Ferrets develop fatal influenza after inhaling small particle aerosols of highly pathogenic avian influenza virus A/Viet...

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Title:
Ferrets develop fatal influenza after inhaling small particle aerosols of highly pathogenic avian influenza virus A/Vietnam/1203/2004 (H5N1)
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Mixed Material
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English
Creator:
Lednicky, John A.
Hamilton, Sara B.
Tuttle, Richard S.
Sosna, William A.
Daniels, Deirdre E.
Swayne, Davia E.
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BioMed Central (Virology Journal)
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Abstract:
Background: There is limited knowledge about the potential routes for H5N1 influenza virus transmission to and between humans, and it is not clear whether humans can be infected through inhalation of aerosolized H5N1 virus particles. Ferrets are often used as a animal model for humans in influenza pathogenicity and transmissibility studies. In this manuscript, a nose-only bioaerosol inhalation exposure system that was recently developed and validated was used in an inhalation exposure study of aerosolized A/Vietnam/1203/2004 (H5N1) virus in ferrets. The clinical spectrum of influenza resulting from exposure to A/Vietnam/1203/2004 (H5N1) through intranasal verses inhalation routes was analyzed. Results: Ferrets were successfully infected through intranasal instillation or through inhalation of small particle aerosols with four different doses of Influenza virus A/Vietnam/1203/2004 (H5N1). The animals developed severe influenza encephalomyelitis following intranasal or inhalation exposure to 101, 102, 103, or 104 infectious virus particles per ferret. Conclusions: Aerosolized Influenza virus A/Vietnam/1203/2004 (H5N1) is highly infectious and lethal in ferrets. Clinical signs appeared earlier in animals infected through inhalation of aerosolized virus compared to those infected through intranasal instillation.
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Lednicky et al. Virology Journal 2010, 7:231 http://www.virologyj.com/content/7/1/231; Pages 1-15
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doi:10.1186/1743-422X-7-231 Cite this article as: Lednicky et al.: Ferrets develop fatal influenza after inhaling small particle aerosols of highly pathogenic avian influenza virus A/Vietnam/1203/2004 (H5N1). Virology Journal 2010 7:231.

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© 2010 Lednicky 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.
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RESEARCHOpenAccess Ferretsdevelopfatalinfluenzaafterinhaling smallparticleaerosolsofhighlypathogenicavian influenzavirusA/Vietnam/1203/2004(H5N1) JohnALednicky 1,4* ,SaraBHamilton 1 ,RichardSTuttle 1,3 ,WilliamASosna 1 ,DeirdreEDaniels 1 ,DavidESwayne 2 Abstract Background: ThereislimitedknowledgeaboutthepotentialroutesforH5N1influenzavirustransmissiontoand betweenhumans,anditisnotclearwhetherhumanscanbeinfectedthroughinhalationofaerosolizedH5N1virus particles.Ferretsareoftenusedasaanimalmodelforhumansininfluenzapathogenicityandtransmissibility studies.Inthismanuscript,anose-onlybioaerosolinhalationexposuresystemthatwasrecentlydevelopedand validatedwasusedinaninhalationexposurestudyofaerosolizedA/Vietnam/1203/2004(H5N1)virusinferrets.The clinicalspectrumofinfluenzaresultingfromexposuretoA/Vietnam/1203/2004(H5N1)throughintranasalverses inhalationrouteswasanalyzed. Results: Ferretsweresuccessfullyinfectedthroughintranasalinstillationorthroughinhalationofsmallparticle aerosolswithfourdifferentdosesof Influenzavirus A/Vietnam/1203/2004(H5N1).Theanimalsdevelopedsevere influenzaencephalomyelitisfollowingintranasalorinhalationexposureto10 1 ,10 2 ,10 3 ,or10 4 infectiousvirus particlesperferret. Conclusions: Aerosolized Influenzavirus A/Vietnam/1203/2004(H5N1)ishighlyinfectiousandlethalinferrets. Clinicalsignsappearedearlierinanimalsinfectedthroughinhalationofaerosolizedviruscomparedtothose infectedthroughintranasalinstillation. Background HumaninfectionscausedbyH5N1highlypathogenic avianinfluenzaviruses(H5N1)thatarosefrom2003onwardshavebeenrareasevidentbyonly500casesconfirmedthrough5July,2010.However,H5N1haveafatalityrateofabout59%[1].Inferrettransmissionmodels, theH5N1viruseswereincons istentintransmissionby directorindirectcontactexpo sureincludingrespiratory droplets,butdirectintranasalexposurecausedmorbidity andsometimes,mortality[2 ,3].Incontrast,the1918 pandemicinfluenzaviruswaseasilytransmissible,especiallyhuman-to-human ,andcausedthedeathsof between20-40millionpeopleworldwideforalethality rateof2.5%,andexperimentalstudiesdemonstratedefficienttransmissionferret-to-ferretbyrespiratorydroplets [4].Thedifferencesintransmissibilityandlethality betweenthetwovirusesisnotfullyunderstood,butthe useofaerosolchallengemayimproveourunderstanding offactorsresponsiblefortransmissionandlethalityof theH5N1viruses. Thereislimitedknowledgeaboutthepotentialroutes anddeterminantsrequiredforH5N1influenzavirus transmissiontoandbetweenhumans,anditisnotclear whetherhumanscanbeinfect edthroughinhalationof aerosolizedcontemporaryH5N1virusparticles.Receptordistributioninthehumanairwayisproposedto restrictefficientinter-humantransmissionofH5N1 influenzavirus[5].Humaninfluenzavirusesspecifically recognize a 2,6-linkedsialicacid(SA)receptors,which aredominantonepithelialcellsintheupperrespiratory tract[5].Incontrast,avianin fluenzavirusesspecifically recognize a 2,3-linkedSAreceptors,whicharelocatedin thelowerrespiratorytract[5,6]andarenoteasily reachedbythelargedroplets(diameterof>10 m) producedbycoughingorsn eezing[7].Asreviewedby Tellier[8],variouspublicationsstatethatlarge-droplet *Correspondence:jlednicky@mriresearch.org 1 EnergyandLifeSciencesDivision,MidwestResearchInstitute,425Volker Boulevard,KansasCity,Missouri64110,USA Fulllistofauthorinformationisavailableattheendofthearticle Lednicky etal VirologyJournal 2010, 7 :231 http://www.virologyj.com/content/7/1/231 2010Lednickyetal;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and reproductioninanymedium,providedtheoriginalworkisproperlycited.

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transmissionisthepredominantmodebywhichinfectionbyseasonalinfluenz aAvirusesisacquiredby humans[7,9,10].However,othersrefertoaerosolsasan importantmodeoftransmissionforinfluenza[11-15].It isalsopossiblethattransmissionoccursthroughdirect contactwithsecretionsorfomiteswithoral,conjunctivalandnasalmucusmembranesbecausetheviruscan remaininfectiousonnonporousdrysurfacesforupto 48hours[16].Sincehumaninfectionswith2003topresentyearH5N1influenzaviruseshasbeenassociated withhighdeathratesandbecausehealthcareworkers cannotasyetbeprotectedbyvaccination,itisimportanttounderstandhowthevirusescanbetransmitted tohumans. Todate,transmissionofH5N1virusestohumanshas beeninefficient,occurred primarilythroughclosecontactwithinfectedbirdsor,inasinglecase,consumption ofrawinfectedduckblood[17].TransmissionofseasonalinfluenzaAvirusesbylargedropletswithoutaccompanyingaerosolshasbeensimulatedbyintranasal dropletinfection[18].ItisassumedthatH5N1infectionsmaybeacquiredthroughdroplettransmission routes,sinceintranasalinoculationofferretswithH5N1 strains(usedasamodelfordropletinfection)canresult inclinicalsignsofsevereinfluenza[3,19-22].Whereas thereissomeevidenceforlimitedhuman-to-human transmissionofH5N1[17,23-26],andtheferretmodel usedasasurrogatefordropletinfectionsuggestsH5N1 infectionscanoccurthroug hdroplets,itisstillunclear whetherdropletinfectionistheprimaryrouteofH5N1 transmissioninhumans.Becausesomeofthecirculating H5N1avianviruseshavedemonstrateduncharacteristic affinityfor a 2,6-linkedSAreceptorsandaretherefore potentiallydangeroustohumans[27,28],itisimportant toevaluatetheirtransmissibilityinasuitableanimal model.Domesticatedferrets( Mustelaputoriusfuro ) havebeenshowntobeanappropriateanimalmodel [29]forstudyofthepathogenicity[19,21]andtransmissibility[30,31]ofinfluenz aviruses.Onthebasisof H5N1viruscelltropismintheirlowerrespiratorytract, ferretshavealsobeenproposedtobeagoodsmall-animalmodelofhumanH5N1pneumonia[6].Since1997, highlypathogenicH5N1viruseshaveevolvedintomultiplegeneticcladesanddifferintheirpathogenicityto mammalianspecies[19,21,32-34].Forexample,some H5N1virusesspreadsystemicallytomultipleorgansof inoculatedferrets[19,21,32]. Wehypothesizedthatclinic allyapparentinfections canarisefrominhalationofaerosolizedH5N1viruses, andtestedourhypothesisusinginhalationexposurestudiesofaerosolizedH5N1inaferretmodel.Inthis report,aerosolsaredefinedassuspensionsinairof smallsolidorliquidparticlesthatremainairbornefor prolongedperiodsoftimesduetotheirlowsettling velocity.Sinceparticles 6 mareincreasinglytrapped intheupperrespiratorytract[35],thesizecut-offof 5 musedbymanyauthorsisalsousedhereinreferencetoaerosols.ThreeavailablerelativelyrecentH5N1s isolatedfromhumansoranimalsfrom2004to2006 thatcausedlowtohighpathogenicityintheiroriginal hosts(Table1)werechosenforaninitialassessmentof pathogenicityinferrets.Ferretswereintranasally instilledwiththeH5N1s.OfthethreeH5N1s,onewas judgedmorevirulentthantheothersandwasaerosolizedusinganose-onlybioaerosolinhalationexposure system(NBIES)thatwerecentlydescribedandvalidated [36].Wereportthatasforintranasalinstillation,inhalationofsmallaerosolparticlesofthatH5N1virusstrain causessevereinfluenzaencep halomyelitisandalethal outcome.Results1.PathogenicityoftheH5N1virusesinferretsfollowing intranasalinoculationThepathogenicityofthethreevirusesdifferedinferrets followingdropletdepositiondirectlyintonasalcavities. Eachviruswasinfectiousateachoftheintranasal(IN) doses(101to104TCID50/ferret).A/Vietnam/1203/2004 (VN/04)causedneurologicalsigns,temperatureelevation,andweightloss(upto21.6%)(Table2),aspreviouslyreported[19-21].Incontrast,whereasferrets inoculatedwithA/Mongo lia/244/2005(MO/05)and A/Iraq/207-NAMRU3/2006(IQ/06)virusesdeveloped fever,theydidnotdevelopneurologicalsigns,andoverallhadlowerweightlosses(upto15.6%)(Table2). NeitherMO/05norIQ/06causedlethalinfectionsand noneoftheanimalsinfectedbythoseviruseshadtobe euthanizedforhumanitarianreasons(Table2).Viruses MO/05andVN/04wereisolatedfrombothnasalwash andrectalswabspecimensatdays3and5p.i.,while IQ/06wasisolatedfromnasalwashesbutnotfromrectalswabspecimens.ThevirusesisolatedinMv1Lucells fromnasalwashesandrectalswabspecimens(Table2) formedcytopathiceffectstypicalforinfluenzaviruses andwereconfirmedasinfluenzaAvirusesbyfirst screeningwithcommercialsolidphaseELISAtest (QuickVueInfluenzaAandBkit,MaterialsandMethods)followedbyRT-PCRandsequencingofrepresentativesamples.Takentogethe r,pathogenicityfollowing intranasalinoculationwasjudged,asgreatesttoleast pathogenic,as:VN/04>MO/05>IQ/06.Fromthese results,VN/04wasthemostvirulentandchosenfor aerosolstudies.2.VirusstabilityinaerosolvehicleThestabilityofVN/04inaerosolvehicle(PBS+0.5% w/vBSAfractionV)wasconfirmed.Afteronehourat roomtemperature,nolossoftiterwasdetectedintheLednicky etal VirologyJournal 2010, 7 :231 http://www.virologyj.com/content/7/1/231 Page2of15

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presenceorabsenceofantifoamagentB(datanot shown).3.InhalationexposureofferretstoVN/04AnimprovedNBIESsystem,slightlymodifiedfromthe originalversion[36]bytheadditionofanadditional pumpattachedtothesamplingsystem(Figure1),functionedasdesignedwithoutmechanicalfailuresorperturbationsofaerosolstream .Ferretholders(prototypes builtforthisproject,Figure2),weredesignedtoaccommodate3-montholdfemaleferrets.Noproblemswere detectedduringinhalationexposure;theanimals ’ faces/ headsdidnotchangecolor(nocyanosisorreddeningof faceorears),suggestingproperoxygenintake,andother signsofstresswerenotobserved.Previoustestsverified thatheattransferfromferretbodyoutoftherestraint tubeswasefficient;neitherheatstressnorelevatedbody temperaturewasdetectedduringinhalationexposure studies.Uponreleasefromtherestrainttubes,the animalsresumednormalbehaviorwithoutincident. Measurementsofthemeanmassaerodynamicdiameter(MMAD)oftheaerosolstreamduringtheexposureperiod(10min)weretakenat30secintervals usingtheAPS.Theresultsforalldosesaresummarized asanaerosolparticlesizelog-probabilityplot(Figure3). Asshown,theMMADrangedfrom3.43-3.5 mwith geometricstandarddeviati ons(GSD)of1.94-2.0over 4doseranges.Foraerosolvehicle(PBS+BSA)alone, thevalueswere:MMADof3.53 m,GSD=2.4.ClinicalobservationsandpathogenicityofVN/04in ferretsfollowingaerosolexposureTheresultsofexposuretoaerosolizedVN/04aresummarizedinTable3.Astypicalfortherange-finding pilotexperimentsperformedhere,thenumbersofanimalsthatwereusedinthisworkaresmall[19-22]but suggestthatseriousclinicalsignsoccursoonerinanimalsexposedtoaerosolizedVN/04thananimals infectedbythesamevirusthroughINinstillation (Table4).Neurologicsignswerealsoapparentina greater%ofanimals.Looses toolsandsheddingofthe liningofthelargeintestinewereevidentbydaytwop.i. intheaerosolgroup,laterintheINgroup.Feverand weightloss(upto-25.95%)weresimilartothose observedfortheINgroupinfectedwithVN/04.Incontrast,thenegativecontrolgroupthatinhaledonlyaerosolvehicleplusantifoamagent(butnovirus)remained clinicallynormalandach ievedanormalweightgain duringthecourseoftheobservationperiod.ThisindicatedthatneitherinhalationofaerosolvehicleorantifoamBcausedthemorbidityandmortalityinthe animalsexposedtoaerosolizedVN/04. Threeorgans(brain,heart,andlung)chosenforvirus titrationweretakenfromthreeanimalsthatreceived presenteddosesof102,103,or104TCID50asaerosolizedinfectiousvirusparticles.Highertiterswere detectedinbrainthaninlungtissues(Figure4).Brain, heart,kidneys,liver,lungs,andspleenwerealsocollectedforhistologyandimmunohistochemistryanalyses fromtwoanimalsthatreceived101and104TCID50as aerosolizedinfectiousviru sparticles,fromoneanimal instilledwith101TCID50asinfectiousvirusparticles, andonenegativecontrolanimalfromtheaerosoland INgroups.BrainlesionsandH5N1viralantigenwere foundinferretsexposedtovirusbyeitheraerosolorIN routes.Theanimalexposedto101virusparticlesby aerosoldemonstratedevidenceofsystemicdisease,with lesionsinliverandspleentissuesat5daysp.i.Incontrast,theanimalthatreceivedthesamedosebyIN routedevelopedneurologicsignssevendayslater,but didnothaveliverorspleenlesions12daysp.i.Among thethreevirus-infectedanimalsforwhichpathologystudieswereperformed,lunglesionswereapparentonlyin theanimalthatinhaledadoseof104aerosolizedvirus particles.Interestingly,grossexaminationrevealedexternalevidenceofmultilobarpneumoniaonlyinthelungs offerretsreceivingdosesof104virusparticlesbyaerosolorINroutes,consistentwithhistologyandimmunohistochemistryresults.Nolesionswerepresentinthe negativecontrolanimalsthatwereadministeredonly PBS(INgroup)orPBS+antifoamagent(aerosol group). Table1VirusstrainsusedincurrentstudyandpreviousdataonferretpathogenicityViruspathogenicity H5N1virusVirusacronymClade andsubcladeaOriginalhost(reference)Ferrets(reference) A/Vietnam/1203/2004VN/041Highb[21]High[19-21] A/Mongolia/244/2005MO/052.2Highc[50]Moderate[20] A/Iraq/207-NAMRU3/2006IQ/062.2Mildd[39]UnknowneaBasedonthephylogeneticanalysisoftheHAgenes[34].bFatalin10-yr-oldhumanmale.cIsolatedfromdeadwhooperswan.dMildillnessin3-yr-oldhumanmale.eNoknownpreviouslypublisheddata.Lednicky etal VirologyJournal 2010, 7 :231 http://www.virologyj.com/content/7/1/231 Page3of15

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Table2OutcomesofINinstillationsofthreedifferentH5N1influenzavirusesinferretsVirusDosea(TCID50 units/ ferret) ClinicalsignsInactivity indexeNeurologicsignsand relatedobservations LethalityhVirustiteriIsolationof H5N1from rectalswab specimens onindicated day postinfection Maximum weightlossb(%) Weightat terminationc(%) MaximumT increased(C) Nasal washeson indicated day postinfection day3day5day3day5 VN/ 04 4.9101-4.21-3.711.52Noneobserved12(t)22++ 4.9101-20.03-20.031.64Ataxiaf;shakingof head 12(e)1-++ 4.9101NA+9.68NA2Noneobserved12(t)---4.9102-2.36-2.362.02Noneobserved12(t)2.93++ 4.9102-9.18-9.181.42Ataxia7(e)1.93++ 4.9103-0.78+1.821.32Noneobserved12(t)1.91.5++ 4.9103-3.85+5.200.72Noneobserved12(t)2.91.9++ 4.9104-21.64-21.642.04Ataxia;convulsionsg5(e)44++ 4.9104-12.16-12.163.02Ataxia;convulsions5(e)43++ MO/ 05 4.9101-1.23-0.821.01NoneobservedNon-lethal3.92.9++ 4.9101-4.95Nochange2.31NoneobservedNon-lethal6.95.9NDj+ 4.9101-15.57-10.752.42NoneobservedNon-lethal33++ 4.9102-3.99+0.590.82NoneobservedNon-lethal45.9++ 4.9102-3.79-2.011.41NoneobservedNon-lethal44++ 4.9103-5.64-3.881.22NoneobservedNon-lethal78++ 4.9103-3.86-2.451.12NoneobservedNon-lethal43.9++ 4.9104-13.58-7.041.72NoneobservedNon-lethal64.7+ND 4.9104-12.85-0.581.82NoneobservedNon-lethal65++ IQ064.9101-7.28+3.640.41NoneobservedNon-lethal43-4.9101-0.24+2.871.61NoneobservedNon-lethal1.91.9-4.9101-0.59+3.42.01NoneobservedNon-lethal31.9-4.9102-1.0+3.391.02NoneobservedNon-lethal1.91.9-4.9102-2.85-1.831.92NoneobservedNon-lethal21.9-4.9103-0.96+0.241.92NoneobservedNon-lethal2.95-4.9103-1.2-1.22.11NoneobservedNon-lethal67-ND 4.9104-5.4-5.442.12NoneobservedNon-lethal65-4.9104-1.16-1.051.82NoneobservedNon-lethal66--aBasedonTCID50inMv1-Lucells.b,cComparedtobodyweightatday0.dComparedtobaselinetemperature.eHighestinactivityindexvalueinoneobservationpriortodeathoreuthanasia.fAtaxia;incoordinationandunsteadiness.gConvulsions;involuntarymuscularcontractions.hLethality;Dayferreteuthanized(e)forhumanitarianreasonsorterminated(t)atendofstudyiValuesforeachanimalareexpressedasvirustiters(log10TCID50/mL)obtainedusingMv1Lucells.jND;Notdeterminedduetodestructionofthecellularmonolayerbyanothervirus.Lednicky etal VirologyJournal 2010, 7 :231 http://www.virologyj.com/content/7/1/231 Page4of15

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DiscussionWedeterminedthatsmallparticleaerosolsofVN/04 werehighlyinfectiousinferrets.Aspreviouslyshown forINinstillation,VN/04wasneurotropicwheninhaled asasmallparticleaerosol.Atlowinhaleddoses(101to 103TCID50unitsofVN/04/ferret),smallparticleaerosolsofVN/04canresultininfectionandresultingbrain lesionswithoutaccompanyinglunglesionsinferrets.In supportofthisnotion,thetiterofVN/04inbraintissueswashigherthanthatdetectedinlungtissuesin animalsthatinhaledaerosolizedvirus.Atahigher inhaleddose(104TCID50unitsofVN/04/ferret),pneumoniaalsooccurs.ThissmallstudysuggeststhatclinicaldiseaseappearsearlierinferretsexposedtoVN/04 byaerosolversusINroutes,thoughseveredisease resultedfrombothroutesofinoculation. TheMMADmeasurementsshowedconsistentparticle delivery(forallfourdosegroups)thatcenteredona sizerangethatshouldberespiredanddepositedinthe lowerrespiratorytractofhumans.Therewaslittle differenceinsizetotheMMADofthecontrolalone, suggestingonevirusparticlewastrappedinthesaltBSAcomplexintheaerosols.Thereisnoformalproof Figure1 SchematicrepresentationoftheNBIES .Componentsoutside(left)andinside(right)thegloveboxaredemarcated. Figure2 Ferretholder .Shownaretheferretrestrainttubewith integralconnectorcone(above)andpushrod["plunger"](below). Lednicky etal VirologyJournal 2010, 7 :231 http://www.virologyj.com/content/7/1/231 Page5of15

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thattheparticlesdetectedbytheAPSindeedcontained virus(thevirusmayhaveaerosolizedasfreevirusparticles),butdevelopmentoflunglesionsanddetectionof virusinlungtissuesprovedeliveryanddepositionof virusinthelungs.Inaddition,thepresenceofbrain lesionswithoutlunglesionsinthelowerdosegroups, suggestsdepositioninpost eriornasalcavityanddirect extensionalongolfactorynervestothebrain.Previously, intranasalinoculationorfeedingMO/05infected chickenmeattoferretsproducedanupperrespiratory infectionwithlocalextensionalongolfactorynervesto olfactorybulbs[20].Similarly,intranasalinoculation withVN/04producedabundantviralantigeninolfactorybulbsofferrets[20]. TheNBIESexposureportflowvelocity(0.234m/s)is relativelylow(~0.52m/hror~0.84km/hr);therefore stresscausedbyairstreamimpactionontheanimal ’ s faceisnotanissue.Moreover,theactualvolumeofair infrontoftheanimals ’ face(approx.12.9ml)issmall andchangesfrequentlyrela tivelytothevolumedelivered/minforeachport( Qport);thus,rebreathingof exhaledairandstallingofaerosolizedviralparticles shouldnotoccur.Thesystemflowrate Qsysof5L/min surpassesthecalculated Vmfor5animalsbyafactorof about2.9withahighestimateof0.345L/minfor Vm, andafactorof5;withavalueof0.2L/min.Thesame valuesapplytoairchanges;at0.345L/min,thenumber ofairchangesrequiredis0.345L/min5/0.101L= ~17.1,sincethereare49.4changes/min,~2.9air changesoccurperbreath,showingthatadequateairflow isgenerated.Adequateairflowisimportantforaccurate dosecalculationsaswellasforthereductionofstress Figure3 Aerosolsizelog-probabilityplotforVN/04 .TheMMADandGSDareindicatedatfourdifferentconcentrationsofvirusandforthe controlsolution. Lednicky etal VirologyJournal 2010, 7 :231 http://www.virologyj.com/content/7/1/231 Page6of15

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duetotheinhalationofincreasedCO2levelsthatoccurs whenairisre-breathed. Astrikingfindinginthispilotstudywithrelativelyfew animalsisthatinfectionacquiredthroughinhalation exposureresultsinmoreabruptclinicalsignsandmay beassociatedwithincreasedprobabilityofdeveloping neurologicdisease.Sincep athologyexam inationswere performedononlythreevirus-infectedanimals,large conclusionscouldnotbedrawnovertherouteofexposureandpathogenesis.Somegeneralconclusions inferredfromourhistology/ immunohistochemistryand virologyworkarethat:(a)histologicchangesmaynot bepresentevenwithhighvirustiterinparticular tissues,and(b)thatbrainlesionsarepossiblewithout lunglesionsinH5N1infectionssuggestingdirect extensionofthevirusfromposteriornasalcavity througholfactorynervesintothebrain,inagreement withapreviousreport[3].Thesefindingsunderscore theneedtoperformpathologyanalysesinconjunction withvirologyanalysestounderstandthecourseof H5N1disease. The50%infectiousdoseinferrets(FID50)andthe FLD50ofVN/04mightbeinferredbutwerenotdeterminedinthiswork(suchataskrequiresmanymore animals).However,itisclearthatthenumberofinfectiousVN/04particlesnecessarytocausefatalinfection is 40.Fromthiswork,itisconcludedthatVN/04is highlyinfectiousthrougha irborneroutesofinfection. Theextentthisoccursinnaturalinfectionswithviruses withinthecladethatincludesVN/04isunclear.Though Table3OutcomesofexposureofferretstoaerosolizedVN/04Presented dosea(TCID50 units/ferret) FerretweightandtemperatureInactivity indexeNeurologicsignsand relatedobservations LethalityjVirustiterkIsolationof H5N1from analswab specimens onindicated day postinfection Max.wt. lossb(%) Wt.at term.c(%) Max.T increased(C) Nasal washeson indicated day postinfection day3day5day3day5 3.2101-11.17-11.171.72Noneobserved12(e)21++ -16.91-16.910.64Unresponsive(moribund)5(e)34++ -25.95-25.951.94AtaxiafConvulsionsgHyper-responsivnesstotactile stimulus 5(e)24++ 3.4102-20.56-20.561.32Ataxia Convulsions Aggression-dementiah4(e)2NA+NA -8.44-8.441.73NA3(d)NANANANA 3.4103-22.59-22.592.14Ataxia; Hind-limbparalysis; Aggression-dementia 4(e)3NA+NA -17.31-17.311.63Ataxia;Convulsions4(e)4NA+NA 3.4104-17.21-17.211.52Ataxia;Convulsions Headtilti4(d)4NA+NA -19.40-19.402.03Ataxia;Headtilt4(e)3NA+NAaBasedonTCID50inMv1-Lucells.bMax.wt.,Maximumweightcomparedtobodyweightatday0.cWt.atterm.,Weightatterminationcomparedtobodyweightatday0.dComparedtobaselinetemperature.eHighestinactivityindexvalueinoneobservationpriortodeathoreuthanasia.fAtaxia;incoordinationandunsteadiness.gConvulsions;involuntarymuscularcontractions.hAggression-dementia;excessivelyaggressivebitingandsnappingofjawsatshadows,inanimateobjectsincludingcagewalls,andatcaretakers.iHeadtilt(torticollis).jDayferretfounddead(d)ordayeuthanized(e)forhumanitarianreasons.kValuesexpressedaslog10TCID50/mLofvirustitersusingMv1Lucells.Lednicky etal VirologyJournal 2010, 7 :231 http://www.virologyj.com/content/7/1/231 Page7of15

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viruswaspresentinnasalwashes,theamountofvirus intheURTislowwithcontemporaryH5N1s.Furthermore,sneezing,whichprimarilyresultsintheformation ofdroplets,wasrarelyobservedintheinfectedanimals. Thus,droplettransmissionmaybelowerthanthat encounteredwithseasonalinfluenzaviruses.Itremains unclearwhyferrettoferrett ransmissionisinefficient withthisvirus;perhapsthevirusisnotpresentinsignificantquantitiesinaerosolsthatmightaccompany sneezesorcoughs.Currentexplanationsforpoor person-to-persontransmissionvary.OnelineofreasoningisthatH5N1sdonothav eviralpolymerasegenes thatfunctionwellincellsoftheupperrespiratorytract. Forexample,Hatta etal .[37]foundthatmutationof Table4Clinicalandbehavioralobservationsinvirus-infectedferrets.Sign/ObservationRangeofday(s)symptomsobservedpostinoculationwithvirusaWS/05 intranasal IRAQ/06 intranasal Viet/04 intranasal Viet/04 aerosol DeathbN/OcN/ODays5-12Days3-5 Softstool/diarrheaN/ON/ODays3-5Days2-5 FeverDays2-7Days2-7Days2-9Days2-5 InappetenceDays2-7Days4-7Days3-12Days2-5 Laboredbreathingd/wheezingDays4-6N/ODay5Days4-5 LethargyDays3-7Days4-5Days3-12Days1-5 NeurologicsignsAggression-dementiaN/ON/ON/ODay4 AtaxiaN/ON/ODays5-12Days4-5 ConvulsionsN/ON/ODay5Days4-5 Head-tiltN/ON/ON/ODay4 Hind-limbparalysisN/ON/ON/ODay4 Shakingofhead(only)N/ON/ODays11-12N/O Shaking(wholebody)orshiveringDays5-6N/ON/ODays2-4 SneezingDays5-6N/ON/ODay2 WeightlossDays1-7Days4-7Days2-12Days1-5 ResolutionDay8onwardsDay8onwardsUncertainN/O Dehydration/ThinDay7N/ODays5-12Days3-5aTen-dayobservationperiodforMO/05andIRAQ/06;twelve-dayforViet/04.bAnimalsfounddeadoreuthanizedforhumanitarianreasonscN/O;notobserved.dLaboredbreathing;animalsexhibitedopen-mouthbreathingwithexaggeratedabdominalmovement. Figure4 Virustitersinbrain,liver,andlungtissuestakenfromthreeanimals Lednicky etal VirologyJournal 2010, 7 :231 http://www.virologyj.com/content/7/1/231 Page8of15

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oneaminoacidinanH5N1 PB2 gene(thePB2protein isacomponentoftheviralpolymerasecomplex) resultedinefficientreplicationofthevirusinupper respiratorytractcells.Usinganon-humanprimate model(Chineserhesusmacaque),Chen etal .[38] showedthatpneumocytesandmacrophagesofthe lowerairway,nottheciliaryepitheliumofthetrachea andbronchi,werethechieftargetcellsinthelungtissue.Theyconcludethat “ predilectionoftheH5N1virus toinfectthelowerairwaysuggeststhatthefailureofthe virustoattachtotheciliaryepitheliumofthetrachea andbronchimaybealimitingfactorinhuman-tohumantransmissibilityoftheH5N1virus ” .Taken together,tropismforcellsoftheLRTtract,andtherarityofsneezing/coughinginferrets,resultinpoortransmissibilityofthevirus.Thisstudypredictsthatpersonto-persontransmissionwi llreadilyoccurifH5N1 acquirestheabilitytoreplicateintheURTandisreadilyaerosolizedorexpelledindroplets.MethodsVirusesH5N1strainsA/Vietnam/1203/2004andA/Mongolia/ 244/2005werefromarchivesoftheSoutheastPoultry ResearchLaboratory,andA/Iraq/207-NAMRU3/2006 wasfromtheNationalBiodefenseAnalysisandCountermeasuresCenter(NBACC),whichobtainedthevirus fromNavalMedicalResearchUnitNo.3(NAMRU-3), Cairo,Egypt[39](Table1).Theviruseswerereceivedas low-passagestocks,andtheiridentityverifiedusingviral genomicsequencing.Ferretswerepre-screenedand wereshowntobenegativeforantibodiestocirculating seasonalinfluenzavirusesA/SolomonIslands/3/2006 (H1N1),A/Wisconsin/67/2005(H3N2),andB/Malaysia/ 2506/2004(allfromAlexanderKlimov,Centersfor DiseaseControlandPrevention).In-vitro cellgrowthandmanipulationsAstheinfectivityofthevirusesinthisworkwashigher ina Mustelavison (mink)lung(Mv1Lu)cellline(validatedattheMidwestResearchInstitute)thaninthe morecommonlyusedMadinDarbycaninekidney (MDCK)celllineusedforinfluenzaviruswork(datato bepresentedelsewhere),Mv1Lucellswereusedto obtainviraltiters.TheMv1Lucellswerepropagatedin ModifiedEagle ’ sMediumwithEarle ’ ssalts(EMEM) supplementedwithL-Alanyl-L-Glutamine(GlutaMAX ™ InvitrogenCorp.,Carlsbad,CA),antibiotics[PSN;penicillin,streptomycin,neomycin(InvitrogenCorp.)],pyruvate(InvitrogenCorp.), non-essentialaminoacids (InvitrogenCorp.),and10%(v/v)gamma-irradiatedfetal bovineserum(HyClone,Pittsburgh,PA).Thecellswere negativebyPCRforthepresenceofmycoplasmaDNA usingaTakaraPCRMycoplasmaDetectionkit(Takara Bio,USA,ThermoFisher).Influenzavirusesweregrown inMv1Lucellsinserum-freeEMEMotherwisesupplementedaspreviouslydescri bedplusL-1-tosylamido-2phenylethylchloromethylketone(TPCK)-treatedmycoplasma-andextraneousvirus-freetrypsin(Worthington BiochemicalCompany,Lakewood,NJ)in5%CO2at 37C(H5N1)or35C(seasonalviruses).TheTPCKtrypsinusedforthisworkhadhigherspecificactivity thanTPCK-trypsinacquiredelsewhereandtherefore usedatafinalconcentration0.1 g/ml.Viruspreparationswereharvestedwhencytopathiceffects(CPE)typicalforinfluenzaviruseswere 80%[40].The50% tissuecultureinfectiousdose(TCID50)werecalculated bytheReed-Muenchmethod[41].ViruspropagationinembryonatingchickeneggsViruswaspropagatedintheallantoiccavityof9to11 day-oldSPFchickenanemiavirus(CAV)-freeembryonatingchickeneggs(ECE)(CRL)[40,42,43].Rapiddetectionofvirusintissue-culturesupernatants andallantoicfluidsAsneeded,acommercialsolidphaseELISAtest(QuickVueInfluenzaAandBkit,QuidelCorp.,SanDiego, CA)wasusedforrapiddetectionofinfluenzaAorB virusesfollowingthemanufacturer ’ sinstructions.FerretsandtheirPre-qualificationforStudiesStudieswereperformedusingdescented,spayed 3-month-oldfemaleferrets(0.5-0.9kg)(TripleF Farms,Sayre,PA)thatwerehousedindividuallyin HEPA-filtered(inletandexhaust)ventilatedindividual cages(Allentown,Inc.,Allentown,NJ).Theanimals lackedsignsofepizooticcatarrhalenteritis,andwere negativebymicroscopyforentericprotozoanssuchas Eimeria and Isospora speciesusingfecasol,asodium nitratefecalflotationsolution(EVSCOPharmaceuticals, Buena,NJ).Theferretswereseronegativebyahemagglutinationinhibition(HAI)assay[43]tocirculating influenzaBvirusesandH1N1,H3N2,andtheH5N1 influenzaAviruses.Prio rtoperformanceoftheHAI assay,theferretseraweretreatedovernightwithreceptordestroyingenzyme(RDE)(DenkaSeikenUSA,Inc., Campbell,CA)at37Ctoinactivatenon-specificHAI activity,thenheatedat56Cfor60minutestoinactivate remainingRDEactivityandcomplementproteins. Roomconditionsforallworkincluded12hr.light cycles,andanaveragerelativehumidityat30%withina roomtemperaturerangebetween64and84F(17.8to 28.9C).Theanimalswerefedpelletedferretfood (TripleFFarms)andwatered adlibitum ,andhoused andmaintainedunderapplicablelawsandguidelines suchastheGuidefortheCareandUseofLaboratory Animals(InstituteofLaboratoryAnimalResources,Lednicky etal VirologyJournal 2010, 7 :231 http://www.virologyj.com/content/7/1/231 Page9of15

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CommissiononLifeSciences,NationalResearchCouncil,NationalAcademicPress,1996)andtheU.S.DepartmentofAgriculturethroughtheAnimalWelfareAct (PublicLaw89-544andSubsequentAmendments),and withappropriateapprovalsfromtheMidwestResearch InstituteAnimalCareandUseCommittee.Bodytemperaturesweremeasuredtwicedailyviasubcutaneously implantableprogrammabletemperaturetransponders (modelIPTT-300,BioMedicDataSystems,Seaford, DE)implantedintheneck.IntranasalinoculationstudiesProceduresbasedonthoseofZitzow etal .[22]were used.Briefly,twelveferretswereusedforeachvirus study:nine(n=9)forvirusinfection,three(n=3)for non-infectedcontrols.Fe rretswereanesthesizedby intramuscularadministrati onofketamineHCl(25mg/ kg)-xylazine(2mg/kg)-atropine(0.05mg/kg),and instilledwithselecteddosesofvirusesinisotonicphosphatebufferedsaline(PBS) with0.5%purifiedbovine serumalbumin(tostabilizetheviruses)andantibiotics. Fifty lofvirussuspensionwasi nstilledintoeachnostril(100 lofvirussuspension/ferret).Twoferretseach wereinoculatedINwith104,103and102TCID50,and threeferretseachwith101TCID50ofvirus(TCID50valuesdeterminedinMv1Lu).Aback-titrationwasperformedonthevirusdosestoverifyviraltitersperdose. ThreeanimalsservedascontrolsandreceivedINdoses ofa1:30dilutionofsterile,non-inoculatedchicken allantoicfluidinPBS.Alltheanimalswerecagedindividuallyandweighedoncedailyforthedurationofthe study.Bodytemperatureswererecordedtwicedaily fromconsciousanimalsthatwerestimulatedandactive foratleastfiveminutes(asthereisarelativelylargevarianceintherestingandactivetemperaturesofferrets). Atemperatureincrease 1.4Coverbaselinewasconsideredsignificant;thebaselinewastheaveragetemperaturefortheentiregroupoverthepre-doseobservation period. Nasalwashesandrectalswabspecimenswerecollectedat3and5dayspost-inoculationwithvirus.Clinicalsignsincludingsneezing(beforeanesthesia), inappetence,dyspnea,andlevelofactivitywereassessed dailyforthedurationofthestudy(8-10days).Inappetencewasjudgedthroughvisualobservationofthefood remaininginthefeederandspilledwithinthesurroundingarea.Ascoringsystem(relativeinactivityindex [RII])basedonthatdescribedbyReuman etal .[44] andasusedbyGovorkova etal. [19]andZitzow etal. [22]wasusedtoassesstheactivitylevelasfollows:0, alertandplayful;1,alertbutplayfulonlywhenstimulated; 2 ,alertbutnotplayfulwhenstimulated;and3, neitheralertnorplayfulwh enstimulated.Theywere alsomonitoreddailyfornasalandoculardischarge, neurologicaldysfunction,andsemi-solidorliquidstools. Neurologicdysfunctionwasdefinedasdevelopmentof motordysfunction(includingparalysisorposterior paresis),convulsions,ataxi a,seizures,anddepression. Ferretswith>25%lossofbodyweightorwithneurologicdysfunctionwereanesth etizedbyintramuscular administrationofketamineHCl(25mg/kg)-xylazine (2mg/kg)-atropine(0.05mg/kg),theneuthanizedwith Beuthanasia-DSpecial(sodiumpentobarbitaland phenytoinsodium)orequi valent(Euthasol)viathe jugularvein.CollectionofnasalwashesandvirustitrationNasalwasheswerecollectedatthesametimeasrectal swabspecimens(onecollectionofeach/day)afteranaesthesiawithketamine(25mg/kg)essentiallyasdescribed byZitzow etal .[22].Briefly,500 lsterileisotonicPBS containing1%bovineserumalbumin,andpenicillin (100U/ml),streptomycin(100 g/ml)andgentamicin (50 g/ml)wasadministered(250 l/nostril)toinduce sneezesinketamine-anaesthesizedferretsondays3and 5post-inoculationwithvirus.Sneezeswerecollectedin aPetridish,anddilutedto1mlwithcoldPBScontainingantibiotics.A100 laliquotofthedilutedmaterial wasinoculatedintoaT25flaskcontainingMv1Lucells andincubatedtoscreenforthepresenceofH5N1virus, andtheremainderstoredat-80C.Samplespositivefor H5N1virusesbythescreenwerethentitratedforfive daysinMv1Lucellsin96-wellmicrotiterplates.CollectionofrectalswabspecimensandvirusdetectionRectalswabspecimenswerecollectedatthesametime asnasalwashes(onecollectionofeach/day)afteranaesthesiawithketamine(25mg/kg)[22].Flockednylon swabspairedwithUniversalTransportMedium(UTM) (bothfromCopanDiagnostics,Inc.,Murrieta,CA)were usedtocollectandtransportanalswabspecimens.The swabswerepre-moistenedwithsterilePBSpriortospecimencollectionfromsedatedanimals,insertedapproximately0.5inches(~1.3cm)intotherectum,retracted, thenswirledin1mlofUTMinthetransporttube.The transporttubeswerevortexedfor1minutetoemulsify thefecalmaterialinUTM.Theemulsifiedmaterialwas diluted1:10inserum-freecompleteEMEMwithtrypsin, 5PSNandFungizone(amphotericinB)(Invitrogen), and0.5%w/vpurifiedBSAfractionV,andleftatroom temperaturefor1hrtoallowthefecalsolidstosettle andtheantibioticstosuppr essbacteriaandfungi.The liquidabovethesettledsolids(nearly10ml)wasthen addedtoMv1LucellsinT75flasksandincubatedfor 1hrat37C.Thereafter,15mlofserum-freemedia containingtrypsinwasadded.Duetospecimenvariabilityinherentwiththeprocedure,noattemptsweremade toquantitatethevirusinth erectalswabspecimens;Lednicky etal VirologyJournal 2010, 7 :231 http://www.virologyj.com/content/7/1/231 Page10of15

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onlyvirusisolationwasatte mpted.However,previous work(performedbyus)indicatedthetiterofVN/04 was>>MO/05inrectalswabspecimens.CollectionandvirustitrationoforgansSelectedorganswerecollectedforvirustitrationfrom animalsthatwerehumanelyeuthanizedafterexhibiting neurologicsignsoratthetimeoftheanimal ’ sdeath. Pooledorgantissuesampleswerecollectedfromeachof thesixlobespresentinferretlungs,fromallpartsof thebrain,andfromtheliver.Alltissuesweresnap-frozenondryiceuponcollectionandstoredat-80Cuntil theywereassayedforviruscontent/quantity.Tissue sampleswereweighedand~0.5ghomogenizedinsterilePBSwithantibioticsand0.5%w/vpurifiedBSA fractionVtoforma10%w/vhomogenizedsuspension. ThehomogenatesweretitratedforfivedaysinMv1Lu cellsinserum-freemediawithtrypsintodeterminethe log10TCID50pergramoftissue.Thelowerdetection limitwasestimatedat101.3TCID50/grtissue.ExposuresystemandgenerationofaerosolsAnose-onlybioaerosolinhalationexposuresystem (NBIES)assembledinaClassIIIIsoGARDGlovebox (TheBakerCompany,Sanford,ME)inanABSL3+ laboratorywasusedforthiswork[36](Figure1). Anose-onlysystemwaschosenforthisstudyover whole-bodyandotherexposureroutesbecause:(a)it minimizesinfectionbynon -inhalationroutes,(b) reducesrequirementsforpost-exposuredecontaminationofanimals(suchasbywipingexteriorofconscious animalwithbleach),(c)minimizespotentialcontaminationofanimalhousingareas,(d)lessenscontamination risksforanimalcareperson nel,and(e)permitstesting athighvirusconcentrationswhileminimizingquantities ofstartingmaterial.Thelatterconsiderationisimportantforcradletograveworkwithselectagents,wherein experimentsarepreferentia llydesignedtoutilizesmall amountsofagent. Ferretrestrainttubeswithpushrods(prototypesbuilt forthisworkbyCHTechnologies,USA,Westwood,NJ) (Figure2)wereusedalongwithamodel3314AerodynamicParticleSizer(APS) Spectrometer(TSIInc.St. Paul,MN).TheAPSisusedtomeasuretheaerosolsize distributioninthetestatmosphereandisoperatedwith AerosolInstrumentManagersoftware,releaseversion 8.0.0.0(TSI,Inc.)runinaDellLatitudeD600computer. A3-jetBioAerosolNebulizingGenerator(BANG),(BGI Inc.,Waltham,MA)wasused(CHTechnologies).The BANGisalowflow,lowdeadspacenebulizerdesigned tooperateintherangeof1to4litersperminutewith apumpedfluid(recirculated)flowthatfeaturesminimal sampleutilization.Itwascho senoverothernebulizers asthemostappropriategenerationdeviceforthe aerosolizationofinfluenzavirus;considerations included:minimalpotentialdamagetoagent,reduced clumpingofvirus,uniformityofdropletsizedistribution,andefficiency(theamountofvirusthatneedsto bepreparedismuchsmallerthanthatrequiredbysimilaraerosolgenerators). Theexposuresystemcontainssamplingportsthatare tappedfor:(a)measurementofaerosolparticlesize,and (b),samplecollectiontoasse sslive-agentaerosolconcentration.Uptothreeanimalswereexposedper experiment[36].Anexposuretimeof10minuteswas used[36].Totalflowthroughtheinhalationsystemwas 5litersperminduringtheexposurescreatedbythe BANG.ThemetricsforusingtheBANGgeneration devicesinassociationwiththeinhalationsystemwas previouslydescribed[36].Exposureconcentration expressedinTCID50/mlwasdeterminedbysamplingof theaerosolstreamusingtwomodel7531midgetimpingers(AGI;AceGlassIncorporated,Vineland,NJ)connectedinseries. Thedynamicairflowthroughtheaerosoldelivery portsonthesystemexceeds3thetotalventilation volumeofallanimalsexposed.Influenzavirusismixed withanon-toxicvehicle(sterilePBSsolutionwith0.5% purifiedBSAfractionV)tohelpmaintainviabilityofthe virusandactasavehicletogeneratethetestaerosol. Thesalinesolutioniswellcharacterizedanditsacute inhalationtoxicityknown;itdoesnotcauseanacute inflammatoryresponseorstimulateexcessmucussecretionleadingtoincreasedmu cociliaryclearance.Thus, theferretsremainsusceptibletochallengeinfection whenthesalinesolutionisinhaledinthequantities usedinthiswork(J.Lednicky,unpublished).Theexposuresystemisoperateddynamicallyatnegativepressure. Priortolive-agentwork,theaerosolsystemwascharacterizedtoassessindividu alparameters,including exposureporttoportaerosolhomogeneity,aerosolconcentrationrampup,concentrationstabilityanddecline, samplemeasurement,expo surelocationtoexposure locationvariation,andsamplingsystemcollectionefficiencies[36].CalculationsforaerosoltransmissionstudiesThepresenteddose D (definedastheinhaleddoseestimatedfromthemultiplicationoftheaerosolconcentrationandthetotalvolumeofairbreathedinbythe animal)isestimatedfromtheferretrespiratoryrateand durationofaerosolexposure.Byconventionusedin aerobiology,()()()() tRtCtftdtexp exp=0,where R refersto respirationrate,Creferstotheconcentrationofaerosolizedagent, f(t) =%ofagentdepositedinthelungs,and texp= exposuredurationtime.Whenthefollowing assumptionsaremade:aconstantminutevolume(Vm) for R(t) ,aconstantlive-agentaerosolconcentrationLednicky etal VirologyJournal 2010, 7 :231 http://www.virologyj.com/content/7/1/231 Page11of15

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(integratedairsampledet erminedconcentrationfor C (t) ,100%depositionfor f(t) ,and t(exp) isfixedatthe timeofexposure,then: D=RCtexp. Theferretrespiratoryminutevolume(Vm),definedas thevolumeofairinhaledorexhaledoveraminute,was estimatedusingGuyton ’ sformula[45],whereBW= bodyweightingr,andthevolumecalculatedinml:VBWBWm== 21021034075../.Ferretsinthisworkrangedfromabout500to900gr. Fora500grferret,log10BW3/4=0.75log10500=2.02. Theantilogof2.02=105.7;therefore,Vm=2.10 105.7=222.0ml/min(0.22L/min).Similarly,fora900 grferret,Vm=345.1ml/min. Sincemostoftheferretswerecloseto500gr,an approximate Vmvalueof0.2L/minwasusedforthis work.TheVmvalueof0.2L/minusedinthisworkwas consistentwithestimatesobtainedbymultiplyingthe ferrettidalvolume(Vt)expressedinmlthebreathing rate(BR)ofconsciousferretsexpressedasbreaths/minute(bpm).Bydefinition,Vt=thevolumeofairinspired orexpiredwitheachnormalbreath,whereasBR=numberofbreaths/minute(bpm) foraconsciousferret.For ferrets,Vt=6.060.30ml,andBR=33-36bpm [46,47].VVBRmt=UsinganaverageVtvalueof6.06mlandanaverage BRof34.5bpm,Vm=209.01ml/min=0.21L/min. Precisionincalculationsofaerosolconcentrationsand estimatesofthenumberofvirusesinhaledperexperimentdependlargelyonthecollectionefficiency/efficacy oftheimpinger(s).Therefore,theimpingersystemmust firstbecharacterizedtoestablishoperationalparameters determinedtoobtaintherequired D .Systemssimilarto oursareoftendesignedwithasingleimpingerandare operatedwiththeassumptionthat>90%oftheaerosolizedmicroorganismsareen trainedduringsamplingof theaerosolflowthroughtheimpinger.Ifthetrueefficiencyis<90%,asignificantundercountoftheaerosol concentrationcanresult,andthiscausesbothanunderestimateoftheinhaleddoseandanoverestimateof virulence(sincethenumberoforganismstocausean infectionisundercounted) .Moreover,thecollection fluidintheimpingermustmaintaintheaerosolized agentinaviable(infectious)mannerandquantification shouldbeforviableagent.Otherwise,quantificationof aerosolizedagentbasedsolelyonbiochemicalorimmunologicalassays(suchasPCRorELISA)mayconfound understandingbymeasuringbothliveandinactivated agents.TheNBIESwasdesi gnedwithadualimpinger arrangementbasedonourpreviousexperience: aerosolizedVN/04isnotcollectedwithhighefficiency withoneimpingeraloneundertheconditionsweused, whereassomeseasonalinfluenzavirusescanbe(data notshown).Thecollectionfluid(PBS+0.5%w/vpurifiedBSAfractionV)wasvalidatedforthiswork(data notshown).Thelengthystepsandprocedurestodetermineimpingercollectionef ficiencywillbepresented elsewhere.Afterestablishingconditionsresultingin> 90%collectionofliveagentattheimpingers,calculationsbasedon(theoretical)100%efficacyofaerosoldisseminationarederivedtosetoperatingparameters: (1)Assuming100%efficiency,thequantityofaerosolizedvirusparticles(VP)foragiven Csiscalculatedas:VP = CQtsmistexp(2)Theconc.ofvirusinimpingerAisdetermined foragiven Cs(3)Theconc.ofvirusinimpingerBisdetermined forthesame Csinstep2 (4)Thevolumesampledbybothimpingers( Vi)is calculatedfor texp(forthiswork,1L/min10min =10L) (5)Assumingevendisseminationbythesystem,the apparentconcentrationofvirus( Capp) intheaerosol streamiscalculatedas:CVappi=+ sum of virus recovered in impingers AB ()/(6)Thevolumedisseminatedbythesystem( Vs)is calculatedas:Vtsexp= System flow rate(7)At100%efficiency,theconcentrationofVPin theaerosolstream( Caero)is:VP/ Vs(8)Thetrueefficiency(expressedas%)ofthesystemis: Capp/ Caero100 (9) D = Capp Vm texpOncetheaboveareestablished,calculationstypically usedinaerobiologycanbemade.Theconcentrationof virusintheaerosolstream,calculatedfromthevirus collectedinimpingers1and2,where Qagi 1+2isthecollectionflowrateinL/minthroughimpingers( agi )1and 2,is: C CV agi CV agi Q agi t expaero= + + [()()] 12 12Thesprayfactor( SF ),definedastheratioofaerosol concentrationtostartingconcentration,isaunitlessLednicky etal VirologyJournal 2010, 7 :231 http://www.virologyj.com/content/7/1/231 Page12of15

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measureusedtopredictaerosolconcentrationfora startingsolution.A SF iscalculatedforarangeofstartingconcentrationsusingthesamenebulizerandflows designedfortheaerosolchallenge;as: SF C aero C neb =where Cneb=concentrationofstartingsolutioninthe BANGreservoir.Anaveragesprayfactor SFavgisthen determinedfromarangeofvirusconcentrations.The predictedrespiratoryvolumeduringexposure( Ve)is calculatedas:VVtemexp= duration of exposure ()Theaerosolconcentration( Caero)neededtoattain D iscalculatedas: VeCaero=D Thestartingconcentration Csisthencalculatedfrom thevaluecalculatedfor SFavgas: SF C aero C savg=Thesystemdisplacementvolume, Vtot,whichisthe volumeofaerosolizedmaterialleavingthenebulizer/ unittime,wasapproximatedusingtheformulabelow, where d referstoinnerdiameterofthetube/cylinder and l isthelength: V tot d li i i=24Thevelocityofairattheanimals ’ nose(theexposure portaerosolflowvelocity)wascalculatedas: Exposureportaerosolflowvelocity Q port A =Finally,thenumberofsystem(total)airchangeswas calculatedas: Airchanges Q sys V tot =AerosolexposurestudiesViruswasdilutedtotheappropriateconcentrationin aerosolvehicle(PBS+0.5%BSAfractionV),towhich wasaddedantifoam0.25%(v/v)molecular-gradeantifoamagentB(Sigma-Aldrich,Inc.,St.Louis,MO).After mixing,4mlofvirus+an tifoamwasplacedinthe reservoir.Similarly,10mlofPBS+0.5%BSAfractionV butwith0.5.%(v/v)antifoamagentBwasplacedinto eachimpinger.Consciousferretswereusedforinhalationstudies.Asfortheintranasalinoculationstudies, ferretswereexposedtoaeros olizedvirusestoattain delivereddosesof101,102,103,or104infectiousvirus particlesovera10minute exposureperiod.Allwork wasperformedexpeditiouslytominimizestress;animals weremovedinandoutoftheferretrestrainttubesrelativelyquickly.Justpriortoexposure,theanimalswere loadedintoferretrestrainttubesandquicklytransportedtotheClassIIIgloveboxhousingtheNBIES. Thetubeswereaffixedontodesignatedinhalationports, theaerosolgenerated,andtheanimalsexposedaccordingtoexperimentaldesign. Uponcompletion,thetubesweredisengagedand placedinatransportbucket.Thebucketwassealed,its outsidesdecontaminated,r emovedfromtheglovebox, andtransportedwithintheABSL-3suitetoananimal room,wherethetubeswereremovedwithinaBSC.The ferretswerethenremovedfromthetubesandplacedin cages(1animal/cage)withintheBSC,andthecages thereafterstackedinracks.Followingaerosolexposure, cage-sideobservationsincludingevaluationofmortality, moribundity,generalhealt handmorbiditywereperformedoncedailyduringthepre-clinicalstageand twicedaily(atapproximate ly8-hourintervals)after symptomsofinfluenzahaddeveloped.Weightandtemperatureweredeterminedoncedaily.NecropsyAllprocedureswereperformedinanABSL3+laboratory.Forschedulednecrops ies,animalswereanesthetizedthenhumanelyeuthanizedasdescribedaboveby trainedtechnicians.Afterconfirmingdeath,theanimals wereprosectedwithinaClassIIA2BSC.Following grossevaluation,tissuesandorganswerecollectedin thisorderspleen,kidneys,intestines,liver,heart,lungs, brain.Toreducehazards,arotarysawwasnotusedto excisethewholebrainsfromskulls.Instead,Deanbone side-cuttingforcepswereused(RoboszSurgicalInstrumentsCompany,Gaithersburg,MD),andtheskullcut frombacktofrontalongthemedialsuturelinesusing long(>5mm)cuttingstrokes.Noteworthy,fragmentationandproductionofairbornebonechipswerecommonwhenothercuttingtoolswereused,especiallywith short(<2mm)cuttingstrokes.HistologyanalysisOrgansandtissues(brain,lungs,liver,spleen,heart,kidney)werecollected,slicedto 5mminthickness,fixed in10%neutralbufferedformalinfor10daystopreserve thetissuesandinactivatetheH5N1viruses,then embeddedinparaffinforsubsequenthistologyand immunochemistryexaminati ons.Formalin-fixedandLednicky etal VirologyJournal 2010, 7 :231 http://www.virologyj.com/content/7/1/231 Page13of15

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paraffin-embeddedtissuesectionswerestainedwith hematoxylinandeosinforhistologicalevaluation,and adjacentsectionsanalyzedbyimmunohistochemistry withaprimaryantibodythatrecognizedtheinfluenzaA nucleoproteinofH5N1virusesaspreviouslydescribed [48].Bio-containmentfacilitiesIn-vitro and invivo experimentswithH5N1viruses wereconductedinUSDA-approvedbiosafetylevel3enhanced(BSL3+)andanimalbiosafetylevel3enhanced(ABSL3+)containmentfacilities,respectively, andrequireduseofpersonalprotectiveequipmentand occupationalhealthmonitoringprogram.Reversetranscription-PCRandvirussequencingViralRNAswereisolatedfromallantoicfluidorcellculturesupernatant(QIAampViralRNAkit;QIAGEN) andtwo-stepreversetranscription-PCRwasdonewith syntheticuniversalandotheroligonucleotideprimers [43,49].ThesequenceswereanalyzedusinganApplied Biosystem3130DNAanalyzerbyusingBigDyeTerminator(v.3.1)chemistryandthesameoligonucleotide primersusedforamplifications.Acknowledgements ThefollowingNBACCscientistsprovidedsignificantguidanceandtechnical inputinthisproject:Drs.MatthewBender,ElizabethLeffel(presentlyat PharmAthene,Annapolis,MD),MichaelKuhlman(presentlyatBattelle MemorialInstitute,OH),RichardKenyonandKennethTucker.Theauthors thankDr.RobertHuebner[DeputyDirectorofInfluenzaandEmerging DiseasesDivisionBiomedicalAdvancedResearchandDevelopment Authority(BARDA),USDepartmentofHealthandHumanServices],whowas involvedintheinitialdesignoftheaerosolexposuresystemandanimal studies,andDr.ClaireCroutch,whoassistedinthedevelopmentofthe ferretmodel.TheprojectteamisgratefultoDr.KevinKing(presentlyat DiagnosticHybrids,Inc.,OH)forassistanceinregulatoryaffairsandsystem validations,andtoDr.BarryAstroff(MRI)forassistanceinestablishinga dedicatedaerosolfacilityforinhalationexposurestudies.Drs.ChadRoy (TulaneUniv.,LA)andJustinHartings(BiaeraTechnologies,MD)arethanked fortheirearlyinputintosystemdesignandequipmentpurchase.The NBACCvirusacquisitionteamisthankedforacquiringandprovidingIQ/06. Dr.RudolphJaegerandBridgettCorbett(CHTechnologies,Inc.)provided invaluableassistanceinequipmentselection,systemdesign,andassembly. ThesafetyoversightofEricJeppesen,manageroftheMRIBiosafety/ BiosuretyOffice,isgreatlyappreciated.UppermanagementatMRIis thankedforprovidingthefacility,classIIIglovebox,andaerosolgenerating equipmentusedinthisproject.ThisprojectwasperformedattheMRI InfluenzaandRespiratoryPathogenResearchCenterinKansasCity,andwas partlyfundedbyacooperativeteamingagreementbetweenMRIandthe USDA/NBACC,byMRIdevelopmentfunds,andbyanMRIInternalResearch andDevelopmentawardtoDr.JohnLednicky. ThetechnicalsupportprovidedbythefollowingindividualsatMRIisgreatly appreciated:CherylNevins,EmilyBuller,SandraLawrence,MirandaRiley, JulieOwells,JaneMorrissey,LindaPoindexter,AmandaYates,MicahHalpern, andBrandiWilson. Authordetails1EnergyandLifeSciencesDivision,MidwestResearchInstitute,425Volker Boulevard,KansasCity,Missouri64110,USA.2SoutheastPoultryResearch Laboratory,AgriculturalResearchService,UnitedStatesDepartmentof Agriculture,Athens,Georgia30605,USA.36006West78thTerrace,Prairie Village,Kansas66208,USA.4Presentaddress:CollegeofPublicHealthand HealthProfessions,UniversityofFlorida,Box100188,101S.NewellDr(HPNP bldg),Suite2150AGainesvilleFlorida32610,USA. Authors ’ contributions JALconceivedtheoverallNBIESdesignincludingchoiceofBANGnebulizer (withtheassistanceofallpartiesmentionedintheAcknowledgments section),interpreteddata,establishedcalculations,providedoversight,and co-preparedthemanuscript;SBHestablishedaccuratevirusquantification procedures,performedin-vitroviruswork,assistedwithdatainterpretation, andhelpedwritethemanuscript;RSThelpeddesignandassemblethe NBIES,establishedprotocolsforinhalationexposurestudies,helpedwith calculations,ledeffortsonthetestingandvalidationoftheNBIES,assisted withdatainterpretation,andco-wrotethemanuscript;WASassistedwith theassembly,testing,andvalidationofthesystem,andoversawanimal work;DEDassistedwithprogrammanagement,in-vitroviruswork,anddata interpretation;DESprovidedvirusstrainsVN/04andIQ/06,technical assistance,helpedinthedesignofexperiments,performedhistopathology andimmunohistochemistryevaluations,assistedininterpretationofthedata, andco-wrotethemanuscript.Allauthorsreadandapprovedthefinal manuscript. 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