2009 A new Sephadex_-based method for removing microbicidal and cytotoxic residues when testing antiseptics against viru

2009·0.64 MB·English

JournalofVirologicalMethods159(2009)217–226
ContentslistsavailableatScienceDirect
Journal of Virological Methods
journal homepage: www.elsevier.com/locate/jviromet
A new SephadexTM-based method for removing microbicidal and cytotoxic
residues when testing antiseptics against viruses: Experiments with a human
coronavirus as a model
ChloéGeller,StéphaneFontanay,ChantalFinance,RaphaëlE.Duval∗
SRSMC,Nancy-University,CNRS,Nancy,France
a b s t r a c t
Articlehistory: Therelativelackofefficientmethodsforevaluatingantisepticantiviralactivity,togetherwithweaknesses
Received18December2008 intheexistingEuropeanStandard(i.e.NFEN14476+A1),underlinestheneedtoseekanewmethodwhich
Receivedinrevisedform23March2009 couldallowamorepreciseevaluationoftheantisepticantiviralactivityofchemicalagents.
Accepted25March2009
Thisprotocolisbasedonanoriginalgel-basedfiltrationmethod,using“in-house”G-25andG-10
Availableonline5April2009
SephadexTMcolumns.Thismethodallowstheneutralizationofboththeactivityandthecytotoxicityofa
largerangeofmolecules,accordingtotheirmolecularsize,inonly1min.
Keywords:
Theviralmodelusedwasthehumancoronavirus(HCoV)229Echosenfor(i)itsincreasingmedical
Antisepticevaluation
interest,(ii)itspotentialresistanceand(iii)itsrepresentingenvelopedvirusesmentionedintheEuropean
SephadexTM
Gelfiltration Standard.
Humancoronavirus First,theprotocolwasvalidatedanditwasdemonstratedthatitwasfullyoperationalforevaluating
Chlorhexidine antiviralantisepticpotentialityandusefultoscreenpotentiallyantisepticmolecules.
Hexamidine Second,chlorhexidine(CHX)andhexamidine(HXM)wereassessedfortheirpotentialanti-HCoV229E
antisepticactivities.Itwasdemonstratedclearlythat(i)HXMhadnoactivityontheHCoV229Eand(ii)
CHXshowedamoderateanti-HCoV229Eactivitybutinsufficienttobeantiseptic.
©2009ElsevierB.V.Allrightsreserved.
1. Introduction pathogenmembersofthisfamilyhavebeenidentified:theHuman
coronavirus,strain229E(HCoV229E)andtheHumancoronavirus,
Antiviralantisepsisanddisinfectionarecrucialforpreventing strainOC43(HCoVOC43).Knowntoberesponsibleforalargepro-
theenvironmentalspreadofviralinfections.Indeed,veryfeweffi- portion of common colds (Hamre and Procknow, 1966; Holmes,
cientandspecifictreatmentsareavailabletocontrolmostofthese 2001;Larsonetal.,1980),theyhavebeenimplicatedinmoreseri-
infections.Furthermore,emergingvirusesandassociateddiseases ousdiseases,alongwiththenewlyidentifiedHCoV:theNL63strain
aswellasnosocomialviralinfectionshavebecomeanimportant (VanderHoeketal.,2004)andtheHKU1strain(Wooetal.,2005).
issueinmedicalfields. Indeed,theseviruseshavebeenrecognizedascausinglowerrespi-
TheCoronaviridaefamily,anenvelopedRNAvirusfamily,isarep- ratorytractinfections,(Vabretetal.,2003;VanEldenetal.,2004).
resentativeexampleofthisissue.Sincethe1960s,onlytwohuman Theyhavealsobeeninvolvedinnosocomialviralinfections,espe-
ciallyinyoungchildrenandneonates(Gagneuretal.,2002a,b;Moes
etal.,2005;Vabretetal.,2008),intheelderly(Falseyetal.,2002;
Nicholsonetal.,1997)andinimmunocompromisedpatients(Pene
Abbreviations: ATS–D,antiseptics–disinfectants;BSA,bovineserumalbumin; etal.,2003).
CC50,50%cytotoxicconcentration;CCID50,50%cellcultureinfectivedose;CHX, Furthermore,theoutbreakofsevereacuterespiratorysyndrome
chlorhexidine;CPE,cytopathiceffect;EBSS,Earle’sbalancedsaltsolution;EDTA, (SARS)in2002–2003,responsibleforthefirstworldwideepidemic
ethylenediaminetetraceticacid;FCS,fetalcalfserum;FW,formulaweight;HCoV, of the 21st century, was due to a newly discovered coronavirus,
humancoronavirus;HXM,hexamidine;IC50,50%inhibitoryconcentration;MEM, theSARSassociatedcoronavirus(SARS-CoV)(Drostenetal.,2003;
minimumessentialmedium;MTT,methylthiazoltetrazolium;NR,neutralred;PBS,
phosphatebufferedsaline;SARS-CoV,severeacuterespiratorysyndromeassociated Ksiazeketal.,2003;Peirisetal.,2003).Thisoutbreakledtoanew
coronavirus;TB,trypanblue. awarenessofthemedicalimportanceoftheCoronaviridaefamily.
∗
Correspondingauthorat:Grouped’EtudedesVecteursSupramoléculairesdu Another interesting feature of coronaviruses is their poten-
Médicament(GEVSM),SRSMC,Nancy-University,CNRS,FacultédePharmacie,5rue tial environmental resistance, despite the accepted fragility of
AlbertLebrun,BP80403,54001NancyCedex,France.Tel.:[phone];
envelopedviruses.Indeed,aHCoV229Esurvivalof70%after72h
fax:[phone].
E-mailaddress:[email protected](R.E.Duval). at20◦Cand50%relativehumidityhasbeenobserved,aswellas
0166-0934/$–seefrontmatter©2009ElsevierB.V.Allrightsreserved.
doi:10.1016/j.jviromet.2009.03.023
218 C.Gelleretal./JournalofVirologicalMethods159(2009)217–226
aHCoV229Esurvivalof90%at6◦Candarelativehumidity≥50%, ATS-D(supplementarydata,Fig.1B).Tovalidatethisprotocoland
whichcouldaccountforairbornetransmissionandforwinterepi- establish its potential limits, antiseptic antiviral activities of two
demicsofHCoV229E,respectively(Ijazetal.,1985).Anotherstudy ATS-D,thechlorhexidine(CHX)andthehexamidine(HXM),were
showed30%and50%ofvirusresidualinfectivityfortheHCoV229E testedonHCoV229E.
andtheHCoVOC43,respectively,after6daysinsuspensioninphos-
phatebufferedsaline(PBS)at37◦Cand30%ofresidualHCoV229E 2. Materialsandmethods
infectivityafter3hofdryingondifferentsupportsfoundinhospi-
talsettings,i.e.aluminium,sterilespongesorlatexsurgicalgloves 2.1. Reagentsanddisposablematerials
(Sizunetal.,2000).Morerecently,numerousstudieshaveunder-
linedanimportantSARS-CoVsurvivalundervariousconditions:in Minimumessentialmedium(MEM;41090.093,fetalcalfserum
serum, in 1:20 diluted sputum, in faeces for at least 96h and in (FCS;10270098),trypsine-EDTA(25300-054),Earle’sbalancedsalt
urineforatleast72h.SARS-CoValsoretainedinfectivityat4◦C,at solution(EBSS;24010043),trypanblue(TB;15250-061)andMEM
roomtemperatureandat37◦Cforatleast2h(Duanetal.,2003; without glutamine and without phenol red (51200-046) were
Rabenauetal.,2005).Furthermore,somestudieshaveshownthe purchased at Invitrogen (Cergy Pontoise, France). For culturing
transferofvirusesfromcontaminatedhosts(hands,nasalmucosa) cells,75cm2 cellcultureflasks(831813)and96-wellflat-bottom
tosurfacesandfromenvironmentalsurfacestohandsandtherefore culture plates (831835) were purchased at Sarstedt (Marnay,
apotentialcontaminationofsusceptiblehosts(Sattaretal.,1993; France). May-Grünwald solution (1014240500) and Giemsa solu-
Wintheretal.,2007).Anotherstudyhasalsoshownalongersur- tion(1092040500)wereboughtfromMerck(Darmstadt,Germany).
vivalofaherpesvirusonhumanskinthanonaninanimatecarrier Chemicalsasdimethylsulfoxyde(D2438),neutralred(NR;N4638),
(i.e.astainlesssteeldisk)(Grahametal.,1996).Thus,considering sodiumdodecylsulfate(S9625)andformaldehydeat36.5%(v/v)
thepotentialresistanceofHCoVforsomehoursintheenvironment, (533998)wereboughtatSigma(SaintQuentinFallavier,France);
theirpathogenesisandtheabsenceofspecifictreatment,aneffi- calcium chloride (CaCl2, 33604.261) at Prolabo, (Fontenay sous
cientskinantisepsis,tostopeventualdisseminationbythehandsof Bois, France) and acetic acid (45726) at Fluka biochemical (Saint
healthcareworkers,isameaningfulwaytofightvirustransmission Quentin Fallavier, France). One milliliter syringes (300013) were
andassociateddiseases. fromBectonDickinson(LePontdeClaix,France)and0.22(cid:2)mfilters
A fundamental point is to evaluate the efficiency of (SLGP033RS) from Millipore Express (Saint Quentin en Yvelines,
antiseptics–disinfectants(ATS-D)onvirusesproperly.Theprinci- France).SephadexTM G-25andtheSephadexTM G-10(17-0010-01
pleofantisepticantiviralactivityevaluationis(i)tocollectviruses and 17-0033-01, respectively) were purchased at GE Healthcare
and the product under test for an appropriately defined and (Saclay, France). Finally, CHX digluconate and HXM diisethionate
precisecontacttime,(ii)toneutralizeproductactivity,i.e.stopping were kindly provided by J.-B. Regnouf-de-Vains (SRSMC, Nancy-
product activity and removing its potential cytotoxicity, and (iii) University,France).
toestimatethelossofviralinfectivityduetotheproductactivity.
These tests require appropriate controls especially to check the 2.2. Cellcultureandvirus
absenceofinterferenceduetothetestitselfonviralinfectivity,the
efficiencyofneutralization,theremovalofcytotoxicity,andalso, Humanembryonicpulmonaryepithelialcells,L-132cells(ATCC
toestablishreproducibleandwelldefinedconditions,e.g.contact CCL-5),kindlyprovidedbyDrH.F.Hildebrand(FacultyofMedicine,
time,temperature,interferingsubstances(Fig.1). Lille,France),weregrowninMEMsupplementedwith5%ofFCS,
Todate,onlyoneEuropeanStandard(NFEN14476+A1)onviru- at37◦Cinahumidifiedatmospherecontaining5%CO2.Theywere
cidalATS-DactivitytestinginhumanMedicinewaspublishedin split2–3timesaweek.Cells,grownin75cm2 cellcultureflasks
January2007(AFNOR,2007).Itspecifiesthetestmethodandmin- were washed 3 times with Phosphate Buffered Saline (PBS) and
imumrequirementstoestablishvirucidalactivityaccordingtothe 0.5mL of trypsine-EDTA was added. Flasks were incubated for a
potentialuseoftheproductstested,e.g.disinfectionofsurfacesand fewminutesat37◦Cwith5%CO2and4.5mLofMEM5%FCSwere
instruments,hygienichandwashorthermo-chemicaldisinfection. addedtoachievecelldissociation.Stockcellswerepreparedwith
Virusstrains,temperatures,contacttimes,interferingsubstances the cellular suspension obtained after trypsinization and mixed
arespecifiedforeachpotentialuse.Accordingtothisstandard,a with MEM containing 10% (v/v) FCS and 10% (v/v) dimethylsul-
productisconsideredtohaveanATS-Dantiviralactivityifitinduces foxyde.Theywerethenaliquotedandconservedat−196◦Cinliquid
alossofinfectivityofatleast4log inviraltitersduringanaccurate nitrogen.
10
contacttime. Human coronavirus, strain 229E (ATCC VR 740), kindly pro-
Some parameters need to be extended to improve antiviral videdbyDr.S.A.Sattar(FacultyofMedicine,Ottawa,Canada)was
ATS-Dactivitytesting.Forinstance,oneproposedmethodinthe cultivated on L-132 cells, in MEM supplemented with 2% FCS, at
EuropeanStandardtoneutralizeproductactivityisa1:10dilutionof 33◦C in a humidified atmosphere containing 5% CO2. 33◦C has
product/virusmixinicedmedium.However,consideringthevery beenshowntobetheoptimalgrowthtemperaturefortheHCoV
largerangeofATS-Dnature(singlemoleculesoramixtureofdif- 229E(Bradburne,1972).Stockviruseswerepreparedbyinfection
ferentones),thismethodisnotalwaysreliableandfurthermore, of75cm2 cellcultureflaskswithaconfluentmonolayerofL-132
it may lengthen contact time. The European Standard suggests cells,notmucholderthan3days.Whenconfluencewasreached,
othermethods,basedongel-filtrationtechniques,usingproducts mediumwasremovedandcellswerewashed3timeswithPBS.Cells
asSephadexTMLH-20,MicrospinTMcolumnsS400HRorMinicon® weretheninoculatedwith1mLofstockvirussuspensiondiluted
concentrators,buttheyleadtoproblemsofcontacttimes,separa- at1:5inEBSSandincubatedforadsorptionvirustime(i.e.1h),at
tioncapacityandcost(supplementarydata,Fig.1). 33◦Cwith5%CO2.Cultureflaskswerethenfilledoutwith11mL
Withtheseaimsinmind,aprotocolwasdevelopedthatallows of MEM 2% FCS and re-incubated at 33◦C with 5% CO2 for 48h,
neutralization,inonly1min,oftheactivityandcytotoxicityofa before the cytopathic effect (CPE, i.e. cell lysis), was completely
largerangeofmolecules,accordingtotheirmolecularsize.Thispro- achieved. Three cycles of freezing/thawing of 15min each were
cedurealsoinvolvesagel-filtration-basedmethodbutuses2other performedinordertoliberatevirions.Mediawerecentrifugedat
typesofSephadexTMgels:SephadexTMG-10andG-25.Theirreten- 2000×gfor5mintoeliminatecellularfragmentsand,supernatants
tion capacities are [100–1000]gmol−1 and [900–5000]gmol−1, containing viruses were aliquoted in cryotubes and conserved
respectively,whichisconsistentwiththemolecularmassofmost at−80◦C.
C.Gelleretal./JournalofVirologicalMethods159(2009)217–226 219
Fig.1. Principleoftheevaluationofantiviralantisepticactivity.
2.3. Endpointdilutionmethodforvirustitration bation period, cells were fixed and stained with May-Grünwald
andGiemsa.Fiftymicroliters/wellofMay-Grünwaldsolutionwere
L-132 cells were seeded at 5×103cells/well in 96-well flat- allowedtofixandstaincellsfor5minandafterawashingstepwith
bottomcultureinMEM5%FCS.Cellcountswereperformedwith tap water, 50(cid:2)L/well of Giemsa solution were added for 15min
theTBexclusionmethod.Cellswereincubatedat37◦Cin5%CO , to achieve the final staining before a last washing step with tap
2
for48htoobtainaconfluentmonolayer.Afterremovingmedium, water.Infectedwellswerecountedunderaphase-contrastinverted
180(cid:2)LoffreshMEM2%FCSwereaddedtoeachwell.Cellswere microscope(Olympus,Rungis,France)andviraltitersor50%cell
theninfectedatamultiplicityof0.2,and10-foldserialdilutions cultureinfectivedose(CCID ),expressedasviralinfectiouspar-
50
werecarriedout.Infectedcellswereincubatedat33◦Cin5%CO ticlesmL−1, were estimated according to the Reed and Muench
2
for 6 days to obtain the optimal CPE. At the end of the incu- method(ReedandMuench,1938).
220 C.Gelleretal./JournalofVirologicalMethods159(2009)217–226
2.4. Moleculestested ThesetestswerealsoperformedwithsolutionsofCHXandHXM
atvariousconcentrationsandaftertheirfiltrationonthe“in-house”
Allmoleculestestedwereprepared,extemporaneously,asaque- SephadexTM columnstoevaluatetheirretentionandtheelimina-
oussolutionsandfilter-sterilizedon0.22(cid:2)mfilters. tionofpotentialcytotoxicity.
2.4.1. Chlorhexidine 2.6. Evaluationofantiviralantisepticactivity
Chlorhexidine (CHX) is a bisbiguanide, used widely as anti-
septic and disinfectant. It has been used at concentrations from 2.6.1. PreparationofSephadexTMmedia
0.05% (m/v), i.e. 5.6×10−4molL−1, in aqueous solutions for skin SephadexTM mediaarearangeofcross-linkeddextrangelsof
damagedisinfectionto0.5%(m/v),i.e.5.6×10−3molL−1,inalco- variable porosity according to the degree of cross-linking. This
holicsolutionsforpre-operativeskindisinfection.ACHXsalt,the property,basedontheprincipleofexclusion-diffusion,allowsthe
CHXdigluconate,wasusedbecauseofitsgreaterwatersolubility separationofdifferentmoleculesaccordingtotheirmolecularsize.
and its commercial use under this form. Its molecular weight is Theyaremanufacturedinabeadformandneedtobesuspendedin
897.80gmol−1.Theaqueoussolutioniscolourless. abuffer.TwodifferentkindsofSephadexTMwereusedinthispro-
tocol,theSephadexTMG-25andtheSephadexTMG-10,whichhave
2.4.2. Hexamidine resolutioncapacitiesof900–5000gmol−1 and100–1000gmol−1,
Hexamidine(HXM)isalsousedwidelyforskindisinfectionand respectively.
externaluse.Itbelongstothearomaticdiamidinegroup.HXMis Accordingtothemanufacturer’sinstructions,SephadexTMG-10
insolubleinwater;thereforeaHXMdiisethionatesaltsolution,of andG-25shouldbesuspendedforminimum3hatroomtemper-
whichthewatersolubilitylimitis55gL−1(Fleurette,1995),isalso ature,inatleast20timestheirvolumeinPBS(i.e.about30gL−1),
usedincommercialformulationsasinthisstudy.Incommercial toequilibratethepHat7andtoallowthegeltoswell.Suspensions
formulations, HXM is used at 0.1% (m/v), i.e. 1.6×10−3molL−1, weresterilizedbyautoclavingandexcessofPBSwaseliminatedto
or at 0.15% (m/v), i.e. 2.4×10−3molL−1. Its molecular weight is obtainasuspensionwithavolumeratioofSephadexTM/PBSof1/1.
606.72gmol−1.Theaqueoussolutioniscolourless.
2.6.2. Preparationofthe“in-house”SephadexTMcolumns
2.5. Cytotoxicityandviabilitytests Columnsweremadeupwitha1mLsyringe,stuffedwithcarded
cotton,associatedwithadrilledEppendorftype1.5mLmicrotube;
MTTviabilityassaysandNRcytotoxicityassayswerecarriedout eachpartwassterilizedbyautoclaving.Thebodyofthesyringewas
toevaluatethecellularimpactofmoleculestestedonL-132cells. filledwith1mLofsterileSephadexTM gel,preparedasdescribed
Inbothassays,L-132cellswereseededinto96-wellcultureplates above.ThesystemwiththesyringeandtheEppendorftubewas
inMEMwith5%FCS.Theywereincubatedat37◦Cwith5%CO for placedintoa50mLconicbottomcentrifugetubetoconservesteril-
2
48hpriortotheadditionoftheappropriatedilutionsoftheproduct ityandcentrifugedat4500×gfor1min.Thedefinitive“in-house”
tobetestedandthenre-incubatedfor24h,48hand168h. SephadexTMcolumnwasthenready(supplementarydata,Fig.2).
For MTT tests, a modified Mosmann’s protocol was used
(Mosmann,1983).Aftermediumremoval,100(cid:2)L/wellofPBSand 2.6.3. Assayforantiviralantisepticactivity
10(cid:2)LofMTTsolutionat5mgmL−1inPBSwereadded.After4hof Ninety-six-well flat bottom culture plates were seeded at
incubationat37◦Cin5%CO ,100(cid:2)Lofsodiumdodecylsulfatewere 5×103cells/wellinMEM5%FCSandincubatedat37◦Cinhumid-
2
addedtodissolveformazandarkbluecrystalsproducedbyreduc- ified atmosphere with 5% CO for 48h to obtain a confluent
2
tion of MTT by succinate mitochondrial deshydrogenase. Plates monolayer.
werere-incubatedfor4handmeasurementswereperformedwith After2daysofincubation,theappropriatedilutionoftheprod-
ascanningmultiwellspectrophotometer(ELISAreader,Multiscan ucttobetestedwaspreparedindistilledwaterandfilter-sterilized
EX, Thermo Fisher Scientific, Saint Herblain, France) using a test ona0.22(cid:2)mfilter.
wavelengthof540nmandareferencewavelengthof690nm.Cell ViralsuspensionsofHCoV229Ewereallowedtothaw;200(cid:2)L
viability was validated, which is proportional to the produced were mixed with 1.8mL of the diluted product and allowed to
quantityofformazancrystalsandexpressedas50%inhibitorycon- stay in contact for the desired contact time. To separate viruses
centration(IC ,molL−1). andtheproduct,500(cid:2)Lofthismixturewereplacedintothe“in-
50
NR assays were performed according to a modified Borenfre- house”SephadexTMcolumn,30sbeforetheendofthecontacttime,
und’sprotocol(BorenfreundandPuerner,1985).Anaqueousstock to leave sufficient time to centrifuge. The system was placed in
solutionat4mgmL−1ofNR,subjectedtoacentrifugationat405×g a 50mL centrifuge tube to maintain sterility and centrifuged at
for10mintoeliminateeventualaggregates,wasprepared.Forthe 405×gfor1min.Thiscentrifugationretainedtheproductaccord-
assay,a“readytouse”solutionofNRat50(cid:2)gmL−1inMEMwithout ingtoitsmolecularsizeandthekindofSephadexTM usedandto
glutamineandwithoutphenolredwaspreparedextemporaneously recovervirusesinthefiltrates.Viraltiterswerethenevaluatedwith
toavoidfineprecipitateswhichcouldformwhenNRismixedwith theendpointdilutionmethod(Section2.3).Thepotentiallossof
medium.Aftermediumremoval,cellswererinsedwith200(cid:2)Lof HCoV infectivity was estimated by the difference between viral
PBS/wellpriortotheadditionof200(cid:2)L/wellof“use”NRsolution. titerofpositivecontrol,whichwasalsosubjectedtofiltrationon
Plates were re-incubated for 3h at 37◦C in 5% CO to allow the the“in-house”SephadexTM columnsbutwithoutthepresenceof
2
uptakeofthedyebylivingcells.Thedye-mediumwasthentaken theproduct;andviraltiterobtainedaftercontactwiththeproduct
off and cells were washed with 200(cid:2)L/well of “A” solution [“A” testedfordesiredcontacttimeandneutralizationbyfiltrationon
solution:4%(v/v)formaldehydeat36.5%(v/v),1%(m/v)CaCl indis- the“in-house”SephadexTMcolumns.
2
tilledwater].ToreleaseNRaccumulatedinlysosomesoflivingcells,
200(cid:2)L/wellof“B”solutionwereadded[“B”solution:1%(v/v)acetic 2.6.4. Controlexperiments
acid,50%(v/v)absoluteethanolindistilledwater].Thequantityof Threetypesofcontrolswereperformedineachexperimentto
NRreleasedwasestimatedbyspectrophotometricmeasurementat validatetheresults:(i)non-retentionofvirusesafterfiltrationon
540nminascanningmultiwellspectrophotometer.Itwaspropor- SephadexTM columns,(ii)neutralizationofthepotentialantiviral
tionaltothecytotoxicityofmoleculestestedandexpressedas50% activityoftheproducttested,and(iii)eliminationofitscytotoxicity.
cytotoxicconcentration(CC ,molL−1). AstheEuropeanStandard(NFEN14476+A1)states,thedifferencein
50
C.Gelleretal./JournalofVirologicalMethods159(2009)217–226 221
viraltiterbeforeandaftertreatment,(i.e.inthiscase,gelfiltration), 2.8. Statisticalanalysis
withoutproducttested,shouldnotexcess0.5log .Toassessthe
10
non-retention of viruses, e.g. HCoV 229E, viral suspensions were Tovalidatethisprocess,statisticalanalysisofthedifferentsteps
mixedwithsteriledistilledwaterforthecontacttimedefinedfor wasundertakenusingStatview®Version5.0.Foralltests,themax-
the experiment, filtered on the “in-house” SephadexTM columns imumacceptableriskwas5%.Toassesshomogeneityofcolumns,
andtitratedasdescribeabove(Section2.3). i.e. volumes of gel after the first centrifugation and volumes
Neutralizationassayswereperformedbyfiltrationof500(cid:2)Lof of filtrate after the second centrifugation, Kolmogorov–Smirnov
thesolutiontobetestedonthe“in-house”SephadexTM columns tests were used to assure distribution normality of each inde-
and 180(cid:2)L of the filtrates were mixed with 20(cid:2)L of viral sus- pendent tested batch, and then Bartlett’s tests were used to
pension. This mixture was then inoculated to the L-132 cells comparevariances.Incaseofvariancesinhomogeneity,anANOVA
monolayers.Theviraltiterobtainedshouldbeequivalenttoviral test was used to compare means of the different batches; in
titer without filtration (i.e. a log difference ≤0.5), since the case of non-homogeneity of variances, a non-parametric test of
10
product should be retained in the column. This experiment also Kruskall–Wallis was used to compare averages. Finally, corre-
verifiedthatfiltratesdidnothaveanyinfluenceonvirusinfectiv- lation studies, using a Fisher transformation, were realised to
ity. estimatewhethertherewasanassociationgelvolumesandeither
Cytotoxicitycontrolswerealsocarriedoutforeachexperiment, filtrates volumes or residual concentrations after filtration on
as a complement to MTT and NR assays undertaken previously. SephadexTM.
These controls were performed by filtration of 500(cid:2)L of the ForUV-spectrophotometry,regressionanalysis(A=f(c))wascar-
product tested and then filtrates were inoculated on L-132 cells. riedouttoestablishspecificspectrophotometricparametersofeach
After6daysofincubation,cellsmonolayerswereobservedunder moleculetested.
an inverted microscope and any sign of cytotoxicity should be
visible.
3. Results
2.7. UV-spectrophotometry 3.1. Validationoftheprotocolforantisepticantiviralactivity
testing
Retentionofthemoleculestestedbythe“in-house”SephadexTM
columns was also confirmed by UV–visible spectrophotometry. 3.1.1. Reproducibilityofthe“in-house”SephadexTMcolumn
The range of dilutions was assayed to establish specific spec- fabricationandretentionofdyemolecules
trophotometric parameters: maximum absorption wavelength As specified in Section 2.6.2, two centrifugation steps were
(cid:2)max,molarabsorptioncoefficientεanddetectionlimits,foreach needed:(i)toprepareoptimizedcolumnsof0.5mLofgel,whether
molecule. forSephadexTMG-25orSephadexTMG-10and(ii)tofilterthedrug-
Dilutions were prepared in distilled water to mimic the test virus mixture to achieve the neutralization step with retention
conditions.SpectrawereperformedonaUV–visiblespectropho- of molecules tested, non-retention of viruses and homogeneous
tometer (UVmc2, Safas, Monaco) within a 180–640nm window. quantityoffiltrates.Differentspeedsandtimesofcentrifugation
Calibrationcurves(A(cid:2)max=f(c))wereestablishedtodeterminethe werethentestedandbestparametersweredetermined:1minat
molarabsorptioncoefficient(ε)foreachmolecule.Differentdilu- 4500×g for the first centrifugation and 1min at 405×g for the
tionsofproductstestedwerepreparedindistilledwaterandfiltered secondcentrifugation.
through the “in-house” SephadexTM columns. Their absorbances The homogeneity of (i) gel volumes obtained after the first
werethenmeasuredat(cid:2)maxtodeterminetheresidualconcentra- centrifugationand(ii)filtratevolumesobtainedafterthesecond
tion. centrifugation;wasfirstestablishedon4independentbatchesof
Table1
UV–visiblespectrophotometricparametersofmoleculestestedandretentionrates.Moleculestestedweresuspendedinsteriledistilledwater(pH7).Calibrationcurves
wereestablishedforeachmoleculeonaUV–visiblespectrophotometerwithina180–640nmwavelengthwindow.Theconcentrationstestedrangedfrom10−7molL−1
to10−2molL−1 exceptfortrypanblue(TB)andneutralred(NR)forwhichconcentrationsrangedfrom10−7molL−1 to4.1×10−4molL−1 andfrom5×10−4molL−1 to
5×10−4molL−1,respectively.Regressionanalyseswerethenperformedtodeterminespecificspectrophotometricparameters:(i)linearitylimits(molL−1),(ii)minimum
detectionlimits(molL−1);withintheselimits,(iii)thespecificmaximalabsorptionwavelength((cid:2)max,nm)and(iv)thespecificmolarabsorptioncoefficient(ε,Lmol−1cm−1).
Retentionrates(seeformulainSection3.1.1)ofeachmoleculebythespecifiedtypeofSephadexTMmediaareindicated,i.e.TBandchlorhexidine(CHX)Retentionratesby
SephadexTMG-25;NRandhexamidine(HXM)RetentionratesbySephadexTMG-10.Cicorrespondstotheinitialconcentrationorconcentrationbeforefiltration.Absorption
wavelengthusedtoestimatetheretentionrateisspecifiedeachtimeitwasnecessary.
Moleculestested Specificspectrophometricparametersforeachmoleculetested Retentionrate
(cid:2)max(nm) ε(Lcm−1mol−1) Linearitylimits(molL−1) Detectionlimit(molL−1) Ci(molL−1) Retentionrates(%)
SephadexTMG-25
TB 234 2.0×104 [2.5×10−6to10−4] 5×10−7 4.1×10−4 589nm:95.11±2.35(n=20)
317 1.1×104 [2.5×10−6to10−4] 7.5×10−7 4.1×10−3 589nm:92.06±3.43(n=20)
589 2.9×104 [5×10−7to7.5×10−5] 5×10−7
CHX 232 2.6×104 [10−6to2.5×10−4] 10−6 10−4,10−3 232nm,255nm:>99a(n=6)
[10−6 232nm:79.73±4.37(n=6)
255 2.5×104 to 10−6 10−2 255nm:81.27±1.28(n=6)
10−4]
SephadexTMG-10
NR 276 3.5×104 [5×10−7to5×10−5] 5×10−7 10−4,10−3 276nm,452nm:>99.5a(n=20)
452 1.0×104 [5×10−7to5×10−5] 5×10−7
HXM 261 2.1×104 [10−6–10−4] 10−6 10−4,10−3 261nm:>99a(n=6)
10−2 261nm:<90b(n=6)
a Belowthedetectionlimit.
b Abovethelinearitylimits.
222 C.Gelleretal./JournalofVirologicalMethods159(2009)217–226
10columnsforeachSephadexTM gel,bystatisticalanalysisusing Table3
Statview®V.5.0(Section2.8). Eliminationofpotentialcytotoxicitybyfiltrationonthe“in-house”SephadexTM
Retention rates of 2 dye molecules were measured: the columns.50%inhibitoryconcentration(IC50)and50%cytotoxicconcentration(CC50)
ofchlorhexidine(CHX)andhexamidine(HXM),withoutfiltration,onL-132cells
TB (960.83gmol−1) for the SephadexTM G-25 and the NR wereevaluatedwithmethylthiazoltetrazolium(MTT)assaysandneutralred(NR)
(288.78gmol−1) for the SephadexTM G-10. To determine this assays,respectively(inbold).IC50 andCC50 of(i)CHXsolutionsat10−3molL−1
rate, the spectrophotometric parameters of TB and NR (Table 1) and10−4molL−1,and(ii)ofHXMsolutionsat10−2molL−1and10−3molL−1,after
filtrationonthe“in-house”SephadexTMG-25andSephadexTMG-10columns,respec-
were established by measuring absorbances of concentrations
tively,werethenevaluated.Results,expressedinmolL−1,representtheaverageof
ranging from 10−7molL−1 to 4.1×10−4molL−1 for TB and from 3independentexperiments.
5×10−7molL−1 to5×10−4molL−1 forNR,followedbyaregres-
Ci 24h 48h 168h
sionanalysis(Section2.8).Theresidualconcentrationsoffiltrates
wereestimatedbymeasuringabsorbancesinUV–visibleandcal- CHX
culating the retention rate according to the formula: retention IC50 Withoutfiltration (4.3±0.6)×10−6 (2.4±1.3)×10−6 (3.9±0.2)×10−6
rate=[(Ci−Cf)/Cf]×100,whereCiwastheinitialconcentrationor 1100−−34mmoollLL−−11 >>1100−−45 >>1100−−45 >>510.6−×510−5
concentrationbeforefiltrationandCfwasthefinalconcentration
ortheresidualconcentrationafterfiltration. CC50 Withoutfiltration (5.6±1.1)×10−6 (2.9±1.3)×10−6 (3.4±0.5)×10−6
10−3molL−1 >10−4 >10−4 >5.4×10−5
RetentionratesobtainedforTBafterfiltrationonSephadexTM 10−4molL−1 >10−5 >10−5 >10−5
G-25 were (95.11±2.35)% and (92.06±3.43)% after filtration of
TB solutions at 4.1×10−4molL−1 and 4.1×10−3molL−1 (which HXM
was the commercial concentration), respectively. For NR solu- IC50 Withoutfiltration (3.8±1.0)×10−5 (1.2±1.4)×10−5 (3.8±2.5)×10−6
tions, either at 10−4molL−1 or 10−3molL−1, after filtration on 10−2molL−1 (5.3±1.4)×10−5 (1.0±0.5)×10−5 (5.9±1.4)×10−6
10−3molL−1 >10−4 >10−4 >10−4
SephadexTMG-10,concentrationswereunderthedetectionthresh-
old.Thus,theretentionratesobtainedwere>99.5%afterfiltration CC50 Withoutfiltration (6.2±1.4)×10−5 (2.5±1.5)×10−5 (5.6±0.1)×10−6
ofNRsolutionsat10−4molL−1and10−3molL−1. 10−2molL−1 (6.2±1.3)×10−5 (3.2±2.2)×10−5 (5.7±0.3)×10−6
10−3molL−1 >10−4 >10−4 >5.5×10−5
Itwasfoundthat,withinarangeof[450–550](cid:2)Lofgelvolumes
afterthefirstcentrifugation,therewasnosignificantvariationin
filtratevolumesandinretentionrates.Infurtherexperiments,all activity,eliminatinganeventualcellularimpactoftheproducton
gelvolumeswereincludedwithintheselimits. L-132cells.MTTandNRassayswerecarriedouttodetermineIC
50
andCC ,respectively.TheIC andCC ofCHXandHXMsolutions
50 50 50
3.1.2. Non-retentionofvirusparticlesbythe“in-house” (withoutfiltration)werefirstdeterminedonL-132cells(Section
SephadexTMcolumns 2.4).Then,IC andCC ofCHXandHXMsolutions,atdifferent
50 50
Basically,the“in-house”SephadexTMcolumnsshouldretainthe concentrationsandafterfiltrationonthe“in-house”SephadexTM
product but not viruses. According to the European Standard NF columns, were determined to make sure of the elimination of
EN 14476+A1 (AFNOR, 2007), the difference between viral titer the eventual cytotoxicity by the filtration on the “in-house”
before and after treatment, i.e. here, after filtration, should not SephadexTMcolumns.Eachexperimentwasrepeated3timesinde-
exceed0.5log10.Toensurethisnon-retentionofviruses,3indepen- pendentlytofindtheIC50 andCC50 correspondingtotheaverage
dentexperimentsforeachSephadexTM typeandforeachcontact resultsofthose3experiments.AsshowninTable3,IC ofCHXonL-
50
time tested, were performed. The HCoV 229E stock suspension 132cellswere(4.3±0.6)×10−6molL−1,(2.4±1.3)×10−6molL−1
wasdiluted1:10insteriledistilledwaterandfilteredonthe“in- and(3.9±0.2)×10−6molL−1at24h,48hand168h,respectively,
house”SephadexTMcolumns.Theviraltiterswerecomparedwith andCC were(5.6±1.1)×10−6molL−1,(2.9±1.3)×10−6molL−1
50
and without filtration (Table 2). For SephadexTM G-10, log10 dif- and(3.4±0.5)×10−6molL−1at24h,48hand168h,respectively.
ferencesbetweenviraltiterswere0.1±0.1,0.3±0.4and0.2±0.2 AfterfiltrationofCHXsolutionsat10−3molL−1 and10−4molL−1
forcontacttimesof0min,30minand60min,respectively(n=3 onSephadexTM G-25columns,CC andIC were>10−4molL−1,
50 50
for each contact time). For SephadexTM G-25, log10 differences according to the detection limit of the method, except at 168h,
betweenviraltiterswere0.2±0.2,0.4±0.2,0.1±0.3,0.1±0.2and whereIC was>5.6×10−5 andCC >5.4×10−5molL−1 afterfil-
50 50
0.0±0.2 for contact times of 0min, 5min, 15min, 30min and trationof10−3molL−1CHXsolution.
60min, respectively (n=6 for each contact time). Consequently, The same experiments were carried out with HXM. IC
50
SephadexTMcolumnsofeachSephadexTMtypedidnotretainHCoV of HXM without filtration were (3.8±1.0)×10−5molL−1,
229Eanddidnotinducealossofinfectivity. (1.2±1.4)×10−5molL−1and(3.8±2.5)×10−6molL−1at24h,48h
and 168h, respectively, and CC were (6.2±1.4)×10−5molL−1,
50
3.1.3. Eliminationofcytotoxicityofmoleculestested(CHXand (2.5±1.5)×10−5molL−1 and (5.6±0.1)×10−6molL−1 at 24h,
HXM)byfiltrationonthe“in-house”SephadexTMcolumns 48h and 168h, respectively. After filtration of HXM solution at
Eliminationofcytotoxicitywasacrucialcontrolbeforeinocula- 10−3molL−1ontheSephadexTMG-10columns,IC andCC were
50 50
tionoffiltratesontothecells.Itallowedtheassessmentofantiviral >10−4molL−1,exceptat168hwhereCC was>5.5×10−5molL−1.
50
Table2
Non-retentionofHCoV229Ebythe“in-house”SephadexTMcolumns.Results,expressedaslog10of50%cellcultureinfectivedose(CCID50),representtheaverageofviraltiters
ofatleast3independentexperiments.Theseassaysallowedtoevaluatethenon-retentionofthehumancoronavirus229E(HCoV229E),foreachSephadexTMtypeandeach
contacttimetested.Viralsuspensionsweredilutedat1:10insteriledistilledwaterforthespecifiedcontacttimebeforefiltrationonthe“in-house”SephadexTMcolumnsand
inoculationtotheL-132cells.nd:notdetermined.
Contacttime(min) SephadexTMG-10 SephadexTMG-25
Withoutfiltration Afterfiltration Difference Withoutfiltration Afterfiltration Difference
0 5.8±0.5(n=3) 5.7±0.6(n=3) 0.1±0.1(n=3) 6.0±0.6(n=6) 5.8±0.6(n=6) 0.2±0.2(n=6)
5 nd nd nd 5.9±0.7(n=6) 5.6±0.6(n=6) 0.4±0.2(n=6)
15 nd nd nd 5.6±0.5(n=6) 5.6±0.4(n=6) ±0.3(n=6)
30 6.7±0.9(n=3) 6.4±0.3(n=3) 0.3±0.4(n=3) 6.1±0.2(n=6) 6.0±0.4(n=6) 0.1±0.2(n=6)
60 6.7±0.9(n=3) 6.4±0.7(n=3) 0.2±0.2(n=3) 6.1±0.2(n=6) 6.1±0.1(n=6) 0.0±0.2(n=6)
C.Gelleretal./JournalofVirologicalMethods159(2009)217–226 223
HXMsolutionat10−2molL−1wasalsoassayed.IC andCC were
50 50
shown to be very close to those without filtration (Table 3) and
thusretentionwasshowntobeincompleteforthisconcentration.
Consequently, the “in-house” SephadexTM columns clearly
demonstratedthattheyefficientlyeliminatecytotoxicityofsolu-
tionsatconcentrations≤10−3molL−1.
3.1.4. CHXandHXMretentionrates(UV–visible
spectrophotometry)afterfiltrationonthe“in-house”SephadexTM
columns
Asfordyemolecules(TBandNR),specificspectrophotometric
parametersandretentionratesofCHXandHXMweredetermined
usingUV–visiblespectrophotometry(Section2.7).Testingdiffer-
ent dilutions of CHX, i.e. from 10−10molL−1 to 10−4molL−1, the
specific spectrophotometric parameters of CHX were established
(Table1).Twomaximumabsorptionwavelengths((cid:2)max)at232nm
and255nmwereobserved,withmolecularabsorptioncoefficients
of2.6×104Lcm−1mol−1and2.5×104Lcm−1mol−1,respectively.
Thesecoefficientswereobtainedbymeansofaregressionanaly-
sisrealizedwithStatview®V.5.0(n=15measurements,p<0.0001).
TherelationA=f(c)waslinearbetween[10−6to2.5×10−4]molL−1
for (cid:2)max=232nm (correlation coefficient=0.998) and between
[10−6 to 10−4]molL−1 for (cid:2)max=255nm (correlation coeffi-
cient=0.996).Thedetectionlimitwas10−6molL−1forbothofthe
2(cid:2)max. These results were consistent with data from the litera-
ture(Bonazzietal.,1995;EuropeanPharmacopeia,2007a;Haand
Cheung,1996;Havlikovaetal.,2007;Hebertetal.,2003),where
UVdetection,especiallyafterhighperformanceliquidchromatog-
raphy,wasdoneeitherat235nmorbetween254nmand258nm.
The retention rates of CHX solutions were then evaluated at dif-
ferentconcentrationsbythe“in-house”SephadexTMG-25columns
(Table1).Threeindependentexperimentswererealizedindupli-
Fig.2. EvaluationofantisepticantiviralactivityofCHXandHXMontheHCoV229E.
cate(n=6).Aretentionrate>99%wasobservedforCHXsolutions Thisfigureshowsthelog10 reductioninviraltitersobtainedaftertheactionof
at10−4molL−1and10−3molL−1.CHXsolutionat10−2molL−1was chlorhexidine(CHX)andhexamidine(HXM)atdifferentconcentrationsandatspec-
notcompletelyretainedandretentionrateswereof(79.7±4.4)%at ifiedcontacttimes:(A)representsthelog10reductioninHumancoronavirus229E
232nmand(81.3±1.3)%at255nm. (HCoV229E)titersobtainedafteractionofCHXat10−3molL−1( )and10−4molL−1
In the same way, HXM was assayed from 10−10molL−1 to o(fH)XanMda(tB)10re−p3rmesoelnLt−s1th(el)o.gT1h0erebdouldctliionneiinnHbCoothVg2r2a9pEhtsitreerpsroebsetanitnsetdheafttherreaschtioolnd
10−3molL−1 (Table1).The(cid:2)max wasobservedat261nmassoci- of4log10reductiontoreachtopretendtoanantiviralantisepticactivity.
ated to a molar absorption coefficient of 2.1×104Lcm−1mol−1,
withalinearrelationshipbetween[10−6 to10−4]molL−1 (n=11, 0.2±0.2,respectively.ThesameassayswerecarriedoutwithHXM
p<0.0001, correlation coefficient=0.980). The detection limit of at 10−3molL−1 and the mean of log difference obtained (n=3
HXM with UV–visible spectrophotometry is 10−6molL−1. These independent assays) was 0.2±0.3. Th10us, potential antiviral anti-
data were consistent with the European Pharmacopeia, which
septicactivitywascorrectlyneutralizedforeachmoleculeandfor
recommendsHXMdetectionat263nm(EuropeanPharmacopeia,
eachconcentrationtested.Furthermore,nointerferencewithvirus
2007b).Hexamidineretentionratesbythe“in-house”SephadexTM
infectivitywasobserved.
G-10 columns were evaluated. They were >99% for HXM at
10−4molL−1 and 10−3molL−1. As for other molecules, retention
limits were reached for HXM at 10−2molL−1 and retention rate 3.2. Evaluationofthepotentialantiviralantisepticactivityof
CHXandHXM
could not be determined accurately because residual concentra-
tionswereabovethelinearitylimitsofdetection,i.e.10−4molL−1.
AssayswereperformedasdescribedinSection2.6.3.Eachassay
Thus,theretentionratewas<90%.
correspondedtotheevaluationofpotentialATSanti-HCoVactivity
ofoneconcentrationofeachmoleculetestedandonecontacttime.
3.1.5. Neutralizationofpotentialantiviralantisepticactivityof Eachassaywasrepeated3independenttimesandincludednec-
productstested essarycontrols,i.e.positiveviruscontrol,non-retentionofHCoV,
Verifyingneutralizationcontrolwasessentialtoassessthestop efficiencyofneutralizationandeliminationofcytotoxicity,tovali-
ofpotentialantiviralactivityandtherespectofthedefinedcontact datethetest(Fig.1).
time.Furthermore,thistestensuredthattherewasnointerference CHX was tested at 2 concentrations, 10−4molL−1 and
ofthefiltrateswithvirusinfectivity(Fig.1).Asrecommendedbythe 10−3molL−1 and each concentration was tested during 4 differ-
EuropeanStandardNFEN14476+A1,thedifferencebetweenvirus entcontacttimes,i.e.5min,15min,30minand60min(Fig.2A).
controlandneutralizationcontrolshouldnotexcess0.5log (Sec- The log reduction in viral titers induced by the action of CHX
10 10
tion2.6.4).Neutralizationcontrolsweredoneforeachindependent at10−4molL−1wasof0.8±0.7,0.5±0.4,1.4±1.5and2.1±1.2for
experiment (each product concentration and each contact time). 5min,15min,30minand60min,respectively.Attheconcentra-
Neutralization assays were performed for CHX at 10−4molL−1 tionof10−3molL−1,CHXinducedareductionof1.4±0.8,2.1±0.4,
and 10−3molL−1. The mean of log differences obtained (n=6 2.4±0.6 and 3.0±0.2 for contact times of 5min, 15min, 30min
10
independent assays for each concentration) were 0.2±0.1 and and60min,respectively.AccordingtotheEuropeanStandardNF
224 C.Gelleretal./JournalofVirologicalMethods159(2009)217–226
EN14476+A1,thelogreductionislowerthan4log ,so,evenifCHX tilledwatercontaining20%ethanolasconservative.Thepresence
10
hadacertainactivityontheHCoV229E,ithadnoATSanti-HCoV ofethanolcouldaffectthetests,emphasizingtheeffectoftheATS-
229Epropertiesintheseconditions. D tested so leading to an overestimation of the product activity.
HXM showed an even worse activity on the HCoV 229E. At The cost was also higher than the “in-house” SephadexTM G-25
10−3molL−1, it induced only a reduction of 0.6±0.5log and orG-10columnsanditsretentioncapacitywasnotwelladapted
10
0.9±0.8log for30minand60minofcontacttime,respectively, (supplementarydata,Fig.1).
10
soveryfarfromthethresholdrecommendedbytheEuropeanStan- The choice of virus strain could also be discussed and has
dard(Fig.2B).Giventheseresults,concentration10−4molL−1and recently been questioned by Ijaz and Rubino (2008). The Euro-
contacttimesof5minand15minwerenottested. pean Standard NF EN 14476+A1 proposed 3 viruses: a poliovirus
(type1,LSc-2ab),anadenovirus(type5)andabovineparvovirus
4. Discussion (Hadenstrain).Polioviruswaschosenforitspotentialhighresis-
tancetochemicalsbuttakingintoaccounttheworldwideprogram
Tofightviralinfections,ATS-Dseemcrucialconsidering:(i)the ofpoliomyelitiseradication,thelegitimacyofthischoiceisques-
relativelackofefficienttreatmentsandvaccinesavailable,and(ii) tionable. In the same way, the choice of B19 parvovirus and
the risk of outbreak of new viral pathogens. Thus, it is essential adenovirusisarguable.AlthoughB19parvovirusisresponsiblefor
toevaluatetheirantiviralactivityproperlyandreliably.Themajor serious but rare materno-foetal infections and also infections in
itemsproposedintheEuropeanStandardNFEN14476+A1were immunocompromisedpatientsand/orpatientswithahaemolytic
followed,suchas(i)contacttimes,(ii)themaximumof0.5log dif- anaemicdisorder,ithasnotbeenshowntobeafrequentnosocomial
10
ferencesbetweencontrolviraltiters(Section2.6.4)tovalidatethe pathogen.Itsinterest,however,liesinitshighresistancetochem-
assaysand(iii)the4log reductiontoassessanantisepticantiviral icalsandtoawiderangeofpHandtemperaturesanditspotential
10
activityofdrugstested. bloodtransmissionroute(Morinetetal.,2003).However,thestrain
However,someparametersofthisstandardneededtobeinten- used in the European Standard is a bovine parvovirus, so there
sified,especiallytheneutralizationmethodandthechoiceofvirus is a question of representativeness. In contrast, Human serovar
strain.Indeed,theneutralizationstepisfundamental:(i)toensure 5 adenovirus is more interesting notably for its responsibility in
aprecisecontacttimeand(ii)toremoveanypotentialcytotoxic- mildupperrespiratorytractinfections,ophthalmologicinfections
itybeforeinoculationtothecells,whichcouldinvolveconfusion andalsogastro-enteritis,particularlyinnew-bornsandyoungchil-
in product activity estimation. In the European Standard NF EN dren.Itcancauserarebutserious(mortalityupto50%)infections
14476+A1, two global techniques are proposed: dilution in iced in immunodepressed patients, especially in bone-marrow trans-
medium and gel filtration techniques, but they involve different planted patients (Carret and Le Faou, 2001). It has also probably
problemssuchaslengtheningofcontacttimes,efficiencyandalso beenchosenforitshighresistanceinenvironmentalconditions.All
cost.Inthedilutionmethod,theneutralizationsteprequires30min, thesevirusesarenakedvirusesandnotalwaysrepresentativeof
inconsistent with recommended contact times for the different majorsrisksencounteredincommunityorinhospitalsettings.
applicationsmentionedintheEuropeanStandard.Also,notallcon- Whenestablishingthisprotocol,thefirstchallengewastoelab-
tacttimesaresuitedtothepotentialuseoftheproduct,e.g.60min orateauniqueprotocolforbothSephadexTMgels,inordertomake
isthemandatorycontacttimefortestingdisinfectantsforsurfaces assays easier to perform. The best parameters for the 2 needed
andinstrumentsandfacultativetimesare5min,15minand30min, centrifugation steps were determined for conserving the steril-
butinpractice,asurfacewouldrarelybedisinfectedduring60min. ityduringtheentireassay.Theseparameterswerefirstvalidated
Furthermore,dilutionhasnotalwaysbeenshowntobeefficient. through statistical analysis, without drugs tested, to ensure the
For instance, the IC and CC of CHX on L-132 cells are higher repeatability of columns yield and to fix the acceptability limits
50 50
than2×10−6molL−1at24h,48hand168h.Soevenaftera1:100 oftheprotocol.Thus,thisprotocolwasmadetoperformneutral-
dilutionofa10−3molL−1solutionofCHX(theEuropeanStandard izationinonly1min,whichwasbetterthanalltheothermethods.
recommendsa1:10dilutionor1:100ifnotefficient),thesolution Therefore,adifferentvirustothoseproposedintheEuropean
remainscytotoxic. Standardwaschosen:theHCoV229E,notonlyforitsmedicalinter-
Nonetheless,theEuropeanStandardalsoproposesgelfiltration estbutalsoforitspresenceintheannexeBoftheEuropeanStandard
techniques to neutralize product activity, using SephadexTM LH- NFEN14476-A1aspotentialcontaminantforsurfaces,instruments
20, an ultrafiltration system (Minicon® Millipore) or pre-packed and hands. Furthermore, it belongs to enveloped viruses, which
columnsMicrospinTMS400HR.Themajorproblemofthesemeth- areabsentfromthestandardandwhichmaybehavedifferentlyto
odsisthelengtheningofcontacttime.Indeed,theyneedatleast nakedvirusesundertheactionofATS-D.Itwasalsochosenforits
2mintoachievetheneutralizationstep,sometimes10minormore, relativeresistanceindifferentconditionstakingintoaccountthat
dependingonthetechniques.Furthermore,separationcapacities envelopedvirusesaremostoftenconsideredfragile.
arenotwelladaptedtothemolecularmassofmostATS-D.Another Duringtheassaystoshowthenon-retentionoftheHCoV229E
drawback of Minicon® concentrators is that they are non-sterile bythe“in-house”SephadexTMcolumns,whetherG-25orG-10,the
devices and do not bear sterilization, so they cannot be used to importanceofpHwashighlightedasanimportantcauseofvariabil-
filterviruses. ityinresults,i.e.inviraltiters.Thesedifficultieswereexperienced,
The American Standard (ASTM E 1482-04) recommends also mainlywithSephadexTM G-10thatneededexcessofsolvent(i.e.
a gel filtration method, using SephadexTM LH-60 or SephacrylTM PBS)whenswellingtoreachneutrality,andalsowithsteriledis-
superfineS-1000,toneutralizevirucidalactivity(ASTM,2004).The tilledwater,owingtothesterilizationprocess,whichofteninvolved
virucidalefficacycriterionfortheASTMstandardisalog reduc- acidification.ThiscouldbeduetoahighsensitivityoftheHCoV
10
tion>3.TheASTMprocedureisclosetotheonedevelopedhere. 229EtothepH.Rapidlossofinfectivityandanaggregationofviri-
However, the centrifugation time: (i) to eliminate the void and onshavebeenshownatpH8and37◦C,whereastheviruswasquite
(ii)toseparatetheproducttestedandvirusesis3mininsteadof stableatpH6and37◦C(Sturmanetal.,1990).SopHshouldbever-
1minwiththe“in-house”SephadexTMcolumns.Thefirstgelpro- ifiedandadjustedtotheneutralitytoallowastandardizationofthe
posed,SephadexTMLH-60,haswelladaptedretentioncapacitiesto protocol.
mostATS-Dmolecularmass(supplementarydata,Fig.1B).Unfortu- Totestthisprotocol,anti-HCoV229EactivityofCHXandHXM
nately,itisnotsoldanymore.Theothergelproposed,SephacrylTM wereevaluatedbecauseoftheirwideuseandtheirmolecularsize,
superfineS-1000,iscommercializedinasuspensionformindis- which allowed testing both types of SephadexTM, G-25 and G-
C.Gelleretal./JournalofVirologicalMethods159(2009)217–226 225
10.First,theywereshowntoberetainedthroughthe“in-house” withinvivoprotocols,usingforinstance,fingertipsofvolunteers.
SephadexTMcolumnsandtheirpotentialcytotoxicitywasremoved. Furthermore,theyarealsousefultoevaluatepathogensurvivalon
Furthermore, the neutralization technique did not interfere with humanskin.Carriertestsneedadilutionsteptorecoverthedried
virusinfectivity. virussuspensionfromthesupportafteractionofachemical.This
Atthisstage,somelimitsofthegelfiltrationmethodinthecon- dilutionstepplaystheroleofneutralizerandensuresthecontact
centration of the product tested appeared. Above concentrations time.Itcouldbeachievedbyadditionofculturemediumorany
of10−2molL−1,moleculeswerenotretainedcompletelyandthere chemical,whichcouldneutralizespecificallyproductactivity(e.g.
wasaphenomenonofcolumnoverloading.However,thisdidnot useofsodiumthiosulfateforneutralizingpovidone-iodine).How-
seemserious,sinceproductsareusedrarelyatthisconcentration ever,thediluentitselfcouldbetoxicforthecells.Thus,thismethod
orabove,notablybecauseoftheirpotentialtoxicity. couldbeappliedinsuchacasetoeliminatethechemicalneutral-
Atthisstage,theanti-coronavirusactivityofCHXandHXMcould izerandinoculatetheviralsuspensionsafely.Thispotentialisalso
beassessed.Thus,theresultsdemonstratedclearlythatHXMhad foundintheASTMstandardE1482-04.
no ATS-D activity on the HCoV 229E, but to our knowledge, lit- Thisstudydescribesaglobalmethodofchemicalneutralization
tle data concerning a possible ATS-D antiviral activity of HXM is whenensuringthenon-retentionofvirusesandthemaintenance
availableintheliterature. oftheirinfectivity,in1min.Therefore,thismethodcouldbeused
InadditiontheresultsshowedthatdespiteactivityontheHCoV totestchemicalsfordifferentapplicationsfromsurfacestowater
229E,CHXcouldnotpretendtohaveATS-DactivityontheHCoV disinfectants.Itcouldbeusedinthehumanmedicalfield,aswell
229EintestingconditionsandaccordingtotheEuropeanStandard. asinveterinaryorindustrial(e.g.foodindustry)applicationsand
However,accordingtotheAmericanStandard,ofwhichtheviru- alsotoscreentheantisepticantiviralpotencyofnewdrugs.
cidal efficacy criterion is a 3log reduction, CHX had an ATS-D
10
antiviral activity at 10−3molL−1 and 60min, but this activity is Acknowledgements
not really representative of field use conditions. In most studies,
CHX has been shown: (i) to be inefficient against naked viruses We sincerely thank Dr. S.A. Sattar (Centre for Research on
(Bernsteinetal.,1990;Kawanaetal.,1997;NarangandCodd,1983; EnvironmentalMicrobiology,FacultyofMedicine,Ottawa,Canada)
Wood and Payne, 1998) and (ii) to be efficient at concentrations for kindly providing us with the HCoV 229E; Dr. H.F. Hildebrand
>10−3molL−1 on enveloped viruses but its activity depends on (Groupe de Recherche sur les Biomatériaux, Laboratoire de Bio-
virusestestedandtestingconditions(Bernsteinetal.,1990;Kawana physique,UPRESEA1049,FacultyofMedicine,Lille,France)who
etal.,1997;PlattandBucknall,1985;TylerandAyliffe,1987;Wood furnished us L-132 cells and also Pr. J.B. Regnouf-de-Vains and
andPayne,1998).Forinstance,CHXreducedHerpessimplexvirus his team (Groupe d’Etude des Vecteurs Supramoléculaires du
viraltitersof>3log10insuspensiontestsforunder3min(Kawana Médicament,UMR7565,FacultédePharmacie,Nancy,France)for
etal.,1997;WoodandPayne,1998)andonlyof1log10onadried providinguswithCHXandHXMmolecules.Wewouldalsoliketo
sampleofHSVin10min(TylerandAyliffe,1987).Itisalsoimportant thankMrs.N.Marshallforeditingthismanuscript.
tonotethatmostantisepticsandotherchemicalsareusedasamix
ofdifferentmoleculesandthediluent,oftenethanol,hasitsown AppendixA. Supplementarydata
virucidal activity. For instance, different formulations containing
chlorhexidinedigluconatewereassayedforATSantiviralactivity.It Supplementarydataassociatedwiththisarticlecanbefoundat
didnotrespondtotheAmericanvirucidalcriterionwhentestedat doi:10.1016/j.jviromet.2009.03.023.
0.008%(m/v),i.e.8.9×10−5molL−1withcetrimideat0.08%(m/v).
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