Differential replicative fitness of the four dengue virus serotypes circulating in Colombia in human liver Huh7 cells

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w w w . e l s e v i e r . c o m / l o c a t e / b j i d

The

Brazilian

Journal

of

INFECTIOUS

DISEASES

Original

article

Differential

replicative

ﬁtness

of

the

four

dengue

virus

serotypes

circulating

in

Colombia

in

human

liver

Huh7

cells

Hernando

Gutiérrez-Barbosa

∗

,

Nadia

Y. Casta ˜neda,

Jaime

E. Castellanos

UniversidadElBosque,GrupodeVirología,Bogotá,Colombia

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received5July2019 Accepted24November2019 Availableonline13December2019

Keywords: Denguevirus Dengueserotypes Replicativeﬁtness Denguestrains

a

b

s

t

r

a

c

t

DenguehasbeenasignificantpublichealthprobleminColombiasincethesimultaneous circulationofthefourdenguevirusserotypes.Thereplicativefitnessofdengueisa bio-logicalfeatureimportantforvirusevolutionandcontributestoelucidatingthebehaviorof viruspopulationsandviralpathogenesis.However,ithasnotyetbeenstudiedin Colom-bianisolates.Thisstudyaimedtocomparethereplicativefitnessofthefourdenguevirus serotypesandunderstandtheassociationbetweentheserotypes,theirinvitroinfection ability,andtheirreplicationintargetcells.WeusedthreeisolatesofeachDENVserotypeto infectHuh-7cellsatanMOIof0.5.Thepercentageofinfectedcellswasevaluatedbyflow cytometry,cellviabilitywasevaluatedbyMTTassay,andthepathogenicityindexwas cal-culatedasaratioofbothparameters.Thereplicativefitnesswasmeasuredbythenumber ofviralgenomecopiesproducedusingquantitativePCRandtheproductionofinfectious viralprogenywasmeasuredbyplaqueassay.WeshowedthatHuh-7cellsweresusceptible toinfectionwithallthedifferentstrainisolates.Nevertheless,thebiologicalcharacteristics, suchasinfectiousabilityandcellviability,werestrain-dependent.Wealsofounddifferent degreesofpathogenicitybetweenstrainsofthefourserotypes,representativeofthe hetero-geneitydisplayedinthecirculatingpopulation.Whenweanalyzedthereplicativefitness usingthemeanvaluesobtainedfromRT-qPCRandplaqueassayforthedifferentstrains, wefoundserotype-dependentbehavior.Thehighestmeanvaluesofreplicativefitnesswere obtainedforDENV-1(log4.9PFU/ml)andDENV-4(log5.28PFU/ml),followedbyDENV-2 (log3.9PFU/ml)andDENV-3(log4.31PFU/ml).Theinternalheterogeneityofthereplicative fitnesswithineachserotypecouldexplainthesimultaneouscirculationofthefourDENV serotypesinColombia.

∗ _{Corresponding}_author_at:_Grupo_de_Virología,_Universidad_El_Bosque,_Av._Carrera₉_No.₁₃₁_A_02,_Bogotá,_Colombia.

E-mailaddress:[email protected](H.Gutiérrez-Barbosa).

https://doi.org/10.1016/j.bjid.2019.11.003

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Introduction

Dengueisapublichealthproblemintropicalandsubtropical regions,whereanestimatedfourbillionpeopleareatriskof sufferingfromthedisease.In2010,390millioncasesofdengue wereestimatedworldwide,ofwhich96millionpresentedwith outwardmanifestations.1_Colombia_is_a_hyperendemic coun-tryfordengue,andalthoughtheprevalenceofserotypeshas changedovertime,thefourDENVserotypeshavecirculated simultaneouslyin30ofthe32departmentsofthecountry.For morethan30years,DENVserotypecirculationhasoccurred withoutdisplacementofanyserotypeandwithhigh variabil-itybetweenthedepartmentsofthecountry.2Thenumberof casesofdengueinColombiaisstillhigh;during2018,44,825 caseswerereported,ofwhich47.4%correspondedtodengue withoutwarningsigns,51.4%todenguewithwarningsigns, and1.2%toseveredengue.3_The_course_of_the_disease_and_the degreeofclinicalcomplicationsarerelatedtothegeneticsand theimmuneresponseofthepatientandtheintrinsic charac-teristicsoftheinfectingviralstrain,4_which_could_explain_the differentepidemiologicaldynamicsobservedworldwide. Dif-ferentfactorscontributetothehighincidenceofthedisease inthecountry,suchashighpopulationdensitiesinruralareas withpoor infrastructure,which favors the presenceofthe vectorAedesaegypti,fluctuationbetweenseasonsofdrought and high rainfall, insufficient methods for vector control, povertywiththeinherentdifficultyofaccesstohealthcare, anunderdeveloped national systemofdiagnosis and clini-calmanagement,hyperendemicityandnaturalchangesinthe relativeabundanceofcirculatingDENVserotypes.5

Dengue virus (DENV) has a complex classification and belongstotheFlaviviridaefamily.Thisvirushasfourtypes (DENV-1, DENV-2, DENV-3, DENV-4) with 62–67% sequence identity.6_The_{classification}_in_four_serotypes_is_based_on_the immuneresponseofthepatienttoprimaryinfectionbyone ofthe serotypes.Primary infection protects against a sec-ondaryinfectionbyahomologousDENVserotypebutconfers partialandtransientprotectionagainstaheterologousDENV serotype.7_{Additionally,}_each_type_of_DENV_is_further_classified intomonophyletictaxonomicgroupsorgenotypes,aswellas subcladesorlineages.8,9 _To_date,_five_genotypes_of_DENV-1, sixofDENV-2,fiveofDENV-3,and fiveofDENV-410–14 _have beendescribed,andtherelationshipbetweentheirpopulation dynamicsandepidemiologicalbehaviorisstillunder investi-gation.

TheepidemiologicalassociationbetweeninfectiousDENV serotypes andthe evolutionofsevere clinicalsymptoms is notconclusive.Previousstudiessuggestacorrelationbetween geographicheterogeneityandspatialdependenceofthe circu-latingviralstrainandtheclinicaloutcomeofDENVdisease. Ameta-analysisfoundthat themostpathogenic serotypes insouthern Asia are DENV-3 followed byDENV-4, DENV-2, andDENV-1,whileincountriesincludingPeru,Cuba,France, Colombia, and Brazil, DENV-2 was described as the most pathogenicstrainfollowedbyDENV-3andDENV-4.15_A_study inNicaraguadescribedDENV-3asthemostpathogenicand DENV-4astheleastpathogenicserotype.16

The replicative ﬁtness of a virus population demon-stratesitsabilitytoproduceinfectiousprogenyinaspeciﬁc

microenvironment.17 _Changes _in _replicative _fitness _profiles mayfavororimpairthetransmissionofthevirus.The diffi-cultyofevaluatingthereplicativefitnessofaviralpopulation duringthe naturaltransmissioncycle hasledtothe devel-opment of in vitro systems that quantify viral titersas an approximationtoreplicativefitness.Thisapproachhasbeen usedindifferentstudies tounderstandtheepidemiological dynamics ofDENV inviral populationreplacementevents. In Nicaragua, a clade replacement of the DENV-2 Asian-American genotypecladeNI-1bycladeNI-2Bwasreported in 2009. Thisreplacement was relatedto a higher replica-tivefitnessofstrainsbelongingtotheNI-2Bclade,whichwas evaluatedinC6/36mosquitocellsandiDCmhuman-derived monocytes.18 _In _Puerto_Rico _in_2011,_there _was_a displace-mentofcladePR-1bycladePR-2BbelongingtoDENV-2.This eventwasassociatedwithadifferentialfitnessbetweenthe cladeswhenthereplicativefitnesswasevaluatedinhuman livercells (Huh-7). Additionally, cladePR-2Bhad higher fit-nessasdemonstratedbyviralinhibitionoftheinterferontype Iresponse.19

IncountrieswherethefourDENVserotypesarein cocircu-lation,suchasColombia,fewstudieshavebeencarriedout to understand the population dynamicsand their possible epidemiologicalrelationship.InColombia,astudyevaluated the replicativefitnessofDENVinC6/36mosquitocells and coloniesofA.aegyptiusingaviralstrainofeachDENVserotype and described a differential fitness between serotypes, in whichDENV-2hadthehighestreplicativecapacityfollowed byDENV-1,DENV-4,andDENV-3.20 _However,_the_use_of_one strain per DENV serotype might not be representative of the fitness ofother viral circulating subpopulations. Addi-tionally,differentevolutionarypatternshavebeendescribed betweenviralpopulationsthatreplicateinmammalianand mosquito cells.Viral progeniesproduced from mammalian cells such as Huh-7 cells accumulate a higher number of mutationscomparedtoC6/36cells.21 _These_mutations_lead to an increase in the genotypic diversity of a viral pop-ulation, and this diversity has a direct relationship with variations in fitnessthat would facilitatethe transmission cycle.22,23

In this study, we evaluated the biological behavior and comparedthereplicativefitnessofColombianstrainsofthe fourDENVserotypesusingthreeclinicalisolatesfrom each serotype and the human hepatocyte cell line Huh-7 as a modelofinfection.Wefoundthatviralcharacteristicssuch as percentageofinfection,effectsoncell viability, and the pathogenicity index are dependent on the strain and not on the serotype, whilethe replicative fitnessis influenced bytheserotypeand hasaninternalheterogeneitybetween strainsthatischaracteristicofanaturaldenguepopulation. Fromtheanalysisofourresults,wewereabletodefineone group,DENV-1andDENV-4strains,withhigherreplicative fit-ness,andasecondgroup,DENV-2andDENV-3strains,with lowerreplicativefitness.Thisstudyconstitutesthefirstreport ofacomplete evaluationofthe replicativefitnessofDENV strainsofeachserotypeinColombia,ahyperendemic coun-try wherethe controland management ofthe disease are complicatedbythesimultaneouscirculationofthefourDENV serotypes.

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Materials

and

methods

Cellcultureandvirus

A.albopictus C6/36HT(HT-hightemperature)cells, immor-talizedhumanliver(Huh-7) cellsand babyhamster kidney ﬁbroblast(BHK-21)cellsweremaintainedinDulbecco´s Modi-ﬁedEagle´sMedium(DMEM,Sigma-Aldrich,St.Louis,MO,USA) andsupplementedwith10%fetalbovineserum(FetalBovine Serum(FBS),Gibco,Sigma-Aldrich,St.Louis,MO,USA).Huh-7 andBHK-21cellsweregrownat37◦Cand5%CO2.C6/36HT

cellswere grownat33◦C and5%CO2. Thevirus strainsof

DENV-1(DENV-1116,DENV-1S33,DENV-1S24),DENV-2 (DENV-2216,DENV-2S3,DENV-2209),DENV-3(DENV-3S7,DENV-3 S8,DENV-3S26)andDENV-4(DENV-4416,DENV-4S29, DENV-4 S32) were isolated from patients or healthy donors and propagatedinC6/36HTcellsaspreviouslydescribed.24 _The low-passagevirus stockswere inoculated inC6/36HT, har-vested3dayspost-infection,aliquotedandstoredat—80◦C untiluse. For this study, previouslystandardized viral iso-latesafter10passages(DENV-1OMS,DENV-2INS16,DENV-3 OMS,DENV-4INSstrains)wereincludedaspositivecontrols forplaqueassay.

Cellinfection

Twentyhourspriortoinfection,Huh-7cellswereseededin 48-wellplatesatadensityof2.5×104_cells_per_well._The_cells

wereinfectedatMOI0.5andincubatedfor1hat37◦Cina 5%CO2atmosphere.Then,theinoculumwasremoved,and

DMEMsupplementedwith10%fetalbovineserum(FBS,Gibco) wasadded.Thecellswerefurtherincubatedfor72hat37◦C in5%CO2andthesupernatantswerecollectedandstoredat

−80◦_C_until_use.

DetectionofviralantigeninHuh-7cellsinfectedwith

DENV

ThesusceptibilityofHuh-7cells toinfection byDENVwas evaluated by an immunoperoxidase assay by detection of viralproteinE.Brieﬂy,20hbeforeinfection,Huh-7cellswere seeded on SPL Life Sciences round coverslips at a density of2.5×104_per _well_and_infected_for₄₈_h_at_an_MOI₁_with

the viral strains DENV-1 116, DENV-2 216, DENV-3 S8 and DENV-4416.Cellswereﬁxedwith4%paraformaldehydefor 30min and then permeabilized with 0.3% Triton X-100 at room temperature. Inactivation of endogenous peroxidase wasperformedwith0.25%H2O2in50%methanol,and

non-speciﬁcbindingsiteswereblockedwith10%goatserum.The cells were incubated withthe DENV monoclonal 4G2 anti-bodyata1:800 dilutionforonehourat37◦C,washed and incubatedwithaperoxidase-coupledanti-mouseIgG.Finally, the reaction was developed with 0.02% H2O2 and 0.075%

diaminobenzidinein0.1MTris-HClbufferatpH7.2and pho-tographed forcell counting under an invertedAxiovert 40 microscope.

Evaluationofthepercentageofinfectedcellsbyﬂow

cytometry

Twentyhourspriortoinfection,1×104_Huh-7_cells_per_well

wereseededin96-wellmicroplatesandmaintainedinDMEM supplementedwith2%FBSand0.5g/mlgentamycin.Huh-7 cellmonolayerswereinfectedatMOI0.5,asmentionedabove for72h,andprocessedforcytometry.Brieﬂy,cellsweretreated withBDCytoﬁx/CytopermTM _Plus _{Fixation/Permeabilization}

Solution Kit with BD GolgiPlugTM _according _to _the

manu-facturer´sinstructionsandthenwashedandincubatedwith 4G2-FITC antibody (donated by the Functional Genetics of Infectious DiseasesUnit,InstitutePasteur, Paris)for30min at4◦C.Afterincubation, cells were washed oncewithPBS (pH7.2), resuspended, andthen analyzedusing aFACScan ﬂow cytometer(BD AccuriTM _C6). _{Mock-infected}_cells _were

usedasnegativecontrols.Atleast1000cellsweregatedby light scatter. Data analysis was performed with FlowJo 10 software.

Cellviability

Cell viability was assessed using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method.Seventy-twohoursafterinfection,Huh-7cell mono-layers were incubated with 100␮L MTT (0.5mg/ml) for1h at37◦Cina5%CO2atmosphereprotectedfromlight.After

incubation,theformazansaltsweredissolvedwithdimethyl sulfoxide for 10min. The absorbance measurements were madeinaTECANInﬁniteM200proreaderat450nm. Unin-fectedcellswereusedasanegativecontrol,andcellstreated with0.1%saponinforﬁveminuteswereusedasapositive control.

Estimationofthepathogenicityindex

Thepathogenicityindexwascalculatedastheratiobetween thecellviabilityvaluesandthepreviouslyreportedpercentage ofinfectionforeachstrain.Strainswithratios>1were classi-ﬁedascytopathic,andstrainswith<1valuewereclassiﬁedas pathogenic.

ReplicativeﬁtnessinHuh-7cells

Twentyhourspriortoinfection,2.5×104_Huh-7_cells_per_well

wereplatedina48-wellplateinmediumsupplementedwith 2%FBSandgentamycin.Then,thecellswere infectedwith eachvirusstrainatMOI0.5intriplicateintwoindependent experiments.Supernatantswereremoved72hafterinfection and stored at−80◦_C _for_subsequent_{quantiﬁcation} _of_viral

genomiccopiesbyRT-qPCRandviralinfectiousparticlesby plaqueformationassay.Themonolayerswereusedto deter-minecellviabilitybyMTT.

QuantiﬁcationofviralRNA

Viral RNAinthe supernatantswasisolated usingaStratec molecular kit (San Francisco, CA, USA). Viral RNA was reverse-transcribedfor60minat37◦CusingM-MLVreverse transcriptase (Promega, USA) and random primers. qPCR wasperformedusingtheDyNAmoHSSYBRGreenqPCRkit

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Fig.1–Reversetranscriptasepolymerasechainreaction (RT-PCR)withviralRNAfromdenguevirusclinicalisolates forgenotypingfragmentsofDENV-1S24,DENV-1116, DENV-S33,DENV-2S3,DENV-2216,DENV-2209,DENV-3 S7,DENV-3S8,DENV-3S26,DENV-4S29,DENV-4416, DENV-S32.Theampliﬁcationproductsoftheexpectedsize (775bp)wereresolvedin2%agarosagelstainedwith ethidiumbromide.

(Thermo, Massachusetts, USA) according to the manufac-turer´sinstructionsusingpreviouslydescribedprimers.25_The qPCRconditionswereasfollows:95◦Cfor15min,followedby 40cyclesof95◦Cfor15s,58◦Cfor1minand72◦Cfor1s, fol-lowedbyameltingcurveanalysis.Forabsolutequantiﬁcation, astandardcurvewasobtainedfromtenserialdilutionsofeach titratedserotype.Theviraltiterforeachstrainwaspreviously determinedbyplaqueassay.RNAfromeachdilutionwas iso-lated,andstandardcurveswithaminimumoffourdilutions weresetup.

Plaqueassay

BHK-21cellswereseededatadensityof1×105_cells/well_for

thetitrationofserotypes2and4and8×104_cells_for_serotypes

1and3perwellina24-wellplate.Thecellsuspensionwas infectedwithserialdilutionsofeachvirus.Fourhoursafterthe infection,DMEM2Xcontaining3%carboxy-methyl-cellulose (SigmaC4888-500G),7.5%sodiumbicarbonate(NaHCO3)and

supplementedwith6%FBS and0.5␮g/ml gentamycinwas added.NaHCO3 wasusedtoreach theoptimalpHforeach

serotype: DENV-1 pH 8.0, DENV-2 pH 7.0, DENV-3 pH 7.6, and DENV-4 pH 7.4. Theinfected cells were incubated for sevendayswithDENV-1andDENV-3,sixdayswithDENV-4 andﬁve dayswithDENV-2 at37◦C and5%CO2. Then,the

cellswere ﬁxedandstained with1%crystal violetsolution in70%methanolfor25min.Thecrystalvioletsolutionwas washed,andthevirustiterwascalculatedusingtheformula PFU/ml=(averagenumberofplaques×dilutionfactor)/ inocu-lumvolume(␮L).

DENVgenotyping

ForthedeterminationofthegenotypeofeachDENVstrain,a regionoftheEgeneofapproximately775bpwasampliﬁedby PCRusingthefollowingprogram:95◦Cfor3min,30cyclesof 95◦Cfor30s,53◦Cfor30s,72◦Cfor30sandﬁnalextension for5minat72◦Cusingpreviouslyreportedprimers.26_The_PCR productsweresequencedbyMacrogenInc.Theresultsofthe sequencingwereanalyzedusingthedatabase“DGV:Dengue GenographicViewer”27

Statistics

Thedataarepresentedasthemedianandinterquartilerange ormean±SDdependingonthedata.Thedatawereanalyzed byaKruskal-WallistestfollowedbyaDunn‘stestof multi-plecomparisonsoranANOVAtestandTukey’smultiplemean comparisonstestaccordingtothenormalityofthedata.All thedatawereanalyzedusingR3.3.0statisticalsoftware,and the graphicswere madeusingPrism7.0a. Statistical signiﬁ-cancewasdeﬁnedasp<0.05.

Results

ColombianDENVstrainsbelongtothegenotypes

previouslyreported

The775bpRT-PCRproductswereefficientlyamplifiedfrom viralRNAfromeachofthestrainsusedinthisstudy(Fig.1). Sequence analysisdeterminedthatall threeDENV-1strains wereclassifiedingenotypeV,theDENV-2strainsinthe Asian-American genotype,theDENV-3 strainsingenotype IIIand the three DENV-4 strains in genotype IIB (Table 1). These dataagreewiththemolecularcharacterizationofDENV pre-viouslyreportedforColombia28–31_and_demonstrate_that_the strainsstudiedforeachserotype belongtothesame geno-type, suggesting that the subsequent biological properties andreplicativefitnessanalysesareinfluencedbystrainand serotypecharacteristicsandnotbyvariationsbetween geno-typesaspreviouslyreported.32

Huh-7cellsaresusceptibletoinfectionbythefourDENV

serotypes

DENVinfectioninHuh-7cellswasconfirmedbythedetection oftheviralEantigeninanimmunocytochemistryassay.In cellsinfectedwiththefourDENVserotypes,thepresenceof theviralEproteinwithacytoplasmicdistributionandno syn-cytiaformationconfirmedthatHuh-7cellsaresusceptibleto DENVinfectionandrepresentacellculturesystemthat sup-portsviralreplication(Fig.2).Theproductionofinfectiousviral progenyafterinoculationdemonstratedthattheinfectionhad spreadthroughthecellmonolayer.Theresultsshowedthatin additiontotheimportanceofhepatocytesasatargetofDENV infectioninthepathophysiologyofthedisease,thesecellsare anadequatemodelforthestudyofthereplicativefitnessof DENV.

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Table1–Detailsofdenguevirusstrains.

Serotype Year Strain Clinicalclassiﬁcation Genotype

DENV-1

2011 S33N Denguewithwarningsigns V

2014 S24 Denguewithwarningsigns V

2016 116 Dengue V

DENV-2

2016 216 Dengue Asian-America

2009 209 Dengue Asian-America

2014 S3 Severedengue Asian-America

DENV-3

2014 S7 Denguewithoutwarningsigns III

DENV-4

2013 S32 Denguewithoutwarningsigns IIB

2016 416 Dengue IIB

2014 S29 Inapparentinfection IIB

ThepatientswerelocatedatCundinamarcadepartment,inthecentralregionofColombia,exceptforthestrainS32isolatedfromthePaciﬁc regionofColombia.

Fig.2–Huh-7cellsarepermissivetoDENVinfectionofthefourserotypes.TheHuh-7cellswereinfectedatanMOI1witha

strainofeachDENVserotype,andat48hpitheviralantigenEwasdetectedbyanimmunoperoxidaseassaywiththe4G-2

antibody.(A)Huh-7cellsnotinfected.(B)SpeciﬁcitycontrolwithoutprimaryantibodyonHuh-7cellsinfectedwithDENV-4

414.Huh-7cellsinfectedwithDENV-1116(C),DENV-2216(D),DENV-3S8(E)orwithDENV-4416(F).

ThepathogenicityandcytopathicityofColombianDENV strainsaredependentonthestrainandnotontheviral serotype

To determine the biological properties of DENV in human hepatocytes,Huh-7cellswereinfectedatanMOIof0.5,and at72hpostinfection(hpi),therateofcell-celltransmission ofthe virus,or pathogenicity, and the effect ofthe infec-tiononcellviability,orcytopathicity,wereevaluated.When thepercentagesofinfectionwerecompared,aheterogeneous

behavior was observedbetween strains ofthe same DENV serotype. TheDENV-1 strainsshowedinfectionpercentages between 27.8% and 38.7%, with no signiﬁcant differences betweenthestrains.Ontheotherhand,thestrainsbelonging totheserotypesDENV-2,DENV-3andDENV-4presented differ-enceswithinserotypes(p<0.05):theDENV-2strainsinfected between15.21%and51.6%,theDENV-3strainsbetween14.0% and46.3%,andinfectionwiththeDENV-4strainsreached per-centagesbetween25.7%and69.2%(Fig.3A,B).Asshownin

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Fig.3–DENVstrain-dependentinfectioncapacityandcytopathicity.TheHuh-7cellswereinfectedwithanMOI0.5ofDENV strains,andcellswereanalyzed72hpost-infection.(A)Infectionpercentagedeterminedtoindividualstrain.(B)Expression ofEantigeninHuh-7cellsinfectedwithDENVofthefourserotypes(twostrainsareshownforeachserotype)byFlow cytometryusingtheantibody4G2-FITC(C)CelldeathpercentageinducedbyDENVinfection.(D)Pathogenicindexforthe12 strainsofDENV.Twoindependentexperimentswiththreereplieswereplotted.AandChorizontallinesindicatemedianof IQR.Dhorizontallinesindicatemeans+/−SD.*p<0.05;**p<0.005;***p<0.0005.

(usingthe MTTassay)and foundthatDENV-1 and DENV-4 strainsinducedthehighestlevelsofcelldeath(24–60%and 39.29–69.02%,respectively). TheDENV-2strains causedless cell death withvalues between −1.7% and 29%, whilethe DENV-3strainsinduced15%and28%celldeath.Thevariability inthecapacitytoinducecelldeathbetweentheDENVstrains showsthattheviralserotypeisnottheonlydeterminingfactor ofthebiologicalcharacteristicsofcirculatingviruses.

Thepathogenicityindexsuggestsaheterogeneous

behaviorbetweenstrainsandDENVthatreﬂectsthe

variabilityfoundincirculatingDENVpopulations

Thepathogenicityindex(PI)wasdeterminedtodeepenthe analysisof howrepresentative the strains included inthis studyareofnaturallycirculatingpopulations.Theﬂow cytom-etryandcellviabilitytestvalueswereusedtocalculatethe indexforeachstrain.Weshowthateachserotypecontains strainswithdifferentPIvalues,ofwhichoneofthreestrains ofeachserotypehadacytopathicPI(PI>1)value,whilethe remainingstrains presenteda pathogenic PI (PI<1) values (Fig.3D).Theseﬁndingssuggestthatthestrainsbelongingto eachserotypeofDENVpresentbiologicalvariabilitythatcould bearepresentativesampleofnaturallycirculatingstrains.

Replicativeﬁtnesscharacteristicsaredeterminedbythe

serotypeandtheviralstrain

The twoexperimental approaches used forthe quantifica-tionofreplicativefitness,qPCRandplaqueassayallowedus todeterminetheinfluenceofbothserotypeandviralstrain factorsontheviralreplicativefitnessofDENVbelongingto thefourserotypes.AbsolutequantificationbyqPCRshowed thatthereplicativefitnesscouldbeinfluencedbytheserotype with some variability betweensome strains. The standard curves for absolute quantificationof replicative fitness for eachviralserotypeareshowninFig.4.Thehighest replica-tivefitnesswasachievedbyDENV-1andDENV-4strainswith values between 5.5 and 6.25 PFU/ml. In contrast, the low-est replicative fitness was recorded for the DENV-2 strains with 3.36–4.86PFU/ml range,followed byDENV-3 with val-uesbetween4.95and5.79PFU/ml(Fig.5A).Theevaluationof thereplicativefitnessbetweenstrainsofthesameserotype showed significant differences betweenthe strains belong-ingtoDENV-2andDENV-3.Forexample,thestrainsDENV-2 209andDENV-2S3hadmeanvaluesof4.78and3.36PFU/ml, respectively(p<0.05),whilethestrainsDENV-3S8andDENV-3 S26hadvaluesof4.95and5.79PFU/ml,respectively(p<0.05). ForserotypesDENV-1andDENV-4,nodifferenceswerefound betweenthestrains.Theseresultsindicatethatthe replica-tive fitnessofDENV as determined byqPCR dependsboth

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Fig.4–StandardcurvesofSYBRGreenRT-qPCRforthetitrationoffourdenguevirusserotypes.SerialdilutionofDENVwas madebeforeRNAextractionandampliﬁcationforeachserotype.(A)StandardcurveforDENV-1.Dilutionsfrom101_to₁₀6

PFU/mlweremadefromtheDENV-1116strain.(B)StandardcurvesforDENV-2.Dilutionsfrom102_to₁₀6_PFU/ml_were_made

fromtheDENV-1216strain.(C)StandardcurveforDENV-3.Dilutionsfrom101to105PFU/mlweremadefromtheDENV-1 S23strain.(D)StandardcurveforDENV-4.Dilutionsfrom103_to₁₀6_PFU/ml_were_made_from_the_DENV-4₄₁₆_strain._Mean

valuesofcyclethresholdscorrespondingatostandardplaqueformingunitsareplotted.Eachcorrelationcoefﬁcientand regressionequationwith95%CIareshown.

onstrain-speciﬁcbiologicalcharacteristicsandonthespeciﬁc characteristicsofeachserotype.

Interestingly, after evaluation of replicative fitness by plaque assay, we again found that fitness is affected by theserotypewithinternalvariabilitybetweenstrainsofthe sameserotype. Inaddition,wereporthere thatthereisan optimalmedia pHfor plaque formationfor each serotype. For example, DENV-1 developed plaques at pH 8.0, DENV-2at pH 6.9–7.0, DENV-3 at pH 7.6, and DENV-4 atpH 7.4, which could represent some specific characteristics of the viral cycle of each serotype (Fig. 5 C). Using the plaque assayapproach,wefoundthat DENV-1and DENV-4strains hadthe highestreplicativefitnesswith valuesbetween3.2 and 5.0 PFU/ml and 4.2–4.5 PFU/ml, respectively. In con-trast, DENV-2 and DENV-3 strains had the lowest values (between 3.3and 4.2 PFU/ml and 2.8–3.42 PFU/ml, respec-tively).Differencesinreplicativefitnessbetweenstrainswere only observed for DENV-1, with 5.1 PFU/ml calculated for strain116,and3.2PFU/mlforstrainS33,whichcorresponded to the upper and lower values of the range for the three strains.Althoughnosignificantdifferencesinreplicative fit-nesswere foundfortheDENV-2and DENV-3 strains(Fig. 5

B),therewasaninternalvariationbasedonqPCR quantifica-tion(Fig.5A).Theseresultsconfirmthatthereplicativefitness isinfluencedbytheviralserotype,althoughthetendencyto internalvariability betweenthe strains ofeach serotype is evident.

ThereplicativeﬁtnessaverageisdifferentbetweenDENV

serotypes

Thereplicativefitnessaverageofeachserotypewasdefined asthe fitnessaverageofthe threeDENVstrainsofeachof the fourserotypesthatwere includedinthestudy.DENV-1 and DENV-4serotypes hadthehighestfitnessvaluesat4.9 and5.2PFU/ml,respectively,buttheywerenotsignificantly different(p>0.05).Incontrast,DENV-2andDENV-3serotypes hadthelowestfitnessvalues,3.9and4.3PFU/ml,respectively, whichwerenotsignificantlydifferent(Fig.6AandB).In addi-tion,eachstrainhadadifferentfitnesspeakaverage,which demonstratestheheterogeneityandinternalplasticityofthe viralpopulationsthatbelongtoeachserotype.Weobserved thattheDENV-4strainshadalowerheterogeneitycompared totheotherthreeserotypes.Theabovedatashowthat replica-tivefitnessisinfluencedbytheviralserotype,althoughthere isaheterogeneouscompositionamongthepopulationsthat compriseeachtypeofDENV(Fig.6A).

Discussion

Thestudyofthereplicativeﬁtnessincirculatingviral popu-lationsand itscorrelationwiththesustainedprevalenceof allserotypesinColombiainadditiontotheirimpactonthe severe manifestations ofthe disease, isas relevant as the effortsdirectedtovectorcontrolandimprovementof

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epidemi-Fig.5–Replicativefitnesscharacteristicsaredeterminedbytheserotypeandtheviralstrain.Huh-7cellswereinfectedat anMOI0.5withanyoneoffourdenguevirusserotypes,andthecellswereanalyzed72hpost-infection.(A)Replicative fitnessquantificationbyqPCR.(B)Replicativefitnessquantificationbyplaqueassay.(C)PlaqueassaywithconditionsofpH overlaymediumandincubationdaysinaserotype-dependentmanner(Crystalvioletstain).Twoindependentexperiments withthreereplicateswhereplotted.HorizontallinesindicatemedianIQR.*p<0.05;**p<0.005;***p<0.0005.

Fig.6–TheaveragereplicativefitnessinHuh7cellsisdifferentialbetweendengueserotypes.(A)TheReplicativefitnessfor eachserotypewasestimatedusingthemeanvaluesofthereplicativefitnessobtainedbybothqPCRandplaqueassay.The meanvaluestothreestrainswiththreereplicatesareplotted.HorizontallinesindicatemeanandSD.*p<0.05;**p<0.005; ***p<0.0005.(B)FitnessLandscapedependsonthedengueserotype.Theheightofthepicsrepresentsthereplicativefitness foreachserotype(meanofreplicativefitnessbyqPCRandplaquesassay).Thevaluesshowedinthe“y,”and“x”axes correspondtothemeanofreplicativefitnessbyqPCRandPlaqueassay,respectively.

ologicalsurveillance.Ourresearchallowedustoestablisha collectionofDENVstrainsofeachofthefourserotypes iso-latedfromtheseraofpatientswithapparentandinapparent infection.ThecollectionhasalownumberofpassagesinA. albopictusmosquitocellsthatguaranteesaphenotype

simi-lartonaturally circulatingviralpopulations.Wefoundthat theinfectiouscapacityandthecytopathiceffectinducedby DENVaredependentontheviralstrainandarenotspeciﬁcally determinedbytheviralserotype,whichwasdemonstratedin theinvitroinfectionofHuh-7humanhepatocytes.We

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classi-fiedtheviralstrainsbythepathogenicityindexproposedin thisstudyandfoundbothcytopathicandpathogenicstrains withinthesameviralserotype.Additionally,ourreportisof greatscientificinterest because itshowed that the Colom-bianDENV strains havea differential replicativefitness by serotypeandheterogeneousstrain-dependentfitnesswithin eachserotype.ThiswasevidencedbytheserotypesDENV-1 andDENV-4displayingthehighestreplicativefitnessfollowed byDENV-2andDENV-3.

DENVreferencestrainsarewidelyusedforvirologicstudies andhavebeenculturedunderdifferentlaboratoryconditions, which results in the accumulation of genetic variations.33 Thesevariationsdonotfaithfullyreflectthebiological behav-iorofnaturalDENVpopulationsinColombia.On theother hand,theimmuneresponseandthehistoryofviralinfections ofthepatienthavebeendescribedasfactorsthatinfluence thepopulationdynamicsofDENV.Theanalysisofvariations inviralproteinsfrom patientswithsecondaryDENV infec-tionshoweduniquevariantsthatdifferfromthosefoundin patientswithaprimaryinfection.Thesevariationswerealso described inproteinE, oneofthe mostimmunogenic pro-teinstowhichtheimmuneresponsemediatedbyantibodiesis directed.34_In_vitro_tests_show_that_a_DENV_population_exposed toan änti-fusionloop äntibodytargetingtheEproteinresults inthe selectionof variants with mutationsin this region, whichallownewpopulationstoevadetheneutralizingeffect ofthisantibody,guaranteeingsubsistenceofthevirus.35

Evidence shows that populationimmunity exerts selec-tionpressureonDENV,whichexplainstheheterogeneityin replicative ﬁtness reported between strains from different regions.36_Colombia_is_a_country_with_exceptional epidemio-logicaldynamicsduetothesimultaneouscirculationofthe four serotypesofDENV, whichfavors successiveinfections withdifferentviralserotypesthroughoutanindividual’slife. ThisepidemiologicalbehaviorleadstoaDENVselectionthat resultsinviralpopulationsthatareuniqueandnot homolo-goustoDENVstrainsoriginatinginothercountries.Ourstudy providesrobustknowledgeontwelverepresentativestrainsof thefourDENVserotypesthatareveryusefulforthe investi-gationoflocalepidemiologicaldynamics.

Inthisstudy,weshowedthatthebiologicalcharacteristics, suchasinfectioncapacityandeffectofinfectiononcell viabil-ity,areinﬂuencedbythestrainandnotbytheviralserotype ofDENV.Previousstudieshavefoundbiological characteris-ticsofDENVthat weredependentonthe viralstrain, such as the ability to regulate the type I interferon response,37 aswell asdifferential NS1 proteinsecretion rates between strains ofthe same serotype.38 _Based _on _the _variable bio-logicalbehaviorbetweenviralstrains,weproposeanindex ofpathogenicity(PI),whichallowsclassiﬁcationoftheviral strainsbased on thephenotypic characteristics that repre-sent critical events in the viral cycle. ThePI of the DENV strainsinthisstudyshowedthattheDENV-2strainsarethe mostphenotypicallyheterogeneous,followedbytheDENV-4, DENV-3,andDENV-1 strains,whichmeans thatpathogenic andcytopathicstrainsexistwithineachserotype.The pheno-typicdiversityfoundamongthelocalDENVstrainsofthesame serotypedemonstratesthecomplexpicturethatwillhaveto beaddressedforthe study ofviral populationsinendemic regions.

Accordingtoourresults,theviralserotypeinfluencesthe replicativefitnessoftheColombianstrainsofthefourDENV serotypes.Previously,itwasshownthattheabilityoftheviral serotypetoformlyticplatesdependedonthepHofthe over-laymediumusedintheplatingassay.39 _The_four _serotypes usedinthisstudyexhibitedthesamepHdependencesince theDENV-1andDENV-3strainsneededbasicmediumoverlay, whiletheDENV-2andDENV-4strainsneededacidicmedium overlay for the optimal development of lytic plaques dur-inginfection.Thisfindingcouldberelatedtothedifferential translocation ofthe NS5 protein inresponse to the extra-cellularpHsincetheNS5proteinofDENV-2 predominantly localizestothenucleusatapHof6.0,whileatpH8.5,itis locatedinthecytoplasm.40_{Additionally,}_the_NS5_viral poly-merase of DENV-2 and DENV-3 predominantly localizes to the nucleus, whilethatofDENV-1 and DENV-4 islocalized to thecytoplasmdue tothe absenceofa nuclear localiza-tion sequences (NLS) inthe NS5 viral polymerase ofthese serotypes.41_Likewise,_the_subcellular_localization_of_the_NS5 proteininDENVserotypeshasbeenassociatedwith differ-encesintheproductionofviralprogenysincemutationsinthe NLSsequencesthatinhibitthetranslocationoftheproteinto thenucleuscauseareductioninviralprogenyproduction.42 ThecellularlocalizationoftheNS5proteindescribedabove could explain the differential replicative abilities observed in this study, in which we found that strains grouped by viral serotype:the DENV-1 andDENV-4 strainsare grouped togetherwiththehighestreplicativefitness,andtheDENV-2 andDENV-3strainsaregroupedtogetherbyalowerreplicative fitness.

Theinfluenceofthe viralserotype onreplicativefitness could alsobeexplainedbydifferencesintheinteractionof theviruswithcellularreceptorsorthetraffickingandrelease ofviralprogeny.Thedifferentialuseofreceptorsamongthe fourDENVserotypeshasbeenpreviouslyreported.The inter-action between the viral bindingprotein (viral attachment protein)andtheexpressionlevelsofreceptorsinthecell mem-braneonthesurfaceofthetargetcellaffecttheentryofviral serotypes.Forexample,polyclonalantibodiesagainstlaminin decreasedDENV-1production,whereastheviralprogenyof DENV-2,DENV-3,andDENV-4wasnotaffected,whichsuggests adifferentialinteractionbetweenviralserotypesandcellular receptors.43_Regarding_the_assembly_of_the_{intracellular}_virus, the interactionoftheN-terminusofthepreMproteinwith thereceptorsKDELR1andKDELR-2hasbeenreported,which iscrucialforthetransportofserotypesDENV-1,-2,and-3but notfortheassemblyofDENV-4.Thus,theinhibitionofKDEL receptorexpressionblocksthe releaseofthevirusesofthe firstserotypes,whichleadstotheaccumulationofviral parti-clesanddeteriorationofthematurationprocess.44_The_use_of morethanonereceptorortheavailabilityofreceptorsincell linesusedintheviralinfectionexperimentscouldinfluence theefficiencyofthematurationandexportofviralprogeny, affectingthereplicativeviralfitnessoftheDENVserotypes.In thiswork,wefounddifferencesbetweenserotypesinterms oftheabilitytoproduceviralprogeny,anditisprobablethat DENV serotypes haveevolved touse different components ofthecellularmachineryintheprocessofviralreplication. Inconclusion,theevidenceshowsthattheDENVserotypes could haveadifferentialreplicativefitness, whichcouldbe

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linkedtotheexpressionofreceptors,aswellastothecellular localizationofviralproteins.

Replicativefitnessisoneofseveral factorsthattogether could explain the hyperendemicity of DENV in Colombia. First, it is possible that the variability in replicative fit-nesswithinviral populations ofthe same serotype results ingreater evolutionaryplasticity, which allows the coexis-tenceofstrainswithvariablereplicativefitnessofthesame serotypeindifferentgeographicalregions.Thissuggeststhat thevariabilitybetweenstrainsdeterminestheprevalenceof one serotype by region. Second, genetic flow events from endemiccountriessuchasVenezuela,45 whichincreasethe geneticandphenotypicvariabilityofpopulations,would pre-venttheestablishmentofoneserotype.Thethirdimportant factor is the high probability of secondary dengue infec-tionscausedbytheantibody-dependentenhancement(ADE) mechanism,whichwouldallowastrainwithaloworhigh replicativefitnesstopersistinthepopulationwithoutdirect competition.46 _Finally, _since _it _has_been _reported _that _84% ofdengueinfections inendemiccountries aremono infec-tions and there are coinfections at a lower percentage,47 theprobabilityofcompetitionbetweenstrainsduring natu-ralinfection islow. Thecombinationofthesefactorscould explainthepresenceofmultiplepeaksofreplicativefitness inpopulationsthatcompriseaviralserotypeand that per-sistdynamicallyovertimeandinendemiccountriessuchas Colombia.

Wereportadifferentialreplicative fitnessbetweenlocal serotypescirculatinginourcountry.Basedonourresults,it wouldbenecessarytoevaluatehowtheserotypeinfluences the immune response induced by DENV. For this evalua-tion,the use ofestablishedand characterizedlocalstrains and an optimal cell culture system that is permissive to infection are critical.This system would allowtracing the kineticsofexpressionandproductionofmultiple inflamma-torycytokines.Additionally,itcouldbepossibletostudythe correlationbetweentheinfectiousviralserotypeandthe clin-icaloutcomeofdengue.

The DENV serotypes most frequently associated with severeclinicalsymptomsinSouthAmericaareDENV-2and DENV-3,followedbyDENV-1andDENV-4.15,16_Since_we_found a high replicative fitness forDENV-1 and -4, it is possible thatthese serotypesdonotinduce anexacerbated inflam-matory response and thus maintain an interaction with the host cell that favors the production of viral progeny and leads to a highly efficient viral replication. In con-trast, strains belonging to serotypes DENV-2 and DENV-3 with lower replicative fitness could induce an aggressive inflammatory response, which could diminish the replica-tivefitnessofthevirus and atthe same timeincreasethe probabilityofasevereclinicaloutcomedirectedbyan out-of-controlimmuneresponse,previouslydescribedasacytokine storm.48

The collection of strains of the four DENV serotypes establishedand characterizedinthisstudy maybeusedin futureresearchonthepathogenesisofseveremanifestations of DENV infection in humans and the role of strains and serotypes in the establishment of circulating serotypes in endemicareas.

Conclusions

The differential replicative fitness between serotypes observed in Colombian DENV strains isolated and char-acterized genotypically and phenotypically in this study reflects the heterogeneityoflocally circulating populations and contributestotheunderstandingofthecomplex phys-iopathologyofthediseaseininfectionscausedbythedifferent serotypes. However, we found that the infectious capacity and thecytopathiceffectaredependentontheviralstrain, which demonstratesthat somebiological characteristicsof the viruses are not shared withinthe same serotype and could contribute, together with specific characteristics of the host, to the severity of the clinical outcome. The cell culture model ofhumanhepatocytes Huh-7is optimalfor viralreplicationinfectionstudies becauseitallowsefficient productionoftheviralprogenybythefourDENVserotypes. The virus collection ofstrains of the four DENV serotypes establishedand characterizedinthis study can beusedin futureresearchonthepathogenesisofseveremanifestations ofDENVinfectioninhumans.

Conﬂicts

of

interest

Thismanuscripthasnotbeenpublishedandisnotunder con-siderationforpublicationelsewhere.Wehavenoconﬂictsof interesttodisclose.ThisstudywasfundedbytheVicerrectoría de Investigaciones-UniversidadElBosque(Project PCI-2015-8327).

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