Hepatitis B Virus RNA in serum and liver tissue ......HBsAg levels in serum. The results also...
Transcript of Hepatitis B Virus RNA in serum and liver tissue ......HBsAg levels in serum. The results also...
HepatitisBVirusRNAinserumandliver
tissue–quantificationusingdigitalPCR
KasthuriPrakash
DepartmentofInfectiousDiseases,InstituteofBiomedicine
SahlgrenskaAcademyatUniversityofGothenburg
Gothenburg,Sweden
Gothenburg2020
Coverillustration:GenesisbyKasthuriPrakash
HepatitisBvirusRNAinserumandlivertissue–quantificationusingdigitalPCR©[email protected](PRINT)ISBN978-91-7833-725-5(PDF)PrintedinGothenburg,Sweden2019PrintedbyBrandFactory
TomydearestNanapaandChemmi
HepatitisBVirusRNAinserumandlivertissue–
quantificationusingdigitalPCR
KasthuriPrakash
DepartmentofInfectiousDiseases,InstituteofBiomedicine
SahlgrenskaAcademyatUniversityofGothenburg
Gothenburg,Sweden
ABSTRACT
Hepatitis B virus (HBV) infection is a global health issue that is responsible for
approximately900,000deathseachyear,byinducinglivercirrhosisandhepatocellular
carcinoma(HCC).AfewmarkersareusedtoclassifyHBVinfectionandmonitortreatment
efficacy,includingHBVDNA,surfaceantigen(HBsAg)andeantigen(HBeAg)inserumas
well asHBVDNA andRNA in liver tissue. The recent discovery of the receptorNTCP
facilitatesinvitrostudiesofHBV.
Theaimsofthisthesiswere(I)tocharacterizeanewmarkerofHBVinfection,HBVRNA
in serum (II) to investigate in vitro the neutralizing effect of HBV encoded subviral
(HBsAg) particles (III) to develop and apply a newmethod to discriminate viral and
integratedDNAinlivertissue(IV)toanalyzefocaldifferenceswithintheliverofHBVand
hepatitisDvirus(HDV)and(V) toexploreHBVRNAprofile in liverbiopsiesbydigital
PCR.
HighlevelsofserumHBVRNAwasfoundinthemajorityof95patientsamplesutilizedin
thisstudy.ThisRNAwasoffullgenomelength,appearedinfractionationtogetherwith
HBV DNA. Sequencing data supported that HBV RNA in serum represents virus-like
particleswithfailingreversetranscriptionofthepregenomicRNA(pgRNA).
Theroleofsubviralparticles(SVP)duringHBVinfectionwasexploredinHepG2-NTCP
cell line. The results support that SVP functions as a decoy to neutralize antibodies
synthesizedbythehost.
A novel droplet digital PCR (ddPCR) methodwas developed and applied on 70 liver
biopsies to quantify circular and linearHBVDNA, in order to estimate the amount of
integratedHBVDNAinthehumangenome.Acomplimentarystudyonthesamematerial
was performed to obtain an RNA profile using ddPCR to amplify six target regions.
Together, these results indicate that integrated DNA represents the majority of
intrahepaticHBVDNAinlatestagesofinfectionandisresponsibleformaintaininghigh
HBsAglevelsinserum.TheresultsalsosuggestthatreducedtranscriptionofpgRNAvia
anovelmechanismmaycontributetolowHBVreplicationinHBeAg-negativephase.
ddPCRanalysisofarangeofHBVmarkerswasusedtostudyfocaldifferencesininfection
in 15-30 pieces of liver explant tissue from six patients with HBV or HDV induced
cirrhosis.Largedifferencesinfocalitywasobservedespeciallyinpatientswithlowdegree
ofviralreplicationorwithHDVcoinfectionandtheresultsalsosupportexpressionofS
RNAfromintegratedHBVDNA.HDVinfectionwaslessfocalwithpresenceofhighHDV
RNAlevelsintheabsenceofHBV.
Insummary,thisthesiscompilationcontributestobetterunderstandingofHBVserum
andtissuemarkersandtheirrelationshiptoreplicationandintegration.
Keywords:hepatitisBvirus,NTCP,HBVDNA,HBVRNA,subviralparticles,integrations,
dropletdigitalPCR
ISBN:978-91-7833-724-8(PRINT)
ISBN:978-91-7833-725-5(PDF)
SAMMANFATTNINGPÅSVENSKA
InfektionmedhepatitB virus (HBV) är ett globalt hälsoproblemsom står för ungefär
900000dödsfallårligengenomattorsakaskrumpleverochlevercancer.Debiomarkörer
som idaganvänds förattklassificeraHBV-infektionenochövervakabehandlingseffekt
inkluderarHBV-DNA,ytantigenetHBsAg,e-antigenetHBeAgiserumsamtHBV-DNAoch
HBV-RNAilevervävnad.NyligenupptäcktesHBV-receptorn,NTCP,vilketunderlättarin
vitro-studieravHBV.
Syftetmeddennaavhandlingvaratt:(I)karakteriseraennybiomarkörförHBV-infektion
(HBV-RNA i serum); (II) undersöka den neutraliserande effekten av HBV-kodade
subviralapartiklar(HBsAg)invitro;(III)utvecklaochappliceraennymetodförattskilja
på viralt och integrerat HBV-DNA i levervävnad; (IV) analysera fokala skillnader i
förekomst av HBV och hepatit D-virus (HDV) i levervävnad; (V) utforska HBV-RNA-
profilerileverbiopsiermeddigitalPCR.
I enmajoritet av serumprover från95 patienter uppmätteshöga nivåer avHBV-RNA.
Detta RNA motsvarade hela HBV-genomets längd och förekom vid fraktionering i
partiklarmedsammadensitetsomdemedHBV-DNA.SekvenseringindikeradeattHBV-
RNA i serummotsvararviruslikapartiklardäromvänd transkriptionavpregenomiskt
RNA(pgRNA)ejfungerat.
FunktionenavHBsAg-bärande subviralapartiklar (SVP) iHBV-infektionundersöktes i
cellinjen HepG2-NTCP. Resultaten stöder hypotesen att HBsAg/SVP minskar den
virusneutraliseradeeffektenavantikropparriktademotvirusetsytprotein.
En ny metod, baserad på droplet digital PCR (ddPCR), utvecklades och användes för
analysav70leverbiopsier.MetodenkvantifieradecirkulärtochlinjärtHBV-DNAföratt
kunna uppskatta mängden integrerat HBV-DNA i det humana genomet. En
kompletterande studie på sammamaterial utfördes för att ta fram virus-RNA-profiler
genom att med ddPCR amplifiera sex olika målregioner. Sammantaget pekar dessa
resultatpåattintegreratHBV-DNAutgördenstörstaandelenavintrahepatisktHBV-DNA
isenarestadieravinfektionen,ochattdettaintegreradeDNAuttryckssåatthöganivåer
avHBsAgiserumbibehålls.ResultatentyderocksåpåattnedregleringavpgRNAviaen
nymekanismskullekunnabidratilllägrereplikationavHBViHBeAg-negativapatienter.
ddPCR-analysav fleraolikaHBV-marköreranvändes förattstudera fokalaskillnader i
HBV-infektioneni15-30bitaravleverexplantatfrånsexpatientermedHBV-ellerHDV-
orsakadskrumplever.Storaskillnader i fokalitetobserveradessärskilt ipatientermed
låggradig virusreplikation och resultaten stöder hypotesen att S-RNA uttrycks från
integrerat HBV-DNA. HDV-infektion (hos två av patienerna) var jämntare utspridd i
levervävnadenmedhöganivåeravHDV-RNAoberoendeavHBV.
Sammanfattningsvis bidrar denna avhandling till bättre förståelse av serum- och
levermarkörervidHBV-infektionochderaskopplingtillreplikationochintegration.
LISTOFPAPERS
The thesis is based on the following studies, referred to in the text by their roman
numerals.
I Kasthuri Prakash, Gustaf E. Rydell, Simon B. Larsson, Maria Andersson, Gunnar
Norkrans,HeléneNorder,MagnusLindh.HighserumlevelsofpregenomicRNAreflect
frequentlyfailingreversetranscriptioninhepatitisBvirusparticles.VirologyJournal
2018;15:86.
II GustafE.Rydell,KasthuriPrakash,HeléneNorder,MagnusLindh.HepatitisBsurface
antigenonsubviralparticlesreducestheneutralizingeffectofanti-HBsantibodieson
hepatitisBviralparticlesinvitro.Virology2017;509:67-70.
III Gustaf E. Rydell, Simon B. Larsson, Kasthuri Prakash, Maria Andersson, Heléne
Norder,KristofferHellstrand,GunnarNorkrans,MagnusLindh.Abundanceofnon-
circularHBVDNAsuggestsintegrationstobethepredominantformofviralDNAin
chronichepatitisB.Manuscript.
IV Kasthuri Prakash, Simon B. Larsson, Catarina Skoglund, Gustaf E. Rydell, Johan
Ringlander, Maria Andersson, Maria Castedal, Heléne Norder, Magnus Lindh.
IntrahepaticfocalityofhepatitisBinfectionanalysedbyquantificationofviralRNAin
multipletissuepiecesfromexplantedlivers.Manuscript.
V Kasthuri Prakash, Simon B. Larsson, Gustaf E. Rydell, Maria Andersson, Johan
Ringlander,GunnarNorkrans,HeléneNorder,MagnusLindh.AnalysisofHBVRNAin
liverbiopsiesbydigitalPCRrevealsdifferencesintranscriptlevels,lengthandorigin
betweeneantigenpositiveandnegativeindividuals.Manuscript.
ABBREVIATIONS
AuAg AustralianAntigen
HBV HepatitisBvirus
HCC Hepatocellularcarcinoma
NA Nucleoside/nucleotideanalogues
rcDNA RelaxedcircularDNA
HBcAg HepatitisBcoreantigen
HBsAg HepatitisBsurfaceantigen
HBeAg HepatitisB“e”antigen
VP ViralParticles
SVP Subviralparticles
NTCP Sodium-taurocholatecotransportingpolypeptide
cccDNA CovalentlyclosedcircularDNA
PreCRNA/PC PrecoreRNA/precore
PgRNA PregenomicRNA
dslDNA DoublestrandedlinearDNA
ssDNA SinglestrandedDNA
ALT Alanineaminotransferase
ORF Openreadingframe
BCP Basalcorepromoter
RT Reversetranscriptase/reversetranscription
HDV Hepatitisdeltavirus
PCR Polymerasechainreaction
qPCR QuantitativePCR
RT-PCR ReversetranscriptasePCR
ddPCR DropletdigitalPCR
cDNA ComplimentaryDNA
TableofContents
1 INTRODUCTIONTOHEPATITISBVIRUS....................................................................................................1
MOLECULARSTRUCTURE..............................................................................................................................................2 VIRALENTRYANDREPLICATION..................................................................................................................................4 SEROLOGICALANDINTRAHEPATICMARKERS..............................................................................................................7 NATURALHISTORYOFINFECTION................................................................................................................................9 INTEGRATIONS............................................................................................................................................................11 GENOMICVARIATIONS................................................................................................................................................12 TREATMENT................................................................................................................................................................15 CELLCULTURE&ANIMALMODELS...........................................................................................................................16
2 INTRODUCTIONTOHEPATITISDELTAVIRUS.......................................................................................18
HDVGENOMEANDREPLICATION.............................................................................................................................18 HBVSUPPRESSIONBYHDV......................................................................................................................................20 TREATMENT................................................................................................................................................................20
3 AIMS...................................................................................................................................................................21
4 MATERIALSANDMETHODS........................................................................................................................22
MATERIALS.................................................................................................................................................................22 METHODS....................................................................................................................................................................23
5 RESULTSANDDISCUSSION..........................................................................................................................28
PAPERI.......................................................................................................................................................................28 PAPERII......................................................................................................................................................................32 PAPERIII....................................................................................................................................................................34 PAPERIV.....................................................................................................................................................................38 PAPERV......................................................................................................................................................................46
6 CONCLUSIONS..................................................................................................................................................52
7 ACKNOWLEDGEMENTS.................................................................................................................................54
8 REFERENCES....................................................................................................................................................57
1
1 INTRODUCTIONTOHEPATITISBVIRUS
In1967,DrBlumbergandhisassociatediscoveredanewproteininserumofpatientsthat
underwentbloodtransfusions,specificallyinAustralianaboriginals.Thisproteinnamed
“Australian Antigen” (AuAg) became the first of many discoveries confirming the
presence of viral hepatitis [1]. In 1970, Dr Davis S Dane described 42nm virus-like
particlesinpatientswithAuAgandnamedthem“DaneParticles”[2]whichisnowknown
asHepatitisBVirus(HBV).
Theviral infection causedbyHBVstill remainsaglobalpublichealth issue in-spiteof
decadesofresearchandavailabilityofapreventivevaccinesince1982.HBVbelongsto
thefamilyofhepatotropicDNAvirusesthatcancausebothacute(infectionclearedwithin
6monthsofexposure)andchronic(infectionthatpersists>6months) infectionof the
liver.TheWorldHealthOrganizationasof2017estimatesthat2billionindividualsareor
have been infected with HBV of which 257 million have a chronic infection [3].
Approximately900,000deathseachyeariscausedbyHBVrelatedcomplicationssuchas
livercirrhosisandhepatocellularcarcinoma(HCC)[4].
HBVcanbe transmittedpercutaneouslyvia sharingofbloodproducts, throughsexual
transmissionorbymeansofvertical transmission, i.e., toanewborn froman infected
mother.AccordingtotheWorldHealthOrganization’s(WHO)statistics,about80-90%of
infantsand30-40%ofchildrenundertheageof6whoareinfectedwithHBVwilldevelop
a chronic disease, but when acquired as adults 95% will clear the infection. The
pathogenesisofcomplicationsinvolvesimmunemediatedkillingofhepatocytescausing
liverinjurywithsubsequentregenerationbyclonalexpansionofhepatocyteseventually
leading to scarification of the liver (fibrosis) and later on cirrhosis and HCC. Other
contributingfactorsforpathogenesisareintegrationofviralDNAintothehostgenome
and oncogenic effects of viral protein [5]. Patients showing signs of progressive liver
damagearegiven long-termtreatmentwithnucleoside/nucleotideanalogues(NA) in
ordertopreventthesecomplications,butwithitarisesthepossibilityforresistantstrains
toemergeviamutation[6-8].
2
DuringthecourseofthisPhD,variousaspectsofHBVinfectionwasinvestigatedinorder
to answer some pressing questions. Interesting associations were explored, and
intriguinghypothesesarepresentedasacompilationwiththehopethatthesestudieswill
aid better understanding of disease progression and assist the clinical community in
developmentofnewtreatmentsforpatientswithHBV.
Molecularstructure
HBVisclassifiedasoneofthesmallestknownDNAvirusesmeasuringapproximately42
nmindiameterbelongingtothefamilyofHepadnaviridaeviruses.Ithasa3.2kbsized
relaxedcircular(rcDNA)genomethatispartiallydoublestranded(ds)withacomplete
minus (-)strandandan incompleteplus (+) strand that is covalentlyboundtoaviral
polymeraseatits5’end.Thisgenomeisenclosedwithinanicosahedralnucleocapsidcore
protein (core antigen,HBcAg) that is surrounded by viral surface protein (hepatitisB
surfaceantigen,HBsAg).
Figure1:HBVparticlecontainingthepartiallydoublestrandedgenomewithcomplete–strandandanincomplete+strandcovalentlyboundtotheviralpolymerase
HBVgenomehasfouroverlappingreadingframes(ORF)thatcodesforviralproteins(i)
precore/coregenefornucleocapsidcoreandsecretory“e”protein(HBeAg)(ii)PreS/S
geneforsmall(SHBsAg),middle(MHBsAg)andlarge(LHBsAg)envelopeproteins(iii)
XgeneforregulatoryXproteinand(iv)polymerasegeneforviralpolymerase.
3
Figure2:HBVgenomethatispartiallydoublestranded.Representedhereare4overlappingreading
framesresponsiblefortranslationofviralproteins.
AlongwithHBVDNAcontainingviralparticles(VP),anexcessproductionof22nmsized
rodandcircularemptysubviralparticles(SVP)comprisedofonlyHBsAgcanbedetected
inthepatient’sserum.
Figure 3: Sphere and rod forms of HBV subviral particles with large, middle and small surface
proteinsthatlackaviralgenome.
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Viralentryandreplication
Although presence of hepadnaviruses in extrahepatic tissues such as lymph nodes,
kidneys, skin and colon have been detected, the only target for these viruses are
hepatocytes [9] because of the presence of cell specific receptors expressed by the
hepatocytes.
The first step in viral entry involves interaction of the pre S1 domain of viral surface
antigen with hepatocyte specific heparin sulphate proteoglycan [10] followed by
attachment to the sodium taurocholate co-transporting polypeptide (NTCP) receptor
[11].Theuptakeofthevirusintothehepatocyteishypothesizedtotakeplaceviaclathrin
or caveolin-1mediated endocytosis throughwhich the encapsidated viral genome is
transportedintothehostcell[12,13].Themicrotubulesinthecytoplasmthentransport
thenucleocapsidcontainingtheviralgenomeandthepolymeraseintothecell'snucleus
where the capsid disassociates and the rcDNA is released [14]. A sequence of highly
precise steps to convert rcDNA into thehighly stablemini chromosomecccDNA takes
place.Theincomplete+strandiselongatedtothe5’endofthe–strand.Afterthisstep,
the polymerase is removed followed by the eliminationof eight terminally redundant
nucleotidesfromthe–strand.Atthisjuncturethe5’andthe3’endsofthe+andthe–
strandcanbeligatedandsupercoiledtoformcccDNA[15].
Ithasbeenestimatedthataround1-50copiesofthecccDNAremainwithineachinfected
hepatocyte as mini chromosomes that serves as a transcriptional template for viral
replication.TranscriptionofcccDNAresultsintheformationoffourmRNAsofsizes3.5kb,
2.4kb,2.1kband0.7kbthataretranslatedintosevenviralproteins(Figure4).The3.5kb
genomic mRNA includes precore (preC) mRNA that is translated into secretory “e
antigen”HBeAgproteinandpregenomicRNA(pgRNA)that is translated intocoreand
polymerase proteins needed for viral replication. They are larger that genome length
because of the presence of terminal redundancies on both 3’ and 5’ ends. The other
transcripts termed subgenomic RNAs are represented by PreS1 mRNA (2.4 kb) that
translatesintoLHBsAg,thePreS2/SmRNA(2.1kb)forMandSHBsAg,andXmRNA(0.7
kb)forregulatoryHBxprotein.
5
Figure 4 : Genomic placements of different ORFs and genomewith polyadenylation at 3’ and 5’
regionsarebothrepresentedinalinearform.AllRNAstranscriptsofdifferentlengthwitha5’cap
anda3’polyadenylation(An)isrepresented.
The pgRNA and precore RNA encompasses two highly conserved sequence regions
termedasepsilon(e)thatformsasecondarystemloopstructureatthe5’and3’regions.
The 5’ e structure directs encapsidation of RNA along with viral polymerase that
undergoesreversetranscriptiontoformfunctionallyinfectiousDNAcontainingvirions
[16-18].However,onlypgRNAisencapsidatedtoforminfectiousDNA[19]whileprecore
RNAiscleavedintheendoplasmicreticulumandissecretedasHBeAg.Figure5describes
highly precise steps required to convert pgRNA into infectious viral genome. These
replication steps generate encapsidated relaxed circular HBV DNA (rcDNA), that can
either(i)acquireHBsAgandbesecretedasviralparticlesor(ii)berecycledwithinthe
hepatocytetoincreaseormaintaincccDNApool[20].
Alongwith themature virion, secretionof empty nucleocapsids, RNA containing viral
particles and to some extent double stranded linear forms (dslDNA) has also been
observed[21,22].Duringplusstrandsynthesis,primertranslocationfailuremaycause
viral genome to remain linear instead of circularizing to form rcDNA. This double
6
strandedlinear(dslDNA)canrecirculateintothehepatocytenucleusandbeintegrated
intothehostchromosome[23].
Figure5:SequenceofstepsfollowingpgRNAencapsidationisrepresented.(A)showslinearpgRNA
within capsid. (B)Bindingof polymerase to thee loopat the5’ end. (C) Synthesis of short oligo
followedbytranslocationofthepolymeraseandtheoligotothedirectrepeat1(DR1)regionwhere
minusstrandsynthesisisinitiated.(D)Minusstrandsynthesisisaccompaniedbydegradationofthe
pgRNAtemplatewhereonlyDR1sequenceandtheshortoligofromthe5’endremains.Thisactsas
precursorfortheplusstrandsynthesisandafteratemplateswitch(representedinE)plusstrand
synthesisbeginsandsubsequentlycircularized(representedinF).
7
Figure6:HBVreplicationcycle
Serological and intrahepatic markers
Clinical staging of HBV infection is primarily done using quantifiable serological and
intrahepaticmarkers.HBViswhatisknownasastealthvirus,thatdependingontheage
atwhichinfectionisacquired,canreplicatefordecadesbeforeimmuneflareisinitiated.
ReplicationofHBVstarts immediatelypost infectionandmatureviralDNAalongwith
HBsAgbecomesdetectableintheblood.PresenceofHBsAgforlongerthan6monthsis
definedclinicallyas“chronicinfection”anditsclearanceisusuallyaccompaniedbythe
appearance of anti-HBs antibodies. Therefore, anti-HBs is an accepted marker to
determine end-point for treatment [24].HBeAg secreted into the blood from infected
hepatocytesisamarkertodetermineviralreplication[25,26].HighHBVDNAlevelsmay
howeverbepresentalsoinpatientsseronegativeforHBeAg,becausemutationsespecially
intheprecoreregion,mayemergeandprecludesynthesisofHBeAg,particularlyinHBV
genotypesB,DorE[27-29].PresenceofHBVRNAinserumfirstreportedin2001has
beensuggestedasadiagnosticmarkersincethen,inparticularformonitoringtheeffect
8
oncccDNAduringantiviraltreatment[30-33].DuringtreatmentofHBVinfectionusing
NA,HBVDNAisreducedtolevelsbelowthequantificationlimit,afterwhichtheeffectof
treatment cannot be assessed. Quantification of HBsAg in serum was proposed to
represent cccDNA,but itwas later found that thiswas found tonotbe true, since the
declineofHBsAglevelswasminimal.SinceHBVRNAisnotdirectlyreducedbydrugsthat
inhibitreversetranscription,itshouldbeusefulasserummarkerrepresentingcccDNA
levelsintheliver[34].
Intrahepatic cccDNA a highly stable mini-chromosome is the template needed to
synthesizeHBVRNAtranscriptsnecessaryforvirusproduction.Reductionofviralload
by two log10 units during HBeAg negative stage is caused by the loss of intrahepatic
cccDNA copies [35, 36]. Induckmodel, it hasbeen found that the number of cccDNA
moleculesmay vary over time,withmeannumber of cccDNAper infected hepatocyte
rangingbetween3and9[36,37].Becauseofitsroleinmaintainingchronicinfectionand
abilitytoavoiddruginducedclearance[38,39],quantifyingcccDNAandunderstanding
its relationship to other viral markers are important. Many approaches have been
designedtoquantifycccDNAintheliver[36,40,41]butbecauseofitspresenceinlow
concentrations, conventional approaches such as southern blot are not adequately
sensitive[42].AchallengefortheseassaysistobeabletodifferentiatebetweencccDNA
fromrcDNA,thereforeapolymerasechainbasedassaywasdesignedtotargetthegapin
plus strand region that would be present only in cccDNA [36, 43]. In addition to
intrahepaticmolecularmarkers suchas cccDNAandHBVDNA immunohistochemistry
canbeperformedtovisualizeHBsAg,HBcAg,HBxAgandpre-Speptidesonlivertissue.
Althoughliverbiopsiesprovidevaluabledata,thisinvasivesamplecollectionmethodhas
to a large extent been replaced by non-invasive techniques such as elastography
assessmentofliverfibrosis,atrendthatwillhamperfutureresearch.Adrawbacktousing
biopsy tissues is the riskof samplingerror,where thematerial analyzed isnota true
representationofthediseaseorinfectionstate.
9
Natural history of infection
DiversenomenclatureshavebeendefinedtodescribetheclinicalphasesofHBVinfection.
Discussed here are phases according to European Association for the Study of Liver
(EASL)guidelinesonHBVinfectionmanagement(i)HBeAgpositivechronicHBVinfection
(ii)HBeAgpositive chronichepatitisB (iii)HBeAgnegative chronicHBVinfection (iv)
HBeAgnegativechronichepatitisB(v)HBsAgnegativephase[24].
1.4.1 HBeAg positive chronic HBV infection / Immune tolerance
HBVinfectionthatisacquiredduringearlychildhood(<2years),usuallythroughvertical
transmission at birth or later horizontal transmission, usually inducesweak immune
responses for several decades. During this phase of HBV infection (often termed the
immunetolerancephase),HBVinfectsalmostall livercellsandreplicatesactivelywith
typically > 107 IU/mL HBV DNA detected in patient serum. The individual remains
asymptomatic at this stage and shows normal to mild elevation of alanine
aminotransferase(ALT)levelswithnormalliverhistologyanddetectableHBeAgdetected
in serum. So far, treatment interventions during immune tolerant phase has not been
recommended[44-48].
1.4.2 HBeAg positive chronic hepatitis B
Typicallyseentolastfromthe3rdto4thdecadepostinfection.Thisphaseischaracterized
byactiveimmunemediatedclearanceofHBVinfection,duringwhich,infectedlivercells
areeradicated,causingelevationinALTlevels.Thedurationandlevelofcellulardamage
totheliverdeterminesifthepatientgetsHBVmediatedfibrosisorcirrhosis.Eventually
most patients seroconvert to anti-HBewith suppressionofHBVDNA.However, some
patients fail to control viral HBV replication thereby progressing to HBeAg negative
hepatitis.
1.4.3 HBeAg negative CHB / inactive carrier
Thedetectionofanti-HBealongwithundetectableorsignificantreductionofviralDNA
andsubstantialreductionornormalizationofALTlevelsinserumofCHBpatientindicates
10
clinicalremission.HBVDNAlevelsvarybetween<2000–20,000IU/mLatthispointand
insomepatientsthiscanleadtoHBsAgseroconversion[49].
1.4.4 HBeAg negative chronic hepatitis
Progressionfrombeinganinactivecarriertoanindividualwithchronicinfectionishighly
variableand isassociatedwith factors likeageatwhich infectionwasacquired,ageof
HBeAgseroconversion,durationofinfectionandgenotype.IncreaseinserumHBVDNA
andALTlevelshavebeenobservedinlongtermfollowupstudiesintheabsenceofHBeAg
[49-53].This ismainlyduetothepresenceofmutations inprecoreregionof theviral
genome that survive as an escape mutant and causes relapse of chronic infection
(discussedindetailunderthetopicgenomicvariations)[54,55].
1.4.5 HBsAg negative phase
DuringthisphasepatientsundergolossofHBsAgwithorwithoutdevelopmentofanti-
HBsalongwithanti-HBcantibodiesandshownormalALTlevelsalongwithundetectable
HBVDNAandpresenceofcccDNAinlivertissue.Insomecasesdevelopmentof“Occult
HBV”takesplace,wherepatientremainsnegative forHBsAg inthepresenceoflowor
undetectablecirculatingviral load.LossHBsAgthatoccurseitherspontaneouslyorby
treatmentinterferencealthoughconsideredas“functionalcure”doesnoteliminatethe
probability of HCC. Development of cirrhosis before HBsAg seroclearance has been
associatedwithHCCincidencetherebyincreasingtheneedforsurveillanceafterreaching
treatmentendpoint[56-58].
11
Figure 7 : History of HBV infection as defined by EASL guidelines. There is no clear distinction
betweenthesephasesandonlyaroughestimateisdefinedhere.
Integrations
SimilaritiesbetweenHBVandretroviruseshavebeendiscussedincludingthepotential
forsuccessfullyintegrateintothehostchromosomes[59].Butunlikeretrovirusesthat
need integration forreplication,HBVintegrationsaredefinedasreplicativedeadends
12
withthepotentialtoproduceviralproteinsevenintheabsenceofactivereplication[60,
61].Primertranslocationfailureduringplusstrandsynthesisresultsintheformationof
double stranded liner DNA (dslDNA)which separates the precore/core gene from its
promoter. Therefore, this form can neither synthesize pregenomic RNA nor generate
capsids.SincedslDNAistheprecursorforintegration[62],onlySgenethatremainsintact
issuspectedtobeactiveandbecapableofproducingHBsAgevenintheabsenceofHBV
replication as indicated by low HBV DNA. This phenomenon was first observed in
hepatocellularcarcinomacell linePLC/PRF/5[63]andhasbeendescribed inarecent
studyconductedonchimpanzees[61].Integrationshavebeenobservedatearlystagesof
infectionandhasbeenassociatedwithincidenceofHCCandaffectsurvivalofinfected
individuals[64].Doublestrandedbreaksinhostchromosomeshavebeenestablishedas
thepreferentialsiteforintegrations[65],wherethehost’scellularmechanismsintegrates
viral into chromosomal DNA damage response (DDR). It has been accepted that DDR
pathways lead to HBV integrations, however the exact methodology by which this is
achievedisdebated.Somestudiessuggestnon-homologousendjoining(NHEJ)[65]for
doublestrandedbreakrepairareresponsibleforviralintegrationswhereasothersargue
thatitoccursviamicrohomology-mediatedendjoining(MMEJ)method[66].
Genomicvariations
1.6.1 Genotypes and Sub-genotypes
The polymerase present in mature HBV lacks proof reading ability causing the virus
withinthecapsidtocontaingenomicsequenceswithvariationsandhasresultedinan
array of viral strains. When the genomic sequences have a variation >8% they are
classified as genotypes andwith variationwithin a genotype between 4-8% they are
defined as sub-genotypes. Ten different genotypes and 30 sub-genotypes have been
identified so far with a distinct geographical distribution pattern. Variations in these
genotypesalsoextendtotheirclinicalandvirologicalfeatures.Largelydiscussedarethe
observationswithregardstodiseaseprogression[67],treatmentresponse,development
of cirrhosis and capability to advance toward HCC [68, 69]. Two studies comparing
genotypesBandCshowedthatthelatterhasthepotentialinassociationwithHBeAgto
cause severe liver disease [27, 70]. Results from a Taiwanese study observed higher
13
frequenciesofcirrhosisandHCCingenotypeCwhencomparedtogenotypeD[69].The
samestudyalsodiscussedlargerincidentsofHCCinnon-cirrhoticpatientscomparedto
genotypeB.InaEuropeanstudycomparinggenotypeAandD,theformerwasseento
causemorechronicinfections[71]andshowedlargerincidentsofremissionpostHBeAg
seroconversionthanthelatter[72].FrequenciesofHBVrelateddeathsarereportedtobe
higherinpatientswithgenotypeFwhencomparedtogenotypeA[72].
Thesestudies furtherpressonthe importanceofunderstandingdifferencescausedby
genotypesonviralload,clinicalmanifestationsandresponsetotreatment;supportbetter
comprehensionandaidinadvancementofclinicalpractices.
Figure8:GeographicaldistributionofHBVgenotypes
Inadditiontothelackofpolymerase’sproof-readingability,geneticvariabilitybecauseof
mutations are a product of immune selective pressure and drug resistance. A study
conductedonwoodchucksin1989approximated1erroroccurringevery107bases[73,
74].Selectionpressurefromimmuneresponsesthattargetwildtypevirusesmayleadto
emergenceofgenomeswithmutations.Mutationshavebeenobservedinall4ORFswith
14
theircontributiontoclinicaloutcomesandattributiontodiseaseprogressionextensively
researched[75].
1.6.2 S ORF mutation
Ahighlyconservedaminoacid(aa)regiontermedas“a”determinantinHBsAgisselected
as a target for vaccine against HBV aswell as for clinical diagnosis of disease status.
Mutationsinthisregionwasfirstdocumentedinapatientthathadverticallytransmitted
HBVevenaftervaccinationwithbothHBVDNAandanti-HBspresenceinserum[76].Lack
ofepitoperecognitioncanthusleadtovaccinefailure[76]andunreliableresultsfrom
diagnosticassays[77].
1.6.3 Precore / core ORF mutation
The precore and core regions are responsible for the production of twoproteins, the
structuralcoreandthesecretory‘e’protein.HBeAgisnotrequiredforviralproduction
buthasbeensuspectedtoserveasadecoytocurbimmuneresponseagainstcore[78],
butimmunetoleranceiseventuallylostandanti-HBeisproducedalongwithreduction
and inmany patients, clearance of HBV DNA. Failure to clear HBV DNA is seenwith
emergence of several mutations in the basal core promoter (BCP) and precore (PC)
regions suggesting selection due to immune pressure [79]. The most common PC
mutationoccursatposition1896whereguanine(G)isreplacedbyadenine(A)(G1896A)
which induces a stop codon in HBeAg sequence and abrogates HBeAg synthesis [80].
Another commonmutation is seenat1899whereguanine (G) is replaced by adenine
G1899AandthismutationwasfoundbothbyitselfandalongwithA1896Tmutation[80].
Mutations in the BCP region, 1762 (A1762T) and 1764 (G1764A) have also been
associatedwithcontributingtothereductioninHBeAgexpressionandiscorrelatedwith
higherHCCincidents[81].LowHBVDNAquantificationsareobservedinpatientswithPC
mutations alongwith lower ALT levels that suggest lesser liver damage compared to
infectionwithwildtypevirus.HoweverBCPmutationssuchasthyminereplacementwith
cytosine (C) at 1753 (T1753C) and also at C1766T reduce HBe secretion via
transcriptional mechanisms and have shown increase in HBV DNA and ALT levels
thereforesuggestinghigherprevalenceofprogressiontowardscirrhosis[82,83].
15
1.6.4 X ORF mutation
The X gene is only found in mammalian hepadnavirus and has important functions
includingabilitytoestablishaninfection[84],promoteviralreplication[85],andsupport
developmentofHCC[86].ThemutationsintheXORFhavebeencorrelatedtoenhanced
progressiontowardslivercirrhosis(LC)andHCC[83,87-89].
1.6.5 Polymerase ORF mutation
Polymerase ORF encodes for the polymerase protein that is encapsidated along with
pgRNAandisresponsibleformanystepsthatarenecessaryforsuccessfulcompletionof
HBVreplicationcycle[90-92].Amutation intheRTregionof thepolymeraseORFcan
dictate and alter replication events. Investigations regarding naturally occurring
mutationshavebeenundertakeninthepastfewyearsbecauseitcouldgiveinformation
about possible drug resistance [93, 94]. This may alter a clinician’s decision about
treatmentofapatientwiththesemutations.Associationsbetweendifferentmutationsin
theRT region and viral load,degree of liver disease have been published [94-96]. RT
inhibitors are commonly used for the treatment of HBV but can induce a selection
pressure that leads to proliferation of drug-resistant HBV strains. One of the most
commonmutations induced by the antiviral drug Lamivudine is seen in the tyrosine-
methionine-aspartate(YMDD)motifofHBVpolymerasearoundnucleotide552[97].This
mutation however is associated with low serum titers of the virus and shows low
probabilitytocauseadverseliverdisease[8].
Treatment
TwoclassesofdrugshavebeenapprovedforthetreatmentofCHB,immunomodulating
agents and nucleoside / nucleotide analogues in order to reduce viral load during
infection to achieve seroconversion of HBeAg and within short duration tominimize
immunemediatedliverdamageandpreventionofadverseeventssuchasLCandHCC.
The drugs used for management of HBV that exists now only manage to keep viral
replicationunder control, and does not eliminate the highly stablemini chromosome,
cccDNA[38,98-100].
16
1.7.1 Immunomodulators
Interferonsarenaturallyproducedproteinsthataresynthesizedbycellsinresponseto
viral infections to induce immune response in retaliation. Immunomodulators suchas
interferonalpha(IFN-a)andPegylatedinterferon(PEG-IFN)areusedinCHBtreatment
becauseofitspotentialtobindtocellularreceptorsandactivateproteinsynthesisneeded
for cellular defense against invading virus [101]. These drugs cannot however be
prescribedtopatientswhoareaffectedbydecompensationoftheliversuchascirrhosis
[102].
1.7.2 Nucleoside / Nucleotide analogues
Drugs under these classifications were initially developed for treatment against
herpesvirus and human immunodeficiency virus (HIV) because of their potential to
impairpolymerasefunctionandcanbeadaptedtoHBVtreatment.Therearecurrentlysix
drugs that are approved for the treatment of CHB, (i) lamivudine (ii) entecavir (iii)
adefovir(iv)tenofovir(v)tenofoviralafenamide(vi)telbivudine.Thesemedicationsare
takenorallyandaresafetousehowever,sincetheyonlyblockRT,longtermtreatmentis
required.Theformationofdruginducedresistantmutationswasamainproblemearlier,
sincelamivudineresistancedevelopedin15-32%ofpatientswithinoneyearoftreatment
[8].Thecurrentlyuseddrugstenofovirandentecavirhowever,havehighbarriersagainst
developmentofresistancemutations[103-105].
Cell culture & animal models
Cellcultureandanimalmodelsfacilitatebetterunderstandingofadiseaseandaidswith
treatmentdevelopment.DuetospecifichostrequirementsforHBV,therearelimitations
onanimalmodelsthatcanbeusedforresearch[106].Someanimalssuchaschimpanzees,
tupaias,ducksandhumanchimericmicehavebeenusedtostudyHBV[107].Ofthese,the
immune responseof chimpanzees are the closest tohumans and itwas precisely this
reasonthatledtotheuseofthismodeltotestefficacyofpreventiveHBVvaccine[108].
CellculturesystemssuchashepatomacelllinesHepG2andHuh7havebeenavailablefor
alongtimenowandhavesupportedresearchregardingtranscriptionandsynthesisof
17
new virion particles [109-111]. Even though they are hepatoma cell lines and are
availableinplenty,theydonotsupportinfectionduetothelackofthereceptorNTCP.
HepRG cells can support infection, however they require complicated differentiation
stepsinafashionsimilartotheinducedhepatocytes(iHep)[112].Comparingthesetwo
celllines,HepRGhaslowerefficiencyofinfectionthaniHepandcansupporttheentirelife
cycleofthevirus.Primaryhumanhepatocytes(PHH)isanothersystemthatisusedto
study HBV infection and although this is the closest condition possible tomimicking
naturalstateofinfection,itislessefficient,moreexpensiveandishugelydependenton
donoravailability[113].NTCPwasrecentlydiscoveredasthecellularreceptorforHBV/
HDVviralentry[11,114].ThereceptorwasthenexpressedintheHepG2hepatomacell
linetomakeitsusceptibletoHBV/HDVinfection.Thisdiscoveryopenedupnewavenues
toexploredifferentfacetsofinfectionandreplication.Althoughthesecellscanbeinfected
there are certain limitations that needs to be addressed. This system requires large
quantitiesofinputanddoesnotpromotelongterminfection;sincethisiscancerouscell
line, it lacksmany of the pathways needed to study host-virus interactions; and post
infectiononlyasmallamountofcccDNAisexpressed;althoughthecellsystemcanbe
infected,itneedsinadditionpolyethyleneglycol(PEG)toenhanceinfectionbypromoting
glycosaminoglycanbindinganddimethylsulphoxide(DMSO)toaidinfection.
18
2 INTRODUCTIONTOHEPATITISDELTAVIRUS
In1977anewantigenassociatedwithHBVwasdiscoveredinpatientswhowereHBsAg
positive [115]. It was later in 1980 using chimpanzees as a model system that delta
antigen was determined to be a defective infectious agent that interferes with HBV
replication[116].Sincethenmanystudieshaveshownhepatitisdeltavirus(HDV)tobea
satellitevirustoHBVthatrequiressurfaceantigenproductionfromHBVtoenvelopeits
genometogainaccessintohepatocytesforsuccessfulinfection[116].
HDVisthesmallestknownRNAvirusthatinfectsmammals[117]andistheonlymember
of the deltavirus genus. Transmission of HDV happens in the same routes as HBV,
percutaneouslyviasharingofbloodproductsorthroughsexualtransmissionorinrare
cases via vertical transmission. A study in 2003 estimated that 5% of the world’s
populationwithHBValsohasHDV[118],butaccordingtoWHOmanycountriesdonot
test for HDV co-infection in HBV positive patients and in countries such asMongolia
where60%ofthepopulationtestedpositiveforHBVmayalsobeinfectedwithHDV[3].
HDVmaybeacquiredasasimultaneousinfectionwithHBVcausingmildtosevereacute
hepatitis,wheredevelopmentintochronicHDVinfectionisrareoccurringinlessthat5%
of the cases [119]. If an individual with chronic HBV is superinfected with HDV,
developmentofchronicHDVinfectionisalmostinevitable,leadingtoLCmoreoftenand
fasterthaninapatientwithHBVmonoinfection[116,120].
HDV genome and replication
HDV is a small single stranded RNA virus with a circular genome containing 1679
nucleotides.With74%intramolecularbasepairing[121], thegenomeformsarodlike
structurethatcanbefoundalongwithdeltaantigen(small,S-dAgandlarge,L-dAg).
Forviralreplication,HDVengagesinamechanismtermedas“thedoublerolling-circle
amplification”whereitusesthehost’scellularpolymerasetosynthesizeacomplimentary
RNAstrandcalledtheantigenomicdRNAandanmRNAwith5’capandapolyadenylated
19
3’endthatcodesforthedeltaantigenprotein[122].Withtheproductionofantigenome,
viralreplicationcontinuesandthevirusissecretedwithanenvelopecontainingHBsAg.
Figure 9: (A) Single stranded HDV with large and small forms of d-antigen. (B) HDV genome
surroundedbyS,MandLsurfaceproteinsfromHBV.
Figure10:HDVreplicationcycleaidedbyHBsAgsynthesisbyHBV.Highlightedinpinkisthe“Rolling
circlemechanism”ofHDVreplication.
20
TwovariantsofdeltaantigenaresynthesizedfromthemRNAandtheyhavetwodistinct
functions. The S-dAg is responsible for viral replication where-as the L-dAg inhibits
replicationandpromotesvirionassembly.Therefore, thebalance in theproductionof
theseproteinsisveryimportanttoestablishasuccessfulinfection.
HBV suppression by HDV
IthasbeenrecordedincellcultureandanimalmodelsystemsthatthepresenceofHDV
reduces the expression ofHBV [123]. Themechanism throughwhich this is achieved
remainsunknown,butincellculturemodelsystemstheL-dAgandS-dAghaveshownthe
abilitytoactivateMxAgenetoincreaseIFN-aresponsetowardsHBVandactivatecellular
immuneproteinsynthesistocontrolinfection[124-126].
Treatment
SinceHBsAg isnecessary for successfulHDV infectionandsubsequent replication, the
currentpreventivevaccineforHBVthattargetsandneutralizesthesurfaceantigenworks
veryeffectivelyagainstHDV[127].Butonceinfectionisactivedthetreatmentoptionsare
quitebleak.SinceHDVonlyrequiressurfaceantigenfromHBV,blockingHBVreplication
isnoteffective[128],andtheonlytreatmentforHDVcurrentlyavailableisINF-a[129].
TreatmentwithINF-ahasshownsignificantimprovementsinhistologicalresponseand
lossofHDVRNAandHBsAginsomepatients[130],butlongtermstudieshavealsoshown
relapseinmostpatientswithfailuretoclearHDV[131].Afewnewtreatmentoptionsfor
chronicHDVinfectionsareunderway,includingMyrcludexBthatblocksHBV/HDVviral
entryintohepatocytes[132],butin-vivoithasbeenshownthatHDVcanpropagatevia
celldivisioneveninthepresenceofthisdrug[133].
21
3 AIMS
Theoverallaimofthisprojectwastoexploreandunderstanddifferentfacetsofchronic
hepatitis B virus infection by means of studying patient serum and liver tissue from
biopsiesandexplantedliver.Specificaimswereasfollows:
PaperI
ToquantifyandcharacterizeHBVRNA in serum in termsof sequence length,particle
association,concentrationandcorrelationswithHBVRNAinlivertissueandHBVDNAin
serum
PaperII
Toinvestigateinvitrotheinfluenceofsubviralparticleoninfectionandexploreitseffects
onanti-HBs.
PaperIII
TodevelopanewmethodtoanalyzeHBVDNAinlivertissueinordertodiscriminateviral
andintegratedDNA.
PaperIV
Toanalyzeintrahepaticfocaldifferencesinthepatientsundergoingtransplantationdue
toHBVorHBV/HDVrelatedliverdisease.
PaperIV
ToexploreHBVRNAprofilesinliverbiopsiesofpatientsrepresentingdifferentstagesof
chronicinfectionbyusingdropletdigitalPCRmethod.
22
4 MATERIALSANDMETHODS
Materials
PaperI:95patientserumfromapreviousstudythatincluded160sampleswasstored
andavailable for furtheranalysis [134].ThesesamplesrepresentbothHBeAgpositive
andnegativegroupsbelongingtogenotypesA,B,CandD.Oneanonymouspatientsample
withHBVDNAconcentrationof108IU/mLthatalsotestedpositiveforHBeAgandHBsAg
wasincludedinthisstudy.
PaperII:TwoanonymousserumsamplesonewithpositiveHBeAgandHBsAgwithHBV
DNAconcentrationwith108IU/mLandtheothernegativeforHBsAgandanti-HBcfrom
avaccinated individualwereselected.Boththesesampleswerealsonegative foranti-
HCV,anti-HIV,anti-HAVIgMandpositiveforanti-HAVIgG.
Paper III : 76 stored liver biopsies out of 160 thatwere included in a previous cross
sectionalstudywereavailableforfurtheranalysis[134]ofwhich70werepositivewith
digitalPCRmethod.ThesampleschosenrepresentedgenotypesA,B,CandD,withliver
damage ranging frommild to severe, either with or without HBeAg. They were also
negative for HIV, hepatitis C or D and were untreated for HBV when biopsy was
performed.
PaperIV:Thisstudyincludedtissuematerialobtainedfrom6patientsundergoingliver
transplantbecauseofHBVrelatedchronicliverdiseasewhereallpatientspresentedliver
cirrhosis. Two patients had HCC; one patient with cirrhosis developed acute liver
decompensationbroughtonbyacute-on-chronichepatitisandpresentedwithveryhigh
serumHBVDNA;twopatientswerealsoco-infectedwithHDV.Sampleswerecollected
duringsurgeryandsubsequentlystoredat-80°Cuntilitwaspreparedforanalysis.
PaperV:76ofthe77liverbiopsiesthatwereusedinpaperIIIwerealsousedforanalysis
inthiswork.
23
Figure11:Schematicrepresentationofpatientsamplesusedindifferentpaperscompiledinthis
thesis.
Methods
4.2.1 Nucleic acid extraction from serum & tissues
Total nucleic acid extractionwas carriedouton both tissue and serumsamples in an
automatedMagNAPureLCsystem(RocheAppliedScience)accordingtomanufacturer’s
protocol. For serum extractionMagNA Pure LC total nucleic acid isolation kit (Roche
AppliedScience)wasusedandfor tissuematerialMagNAPureLCDNAisolationkit II
(Roche)wasused.
4.2.2 DNase and RNAse treatment
A portion of the total nucleic acid extracted was treated with TURBO DNA-free kit
(ThermoFisherScientific)inarigoroustwo-stepprotocolaccordingtomanufacturer’s
instructiontoremovecontaminatingDNA,forRNAanalysis.
24
ASerumsamplewas treatedwithRNAse (ThermoFisherScientific)prior toandpost
MagNAPureLC(RocheAppliedScience)extractiontoquantifyfreeHBVRNAcontentin
serum.
4.2.3 Polymerase chain reaction
MolecularbiologytechniqueshavecomealongwaysinceestablishingtheWatson-Crick
modelin1953.Thepolymerasechainreaction(PCR)methodwasinventedin1983and
allowsamplificationofspecificDNAsegmentofinterest.Itwasbasedontheidentification
ofthespeciesThermusaquaticusin1969thatrequiresatemperatureof70–79°Cforits
growth[135],andtheisolationofitspolymerasetermedasthe“TaqPolymerase”in1976
[136],anenzymethatcouldwithstandhightemperatures.Thisenzymeisusedtocopya
specificDNA sequence in a PCR reaction that is supplementedwith (i) a target to be
amplified(ii)oligonucleotidesequencescalledprimerthatarespecifictothetarget(iii)
nucleotidesneededforcreatingnewtargetsequences(iv)Taqpolymerasetoplaceeach
nucleotideintherightorderforsequenceextension.
4.2.3.1 Reverse Transcriptase PCR
TheonlydifferencebetweenastandardPCRandareversetranscriptasePCR(RT-PCR)is
the template used for amplification. The former uses double stranded DNA (dsDNA)
whereasthelatterusesRNAasstartingmaterial.DuringanRT-PCR,anenzymetermed
asreversetranscriptaseisusedtocreateacomplimentaryDNA(cDNA)strandtotheRNA.
ThisenzymealsocontainstheRNAseHactivitywheretheRNAtemplateisdegradedafter
copying.Now,usingthenewcDNAastemplateasecondstrandissynthesizedtohavea
completedsDNA.Amplificationof thisnewlysynthesizedDNAiscarriedoutusingthe
principlesofstandardPCR.
4.2.3.2 Quantitative PCR and quantitative RT PCR
Aquantitative PCR (qPCR) is amethod that iswidely used in clinical diagnostics and
research alike. There are typically twomethods available to quantify the sequence of
interest,(i)dye-basedmethodand(ii)probe-basedmethod.Inthefirstmethod,agreen
fluorescent dye that intercalates with all double stranded products is used and the
increaseinfluorescenceismeasuredateachcycle[137].Thetechniquethatisextensively
used inthisstudy isprobebasedPCRmethodthat justlikethedye-basedmethodcan
25
measurein“realtime”theamplificationoftargetsequence.Thisproceduremakesuseof
astrandspecificprobethatistaggedwithafluorophoreononeendandaquencheron
theother.Whenkeptincloseproximity,thequencherquenchesthatfluorescenceemitted
bythefluorophore.Thepolymeraseinadditiontoaddingspecificnucleotidesforstrand
extensionhasa second responsibility to removeanydouble strandedsequences in its
pathand replace itwithavailablenucleotidesthis is termedas “exonucleaseactivity”.
Therefore, when it encounters the probe, it cleaves the probe thereby separating the
fluorophorefromthequencherandthefluorescencenowemittedcanbecapturedand
measured.Anadvantagetousingthistechniqueistheabilitytomeasuremultipletargets
inthesamereactionwhichwaspursuedonpaperIVandVusingthedigitalPCRmethod.
ForanRNA target, reverse transcriptase (RT)enzymewas included to create the first
cDNAstrandandthestandardPCRprotocolwasfollowed.
4.2.3.3 Digital PCR
ThetermDigitalPCR(ddPCR)wascoinedin1999whentworesearchersBertVogelstein
andKennethKinzlerstudyingraremutationsincolorectalcancerdevelopedaPCRsystem
wheremultiplereactionswereusedtostudyasingletargetfromthesamepatientsample
[138]. The disadvantage with a qPCR system is that it requires a standard curve to
determine the initial quantity that was present in the reaction which means small
variationsinthesamplesgounnoticed.Toavoidthis,VogelsteinandKinzlerhybridized
twofluorescentprobestotheamplifiedproductonethatbindstothespecifictargetand
thesecondthatbindstoallsequencesasacontrolandmeasuredthe fluorescence.By
doing this they obtained an absolute quantification of the mutation. This came with
certaindisadvantagesliketimerequiredfortheanalysisandtheneedforlargequantity
of starting material. The simplified commercialized procedure now enables the
separationofeachreactioninto20,000nanodropletsusingwater-emulsiontechnology.
FollowingastandardPCR,targetsamplifiedisassessedbyanautomatedsystemwhere
the fluorescence ismeasuredwithineachdroplet.UsingPoisson statisticsanabsolute
quantificationofthetargetcanbeobtained.
4.2.4 Nycodenz fractionation
FractionationofserumsampleinpaperItoshowthepresenceofviralRNAwithincapsids
andinpaperIItoseparateviralandsubviralparticleswascarriedoutusingNycodenz–
26
asubstanceusedcommonlyforisolationofmanycelltypes.Thissubstanceiswidelyused
becauseofitsnon-cytotoxicnature,abilitytobeautoclavedwithoutcompromiseandnon-
interferencewithdownstreamanalysis [139-141].Thismethodwasutilized inpaper I
and II where different products synthesized by HBV infection present in serum was
separated in different Nycodenz fractions depending on their density. The separated
fractions were then subjected to analysis such as TaqMan PCR, RT PCR and in-vitro
infectionofHepG2NTCPcells.
4.2.5 Sanger sequencing
ExtractedHBVRNAsequencefrompatientserumwasinthepresenceofRTenzymeand
areverseprimerthattargetsthepoly-Atailatthe3’end,usedtocreateacomplimentary
cDNAstrand.ThecDNAwasthensubjectedtotwoPCRsthattargetthe5’and3’regions
ofthecDNAandtheampliconsobtainedweresequencedusingSangermethodtoidentify
mutations that may contribute to the presence of RNA in serum where reverse
transcriptionhasnotoccurred.
4.2.6 In-vitro infection
Invitrocellculturetechniquesarebeingdevelopedrigorouslytoefficientlyreplicatean
infectionoutsidethehostinordertoimproveunderstandingofthecausativeagentand
developappropriatetreatment.HepG2NTCPcelllinewasrecentlyestablishedafterthe
discoveryofthereceptorNTCPneededforHBVtogainentryintothecells[114].HepG2
cellsstablytransfectedwithHBVspecificreceptorNTCPwasmaintainedat37°Cunder
5% CO2 in Dulbecco’s modified Eagles medium (DMEM) containing high glucose
(Invitrogen).InaccordancewiththeprotocoldefinedbyNietal.,in2014,themediaused
for culture maintenance and subsequent infection was substituted with 10% FBS,
100U/mL penicillin, 0.1 mg/mL streptomycin and 2mM L-glutamine [11]. Using a
hemocytometercellswerecountedandseededtogiveapproximately1.25*105cellsper
well.24hourslaterwhencellsreachedoptimumconfluency,HBVserumsamplewithor
withoutpriorincubationwithanti-HBsantibodieswasaddedtothecellsalongwith2.5%
dimethylsulfoxide(DMSO)(Sigma)and4%polyethyleneglycol800(PEG800)(Sigma)
andincubatedtostartinfectionexperiment.Thecellswerethenwashedthreetimeswith
DMEMafter24hoursandreplacedwithfreshculturemedia(CM).Everyseconddaythe
27
CMfromeachwellwascollectedandstoredfordownstreamanalysisandreplacedwith
freshCMspikedwith2.5%DMSOuntiltheendoftheexperiment.
4.2.7 Architect Assay
ThistechniquewasusedtoquantifyHBeAgsecretedbyinfectedHepG2NTCPcells.Inthis
two-stepimmunoassay,thesampleisfirstfloodedwithanti-HBecoatedmicroparticles
followedby introductiontoacridiniumlabeledconjugatedanti-HBe.Awashingstep is
includedafterbothstagestoremoveanyunboundproducts.Thechemiluminescencethat
isproducedbythebound,labelledtargetiscapturedandcomparedtosignalcutoffratio
measuredduringcalibrationoftheinstrumenttoobtainaquantificationofHBeAginCM
frominfectedcells.
28
5 RESULTSANDDISCUSSION
Paper I
NAsusedforCHBtreatment,blockpolymeraseactivitythatleadstorapidreductionof
HBVDNA,aserologicalmarkerquantifiedtomonitordiseaseprogression[24]aswellas
a predictor for liver cirrhosis andHCCdevelopment [142].HBVRNAhowever, is not
affectedbythetreatmentandthereforecanbeusedasamarkeroftheinfectioninthe
liver[31-33]inadditiontoserumHBsAg[143-145].
Inorder to characterizeHBVRNAdetected in serum,density-based fractionationwas
performedusingsixconcentrationsofNycodenzgradients(8,16,25,33,42,and50wt%)
followedbyquantificationusingrealtimePCR.HBVRNAandDNAparticlesweredetected
inthesamefractionssuggestingsimilardensities(figure12A).Anearlierstudyusing
sucrosegradienttofractionateHBVDNAandRNApostentecavirtreatmenthasshown
similar results [146]. Since mature HBV virions are present within nucleocapsids,
treatmentwithdetergentTween-80disrupted these capsids causing thepeak fraction
containingHBVDNA tomove toward higherNycodenz concentration (figure 12B). A
similarpatterninRNAsuggestedpresenceofRNAwithinenvelopedcapsids(figure12
B).
Figure12:(A)NycodenzgradientcentrifugationofserumsampleshowingseparationofHBVDNA
andRNA(B)NycodenzfractionationofHBVpositiveserumafterTween-80treatment.
HBVDNAandRNAwerequantifiedinserumcollectfrom95patientschronicallyinfected
withHBV.ThelevelsofHBVRNApresentweresimilartoHBVDNAinbothHBeAgpositive
andnegativepatientgroups(figure13A).
29
Figure13:(A)correlationbetweenHBVDNAandRNApresentinserum(B)GenotypeDshowing
significantlyhigherratiobetweenHBVDNAandRNAincomparisonwithnon-genotypeDsamples
(p=0.0001).
Sequencingwasusedtocharacterizethe3’partofHBVRNAfromserum.FirstcDNAfrom
wassynthesizedbyusingaprimerwithtagsequencetargetingthepolyadenylated3’end.
Thiswasfollowedupwithaforwardprimeratnt1550andareverseprimertargetingtag
sequence inserted during cDNA synthesis. Agarose gel electrophoresis of the PCR
products(figure14A) indicatedthatonlyasmallproportionofRNAwasprematurely
polyadenylated. In order to assess if the HBV RNA was of full length, real time PCR
targetingcoreat5’regionandXat3’regionwasperformedonthecDNAgeneratedbythe
poly-Treverseprimer(figure14B).Theresultsshowingsimilarquantitiessuggestthat
theHBVRNApresentinserumwasoffull(genome)length.
FromtheseresultsweconcludedthatHBVRNAinserumwaspgRNAinsidecapsidsthat
probablywere enveloped, likely because the reverse transcription step had failed. To
study if this might be due to mutations in the e sequence, Sanger sequencing was
performedon3samplesaftersynthesizingcDNAusingareverseprimertargetingthe3’
polyadenylationregion.Acorrectesequenceisnecessaryforencapsidation[17,147]and
toinitiatereversetranscription[18].Nomutationsthatwouldinfluencethefunctionof
the e sequencewas detected. Yet, sequence variationmight be of importance for the
failureofreversetranscription,becausetheratiobetweenHBVDNAandHBVRNAwas
higheringenotypeDincomparisontogenotypeA,BorC(figure13B).
30
Figure14:(A)AgarosegelelectrophoresisshowingPCRproductsobtainedfromserumsample(B)
Real time PCR targeting 5’ and 3’ genomic sequences (core and X) after cDNA targeting
polyadenylatedtailat3’region.
Figure15:LowcorrelationbetweenpgRNAinlivertoHBVRNApresentinserumforHBeAgpositive
andnegativepatients.
ThelevelsofHBVRNAinlivertissueandserumcorrelatedsignificantly,althoughlessin
theHBeAg-negativegroup(Figure15).Comparingresultsinfigure13Atodatainfigure
15,showsthatthecorrelationbetweenlevelsofHBVDNAandHBVRNAinserumwas
strongerthanbetweenpgRNAinliverandHBVRNAinserum.Thisshowsthatsecretion
of virus-like particles with HBV RNA is linked to secretion of mature viral particles
(containingHBVDNA),ratherthanrepresentingintrahepaticRNAlevels.
31
In conclusion, the results presented in this article represent high concentrations of
encapsidated, full genomic length RNA in serum and encourages further research to
explainfailureofreversetranscription.
32
Paper II
ExcessproductionofSVPduringHBVinfectionincomparisontogenomecontainingVP
hasbeenobservedpreviouslyattherateof2000SVPforeveryVP[141,148].BothVP
andSVPcontain theenvelopeproteinHBsAg,however the ratioofpreS1domainthat
recognizesthecellspecificreceptorNTCPishigherinVPincomparisontoSVP.Excess
SVPproductionhasbeenproposedtoactasadecoytoreduceeffectofanti-HBsantibody,
butnoexperimentalprooftosupportthishypothesisisavailable.
Inorder to study theeffectsof SVP,Nycodenz fractionationofpatient serumshowing
signsofhighviralreplicationwascarriedouttoseparateSVPfromVP, tobeusedfor
subsequentinfectiononHepG2-NTCPcells.Serumfromanindividualwithhighanti-HBs
levelsafterbeingvaccinatedforHBVwasalsousedinthisstudytoassesseffectsofSVP
onhost antibodies.HBeAgproducedandsecretedby infectedHepG2-NTCPcellswere
measuredtoquantifyinfectivity.
Anti-HBswasseriallydilutedandpreincubatedwith(i)unfractionatedserum(containing
bothVPandSVP),(ii)VPand(iii)mixtureofVP+SVPin1:1ratio.IncubationofVPwith
anti-HBsshowedamarkedreductionintheinfectionofHepG2-NTCPcells(lowerHBeAg
levelsforthestraightascomparedwiththedottedlinesinfigure16).Inthepresenceof
subviralparticles(VP+SVP), thisneutralizingeffectofanti-HBswasreduced, iehigher
concentration of anti-HBs was required to reduce infection. This effect was more
pronounced in unfractionated serum (which likely contained SVP in much higher
concentrationsthanVP(figure16AandB).
TostudyifSVPcanalsoblockVPentry,differentconcentrationsofSVPweremixedwith
fixedVPconcentrationsinthepresenceorabsenceofanti-HBsantibodies(table1Aand
B).SVPneutralizedactivityofanti-HBsby55%ataconcentrationof200IU/mL,whereas
SVP alone, in the absence of anti-HBs, showed minimum reduction in infectivity,
suggestingthatcompetitionbetweenSVPandVPforbindingandcellularentryisnotof
significant importance. This result is comparable to previously published datawhere
artificiallysynthesizedSVPdidnotblockVPentry[149],butafewstudiesconductedon
33
DuckHBVhaveexhibitedSVPspotentialtobothneutralizeanti-HBsandcompeteforviral
entry[150,151].
Figure16:Serialdilutionofanti-HBsconcentrationstartingfrom(A)10,000IU/Lor (B)30,000
IU/L. Straight and dotted lines represent presence or absence of anti-HBs antibodies. Infection
potentialwasquantifiedusingHBeAgsecretedbycells.
Table1:ExperimentaldesignandtabulatedresultshowinghighercapacityofSVPtoadsorbanti-
HBs(A)thanblockentryofVP(B).
In conclusion, observations from this study suggest that themain role of SVP during
infectionistoactasadecoyandneutralizeantibodiesproducedbythehost.
34
Paper III
Inthisarticleamethodwasfirstdevelopedtoquantifythecircularandlinearformsof
intrahepaticDNA fromCHBpatient biopsies. Nucleotide (nt) position 1830has been
recognizedasapreferentialsiteforHBVintegrationsandthereforeaPCRspanningthis
region would differentiate between integrated and non-integrated transcripts. A
discriminatingPCRassaywassetupusingtwosetsofprimers,onespanningnt1830(for
circularDNA)andanotherplacedintheXregionthatwouldamplifyallformsofHBVDNA
(circularandlinear).
Figure17 :DiscriminatingPCRusing forwardprimerat nt 1550 in combinationwitha reverse
primeratnt1627toamplifyallHBVDNAforms,andforwardprimeratnt1776incombinationwith
reverseprimeratnt1924toamplifyonlycircularformsofHBVDNA.
ThisdiscriminatingPCRwasappliedon70liverbiopsiesfrompatientsindifferentstages
ofchronicinfection.TheresultsrevealedthatlinearDNAconstitutedalargefractionof
thetotalintrahepaticHBVDNAinbothHBeAgpositive(54%)andnegativegroup(89%).
The variation between these two groups were statistically significant (P<0.0001)
indicatinghigherquantityoflinearDNAine-negativepatients(figure18).
Controlexperimentsonserumand liversamplesshowedthat linearHBVDNAin liver
tissue (but much less in serum) was sensitive to degradation by endonuclease
35
(benzonase), indicating that it was not encapsidated but rather represents HBV DNA
integratedinthechromosomalDNA.
Figure18:PercentageoflinearDNAinHBeAgpositiveandnegativeHBVpatients
Figure19:PercentageofintegratedHBVDNAoutoftotalintrahepaticDNA.
To calculate the fraction of integrated HBV DNA out of total intrahepatic DNA, two
assumptionweremade:(i)dslDNA(notintegrated)isapproximately20%ofrcDNAand
(ii)allcircularDNAisrcDNA(cccDNAismuchlowerthanrcDNA).Theseassumptionsare
supportedbypublisheddata, includingstudiesestimatingcccDNAtobe<10%of total
circularDNAinliver[36,39,142].ByusingtheformulaIntegratedDNA=totallinearDNA
–dslDNA(assumedtoconstitute20%oftotalrcDNA),integrationinHBeAgpositiveand
negativepatientswerecalculatedtoconstitute46%and87%oftotalintrahepaticlinear
36
DNA respectively (figure 19). Together, these experiments indicate presence of
integrationsinlargeproportionsoccurringveryearlyduringHBVinfection.
Previously published large scale study using inverse PCR technique has estimated
presenceof integrations inapproximatelyabout1%of total livercells[152].However
due to technical challenges, inverse PCR is said to detect only about 10% of total
integration events [153] which suggests approximately 10% of liver cells carry
integration.Thisislowerthanourestimates,butalmostatasimilarlevel.
Thehighdegreeof integration, inparticular inHBeAg-negativepatients,supports that
expressionofintegratedHBVDNAmightcontributetomaintaininghighlevelsofserum
HBsAginpatientswithlowrateofreplication.SuchhighHBsAglevelshavebeennoticed
in clinical diagnostics for a long time [60], and illustrates the striking difference in
immunemediatedreductionofHBVDNAandHBsAg.Inoursamplesthiswasobserved
asalarge(thousand-fold)differenceintheHBsAg/HBVDNAratioinHBeAg-positiveas
comparedwithHBeAg-negativepatients.AsshowninFigure20thisHBsAgtoHBVDNA
ratioinserumsignificantlycorrelatedwiththefractionoftotalHBVDNAinlivertissue
thatwaslinearHBVDNA,likelyintegratedinchromosomalDNA.Thisfindingsupports
that integrated DNA significantly contributes to HBsAg production in HBeAg-negative
patients.
Figure 20 : Significant correlation (p<0.0001) between ratio of HBsAg andHBVDNA in HBeAg
positiveandnegativepatientserumandproportionofintegrationindicativeoflinearDNAinbiopsy.
37
Inconclusion,thisstudydescribesanovelandreliabletechniquetoquantifyintegrations.
Inaddition,thisstudyalsocontributesexperimentalproofusingnaturalHBVinfectionin
patients,tosupportthehypothesisthatintegrationscontributetomaintenanceofHBsAg
evenduringlowreplicationrates.
38
Paper IV
Liverbiopsieswerewidelyusedinthepasttoestimateintrahepaticinjuryandanalyze
HBVmarkersbyimmunohistochemistrystainingormolecularmethods.Inthisstudy,15-
30livertissuefrom6patientsundergoingtransplantationforHBVrelatedliverdisease
wasanalyzed.TheaimofthestudywastoinvestigatetherelationbetweendifferentHBV
markers, in particular those representing integration in the host genome, that was
previously hypothesized to be variable by Lindh et al. [154]. This strategy was also
employedtoestimatetheriskofsamplingerror,thatis,theriskthatananalyzedpieceof
livertissueisnotrepresentativeoftheinfectionstatusinthewholeliver
Allpatientswereonantiviral therapyofshortorlongduration.Patients1-4hadHBV-
induced livercirrhosis,but the infectionactivitydifferedgreatly.Patients1and3had
recentlyexperiencedreactivationofhepatitiswithserumlevelsofHBVDNAat≈8log
IU/mL prior to initiation of entecavir or tenofovir treatment three weeks and four
months, respectively, before transplantation. Patient 2 had an infection thathad been
highlyactiveforalongtimeandhadrelativelyhighlevelsofHBVDNAinserumasaresult
ofantiviralresistance.Patient4hadlow-activeinfectionandhadbeenontenofovirfor>
twoyears.Patients5and6hadHBV/HDVco-infectionwithlowHBVDNAandrelatively
highHDV-RNAlevelsinserum.
As shown in Figure 21, the degree of infection focality differed markedly between
patients;betweenviruses;andbetweendifferentHBVviralmarkers.ForHBVinfection,
themostabundantmarkerwasSRNA,followedbycoreRNA,totalHBVDNAandcccDNA.
Patient 1 expressed the highest levels of all HBV markers, and had relatively small
variation in the distribution of infection (all markers detected in all pieces, with CV
rangingbetween4.4%and8.6%).Patients2and3(P3)hadlowerlevelsandhigherCV
values forbothHBVDNAandRNA,but thesemarkerswerestilldetected inallpieces.
Patient4(P4)(ontenofovir for>2yearsbeforetransplantation)had lowHBVDNAin
mostpiecesandmuchhigherCVvalues(Table2).
39
Figure21:Quantificationof(A)totalHBVRNAmeasuredbyStranscriptPCRand(B)coreRNA(C)DeltaRNAinpatient5(inblue)andpatient6(in
brown).
40
Patient1 Patient2 Patient3 Patient4Indicationfortransplantation Acuteliverfailure,
cirrhosisCirrhosis HCCandcirrhosis Cirrhosis
Ageattransplantation 43.6 50.5 54.8 34Geographicorigin EastAfrica Balkan MiddleEast EastAfricaAntiviraltreatment Entecavir, Lamivudine, Tenofovir Tenofovir
3weeks resistancea 4months >2yearsHBsAgserum(logIU/mL) 4.16 3.79 3.61 1.00HBVDNAserum(logIU/mL) 6.64b 5.55 2.09c NegLivertissuedata
Piecesanalyzed 20 30 25 20
Cellsperpiece,median(IQR) 2735 1962 2900 5940
HBVDNA,logcopies/1000cells Total(Starget) 3.69(3.07-4.24) 2.41(1.66-2.83) 1.32(0.59-1.72) 0.14(-0.32-1.72)Coefficientofvariation 8.6% 11% 22% 135%PiecesPCRpositive 20(100%) 30(100%) 25(100%) 16(80%)
cccDNA,logcopies/1000cells 3.27(2.78-3.66) 1.04(0.31-1.91) 0.84(-0.02-1.45) <-1
Coefficientofvariation 7.3% 33% 48%
PiecesPCRpositive 20(100%) 30(100%) 24(96%) 0
HBVRNA,logcopies/1000cells Starget 5.40(4.96-5.89) 4.24(2.75-4.83) 2.65(0.69-3.82) 0.15(<-1-2.54)Coefficientofvariation 4.4% 12% 30% 187%PiecesPCRpositive 20(100%) 30(100%) 25(100%) 12(60%)Coretarget 5.29(5.11-5.45) 2.78(2.55-2.97) 2.24(1.12-2.81) <-1Coefficientofvariation 4.7% 10% 40% 110%PiecesPCRpositive 20(100%) 30(100%) 25(100%) 9(45%)
aM204VmutationinthereversetranscriptaseregionofHBV. bTheHBVDNAlevelwas8.19logIU/mLwhenentecavirtreatmentwasinitiated3weeksbeforetransplantation.cTheHBVDNAlevelwas7.90logIU/mLwhentenofovirtreatmentwasinitiated4monthsbeforetransplantation.Medianand(range)values.CV,Coefficientofvariation=standarddeviation/mean.
Table2:CharacteristicsoffourlivertransplantedpatientswithHBVinfectionandresultsfromddPCRanalysesofliverexplanttissuepiec
41
Patient5 Patient6
Indicationfortransplantation Cirrhosis HCCandcirrhosis
Ageattransplantation 53.3 47.2
Sex Male Male
Geographicorigin MiddleEast MiddleEast
Antiviraltreatment Tenofovir2years Entecavir2months
HBsAgserum(logIU/mL) 3.27 2.86
HBVDNAserum(logIU/mL) Neg Neg
HDVRNAserum(logcopies/mL) 4.99 3.94
Livertissuedata
Piecesanalyzed 20 15
Cellsperpiece,median(IQR) 2130 4721
HBVDNA,logcopies/1000cells
Total(Starget) 1.03(0.43-2.29) <1(-0.08-1.87)
Coefficientofvariation 45% 86%
PiecesPCRpositive 19(95%) 7(47%)
cccDNA,logcopies/1000cells <-1 <-1
Coefficientofvariation
PiecesPCRpositive 0 0
HBVRNA,logcopies/1000cells
Starget 2.85(1.31-4.10) 1.91(1.12-3.74)
Coefficientofvariation 29% 42%
PiecesPCRpositive 20(100%) 14(93%)
Coretarget 0.75(0.13-1.38) <-1
Coefficientofvariation 68% 18%
PiecesPCRpositive 4(20%) 2(13%)
HDVRNA,logcopies/1000cells 6.70(5.26-7.28) 4.28(3.82-4.99)
Coefficientofvariation 11% 8%
PiecesPCRpositive 19(95%) 15(100%)Medianand(range)values.Coefficientofvariation,CV=standarddeviation/mean,calculatedforlevelsinsampleswithdetectedtarget.
Table3:CharacteristicsoftwolivertransplantedpatientswithHBV/HDVinfectionandandresults
fromddPCRanalysesofliverexplanttissuepieces
Patients5and6(P5andP6)hadco-infectionwithHDVlowHBVDNAandrelativelyhigh
HDVRNAinserum.Inlivertissue,cccDNAwasundetectedinallpieces,HBVDNAand
coreRNAwasloworundetected.Yet,SRNArangedfrommoderatetohighlevelsinall
pieces.HDVRNAwasdetectedathighlevelsinallpieces(figure21andtable3).
42
The findings in the HDV cases agrees relatively well with those in a previous study
comparingintrahepaticmarkersin21HBV/HDVco-infectedand22HBVmonoinfected
patients[155].Inthatstudy,livertissuefromHDV-infectedpatientshadlowerlevelsof
cccDNA, lower pgRNA/cccDNA and higher S RNA/cccDNA than patients with HBV
monoinfection.
TheresultsshowsignificantfocaldifferencesinthedistributionandactivityofbothHBV
and HDV and demonstrates the risk involvedwhen analyzing a single tissue piece to
quantitativelyrepresentintrahepaticlevelsespeciallyinpatientswithlowviralload.The
riskofobtainingfalsenegativeresultswasrelativelylowexceptforHBVmarkersintissue
withlowHBVload(suchasoftenseenwhenthereisHDVco-infection)
In the following figures,markersarepresentedpairwise inordertoshowcorrelations
andtodisplaydifferencesinmoredetailbyshowingvaluesforindividualpieces.Figure
22showsthatHBVcoreRNAandSRNAvaluescorrelatedstronglyinpiecesfromPatients
1 and 3. This probably reflects that in these patients, core and S RNA were mainly
transcribedfromcccDNA,andatsimilarratesindifferentpieces.Bycontrast,thelevels
didnotcorrelateinpiecesfromPatients2,4,5and6.InPatient2,coreandSRNAwere
bothpresentinrelativelyhighamounts,butwithoutcorrelation.InPatient4thelackof
correlationmighttosomeextentbeduetothepooreranalyticalprecisionforlowtarget
levels.InPatients5and6(withHDVco-infection)coreRNAwasundetectableormuch
lowerthanSRNA.
Figure23showsreflectionofcccDNAonHBVRNAlevels(in thethreepatientswhere
cccDNAwasdetected,P1-3).Overall,coreRNAcorrelatedstronglywithcccDNA,whereas
forSRNAacorrelationwasonlyobservedinP1andP3butnotinP2.
Infigure24thesamedataispresentedasRNAcopiespercccDNA.InPatient1whohada
highlyactiveinfectionthecoreandSRNAlevelspercccDNAweresimilarandatlevels
around100RNAcopiespercccDNAwithlittlevariationbetweenthepieces.InPatient2
thecoreRNAlevelswereslightly lowerwithrelatively littlevariation,whereasSRNA
levelsweremuch higher andwith large variation, from60 to8000 SRNA copies per
cccDNA.
43
Figure22.HBVcoreRNAandSRNAin15-30piecesoffromfourpatients(P1-P4)withHBV(A),and
twopatients(P5-P6)withHDV/HBVcoinfection(B).Thevaluesareinlog10copies/1000cells.
Figure23:Correlationsbetween(A)cccDNAandcoreRNAand(B)cccDNAandSRNAinpiecesof
explantedliverfromP1-3.Thevaluesareinlog10copies/1000cells.
SincecccDNAistheonlytemplateforcoreRNAwhereasSRNAcanbetranscribedfrom
cccDNA or from integrated sequences [63, 156], the results suggest that a large
proportionof theSRNA inPatient2 (andtosomeextent inPatient3)maybedue to
transcriptionfromintegratedHBVDNA.Thisobservationfitsbothwithourfindingsin
Paper 3 and with data from a recent publication on experimental HBV infection in
chimpanzees[61].
44
Figure24:LevelsrepresentedinP1-3ascopies/cccDNA.
In thetwopatientswithHDVco-infection, therewasnocorrelationobservedbetween
HDVRNAandHBVSRNA.PresenceofhighlevelsofHDVRNAwasobservedevenwhenS
RNAlevelswerelow,asshownin figure25A.Accordingly,asshownin figure25B,the
HDV RNA:S RNA ratio was highly different, spanning from 10:1 to >100,000:1. This
findingsuggeststhatproductionofHDVRNAisindependentofHBsAgandthatHDVRNA
might be produced also in cells that do not produceHBsAg. Thus, it seems as ifHDV
infectionmaybepresentinhepatocytesindifferentforms:(i)atrueco-infectiontogether
withreplicatingHBVwhichprovidesHBsAgfromcccDNA;(ii)incellswithoutreplicating
HBVbutwithintegratedHBVDNAthatprovidesHBsAgsothatHDVvirusparticlescan
beformedandsecreted;(iii)HDVRNAincellswithoutHBsAgproduction,replicatingand
translatedtodeltaantigenbutnotsecretedsinceHBsAgisnotprovided.Thismodelis
supportedbydatafrompreviouspublications.Inonestudy,HBsAgfromintegrationswas
showntosupportHDVreplicationinvitro[157],andarecentstudyreportedpersistence
of HDV during hepatocyte replication and its presence in daughter cells that did not
containHBV[133].
Patient 1 Patient 2 Patient 3
45
Figure25:(A)LackofcorrelationbetweenHDVRNAandHBVSRNAinP5andP6(B)Variedratio
betweenHDVRNAandHBVSRNAinP5andP6.
The model proposes that only a minority of cells capable of producing HBsAg (from
cccDNA or transcripts integration) are required for production of high quantities of
intrahepaticHDVRNA(asseenbythelackofcorrelationbetweenHBVSRNAandHDV
RNA).Thismayhaveclinicalimplications.ItwouldmeanthatproductionofHDVvirus
particlesmay occur in a small proportion of hepatocytes that produces HBsAg (from
cccDNAorintegrations),butthatHDVRNAfromtheseparticlesmightenteramuchlarger
number of hepatocytes and drastically increase the production of HDV antigens and
expressionoftheirepitopesonthecellsurfacetoprovokeharmfulimmuneresponses.
ThismodelwouldnotcontradicttheassociationbetweenHDVRNAinserumandliver
damage,butratherproposeamechanismbywhichthepathogeniceffectsofsecretedHDV
couldbeamplified.Iftrue,thismodelmayhelptounderstandmechanisminvolvedinthe
development of severe necroinflammation that is often seen during HDV infection.
Furtherresearchisrequiredtoclarifythesehypotheses.
Inconclusion,theanalysisofmultiplepiecesoflivertissueshowswiderangeoffocality
within the same infected liver especially in individuals with low viremia; shows
integrationstobeasourceofHBVSRNAespeciallyinHDVcoinfectedpatientswithlow
HBVviremia;andalsoproposesnovelmechanismstopavewayfor futureresearchto
understandaggravatedliverinjuryduringHBV/HDVco-infection.
46
Paper V In this article, biopsymaterial representing bothHBeAg positive andHBeAgnegative
infectionin76patientswereusedtodescribeRNAprofileofthedifferenttranscriptsseen
during HBV infection. Levels of the transcriptswere quantified to also distinguish its
source(cccDNAorintegratedHVDNA).
ThePCRtargetsareshowninfigure26.ThesePCRsinvolveamplificationofoverlapping
targets, anddropletdigitalPCR techniquegivesamethodological advantageover real
timePCRsinceabsolutequantificationcanbeobtainedwithoutamplificationefficiency
bias.
Figure26:PCRstrategytoquantifydifferentRNAspecies.
Figure27showslevelsofallRNAspeciesmeasuredinthe76biopsies,demonstratingthat
theRNAprofiledifferedsignificantlybetweenHBeAgpositiveandnegativepatients.In
particular,HBeAg-negativepatientshadlowerlevelsofpgRNA,coreRNAand3’precore
regions, a finding that agreeswellwith recent transcriptomedata from liver biopsies
takenfromexperimentallyinfectedchimpanzees[61].
47
Figure26:IntrahepaticHBVRNAprofilesinpatientspositiveornegativeforHBeAg.Theboxplots
showmedianlevelsandinterquartilerangesbydigitalPCRassaystargetingdifferentsegmentsof
thegenome.ThelevelsofHBsAgandHBVDNAinserumareshownforcomparison.Thewhiskers
show10thand90thpercentile.PC,precore;pg,pregenomic..***,p<0.0001;**,p<0.001.
Figure27showslevelsofcoreandSRNAfrom76patients.TheHBeAg-positivepatients
hadsimilarlevelsofcoreandSRNA.Bycontrast,HBeAgnegativepatientshad10-100
timeslowercorethanSRNA,illustratedinthefigurebythedistancefromthedottedline.
The difference could be due to specific transcriptional down- regulation of core RNA
synthesis.EvidenceofepigeneticregulationinHBVhasbeenpresented[158],butsofar
nodatasuggestsspecificcontrolofcoreRNA.
QuantificationofcoreRNAwasperformedusingtwoadditionalprimers,targetingits5’
part(“pgRNA”)oratargetregiondownstreamofthecoregene.Figure28Ashowslower
levelsofpgRNA(5’coreRNA)thancoreRNA,inparticularinHBeAgnegativepatients.
Figure 29 presents the levels from 28A as box plots, and with the core RNA values
obtained after subtraction of pgRNA (performed because the values by the core PCR
detectsalsothepgRNAmolecules).ThisplotclearlydemonstrateslowerpgRNAlevelsin
HBeAg-negativepatients.
48
Figure27:CorrelationbetweencoreandSRNAinHBeAgpositiveandnegativepatients.
Sincethetranscriptslackthe5’partcontainingtheeregionessentialforencapsidation
andinitiationofreversetranscription,thesecannotserveaspregenomicRNAandmay
contribute to explain lower level of HBV replication observed in HBeAg negative in
comparisonwithHBeAgpositivepatients[24].
Primers targeting the 3’ precore region (the 3’ redundancy) should amplifies the 3’
terminalpartofcoreRNA.Thefindingthatthelevelsby3’precorePCRwereloweras
comparedtocore(figure28B)suggestthatpartofthecoreRNAisshorterbecausean
upstreampolyadenylationsignalatnt1930wasused[159,160].Degradationofthe3´
partmightbeanalternativeexplanationtothisobservedreduction.
All RNA that are transcribed from cccDNA should contain the terminal 3’ redundancy
representingnt1830-1927.Therefore,ifcccDNAwasthesourceofSRNA,amplification
bytheSRNAand3’PCassaysshouldgivesimilarresults.AsshowninFigure30there
was a striking discrepancy,withmuch lower3’ RNA than and SRNA levels inHBeAg
negative than in HBeAg positive patients. The lower 3’ levels might be due to RNA
degradation,butthefindingthat3’RNAlevelswerealmostashighasSRNAinHBeAg-
positivepatientsargueagainst thispossibility.ThediscrepancybetweenSand3’RNA
wasgreatestinHBeAg-negativepatientswithlowerlevelsofreplication.Inthesecases,
49
3’RNAwas3log10unitslowerthanS,indicatingthat>99%oftheSRNAwasderivedfrom
integratedHBVDNA.
Figure28:RNAlevelsdetectedby(A)coreand5’core(pgRNA)assaysand(B)coreand3’PCRNA.
Figure29:Boxplotshowingthesamemarkersasinfigure27A,butwithcoreRNAvaluesobtained
aftersubtractionofpgRNAvalues.
pgRNA core RNA pgRNA core RNA0
2
4
6
8
Log
copi
es/1
000
cells
HBeAg+ HBeAg–
50
Figure30:Correlationbetween3’andSRNAtranscriptsinHBeAgpositiveandnegativepatients.
ToexploretheputativeimportanceofcccDNAandintegratedHBVDNAassourcesofHBV
DNAandHBsAg inbloodwecompared the ratiosof thesemarkers in liver tissueand
serum.Figure31AshowsratiobetweencccDNAandtotalintrahepaticDNAcorrelating
withratiobetweenpgRNA(thatcomesfromcccDNA)andXRNA(whichcancomefrom
bothcccDNAandintegratedDNA).Thesignificantcorrelationfitswiththeexplanationof
cccDNAbeingthesourceforpgRNAsynthesis.Figure30Bshowsthatinthenextstep,the
ratio between serumHBVDNA andHBsAg correlatewith intrahepatic proportions of
betweenpgRNAandXRNA.Theseresultsagreewellwiththeideathatalargeproportion
of HBsAg synthesized in HBeAg negative patients are a product of integrations, in
accordancewitharecentpublicationwheretranscriptomeanalysisinchimpanzeemodel
systemwasusedtostudyintegrations[61].
51
Figure 30. Correlations between (A) the core RNA/total HBV RNA ratio and the ratio between
cccDNAandtotalintrahepaticHBVDNA,and(B)thecoreRNA/totalHBVRNAratioandtheserum
HBVDNA/serumHBsAgratio.
AnalysesofmultiplebiopsiesandexplorationofRNAtranscriptratiobymeansofdigital
PCR has provided valuable support to the theory that most of the HBsAg in HBeAg
negative patients have integrated transcripts as their source. The results also show
presenceofintrahepaticHBVcoreRNAthatlacksthe5’orboth5’and3’end,eliminating
thepossibilitytofunctionaspgRNA.Thisishypothesizedtobecontributetothereduction
ofHBVreplicationinpatientswithHBeAgnegativeinfection.
A
B
y=0.75x+0.57R²=0.31p<0.0001
-4
-3
-2
-1
0
1
-6 -4 -2
pgRN
A/total(X)RNA
cccDNA/totalintrahepaGcHBVDNA
y=1.17x+3.86R²=0.57p<0.0001
-2
-1
0
1
2
3
4
5
6
-4 -3 -2 -1 0 1
SerumHBV
DNA/HB
sAgraGo
pgRNA/total(X)RNA
52
6 CONCLUSIONS
HepatitisBvirus(HBV)infectionhasthepotentialtocausesevereliverdamageincluding
cirrhosis and hepatocellular carcinoma (HCC) which is predicted using diagnostic
markerssuchasHBVDNA,HBsAgandHBeAginserumandHBVDNAandRNAinliver
tissue.Nucleoside/nucleotideanaloguetreatmentscurrentlyavailableeffectivelyblocks
viral replication but fails to clear reminiscent cccDNA in liver which can reactivate
replicationwhentreatmentisterminated.
Duetorapidreductioninviralreplication,HBVDNAinserumcannotbeusedtoevaluate
thelong-termeffectoftreatmentonintrahepaticcccDNA.HBVRNAinserumhasbeen
proposed as a potentialmarker for this purpose since it is not directly influenced by
treatment.Inpaper1,thismarkerwascharacterized.Analysisofserumfrom95patients
indicate that HBV RNA in serum is encapsidated and of full genomic length RNA and
possiblylessabundantingenotypeD.
Subviral particles (SVP) in the ratio of >10,000:1 to viral particles (VP) during active
replication has been proposed to act as a decoy to host antibodies (anti-HBs) against
HBsAg.Thefirstexperimentalsupportforthishypothesiswasdescribedinpaper2,in
resultsobtainedusingarecentlydevelopedcelllineHepG2-NTCP.ItwasshownthatSVP
significantly reduced theneutralizingeffectof anti-HBs in in vitro infection,buthad a
rathersmallcompetingimpactonbindingtotheviralspecificreceptor.
DuringHBVreplication,adoublestranded linear formof thegenome(dslDNA)rather
thanthe functionalcircular form, isproducedinminority,andmaybecomeintegrated
intothehostgenome.Anovelstrategytoestimate integrationbymeansofmeasuring
circularandlinearformsofHBVDNAinliverusingdigitalPCRwasdevelopedandapplied
on liver biopsies. A panel of digital PCR assays were also developed to obtain a
quantitativeprofileofHBVRNAinthesebiopsies.Theresultsfromtheseanalysessuggest
thatamajorityofHBVDNAfoundinliverduringlateinfectionstageisinintegratedforms
thatcontributestomaintaininghighlevelsofHBsAgintheserumevenduringlowHBV
replication. In addition, results suggesting a novelmechanism associating low pgRNA
transcriptiontoreducedreplicationduringHBeAgnegativestatewereobtained.
53
Inaseparatestudy,focaldifferencesinthedistributionofHBVinfectionintheliverwere
studiedusingliverexplantmaterialfrompatientsundergoingtransplantationduetoHBV
orHDVinducedliverdisease.Byanalyzingmultipletissuepiecesextensivedifferencesin
HBV viral load and transcription (a hundred-fold or more between some pieces),
especiallyinpatientswithlowviremiaorwithHDVcoinfectionwerefound.Highlevels
ofSRNAintheabsenceofcccDNAorcoreRNAsupporttheexpressionofHBsAgfrom
integratedHBVDNA.ThelevelsofHDVRNAweregenerallyhighwithlowerdegreeof
focaldifferences,andwithoutcorrelationtoSRNAlevels.WeproposethatpartialHDV
replicationandexpressionofdeltaantigensmayoccuralsoinhepatocytesthatarenot
co-infected by HBV or even express HBsAg from integrations, and that this might
contribute to the severe necroinflammation that is observed in many HDV infected
patients.
Thecompiledworkspresentedinthisthesisaddressimportantquestionsandproposes
novel mechanisms that hopefully will aid further research and contribute to the
understandingofinfectionscausedbyHBVandHDV.
54
7 ACKNOWLEDGEMENTS
Iwouldliketotakethisopportunitytothankeveryonewhohasbeenapartofmylifeand
madethisPhDpossible.
FirsttomysupervisorMagnusLindhforgivingmethiswonderfulopportunitytopursue
research,forallthevaluableguidanceandallthemidnightemailswithoutwhichthese
projectswouldhavebeenimpossible.Thankyoufortakingthetimetoteachandinspire
meinspiteofyourimpossiblybusyschedule!
Tomyco-supervisorHeléneNorder,thankyouforthesupport,wonderfulinsightsand
suggestionsonallmyprojects.YourpassionforresearchissomethingIadmire.Youare
awonderfulinspirationtoallresearchers.
Tomyco-supervisorSimonLarsson,fornotjusthelpingmewithmyprojectsbutalsofor
being therewhenever I needed help! Thank you for being awonderfulmentor and a
friend.
ToMariaAndersson,thankyouforintroducingmetowaysofVirologen,forteachingme
allthelabwork,foryourpatienceandhelpwithallmyprojects.Butmorethananything,
thankyouforbeingagoodfriend.
ToGustafRydell, forbeingagreatmentorand teachingme tobemeticulouswithmy
research.Thankyouforallthediscussions,foransweringmyquestionsnomatterhow
sillytheywereandforgreatcollaborationonallmyprojects.
ToEbbaSamuelsson,mypartnerincrimeatVirologenandtomyfellow“Indian”(well,
youaremoreIndianthanIcouldeverbe!)thankyouforeverything!Thememorieswe
sharedissomethingIwillcherishfortherestofmylife.Thankyouforallthechatsover
coffeeandforthe“Mondayicecream”tradition.These4yearsofmyPhDwouldnothave
beenpossibleifyouweren’tthereencouragingmeon.
ToGiorgiaOrtolani,itissaidthatgreatfriendsarehardtocomeby.Boy!AmIfortunate
tohavefoundyou!Thankyousomuchforbeingapartofmylife,forencouragingand
cheeringmeonalways!
55
ToSofiaBrunet,thankyouforbeingawonderfulfriend,forhearingoutallmyfrustrations
andnever-endingsupport.
ToRickardNordén,apersonwhoIhavegreatregardfor.Thankyouformakingtimeto
teachme,forallthegreatadvicesandforyourneverwaveringsmilesthatsays“you’re
gonnabefine!!!!”
ToJohanRinglander,thankyouforallthehelponmyprojectsandforlivelydiscussions.
Iwillcherishyourfriendshipforever!
ToMichaelKaan,thecoolestbossIhaveeverknown!Ithasbeenanabsolutepleasure
knowingyou.Theenthusiasmandvigoryouhaveforresearchisinfectious!Thankyou
fortheadvices,suggestionsandforallthefun!
ToThomasBergström,thankyouverymuchforallthesuggestionsandkindwords,for
encouragingbuddingresearcheslikemeandmotivatingustodobettereachday!
ToHaoWang,thepersonwhoIbuggedthemostthesefouryears!YoustartedyourPhD
onemonthbeforeIdid,whichmeansIwenttoyoutofindouteverythingonemonthin
advance!Forthoseofyouwhodon’tknow, that’sa lotttttttofquestionsheanswered!
ThankyouHao,forbeingsuchagreatfriendanddealingwithmycraziness!
Tomyco-authorsGunnerNorkrans,KristofferHellstrand,CatarinaSkoglundandallthe
staffattransplantationteam,althoughIhaven’tmetmostofyou,yourcontributionsto
thisprojectweretremendous!Thankyou!
My heartfelt thank you to Maria Johansson, Carolina Gustafsson, Anette Roth, Marie
Karlsson, Freddy Saguti, Johanna Said, Kristina Elfving, Edward Trybala, Theogene
Twagirumugabe,EricSeruyange,CharlsHannoun,PeterNorberg,NancyNenonen,Anna
Lundin, Charlotta Ericsson, Linn Persson, Kristina Nyström, Ka-Wei Tang, Hedvig
Engström,JacksonThomas,JoelGustafsson,SebastianMalmström,ShadiGeravandi,Priti
Gupta,GianlucaTripodi,YarongTian,MilaAdmekandsomanyothers!Thiswouldhave
beeninsurmountableifnotforthecontributionofeachoneofyou!Sayingthankyouonce
56
isnotenough.Youhaveallwelcomedmeintoyourlivesandmademefeelathomehere.
ForthatIwillbeeternallygrateful!
IwouldliketogivemyspecialthankstoeveryoneattheKvant,DetektionandHepatitlab
especially to Silviu Nitescu and Cvetla Spirovska taking the time to helpmewithmy
projectsandforMAKINGMESPEAKSWEDISH!TusenTackförallhjälpochuppmuntran!
TomydearestMaaruthyKumarYelleswarapuVenkataSathya(Yes,Ididwriteyourfull
name!),Ioweallmysuccesstoyoumybelovedfriend!Iwillforevercherishourgoodold
undergrad days! Phewwhat fun itwas! I don’t think I would have evermade it past
“ChemicalEngineering”ifnotforyou,I’dprobablystillbetrying.Thankyouforbeinga
partofmylifeandforeverythingelse!
ToKeerthana,Arjun,Sunith,NeethiandSiddharthyouareallmuchmorethanfriendsto
me,myfamilyfarawayfromhome.Forallthecrazydaysandwonderfulmemories,thank
youforbeingthereformethrougheverything!
Tomydearfamily,SushilaAmma,PrakashAppa,Gayu,sweetlittleYug,dearestAro,my
grandmotherSulo,mysecondparentsBalaAmmaandSureshAppaandtherestofmy
hugeeeeeefamilybackinIndia,thankyouisnotenoughtoexpressmygratitudetoyou.
Thestrengththatallofyougivemeisunmatchedandiswhatmadethispossible.
OneofmyfavoritequotesfromHarryPotterreads“Onewhowelovenevertrulyleaves
us”.IhavenowordstoexpresstheloveandadorationIhaveformydearestgrandparents
NanapaandChemmi.TheyarethetrueinspirationbehindeverythingIhaveachievedand
willachieveinthefuture.IfIgrowuptobehalfthepersonyouwere,Iwouldconsider
myselflucky!Thankyouforteachingmethevalueofeducation.
Lastbutnottheleast,mydearlovinghusbandAshwinwhohasbeenwaitingfouryears
to read this part! How can I ever begin to thank you for all that you have done?
Nevertheless,Iamgoingtotry.Thankyouforbeingwithmethroughallthegoodtimes
andthebad,foralltheencouragementandloveforgivingmeashouldertoleanon,for
sharingmyhappinessandsadnessalike!Thankyouforbeingyou.
57
8 REFERENCES
1. Blumberg,B.S.,H.J.Alter,andS.Visnich,A"New"AntigeninLeukemiaSera.JAMA,1965.191:p.541-546.
2. Dane,D.S.,C.H.Cameron,andM.Briggs,Virus-LikeParticlesInSerumOfPatientsWithAustralia-Antigen-AssociatedHepatitis.TheLancet,1970.295(7649):p.695-698.
3. WHO,GlobalHepatitisReport2017.Geneva:WorldHealthOrganization,2017.4. Hutin,Y.,S.Desai,andM.Bulterys,PreventinghepatitisBvirusinfection:milestones
andtargets.BullWorldHealthOrgan,2018.96(7):p.443-443A.5. Chisari, F.V., M. Isogawa, and S.F. Wieland, Pathogenesis of hepatitis B virus
infection.Pathologie-biologie,2010.58(4):p.258-266.6. Nafa,S.,etal.,EarlydetectionofviralresistancebydeterminationofhepatitisBvirus
polymerase mutations in patients treated by lamivudine for chronic hepatitis B.Hepatology,2000.32(5):p.1078-88.
7. Yuen,M.F., et al.,Factors associatedwith hepatitis B virusDNA breakthrough inpatientsreceivingprolongedlamivudinetherapy.Hepatology,2001.34(4Pt1):p.785-91.
8. Lai, C.L., et al., Prevalence and clinical correlates of YMDD variants duringlamivudine therapy for patients with chronic hepatitis B. Clin Infect Dis, 2003.36(6):p.687-96.
9. Mason,A., et al.,HepatitisB virus replication indiverse cell typesduring chronichepatitisBvirusinfection.Hepatology,1993.18(4):p.781-9.
10. Schulze,A.,P.Gripon,andS.Urban,HepatitisBvirusinfectioninitiateswithalargesurface protein-dependent binding to heparan sulfate proteoglycans. Hepatology,2007.46(6):p.1759-68.
11. Ni,Y.,etal.,HepatitisBandDVirusesExploitSodiumTaurocholateCo-transportingPolypeptide for Species-Specific Entry into Hepatocytes. Gastroenterology, 2014.146(4):p.1070-1083.e6.
12. Huang, H.C., et al., Entry of hepatitis B virus into immortalized human primaryhepatocytesbyclathrin-dependentendocytosis.JVirol,2012.86(17):p.9443-53.
13. Macovei,A.,etal.,HepatitisBvirusrequiresintactcaveolin-1functionforproductiveinfectioninHepaRGcells.JVirol,2010.84(1):p.243-53.
14. Rabe,B.,D.Glebe,andM.Kann,Lipid-MediatedIntroductionofHepatitisBVirusCapsidsintoNonsusceptibleCellsAllowsHighlyEfficientReplicationandFacilitatestheStudyofEarlyInfectionEvents.JournalofVirology,2006.80(11):p.5465.
15. Nassal,M.,HepatitisBviruses:Reversetranscriptionadifferentway.VirusResearch,2008.134(1-2):p.235-249.
16. Knaus, T. andM. Nassal,The encapsidation signal on the hepatitis B virus RNApregenomeformsastem-loopstructurethatiscriticalforitsfunction.Nucleicacidsresearch,1993.21(17):p.3967-3975.
17. Pollack, J.R. andD. Ganem,AnRNA stem-loop structure directs hepatitis B virusgenomicRNAencapsidation.JVirol,1993.67(6):p.3254-63.
18. Nassal,M.andA.Rieger,AbulgedregionofthehepatitisBvirusRNAencapsidationsignalcontains thereplicationoriginfordiscontinuousfirst-strandDNAsynthesis.Journalofvirology,1996.70(5):p.2764-2773.
58
19. Nassal,M.,M.Junker-Niepmann,andH.Schaller,TranslationalinactivationofRNAfunction: discrimination against a subset of genomic transcripts during HBVnucleocapsidassembly.Cell,1990.63(6):p.1357-63.
20. Ko,C., et al.,HepatitisB virusgenome recyclinganddenovo secondary infectioneventsmaintainstablecccDNAlevels.JHepatol,2018.69(6):p.1231-1241.
21. Zhao, X.L., et al., Serum viral duplex-linear DNA proportion increases with theprogressionofliverdiseaseinpatientsinfectedwithHBV.Gut,2016.65(3):p.502-11.
22. Beck, J. and M. Nassal, Hepatitis B virus replication. World journal ofgastroenterology,2007.13(1):p.48-64.
23. Yang, W. and J. Summers, Integration of Hepadnavirus DNA in Infected Liver:EvidenceforaLinearPrecursor.JournalofVirology,1999.73(12):p.9710.
24. EASL 2017 Clinical Practice Guidelines on the management of hepatitis B virusinfection.JHepatol,2017.67(2):p.370-398.
25. Milich,D.andT.J.Liang,ExploringthebiologicalbasisofhepatitisBeantigen inhepatitisBvirusinfection.Hepatology,2003.38(5):p.1075-86.
26. Magnius,L.O.andA.Espmark,Anewantigencomplexco-occurringwithAustraliaantigen.ActaPatholMicrobiolScandBMicrobiolImmunol,1972.80.
27. Lindh, M., et al., Core Promoter Mutations and Genotypes in Relation to ViralReplicationandLiverDamageinEastAsianHepatitisBVirusCarriers.TheJournalofInfectiousDiseases,1999.179(4):p.775-782.
28. Rodriguez-Frias, F., et al., Hepatitis B virus infection: precore mutants and itsrelationtoviralgenotypesandcoremutations.Hepatology,1995.22(6):p.1641-7.
29. Suzuki, S., et al., Distribution of hepatitis B virus (HBV) genotypes among HBVcarriers in the Cote d'Ivoire: complete genome sequence and phylogeneticrelatednessofHBVgenotypeE.JMedVirol,2003.69(4):p.459-65.
30. Breitkreutz,R.,etal.,HepatitisBVirusNucleicAcidsCirculatingintheBlood.AnnalsoftheNewYorkAcademyofSciences,2001.945(1):p.195-206.
31. Rokuhara,A.,etal.,HepatitisBvirusRNAismeasurableinserumandcanbeanewmarker for monitoring lamivudine therapy. Journal of Gastroenterology, 2006.41(8):p.785-790.
32. vanBommel,F.,etal.,SerumhepatitisBvirusRNAlevelsasanearlypredictorofhepatitis B envelope antigen seroconversion during treatment with polymeraseinhibitors.Hepatology,2015.61(1):p.66-76.
33. Jansen,L.,etal.,HepatitisBVirusPregenomicRNAIsPresentinVirionsinPlasmaandIsAssociatedWithaResponsetoPegylatedInterferonAlfa-2aandNucleos(t)ideAnalogues.JournalofInfectiousDiseases,2016.213(2):p.224-232.
34. Su, Q., et al., Circulating Hepatitis B Virus Nucleic Acids in Chronic Infection.RepresentationofDifferentlyPolyadenylatedViralTranscriptsduringProgressiontoNonreplicativeStages,2001.7(7):p.2005-2015.
35. Volz,T.,etal.,ImpairedintrahepatichepatitisBvirusproductivitycontributestolowviremiainmostHBeAg-negativepatients.Gastroenterology,2007.133(3):p.843-52.
36. Laras, A., et al., Intrahepatic levels and replicative activity of covalently closedcircularhepatitisB virusDNA in chronically infectedpatients.Hepatology,2006.44(3):p.694-702.
37. Zhang,Y.Y.,etal.,Single-cellanalysisofcovalentlyclosedcircularDNAcopynumbersinahepadnavirus-infectedliver.ProcNatlAcadSciUSA,2003.100(21):p.12372-7.
59
38. Werle-Lapostolle, B., et al., Persistence of cccDNA during the natural history ofchronichepatitisBanddeclineduringadefovirdipivoxiltherapy.Gastroenterology,2004.126(7):p.1750-8.
39. Wursthorn, K., et al.,Peginterferon alpha-2b plus adefovir induce strong cccDNAdeclineandHBsAgreductioninpatientswithchronichepatitisB.Hepatology,2006.44(3):p.675-84.
40. Wong,D.K.,etal.,QuantitationofcovalentlyclosedcircularhepatitisBvirusDNAinchronichepatitisBpatients.Hepatology,2004.40(3):p.727-37.
41. Tuttleman, J.S., C. Pourcel, and J. Summers, Formation of the pool of covalentlyclosedcircularviralDNAinhepadnavirus-infectedcells.Cell,1986.47(3):p.451-460.
42. Cai,D.,etal.,AsouthernblotassayfordetectionofhepatitisBviruscovalentlyclosedcircularDNAfromcellcultures.Methodsinmolecularbiology(Clifton,N.J.),2013.1030:p.151-161.
43. He,M.-L.,etal.,AnewandsensitivemethodforthequantificationofHBVcccDNAbyreal-time PCR. Biochemical and Biophysical Research Communications, 2002.295(5):p.1102-1107.
44. Marrone,A.,etal.,Three-phasesequentialcombinedtreatmentwithlamivudineandinterferoninyoungpatientswithchronichepatitisB.JViralHepat,2005.12(2):p.186-91.
45. D'Antiga, L., et al., Combined lamivudine/interferon-alpha treatment in"immunotolerant" children perinatally infected with hepatitis B: a pilot study. JPediatr,2006.148(2):p.228-233.
46. Poddar,U.,etal.,Cureforimmune-toleranthepatitisBinchildren:isitanachievabletargetwithsequentialcombotherapywithlamivudineandinterferon?JViralHepat,2013.20(5):p.311-6.
47. Zhu,S.,etal.,AntiviraltherapyinhepatitisBvirus-infectedchildrenwithimmune-tolerant characteristics: A pilot open-label randomized study. J Hepatol, 2018.68(6):p.1123-1128.
48. Lal,B.B.,etal.,ImmunotolerantchildrenwithchronichepatitisB–Totreatornot–Thedilemmacontinues.JournalofHepatology,2018.69(4):p.979-981.
49. Hadziyannis,S.J.andG.V.Papatheodoridis,HepatitisBeantigen-negativechronichepatitisB:naturalhistoryandtreatment.SeminLiverDis,2006.26(2):p.130-41.
50. Hsu, Y.S., et al., Long-term outcome after spontaneous HBeAg seroconversion inpatientswithchronichepatitisB.Hepatology,2002.35(6):p.1522-7.
51. Chen,Y.C.,C.M.Chu, andY.F.Liaw,Age-specificprognosis following spontaneoushepatitisBeantigenseroconversioninchronichepatitisB.Hepatology,2010.51(2):p.435-44.
52. Zacharakis,G.H.,etal.,NaturalhistoryofchronicHBVinfection:acohortstudywithupto12yearsfollow-upinNorthGreece(partoftheInterregI-II/EC-project).JMedVirol,2005.77(2):p.173-9.
53. Bortolotti, F., et al.,ChronichepatitisB in childrenafter eantigen seroclearance:finalreportofa29-yearlongitudinalstudy.Hepatology,2006.43(3):p.556-62.
54. Tong,S.P.,etal.,ActivehepatitisBvirusreplication in thepresenceofanti-HBeisassociatedwithviralvariantscontaininganinactivepre-Cregion.Virology,1990.176(2):p.596-603.
55. Raimondo,G.,etal.,LatencyandreactivationofaprecoremutanthepatitisBvirusinachronicallyinfectedpatient.JHepatol,1990.11(3):p.374-80.
60
56. Huo,T.I.,etal.,Sero-clearanceofhepatitisBsurfaceantigeninchroniccarriersdoesnotnecessarilyimplyagoodprognosis.Hepatology,1998.28(1):p.231-6.
57. Chen,Y.C.,etal.,PrognosisfollowingspontaneousHBsAgseroclearanceinchronichepatitisBpatientswithorwithoutconcurrentinfection.Gastroenterology,2002.123(4):p.1084-9.
58. Simonetti,J.,etal.,ClearanceofhepatitisBsurfaceantigenandriskofhepatocellularcarcinomainacohortchronicallyinfectedwithhepatitisBvirus.Hepatology,2010.51(5):p.1531-7.
59. Leoni, M.C., et al., HIV, HCV and HBV: A Review of Parallels and Differences.Infectiousdiseasesandtherapy,2018.7(4):p.407-419.
60. Omata,M.,etal.,CorrelationofhepatitisBvirusDNAandantigens in the liver.Astudyinchronicliverdisease.Gastroenterology,1987.92(1):p.192-6.
61. Wooddell, C.I., et al., RNAi-based treatment of chronically infected patients andchimpanzeesrevealsthatintegratedhepatitisBvirusDNAisasourceofHBsAg.SciTranslMed,2017.9(409).
62. Chakraborty, P.R., et al., Identification of integrated hepatitis B virus DNA andexpressionofviralRNAinanHBsAg-producinghumanhepatocellularcarcinomacellline.Nature,1980.286(5772):p.531-3.
63. Edman,J.C.,etal.,IntegrationofhepatitisBvirussequencesandtheirexpressioninahumanhepatomacell.Nature,1980.286(5772):p.535-538.
64. Sung, W.K., et al., Genome-wide survey of recurrent HBV integration inhepatocellularcarcinoma.NatGenet,2012.44(7):p.765-9.
65. Bill, C.A. and J. Summers, Genomic DNA double-strand breaks are targets forhepadnaviralDNAintegration.ProceedingsoftheNationalAcademyofSciencesoftheUnitedStatesofAmerica,2004.101(30):p.11135-11140.
66. Zhao, L.-H., et al., Genomic and oncogenic preference of HBV integration inhepatocellularcarcinoma.Naturecommunications,2016.7:p.12992-12992.
67. Malmstrom,S.,etal.,Genotypeimpactonlong-termvirologicaloutcomeofchronichepatitisBvirusinfection.JClinVirol,2012.54(4):p.321-6.
68. Orito, E. and M. Mizokami, Differences of HBV genotypes and hepatocellularcarcinomainAsiancountries.HepatolRes,2007.37(s1):p.S33-5.
69. Kao, J.H., Hepatitis B virus genotypes and hepatocellular carcinoma in Taiwan.Intervirology,2003.46(6):p.400-7.
70. Shiina, S., et al.,RelationshipofHBsAg subtypeswithHBeAg/anti-HBe statusandchronic liverdisease.Part I:Analysisof1744HBsAgcarriers.AmJGastroenterol,1991.86(7):p.866-71.
71. Mayerat, C., A. Mantegani, and P.C. Frei,Does hepatitis B virus (HBV) genotypeinfluencetheclinicaloutcomeofHBVinfection? JViralHepat,1999.6(4):p.299-304.
72. Sanchez-Tapias,J.M.,etal.,InfluenceofhepatitisBvirusgenotypeonthelong-termoutcomeofchronichepatitisBinwesternpatients.Gastroenterology,2002.123(6):p.1848-56.
73. Girones,R. andR.H.Miller,Mutation rateof thehepadnavirusgenome.Virology,1989.170(2):p.595-7.
74. Locarnini, S.,Hepatitis B viral resistance: mechanisms and diagnosis. Journal ofHepatology,2003.39:p.124-132.
75. Caligiuri,P.,etal.,OverviewofhepatitisBvirusmutationsandtheirimplicationsinthemanagementof infection.World journalofgastroenterology,2016.22(1):p.145-154.
61
76. Zanetti,A.R.,etal.,HepatitisBvariantinEurope.Lancet,1988.2(8620):p.1132-3.77. Hossain,M.G.andK.Ueda,InvestigationofaNovelHepatitisBVirusSurfaceAntigen
(HBsAg) Escape Mutant Affecting Immunogenicity. PLOS ONE, 2017. 12(1): p.e0167871.
78. Dandri,M. andS. Locarnini,New insight in the pathobiology of hepatitis B virusinfection.Gut,2012.61(Suppl1):p.i6.
79. Inoue,J.,T.Nakamura,andA.Masamune,RolesofHepatitisBVirusMutationsintheViral Reactivation after Immunosuppression Therapies. Viruses, 2019. 11(5): p.457.
80. Carman, W.F., et al., MUTATION PREVENTING FORMATION OF HEPATITIS B eANTIGENINPATIENTSWITHCHRONICHEPATITISBINFECTION.TheLancet,1989.334(8663):p.588-591.
81. Baptista, M., A. Kramvis, and M.C. Kew, High prevalence of 1762(T) 1764(A)mutationsinthebasiccorepromoterofhepatitisBvirusisolatedfromblackAfricanswithhepatocellularcarcinomacomparedwithasymptomaticcarriers.Hepatology,1999.29(3):p.946-53.
82. Parekh,S.,etal.,Genomereplication,virionsecretion,andeantigenexpressionofnaturallyoccurringhepatitisBviruscorepromotermutants.JVirol,2003.77(12):p.6601-12.
83. Malik,A.,etal.,HepatitisBvirusgenemutationsinliverdiseases:areportfromNewDelhi.PloSone,2012.7(6):p.e39028-e39028.
84. Dandri,M.,P.Schirmacher,andC.E.Rogler,WoodchuckhepatitisvirusXproteinispresent in chronically infected woodchuck liver and woodchuck hepatocellularcarcinomaswhich are permissive for viral replication. Journal of virology, 1996.70(8):p.5246-5254.
85. Reifenberg,K.,etal.,ThehepatitisBvirusXproteintransactivatesviralcoregeneexpressioninvivo.Journalofvirology,1999.73(12):p.10399-10405.
86. Kim,C.M.,etal.,HBxgeneofhepatitisBvirusinduceslivercancerintransgenicmice.Nature,1991.351(6324):p.317-20.
87. Ghosh,S.,etal.,Trackingthenaturallyoccurringmutationsacross the full-lengthgenomeofhepatitisBvirusofgenotypeDindifferentphasesofchronice-antigen-negativeinfection.ClinMicrobiolInfect,2012.18(10):p.E412-8.
88. Wang, Q., et al.,Analysis of hepatitis B virus X gene (HBx)mutants in tissues ofpatientssufferedfromhepatocellularcarcinomainChina.CancerEpidemiol,2012.36(4):p.369-74.
89. Shinkai,N.,etal.,InfluenceofhepatitisBvirusXandcorepromotermutationsonhepatocellular carcinoma among patients infected with subgenotype C2. J ClinMicrobiol,2007.45(10):p.3191-7.
90. Jones,S.A.,etal.,InVitroEpsilonRNA-DependentProteinPrimingActivityofHumanHepatitisBVirusPolymerase.JournalofVirology,2012.86(9):p.5134-5150.
91. Wang,G.H.andC.Seeger,ThereversetranscriptaseofhepatitisBvirusactsasaproteinprimerforviralDNAsynthesis.Cell,1992.71(4):p.663-70.
92. Gong,Y.,etal.,Evidencethatthefirststrand-transferreactionofduckhepatitisBvirusreversetranscriptionrequiresthepolymeraseandthatstrandtransferisnotneededfortheswitchofthepolymerasetotheelongationmodeofDNAsynthesis.JGenVirol,2000.81(Pt8):p.2059-65.
93. Zhang, X., et al., Potential resistant mutations within HBV reverse transcriptasesequences in nucleos(t)ide analogues-experienced patients with hepatitis B virusinfection.ScientificReports,2019.9(1):p.8078.
62
94. Kim,J.-E.,etal.,Naturallyoccurringmutationsinthereversetranscriptaseregionofhepatitis B virus polymerase from treatment-naïve Korean patients infectedwithgenotypeC2.Worldjournalofgastroenterology,2017.23(23):p.4222-4232.
95. Xu, J.,et al.,Pre-existingmutations inreversetranscriptaseofhepatitisBvirus intreatment-naiveChinesepatientswithchronichepatitisB.PLoSOne,2015.10(3):p.e0117429.
96. Liu, B.M., et al., Characterization of potential antiviral resistance mutations inhepatitis B virus reverse transcriptase sequences in treatment-naive Chinesepatients.AntiviralRes,2010.85(3):p.512-9.
97. Bartholomew, M.M., et al., Hepatitis-B-virus resistance to lamivudine given forrecurrentinfectionafterorthotopiclivertransplantation.Lancet,1997.349(9044):p.20-2.
98. Moraleda, G., et al.,Lack of effect of antiviral therapy in nondividing hepatocytecultures on the closed circular DNA of woodchuck hepatitis virus. J Virol, 1997.71(12):p.9392-9.
99. Dandri, M., et al., Increased hepatocyte turnover and inhibition of woodchuckhepatitisBvirusreplicationbyadefovirinvitrodonotleadtoreductionoftheclosedcircularDNA.Hepatology,2000.32(1):p.139-46.
100. Bowden,S.,etal.,Covalentlyclosed-circularhepatitisBvirusDNAreductionwithentecavirorlamivudine.WorldJGastroenterol,2015.21(15):p.4644-51.
101. Rijckborst, V. and H.L.A. Janssen,The Role of Interferon in Hepatitis B Therapy.Currenthepatitisreports,2010.9(4):p.231-238.
102. Tan,G.,etal.,WhenHepatitisBVirusMeetsInterferons.Frontiersinmicrobiology,2018.9:p.1611-1611.
103. Zoulim,F.,HepatitisBvirusresistancetoantiviraldrugs:wherearewegoing?Liverinternational:officialjournaloftheInternationalAssociationfortheStudyoftheLiver,2011.31Suppl1:p.111-116.
104. Cho, W.H., et al., Development of tenofovir disoproxil fumarate resistance aftercompleteviralsuppressioninapatientwithtreatment-naïvechronichepatitisB:Acasereportandreviewoftheliterature.Worldjournalofgastroenterology,2018.24(17):p.1919-1924.
105. Yamada,N.,etal.,ResistancemutationsofhepatitisBvirusinentecavir-refractorypatients.Hepatologycommunications,2017.1(2):p.110-121.
106. Guo, W.-N., et al., Animal models for the study of hepatitis B virus infection.Zoologicalresearch,2018.39(1):p.25-31.
107. Dandri,M. and J. Petersen,Animalmodels of HBV infection. Best Pract Res ClinGastroenterol,2017.31(3):p.273-279.
108. McAuliffe, V.J., R.H. Purcell, and J.L. Gerin,Type B hepatitis: a review of currentprospectsforasafeandeffectivevaccine.RevInfectDis,1980.2(3):p.470-92.
109. Sells,M.A.,M.L.Chen,andG.Acs,ProductionofhepatitisBvirusparticlesinHepG2cells transfectedwith cloned hepatitis B virusDNA. Proceedings of the NationalAcademyofSciences,1987.84(4):p.1005-1009.
110. Tsurimoto,T.,A.Fujiyama,andK.Matsubara,Stableexpressionandreplicationofhepatitis B virus genome in an integrated state in a human hepatoma cell linetransfectedwiththeclonedviralDNA.ProcNatlAcadSciUSA,1987.84(2):p.444-8.
111. Yaginuma,K.,etal.,HepatitisBvirus(HBV)particlesareproducedinacellculturesystembytransientexpressionoftransfectedHBVDNA.ProceedingsoftheNationalAcademyofSciences,1987.84(9):p.2678.
63
112. Gripon, P., et al., Infection of a human hepatoma cell line by hepatitis B virus.ProceedingsoftheNationalAcademyofSciences,2002.99(24):p.15655.
113. Galle,P.R.,etal.,InvitroexperimentalinfectionofprimaryhumanhepatocyteswithhepatitisBvirus.Gastroenterology,1994.106(3):p.664-73.
114. Yan, H., et al., Sodium taurocholate cotransporting polypeptide is a functionalreceptorforhumanhepatitisBandDvirus.Elife,2012.1:p.5233.
115. Rizzetto,M.,etal.,Immunofluorescencedetectionofnewantigen-antibodysystem(delta/anti-delta) associated to hepatitis B virus in liver and in serum of HBsAgcarriers.Gut,1977.18(12):p.997-1003.
116. Rizzetto,M.,etal.,TransmissionofthehepatitisBvirus-associateddeltaantigentochimpanzees.JInfectDis,1980.141(5):p.590-602.
117. Wang,K.S.,etal.,Structure,sequenceandexpressionofthehepatitisdelta(delta)viralgenome.Nature,1986.323(6088):p.508-14.
118. Farci,P.,Deltahepatitis:anupdate.JHepatol,2003.39Suppl1:p.S212-9.119. Caredda,F.,etal.,Prospectivestudyofepidemicdeltainfectionindrugaddicts.Prog
ClinBiolRes,1983.143:p.245-50.120. Smedile,A.,etal.,INFLUENCEOFDELTAINFECTIONONSEVERITYOFHEPATITIS
B.TheLancet,1982.320(8305):p.945-947.121. Kuo,M.Y., et al.,Molecular cloning of hepatitis delta virus RNA froman infected
woodchuckliver:sequence,structure,andapplications.JVirol,1988.62(6):p.1855-61.
122. Branch, A.D. and H.D. Robertson,A replication cycle for viroids and other smallinfectiousRNA's.Science,1984.223(4635):p.450-5.
123. Wu,J.C.,etal.,ProductionofhepatitisdeltavirusandsuppressionofhelperhepatitisBvirusinahumanhepatomacell line. Journalofvirology,1991.65(3):p.1099-1104.
124. Williams,V.,etal.,HepatitisdeltavirusproteinsrepresshepatitisBvirusenhancersandactivatethealpha/betainterferon-inducibleMxAgene.JGenVirol,2009.90(Pt11):p.2759-67.
125. Krogsgaard,K.,etal.,Delta-infectionandsuppressionofhepatitisBvirusreplicationinchronicHBsAgcarriers.Hepatology,1987.7(1):p.42-5.
126. Farci,P.,etal.,Acuteandchronichepatitisdeltavirus infection:director indirecteffectonhepatitisBvirusreplication?JMedVirol,1988.26(3):p.279-88.
127. Koff, R.S., Hepatitis vaccines: recent advances. International Journal forParasitology,2003.33(5):p.517-523.
128. Farci,P.,etal.,TreatmentofchronichepatitisD.Journalofviralhepatitis,2007.14Suppl1:p.58.
129. Farci,P.andG.AnnaNiro,CurrentandFutureManagementofChronicHepatitisD.Gastroenterology&hepatology,2018.14(6):p.342-351.
130. Farci,P.,etal.,TreatmentofchronichepatitisDwithinterferonalfa-2a.NEnglJMed,1994.330(2):p.88-94.
131. Lau,D.T.,etal.,Resolutionofchronicdeltahepatitisafter12yearsofinterferonalfatherapy.Gastroenterology,1999.117(5):p.1229-33.
132. Blank,A.,etal.,First-in-humanapplicationofthenovelhepatitisBandhepatitisDvirusentryinhibitormyrcludexB.JHepatol,2016.65(3):p.483-9.
133. Giersch, K., et al.,Hepatitis delta virus persists during liver regeneration and isamplifiedthroughcelldivisionbothinvitroandinvivo.Gut,2019.68(1):p.150-157.
64
134. Lindh,M., et al.,Hepatitis B virus DNA levels, precoremutations, genotypes andhistologicalactivityinchronichepatitisB.JViralHepat,2000.7(4):p.258-67.
135. Brock,T.D.andH.Freeze,Thermusaquaticusgen.n.andsp.n.,anonsporulatingextremethermophile.JBacteriol,1969.98(1):p.289-97.
136. Chien, A., D.B. Edgar, and J.M. Trela,Deoxyribonucleic acid polymerase from theextremethermophileThermusaquaticus.Journalofbacteriology,1976.127(3):p.1550-1557.
137. Kralik,P.andM.Ricchi,ABasicGuidetoRealTimePCRinMicrobialDiagnostics:Definitions,Parameters,andEverything.Frontiersinmicrobiology,2017.8:p.108-108.
138. Vogelstein,B.andK.W.Kinzler,DigitalPCR.ProceedingsoftheNationalAcademyofSciences,1999.96(16):p.9236.
139. Rickwood,D.,T.Ford,andJ.Graham,Nycodenz:Anewnonioniciodinatedgradientmedium.AnalyticalBiochemistry,1982.123(1):p.23-31.
140. Ford, T.C. and D. Rickwood, Formation of isotonic Nycodenz gradients for cellseparations.AnalyticalBiochemistry,1982.124(2):p.293-298.
141. Desire,N.,etal.,DefinitionofanHBsAgtoDNAinternationalunitconversionfactorbyenrichmentofcirculatinghepatitisBvirusforms.JViralHepat,2015.22(9):p.718-26.
142. Chen,C.-J.,RiskofHepatocellularCarcinomaAcrossaBiologicalGradientofSerumHepatitisBVirusDNALevel.JAMA,2006.295(1):p.65.
143. Larsson,S.B.,etal.,HBsAgquantificationforidentificationofliverdiseaseinchronichepatitisBviruscarriers.Liverinternational:officialjournaloftheInternationalAssociationfortheStudyoftheLiver,2014.34(7):p.e238-45.
144. Thompson,A.J.,etal.,SerumhepatitisBsurfaceantigenandhepatitisBeantigentiters:diseasephaseinfluencescorrelationwithviralloadandintrahepatichepatitisBvirusmarkers.Hepatology,2010.51(6):p.1933-44.
145. Cornberg,M.,etal.,TheroleofquantitativehepatitisBsurfaceantigenrevisited.JHepatol,2017.66(2):p.398-411.
146. Wang, J.,et al.,SerumhepatitisBvirusRNAisencapsidatedpregenomeRNAthatmaybeassociatedwithpersistenceofviralinfectionandrebound.JHepatol,2016.65(4):p.700-710.
147. Junker-Niepmann, M., R. Bartenschlager, and H. Schaller, A short cis-actingsequenceisrequiredforhepatitisBviruspregenomeencapsidationandsufficientforpackagingofforeignRNA.Emboj,1990.9(10):p.3389-96.
148. Gerlich,W.H.,MedicalVirologyofHepatitisB:howitbeganandwherewearenow.Virologyjournal,2013.10(1):p.239-25.
149. Chai,N.,etal.,PropertiesofsubviralparticlesofhepatitisBvirus.Journalofvirology,2008.82(16):p.7812-7817.
150. Klingmuller,U.andH.Schaller,Hepadnavirusinfectionrequiresinteractionbetweentheviralpre-Sdomainandaspecifichepatocellularreceptor.JVirol,1993.67(12):p.7414-22.
151. Bruns, M., et al., Enhancement of Hepatitis B Virus Infection by NoninfectiousSubviralParticles.JournalofVirology,1998.72(2):p.1462.
152. Mason, W.S., et al., HBV DNA Integration and Clonal Hepatocyte Expansion inChronicHepatitisBPatientsConsideredImmuneTolerant.Gastroenterology,2016.151(5):p.986-998.e4.
153. Tu,T.,etal.,HepatitisBVirusDNAIntegrationOccursEarlyintheViralLifeCycleinan <em>In Vitro</em> Infection Model via Sodium Taurocholate
65
CotransportingPolypeptide-DependentUptakeofEnvelopedVirusParticles.JournalofVirology,2018.92(11):p.e02007-17.
154. Lindh,M.,G.E.Rydell,andS.B.Larsson,ImpactofintegratedviralDNAonthegoaltoclearhepatitisBsurfaceantigenwithdifferenttherapeuticstrategies.CurrOpinVirol,2018.30:p.24-31.
155. Pollicino,T.,etal.,ReplicativeandTranscriptionalActivitiesofHepatitisBVirusinPatientsCoinfectedwithHepatitisBandHepatitisDeltaViruses.JournalofVirology,2011.85(1):p.432.
156. Twist,E.M.,etal.,IntegrationpatternofhepatitisBvirusDNAsequencesinhumanhepatomacelllines.Journalofvirology,1981.37(1):p.239-243.
157. Freitas,N.,etal.,EnvelopeProteinsDerivedfromNaturallyIntegratedHepatitisBVirusDNASupportAssemblyandReleaseofInfectiousHepatitisDeltaVirusParticles.JournalofVirology,2014.88(10):p.5742.
158. Levrero,M.,etal.,ControlofcccDNAfunctioninhepatitisBvirusinfection.JournalofHepatology,2009.51(3):p.581-592.
159. Freitas,N., et al.,RelativeAbundanceof Integrant-DerivedViralRNAs in InfectedTissuesHarvestedfromChronicHepatitisBVirusCarriers.JVirol,2018.92(10).
160. Schutz, T., A. Kairat, and C.H. Schroder, DNA sequence requirements for theactivationofaCATAAApolyadenylationsignalwithinthehepatitisBvirusXreadingframe:rapiddetectionoftruncatedtranscripts.Virology,1996.223(2):p.401-5.