MEDICINA - Volumen 70 - Nº 6, 2010518

ORIGINAL ARTICLE

ISSN 0025-7680

MEDICINA (Buenos Aires) 2010; 70: 518-523

Med. 6-18 2da. sal.

MolECulAr ChArACtErIzAtIoN of sEVErE AND MIlD CAsEs of INfluENzA A(h1N1) 2009 strAIN froM ArgENtINA

ELSA BAumEISTER1, GuSTAvO PALACIOS2, DANIEL CISTERNA1, ALExANDER SOLOvyOv2, 3, JEffREy HuI2, NAzIR SAvJI2, ANA vALERIA BuSSETTI2, ANA CAmPOS1, ANDREA PONTORIERO1, OmAR J. JABADO2, CRAIG

STREET2, DAvID L. HIRSCHBERG2, RAuL RABADAN3, vIRGINIA ALONIO1, vIvIANA mOLINA1,STEPHEN HuTCHISON4, mICHAEL EGHOLm4, W. IAN LIPkIN2

1Instituto Nacional de Enfermedades Infecciosas, ANLIS Dr. Carlos G. Malbrán, Buenos Aires, Argentina,2Center for Infection and Immunity, Mailman School of Public Health, Columbia University, New York, New York, USA,

3Department of Biomedical Informatics, Center for Computational Biology and Bioinformatics, Columbia University, New York, New York, USA, 4454 Life Sciences, Branford, Connecticut, USA

Abstract WhileworldwidepandemicinfluenzaA(H1N1)pdmcasefatalityrate(CFR)was0.4%,Argentina’s was4.5%.Atotalof34strainsfrommildandseverecaseswereanalyzed.Afullgenomesequencingwascarriedouton26ofthese,andapartialsequencingontheremainingeight.WeobservednoevidencethatthehighCFRcanbeattributedtodirectviruschanges.Noevidenceofre-assortment,mutationsassociatedwithresistancetoantiviraldrugs,orgeneticdriftthatmightcontributetovirulencewasobserved.AlthoughthemutationD225Gassociatedwithseverity in the latest reports from theUkraineandNorway isnotobservedamong theArgentinestrains,anaminoacidchangeinthearea(S206T)surroundingtheHAreceptorbindingdomainwasobserved,thesamepreviouslyestablishedworldwide.

key words: H1N1pdm,influenza,Argentina

Resumen Caracterización molecular de cepas de influenza A (H1N1) 2009 de casos leves y graves de la Argentina.Mientrasquelatasadeletalidad(CFR)para(H1N1)pdmentodoelmundoeradel0.4%,enlaArgentinalamortalidadobservadafuede4.5%.Lasecuenciacióndelgenomacompletode26cepasdevirusargentinosdeinfluenzaA(H1N1)pdmdecasoslevesygravesyde8cepassecuenciadasparcialmentenomostróevidenciadequelaelevadatasade letalidadsepuedaatribuirdirectamenteacambiosenelvirus.Noseencontraronhallazgosderecombinación,demutacionesasociadasconlaresistenciaalosmedicamentosantiviralesnidevariacionesgenéticasquepuedancontribuiralavirulenciaobservada.SibienlamutaciónD225Gasociadacon lagravedad,comunicadaen informesprocedentesdeUcraniayNoruega,nosehaencontradoenlascepasargentinasestudiadas,sehaobservadouncambioaminoacídicoenlaregión(S206T)entornoaldominiodelsitiodeuniónalreceptorenlaHA,elmismohalladoencepasdistribuidasalrededordelmundo.

Palabras clave: H1N1pdm,influenza,Argentina

Recibido:26-I-2010 Aceptado:27-IX-2010

Postal address:Dra.ElsaBaumeister,InstitutoNacionaldeEnferme-dadesInfecciosas,ANLISDr.CarlosG.Malbrán,Av.VélezSarsfield563,1281BuenosAires,ArgentinaFax:(54-11)4301-1035 e-mail:ebaumeister@anlis.gov.ar

OnJune11,2009,inacknowledgementofsustainedglobalhumantohumantransmission,theWHOdeclaredthatthenovelinfluenzaAvirus(H1N1pdm)waspandemic.AlthoughH1N1pdmhasnotbeenreportedasmoreviru-lentthanseasonalH1N1influenza,itshightransmissibilityinanimmunologicallynaïvepopulationimpliespotentialforsubstantialmorbidityandmortalityandmandatesclosesurveillance for evolution toward increased virulence.

Basedonworldwidesurveillancedataandmathematicalmodeling,thecasefatalityrate(CFR)ofH1N1pdmwasestimatedat0.4%1, 2. Incontrast,althoughthefirstcaseinArgentinawasdetectedinMay17,3056caseswith137deathswerereportedbyJuly16,representingacomputedCFRof4.5%2.

Thepathogenesisofinfectiousdiseasesistheresultof the interactionbetween thepathogen, thehost andtheenvironment(e.g.secondaryinfections,healthcaresystem).Althoughit is likelythatseveritywillvaryfromcountrytocountrydependingonhealthcareresourcesandthepublichealthmeasuresadoptedtomitigateimpact,asecondpossiblecauseforasignificantlyhighermortalityratemaybechangesinthevirusitself.Influenzavirusesmaychangeinvirulenceandhostrangeasafunctionof

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reassortmentwithstrainsthatcontainvirulencefactors3, orthroughgeneticdrift.ToinvestigatethesepossibilitiesasexplanationsfortheincreasedCFRinArgentinawesequencedthegenomesofH1N1pdmvirusobtainedfromcasesofmildandseveredisease.

materials and methods

Samples collected included: 50 nasal swabs containingH1N1pdmand50virus isolates.Severecasesweredefinedas having a fatal outcome (n = 21) or requiring ventilationsupport(n=15),(Table1).SampleshereanalyzedhadbeenroutinelycollectedforuseinapprovedH1N1testsatINEI-AN-LISMalbrán.Sampleswereidentifiedbeforebeingre-analyzedatColumbiaUniversity.Datawereanalyzedanonymously.

RNAwasisolatedusingtrireagent (Molecular research Center,Cincinnati,OH,USA).DNAwasremovedfromRNApreparationsby treatmentwithDNase I (DNA-free,Ambion,Austin,TX,USA).Reversetranscription(RT)reactionswereperformed using the Superscript II Reverse Transcriptase(Invitrogen,Carlsbad,CA,USA).

Two protocols were used to amplify template for 454pyrosequencing (Roche Life Sciences, CT, USA). In oneprotocol, first strand synthesiswas initiatedwith a randomoctamerlinkedtoanspecificartificialprimersequenceknownas the sequence independent amplification (SIA) primer4; a modifiedprotocolwasalsousedwhereinfirststrandsynthesiswasinitiatedwiththeSIAprimerdopedwithaprimermixturecontaining the same specific sequence linked to influenzavirus A (FLUAV), influenza virus B (FLUBV) and influenzavirusC(FLUCV)sequencesrepresentingtheconservedter-miniof influenzavirusgenomesegments5.Randomlibrariesprepared with thismethod were pyrosequenced. The rawsequencing reads were trimmed and analyzed using GSReferenceMapper (RocheLifeSciences,CT,USA)againstthereferencesequenceA/H1N1/4/California/2009(accessionnumbersFJ966079–FJ966086).

Alternatively, random primed cDNAwere amplifiedwithprimers spanning thewhole genome of H1N1pdm for con-ventional Sanger sequencing.Primerswere designed usingSCPrimer(http://scprimer.cpmc.columbia.edu)6toamplify800bp regions of theH1N1pdm genome. A 500 bp staggeredoverlap strategy was employed to improve fidelity of thesequences. Terminal sequenceswere addedbyPCRusingauniversal influenzaAprimer, targeting theconservedviraltermini(5’ATATCGTCTCGTATTAgt AgA AAC AAg g,)7 combinedwith specific primers positioned near the ends ofthegenomesegments.Real-timePCRSYBRGreenassays(Applied Biosystems, Foster City, CA) were conducted todetermine the viral load in each sample (primers availableuponrequest).Thelibrariesyieldedbetween2475and42188non-hostsequencereads.Mappingindicatedcoveragerangingfrom21%to99%distributedalongtheinfluenzavirus8seg-ments.Virussequencerepresentationwasdirectlyproportionaltoviralload.Atiterofatleast20000genomecopies/reactionwasnecessarytoachieve>90%coverage.

For the clustering analysiswe performed thePrincipalComponentAnalysis(PCA)followedbyconsensusK-meansclustering.Aconsensusclusteringoutofthe1000individualK-means trials was built. Parsimony tree was built of the8concatenatedsegmentsusingthednaparsprogramfromthe PHYLIP package8. Clusters previously found with theK-means algorithm correspond to particular clades in thetree.

Results

Twenty-sixcompletegenomesandeigthpartialgenomescontainingcompleteHAandNAgenewereobtained(Ta-ble1).Theywereanalyzedforre-assortment,presenceofmutationsthatconferantiviralresistanceandphylogeneticclustering.All26genomesobtainedcontainedtheS31Nmutation in theM2protein,whichconferscross-resist-ancetotheadamantineclassofanti-influenzadrugs.Themostcommonlydetectedmutationinoseltamivir-resistantviruses(H274Y)wasnotdetectedinanyofthegenomes.NoneoftheArgentineangenomescontainthemutationD225G inwithin the influenzaHAreceptorbindingsite(Fig.1).Nore-assortmentwithregularseasonalinfluenzastrains(eitherH1N1orH3N2)wasdetected.

ByanalysisoftheeigenvectorsofPCA,weselected17singlenucleotidepolymorphims(SNPs)thatreliablyclas-sified5clusters,whereinthedifferencebetweenanypairofclusterswasatleast2SNPs(Table2).Eachoftheclus-tersisnamedaftertheearliestisolatethatbelongstoit.OuranalysisidentifiesfivedifferentclustersofH1N1pdmsequences.Cluster 1 is composed of sequences firstidentifiedinMexicoinearlyAprilandsequencesobtainedshortlythereafterintheUS,Canada,ChinaandEurope.Cluster2containssequencesdetectedinCalifornia,USinearlyAprilandtheirrelatives,isolatedintheUS,Mexico,Canada,DominicanRepublic,Japan,ChinaandEurope.Cluster3isdefinedbysequencesobtainedinJapaninthesecondhalfofMay.Cluster4iscomposedofisolatesfromNewEngland,USandCanadaobtainedinMayandJune.Cluster5isthelargestcluster,datesbacktotheearliest isolates fromMexico (April 19, 2009) andhaswidegeographicalspread,includingMexico,theUnitedStates,Argentina,Canada,ChinaandseveralEuropeancountries.SpecimensfromArgentinabelongtocluster5.Itisdistinguishedby6mutations:G2163A(PB2)G658A(HA)G1248A(NP)G492A(MA)G600A(MA)A367G(NS)(Fig.2).TheArgentinean isolates (except for #8989) contain acharacteristicpolymorphicmarkerT1623CinthePAseg-ment.Thissubstitutiondoesnotchangetheaminoacid.

Discussion

PCAanalysesrevealedthatallArgentineanisolatesarerelatedsuggestingarecentcommonorigin,butgiventhatthenewersplits(especiallythoseintheCluster5)cannotbe resolved completely,we cannot determinewhethertherewasasingleH1N1pdmintroductioninArgentinaorseveraleventsinvolvinghighlyrelatedviruses.Interest-ingly, oneof thephylogenetically informative sites thatdefinealargesub-cladeincluster5isthetransversionA-Tinposition658,thatresultsinaThr-Serchangeinposition206(H3numbering).Thischangeoccursinthedefined

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receptor-bindingsiteofFLUAVH1N1.Sincethechangeisobservedinbothclinicalsamplesandvirusisolates,itcannotbeattributedtoselectionincellculture.Ithasbeenmodeledextensivelyandthesitesofinteractionbetweenthesialicbasedcarbohydratesandthehemagglutininarewell characterized9-13.Moreover, this same region hadbeendescribedasoneofthemoresignificantantigenicepitopesofH1-subtypeinfluenzaAvirusandrelatedwithvaccineefficacy14.Threesecondarystructureelements,the190helix(residues190-198);the130loop(residues135to138),andthe220loop(residues221to228)formthesidesofthereceptor-bindingsubdomain.Changesinthisarea(D225G,D190E,K222L9, 11)orothersoutside

of thisareathatresult inachange in theconformationof thedomain (e.g.Thr18913), result in changes in theaffinityofthereceptor-bindingsiteandarebelievedtoberesponsibleforhuman,swineoravianhostsadaptation.In addition, the substitutionK222L results in completeabolitionofbinding9.Ofnote,themutationD225GinthissameareahasalsobeenobservedinfatalcasesfromtheUkraineandNorway15, 16.GiventhelimitedavailabilityofdataontheH1N1pdmstrains,wehavenotbeenabletodemonstrateifthischangeispositivelyselected.

Insummary,sequencing resultsof26completege-nomesofH1N1pdmvirusobtained fromcasesofmildandseverediseaseindicatecontinuousvirusevolutionby

TABLE1.–Data for the specimens presented in this study

Sample Date Age Sex Location,Province Symptoms Sample Accession

7649 15-Jun 63yrs. F SaltaCity,Salta mild isolate CY047776-837785 15-Jun 2mth. M Unknown,BuenosAires severe isolate CY047784-917937 17-Jun Unknown Unknown L.Zamora,BuenosAires mild isolate CY047792-997953 17-Jun 46yrs. F Neuquén,Neuquén mild isolate CY047800-077967 17-Jun 35yrs. F Quilmes,BuenosAires mild isolate CY047808-157980 17-Jun 25yrs. F CityofBuenosAires mild isolate CY047816-238019 18-Jun 10yrs. F CityofBuenosAires mild isolate CY047824-318551 19-Jun 26yrs. M Ezpeleta,BuenosAires severe isolate CY047832-398574 21-Jun 18mth. F C.Suárez,BuenosAires severe isolate CY047840-478673 19-Jun 66yrs. F Bariloche,RioNegro mild isolate CY047848-558989 21-Jun 16yrs. F Olavarria,BuenosAires mild isolate CY047856-638994 22-Jun 30yrs. M Olavarria,BuenosAires mild isolate CY047864-719004 22-Jun 12yrs. F SanPedro,BuenosAires mild isolate CY047872-799180 23-Jun 8yrs. M GeneralPico,LaPampa mild isolate CY047880-879333 20-Jun 58yrs. F RioGallegos,SantaCruz mild isolate CY047888-959384 23-Jun 20yrs. M Neuquén,Neuquén mild isolate CY047896-039452 22-Jun 9mth. M CityofBuenosAires mild isolate CY047904-119576 23-Jun 9mth. F Unknown,BuenosAires severe isolate CY047912-079579 23-Jun 5yrs. M Unknown,BuenosAires severe isolate CY047920-279583 24-Jun 5yrs. Unknown Unknown,BuenosAires severe isolate CY047928-359588 24-Jun 1mth. F Unknown,BuenosAires severe isolate CY047936-439597 24-Jun 1yrs. F Unknown,BuenosAires severe isolate CY047944-519705 24-Jun 9yrs. F PuertoMadryn,Chubut mild isolate CY047952-599711 24-Jun 36yrs. Unknown Ushuaia,TierradelFuego mild isolate CY047960-679721 24-Jun 52yrs. F Ushuaia,TierradelFuego mild isolate CY047968-7512295 27-Jun Unknown Unknown Jujuy,Jujuy mild NS* CY047752-5312508 27-Jun Unknown Unknown Neuquén,Neuquén mild NS* CY047754-5513777 30-Jun 50yrs. M Cipolletti,RioNegro mild NS* CY047756-6314044 1-Jul Unknown Unknown Tucumán,Tucumán mild NS* CY047764-6514192 3-Jul 19mth. Unknown Unknown,BuenosAires severe NS* CY047766-6714332 3-Jul 1mth. Unknown CityofBuenosAires severe NS* CY047768-6914591 3-Jul 39yrs. M S.Fernando,BuenosAires severe NS* CY047770-7115765 6-Jul Unknown M Cipolletti,RioNegro mild NS* CY047772-7318426 9-Jul 48yrs. M CityofBuenosAires severe NS* CY047774-75

*NS: nasal swab

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drift,butshownoevidencethattheArgentineanhighcasefatalityratecanbeattributedtochangesinviralvirulenceorresistancetoantivirals.

Acknowledgements:Wewouldliketothankalltheperson-neloftheINEI-ANLISDr.CarlosG.Malbrán.Thisworkwouldnothavebeenpossiblewithouttheirhelpduringthepeakofthe

outbreak.Especially,wewould liketothankall thepersonnelof theVirologyDepartment,wherethemajorityof thesamplehandlingandtestingwasperformed.Thisworkwassupportedby theNational Institutes of Health awards AI051292,U54AI57158(NortheastBiodefenseCenter),NO1AI30027,Google.org,theUnitedStatesDepartmentofDefenseandtheMinistryofScience,TechnologyandProductiveInnovationofArgentina,ANLISInstitutionalStrengtheningPlan.

TABLE2.–Single Nucleotide Polymorphisms (SNPs) used to classify 5 clusters

Locus Consensus Cluster1 Cluster2 Cluster3 Cluster4 Cluster5

888(PB2) C * * * * T1711(PB2) T * * C * *1945(PB2) G * * A * *603(PB1) G * * * * A636(PB1) G * * A * *2000(PB1) T * * C * *2076(PB1) C * * * * T936(PA) G * * A * *1741(PA) A C * * * *4(HA) A * * * * G1408(HA) T C C C * *298(NP) A G G * * *1118(NP) C T * * * *1143(NP) A G G * * *316(NA) A G G * * *742(NA) G A A A * *659(NS) G * * A * *

*: no change compared with the consensus

Fig.1.–Aminoacidalignment(inone-lettercode)withintheinfluenzaHAreceptorbindingsite(position171-237;H3number-ing).Positionsthathadbeenimplicatedinreceptorspecificityareindicatedby^.Positions183,190,222,225,226,2279-11,

13 and 189, 193 and 1959aremarked.Positionswithaminoacidsequencechangesarehighlighted.Theboxoverposition206highlightstheaminoacidchangeinCluster5HAsegments.Originalhostandtypeofreceptorbindingspecificityarealsoindicated;Swine(H)arehumanisolatesofinfluenzavirusofswineorigin.

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Fig.2.–Phylogeneticanalysisof theH1N1pdmsequencesusingtheMaximumParsimonymethod.ForbuildingthetreeweusethePHYLIPpackage8,andforvisualizingitweusetheDendroscopeprogram17.

Conflicts of interest: The fundingorganizationshadnoroleinstudydesign,datacollectionandanalysis,decisiontopublish, or preparation of themanuscript. S.Hutchison andM.Egholmare454 Life Sciencesemployees.

References

1. FraserC,DonnellyCA,CauchemezS,etal.PandemicpotentialofastrainofinfluenzaA(H1N1):earlyfindings.Science2009;324:1557-61.

2. VaillantL,LaRucheG,TarantolaA,BarbozaP.Epide-miologyof fatal casesassociatedwithpandemicH1N1influenza2009.Euro Surveill 2009; 14(33), pii: 19309

3. ZamarinD,OrtigozaMB,PaleseP.InfluenzaAvirusPB1-F2 protein contributes to viral pathogenesis inmice. J Virol2006;80:7976-83.

4. PalaciosG,QuanPL,JabadoOJ,etal.Panmicrobialoli-gonucleotidearray fordiagnosisof infectiousdiseases.Emerg Infect Dis2007;13:73-81.

5. QuanPL,PalaciosG,JabadoOJ,etal.Detectionofres-piratoryvirusesandsubtypeidentificationofinfluenzaA

virusesbyGreeneChipRespoligonucleotidemicroarray.J Clin Microbiol2007;45:2359-64.

6. JabadoOJ,PalaciosG,KapoorV,etal.GreeneSCPrimer:arapidcomprehensivetoolfordesigningdegeneratepri-mersfrommultiplesequencealignments.Nucleic Acids res2006;34:6605-11.

7. HoffmannE,StechJ,GuanY,WebsterRG,PerezDR.Universalprimersetforthefull-lengthamplificationofallinfluenzaAviruses.Arch Virol2001;146:2275-89.

8. Felsenstein J. PHYLIP (Phylogeny InferencePackage)version3.69.Distributedbyauthor;2009.

9. StevensJ,BlixtO,GlaserL,etal.Glycanmicroarrayanaly-sis of the hemagglutinins frommodern and pandemicinfluenzavirusesrevealsdifferent receptorspecificities.J Mol Biol2006;355:1143-55.

10. StevensJ,CorperAL,BaslerCF,TaubenbergerJK,Pa-lese P,Wilson IA. Structure of the uncleaved humanH1hemagglutinin fromtheextinct1918 influenzavirus.Science2004;303:1866-70.

11. TumpeyTM,MainesTR,VanHoevenN,etal.Atwo-aminoacid change in the hemagglutinin of the 1918 influenzavirusabolishestransmission.Science2007;315:655-9.

SEqUENCING of H1N1 PDM froM ArGENtINA 523

12. ChildsRA,PalmaAS,WhartonS,etal.Receptor-bindingspecificity of pandemic influenzaA (H1N1) 2009 virusdeterminedbycarbohydratemicroarray.Nat Biotechnol 2009;27:797-9.

13. GamblinSJ,Haire LF,RussellRJ, et al. The structureand receptor binding properties of the 1918 influenzahemagglutinin.Science2004;303:1838-42.

14. DeemMW,PanK. The epitope regions ofH1-subtypeinfluenzaA,withapplicationtovaccineefficacy.Protein Eng Des Sel2009;22:543-6.

15. ShenJ,MaJ,WangQ.EvolutionarytrendsofA(H1N1)influenza virus hemagglutinin since 1918.PLoS one 2009;4(11):e7789.

16. ProMED-mail.MutationofpandemicinfluenzaA(H1N1)2009 virus inNorway.ProMED-mail 2009, Fri 20Nov2009.

17. HusonDH,RichterDC,RauschC,DezulianT,FranzM, Rupp R. Dendroscope: An interactive viewer forlarge phylogenetic trees.BMC Bioinformatics 2007; 8:460.

- - - -NYAS: What about the importance of the impact factor in scientific publishing?¿Cual es la importancia del factor de impacto en la publicación científica?

Varmus: The impact factor is a completely flawed metric and it’s a source of a lot of unhap-piness in the scientific community. Evaluating someone’s scientific productivity by looking at the number of papers they published in journals with impact factors over a certain level is poisonous to the system. A couple of folks are acting as gatekeepers to the distribution of information, and this is a very bad system. It really slows progress by keeping ideas and experiments out of the public domain until reviewers have been satisfied and authors are allowed to get their paper into the journal that they feel will advance their career.

Elfactordeimpactoesunaviciadamétricayfuentedemuchainfelicidadenlacomuni-dadcientífica.Evaluarlaproductividadcientíficadealguienmirandoelnúmerodeartículosquepublicaronenrevistasconfactoresdeimpactoporsobreunciertonivelesvenenosopara todoelsistema.Unparde tiposactúancomoguardianesde ladistribuciónde lainformación,yestoesunmuymalsistema.Enrealidadretardaelprogresomanteniendoideasyexperimentosfueradeldominiopúblicohastaquelosrevisoressehansatisfechoyalosautoresselespermitepublicarsuartículoenlarevistaqueelloscreenquelosharáavanzarensucarrera.

harold Varmus

AdrienneJ.Burke(Entrevistadora).Spreading Science Knowledge far and Wide. Conversa-tions with seven Science 2.0 pioneers. New York Academy of Sciences Magazine, Spring 2010,p16-8.[HaroldVarmusrecibióelPremioNobeldeFisiologíayMedicina(conJ.MichaelBishop)en1989.FueDirectordelNacional Institute of Health,PresidentedelMemorial Sloan Kettering Cancer Center y,desdejuliodel2010,DirectordelNacional Cancer Institute.Es

Co-fundadoryChairman of the BoarddelaPublicLibraryofScience(PLoS)]