Isolation of infectious chikungunya virus and dengue virus using anionic polymer-coated magnetic beads

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Isolation of infectious chikungunya virus and dengue

virus using anionic polymer-coated magnetic beads

Sirilaksana Patramool, Eric Bernard, Rodolphe Hamel, Luplertlop Natthanej,

Nathalie Chazal, Pornapat Surasombatpattana, Peeraya Ekchariyawat, Simon

Daoust, Supatra Thongrungkiat, Frédéric Thomas, et al.

To cite this version:

Sirilaksana Patramool, Eric Bernard, Rodolphe Hamel, Luplertlop Natthanej, Nathalie Chazal,

et al..

Isolation of infectious chikungunya virus and dengue virus using anionic

polymer-coated magnetic beads.

Journal of Virological Methods, Elsevier, 2013, 193 (1), pp.55-61.

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ContentslistsavailableatSciVerseScienceDirect

Journal

of

Virological

Methods

j o ur na l h o me pa g e :w w w . e l s e v i e r . c o m / l o c a t e / j v i r o m e t

Isolation

of

infectious

chikungunya

virus

and

dengue

virus

using

anionic

polymer-coated

magnetic

beads

夽

Sirilaksana

Patramool

a

_,

_Eric

_Bernard

b

_,

_Rodolphe

_Hamel

a

_,

_Luplertlop

_Natthanej

c

_,

_Nathalie

_Chazal

b

_,

Pornapat

Surasombatpattana

a

_,

_Peeraya

_Ekchariyawat

a

_,

_Simon

_Daoust

a

_,

_Supatra

_{Thongrungkiat}

d

_,

Frédéric

Thomas

a

_,

_Laurence

_Briant

b,∗∗

_,

_Dorothée

_Missé

a,∗

a_Laboratoire_MIVEGEC,_UMR₂₂₄_{IRD/CNRS/UM1,}_Montpellier,_France

b_Centre_d’étude_d’agents_Pathogènes_et_{Biotechnologies}_pour_la_Santé,_CNRS-UMR_{5236/UM1/UM2,}_Montpellier,_France c_Department_of_Tropical_Hygiene,_Faculty_of_Tropical_Medicine,_Mahidol_University,_Bangkok,_Thailand

d_Department_of_Medical_Entomology,_Faculty_of_Tropical_Medicine,_Mahidol_University,_Bangkok,_Thailand

Articlehistory:

Received30October2012

Receivedinrevisedform11April2013 Accepted24April2013

Available online 10 May 2013 Keywords: Arbovirus Chikungunya Dengue Magneticbeads Viro-Adembeads

a

b

s

t

r

a

c

t

Mosquitoes-bornevirusesareamajorthreatforhumanpopulations.Amongthem,chikungunyavirus (CHIKV)anddenguevirus(DENV)causethousands ofcasesworldwide.Therecentpropagationof mosquitovectorscompetenttotransmitthesevirusestotemperateareasincreasestheirpotentialimpact onsusceptiblehumanpopulations.Thedevelopmentofsensitivemethodsallowingthedetectionand isolationofinfectiousvirusesisofcrucialinterestfordeterminationofviruscontaminationinhumans andincompetentmosquitovectors.However,simpleandrapidmethodallowingthecaptureofinfectious CHIKVandDENVfromsampleswithlowviraltitersusefulforfurthergeneticandfunctional characteri-zationofcirculatingstrainsislacking.Thepresentstudyreportsafastandsensitiveisolationtechnique basedonviralparticlesadsorptiononmagneticbeadscoatedwithanionicpolymer,poly(methylvinyl ether-maleicanhydrate)andsuitableforisolationofinfectiousCHIKVandDENVfromthefourserotypes. Startingfromquitereducedbiologicalmaterial,thismethodwasaccuratetocombinewithconventional detectiontechniques,includingqRT-PCRandimmunoblottingandallowedisolationofinfectious parti-cleswithoutresortingtoastepofcultivation.Theuseofpolymer-coatedmagneticbeadsisthereforeof highinterestforrapiddetectionandisolationofCHIKVandDENVfromsampleswithreducedviralloads andrepresentsanaccurateapproachforthesurveillanceofmosquitovectorinareaatriskforarbovirus outbreaks.

1. Introduction

Chikungunya virus (CHIKV) and dengue virus (DENV) are

arboviruses transmitted to humans by Aedes sp. mosquitoes.

DenguefevercausedbyDENVisthemostcommonarboviral

dis-easeinhumans,with50millionannualcasesinmorethan100

countries,and2.5billionpeopleatrisk(WHO,2012).About500,000

personsrequirehospitalizationeveryyearfordenguehemorrhagic

夽 Thisisanopen-accessarticledistributedunderthetermsoftheCreative Com-monsAttribution-NonCommercial-NoDerivativeWorksLicense,whichpermits non-commercialuse,distribution,andreproductioninanymedium,providedthe originalauthorandsourcearecredited.

∗ Corresponding author at: Laboratoire MIVEGEC, Maladies Infectieuses et Vecteurs: Ecologie, Génétique, Evolution, Control UMR 224 IRD/CNRS/UM1 Montpellier,France.Tel.:+33467416381.

∗∗ Correspondingauthorat:Centred’étudesd’agentsPathogèneset Biotechnolo-giespourlaSanté,CPBS,UMR6236CNRS/UM1,Montpellier,France.

E-mailaddresses:laurence.briant@cpbs.cnrs.fr(L.Briant),dorothee.misse@ird.fr

(D.Missé).

feverand2.5%ofcasesarefatal(WHO,2012).Intherecentyears,

CHIKVhasreemergedin Africaand spread totheIndian Ocean

areaandtoIndiawhereitcausedthousandsofcases(Dasetal.,

2007;Renaultetal.,2007;Soumahoroetal.,2011).AsAedesaegypti

andAedesalbopictusmosquitoes,knownascompetentvectorsfor

CHIKVandDENVdisseminationtohuman,arespreading

world-wide,includingtotemperateareas,bothvirusesrepresentaglobal

threattopublichealth(Charreletal.,2007).Thepotentialthreat

of arboviruses for countrieswhere mostindividuals bearnaïve

immunesystemshasbeenillustratedintherecentyearsbythe

limitedCHIKVoutbreakinEmilie-RomagnainItaly(Angelinietal.,

2008)andbythedetectionofautochtonousDENVandCHIKV

infec-tioninEurope(Gouldetal.,2010;LaRucheetal.,2010).Inthis

context,theearlydetectionofarbovirusesthroughsurveillanceof

insectpopulationsiscriticaltoprovidewarningofpotentialdisease

incursionandforresolvingtheemergenceofsuchepidemicsinthe

future.

ClassicaltechniquesusedfordetectionofCHIKVandDENVin

biologicalsamplesincludereversetranscription(RT)-polymerase

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56 S.Patramooletal./JournalofVirologicalMethods193 (2013) 55–61

chainreaction(PCR),enzyme-linkedimmunoassays,

immunoﬂuo-rescenceassays(ELISA)and serologicaltestsallowingdetection

of speciﬁc IgM or IgG production in exposed humans. While

growthincellculturerepresentsthe“gold-standard”forviral

iso-lation and allows the detection of any virusthat replicates in

thecells,theprocedurerequirestime forthevirustogrowand

forsubsequentidentiﬁcation.Totheopposite,nucleicacid-based

techniques,especiallyRT-PCR,havetheadvantageofspeed,

speci-ﬁcityandsensitivityfordetectionoftheviralRNA.Nevertheless,

thesemethodsdonotenabletofurtherisolateinfectiousparticles,

becausesomereagentsusedforproteinornucleicacids

extrac-tion,includingdetergentsandphenol-chloroform,arewellknown

toinactivateenvelopedviruses.Thedevelopmentofareally

sensi-tivemethodaccuratetoisolatelowamountsofinfectiousparticles

willallowtherapid accesstocirculating strainsofviruses and

isthereforeofinterestfor researchers,includingvirologistsand

entomologistsinterestedintheisolationandfunctional

character-izationofarthropod-bornevirusesfromcirculatingmosquitoes.

Inthisstudy,magneticbeadscoatedwithananionicpolymer,

poly(methylvinylether-maleicanhydrate)[poly(MVE-MA)]were

usedtocapturearbovirusparticlesincludingDENVfromthefour

serotypesandCHIKVinthemediumofinfectedcellcultures.This

methodtherefore representsa newrapid and efﬁcientmethod

forarbovirusescapture, puriﬁcationandconcentrationthatisof

interestfor isolationand furtherphenotypiccharacterizationof

infectiousparticlesfromareducedamountofinfectedbiological

material.

2. Materialsandmethods

2.1. Cells

TheC6/36celllinederivedfromA.albopictuswasgrownin

Mini-mumessentialMedium(MEM)(Invitrogen,France),supplemented

with10%fetalcalfserum(FCS,Lonza,Basel,Switzerland)at28◦C.

TheCCL-125celllinederivedfromA.aegyptiwasculturedinEagle’s

MinimalEssentialMedium(EMEM),supplementedwith20%FCS

and1%glutamineat28◦C.

HEK293Thumanepithelialcells weremaintainedat 37◦C in

DMEM(Lonza,Basel,Switzerland)containing10%inactivatedfetal

calfserumand1%antibiotics.BHK-21andVerocellsusedforvirus

productionandtitrationwereculturedundersimilarconditions.

2.2. Productionofviralstocksandtitration

The pCHIKicsubgenomic clonecontaining the entire CHIKV

genome (37997 strain) and a green ﬂuorescent protein (GFP)

sequencefusedtothe3endofthenonstructuralgeneswaskindly

providedbyS.Higgs(UTMB,Galveston)(Tsetsarkinetal.,2006).

Theinfectiousclonewastranscribedinvitro fromtheSP6

pro-moterusingthemMESSAGEmMACHINEkit(Ambion,SaintAubin,

France)accordingtomanufacturer’sinstructions.RNA(0.5␮g)was

thenelectroporatedintoBHK-21cells(5×106₎_derived_from

ham-sterkidneyﬁbroblasts(ATCC#CCL-10TM₎_with₂_pulses_at_1.5_kV,₂₅

Fand˛ω.Aftertwodays,cellculturesupernatantwasharvested,

ﬁlteredonto0.22mﬁltersandpropagatedintheC6/36cellline

derivedfromA.albopictusaspreviouslydescribed(Gayetal.,2012).

After2days,culturesupernatantwasﬁltered,aliquotedandstored

at₋₈₀◦C.Viralstocksweretitteredusingplaqueassayformation

performedonVerocells,as previouslyreported(Bernardetal.,

2010).ThefourdengueserotypesDENV1(Hawaiistrain)(Halstead

etal.,1970),DENV2(16681strain)(Halsteadetal.,1970),DENV3

(H87strain)(Halsteadetal.,1970)andDENV4(814669strain)(Yao

etal.,2003)werealsopropagatedinC6/36cellsusingsimilar

cul-tureconditions.

2.3. Viruscapture

Virus capture was performed using Viro-Adembeads

(Ademtech, Pessac, France) following manufacturer’s

instruc-tions. Virus-containing supernatants were serially dilutedfrom

109 _to₁₀0_pfu/ml_with_serum _free _medium._Brieﬂy, ₄₀_␮l

Viro-Adembeadswerewashedtwicewithbindingbuffer,mixedwith

40␮lculturesupernatantand360␮lofserumfreemediumand

incubatedfor20minatroomtemperature.Thetubesweresetina

magneticﬁeldfor1minusingtheAdem-MagSVmagneticdevice

(Ademtech,PessacFrance).Thesupernatantswerediscardedand

thebeads-virusescomplexeswerewashedthreetimeswithserum

free medium. Then, thecomplexes wereresuspended either in

25␮lserumfreemediumwhenusedininfectionassaysandPCR

experimentsordilutedin25␮lofRIPAbufferforproteinsanalysis

byimmunoblotting.

2.4. Experimentalinfections

Thebeads-virusescomplexeswereuseddirectlyforcell

infec-tionofC6/36orCCL-125cells.105_cells_were_seeded_in_24-wells

platesinappropriatemedium.After24hinculture,beads-virus

complexeswereaddedtothesupernatantandtheinfectionwas

allowedtoproceedfortheindicatedtime.

2.5. Westernblotting

SamplesinRIPAbufferwereresuspendedin25␮lofgel-loading

buffercontaining90mMTris–Cl(pH6.8),10%2-mercaptoethanol,

2%SDS,0.02%bromopheolblueand20%glycerolandboiledfor

5min. Proteins were separated ona 12% SDS-PAGE and

trans-ferredtoapolyvinyldenediﬂuoride(PVDF)membrane(Millipore,

Molsheim, France). After proteins transfer, the membrane was

saturated with 5% skim milk in PBS for 1h at room

tempera-ture and incubated with mAbs hybridizing withCHIKV capsid

(Greiser-Wilke et al., 1989) or with 4E11 anti-DENV envelope mAbs(Cockburnetal.,2012).AfterthreewashesinPBScontaining

0.1%Tween-20,themembraneswereincubatedwithsecondary

antibodies conjugated to horseradish peroxidase and revealed

usingSuperSignalWestPicoChemiluminescentSubstrate(Thermo

FischerScientiﬁc,Illkirch,France).

2.6. RNAextraction

ViralRNAinculturesupernatantwasisolatedusingtheQIAamp

viralRNAminikit(QIAGEN,Courtaboeuf,France). TheRNAwas

resuspendedin30␮lofRNAsefreedistilledwaterandstoredat

−80◦_C_until_used._RNA_isolation_from_{virus-Viro-Adembeads}

com-plexeswasperformedaspreviouslydescribedusingTriReageant

andphenol-chloroformextraction(Fenardetal.,2009).

2.7. RT-PCRofDENV2negativestrand

DENV2negativestrandRNAwasampliﬁedbysemiquantitative

RT-PCRaspreviouslydescribed(Surasombatpattanaetal.,2011).

Brieﬂy,0.5␮gofRNAwasconvertedtocDNAwithM-MLVReverse

Transcriptase(Promega,Charbonnières-Les-Bains,France)witha

DENV-speciﬁc primer according to manufacturer’s instructions.

Then,PCRwascarriedoutonthecDNAusingTaqDNAPolymerase

(RocheDiagnostics,Meylan,France).Eachreactionof50␮l

con-tained200nMofspeciﬁcprimers(seeTable1).Theampliﬁcation

programwasperformedunderthefollowingcondition:one

dena-turationcycleat95◦Cfor2minfollowedby40cyclesof95◦C15s,

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Table1

NucleotidesequencesofprimersusedforPCRdetectionofDENV1,DENV2,DENV3,DENV4andCHIKV.

Virus Oligonucleotidesequence Protein Position Productsize

DENV1 5

_TCA_ATA_TGC_TGA_AAC_GCG_CGA_GAA_ACC_G₃

Structuralpolyprotein 132–160 482bp

5_CGT_CTC_AGT_GAT_CCG_GGG_(A,G)C₃ _596–614

DENV2 5

_GCA_GAA_CCT_CCA_TTC_GGA_GAC_AGC_TAC_AT₃

Envelopeglycoprotein 2024–2052 392bp

5_AGC_TCA_CAA_CGC_AAC_CAC_TAT₃ _2406–2427

DENV3 5

_TCA_ATA_TGC_TGA_AAC_GCG_CGT_GAA_ACC_G₃

Structuralpolyprotein 117–144 290bp

5GTAACATCATCATGAGACAGAGC3 385–407

DENV4 5TCAATATGCTGAAACGCGCGAGAAACCG3 Structuralpolyprotein 136–163 392bp

5CTCTGTTGTCTTAAACAAGAGA3 506–527

DENV2forqPCR 5GGAAGGAGAAGGACTGCACA3 NS5 9091–9109

104bp

5_{ATTCTTGTGTCCCATCCTGCT}₃ _NS5 _9175–9195

Probe/ROXDENV2 5_[FAM]_{TAAGAGACGTGAGCAAGAAAGAGGGAGGAG}_[TAMRA]₃ _NS5 _9125–9150

CHIKVforqPCR 5GGCAGTGGTCCCAGATAATTCAAG3 nsP2 1682–1706 107bp

5_{GCTGTCTAGATCCACCCCATACATG}₃ _1764–1789

T7-Denv-F 5_{TAATACGACTCACTATAGGGGGAAGGAGAAGGACTGCACA}₃ _T7_promoter-NS5 _9091–9109 ₁₂₄_bp

Denv-R 5_{ATTCTTGTGTCCCATCCTGCT}₃ _NS5 _9175–9195

for2min.Theresultingfragmentof392bpforthenegativestrand wasevidencedbyelectrophoresisinagarosegels.

2.8. DENV-2quantitativerealtimeRT-PCR

TheMaximaTM_Probe/ROX_qPCR_Master_Mix₍₂_×)_(Fermentas,

Saint-RémyLesChevreuses,France)wasusedinallqPCR exper-iments.Eachreactionof25␮lcontained400nMofeachprimer, 250nMofspeciﬁcprobeand1×MaximaTM_Probe/ROX_qPCR

Mas-ter Mix. Primers and probe sequences were already described

(Kong et al.,2006)and arelistedin Table1.Ampliﬁcationwas

performedinanApplied Biosystem7300systemunderthe

fol-lowingconditions:95◦Cfor10minfollowedby40ampliﬁcation

cyclesof95◦Cfor15s,60◦Cfor15sand72◦Cfor30s.Real-time

datawereanalyzedusingtheSDSsoftware(ThermoFischer

Sci-entiﬁc,Illkirch,France). ViralRNAwasquantiﬁedbycomparing

thesample’sthresholdcycle(Ct)valueswithaDenguevirusRNA

standardcurvewhichwasobtainedasfollows:ﬁrstly,totalviral

RNAwaspuriﬁedfromaninfectedcultureusingtheQIAampViral

RNA kit (QIAGEN, Courtaboeuf, France). Then, standard RT-PCR

wascarriedoutusing aprimer containingtheT7-Denv-F5 and

Denv-R5primersaspreviouslydescribed(Luplertlopetal.,2011;

Surasombatpattanaetal.,2012).ThePCRproduct,containingtheT7

promotersequencewasusedtogenerateDENVRNAfragmentsby

invitrotranscriptionusingtheMAXIscriptkit(Ambion,SaintAubin,

France).Then,RNAwaspuriﬁedbyprecipitationinsodiumacetate

andabsoluteethanol.TheamountofRNAgeneratedwas

deter-minedbyspectrophotometryandconvertedtomolecularcopies

usingthefollowingformula:

Ymolecules/␮l= Xg/LRNA

transcript length(bp)×340×6.02×10

23

RNAstandards containing1.21×1010_–1.21_×₁₀3 _RNA_copies

wereusedtoconstructastandardcurve.

2.9. RealtimeRT-PCRampliﬁcationofCHIKVRNA

0.5␮gofRNAwereconvertedtocDNA withM-MLVReverse

Transcriptase(Promega,Charbonnières-Les-Bains,France)withan

oligodT(12:18) primer(Invitrogen, Carlsbad, USA)accordingto

manufacturer’sinstructions.PCRampliﬁcationwascarriedouton

100ngcDNAinareactionmixcontaining0.4␮Mofeachprimer,

and2_␮lSYBRGreenmasterampliﬁcationmix(FaststartDNA

Mas-terplusSYBRGreenIampliﬁcationkit,RocheDiagnostics,Meylan,

France).Foreachampliﬁcation,acontrolreactionwasperformed

inwhichDNAsamplewasreplacedbywater.Reactionswere

sub-jectedtoaﬁrstcycleof10minat95◦Cfollowedby40ampliﬁcation

cyclesof15sat95◦C;15sat65◦Cand20sat72◦ContheRotorGene

system(LabgeneScientiﬁc,Châtel-St-Denis,France).Fluorescence

signalwasrecordedattheendofeachcycle.Astandardcurvewas

generatedfrom101_to₁₀5_copies_of_pCHIKic_plasmid._Primers_used

forampliﬁcationareshowninTable1.

3. Results

Anionic polymer, poly(methyl vinyl ether-maleic anhydrate

(Poly(MVE-MA) hasbeen reportedtobind thesurface of some

virus particles (Sakudo and Ikuta, 2008, 2012; Sakudo et al.,

2011).The present studywas designed to investigate whether

[poly(MVE-MA)]-coatedmagneticbeadsareapplicableforcapture

andisolationofinfectiousCHIKVandDENVfromthefourserotypes.

Tothisend,culturemediumofCHIKV-orDENV-infectedmosquito

cellsormediumfrommock-infectedcellsusedascontrolsample

weresubjectedtoincubationwithViro-Adembeadsfollowingthe

manufacturer’sprotocol(seeschematicrepresentationinFig.1A).

Brieﬂy, viralpreparations were incubatedwithmagnetic beads

undergentleagitationandthenseparatedfromthesupernatant

by applying a magnetic ﬁeld. Complexes recovered from

sam-plesbyanionicmagneticbeadswereresuspendedinlysisbuffer,

subjectedtosodiumdodecylsulfate(SDS)-polyacrylamidegel

elec-trophoresis(PAGE),transferredontomembranesandrevealedwith

antibodiesdirectedeithertoCHIKVcapsidortoDENVenvelope

proteins.Foreachdilution,asamplecorrespondingtothenative

viralpreparationusedintheisolationassaywasruninparallel.

WhileCHIKVorDENVproteinswereundetectablefromthenative

samples(Fig.1BandC),a31kDabandwasrevealedfrom

CHIKV-positivesamplesincubatedwithVirobeads,usinganti-capsidmAbs

(Fig.1B).Similarly,a50kDabandcorrespondingtoDENVenvelope

wasdetectedfrommagneticbeadsisolatedcomplexes(Fig.1C).

Accordingly, in our conditions, proteinsfrom CHIKV and from

DENVcouldbesuccessfullyisolatedfromvirus-containing

suspen-sions.Moreover,envelopeglycoproteinsfromDENV1andDENV2

serotypesweresuccessfullyisolated.

Next,RT-PCRampliﬁcationwasusedtodeterminethe

capac-ityofmagneticbeadstoenrichthesampleinviralgenomicRNA

(Fig.2).Isolationexperimentswereperformedstartingfromserial

dilutionofviralsuspensionscontainingCHIKVorDENVparticles

ofeachofthefourserotypesnormalizedaccordingtotheir

infec-tioustiterdeterminedbyplaqueformingassays.Afterisolation,

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Fig.1.CaptureofCHIKVandDENVantigensbyanionicpolymer-coatedbeads.(A)SchematicrepresentationofexperimentalproceduresusedforcaptureofCHIKVorDENV particlesfromculturesupernatant.(BandC)DetectionofCHIKVorDENVantigensinvirus-beadscomplexes.BeadsfractionrecoveredafterincubationwithCHIKV-positive (B)orDENV1-orDENV2-infectedsamples(C)weresubjectedtoimmunoblotandrevealedwithmAbsspeciﬁcforCHIKVcapsidorforDENVenvelopeglycoproteins.Ineach experiment,thesupernatantofmock-infectedculture(NI)andthenon-capturedvirus-positivesample(Sup)wereruninparallel.Molecularweightmarkersareshowon theleft.Dataarerepresentativeofthreeindependentexperiments.

toRT-PCRampliﬁcationusingprimersspeciﬁc forCHIKVor for

DENVserotypes(seeTable1foroligonucleotidesequences).The

expectedampliﬁcationproductssizeswere107bpforCHIKVand

482bp,392bp,290bpand392bpforDENV1,DENV2,DENV3and

DENV4,respectively.StartingfromCHIKVorDENV suspensions,

RT-PCRanalysisallowedthedetectionofasinglebandwiththe

expectedsize.DENV,RNA wasefﬁcientlyisolated startingfrom

DENVfromthefourserotypes(Fig.2B).Therefore,thebead

frac-tionwhereviralantigensweredetectedalsocontainedtheRNA

genome.Nexttoevaluatetheefﬁciencyofmagneticbeads

puriﬁ-cationmethodgenomicRNAcontainedinvirus-beadscomplexes

werecomparedwiththatpresentinthestartingsample.Tothis

end, viralsuspensions containing increasingamounts of CHIKV

or DENV2 particles ranging from 102 _to ₁₀6_pfu _were _diluted

in400␮l serumfree medium and incubatedwithVirobeads in

conditions described in Section 2. After extraction of

beads-associatednucleicacids,realtimeRT-PCRampliﬁcationwascarried

outandRNAgenomescopieswerequantiﬁedinthebeadfraction

andintheinitialsample.AsshowninFig.3,RNAcopynumbers

detectedfromCHIKVsamplecorrespondedto19–27%oftheinitial

input.ForDENVefﬁciencyofvirusrecoverydeterminedaccording

togenomicRNAcopynumberspresentinthestartingsampleand

inthevirus-beadspreparationevidencedwascomprisedbetween

97%and56%.Thereasonsaccountingforthediscrepanciesbetween

thetwovirusesremainunknown.

CHIKV and DENV are propagated through alternate

replica-tioninmosquitoesandhumanhosts.Aspolymer-coatedmagnetic

beadsmaybeofinterestforisolationofviralparticlesfromhuman

bloodsamples,thecapacityofViro-AdembeadstocaptureCHIKV

capture and DENV particles in serum containing medium was

Fig.2.DetectionofgenomicCHIKVorDENVRNAcapturedonanionicpolymer-coatedmagneticbeads.SupernatantofcellculturesinfectedwithCHIKV(A)orwithDENV1, DENV2,DENV3orDENV4(B)weresubjectedtoserialdilutionstogeneratesuspensionscontaining107_to₁₀1_pfu/ml_and_incubated_with_anionic_{polymer-coated}_beads._After

separation,totalRNAwasextractedandanalyzedbyRT-PCRusingoligonucleotideprimersspeciﬁcforeachviralserotype.ThesizeofPCRproductsisindicated.Ampliﬁcation productsobtainedeitherfromtheinitialsupernatantorfollowingincubationofVirobeadswithmock-infectedsupernatantsareshownascontrols.Alldatapresentedare representativeofthreeindependentexperiments.

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Fig.3. QuantitativeanalysisofRNAinCHIKVandDENV2capturedonanionic polymer-coatedmagneticbeads.SerialdilutionsofCHIKV(A)orDENV(C) prepa-rationscontaining106_to₁₀3_pfu/ml_and_virus-beads_complexes_isolated_from_the

correspondingpreparations(B)and(C)weresubjectedtoqRT-PCRquantiﬁcationof genomicRNAlevels.Valuesarerepresentativeoftwoseparateexperiments.

investigated. Viral suspension containing 106 _to ₁₀2_pfu _CHIKV

orDENV2weresupplementedwithhumanserumcollectedfrom

anuninfectedpatientandprocessedformagneticbeadsisolation

asdescribedpreviously.Then, viralgenomicRNApresentinthe

virus-beadscomplexeswasdetectedbyrealtimeRT-PCR.Inthese

conditions,noRNAcouldbedetectedinanysampleregardlessof

theconcentrationofDENV orCHIKV presentin theinitialviral

suspension(Fig.4).Viralsuspensionsusedinthisexperiment

cor-respondtothoseusedinFig.3.Therefore,thepresenceofhuman

serumabolishedthecapacityofpolymer-coatedbeadsto

concen-trateviralparticles.

Finally, the infectivity of the fraction captured with

Viro-AdembeadsfromCHIKV orDENV suspensions wasdetermined.

ComplexesrecoveredfromCHIKVsampleswereincubatedwith

theC6/36cellline,anA.albopictuscelllinesusceptibletoCHIKV

Fig.4.Viralparticlesrecoveryisinhibitedbyhumanserum.Thecapacityof Viro-AdembeadstoisolateCHIKV(A)orDENV2(B)particlesinthepresenceofnormal humanserumwasdeterminedstartingformserialdilutionsofviralparticles.Viral RNAcontainedinthecomplexeswasquantiﬁedbyrealtimeRT-PCR.Valuesarethe meanofduplicateexperiments.

(Gayetal.,2012).Infectionoftheculturewasanalyzedfor

expres-sionofthevirus-encodedGFPreportergeneatdays1,3and6post

exposure(Fig.5A).Thisreportergene,locatedatthe3endofthe

nonstructuralproteins,isexpresseduponsynthesisoftheCHIKV

replicationcomplex.GFPﬂuorescencewasdetectedafter1dayin

culture.AlmostallthecellswereGFP-positiveafter3days,attesting

forCHIKVreplicationandpropagationintheculture.Basal

ﬂuo-rescenceleveldetectedfollowingincubationofC6/36 cellswith

complexesformedbybeadsandsupernatantofmock-infectedcells

isshownascontrol.Similarexperimentswererepeatedusingthe

LR-OPY1CHIKVstrainisolatedfromtherecentReunionoutbreak

(Tsetsarkinet al., 2006).Data produced withthis variant were

strictlyidenticalthoseobtainedwiththe37997CHIKVreference

strain(datanotshown).Thereleaseofviralparticlesin culture

supernatant wasinvestigated. qRT-PCRanalysisof supernatants

collectedfromCHIKV-infectedC6/36cellsevidencedthepresence

ofviralRNA,attestingthatviralparticlesarereleasedathighlevels

intheculturemedium(Fig.5B).Infectionassayswererepeatedwith

complexescapturedfromDENV2viralsuspensions(Fig.5C).Asa

DENVrecombinantvirusexpressingaGFPreportergenewasnot

available,infectionofC6/36cellsincubatedwithDENV-Virobeads

complexeswasmonitoredbyRT-PCRampliﬁcationofenvelope

gly-coproteingene startingfromRNAisolatedafterlysisof thecell

culture.A392bpampliconcorrespondingtoDENVminus-strand

RNAandindicativeofDENVreplicationwasdetectedafter24hin

culture.IdenticalresultswereobtainedusingtheCCL-125cellline

originatingfromA.aegypti.qRT-PCRanalysisofculturesupernatant

attestedforthecapacityoftheculturetoreleaseDENV2particles

(Fig.5D).Altogethertheseresultsattestfortheinfectivityof

com-plexesisolatedfromCHIKVandDENV2suspensionusinganionic

polymer-coatedmagneticbeads.

4. Discussion

Thisstudyreportstheuseofmagneticanionicpolymer-coated

beadsissuitablefortheisolationofintactCHIKVandDENV

parti-cles.Accordingtoourresults,thismethodisapplicabletothefour

DENVserotypesandtotheprototypalAfricanCHIKV(37997strain)

aswellastotheLR-OPY1CHIKVisolatewithgenetic

characteris-ticsofthestrainthatrecentlyreemergedduringthe2005–2006

outbreakintheIndianOceanarea.Thisisolationtechnologycanbe

combinedwithclassicaldetectionmethods,includingimmunoblot

analysisof viralproteinsand quantitativeampliﬁcationof viral

genomes.Inaddition,capturedparticlesareinfectiousand

prop-agateefﬁcientlyinafreshculture.Accordingly,thecombination

ofapproachesreportedhereincouldserveasausefulstrategynot

onlyforidentiﬁcationofpositivesampleswithlowviralburden,

butalsoforsimultaneousisolationofinfectiousparticlesfroma

singlesample.Goldstandardmethodsusedtoconcentrateviral

particlesgenerallyrelyontheuseofultracentrifugationandPEG

precipitation;nevertheless,thesemethodsdisplayseveralcaveats.

Indeed,PEGprecipitationdoesnotallowtheproductionof

ultra-puriﬁed viralparticles.Thismaybea majorlimitationin some

typeofstudies.Mainly,thepresenceofcontaminatingmaterial,

i.e.derivedfromserumaddedtotheculturemedium,may

cre-atea bias fortranscriptomic and proteomic analysisof cellular

responseselicitedbythevirusintargetcells. Incontrast,

ultra-centrifugationondensitygradientorevenoncushiongenerally

leadstotheproductionofultrapureviralpreparations.However,

insomecases,viralparticlesmaynotsupportwellrepeated

ultra-centrifugationsteps.Virusescollectedintheseprotocolsmayshow

adecreasedinfectivityduetothelossofenvelopeglycoproteinsor

tothealterationofviralparticlesstructure.Thisisspeciallythecase

(7)

Fig.5.Efficientrecoveryofinfectiousarbovirusparticlesusingpoly(MVE-MA)-coatedmagneticbeads.(A)Complexesformedbypoly(MVE-MA)-coatedmagneticbeadsand CHIKVparticleswereincubatedwiththeCHIKV-permissiveC6/36cellline.ViralreplicationwasmonitoredbydirectvisualizationofGFPreportergeneexpressioninthe cellcultureatday1,3and6post-exposure.Culturesexposedtobeadsincubatedwithsupernatantfrommock-infectedcellsareshownascontrol.(C)InfectivityofDENV2 particlesrecoveredusinganionicpolymer-coatedmagneticbeadswasmonitoredbyincubatingthevirus-beadscomplexeswithC6/36orCCL-125cultures.After6daysin culture,cellswerelysedandRNAwasextracted.ThereplicativecomplexespresentinthecellswereevidencedbyRT-PCRamplificationofDENV2RNAnegativestrand.Data arerepresentativeofthreeindependentexperiments.Thesupernatantsofcellculturepresentedin(A)and(C)werecollectedatday6postinfection.ViralRNAwasextracted, reversetranscribedandquantifiedbyrealtimePCRamplificationusingspecificprimers.

polymer-coatedbeadssimultaneouslyallowstheisolationofpure

preparationsofinfectiousparticles.

Virus captureusing anionic polymer-coated magnetic beads

haspreviously beenshown tobe applicabletothe isolation of

variousviruses,includingrespiratorysyncytialvirus,humanand

avian inﬂuenza viruses,cytomegalovirus, herpes viruses, borna

diseasevirusand humanimmunodeﬁciency virus, vacciniaand

rotavirus(Sakudoetal.,2009a,b,2011;Hatanoetal.,2010;Sakudo

andIkuta,2012).Therefore,thistechnologyhasabroadpotential

forthe isolation of diverseviruses.Theresults reportedherein

indicatethatthis techniqueis suitable forisolation ofDENV or

CHIKVtwoviruseswithhighepidemicimpactintropicalcountries

and that are subjected to an epidemic vigilance in temperate

areas. According the present study, this method is of interest

for the detection of DENV and CHIKV viruses. The possibility

tousepolymer-coated beads puriﬁcation methodfor diagnosis

of exposed patients was also considered. Human serum was

foundtoinhibitCHIKVandDENVisolation.Thisinhibitoryeffect

mayreﬂectthecapacityofserumcomponentssuchasalbumin,

to saturate the magnetic beads. Accordingly, when applied to

humanblood samples, theuseof magnetic Virobeads must be

combinedwithdepletionmethodscapabletodecreasealbumin

levelsinthestartingsample.Morelikely,thesigniﬁcantcapacityof

Virobeadstoenricharbovirusparticlesinsamplescontaininglow

amountsofstartingmaterialisofparticularinteresttobeapplied

to surveillance of arthropod vectors in order to provide early

detectionofinvadingvirusesorforfunctionalstudiesofDENVor

CHIKVstrainsvectoredbymosquitoesinvariousgeographicareas.

Thisnon-destructivemethodisofmajorinterestforavarietyof

applicationsincludinggeneticanalysisofcirculatingarboviruses,

especiallywhenisolatedfromsamplescontaininglowamountsof

viralparticles.Itwillpermitcouplingthedetectionandfunctional

studiesofthecorrespondingvirusesisolatedfrominfectedinsects.

Recently,CHIKVandDENV-2havebeenreportedtobe

respon-sibleforalargesimultaneousoutbreakcenteredonGabon(Caron

etal.,2012).A.albopictusmosquitoes,themainvectorforDENVand

CHIKVinrecentoutbreaks,canbeexperimentallyorallycoinfected

bythesetwoviruses(Vazeilleetal.,2010)andthesimultaneous

presenceofCHIKVandDENV-2wasrecentlydetectedina

wild-caughtmosquito(Caronetal.,2012).Inthiscontext,isolationwith

anionicpolymer-coatedbeadsappearsasausefulstrategyto

mon-itorthestatusofcirculatingmosquitoesinregionatriskforDENV

and/orCHIKVoutbreak.Thereducedcost,thegoodsensitivityand

therapidityofthistechnologyallowingfasterisolationthan

tra-ditional techniques areespecially appropriate for simultaneous

analysisofalargenumberofsamples.Obviously,besidesits

inter-estforepidemiologicalvigilanceanddiagnosis,thisstrategywill

represent,in thefuture,aninterestingwaytoinvestigate

virus-hostinteractionsengagedduringearlyreplicationstepsofCHIKV

andDENVinmosquitoesandinhumans.

Acknowledgements

DrPhilippeDesprèsisespeciallyacknowledgedforthekindgift

of4E11antibody.Thisworkwassupportedbygrantsfromthe

FrenchResearchAgency“AgenceNationaldelaRecherche”

(8)

References

Angelini,P.,Macini,P.,Finarelli,A.C.,Pol,C.,Venturelli,C.,Bellini,R.,Dottori,M., 2008.ChikungunyaepidemicoutbreakinEmilia-Romagna(Italy)during sum-mer2007.Parassitologia50,97–98.

Bernard,E.,Solignat,M.,Gay,B.,Chazal,N.,Higgs,S.,Devaux,C.,Briant,L.,2010.

Endocytosisofchikungunyavirusintomammaliancells:roleofclathrinand earlyendosomalcompartments.PLoSONE5,e11479.

Caron,M.,Paupy,C.,Grard,G.,Becquart,P.,Mombo,I.,Nso,B.B.,KassaKassa,F., Nkoghe,D.,Leroy,E.M.,2012. Recentintroductionandrapiddisseminationof chikungunyavirusanddenguevirusserotype2associatedwithhumanand mosquitocoinfectionsinGabon,centralAfrica.Clin.Infect.Dis.55,e45–e53.

Charrel,R.N.,deLamballerie,X.,Raoult,D.,2007. Chikungunyaoutbreaks–the globalizationofvectorbornediseases.N.Engl.J.Med.356,769–771.

Cockburn,J.J.,NavarroSanchez,M.E.,Fretes,N.,Urvoas,A.,Staropoli,I.,Kikuti,C.M., Coffey,L.L.,ArenzanaSeisdedos,F.,Bedouelle,H.,Rey,F.A.,2012. Mechanism ofdenguevirusbroadcross-neutralizationbyamonoclonalantibody.Structure 20,303–314.

Das,S.,Kolher,R.P.,Mane,B.G.,Singh,J.P.,Singh,A.P.,2007.Chikungunyaepidemic: globalandIndianscenario.J.Commun.Dis.39,37–43.

Fenard,D.,Houzet,L.,Bernard,E.,Tupin,A.,Brun,S.,Mougel,M.,Devaux,C.,Chazal, N.,Briant,L.,2009. UracilDNAglycosylase2negativelyregulatesHIV-1LTR transcription.NucleicAcidsRes.37,6008–6018.

Gay,B.,Bernard,E.,Solignat,M.,Chazal,N.,Devaux,C.,Briant,L.,2012.pH-dependent entryofchikungunyavirusintoAedesalbopictuscells.Infect.Genet.Evol.12, 1275–1281.

Gould,E.A.,Gallian,P., De Lamballerie,X., Charrel,R.N.,2010. First casesof autochthonousdenguefeverandchikungunyafeverinFrance:frombaddream toreality!Clin.Microbiol.Infect.16,1702–1704.

Greiser-Wilke, I., Moenning, V., Kaaden, O.R., Figueiredo, L.T., 1989. Most alphavirusesshareaconservedepitopicregionontheirnucleocapsidprotein.J. Gen.Virol.70(Pt3),743–748.

Halstead,S.B.,Udomsakdi,S.,Simasthien,P.,Singharaj,P.,Sukhavachana,P.,Nisalak, A.,1970.Observationsrelatedtopathogenesisofdenguehemorrhagicfever.I. Experiencewithclassiﬁcationofdengueviruses.YaleJ.Biol.Med.42,261–275.

Hatano,B.,Kojima,A.,Sata,T.,Katano, H.,2010. Virus detectionusing Viro-Adembeads,arapidcapturesystemforviruses,andplaqueassayinintentionally virus-contaminatedbeverages.Jpn.J.Infect.Dis.63,52–54.

Kong,Y.Y.,Thay,C.H.,Tin,T.C.,Devi,S.,2006.Rapiddetection,serotypingand quanti-tationofdenguevirusesbyTaqManreal-timeone-stepRT-PCR.J.Virol.Methods 138,123–130.

Luplertlop,N.,Surasombatpattana,P.,Patramool,S.,Dumas,E.,Wasinpiyamongkol, L.,Saune,L.,Hamel,R.,Bernard,E.,Sereno,D.,Thomas,F.,Piquemal,D.,Yssel,H., Briant,L.,Missé,D.,2011. Inductionofapeptidewithactivityagainstabroad spectrumofpathogensintheAedesaegyptisalivarygland,followinginfection withdenguevirus.PLoSPathog.137(1),e1001252.

LaRuche,G.,Souarès,Y.,Armengaud,A.,Peloux-Petiot,F.,Delaunay,P.,Desprès,P., Lenglet,A.,Jourdain,F.,Leparc-Goffart,I.,Charlet,F.,Ollier,L.,Mantey,K.,Mollet,

T.,Fournier,J.P.,Torrents,R.,Leitmeyer,K.,Hilairet,P.,Zeller,H.,VanBortel,W., Dejour-Salamanca,D.,Grandadam,M.,Gastellu-Etchegorry,M.,2010.Firsttwo autochthonousdenguevirusinfectionsinmetropolitanFrance.EuroSurveill. 15,19676,September2010.

Renault,P.,Solet,J.L.,Sissoko,D.,Balleydier,E.,Larrieu,S.,Filleul,L.,Lassalle,C., Thiria,J.,Rachou,E.,deValk,H.,Ilef,D.,Ledrans,M.,Quatresous,I.,Quenel,P., Pierre,V.,2007. AmajorepidemicofchikungunyavirusinfectiononReunion Island,France,2005–2006.Am.J.Trop.Med.Hyg.77,727–731.

Sakudo,A.,Baba,K.,Tsukamoto,M.,Ikuta,K.,2009a. Useofanionicpolymer, poly(methylvinylether-maleicanhydride)-coatedbeadsforcaptureof respi-ratorysyncytialvirus.Bioorg.Med.Chem.Lett.19,4488–4491.

Sakudo,A.,Baba,K.,Tsukamoto,M.,Sugimoto,A.,Okada,T.,Kobayashi,T.,Kawashita, N.,Takagi,T.,Ikuta,K.,2009b.Anionicpolymer,poly(methylvinylether-maleic anhydride)-coatedbeads-basedcaptureofhuman inﬂuenzaAandBvirus. Bioorg.Med.Chem.17,752–757.

Sakudo,A.,Ikuta,K.,2008. EfﬁcientcaptureofinfectiousH5avianinﬂuenzavirus utilizingmagneticbeadscoatedwithanionicpolymer.Biochem.Biophys.Res. Commun.377,85–88.

Sakudo,A.,Ikuta,K.,2012.Atechniqueforcapturingbroadsubtypesandcirculating recombinantformsofHIV-1basedonanionicpolymer-coatedmagneticbeads. Int.J.Mol.Med.30,437–442.

Sakudo,A.,Tanaka,Y.,Ikuta,K.,2011.Captureofinfectiousbornadiseasevirususing anionicpolymer-coatedmagneticbeads.Neurosci.Lett.494,237–239.

Soumahoro,M.K.,Boelle,P.Y.,Gauzere,B.A.,Atsou,K.,Pelat,C.,Lambert,B.,LaRuche, G.,Gastellu-Etchegorry,M.,Renault,P.,Sarazin,M.,Yazdanpanah,Y.,Flahault, A.,Malvy,D.,Hanslik,T.,2011.ThechikungunyaepidemiconLaReunionIsland in2005–2006:acost-of-illnessstudy.PLoSNegl.Trop.Dis.5,e1197.

Surasombatpattana,P.,Hamel,R.,Patramool,S.,Luplertlop,N.,Thomas,F.,Desprès, P.,Briant,L.,Yssel,H.,Missé,D.,2011. Denguevirusreplicationininfected humankeratinocytesleadstoactivationofantiviralinnateimmuneresponses. Infect.Genet.Evol.11,1664–1673.

Surasombatpattana,P.,Patramool,S.,Luplertlop,N.,Yssel,H.,Missé,D.,2012.Aedes aegyptisalivaenhancesdenguevirusinfectionofhumankeratinocytesby sup-pressinginnateimmuneresponses.J.Invest.Dermatol.132(8),2103–2105,

http://dx.doi.org/10.1038/jid.2012.76.

Tsetsarkin,K.,Higgs,S.,McGee,C.E.,DeLamballerie,X.,Charrel,R.N., Vanlanding-ham,D.L.,2006.Infectiousclonesofchikungunyavirus(LaReunionisolate)for vectorcompetencestudies.VectorBorneZoonoticDis.6,325–337.

Vazeille,M.,Mousson,E.,Martin,E.,Failloux,A.B.,2010. Orallyco-infectedAedes albopictusfromLaRéunionIsland,IndianOcean,candeliverbothdengueand chikungunyainfectiousparticleintheirsaliva.PLoS Negl.Trop.Dis.4(6), e706.

WorldHealthOrganization(WHO),2012.WorldHealthStatistics.WHO,Geneva, Fact sheet no. 117. Available from: http://www.who.int/gho/publications/ worldhealthstatistics/ENWHS2012Full.pdf

Yao,H.,Fang,M.,Zhao,W.,Duan,F.,Lin,L.,Chen,C.,2003. NS2a–NS2bsequence analysisofDEN-4virusstrains.SoutheastAsianJ.Trop.Med.PublicHealth34, 554–558.