Protocol for the assessment of viral retention capability of membranes

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_{Pierre, Gwenaelle and Furiga, Aurélie and Bergé,}

Mathieu and Roques, Christine and Aimar, Pierre and Causserand,

Christel (2011) Protocol for the assessment of viral retention capability of

membranes. Journal of Membrane Science, vol. 381 (n°1-2). pp. 41-49.

ISSN 0376-7388



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Protocol

for

the

assessment

of

viral

retention

capability

of

membranes

Gwenaëlle

Pierre

a,c,1

_{, Aurélie}

_Furiga

b,c,1

_{, Mathieu}

_Berge

b,c

_{, Christine}

_Roques

b,c

_,

Pierre

Aimar

a,c

_,

_Christel

_Causserand

a,c,∗

a_{UniversitédeToulouse,INPT,UPS,LaboratoiredeGénieChimique,118RoutedeNarbonne,F-31062Toulouse,France} b_{UniversitédeToulouse,INPT,UPS,LaboratoiredeGénieChimique,35ChemindesMaraîchers,F-31062Toulouse,France} c_{CNRS,LaboratoiredeGénieChimique,F-31030Toulouse,France}

a

b

s

t

r

a

c

t

AseriesofexperimentshasbeencarriedouttodeterminetheLogremovalvalue(LRV)ofMS2 bacterio-phagessuspendedinvariousbuffers(osmosedwater,tapwater,aqueoussolutionsofNaClandphosphate buffersolution)duringfiltrationthroughhollowfibermembranesmadeofcelluloseacetate.Viral concen-trationsinpermeateandretentateweredeterminedusingtwodifferentmethods,namelyplaqueforming unit(PFU)counting,whichrevealsonlyinfectiousparticlesandquantitativeRT-PCRwhichdetectsthe total(infectious+inactivated)numberofviralgenomesregardlessoftheirinfectivity.

Fromthisexperimentalstudy,weproposeguidelinesforpreparingthechallengingsolutionsand measuringtheirconcentrationwhichensureareliableassessmentofthemembraneperformance.

1. Introduction

Membranetechnologiesusedindrinkingwaterproductionand wastewatertreatmentprovideaneffectivebarriertopathogens such as virusesaslong asthemembrane integrity is not com-promised [1,2].The development of a virus challengetest is a prerequisite tothe assessment of the capacity of a membrane toretain viruses.Such test requires theselection of test parti-cles,ofa buffer, ofquantificationmethods andof experimental conditions for which the data collected would be meaning-ful.

Although viruschallenge tests are commonly performed by water authorities and companies supplying water disinfection units,nowellestablishedprotocolis availabletoday. Moreover, intheliteratureonthissubject,therangeofoperatingconditions is diverse and the trends reported during filtration are some-timesincontradictionwitheachother.Forexample,thesolvent forsuspendingphagesisnotclearlyestablished.Langletetal.[3]

usephosphatebufferatlowconcentration(typicallyat0.2mM), whereasothersusedistilledwater[4]ormilli-QTM_water[5].

Fil-trationtimeduringwhichthefeedconcentrationremainsconstant

∗ Correspondingauthorat:UniversitédeToulouse,INPT,UPS,Laboratoirede GénieChimique,118RoutedeNarbonne,F-31062Toulouse,France.

E-mailaddress:caussera@chimie.ups-tlse.fr(C.Causserand).

1_{Theseauthorscontributedequallytothiswork.}

isaparametertakenintoaccountinsomeworks,butnotalways clearlyspecified.Ackeretal.[6]recommendafiltrationtimeshorter than6minwithoutgivingfurtherdetails,whichmakesdifficult thecompletionofmembranecharacterizationexperimentsandthe collectionofmultiplesamples.Langletetal.[3]donotspecifytheir filtrationtimebutexplainthatitdependsonthepermeabilityof thetestedmembranes;theyrecommendthefiltrationof400mLof viralsuspensionasaminimum,butthemembraneareausedisnot reported.

Thecapacityofamembranesystemtoreducethebacteriaor viruscontentinastreamisgenerallyquantifiedbythelogarithmic reductionvalue(LRV)definedbyEq.(1):

LRV =Log10



_C r(t) Cp(t)



(1)

withCr(t),virusconcentrationinretentateandCp(t),virus

concen-trationinpermeateattimet.

Inmoststudies,theconcentrationsarethenumbersof infec-tiousvirusespermillilitresdeterminedinpermeateandretentate samples,byplaqueformingunit(PFU)counting.Someauthors sus-pectthismethodtoleadtoanoverestimationofthevirusremoval becauseofpossibleoccurrenceofvirusaggregationinthe perme-atecompartment[3],butaggregationmayoccurintheretentateas well.Inaddition,PFUcountingisaverytime-consumingmethod, whichtakesabout24htogetaconcentrationinasample.

A promising alternative method for counting viruses,is the quantitativereversetranscriptase-polymerasechainreaction (qRT-PCR),whichisclassicallyemployedinmolecularbiologytodetect theviral genome, and has beenreported to bea relevant one fortheevaluation oftheretrovirusremovalbychromatography by Lau et al. [7]. This method seems tobe the mostsensitive ofthespecific tools.On theotherhandqRT-PCR isreportedto detect inactivatedviruses [8]. As a consequence, the detection ofvirusesbasedonqRT-PCRmightleadtofalsepositiveresults

[9].

The virussurrogate usedfor thechallenge tests maybe an issue.Bacteriophagesinfectingcoliformbacteriahavebeen consid-eredaspossibleindicatororganismsforentericvirusesinsurface andgroundwaterscontaminated withfecalmaterial[10,11].As aconsequencetheyareoftenusedassurrogatestoevaluatethe pathogenicvirusremovalefficiencyoffiltrationmembranesused forwatertreatment.Thebacteriophagemostcommonlyusedby scientists and industrialists to challenge membranes is MS2, a nucleicacid(single-strandedRNA)virus[e.g.12,13].The advan-tagesofMS2aremany:itisoneofthesmallestviruses(23–30nm indiameter)then abletorevealsmalldefectsor pores,closein sizeandshape(icosahedralcapsid)toenterichepatitisAvirusand poliovirus,non-pathogenicandrelativelyinexpensive.

An analysis of the relevant literature shows, not necessar-ily explicitly, that virusaggregation, adsorption or inactivation interfereswiththeassessment ofthevirusremovalcapacity of membranes.BeforethedevelopmentofthePCR technique,PFU countingwastheonlywayfor checkingthepresenceofviruses inamedium.DecreasesinPFUvalueswereinterpretedintermsof “virusinactivation”.

Thompsonetal.[14]showthatMS2inactivationistheresult ofexposuretosurfaceforcesatthedynamicair–water–solid inter-face.Moreover,MS2isincreasinglyinactivatedduringmixingin polypropylene tubes as theionic strength of the suspension is raised.MS2inactivationisminimalwhentheair–waterinterfaceis completelyeliminatedfrompolypropylenetubes.Allbatch exper-iments performed withglass tubes demonstrate nosubstantial inactivationofMS2.Thesesauthorsconcludethatviral inactiva-tioninsimpledynamicbatchexperimentsisdependentupon(i) thepresenceofadynamicair–water–solid interface(wherethe solidisahydrophobicsurface),(ii)theionicstrengthofthe sus-pension,(iii)theconcentrationofsurfaceactivecompoundsinthe suspension,and(iv)thetypeofvirusused.

Achangeininfectivitycanhoweverbetheconsequenceofat leastthreemechanisms:virusinactivation,adsorption/adhesionto thewallsoftheequipment,aggregation(asanaggregateofseveral virusesproducesonlyone“plaque”).

Theassessmentofadsorptiononanysolidsurfaceofthetesting equipmentorofthemembraneisofmajorimportanceconsidering theverylowconcentrationofvirusesinvolvedinfiltrationtests,in particularinthepermeate.Theknowledgeofadsorptionkinetics allowstoevaluatethetimetoreachsaturationwithoutwhichan accurateevaluationofvirusretentionbythemembranewouldnot bepossible[15,16].Virusadsorptionisenhancedwhenparticles andmembranechargesareoppositeinsignorsmallinmagnitude. MS2phageshaveanisoelectricpoint(IP)of3.9at100mMionic strength[12],whichsuggestsasignificantnegativechargecarried bythevirusatneutralpHandplaysagainstadsorptionof bacte-riophagesontonegativelychargedmembranesasthoseclassically usedinwatertreatmentprocesses.Ionicstrengthofthefluidalso playsacriticalroleonadsorptionofvirusestosurfacesaswellas ontheiraggregation.Thepresenceofdiandtrivalentcations pro-motesadsorptionofMS2virusesontomembranesbyinfluencing electrostaticinteractions[17].

AggregationofMS2particlesisnotobservedforpHhigherthan theisoelectricpointof theparticle(pH3.9)andionicstrengths

for whichinterparticularrepulsiveelectrostatic interactions are expectedtobesufficientlyscreened(1–100mMNaNO3)[12,18].

Operating at neutral pH then allowed overcoming the aggre-gation process. On the other hand, Langlet et al. [18] clearly show that MS2 phages exhibit significant aggregation for pH <IP, conditions for which aggregates up to a few micrometers in size are observed. Langlet et al. [12] show that Qb, which is another potential virus surrogate, suspended in solutions of largeelectrolyteconcentrationsaggregateover thewholerange of pH from 1.5 to 7.5. This behavior is in favor of choosing MS2 as model particlefor virus challengetest as compared to Qb.

So,althoughtheimpactsoffactorssuchasionicstrength,virus concentrationandfiltrationtimeonthevirusretentionby mem-braneshaveoftenbeenreported,studiesontheinfluenceofthese parametershavenotconductedsofarinasystematicway.Froma technicalpointofview,acharacterizationexperimentmustallow timetotakeseveralpermeatesamples,andtheconcentrationof thechallengingsolutionmustremainashighaspossibleduringthe test.IfoneaccountsfortherecommendationbytheU.S. Environ-mentalProtectionAgency[19]regardingthecontrolofthequality oftreatedsurfacewaterbymembranefiltration,virusfeed con-centrationhastobesufficientlyhightoallowthedemonstrationof upto6.5Logremovalifthesurrogateisremovedtothedetection limit(Eq.(1)).Inaddition,consideringthattheliteraturereports variationsinvirusconcentrationsbyseveralordersofmagnitude overthetimeofanexperiment(onetoafewhours),wehaveto setanacceptablelimit ofsuchvariation.In thepresent project, wehavethereforeconsideredthatifthevirusconcentrationinthe retentatedecreasesbymorethan90%(oneorderofmagnitude), thentheconditionshavetoomuchchangedforbeingconsidered asacceptable.

Theaimofthisstudywasthentodefineexperimentalconditions allowingareliabledeterminationofthevirusretentioncapacity ofa membrane used inwater treatment. Specificattentionhas beenpaidtotheeffectsofaggregation,adsorptionandinactivation ofvirusesduringfiltration.Viralconcentrationsinpermeateand retentateweredeterminedusingtwodifferentmethodsaccording toapreviousstudy[21],namelyplaqueformingunit(PFU)method andqRT-PCRwithRNAextraction.

Wefirstmonitorthechangesinvirusconcentrationovertime intheretentatecircuit,thenchecktheroleofthesomeselected buffers.Fromtheseexperimentalobservations,wepropose guide-linesforareliabledeterminationofthevirusretentioncapacity (LRV)ofamembranesystem.

2. Materialsandmethods 2.1. Bacteriophagestockpreparation

AlltestswereperformedwithMS2phage(ATCC15597-B1)and EscherichiacoliW1485(ATCC12435)ashostbacteria,obtained fromInstitutPasteur(Paris).Thereplicationmethodisdescribedin apreviouspaper[20].Thephagestocksuspension(1011_PFUmL−1₎

wascharacterizedintermsofshapeandsizebytransmission elec-tronmicroscopy(TEM)anddynamiclightscattering(DLS).Despite thefairlyhighviralconcentration,isolated andnon-aggregated viruseswereobtainedafteramplification.Thereproducibilityof thesuspensionswascheckedintermsofconcentrationandsize

[21]. Dynamic light scattering revealed a single size distribu-tionpeakwitha z-averagedhydrodynamic diameterof26.0nm whereasnegativelystainedpreparations(TEM)showeda diame-terofapproximately30nm.Theseresultsareinagreementwith previousstudies[12],whichreportthediameterofthespherical MS2tobe30nm.

2.2. Bacteriophageassays

2.2.1. Quantificationofinfectiousvirusesbycellculture:PFU method

Theplaqueassayprocedureusedtodeterminethe concentra-tioninphagesisasdescribedinFurigaetal.[20].Theonlydifference isthatinthepresentstudy,1mLofbacteriophagesamplewas col-lectedandmixedto9mLofE.colisuspensionwhenFurigaetal. combined0.1mLofbacteriophagesamplewith0.9mLofE.coli. Asaconsequence,inthepresentwork,thedetectionlimitofthe plaqueassaywhichcorrespondstothesmallestamountofphages thatcouldbedetectedbutnotnecessarilyaccuratelyquantified (resultsnot reproducible)was1PFUmL−1 _{whereas thesmallest}

amountofphagesthatcouldbequantified(reproducibleresults) was30PFUmL−1_{.SamplesweredilutedwhennecessaryusingPBS}

(9gL−1_NaCl,0.8gL−1_Na

2HPO4,0.1gL−1KH2PO4;Lonza,Verviers,

Belgium)inordertodecreasetheconcentrationinbacteriophage below300PFUmL−1_{whichistheconcentrationthatcaneasilybe}

countedonaplatewiththenakedeye.PBSwaspreferredtoother buffersinanefforttopromoteviralsuspensionstability.

2.2.2. QuantificationofviralgenomebyqRT-PCRmethod

WedefinetheconcentrationmeasuredbytheqRT-PCRmethod asthetotalviralRNAconcentration.

TheviralRNAwasextractedusingtheQIAamp®_ViralRNAMini

kit(Quiagen,Courtaboeuf,France)accordingtothemanufacturer’s instructions.Extractionwasperformedfrom140mLofviral sus-pension(standardorsamples).TheextractedRNAwaselutedin 60mLofbufferandimmediatelystoredat−20◦_C.qRT-PCR

condi-tionsusedforMS2detectionandquantificationaredescribedin

[20].Inthesamplingconditionsdescribedintheprevioussection, thedetectionlimitoftheqRT-PCRwas101_equiv.PFUmL−1_andthe

quantificationlimitwas102_equiv.PFUmL−1_[20].

2.3. Membranes

Testswereconductedusingultrafiltrationmembranesprepared forthisproject.Theseareinnerskinnedhollowfibers(molecular weightcut-off 100kDa –permeability 142±54Lh−1_m−2_bar−1₎

madeofcelluloseacetate.15hollowfibers(0.93and1.66mmof internal andexternal diameterrespectively)wereassembled in abench-scalemoduleof300mminlengthand8mmininternal diameter.Anewmodulewasmadeforeachexperiment.The mem-braneeffectiveareapermodulewas91±5cm2_{.Theintegrityof}

eachmodulewastestedpriortoanyexperiment:themodulewas firstfilledwithdistilledwater,thencompressedairwasinjected intheretentatecompartmentat1±0.005barinaclosedcircuit andthetransparentmoduleshellallowedtocheckforbubbling.A modulewasconsideredintegerwhennobubblewasdetectedat nakedeye.Themembranepermeabilitytodistilledwater,Lp,was

determinedbeforeandafterthefiltrationofbacteriophage suspen-sioninordertocheckformembranefoulingaccordingtotheDarcy law:

J=Q

A =Lp1P (2)

whereJ,fluxdensity[Lh−1_m−2_{],Q,filtrationflowofpuresolvent}

[Lh−1_{],A,membraneeffectivearea[m}2_],L

p,membranehydraulic

permeability[Lh−1_m−2_bar−1_{]and1P,trans-membranepressure}

[bar].

Fivevaluesofappliedpressurewheresystematicallyusedfor thepermeability determination.Thepermeability is givenfor a temperatureof20◦_{C,asdatawerecorrectedwhennecessaryfor}

theeffectoftemperatureonthewaterviscosity[22]usingEq.(3), validfor0◦_C<T<30◦_C:

Lp (20◦C)=Lp(T)exp(−0.0239(T−20)) (3)

whereTisthetemperature[◦_C].

Accordingtoa Frenchstandard[23],for thecharacterization ofultrafiltrationormicrofiltrationmembranes,thelossin perme-abilitybyfoulingduringthetesthastobesmallerthan30%fora retentionmeasurementtobeconsideredasvalid.Forall experi-mentsconductedinthisstudy,wecheckedthatthelossinLpafter

bacteriophagefiltrationmetthiscriterion.

Inordertoavoidcrosscontamination,anewmodulewasused for eachexperiment, firstrinsedwithdistilled waterin normal filtrationmode,thenbackwashedwithasodiumhypochlorite solu-tionat200ppmtotalfreechlorineduring10min.Themodulewas thenfilledwiththehypochloritesolutionandafter30min,was thoroughlyrinsedwithsterilewater.

2.4. Ultrafiltrationset-upandprocedure

Experimentswereperformedusingalaboratorycross-flow fil-trationapparatus.Thefeedtankwasa5Lglassvesselasprevious studiesshowedthatnosubstantiallossbyadsorptionofMS2was observedwhenusingglassware[14,21]andthepumpwasof posi-tivedisplacementtype(PCMP2MGI;Moineau,Vanves,France).The feedtankwasjacketed,whichallowedthetemperaturetobe con-trolled(Fig.1).Themoduleshellwasmadeofpolyvinylchloride andtubingofpolyamide.

Experimentswereconductedover2hincross-flowconditions under constanttransmembrane pressure(0.5bar), at controlled temperature20◦_C±2◦_{C andatthenaturalpHofthewater(pH}

7±0.5).2Loffeedsuspensionwasobtainedbydilutingthe bac-teriophagestocksolutionin variousmedia(osmosedwater, tap watermicrofilteredthrough0.2mmfilters,distilledwater contain-ing1gL−1_{NaCl,tapwatercontaining5gL}−1_{NaCl,distilledwater}

containing 9gL−1 _{NaCl and PBS).Permeate and retentatewere}

recycledintothefeedtank.Samples(1mL)weretakenperiodically andassayedusingthetwomethodspresentedinSections2.2.1and

2.2.2.Thesampleswerestoredinthedarkat4◦_{Cwhentheywere}

analyzedwithinaday(typicallybyPFUcounting),orpreservedat −80◦_{CuntilqRT-PCRanalysiswasperformed.}

Inourexperiments,thequantificationlimitofthePFUmethod being 30PFUmL−1_{, the minimum initial concentration chosen}

forthefeedsuspensionwas108_PFUmL−1 _{inordertoallowfor}

thedemonstrationofupto6.5Logremoval,accordingtoEq.(1)

(Log10[108/30]=6.5).

Inaddition,whennoviruswasdetectedinapermeatesample, thepermeateconcentrationwastakenequalto30PFUmL−1_inEq.

(1),thevaluethenobtainedwasconsideredastheminimumLRV thatcouldbeclaimedintheconditionsoftheexperiment.Thetrue LRVvaluewasequaltoorlargerthanthisone.

Experiments were at least duplicated and the difference betweentwoLRVwas<0.5.Theequipmentwascleanedaftereach experimentbycirculatinga 200ppm sodiumhypochlorite solu-tion,butusedmembranemoduleswerediscarded.Thesystemwas thenrinsedwithdistilledwateruntilnohypochloritewasdetected inpermeateandretentatestreams.Forthis, thetotal free chlo-rineconcentrationintherinsingsolutionwasassayedbyadding DPDfreechlorinereagent(HACH14070-14099Pk/100)toitand measuringtheabsorbanceat530nm(HACH2400).

2.5. Virusinactivationduringfiltration

Weusedthesamemodelfordescribingthevirusinactivation kineticsasGassilloudetal.[24](Eq.(4))i.e.afirstorderreaction: Log10



_C r(t) Cr(0)



=−ait (4)

Fig.1. Diagrammaticviewoftheexperimentalsetup.

withai,inactivationrateconstant,t,time,Cr(0),virusconcentration

inretentateattime0andCr(t),virusconcentrationinretentateat

timet.

From Eq. (4), Gassilloud et al. [24] calculate T90, the time requiredfortheinfectivitytobereducedby90%(or1Log10).This

approachusedwiththePFUmethodcharacterizesinactivationbut doesnotdiscriminatepureinactivationfromadhesionor aggrega-tion.Thispointisdiscussedfurtherbelow.

TheinactivationrateconstantaiofMS2phageswasobtained

fromtheplotofLog10(Cr(t)/Cr(0))versustime. Thefiltrationtest

duration(dfiltration)correspondingtothetimeatwhichthe

con-centrationininfectiousparticlesMS2decreasedby1Log10inthe

retentate(namedT90byGassilloudetal.[24]),wasthensimply givenby:

dfiltration=

1

ai (5)

3. Resultsanddiscussion

The virus challenge filtration test requires the definition of experimentalconditionswhichmostfavorthevirustransmission. Thismeansthatvirusesshouldnotaggregate,adsorbonthe exper-imentalset-up(tank,membranemodules,pipes,etc.)duringthe testandthatinactivationshouldbeaslimitedaspossible.Inorder toevaluatetheextendofeachphenomenon;thesuspensionwas firstcirculatedinanemptyfiltrationmoduleshell.

3.1. Preliminaryexperimentsconductedwithanemptymodule shell

Aseriesofexperimentswasperformedbycirculatinga MS2 suspensionprepared in osmosedwater in thefiltrationsystem containing an empty module shell (without membrane). The retentatewas recirculatedand analyzed over time by PFU and qRT-PCR. We observed in these preliminaryexperiments (data notshown)thattheinfectiousvirusconcentrationdecreasedfrom 9.2×107_{toaround7×10}1_PFUmL−1_{within60min.Inthesame}

time, the concentration in viral genomes determined by qRT-PCRdecreasedfrom5.2×108 _to2×108_equiv.PFUmL−1 _(andto

8.23×107_equiv.PFUmL−1 _{over 240min). This decrease can be}

partlyattributedtovirusadsorptionontheexperimentalset-up

[15]. In order tocompensate this virusloss by adsorption, we decidedtoadd 2mLof virusstocksuspensionat1011_PFUmL−1

tothefeedtank,15minafterthebeginningofeachfiltrationrun,

accordingtoaprotocolproposedbyUraseetal.[13].This addi-tioncalled“doping”intherestofthispaper,allowedtorestaure theinfectiousvirusconcentrationbacktocloseto108_PFUmL−1_,

anditseffectonthestabilizationofinfectivityisshowninFig.2. Whenvirusesweredetectedinthepermeatetheirconcentration wasalmoststableafter10–15minoffiltration.Wethenassumed thatadsorptiononthewallsofthepermeatecircuithadreached saturation.

InFig.2wecomparethephageconcentrationovertimeduring inthefiltrationsystemwithoutmembrane.Amorerapiddeclinein infectiousMS2particlesconcentrationwasobservedinPBS(ionic strength182mM)thanin osmosedwater,leading toa concen-trationaround2×102_PFUmL−1_{30minafterdoping.Despitethe}

dopingat15min,thetotalviralRNAdecreasedby1Login105min afterdopingathighionicstrength(182mM),whenitwasalmost stableinosmosedwater.

AccordingtoGassilloudandGantzer[15],alossininfectious virusinanaqueousmedium asmeasuredbyPFUcountingcan resultfrominactivation,adhesiontotheexperimentalset-upand aggregation, whereas for viral genomes, the loss measured by RT-PCRcanonlyresultfromadhesion.Asaconsequence,several hypothesescouldexplainourobservations:

Total viral RNA of MS2 – Osmosed water Infectious MS2 particles – Osmosed water Total viral RNA of MS2 – PBS Infectious MS2 particles –PBS A 1E+00 1E+01 1E+02 1E+03 1E+04 1E+05 1E+06 1E+07 1E+08 1E+09 150 135 120 105 90 75 60 45 30 15 0 Cr (t) (P FU/mL or equiv. PFU/mL) time (min)

Total viral RNA of MS2 after doping-Milli-Q water Infectious MS2 particles after doping- Milli-Q water Total viral RNA of MS2 after doping-PBS MS2 infectious particles after doping-PBS

Doping

Fig.2. ConcentrationintotalviralRNA(inequiv.PFUmL−1_{,qRT-PCRmethod)and}

ininfectiousMS2particles(inPFUmL−1_{,PFUmethod).Thesuspensioniscirculated}

inthefiltrationsystemwithoutmembrane(emptymoduleshell).Phagesarediluted inmilli-QTM_{waterorPBS.Originoftimewastakenatthebeginningofthetestwhen}

1E+00 1E+01 1E+02 1E+03 1E+04 1E+05 1E+06 1E+07 1E+08 0 15 30 45 60 75 90 105 120 Cr( t) (PFU/m L o r equiv.PFU/mL time (min)

Total viral RNA in retentate -PBS Total viral RNA in permeate -PBS Infectious MS2 particles in retentate -PBS

Doping

Fig.3. ConcentrationintotalviralRNAandininfectiousMS2particlesduringa2h filtrationtestwith“doping”at15min.SolventwasPBS(n=2).

(i)Thepresenceofsaltsmightfavorthedamageoftheviruscapsid bythepump,theninactivatingtheviruses.

(ii)Thepresenceofsaltsmightfavorinfectiousphagesaggregation inthefiltrationsystem,resultinginadecreaseinthenumber ofinfectiousparticlescountedbyPFUwhereasinstatic con-ditions[20]thesuspensionpreparedinPBSwasstableover 2h.

(iii) ThegenomereductionshownbyqRT-PCRsuggestsastronger adsorptiononthewallsofthefiltrationsystemcausedbythe combinedeffectsofionicstrengthandparticlesinactivation. Electrostatic repulsionsare expectedtobescreenedbythe presenceofsaltinsolutionthusleadingtoanincreaseinvirus adsorptiononsurfaces,bothofthem beinggenerally nega-tivelychargedasmentionedintheintroduction.

3.2. Aggregation,adsorptionandinactivationofbacteriophages duringafiltrationtest

Aggregation,adsorptionandinactivationofMS2particles dur-inga filtrationtest werestudiedseparatelysoastodistinguish whichassumptionsamongstthosepreviouslymentioned,isvalid inordertoaccountforthesephenomenaincalculatingtheLRV. 3.2.1. Aggregation

IftheimportantreductionininfectiousMS2particles concentra-tionathighionicstrength(182mM)wereduetoviralaggregation, thenweshouldexpectalowertotalviralRNAconcentrationinthe permeateasalargeaggregatewasexpectedtobemorerejectedby themembranethanasinglephage(theconcentrationininfectious particlesinthepermeatebeingzero,wecouldnotmakea compar-isonwiththatone).However,nodecreaseintotalviralRNAover timewasobservedinthepermeatebyqRT-PCR(Fig.3).We con-cludethatifMS2aggregationintheretentatewassignificant,it wouldimpactthevirusretentionbythemembrane,andhenceits concentrationinthepermeate.Therefore,onecanconsiderthatin ourconditions,themembranewaschallengedwithasuspensionof trulydispersedinfectiousviruses.Theseresultsareinaccordance withthoseofLangletetal.[3].

3.2.2. Adsorption

Theinfluenceofabufferonadsorptionduringfiltrationcanbe quantifiedbymonitoringthechangesintotalviralRNAdetermined byqRT-PCR.InFig.4,theLog10(Cr(t)/Cr(0))wasplottedversustime

accordingtoEq.(4).Theoriginoftimeandconcentrationwastaken justafterdoping.

Adsorptionseemstobemoreimportantinthepresenceofsalts inthesuspension.Thehighertheionicstrength,thesteeperthe totalviralRNAdecrease.Thisisconsistentwithascreeningof

repul--2 -1 0 1 0 15 30 45 60 75 90 105 L o g10 (C r( t) /C r( 0) ) time (min) Tap water

Distilled water containing 1 g/L NaCl Tap water containing 5 g/L NaCl PBS

Fig.4. RetentateconcentrationintotalRNAviral(qRT-PCRmethod)duringfiltration testofsuspensionsinvariousmedia.The“doping”wastakenastheoriginoftime, theconcentrationCr(0)wasthenequaltothetotalamountofviralRNAafterdoping

(n≥2).

siveelectrostaticinteractionsbythepresenceofsalt.However,the maximumdecreaseoverthefiltrationrunof105minwhichwas observedwithPBS(182mM),wasonly0.4Log10.Theexperiment

conductedinthesameconditionsbutwithouthollowfibersinthe moduleshell(Section3.1)showedadecreasebyaround1Log10

overthesametimeof105minafterdoping(symbolAinFig.2). ThelossintotalviralRNAwaslessimportantinthepresenceof membranesinthemoduleprobablybecauseinthiscasethecontact areabetweenthesuspensionandtubingwasreduced,thephages retainedbythemembranebeingconfinedintheretentate com-partmentandnotcomingintocontactwiththepermeatecircuit andmoduleshell.

3.2.3. Inactivation

Fig.5reportsthechangesinretentateconcentrationof infec-tiousparticlesovertimeforvariousbuffers(sameasinFig.4).We couldascribethedecreaseinPFUmL−1_{toadecreasein}

concentra-tioninisolatedinfectiousMS2phagesinsuspensionasaggregation andadsorptionphenomena havebeendismissed.We observea highrateofinactivationathighionicstrengths(I),startingfromthe nominalvirusconcentration,whereasatlowI,wehaveasharp ini-tiallossininfectivity,thenaveryslowinactivation.Furigaetal.[20]

reportanimmediatedecreaseininfectivityatlowionicstrengthby about1Log,whereastheydonotobservesuchaninactivationover timewhenworkinginstirredvessels(withnopumpingdevices then).Theseresults[20]shownthatduringfiltrationtestsusing high-ionic-strengthsolutions,theinactivationphenomenon was differentfromtheosmoticstressone: theviruscapsidwasnot totallybroken,asnofreeRNAwasdetectedbyqRT-PCRperformed withouttheRNAextractionstep.Asa consequence,theparticle sizewasnotsignificantlymodifiedandthebehaviorofviruses dur-ingfiltrationwassimilarwithregardstothesievingmechanism. ThisassumptionwassupportedbytheresultsshowninFig.7D andD′_{asthedecreaseininfectiousMS2intheretentateovertime}

(Fig.7D)wasnotaccompaniedbyanincreaseintotalviralRNAin thepermeate(Fig.7D′_).

Ontheotherhand,accordingtoThompsonetal.[14]the pres-enceofsaltsleadstoanincreaseoftheparticlesattractiontothe air–water–solidinterfaceduetoadecreaseofthethicknessofthe electrostaticdoublelayeraroundtheparticles.Thisgreatervirus sorptionattheair–water-interfaceresultsinexposureofphages toinactivatingforces.Itisobviousthatthiseffectismoremarked whenthevirusishydrophobiclikeMS2butwecanalsoexpecta greaterinactivationthantheinterfaceisrapidlyrenewed,asitwas thecaseduringourfiltrationtests.

y = -0.0138x R² = 0.9487 y = -0.0313x R² = 0.9897 y = -0.0600x R² = 0.8990 y = -0.0997x R² = 0.9921 y = -0.1748x R² = 0.8409 -9 -8 -7 -6 -5 -4 -3 -2 -1 0 1 0 30 60 90 120 150 180 210 L o g10 (C r( t) /C r( 0) ) time (min) Tap water

Distilled water containing 1 g/L NaCl Tap water containing 5 g/L NaCl Distilled water containing 9g/L NaCl PBS

Fig.5.RetentateconcentrationininfectiousMS2(PFUmethod)duringfiltrationofsuspensionsinvariousmedia.The“doping”wastakenastheoriginoftime,the concentrationCr(0)wasthenequaltothetotalamountofviralRNAafterdoping(n≥2).

Table1

Inactivationrateconstantai(Eq.(4))anddfiltration(Eq.(5)),timeatwhichtheconcentrationofinfectiousMS2hasdecreasedby1Log10,whenphagesweresuspendedin

differentmedia.

Quantification Ionicstrength,I(mM) ai(s−1) ai(min−1) dfiltration(min)(calculated)

Inactivation–tapwater 3 2.30×10−4 _{0.0138 72}

Inactivation–distilledwater+1gL−1_{NaCl 17 5.22×10}−4 _{0.0313 32}

Inactivation– tapwater+5gL−1_{NaCl 86 10.0×10}−4 _{0.0600 17}

Inactivation–distilledwater+9gL−1_{NaCl 155 16.6×10}−4 _{0.0997 10}

Inactivation– PBS 182 29.1×10−4 _{0.1748 0}

3.2.4. Durationofthefiltrationtest

Wecalculatedaninactivationratecoefficientaiastheslopeof

theplotofLog10(Cr(t)/Cr(0))versustimeforeachbuffer(Fig.5).The

filtrationtestdurationd_filtration(Table1)wasdeducedbyusingEq.

(5).Thehighertheionicstrength,thelargertheinactivationrate coefficientaiandthereforetheshorterdfiltration.InPBS,

immedi-atelyafterdoping,over90%ofthephagesinitiallypresentinthe retentatewerenolongerinfectious,thereforetheevaluationofthe virusretentionwastechnicallynotreliable.Atlowionicstrengths, andtypicallyintapwater,thefiltrationmightbecarriedoutfor 70–80min.

Wehaveplottedthefiltrationtestdurationdfiltrationasa

func-tionoftheionicstrengthofthephagesuspensionin Fig.6 and found that the empirical Eq. (6) fits the data with a correla-tion coefficient of 0.96, for ionic strengths between3mM and 180mM:

dfiltration=−16LnI+85 (6)

Tosumup,whenPFUcountingisusedtoassessphage concen-trationinpermeateandretentate,virusretentionbyamembrane shouldbedeterminedwithinthecharacteristictimedfiltration.When

theqRT-PCRmethodisused,theobservedlossintotalviralRNAin retentateislessthan1Log10evenafter105minoffiltration

what-everthebufferinvestigated(Fig.4);thereforethedurationofthe filtrationtestcanbelongerinthiscase,butwehavetokeepinmind thattheparameterwhichismeasuredthenisnottheinfectivityof thepermeate.

3.3. LRVassessment

3.3.1. Permeateandretentateconcentrationsduringfiltration

Fig.7showsMS2concentrationsinpermeateandretentate col-lectedduring2hfiltrationtests,determinedbyPFUandbyqRT-PCR methods.Datafromsamplescollectedafterdopingonlyareshown. InfectiousMS2phage.ThedashedverticallinesinFig.7represent dfiltration.Asdiscussedbefore,datafromexperimentsinPBSwere

notvalid.Intapwater,conditionswereacceptableforca.70min,

y = -16 ln(x) + 85 R² = 0,9614 0 10 20 30 40 50 60 70 80 0 20 40 60 80 100 120 140 160 180 200 dfi lt r a ti o n (m in -1) Ionic Strength (mM)

Fig.6. Inactivationcoefficientai(s−1)asafunctionofionicstrengthofthe

whereas,thetimeforsamplingwasmuchshorterif5gL−1_NaCl

wasadded.

WecouldnotdetectanyinfectiousMS2inpermeateathigh saltconcentrations.Thiscanbeexplainedbytherapiddecrease in infectiousMS2 concentrationin theretentate. ForNaCl con-centrations below 5gL−1_{, the virus concentration in permeate}

wasbelowthePFUquantificationlimit(30PFUmL−1_)orslightly

above.

Total viralRNAconcentrations.TheamountoftotalviralRNA intheretentatechangedwithinarangeconsideredasnegligible (seeFig.4andcomments)duringa2hfiltrationrun,whateverthe buffer.Thusinourconditions,theexperimentsaimedat determin-ingtheLRVintotalviralRNAcouldlast105minandevenmore afterdoping.ThepermeateconcentrationintotalviralRNAwas below thequantificationlimit ofqRT-PCR (102_equiv.PFUmL−1₎

whenthefiltrationwasperformedatlowionicstrength(below 86mMor5gL−1_{NaCl),butabovethislimitinPBS,typicallyaround}

103_equiv.PFUmL−1 _(Fig.7D′_{).Intheseconditions,thepermeate}

concentrationwasalmoststableafter10–15minoffiltration.From theseresultswehaveassumedthistimesufficienttoreach satura-tionofpermeatecircuit(fromthepermeatesideofthemembrane tothepermeatesamplingpoint)intermofvirusadsorption.

Inthepresenceofsalts,alargeramountofnon-infectiousviruses (because nodetectablebyPFU)(Fig.7D and D′_)werefoundin

thepermeate,suggestingthatnon-infectiousvirusestransferwas facilitatedbyhighionicstrengths.

3.3.2. LRVcomparison

Asforanexample,LRVswerefinallydeterminedforMS2 dis-persed in various buffers (Fig. 8). The reported values are an averageofatleasttworeproducibletestsperformedontwo dif-ferent modules over dfiltration for infectious phageand over 2h

offiltrationfortotalviralRNA.Whennophagewasdetectedin thepermeate orwhen the concentrationwasbelow the quan-tificationlimitoftheanalyticalmethod,permeateconcentrations in infectious MS2 and in total viral RNA were taken equal to 30PFUmL−1_and102_equiv.PFUmL−1_{respectivelyfortheLRV}

cal-culation.

TheLRVsreportedinFig.8aretheminimumvaluesthatcan beclaimedinourconditionsaccountingfordfiltration(forinfectious

MS2),retentateconcentrationandquantificationlimitsofPFUand qRT-PCRmethodsforthepermeateexceptforPBS(Fig.7D′_).

The minimumLRV for infectious MS2 decreased slightly for ionicstrengthsbetween3mM(tapwater)and155mM(distilled water+9gL−1_NaCL).

LRVintotalviralRNAwasindependentoftheionicstrength upto86mMandslightlydecreasedforhighersaltconcentrations. Forionicstrengthsabove155mM,theconcentrationininfectious

1E+ 1E+ 1E+ 1E+ 1E+ 1E+ 1E+ 1E+ 1E+ 1E+ C o n ce n tr a ti o n ( P F U /m L )

(A)

1E+00 1E+01 1E+02 1E+03 1E+04 1E+05 1E+06 1E+07 1E+08 1E+09 C o n ce n tr a ti o n ( P F U /m L ) C C

(B)

_Cr Cp 00 01 02 03 04 05 06 07 08 09 0 15 30 Cr (virus Cp (viru 0 1 2 3 4 5 6 7 8 9 0 15 30 r (virus infectieux p (virus infectieu

d Cr (infec Cp (infec r (infectious MS2) p (infectious MS2 0 45 60 75 time (min) s infectieux) - Ea s infectieux) - Ea q df 45 60 75 time (min)

x) - Eau distillée ux) - Eau distillée

dfiltration

quantif

ctious MS2) – tap w ctious MS2) – tap

) – distilled water 2) – distilled water

5 90 105 120 u du réseau au du réseau

quantification limit

filtration 90 105 120 + 1 g/L NaCl e + 1 g/L NaCl fication limit water water +1g/L NaCl +1g/L NaCl 0 1E+0 1E+0 1E+0 1E+0 1E+0 1E+0 1E+0 1E+0 1E+0 1E+0 C o n ce n tr a ti o n ( eq u iv .P F U /m L )

(A')

1E+00 1E+01 1E+02 1E+03 1E+04 1E+05 1E+06 1E+07 1E+08 1E+09 0 C o n c en tr a ti o n ( eq u iv .P F U /m L ) Cr Cp

(B')

Cr Cp 00 01 02 03 04 05 06 07 08 09 0 15 30 Cr (virus Cp (virus 0 15 30 4 (virus totaux) - E (virus totaux) - E Cr (total Cp (total

r (total viral RNA) p (total viral RNA)

45 60 75

time (min)

totaux) - Eau du s totaux) -Eau du

q

5 60 75 9

time (min)

Eau distillée + 1 Eau distillée + 1 g

quantific

viral RNA) – tap l viral RNA) – tap

)–distilled water + )–distilled water +

90 105 120 u réseau u réseau

quantification limit

90 105 120 g/L NaCl g/L NaCl cation limit water water + 1 g/LNaCl + 1 g/L NaCl

Fig.7.InfluenceofthetypeofbufferoninfectiousMS2andviralRNAconcentrationsinretentateandpermeateduringfiltration.(A)Tapwater,(B)distilledwatercontaining 1gL−1_{NaCl,(C)filteredtapwatercontaining5gL}−1_{NaCland(D)PBS).The“doping”wastakenastheoriginoftime(n≥2).}

1E+00 1E+01 1E+02 1E+03 1E+04 1E+05 1E+06 1E+07 1E+08 1E+09 0 15 30 45 60 75 90 105 120 C o n c en tr a ti o n ( P F U /m L ) time (min)

Cr (virus infectieux)

-dfiltration

quantification limit

dfiltration

quantification limit

dfiltration

quantification limit

(C)

1E+00 1E+01 1E+02 1E+03 1E+04 1E+05 1E+06 1E+07 1E+08 1E+09 0 15 30 45 60 75 90 105 120 C o n ce n tr a ti o n ( e q u iv .P F U /m L )

Cr (virus totaux) Eau du réseau + 5 g/L NaCl

(C')

quantification limit

1E+00 1E+01 1E+02 1E+03 1E+04 1E+05 1E+06 1E+07 1E+08 1E+09 0 15 30 45 60 75 90 105 120 C o n c e n tr a ti o n ( PF U /m L )

Cr (virus infectieux) - PBS Cp (virus infectieux) -PBS

(D)

quantification limit

1E+00 1E+01 1E+02 1E+03 1E+04 1E+05 1E+06 1E+07 1E+08 1E+09 0 15 30 45 60 75 90 105 120 C o n c e n tr a ti o n ( e q u iv .P F U /m L ) Cr (virus totaux) -PBS Cp (virus totaux) -PBS

(D')

quantification limit

Cr (infectious MS2) –PBS Cp (infectious MS2) –PBS

Cr(total viral RNA) –tapwater + 5 g/L NaCl total viral RNA) –tapwater

Cr (total viral RNA) –PBS Cp (total viral RNA) –PBS Cr (infectious MS2) –tap water

Cp (infectious MS2) –tapwater

time (min)

+5g/L NaCl

+5g/L NaCl Cp ( + 5 g/L NaCl

time (min) time (min)

Fig.7. (Continued).

Fig.8. MinimumLRVsininfectiousMS2andtotalviralRNAthatcanbeclaimedinourconditionsaccountingfordfiltration(forinfectiousMS2),retentateconcentrationand

quantificationlimitsofPFUandqRT-PCRmethodsforthepermeate(exceptforPBSFig.7D′

phagesdecreasedveryrapidly,andthecorrespondingLRVcould notbecalculated.At155mM,dfiltrationwasequalto10min,andthe

LRVininfectiousphagescouldbecalculatedduringthisperiodonly. Therefore,theLRVsdeterminedbyqRT-PCRover2hoffiltration (5.1forI=155mMand5forI=182mM)correspondtotheremoval ofinactivatedphages.Thisinformationisrelevantwithregardsto publichealthissues,especiallywhentheriskofvirusreviviscence isconcerned.

Amaximumdifferenceof0.7Log10wasobservedbetweenthe

LRVdeterminedbyPFUandqRT-PCRmethodsforionicstrengths strictly different (aboveor below)from 86mM, The difference betweenLRV’sdeterminedbythetwo methodsremainswithin theexperimentaluncertainty,exceptmaybewhentapwaterwas used,whereasLangletetal.[3]reportadifferenceof1Log10intheir

conditions(0.2mMPBSsolution).At86mMwhichwasthe “min-imum”ionicstrengthallowingtopreservetheinfectivityofthe initialsuspension[20],thedifferencebetweentheLRV’swas min-imum.Whendilutionbuffershavelowerorhigherionicstrengths, bothmethodsshouldbeused.

Ifonlyonequantificationtechnique(oftenthetraditionalPFU method)isavailable,thisstudysuggeststheuseofa low-ionic-strengthsolutionwhichallowstomaintaintheinfectivityofthe initialsuspensionwhilegivingenoughtimetorunthefiltration experiment. From a practical point of view, filtered tap water provedtobeaveryacceptabledilutionmedium.

4. Conclusion

TheminimumLRVwhichcanbeclaimedfromthedatacollected afteramembranecharacterizationtestislimitedby:

•_{Theminimumvirusconcentrationintheretentateduringthetest.} •_{Thedetectionlimitoftheviruscountingmethod,whenapplied}

tothepermeate.

Amongstthetwomainmethodsavailableforviruscounting,PFU countinghasalowerdetectionlimitthanqRT-PCRanditenlarges therangeofLRV’swhichcanbeclaimed.Itiswellestablished,and weconfirmhere,thatthevirusconcentrationinasolutionusedto characterizefiltrationmembranesmaydramaticallychangeduring thetestandweconsiderthatthisshouldbeaccountedforwhen assessingmembraneLRVs,unlessthetestisinvalidatedbecausethe conditionsexperiencedbythemembranechangetoomuchduring thetest.

We suggest to collect the data used for assessing a mem-brane/modulevirusLRVwithinaperiodoftimeduringwhichthe changeinvirusconcentrationremainswithinsomelimits(here, 100–10%).Weshowthatthismaximumdurationchangeswiththe typeofbufferusedtodilutethevirusstocksolution,anddecreases withitsionicstrength(suchasinEq.(6)).

Implementingtherecommendationstotheassessmentofa cel-lulose acetate membrane showed that there wasno difference inourconditionsbetweentheLRV’sfoundbyPFUandqRT-PCR, and thattheLRV slightly decreasedwhen theionicstrength of thebufferincreased.However,operatingwithlowionicstrength buffers gives much more time for running the filtration tests, and filteredtap water proved in ourcase welladapted to this purpose.

Acknowledgements

FinancialsupportfromthePRECODDResearchProgramofthe FrenchNationalResearch Agencyisgratefully acknowledgedas

wellasthecontributionofPOLYMEMSA,AQUASOURCEandVEOLIA WATERScompanies.

TheauthorsthankMarieGloriesforhertechnicalcontribution inthesamplesanalysis.

References

[1]A.J.Gijbertsen-Abrahamse,E.R.Cornelissen,J.A.M.H.Hofman,Fiberfailure fre-quencyandcausesofhollowfiberintegrityloss,Desalination194(1–3)(2006) 251–258.

[2]T.Urase,K.Yamamoto,S.Ohgaki,Effectofporesizedistributionof ultrafiltra-tionmembranesonvirusrejectionincrossflowconditions,WaterSci.Technol. 30(1994)199–208.

[3] J.Langlet,L.Ogorzaly,J.-C.Schrotter,C.Machinal,F.Gaboriaud,J.F.L.Duval,C. Gantzer,EfficiencyofMS2phageandQbphageremovalbymembranefiltration inwatertreatment:applicationofreal-timeRT-PCRmethod,J.Membr.Sci.236 (1)(2009)111–116.

[4] G.Herath,K.Yamamoto,T.Urase,Removalofvirusesbymicrofiltration mem-branesatdifferentsolutionenvironments,WaterSci.Technol.40(4–5)(1999) 331–338.

[5]K.H.Oshima,T.T.Evans-Strickfaden,A.K.Highsmith,E.W.Ades,Theremoval ofphagesT1andPP7,andpoliovirusfromfluidswithhollowfiberultrafilters withmolecularweightcut-offsof50000,13000and6000,Can.J.Microbiol. 41(1995)316–322.

[6]C.L.Acker,C.K.Colvin,B.J.Marinas,J.C.Lozier,Assessingtheintegrityofreverse osmosisspiral-woundmembraneelementswithbiologicalandnon-biological surrogatesindicators,in:AmericanWaterWorksAssociationMembrane Tech-nologyConference,2001.

[7]A.S.L.Lau,Y.S.Lie,L.A.Norling,J.Sernatinger,M.Dinowitz,C.J.Petropoulos,Y. Xu,Quantitativecompetitivereversetranscription-PCRasamethodtoevaluate retrovirusremovalduringchromatographyprocedures,J.Biotechnol.75(2–3) (1999)105–115.

[8]S.R.Vaidya,U.K.Kharul,S.D.Chitambar,S.D.Wanjale,Y.S.Bhole,Removalof hepatitisAvirusfromwaterbypolyacrylonotrile-basedultrafiltration mem-branes,J.Virol.Methods119(1)(2004)7–9.

[9]M.D.Sobsey,D.A.Battigelli,G.A.Shin,S.Newland,RT-PCRamplificationdetects inactivatedvirusesinwaterandwastewater,WaterSci.Technol.38(1)(1998) 91–94.

[10]H.Leclerc,S.Edberg,V.Pierzo,J.M.Delattre,Bacteriophagesasindicatorsof entericvirusesandpublichealthriskingroundwaters,J.Appl.Microbiol.88 (2000)5–21.

[11]F.Lucena,X.Mendez,A.Moron,E.Calderon,C.Campos,A.Guerrero,M. Carde-nas,C.Gantzer,L.Shwartzbrood,S.Skraber,J.Jofre,Occurrenceanddensitiesof bacteriophagesproposedasindicatorsandbacterialindicatorsinriverwaters fromEuropeandSouthAmerica,J.Appl.Microbiol.94(2003)808–815. [12]J.Langlet,F.Gaboriaud,J.Duval,C.Gantzer,Aggregationandsurfaceproperties

ofF-specificRNAphages:implicationformembranefiltrationprocesses,Water Res.42(2008)2769–2777.

[13] T.Urase,K.Yamamoto,S.Ohgaki,Evaluationofvirusremovalinmembrane separationprocessesusingcoliphageQb,WaterSci.Technol.28(7)(1993) 9–15.

[14] S.Thompson,M.Flury,M.Yates,W.A.Jury,Roleoftheair–watersolidinterface inbacteriophagesorptionexperiments,Appl.Environ.Microbiol.64(1998) 304–309.

[15]B.Gassilloud,C.Gantzer,Adhesion–aggregationandinactivationofPoliovirus 1ingroundwaterstoredinahydrophobiccontainer,Appl.Environ.Microbiol. 71(2)(2005)912–920.

[16]B.Gassilloud,L.Huguet,A.Maul,C.Gantzer,Developmentofaviral concentra-tionmethodforbottledwaterstoredhydrophobicsupport,J.Virol.Methods 142(1–2)(2007)98–104.

[17]S.Farrah,ChemicalfactorsinfluencingadsorptionofbacteriophageMS2to membranefilters,Appl.Environ.Microbiol.43(1982)659–663.

[18]J.Langlet,F.Gaboriaud,C.Gantzer,EffectsofpHonplaqueformingunitcounts andaggregationofMS2bacteriophage,J.Appl.Microbiol.103(5)(2007) 1364–5072.

[19]USEPA2005,PartII,EnvironmentalProtectionAgency,NationalPrimary Drink-ingWaterRegulations:LongTerm2EnhancedSurfaceWaterTreatmentRule. [20]A.Furiga,G.Pierre,M.Glories,P.Aimar,C.Roques,C.Causserand,M.Berge, EffectsofionicstrengthonbacteriophageMS2behavior:implicationsonthe assessmentofvirusretentionbyultrafiltrationmembranes,Appl.Environ. Microbiol.77(1)(2011)229–236.

[21]G.Pierre,C.Causserand,C.Roques,P.Aimar,AdsorptionofMS2 bacterio-phageonultrafiltrationmembranelaboratoryequipments,Desalination250 (2)(2010)762–766.

[22]HandbookofChemistryandPhysics,83rded.,EditorDavidLide,2002. [23]FrenchStandardNFX45-103AFNORAssociationFranc¸aisedeNormalisation,

1997.

[24]B.Gassilloud,L.Schwartzbrod,C.Gantzer,Presenceofviralgenomesinmineral water:asufficientconditiontoassumeinfectiousrisk?Appl.Microbiol.69(7) (2003)3965–3969.