Identification of hepatitis B virus A1762T/G1764A double mutant strain in patients in Southern Brazil

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

Identification

of

hepatitis

B

virus

A1762T/G1764A

double

mutant

strain

in

patients

in

Southern

Brazil

Adaliany

Cecília

da

Silva

Souza

_,

_Giórgia

_de

_Souza

_Marasca

_,

Nélson

Alexandre

Kretzmann-Filho

_,

_Aline

_Dall-Bello

_,

_Dimas

_Alexandre

_Kliemann

_,

Cristiane

Valle

Tovo

_,

_Ana

_Beatriz

_Gorini

_da

_Veiga

a_{UniversidadeFederaldeCiênciasdaSaúdedePortoAlegre(UFCSPA),ProgramadePos-Graduac¸ãoemMedicina:Hepatologia,Porto} Alegre,RS,Brazil

b_{HospitalNossaSenhoradaConceic¸ão(HNSC),PortoAlegre,RS,Brazil}

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Articlehistory:

Received18January2017 Accepted11May2017 Availableonline9June2017

Keywords:

HepatitisB HBV

A1762T/G1764Amutation

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Infection byhepatitis Bvirus(HBV)isaworldwidepublichealthproblem.ChronicHBV infectionwithhighviralreplicationmayleadtocirrhosisand/orhepatocellularcarcinoma. MutantHBVstrains,suchastheHBVA1762T/G1764Adoublemutant,havebeen associ-atedwith poorprognosisandhigherriskofthepatientfordevelopingcirrhosisand/or hepatocellularcarcinoma.ThisstudyanalyzedthepresenceoftheHBVA1762T/G1764A doublemutantinpatientswithchronicHBVanditsassociationwithclinicalparameters suchasviralload,aminotransferases,andHBVantigens.Atotalof49patientswithchronic hepatitisBwereincludedinthestudy,andtheHBVA1762T/G1764Adoublemutantstrain wasdetectedinfoursamples(8.16%)bypolymerasechainreactionfollowedbyrestriction fragmentlengthanalysis(PCR-RFLP).Theviralloadwasnotsignificantlydifferentbetween patientswithorwithoutthedoublemutantstrain(p=0.43).Ontheotherhand,carriers oftheHBVA1762T/G1764AdoublemutanthadhigherlevelsofALT(p=0.0028),whileAST levelsdidnotdifferbetweengroups(p=0.051).Inthisstudy,75%ofthesampleswiththe HBVA1762T/G1764AdoublemutationwereHBeAgnegativeandanti-HBepositive, reflect-ingseroconversioneventhoughtheystilldisplayedhighviralloads.Ourstudyhasshown thattheHBVA1762T/G1764AdoublemutantstraincirculatesinBrazilianpatients,andis associatedwithelevatedlevelsofALTandHBeAgseroconversion.

©2017SociedadeBrasileiradeInfectologia.PublishedbyElsevierEditoraLtda.Thisisan openaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/ by-nc-nd/4.0/).

Introduction

InfectionbyhepatitisBvirus(HBV)isapublichealthproblem. Despitebeingadiseaseforwhichthereisvaccineavailable,

∗ _{Correspondingauthor.}

E-mailaddress:anabgv@ufcspa.edu.br(A.B.GorinidaVeiga).

it is estimatedthat two billion people are infected world-wide, out of which about 350 million have serological markers ofactive infection.1 _{Carriersof HBV may develop}

cirrhosisand/orhepatocellularcarcinoma(HCC),whichmay be related to mutations in specific regions of the HBV

http://dx.doi.org/10.1016/j.bjid.2017.05.002

1413-8670/©2017SociedadeBrasileiradeInfectologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

genome and is responsible for over one million deaths annually.2

TheareaswithhighprevalenceofHBV(>8%)are concen-tratedinSoutheastAsia,China,the Philippines,Africa,the AmazonBasin,andtheMiddleEast;areaswithintermediate prevalence(2–7%)areregionsofEasternEurope,CentralAsia, Japan,Israel,andRussia;andareaswithlowprevalence(<2%) areWesternEurope,Australia,NewZealand,NorthAmerica, andSouthAmerica.3,4_{InBrazil,thedistributionofHBVisquite}

heterogeneous;itismoreprevalentinthenorthernregionof thecountry,coveringthestatesofAcre,Amazonas,Rondônia, andRoraima,andlessprevalentintheSouth.From 1999to 2011,ofthe120,343confirmedcasesofhepatitisBthatwere reportedinBrazil,31.6%(38,007cases)wereintheSouthern region,withatotalof1,693casesreportedinthecityofPorto Alegre,RioGrandedoSul.5,6

HBVpresentseightgenotypes(A,B,C,D,E,F,G,andH), whichdifferintheirnucleotidesequences.7–11_{InBrazil,A,B,D,}

andFgenotypesarefound,12,13_{themostprevalentbeing}

geno-typeA,accountingfor48.5%ofallcases,followedbygenotype Din38.5%.5,12–14_{HBVgenotypeDisthemostprevalent}

geno-typeinthestateofRioGrandedoSul,Brazil,beingfoundin approximately63%ofthecases,followedbygenotypeA(32%); genotypeFrepresentsonly5%ofallcases.7,12

HBVisanenvelopedvirusofthefamilyHepadnaviridae; itspartiallydouble-strandedcircular DNAgenomecontains approximately3200nucleotides(3.2kb),arrangedin overlap-pinggenes.GeneShasthreeregions,Pre-S1, Pre-S2andS, eachcoding foroneofthesurfaceglycoprotein–S(small), L(large),andM(middle),respectively,andalsofortheHBVs antigen(HBsAg);geneC,withregionspre-coreandcore,codes forantigenscande(HBcAgandHBeAg,respectively);geneP codestheDNApolymerase;andgeneXcodeforthexantigen (HBxAg).2,5,15–18

MutationsincertainregionsoftheHBVgenome,suchasin thepre-coreandinthebasalcorepromoter(BCP),maydisrupt theexpressionofviralproteins.TheBCPregionislocatedin theXgene,whichinadditiontocodingforHBxAg,italso reg-ulatestheexpressionofHBeAg;patientscarryingHBVstrains withmutationsinthisregionoftenappearasHBeAg-negative. Notably,eventhoughtheyappeartohaveseroconverted,these patientsmighthaveanunfavorableprognosis.19–21

Amongthe mutationsdescribed in the BCP region, the HBV A1762T/G1764A double mutationis worth ofnote for itssignificantclinicalimportance:patientswiththismutant strain showed an increased level of viral replication, and decreaseorabsenceofHBeAg.DespitebeingHBeAg-negative, these patients may rapidly develop liver cirrhosis, which installsprogressivelyanddespitebeingoftenasymptomatic, itmayprogresstoHCC.22–29_{TheHBVA1762T/G1764Adouble}

mutationislocatedintheBCPregionandcausesanamino acidsubstitutionfrom lysine(K)tomethionine(M)at posi-tion 1762, and from valine (V) to isoleucine (I)at position 1764.30,31

The aim ofthis study was to assess the circulation of HBV strains with the A1762T/G1764A double mutation in patientsfromPortoAlegre,RioGrandedoSul.Thepresenceor absenceofthedoublemutantwascorrelatedwithviralload, aminotransferaseslevels,aswellaswithHBeAgandanti-HBe parameters.

Material

and

methods

This was a cross-sectional study, which followed-up HBV patientsreceivingcareattheInfectiousDiseasesAdultClinic oftheNossaSenhoradaConceic¸ãoHospitalinPortoAlegre, Brazil,andfromtheGastroenterologyClinicofSantaCasade Porto AlegreHospitalComplex,Brazil,fromNovember2012 toDecember2013.ThisstudywasapprovedbytheResearch EthicsCommittee(CEP-UFCSPA),ProtocolNumber575.347.

All patientsincluded in this study were HBsAg-positive for more than six months, with viral load higher than 200copies/ml, and agreeing to participate in the study by signingtheInformedConsentForm.Exclusioncriteriawere HBsAg-negative,alcoholconsumptionofmorethan20g/day (forwomen)or30g/day(formen),coinfectionwithhepatitis Dvirus(HDV),autoimmunehepatitisandNASH,patientson drugtreatmentforhepatitis,andpatientswhowereunableto readandsigntheInformedConsentForm.Eachsamplegained anumericalsequenceandwasde-identified,andadatabase wascreatedincludingthemainlaboratorytests:biochemical parameters,HBVviralload,HBVantigens,anti-HBV antibod-ies,andaminotransferases.Suchinformationwasabstracted fromphysicalandelectronicrecordsofeachpatienttogether withtheircoadministredmedications.

All selectedpatientsweresubmittedtoblood collection, which was carried out at the hospital by using vacuum venipuncture in 4ml tubes containing EDTA. Thesamples were transported to the Molecular Biology Laboratory of Universidade Federalde Ciênciasda Saúdede Porto Alegre (UFCSPA),wheremolecularanalyseswereperformed.

Molecularanalysis

Analiquotof500␮lofbloodwascentrifugedforfiveminutesat 500×gatroomtemperature;plasmawasseparatedina1.5ml tube.Allplasmasampleswerenumberedandwerestoredat −80◦_{CuntilviralDNAextraction.}

ViralDNAwasextractedwithPureLinkTM_viralDNA/RNA

Kit (Invitrogen Life Technologies), quantified in a Nan-oDropinstrument(NanoDropTechnologies,Wilmington,USA) and used as template in PCR. The primers used in the PCR were the forward primer P2-1 that anneals at posi-tion 1652–1672 (5-CACAAGAGGACTCTTGGACT-3), and the reverse primer P2-2that anneals atposition1940–1959 (5 -GGCAAAAAAGAGAGTAACTC-3). PCR was performed with GoTaq® Master Mix(Promega)and 0.05␮M ofeachprimer, inafinalvolumeof25␮l.Reactionconditionswere95◦Cfor fiveminutes;40cyclesof95◦Cforoneminute,55◦Cforone minute,72◦Cforoneminute;finalextensionat72◦Cforfive minutes.ThePCRproductwasanalyzedbyelectrophoresison 1.5%agarosegel.32,33

ThePCRproductpresentedafragmentsizeof307bp,which wassubjectedtoRFLPwithSau3AIenzyme,whichcleavesthe nucleotidesequencewiththeA1762T/G1764A(G/ATC)double mutation.Therefore,whentheA1762T/G1764Adouble muta-tionispresent,Sau3AIenzymecleavestheamplicon generat-ingtwofragmentsof197pband110pb.FortheRFLP,10␮lof

thePCRproductwascleavedwith10unitsofSau3AIenzymein afinalvolumeof50␮l.Thereactionwasincubatedat37◦Cfor 100minandinactivatedat65◦Cfor20min.TheRFLPproduct wasanalyzedbyelectrophoresison2%agarosegel.

Statisticalanalysis

Quantitativevariablesweredescribedastheminimum, max-imum, mean, standard deviation, and compared between groupswithStudent’sttest.Categoricalvariableswere ana-lyzedbythechi-squaretest.Thesignificancelevelwas5.0%.

Results

Atotalof49patientswereincludedinthisstudy,21females (42.85%)and28males(57.14%).Agerangedfrom19through 79years-old(averageof50.48±12.76).

Resultsofthebiochemicalanalysis,HBVviralload,HBV antigen, anti-HBV antibodies, and aminotransferase tests were abstracted from the physical and electronic records ofeach patient. Divergence occurs in the total number of samplesforsomeparameters,duetounavailabilityofsome patientrecords.

ThemainantigensandantibodiesusedforHBVevaluation wereHBsAg,HBeAg,anti-HBs,andanti-HBe.Table1 summa-rizesthereactivityofpatients’seratoeachofthesemarkers. Allpatientswere positivefortheHBsAgand noneofthem displayedantibodiesagainstthisantigen.RegardingHBeAg, whichisamarkerofviralreplication,25.58%ofthepatients wereHBeAg-positive,and71.73%ofthepatientshad serocon-vertedasshownbytheanti-HBelevels.Twopatientswerealso coinfectedwithHIV,andfourwithHCV.

Analysisofliverbiochemicalparametersshowedthatthe majorityofpatientshadnormallevelsofaminotransferases, bilirubin,andcreatinine,exceptfordirectbilirubin,whichwas alteredin39.13%ofthepatients(Table2).Also,five(11.63%)

patientshaveincreasedcreatininelevels,butallofthemwere onconservativetreatment.

All samples were successfully amplified by PCR, using primersspecificfortheBCPregionofHBVwherethedouble mutationislocated,generatingfragmentsof307pb.Analysis byRFLPrevealedthatfour(8.16%)patientscarriedtheHBV A1762T/G1764Adoublemutant,asshownbythepresenceof DNAfragmentsof197pband110pb;thefragmentof307pb wasalsovisibleinthesesamples(notshown).

Toverifyapossibleassociationbetweenthepresenceof theA1762T/G1764Adoublemutantandtheriskof develop-ingchronichepatitis,viralloadandaminotransferaseswere analyzedinthesepatients.AsshowninTable3,therewasno significantdifferenceinthemeanviralloadbetweenpatients carryingthedoublemutantandpatientswithoutthemutant strain. Aspartate aminotransferase (AST) levels showed a larger varianceinpatients withoutthe doublemutant,but the mean AST levels were higher in patients with HBV A1762T/G1764A double mutant. However, such differences were not significant (p=0.051). On the other hand,alanine aminotransferase (ALT) levels were significantly higher in patientscarryingthedoublemutant(p=0.0028).

TheHBeAgandanti-HBewereanalyzedin42and46 sam-ples, respectively,includingthefour sampleswiththeHBV A1762T/G1764A double mutation. According to the results shown in Table 4, the serological profile of samples with the HBV A1762T/G1764Adoublemutationwas25% HBeAg-positive, 75% HBeAg-negative, 75% anti-HBe-positive, and 25% anti-HBe-negative. Regarding the samples without the presenceofthe doublemutation,theywere26.31% HBeAg-positive, 73.68% HBeAg-negative, 71.42% anti-HBe-positive, and 28.57%anti-HBe-negative. TheratesofHBeAg and the anti-HBewerenotsignificantlydifferent.

Discussion

HBV infection is a worldwide public health problem, even thoughitisadiseasethatcanbepreventedbyvaccination.

Table1–MainserologicalparametersinpatientswithchronichepatitisB. HbsAg n(%) HbeAg n(%) Anti-HBs n(%) Anti-HBe n(%) Positive 49(100%) 11(25.58%) 0 33(71.74%) Negative 0 32(74.42%) 49(100%) 13(28.26%) Total 49(100%) 43(100%) 49(100%) 46(100%)

HBsAg,hepatitisBsurfaceantigen;HBeAg,hepatitisBeantigen;anti-HBs,antibodyagainstthesurfaceantigen;anti-HBe,antibodyagainstthe eantigen.

Table2–MainbiochemicalparametersinpatientswithchronichepatitisB. AST (40U/L) ALT (40U/L) BT (0.20–1.00mg/dl) BD (0.0–0.20mg/dl) BI (0.20–0.80mg/dl) Creat (0.40–1.4mg/dl) Normal 38(79.17%) 36(73.47%) 38(80.85%) 28(60.87%) 41(89.13%) 38(88.37%) Abnormal 10(20.83%) 13(26.53%) 9(19.15%) 18(39.13%) 5(10.87%) 5(11.63%) Total 48(100%) 49(100%) 47(100%) 46(100%) 46(100%) 43(100%)

AST,aspartateaminotransferase;ALT,alanineaminotransferase;BT,totalbilirubin;BD,directbilirubin;BI,indirectbilirubin;Creat,creatinine. Normalreferencevaluesforeachparameterareshown.

Table3–MeanvaluesofHBVviralloadandaminotransferasesinpatientswithchronichepatitisBaccordingtothe presenceofthedoublemutationA1762T/G1764AinHBV.

Without mutation n(%) A1762T/G1764A n(%) p-Value

HBVviralload,copies(mean±SD) 6,320,278±27,373,142 3,478,000±5,212,795 0.43

ALT,U/L(mean±SD) 36.53±35.82 101.50±86.91 0.0028

AST,U/L(mean±SD) 37.70±47.14 79.25±14.66 0.051

Table4–AnalysisofHBsAgandanti-HBeinsamplesfrompatientswithchronichepatitisBwithandwithoutthedouble mutationA1762T/G1764A. Withoutmutation n(%) A1762T/G1764A n(%) HBeAg-positive 10(26.31%) 1(25.0%) HBeAg-negative 28(73.68) 3(75.0%) Anti-HBe-positive 30(71.42%) 3(75.0%) Anti-HBe-negative 12(28.57%) 1(25.0%)

The presence of mutant viral strains may aggravate the disease;ofnote, theHBV A1762T/G1764Adoublemutantis associatedtocomplicationsleadingtothedevelopmentof cir-rhosisand/orHCC.2,31,32_{ThestudyofHBVandimprovements}

oftechniquestoeasilyidentifytheHBVA1762T/G1764A dou-blemutationaswellasitscorrelationwithbiochemicaland serologicaldatacancontributetoanimprovedunderstanding ofhepatitisB.29,30,32

Inthis study,theserologicaldataofthe majorantigens andantibodiesrelatedtoHBVwere analyzedtogether with thepresenceoftheHBVA1762T/G1764Adoublemutation.All patientsinthisstudy werepositivefortheHBsAgformore than sixmonthsand werenotusing any antiviraltherapy, thereforeHBsAgreactivitywas100%asexpected.

Other markers for hepatitis B are HBeAg, which indi-cates viral replication, while the antibody anti-HBe is an indicator of seroconversion. In this study, 74.41% of the samples were HBeAg negative and 71.73% anti-HBe posi-tive, representing patients who had seroconverted. Other biochemical parameters were normal in most of the samples.

Inthepresentstudy,therewerefourpatients(8.16%) pos-itivefortheHBVA1762T/G1764Adoublemutant,asrevealed byDNAfragmentsof197pband110pbafterincubationwith Sau3AIenzyme.Ofnote,the307pbDNAfragmentwasalso observedinthesesamples,suggestingthatHBVstrains with-outthismutationwaspresentinthesepatientstogetherwith thedouble-mutantstrain.Infact,thedouble-mutantstrainis usuallyfoundinverylowlevelsinpatientsinfectedwithHBV, areasonwhymostanalysesfailtodetectthemutantinthese samples.29,32,33 _{Nonetheless,identification ofthe mutantis}

importantconsideringitspotentialtoincreasetheriskof cir-rhosisandHCC.

HBVviralloadwascomparedbetweensampleswithand without the HBV A1762T/G1764A double mutation,and no significant difference was found (p=0.43) probably due to the low number ofsamples with the HBV A1762T/G1764A double mutation. Nevertheless, studies suggest that the HBV A1762T/G1764A doublemutationis directly related to increasedviralreplicationand thereforethe progressionof liverdisease.29,32,33

Theaminotransferases(ASTandALT)werealsoanalyzed inpatientswithandwithouttheHBVA1762T/G1764Adouble mutation. For the ASTparameter, therewas no significant difference, but there was a clear trend for a significant difference (p=0.051). On the other hand, ALT levels were significantly higherinpatients carryingthe doublemutant (p=0.0028).Giventheresultsfoundhereandinotherstudies, aminotransferases may be used as an indication for the possible presence of strains with the HBV A1762T/G1764A doublemutation.29,30

TheseroconversionofHBeAgtoanti-HBeinsampleswith active viral replication is directly related to the presence ofmutationsinHBV intheBCPregion, especiallythe HBV A1762T/G1764Adoublemutation.Usuallytheseroconversion process inpatientswithviralreplicationisassociatedwith poor prognosis ofthe disease.34 _{In this study,}

seroconver-sion was observed in 75% of the patients with the HBV A1762T/G1764A strain; the patientthat didnot show sero-conversionwasthesamethatshowedhighviralreplication accordingtohismedicalrecords.

TheincidenceofhepatitisBisrelativelylowinPorto Ale-gre,thereforealownumberofpatientscouldbeenrolledin thepresentstudy.Inaddition,duetothelackofbiochemical datainsomepatients’electronicrecords,someparameters could not be analyzed for all patients. Another limitation wasthedifficulty inestablishingagoodmolecularassayto detectthedoublemutantstrain. Eventhoughsomestudies havereportedtheuseofreal-timePCR(qPCR)todetectthe A1762T/G1764A doublemutation, mostpatients that carry the double mutant also have non-mutant strains, which are usuallyinhigherconcentrations;therefore,detectionof the mutantstrain ishamperd bynon-mutantamplification products.35

Inconclusion,inSouthernBrazilHBVA1762T/G1764A dou-ble mutant strain circulatesin low levels in patients with apparent seroconversion. This strain was associated with alteredlevelsofALTandseroconversionofHBeAgtoanti-HBe inpatientswithliverdisease.Consideringthatthisstrainhas beenassociatedwithahigherriskofcirrhosisand/orHCCin patientswithhepatitisB,itisimportanttoverifythepresence ofthisHBVdoublemutantinpatientsintheregion.

Financial

support

A.C.S.SouzaandG.S.MarascaholdafellowshipfromCAPES (MinistryofEducation,Brazil).AnaB.GorinidaVeigahelda fel-lowshipfromCNPq(ProductividadeemPesquisa)duringthe project

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

WethanktheGraduatePrograminMedicine:Hepatologyat UFCSPAandtheHospitalNossaSenhoradaConceic¸ão(HNSC).

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