissuecult ureplateswereinoculated with0.2mlofvirusseriallydilutedinserum-freecompleteEMEMcontainingTPCK-treatedtrypsin.Virus wasadsorbedtocellsfor1hrat37C(H5N1)or35C (seasonalinfluenzaviruse s)withrockingevery15min (2009pandemicinfluenzaH1N1wereadsorbedfor2h at35C).Aftervirusadsorption,thecellswerewashed withserum-freeEMEMandthewellsoverlaidwith 3ml/wellofprimaryoverlayconsistingof1.6%w/v agarose(InvitrogenCorp.) mixed1:1withserum-free 2XEMEM(Lonza,Walkersville,MD)containingantibioticsandTPCK-trypsin.WiththeexceptionofH1N1 pandemicinfluenza2009vir uses,plateswereinverted andincubatedfor3daysattemperaturesappropriate fortheviruses,thenoverlaidwith2mlofsecondary overlayof1.6%w/vagarosemixed1:1with2XEMEM containing0.14mg/mlneutralred(catalognumber N2889,Sigma-Aldrich,St.Louis,MO),theplates inverted,andincubatedfortwoadditionaldaystovisualizeplaques.Thepandemic2009H1N1virusesrequired longerincubationtimes:3daysaftertheprimaryoverlay (performedasdescribedabov e),thecellswereoverlaid 2ml/wellof1.6%w/vagarose(InvitrogenCorp.)mixed 1:1withserum-free2XEMEM(Lonza)containingantibioticsandTPCK-trypsin,inverted,andincubatedfor another2days.Theyweresubsequentlyoverlaidwith 2mlofsecondaryoverlaywithneutralredasdescribed above,andtheplatesincubatedfor2additionaldaysto visualizeplaques.Acknowledgements PartofthisworkwasperformedattheMidwestResearchInstitute(MRI)in KansasCityinsupportofacooperativeteamingagreementbetweenMRI andtheUnitedStatesDepartmentofAgriculturetogetherwiththeNational BiodefenseAnalysisandCountermeasuuresCenter.TheauthorsthankDr. DavidSwayne,SoutheastPoultryResearchLaboratory,AgriculturalResearch Service,UnitedStatesDepartmentofAgriculture,Athens,Georgia,for providingtwooftheH5N1strainsusedforthisworkandforhelpful scientificinsightsanddiscussion.ThefollowingscientistsworkingatNBACC providedtechnicalinput:Drs.MatthewBender,ElizabethLeffel(nowat PharmAthene,Annapolis,MD),andRichardKenyon.Finally,theNBACCvirus acquisitionteamisthankedforacquiringandprovidingA/Iraq/207NAMRU3/2006(H5N1). Authordetails1EnergyandLifeSciencesDivision,MidwestResearchInstitute,425Volker Boulevard,KansasCity,Missouri64110,USA.2KCBio,LLC,2111E.SantaFe, Suite312,Olathe,KS66062,USA.3UniversityofKansasMedicalCenter,3901 RainbowBlvd.MS1018,KansasCity,KS66103,USA.4Environmentaland GlobalHealthDepartment,CollegeofPublicHealthandHealthProfessions, UniversityofFloridaatGainesville,Box100188,Gainesville,FL32610,USA. Authors contributions SBHestablishedaccuratevirusquantificationprocedures,performed plaqueandTCID50assays,assistedwithdatainterpretation,propagated MDCKandMv1Lucells,providedphotographsrelatedtocellmorphology andplaqueassays,performedtrypsindoseresponseanalyses,engagedin Table3Titersofegg-grownseasonaland2009pandemicinfluenzavirusesinMDCKandMv1LucellsaVirus MDCKcells Mv1Lucells Log10TCID50/mlLog10PFU/mlLog10TCID50/mlLog10PFU/ml A/NWS/1933(H1N1)7.5.27.2.28.1.28.2 A/PuertoRico/8/1934(H1N1) 7.2.3 7.1.2 8.3.2 8.1.2 A/NewCaledonia/20/1999(H1N1) 8.1.2 NDb8.5.2 ND A/California/04/2009(H1N1) 7.2.2 7.2 7.8.2 7.7.2 A/California/07/2009(H1N1) 7.3.1 7.2.2 7.4.1 7.3.5 A/NewYork/18/2009(H1N1) 7.9.2 ND 8.2.3 ND A/HongKong/8/1968(H3N2) 8.1.4 ND 8.8.2 ND A/NewYork/55/2004(H3N2) 7.8.2 ND 8.6.2 ND A/Wisconsin/67/2005(H3N2) 7.9.1 8.1.1 8.9.2 8.8.2aTCID50andplaqueassaydatafromthreeseparateexperiments.bND,notdeterminedintheseseriesofexperiments.Hamilton etal VirologyJournal 2011, 8 :66 http://www.virologyj.com/content/8/1/66 Page8of9

PAGE 9

trouble-shootingandcell-banking,andhelpedwritethemanuscript;DEW performedplaqueandTCID50assays,assistedwithdatainterpretation, propagatedMDCKandMv1Lucells,engagedintrouble-shootingand cell-banking,establishedcell-growthcurves,performedplasmocin-related procedures,mycoplasmatests,CASYanalyses,cellbanking,provided figuresrelatedtoCASYanalyses,andhelpedwritethemanuscript;BTW andMKOassistedwithplaqueandTCID50assays,propagatedMDCKand Mv1Lucells,engagedincell-banking,establishedcell-growthcurves, performedplasmocin-relatedprocedures,mycoplasmatests,andCASY analyses;JMMpropagatedMDCKandMv1Lucells,engagedincellbanking,establishedcell-growthcurves,andestablishedmanyrecordkeepingandmonitoringprotocols;DEDperformedplaqueandTCID50assays,assistedwithdatainterpretation,performedtrypsindoseresponse analyses,engagedintrouble-shooting,andmanagedmostprograms relevanttotheworkofthismanuscript;JALconceivedoftheuseofMv1 Lucells,wasinvolvedinmostaspectsofthiswork(excludingCASY analysesandplasmocintreatments)andco-preparedthemanuscript.All authorsreadandapprovedthefinalmanuscript. Competinginterests Theauthorsdeclarethattheyhavenocompetinginterests. Received:22December2010Accepted:11February2011 Published:11February2011 References1.LiIW,ChanKH,ToKW,WongSS,HoPL,LauSK,WooPC,TsoiHW,ChanJF, ChengVC,ZhengBJ,ChenH,YuenKY: Differentialsusceptibilityof differentcelllinestoswine-origininfluenzaAH1N1,seasonalhuman influenzaAH1N1,andavianinfluenzaAH5N1viruses. JClinVirol 2009, 46 :325-330. 2.Schultz-CherryS,Dybdahl-SissokoN,McGregorM,HinshawVS: Minklung epithelialcells:uniquecelllinethatsupportsinfluenzaAandBvirus replication. JClinMicrobiol 1998, 36 :3718-3720. 3.LednickyJA,HamiltonSB,TuttleRS,SosnaWA,DanielsDE,SwayneDE: Ferretsdevelopfatalinfluenzaafterinhalingsmallparticleaerosolsof highlypathogenicavianinfluenzavirusA/Vietnam/1203/2004(H5N1). VirolJ 2010, 7 :e231. 4.MarcusPI,NgunjiriJM,SekellickMJ: Dynamicsofbiologicallyactive subpopulationsofinfluenzavirus;plaque-forming,noninfectiouscellkilling,anddefectiveinterferingparticles. JVirol 2009, 83 :8122-8130. 5.MarcusPI,RojekJM,SekellickMJ: Interferoninductionand/orproduction anditssuppressionbyinfluenzaviruses. JVirol 2005, 79 :2880-2890. 6.NayakDP,ChambersTM,AkkinaRK: Defective-interfering(DI)RNAsof influenzaviruses:origin,structure,expression,andinterference. CurrTop MicrobiolImmunol 1985, 114 :103-151. 7.NgunjiriJM,SekellickMJ,MarcusPI: ClonogenicassayoftypeAinfluenza virusesrevealsnoninfectiouscell-killing(apoptosis-inducing)particles. JVirol 82 :2673-2680. 8.ReinaJ,Fernandez-BacaV,BlancoI,MunarM: ComparisonofMadin-Darby caninekidneycells(MDCK)withagreenmonkeycontinuouscellline (Vero)andhumanlungembryonatedcells(MRC-5)intheisolationof influenzaAvirusfromnasopharyngealaspiratesbyshellvialculture. JClinMicrobiol 1997, 35 :1900-1901. 9.SchepetiukSK,KokT: TheuseofMDCK,MEK,andLLC-MK2celllineswith enzymeimmunoassayfortheisolationofinfluenzaandparainfluenza virusesfromclinicalspecimens. JVirolMethods 1993, 42 :241-250. 10.SwayneDE,SenneDA,SuarezDL: Avianinfluenza. In Isolationand IdentificationofAvianPathogens. 5edition.Editedby:Dufour-ZavalaL, SwayneDE,GlissonJR,PearsonJE,ReedWM,JackwoodMW,WoolcockPR. Jacksonville,Florida:AmericanAssociationofAvianPathologists; 2008:128-134. 11. WorldHealthOrganizationManualonAnimalInfluenzaDiagnosisand Surveillance. [http://www.who.int/vaccine_research/diseases/influenza/ WHO_manual_on_animal-diagnosis_and_surveillance_2002_5.pdf]. 12.HuangYT,TurchekBM: MinkLungCellsandMixedMinkLungandA549 CellsforRapidDetectionofInfluenzaVirusandOtherRespiratory Viruses. JClinMicrobiol 2000, 38 :422-423. 13.MatsuuraY,YanagawaR,NodaH: Experimentalinfectionofminkwith influenzaAviruses. ArchVirol 1979, 62 :71-76. 14.HamiltonSB,DanielsDE,SosnaWA,JeppesenER,OwellsJM,HalpernMD, McCurdyKS,RaynerJO,LednickyJA: Gas-permeableethylenebagsforthe smallscalecultivationofhighlypathogenicavianinfluenzaH5N1and othervirusesinembryonatedchickeneggs. VirolJ 2010, 7(1) :23. 15.SzretterKJ,BalishAL,KatzJM: Influenza:Propagation,quantification,and storage. In Currentprotocolsinmicrobiology.Volume1,Chapter15. New Jersey:JohnWiley&Sons,Inc;2006:Unit15G.1. 16.ReedLJ,MuenchH: Asimplemethodforestimatingfiftypercent endpoints. AmJHyg 1938, 27 :493-497.doi:10.1186/1743-422X-8-66 Citethisarticleas: Hamilton etal .: HighertitersofsomeH5N1and recenthumanH1N1andH3N2influenzavirusesinMv1Luvs.MDCK cells. VirologyJournal 2011 8 :66. Submit your next manuscript to BioMed Central and take full advantage of: Convenient online submission Thorough peer review No space constraints or color gure charges Immediate publication on acceptance Inclusion in PubMed, CAS, Scopus and Google Scholar Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Hamilton etal VirologyJournal 2011, 8 :66 http://www.virologyj.com/content/8/1/66 Page9of9