Phylogenetic analysis of the emergence of main hepatitis C virus subtypes in São Paulo, Brazil

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ww 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

Phylogenetic

analysis

of

the

emergence

of

main

hepatitis

C

virus

subtypes

in

São

Paulo,

Brazil

Anna

Shoko

Nishiya

a,b,∗

_,

_César

_de

_Almeida-Neto

a,c

_,

_Camila

_Malta

_Romano

d

_,

Cecília

Salete

Alencar

b,e

_,

_Suzete

_Cleusa

_Ferreira

a,b

_,

_Claudia

_{Di-Lorenzo-Oliveira}

f

_,

José

Eduardo

Levi

a

_,

_Nanci

_Alves

_Salles

a

_,

_Alfredo

_{Mendrone-Junior}

a

_,

Ester

Cerdeira

Sabino

b,g

a_Fundac¸ão_{Pró-Sangue/Hemocentro}_de_São_Paulo,_São_Paulo,_SP,_Brazil

b_Infectious_Diseases_Division_(DIPA),_Universidade_Federal_de_São_Paulo_(UNIFESP),_São_Paulo,_SP,_Brazil c_Discipline_of_Medical_Science,_Faculdade_de_Medicina,_Universidade_de_São_Paulo_(USP),_São_Paulo,_SP,_Brazil d_Laboratory_of_Virology,_Department_of_Infectious_and_Parasitic_Diseases,_Instituto_de_Medicina_Tropical_de_São_Paulo, FaculdadedeMedicina,UniversidadedeSãoPaulo(USP),SãoPaulo,SP,Brazil

e_Lim₀₃_Medical_Research_Laboratory,_Hospital_das_Clínicas,_Faculdade_de_Medicina,_Universidade_de_São_Paulo_(USP), SãoPaulo,SP,Brazil

f_Universidade_São_João_Del_Rei,_São_João_Del_Rei,_MG,_Brazil

g_Department_of_Infectious_Disease,_Faculdade_de_Medicina,_Universidade_de_São_Paulo_(USP),_São_Paulo,_SP,_Brazil

a

r

t

i

c

l

e

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f

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Articlehistory:

Received6January2015

Accepted14June2015

Availableonline19August2015

Keywords: Growthrate HCV Phylogeneticanalysis Subtypes

a

b

s

t

r

a

c

t

Background:ItisrecognizedthathepatitisCvirussubtypes(1a,1b,2a,2b,2cand3a)

origi-natedinAfricaandAsiaandspreadworldwideexponentiallyduringtheSecondWorldWar

(1940)throughthetransfusionofcontaminatedbloodproducts,invasivemedicalanddental

procedures,andintravenousdruguse.TheentryofhepatitisCvirussubtypesintodifferent

regionsoccurredatdistincttimes,presentingexponentialgrowthratesoflargerorsmaller

spread.Ourstudyestimatedthegrowthandspreadofthemostprevalentsubtypescurrently

circulatinginSãoPaulo.

Methods:Atotalof465non-structuralregion5BsequencesofhepatitisCviruscoveringa

14-yeartime-spanwereusedtoreconstructthepopulationhistoryandestimatethepopulation

dynamicsandTimetoMostRecentCommonAncestorofgenotypesusingtheBayesian

MarkovChainMonteCarloapproachimplementedinBEAST(Bayesianevolutionaryanalysis

bysamplingtreesoftware/program).

Results:EvolutionaryanalysisdemonstratedthatthedifferenthepatitisCvirussubtypeshad

distinctgrowthpatterns.TheintroductionofhepatitisCvirus-1aand-3awereestimated

tobecirca1979and1967,respectively,whereashepatitisCvirus-1bappearstohaveamore

anciententry,circa1923.HepatitisCvirus-1bphylogeniessuggestthatdifferentlineages

circulateinSãoPaulo,andfourwell-supportedgroups(i.e.,G1,G2,G3andG4)wereidentiﬁed.

HepatitisCvirus-1apresentedthehighestgrowthrate(r=0.4),butitsspreadbecameless

markedafterthe2000s.HepatitisCvirus-3agrewexponentiallyuntilthe1990sandhadan

∗ _{Corresponding}_author_at:_Departamento_de_Biologia_Molecular,_Fundac¸ão_{Pró-Sangue/Hemocentro}_de_São_Paulo,_Av._Dr._Enéas_Carvalho

deAguiar155,primeiroandar,05403-000,SãoPaulo,SP,Brazil.

E-mailaddress:anishiya@hotmail.com(A.S.Nishiya).

http://dx.doi.org/10.1016/j.bjid.2015.06.010

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intermediategrowthrate(r=0.32).Anevidentexponentialgrowth(r=0.26)wasfoundfor

hepatitisCvirus-1bbetween1980andthemid-1990s.

Conclusions: Afteraninitialperiodofexponentialgrowth,theexpansionofthethreemain

subtypesbegantodecrease.HepatitisCvirus-1bpresentedinﬂatedgeneticdiversity,and

itstransmissionmayhavebeensustainedbydifferentgenerationsandtransmissionroutes

otherthanbloodtransfusion.HepatitisCvirus-1aand-3ashowednogroupstratiﬁcation,

mostlikelyduetotheirrecententry.

Introduction

HepatitisCvirus(HCV)wasidentiﬁedbyChooetal.in19891

and is currently amajor cause of chronic hepatitisin the

world,reaching150millioncarriers(2–3%oftheworld

pop-ulation),withapproximately3–4millionnewinfectionsand

350,000deathsannually.2_HCV_is_thought_to_have_originated

inWestAfricaorSouthernAsia,regionswhereendemic

geno-typesremainedforhundredsofyears(between500and1000

years),withrelativelylowtransmissionrates,mainlyvia

inef-ﬁcientroutes,suchassexualorverticaltransmission,andby

practicessuchascircumcision,excision,andscariﬁcation.3–8

Representatives from these endemicgenotypes show high

geneticvariationamongstrainsand arefoundinrestricted

geographicregions, including genotypes1and 4inCentral

Africa,2inWestAfrica,5and7inCentral/SouthernAfrica,

and3and6ontheIndiansub-continentandinSouthandEast

Asia.6,7,9–12_Some_subtypes_of_endemic_regions_have_spread_to

differentregionsandexpandedglobally.Theepidemic

sub-types(1a,1b,2a,2b,2c,and 3a) thenspread exponentially

duringandaftertheSecondWorldWar(1940)whentherewas

anincreaseofveryefﬁcientnewwaysoftransmission,such

asthetransfusionofcontaminatedbloodandbloodproducts,

invasivemedicalanddentalprocedures,andalsointravenous

drug use (IVDU).13–15 _In _addition _to_their _wide _distribution

intheworld,thesesubtypesarecharacterizedbytheirhigh

prevalenceandlowgeneticvariation.3,16,17

TheriskofHCV transmissionviablood transfusionwas

strikinglydecreasedaftertheintroductionofanti-HCV

sero-logic screening tests for blood donors. Additionally, the

introductionofnucleicacidtesting(NAT)forHCVscreening

furtherreducedtheriskoftransfusion-transmittedHCV.18,19

Incontrast,theuseofintravenousdrugsremainsoneofthe

majorriskfactorsforHCVinfection,20_and_sexual

transmis-sion,whichgenerallyshowedloworinefﬁcienttransmission

inthegeneralpopulationhasalsobeenassociatedwith

prac-ticesthatleadtomucosaltraumaandpresenceofulcerative

genitaldiseases.21,22

InBrazil,theprevalenceofHCVamongdrugusers may

reach36%.23–25 _In_the_HIV_co-infected_population, _it_ranges

between 18% and 31%.26,27 _In _the _general _population, _the

prevalence is approximately 1.5%28–30 _and _may _vary _from

0.19%to1.2%inblooddonors.31–36

InSão PauloState (Brazil),a predominance ofepidemic

subtypesisobserved.HCV-1acorrespondsto32%–34.4%ofall

infectedsubjects,whileHCV-1bisdetectedin36.2%–45.5%,

andHCV-3ain18%–24.2%.Othersgenotypessuchas4and5

arerarelydetected.37–39 TheentryofthedifferentHCV

sub-typesinto SãoPauloappearstohaveoccurred ondifferent

dates, andeach subtypeappearstohavegrown atdistinct

rates.38,40,41_In_this_study,_we_sought_to_estimate_the_growth

andspreadofthemostprevalentsubtypesandtoinvestigate

thecurrentsituationofthesesubtypesinSãoPaulo.

Materials

and

methods

Studypopulation

Samplesfromtwodifferentstudieswereanalyzed,

compris-ingaperiodof14years(1997–2011):580partialsequencesof

thenon-structuralregion5B(NS5B)(IDsGQ490493–GQ491027)

ofpatientsfromSãoPauloState38_and₁₇₀_partial_sequences_of

NS5BfromblooddonorsofFundac¸ãoPró-Sangue/Hemocentro

de São Paulo (IDs KF523955–KF524152).37 _The _ﬁrst _study

included patients from four different cities of São Paulo

State (Ribeirão Preto, São José do Rio Preto, São Bernardo

doCampo,andfromtworeferencecentersforHCV

surveil-lance and treatment inSão Paulo city) thatwere collected

between 1997and 2006(IDsGQ490493–GQ491027). The

fre-quencyofmajorriskfactorswere 36%ofblood transfusion

history, 18%injectingdrugusers,and 32%thatdidnot

dis-closeanyriskfactor.38 _The_second_study _was_conducted_in

blood donorsscreenedfrom September2007toJuly2011in

SãoPaulocityandalthoughconsideredtobeapopulationwith

lowriskbehavior11%reportedbloodtransfusion,6%

inject-ing druguse, and 20%norisk factorsassociatedwithHCV

infection.37 _The_NS5B_sequences_were_aligned_using_Clustal

Xandgenotypedbymaximumlikelihoodanalysisusingthe

GARLiprogram(GeneticAlgorithmforRapidLikelihood

Infer-ence).Fromthis,threedistinctdatasetscontainingonlySão

Paulosequenceswerebuilt,HCV-1a(n=98),HCV-1b(n=218)

andHCV-3a(n=149),toinvestigatethepopulationhistoryof

thesamples.Othersequenceswereremovedfromthestudy.

Additionally,adatasetcontaining70non-structuralregion3

(NS3)sequences(708nucleotideslong)generatedinthesame

studyfromHCV-1b-infectedblooddonorswasconstructedfor

furtheranalysis(IDsKF524133–KF524257).

HCVphylodynamics

ThepopulationdynamicsandTMRCA(TimetoMostRecent

Common Ancestor) for the three genotypes were

investi-gatedusingtheBayesianMarkovChainMonteCarlo(MCMC)

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constrainedBayesianskyride(BSK)coalescent wasused as

atreepriorunder arelaxed (uncorrelated)molecularclock

withthe best model ofnucleotide substitution (GTR+G+I)

estimatedinMODELTEST.43_The_substitution_rate_was_set_as

previouslyestimatedforeach genotype.38 _MCMC_runs

con-sisting of 50 million generations (with 10% burn-in) were

undertaken toobtain parameter convergence. Amaximum

cladecredibility(MCC)treewasobtainedbysummarizingthe

50,000treeswithbranchlengthsofnucleotidesubstitution

(after excluding 10% of the burn-in) using Tree Annotator

v.1.7.2.42 _The _phylogenetic _trees _were _visualized _in_FigTree

v.1.2.2(availableat:http://tree.bio.ed.ac.uk/software/ﬁgtree).

Wealsoaimedtoestimatetherateofpopulationgrowth(r)

underthedemographicmodel(exponentialandlogistic

pop-ulationgrowth)thatbestﬁteachsubtypedataset,withmodel

comparisonsundertakenusingBayesFactorcomparison.Inall

cases,theconvergenceofparametersduringtheMCMCruns

wasinspectedwithTracerv.1.4,42_with_{uncertainties}_depicted

as95%highestprobability(HPD)intervals.

Potentialriskfactors involvedinHCVtransmissionsuch

asahistoryofbloodtransfusion,tattooing,intravenousdrug

use,occupationalexposure,and sexualbehaviorwere

ana-lyzedinadditiontodemographicssuchasage,sex,race,and

education.

Statisticalanalysis

Thecharacteristicswereevaluatedaccordingtothesubgroups

belongingtosubtype1bofblooddonors.TocomparetheG1-G4

subgroupsaccordingtosomedemographiccharacteristicsand

relatedriskbehaviors,weusedchi-squaredtestofsigniﬁcance

levelof5%.

Results

WeanalyzedpartialsequencesoftheNS5Bgenebelonging

tothemostprevalentsubtypesinourpopulation,subtypes

1a,1band3a.Aftergenotypingusingworldwidesequences

asreferences,weexcludedalltaxathatclusteredwith

sam-plesfromoutsideBrazil.Thiswasperformedtoreducethe

effectofanyphylogeographicstructureonthefurther

analy-sisofthetransmissiondynamics.Byincludingonly‘Brazilian’

sequences,theevolutionaryanalysisdemonstratedthatthe

differentsubtypeshavedistinctgrowthpatterns,as

demon-stratedbythetreeshapes,andalsoconﬁrmedtheprevious

observationthattheywereintroducedintoSãoPauloat

dif-ferenttimes.TheTMRCAsforHCV-1aand-3awereestimated

tobecirca1979and1967,respectively,andHCV-1bappeared

tohaveamoreanciententry,circa1923,whichisverysimilar

tothepreviousestimate38₍_Table₁_).

DifferentfromwhattheHCV-1aphylogenysuggests,the

HCV-3aand HCV-1bphylogeniesindicate thatdifferent

lin-eages circulated in São Paulo (Fig. 1). However, although

no support was observed for HCV-3a lineages, four

well-supportedgroups,namedGroups1–4(i.e.,G1,G2,G3andG4)

wereidentiﬁedforHCV-1b(Fig.1).Toverifytheconsistency

oftheHCV-1bsubgroups,we investigatedthe phylogenetic

pattern ofthis subtypeusing the NS3 sequences obtained

fromthesamesamplesgeneratedinthisstudy.Similartothe

Table1–HCVsubtypesandsubgroupTimetoMost RecentCommonAncestor(TMRCA)andrateof populationgrowth.

Subtype Samples(n) TMRCA(upperandlower) Growth rate(r) 1a 98 1979(1967–1987) 0.4 3a 149 1967(1955–1980) 0.32 1ball 218 1923(1844–1967) 0.26 1bG1 79 1966(1947–1981) 0.3 1bG2 90 1972(1959–1983) 0.4 1bG3 6 1983(1975–1991) – 1bG4 36 1973(1955–1985) 0.26

TMRCA,TimetoMostRecentCommonAncestor;r,rateof popula-tiongrowth.

ThegrowthratewasnotestimatedforG3becauseitincludesonly sixsequences.

structureobtainedfortheNS5gene,NS3alsodepictedfour well-supportedgroups(Fig.1).

Becausetheobservedgroupswereveryconsistentinboth

phylogenies(NS5BandNS3), wealsoestimatedthe timeof

theiremergence.Assumingasapriorthesameevolutionary

rateforallsubgroupsandusingtheNS5dataset,wefound

that the subgroupsdidnotdiffersigniﬁcantly accordingto

theiremergenceinSãoPaulo(becauseupperandlower

val-uesoverlappedeachother).Nevertheless,themedianvalues

suggestedthatGroup1wastheﬁrsttospreadinthis

popula-tion(late1960s),followedbyGroups2and4,whichemerged

atthesametimearoundthebeginningofthe1970s(Table1).

These subgroups(G1–G4) were notassociatedwith gender,

age,ethnicity,oreducationlevel,andtherewasnoassociation

betweenthesubgroupsandthedifferentexposurecategories,

exceptforG2,whichhadenteredin1972andwasassociated

withahistoryofbloodandbloodproducttransfusionin62.5%

(5/8;p=0.043)ofthecarriers.

WealsoestimatedthegrowthrateforHCV-1a,-1b(totaland

subgroups)and-3ausingBEAST(Bayesianevolutionary

anal-ysisbysamplingtreesoftware/program)basedonthebest-ﬁt

demographic modelforeach NS5 dataset.Theexponential

growth modelundertheexponentialrelaxed(uncorrelated)

molecularclockwasthebestforalldatasetsaccordingtoBayes

Factor.HCV-1apresentedthehighestgrowthrateamongthe

subtypes(r=0.4),butaccordinglytotheskyrideplot(Fig.1),the

spreadhasbecomelessmarkedafterthe2000s.HCV-3a,which

islikelytohaveenteredSãoPaulosomeyearsbeforeHCV-1a,

grewexponentiallyuntil1990andhadanintermediategrowth

rate (0.32)in comparisontoHCV-1a and -1b (Table 1).The

HCV-1bskyrideclearlyshowedanevidentexponentialgrowth

between1980andthemid-1990s,asalreadydemonstratedby

others.15,38

Discussion

HCV-3aand-1bdisplaymultiplelineagesinSãoPaulo,

indi-cating the simultaneous dissemination ofmultiple strains.

However,onlytheHCV-1blineagesarewellsupported.

HCV-1bshowedfourdistinctsubgroups,hereinnamedG1toG4,in

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A

C

HCV-1a 44-9 ya 39.2 ya 27.8 ya 38.2 ya G4 G1 G2 G3 HCV-1a HCV-3a HCV-1b HCV-1b HCV-3a

B

D

Fig.1–(A)HCV-1aand(B)HCV-3aMCCtreesshowingtheclassicalstar-shapedphylogenies.(C)HCV-1bMCCtreeshowing

fourwell-supportedgroupsnamedGroups1–4.(D)SkyrideplotshowingthepopulationdynamicsandTMRCAfortheNS5B

HCVregionofsubtypes1a,-1band-3a.MCC,maximumcladecredibility;TMRCA,timetomostrecentcommonancestor.

subgroupsofsubtype1b.Interestingly,Lampeetal.41 _found

thatthoselineageswereassociatedwithgeographicallocation

(westernandsoutheastregionsofBrazil),andoursequences

obtainedfrom SãoPauloalsoreﬂectsuchgeneticisolation.

Althoughnoevidentassociationbetweenclinicalor

epidemi-ologicalrelationshipsforsuchgroupswasfound,wespeculate

thattheselineagescirculatedwithinparticulartransmission

groupsrestrictedbyfactorsyettobedetermined.Therewas

alsonoassociation betweensubgroup1band age,and the

mostlikelyhypothesisisthatthecirculationoftheselineages

occurredoveralongperiodfromthebeginningofthe20th

cen-tury(oldestsubtype),whichcouldhaveresultedina“dilution”

inthetransmissionofthissubtypefordifferentriskgroups

anddifferentagegroups.UnlikeHCV-1a,thelatestentryin

ourpopulationshowedasimilartransmissionwithingroups

thatsharethesamehabitsandthesameagegroup.38_The_data

presentedinthisworkappeartoberobustbecausetheyare

consistentwithpreviousestimatesthatusedsequences

com-prisingashortertimespan(nineyears).Thelargernumberof

sequencesaswellthetimespan(14years)didnotalterthe

growthratesorcoalescenttimesofthemainHCVsubtypesin

SãoPaulo.

In fact, the ﬁndings for HCV-1b (especially the periods

ofexponential growth and decrease) coincidewith

histori-cal events such as the foundation of the ﬁrst blood bank

andimprovementsinbloodtransfusionservices,1940–1960,44

and thesimultaneous increaseinmedicalproceduressuch

ashemodialysisin1960.45 _Conversely,_measures_to_prevent

transmissibleagentsinblood,suchashepatitisBvirusand

human immunodeﬁciency virus, inthe late 1980s and the

obligatoryserologicalscreeninginbloodbanksforHCVin1993

contributedtothereductionandstabilizationoftheepidemic

after1990.Theobservedpatternofexpansionisverysimilar

tothatfoundbyMagiorkinisetal.15_in_the_USA,_where_subtype

1bshowedaTMRCAof1922andanexponentialincrease.After

theSecondWorldWar,transfusionsandinvasivemedical

pro-cedures as well as therapeuticinjections were widespread

untilthe1980s.Atthistime,anincreasedawarenessof

par-enteralriskandtheselectionandscreeningofblooddonorsfor

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theriskofHCVtransmission,evenbeforetheimplementation

oftheanti-HCVtestin1990.19

Althoughintroducedmorerecentlythanthesubtype-1b,

HCV-3aspreadexponentiallyuntil1990;theworldwide

sub-type3aepidemicmostlikelybeganinthemid-20thcentury,

hadacommonoriginandquicklyspreadgloballyamongdrug

users.46,47_Some_studies_have_associated_genotype₃_with_IVDU

andalsowiththeuseofstimulantmedications(IVnon-illicit

drugssuchasGluconergan) inthe 1970s.48_The_association

withthese groups would mostlikely explainthe high

fre-quencyofthissubtypeintheoldergroup(>40years)inthis

population.37_The_decline_in_needle_sharing_and_the_frequency

ofinjectionsandincreasingeducationalmeasuresmighthave

contributedtoreducingthisriskofparenteralexposureinthe

early1990s.49

HCV-1a was found to be the most recently introduced

subtypeinourregion andshowed thehighestgrowth rate

comparedwithother subtypes.Unlikesubtype1bthathad

adeclineofthespreadaftermandatoryserologicalscreening

forHBV,HIV,andHCVattheendofthe1980sinBrazilianblood

banks,subtype1ashowedanincreaseinthespreadas

demon-stratedinexponentialexpansioncurve(inSkyrideplot)inthe

mid-1990s,whichcontinueduntil2005.So,themainrouteof

HCV-1atransmissionisprobablynotbloodtransfusion,but

UDIV.

Someauthors haveassociated subtype1awith the use

ofintravenousdrugs,15,40,48,49_and_even_young_individuals_or

short-timedrugusers(recent)couldbemoreassociatedwith

needlesharingandcontributingtothespreadofthissubtype

1ainthisagegroup.24,50_This_is_in_accordance_with_an_increase

inthefrequencyofsubtype1aamongyoungerdonors(<30

years).37

Ourdataclearlyshowthatafteraninitialperiodof

expo-nentialgrowth, the expansion ofthe threemain subtypes

begantodecreaseasaconsequenceoftheintroductionof

anti-HCVscreeningtestsandpoliticalandeducationalmeasures

avoidingneedlesharingamongIVDUs.Thedelayobservedin

BSKforHCV-1bdeclineappearstobetheconsequenceoftwo

possibleandnon-exclusivehypotheses:(i)theinﬂatedgenetic

diversityinthetreecausedbytheconcomitantcirculationof

atleastfourmonophyleticstrains,and(ii)asHCV-1bisthe

mostancientandmostprevalentsubtypeinSãoPaulo,where

itwasﬁrstspreadbytransfusionrouteanditstransmission

wassustainedbydifferentgenerations throughroutesthat

allowedspreadof4lineages(G1–G4)forminggroupsrestricted

byfactorsyettobedetermined.HCV-1aand-3ashowedlow

geneticvariation,inaccordancewiththeirrecententry.

Conﬂict

of

interest

Theauthorsdeclarenoconﬂictsofinterest.

Acknowledgements

This study was supported by CAPES (Coordenac¸ão

de Aperfeic¸oamento de Pessoal de Nível Superior) of

Brazilian Ministry of Education and by Fundac¸ão

Pró-Sangue/HemocentrodeSãoPaulo.

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s

1.ChooQL,KuoG,WeinerAJ,OverbyLR,BradleyDW,Houghton M.IsolationofacDNAclonederivedfromablood-borne non-A,non-Bviralhepatitisgenome.Science.

1989;244:359–62.

2.WHO.HepatitisC.N◦164Fs,ed,vol.2014;2012.Available:

http://www.who.int/mediacentre/factsheets/fs164/en/

[accessedJuly2014].

3.PybusOG,CharlestonMA,GuptaS,RambautA,HolmesEC, HarveyPH.TheepidemicbehaviorofthehepatitisCvirus. Science.2001;292:2323–5.

4.ShepardCW,FinelliL,AlterMJ.Globalepidemiologyof hepatitisCvirusinfection.LancetInfectDis.2005;5:558–67.

5.SimmondsP.GeneticdiversityandevolutionofhepatitisC virus–15yearson.JGenVirol.2004;85Pt11:3173–88.

6.SimmondsP,BukhJ,CombetC,etal.Consensusproposalsfor auniﬁedsystemofnomenclatureofhepatitisCvirus genotypes.Hepatology.2005;42:962–73.

7.SimmondsP.TheoriginofhepatitisCvirus.CurrTop MicrobiolImmunol.2013;369:1–15.

8.SmithDB,SimmondsP.Review:molecularepidemiologyof hepatitisCvirus.JGastroenterolHepatol.1997;12:522–7.

9.MellorJ,HolmesEC,JarvisLM,YapPL,SimmondsP. InvestigationofthepatternofhepatitisCvirussequence diversityindifferentgeographicalregions:implicationsfor virusclassiﬁcation.TheInternationalHCVCollaborative StudyGroup.JGenVirol.1995;76Pt10:2493–507.

10.JeannelD,FretzC,TraoreY,etal.Evidenceforhighgenetic diversityandlong-termendemicityofhepatitisCvirus genotypes1and2inWestAfrica.JMedVirol.1998;55:92–7.

11.CandottiD,TempleJ,SarkodieF,AllainJP.Frequentrecovery andbroadgenotype2diversitycharacterizehepatitisCvirus infectioninGhana,WestAfrica.JVirol.2003;77:7914–23.

12.NdjomouJ,PybusOG,MatzB.Phylogeneticanalysisof hepatitisCvirusisolatesindicatesauniquepatternof endemicinfectioninCameroon.JGenVirol.2003;84Pt 9:2333–41.

13.PybusOG,MarkovPV,WuA,TatemAJ.Investigatingthe endemictransmissionofthehepatitisCvirus.IntJParasitol. 2007;37:839–49.

14.HauriAM,ArmstrongGL,HutinYJ.Theglobalburdenof diseaseattributabletocontaminatedinjectionsgivenin healthcaresettings.IntJSTDAIDS.2004;15:7–16.

15.MagiorkinisG,MagiorkinisE,ParaskevisD,etal.Theglobal spreadofhepatitisCvirus1aand1b:aphylodynamicand phylogeographicanalysis.PLoSMed.2009;6:e1000198.

16.SimmondsP,McOmishF,YapPL,etal.Sequencevariabilityin the5non-codingregionofhepatitisCvirus:identiﬁcationof anewvirustypeandrestrictionsonsequencediversity.JGen Virol.1993;74Pt4:661–8.

17.WHO.GlobalsurveillanceandcontrolofhepatitisC.Reportof aWHOConsultationorganizedincollaborationwiththeViral HepatitisPreventionBoard,Antwerp,Belgium.JViralHepat. 1999;6:35–47.

18.DonahueJG,MunozA,NessPM,etal.Thedecliningriskof post-transfusionhepatitisCvirusinfection.NEnglJMed. 1992;327:369–73.

19.BuschMP,KleinmanSH,NemoGJ.Currentandemerging infectiousrisksofbloodtransfusions.JAMA.2003;289:959–62.

20.WasleyA,MillerJT,FinelliL.Surveillanceforacuteviral hepatitis–UnitedStates,2005.MMWRSurveillSumm. 2007;56:1–24.

21.DantaM,BrownD,BhaganiS,etal.Recentepidemicofacute hepatitisCvirusinHIV-positivemenwhohavesexwithmen linkedtohigh-risksexualbehaviours.AIDS.2007;21: 983–91.

(6)

22.TohmeRA,HolmbergSD.Issexualcontactamajormodeof hepatitisCvirustransmission?Hepatology.2010;52: 1497–505.

23.LopesCL,TelesSA,Espirito-SantoMP,etal.Prevalence,risk factorsandgenotypesofhepatitisCvirusinfectionamong drugusers,Central-WesternBrazil.RevSaudePublica. 2009;43Suppl.1:43–50.

24.OliveiraML,HackerMA,OliveiraSA,etal.Theﬁrstshot:the contextofﬁrstinjectionofillicitdrugs,ongoinginjecting practices,andhepatitisCinfectioninRiodeJaneiro,Brazil. CadSaudePublica.2006;22:861–70.

25.SilvaMB,AndradeTM,SilvaLK,etal.Prevalenceand genotypesofhepatitisCvirusamonginjectingdrugusers fromSalvador-BA,Brazil.MemInstOswaldoCruz. 2010;105:299–303.

26.Mendes-CorreaMC,BaroneAA,GuastiniC.HepatitisCvirus seroprevalenceandriskfactorsamongpatientswithHIV infection.RevInstMedTropSaoPaulo.2001;43:15–9.

27.WolffFH,FuchsSC,BarcellosNN,etal.Co-infectionby hepatitisCvirusinHIV-infectedpatientsinsouthernBrazil: genotypedistributionandclinicalcorrelates.PLoSONE. 2010;5:e10494.

28.FocacciaR,daConceicaoOJ,SetteHJr,etal.Estimated PrevalenceofViralHepatitisintheGeneralPopulationofthe MunicipalityofSaoPaulo,MeasuredbyaSerologicSurveyof aStratiﬁed,RandomizedandResidence-BasedPopulation. BrazJInfectDis.1998;2:269–84.

29.ZarifeMA,SilvaLK,SilvaMB,etal.PrevalenceofhepatitisC virusinfectioninnorth-easternBrazil:apopulation-based study.TransRSocTropMedHyg.2006;100:663–8.

30.PereiraLM,MartelliCM,MoreiraRC,etal.Prevalenceandrisk factorsofHepatitisCvirusinfectioninBrazil,2005through 2009:across-sectionalstudy.BMCInfectDis.2013;13:60.

31.deAlmeida-NetoC,SabinoEC,LiuJ,etal.Prevalenceof serologicmarkersforhepatitisBandCvirusesinBrazilian blooddonorsandincidenceandresidualriskoftransfusion transmissionofhepatitisCvirus.Transfusion.2013;53: 827–34.

32.SallesNA,SabinoEC,BarretoCC,BarretoAM,OtaniMM, ChamoneDF.Thediscardingofbloodunitsandthe prevalenceofinfectiousdiseasesindonorsatthePro-Blood Foundation/BloodCenterofSaoPaulo,SaoPaulo,Brazil.Rev PanamSaludPublica.2003;13:111–6.

33.RosiniN,MousseD,SpadaC,TreitingerA.Seroprevalenceof HbsAg,Anti-HBcandanti-HCVinSouthernBrazil,1999–2001. BrazJInfectDis.2003;7:262–7.

34.BrandaoAB,FuchsSC.RiskfactorsforhepatitisCvirus infectionamongblooddonorsinsouthernBrazil:a case-controlstudy.BMCGastroenterol.2002;2:18.

35.ValenteVB,CovasDT,PassosAD.HepatitisBandCserologic markersinblooddonorsoftheRibeiraoPretoBloodCenter. RevSocBrasMedTrop.2005;38:488–92.

36.NascimentoMC,MayaudP,SabinoEC,TorresKL,Franceschi S.PrevalenceofhepatitisBandCserologicalmarkersamong ﬁrst-timeblooddonorsinBrazil:amulti-centerserosurvey.J MedVirol.2008;80:53–7.

37.NishiyaAS,deAlmeida-NetoC,FerreiraSC,etal.HCV genotypes,characterizationofmutationsconferringdrug resistancetoproteaseinhibitors,andriskfactorsamong blooddonorsinSaoPaulo,Brazil.PloSONE.2014;9:e86413.

38.RomanoCM,deCarvalho-MelloIM,JamalLF,etal.Social networksshapethetransmissiondynamicsofhepatitisC virus.PLoSONE.2010;5:e11170.

39.LeviJE,TakaokaDT,GarriniRH,etal.Threecasesofinfection withhepatitisCvirusgenotype5amongBrazilianhepatitis patients.JClinMicrobiol.2002;40:2645–7.

40.NakanoT,LuL,LiuP,PybusOG.Viralgenesequencesreveal thevariablehistoryofhepatitisCvirusinfectionamong countries.JInfectDis.2004;190:1098–108.

41.LampeE,Espirito-SantoMP,MartinsRM,BelloG.Epidemic historyofhepatitisCvirusinBrazil.InfectGenetEvol. 2010;10:886–95.

42.DrummondAJ,RambautA.BEAST.Bayesianevolutionary analysisbysamplingtrees.BMCEvolBiol.2007;7:214.

43.PosadaD,CrandallKA.MODELTEST:testingthemodelofDNA substitution.Bioinformatics.1998;14:817–8.

44.JunqueiraPC,RosenblitJ,HamerschlakN.HistoryofBrazilian Hemotherapy.RevBrasHematolHemoter.2005;27:201–7.

45.NoronhaIL,SchorN,CoelhoSN,etal.Nephrology,dialysis andtransplantationinBrazil.NephrolDialTransplant. 1997;12:2234–43.

46.MoriceY,CantaloubeJF,BeaucourtS,etal.Molecular epidemiologyofhepatitisCvirussubtype3aininjectingdrug users.JMedVirol.2006;78:1296–303.

47.PybusOG,CochraneA,HolmesEC,SimmondsP.Thehepatitis Cvirusepidemicamonginjectingdrugusers.InfectGenet Evol.2005;5:131–9.

48.OliveiraML,BastosFI,SabinoRR,etal.DistributionofHCV genotypesamongdifferentexposurecategoriesinBrazil.Braz JMedBiolRes.1999;32:279–82.

49.OliveiraMdeL,BastosFI,TellesPR,etal.Epidemiologicaland geneticanalysesofHepatitisCvirustransmissionamong young/short-andlong-terminjectingdrugusersfromRiode Janeiro,Brazil.JClinVirol.2009;44:200–6.

50.MillerER,HellardME,BowdenS,BharadwajM,AitkenCK. MarkersandriskfactorsforHCV,HBVandHIVinanetwork ofinjectingdrugusersinMelbourne,Australia.JInfect. 2009;58:375–82.