Hepatitis D review from the Lancet

8/12/2019 Hepatitis D review from the Lancet

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www.thelancet.com Vol 378 July 2, 2011 73

Hepatitis delta virus

Sarah A Hughes, Heiner Wedemeyer, Phillip M Harrison

Hepatitis delta virus (HDV) is a small, defective RNA virus that can infect only individuals who have hepatitis B virus(HBV); worldwide more than 15 million people are co-infected. There are eight reported genotypes of HDV withunexplained variations in their geographical distribution and pathogenicity. The hepatitis D virion is composed of acoat of HBV envelope proteins surrounding the nucleocapsid, which consists of a single-stranded, circular RNAgenome complexed with delta antigen, the viral protein. HDV is clinically important because although it suppressesHBV replication, it causes severe liver disease with rapid progression to cirrhosis and hepatic decompensation. Therange of clinical presentation is wide, varying from mild disease to fulminant liver failure. The prevalence of HDV isdeclining in some endemic areas but increasing in northern and central Europe because of immigration. Treatmentof HDV is with pegylated interferon alfa; however, response rates are poor. Increased understanding of the molecularvirology of HDV will identify novel therapeutic targets for this most severe form of chronic viral hepatitis.

IntroductionHepatitis delta virus (HDV) is a small, defective RNAvirus that is related more to plant viroids than to otherhuman pathogens. It can propagate only in an individualwho has coexistent hepatitis B virus (HBV), either aftersimultaneous transmission of the two viruses, or viasuperinfection of an established HBV carrier.1 Clinicalexpression of HDV is wide, and although it sometimesfollows a benign course, the disease is clinicallyimportant. Studies have consistently shown that mostpatients with HBV and HDV co-infection have moresevere liver disease,2,3 more rapid progression tocirrhosis,4,5 and increased hepatic decompensation and

death

6,7

than do those with HBV infection alone. Advanceshave improved our understanding of the transmission,replication, and pathogenesis of the virus; however, wedo not yet fully understand the mechanisms by which itcauses such severe liver disease. In this Seminar, we willreview the biology, pathogenesis, epidemiology, naturalhistory, clinical presentation, and management of thisdisease. We discuss how the most recent evidence mighthelp in the design of novel therapeutic agents for HDV,the most severe of all the chronic viral hepatitides.

Historical perspectiveRizzetto and colleagues8 discovered HDV in the mid-1970s while investigating a group of patients with HBV

who had severe hepatitis. They showed a novel antigen-antibody system, which they called delta-antigen anddelta-antibody, and noted that this system occurred onlyin patients with HBV and was associated with severeliver disease. The disease was later associated with aparticle consisting of an RNA genome of low molecularweight that was encapsidated by HBV envelope proteins.9This particle was termed the delta agent, or HDV, andwas classified under the Deltavirus genus.

Viral structureThe HDV virion is a small, spherical particle of about36 nm in diameter. It is composed of an outer coatcontaining the three HBV envelope proteins termedlarge, medium, and small hepatitis B surface antigen

(HBsAg),10 and host lipids surrounding an innernucleocapsid, consisting of a single-stranded, circularRNA of 1679 nucleotides and about 200 molecules ofhepatitis D antigen (HDAg) per genome.11,12 Because ofthe high GC content of the nucleotide sequence, 12 thecircular genome, which is unique to animal viruses andmore closely related to plant viroids than to humanpathogens,13 can fold into an unbranched, rod-likestructure with 74% intramolecular base-pairing.

Virus life cycleThe HDV receptor on the human hepatocyte remainsunidentified, but is thought to be the same as that of

HBV because of the shared identity of their outer coat.HDV infectivity is dependent upon a receptor-bindingdomain in the N-terminal region of the pre-S1 moiety oflarge HBsAg.14,15 For HDV infectivity, this domainrequires modification by myristoylation.16Fine mappinghas identified aminoacid residues 915 as the receptorbinding site.17A second infectivity region in the antigenicloop of all three envelope proteins is also required forinfectivity,18,19 but whether the antigenic loop and thepre-S1 determinants act synergistically or independentlyin viral entry is unclear.

The virus is uncoated after entering the hepatocyte,and a signal in HDAg translocates the nucleocapsid tothe nucleus.20 The antigen has no RNA polymerase

Lancet2011; 378: 7385

Published Online

April 20, 2011

DOI:10.1016/S0140-

6736(10)61931-9

Institute of Liver Studies, Kings

College Hospital, London, UK

(S A Hughes MBBCh);

Transplantation Immunology

and Mucosal Biology, Kings

College London, London,

UK (P M Harrison MD);

andDepartment of

Gastroenterology, Hepatologyand Endocrinology, Hannover

Medical School, Hannover,

Germany(H Wedemeyer MD)

Correspondence to:

Dr Phillip M Harrison,

Transplantation Immunology

and Mucosal Biology, Kings

College London, Denmark Hill

Campus, London SE5 9PJ, UK

[email protected]

Search strategy and selection criteria

We searched PubMed, Embase, and the Cochrane Library, with

no date or language restrictions. We used the search terms

hepatitis delta virus, hepatitis D virus, and Delta hepatitis,

in combination with the following terms: replication,

epidemiology, genotype, transmission, clinical

presentation, pathogenesis, diagnosis, and treatment. We

largely selected publications in the past 5 years. We searched the

reference lists of articles identified by this strategy, and selected

references that were most relevant. Abstracts from international

congressional proceedings in the past 3 years that were thought

to add key information were also included.


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74 www.thelancet.com Vol 378 July 2, 2011

activity; to replicate its genome, the virus hijacks the

cellular RNA polymerases of the host, which might thentreat the genome as double-stranded DNA because of itsfolded, rod-like structure.21Three forms of RNA are madein the host during replication: circular genomic RNA,circular complementary antigenomic RNA, and a linearpolyadenylated antigenomic RNA of 08 kb, which is themessenger RNA (mRNA) containing the open readingframe of the HDAg.

Initial studies implicated RNA polymerase II in HDVreplication; however, one study has shown that RNApolymerases I and III also interact with HDV RNA,suggesting a more complex reliance on several hostpolymerases.22 Evidence suggests that synthesis of thedifferent RNA species occurs in different subcellular

locations, mediated by distinct cellular polymerases:synthesis of antigenomic RNA occurs in the nucleolusmediated by RNA polymerase I, whereas synthesis ofgenomic RNA takes place more diffusely in thenucleoplasm by RNA polymerase II.23Replication of thecircular HDV RNA template occurs via a rollingmechanism that is unique to animal viruses but analogousto that of plant viroids. HDV RNA is first synthesised as alinear molecule, potentially containing many copies ofthe genome, but in the genomic and antigenomic RNA, asequence of 85 nucleotides acts as a ribozyme, which self-cleaves the linear HDV RNA into monomers.24,25 Thesemonomers are then ligated to form circular RNA (theinvolvement of a host RNA ligase remains controversial).Ribozymes are regarded as a feature of plant viroids,26buta self-cleaving RNA sequence in an intron of the CPEB3gene is structurally and biochemically related to the HDVribozymes; therefore, HDV might have arisen from thehuman transcriptome.27

HDAg is the only protein that is known to be encodedby the HDV genome. It consists of two isoforms: the27 kDa large-HDAg with 214 aminoacids; and a 24 kDasmall-HDAg with 195 aminoacids.28 The N-terminalsequence of the two isoforms is the same; they differ byonly 19 aminoacids at the C-terminus of the large-HDAg.The open reading frame of the antigenomic RNAgenerates both isoforms because of heterogeneity in the

RNA at codon 196.28A UAG stop codon at this positionleads to translation of small-HDAg; however, RNAediting by the cellular enzyme adenosine deaminase-1changes the sequence to UGG and, consequently, thelonger large-HDAg is produced.12,28,29 Small-HDAgreturns to the nucleus and supports viral replication,30,31whereas large-HDAg is a negative regulator of HDVreplication and is essential for virion assembly.32Therefore, RNA editing is central to the replication cycleof HDV because it controls the levels of each isoform,and consequently, the balance between viral synthesisand particle assembly.

Post-translational modification of large-HDAg, espe-cially prenylation of the cysteine residue at the C-terminus,is integral to its ability to bind HBsAg and assemble the

viral particle.33 Phosphorylation of a serine residue at

position 177 of small-HDAg increases replication ofantigenomic RNA by interacting with RNA poly-merase II,34,35 whereas the sumoylation of small-HDAgenhances the synthesis of genomic RNA and mRNA, butnot of antigenomic RNAproperties that are alsoascribed to acetylation.36Methylation of small-HDAg byarginine methyltransferase at arginine-13 (an RNA-binding domain), is essential for translocation of small-HDAg to the nucleus, and for replication of theantigenomic RNA strand to form the genomic RNAstrand.37 Hence, post-translational modifications deter-mine the balance of the viral life cycle, and presentattractive novel therapeutic targets for drug development.In the nucleus, molecules of large-HDAg form complexes

with small-HDAg and new constructs of genomic RNA,and these complexes are exported to the Golgi membranesby a signal in the C-terminus of large-HDAg. Once there,these complexes associate with HBV envelope proteins tocreate an infectious virion.14,38Interaction of the C-terminusof large-HDAg with the clathrin heavy-chain in the trans-Golgi network is essential for viral assembly.39Figure 1 isa schematic representation of the delta particle and itslife cycle.

Viral heterogeneityThe sequence of the HDV RNA genome is highlyvariable, and there is divergence of up to 16% within thesame genotype, compared with 2040% between differentgenotypes. Even in one individual the virus is a pool ofclosely related quasispecies.40 This divergence is partlydue to the scarcity of proofreading ability of RNApolymerases. The average mutation rate for the non-coding region of HDV is about 35210 base sub-stitutions per genome site per year, whereas for the HDVcoding region, it is about 14910 for non-synonymoussubstitutions, and 06710 for synonymous sub-stitutions.41 However, heterogeneity is not uniform forthe entire coding region of the genome; the self-cleavingribozyme sequence and the RNA-binding domain ofHDAg are highly conserved, whereas the C-terminalregion of large-HDAg is very divergent. Historically,

HDV genotyping was achieved by either immuno-histochemical analysis of liver tissue42 or by restrictionfragment length polymorphism of PCR products,43 andthe virus was thought to have evolved into three majorgenotypes that differed in their global distribution.44Genotyping by direct sequencing and analysis ofmolecular phylogenetic trees has shown that HDV existsas at least eight different clades, four of which seem to beof exclusively African origin.45,46

EpidemiologyOf the 350 million chronic carriers of HBV worldwide,more than 15 million have serological evidence ofexposure to HDV.47Traditionally, the regions with highrates of HDV carriage where the virus is endemic are


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central Africa, the Horn of Africa, the Amazon Basin,eastern and Mediterranean Europe, the Middle East, andparts of Asia.48 Rates of HDV infection are generallyhighest in regions where HBV is endemic, but there areexceptionseg, HDV co-infection is uncommon inVietnam and Indonesia.49,50In Chinaa large reservoir ofHBV infectionHDV prevalence varies widely betweenprovinces despite a high prevalence of HBV. In one studyfrom Hong Kong, carriage of HDV was almost universalin intravenous drug users who were HBsAg positive,contrasting with low rates in non-drug users.51

HDV genotype 1 is prevalent worldwide,52 whereasgenotype 2 (previously termed genotype-2a) is found inJapan, Taiwan, and the Yakutia region of Russia.5355Genotype 3the most divergent genotypeis common

in the Amazon Basin,56whereas genotype 4 (previously2b) is found in Taiwan and Japan.54,57HDV genotypes 58are found in individuals of African origin, including inthose who have migrated to northern Europe.45,46Figure 2shows the worldwide prevalence of HDV and distributionof its genotypes.

Longitudinal studies have shown a decrease in HDVprevalence in some endemic areas, such as in Italy, whereinfection in HBsAg carriers has fallen from 246% in1983, to 14% in 1992, and 83% in 1997.5860Infection ratesare especially reduced in younger patients, and evidencesuggests that in Italy HDV infection is restricted toageing cohorts who were infected in the 1980s. In thepast three decades, reductions in HDV prevalence havealso been reported in Spain, Taiwan, and Turkey.6163

Vaccination programmes for HBV have probablycontributed substantially to the decline in HDV in theseregions, but additional factors, including increasedawareness of the virus and its mode of transmission,have led to better implementation of preventive measures,such as the change to disposable needles, syringes, andother medical equipment, and a general improvement insocioeconomic conditions.

There are caveats to the assertion that HDV prevalenceis declining. First, developing countries have not alwayshad the same measures of HBV control, and have notshown the same improvements in socioeconomicconditions as have developed countries. Data forprevalence in these regions are sparse, but cross-sectionalstudies have shown that rates of HDV co-infection in

carriers of HBsAg remain greater than 10%, andsometimes as high as 70% in Nigeria, Gabon, India,Pakistan, Iran, the western Brazilian Amazon, Tajikistan,and Mongolia.6471 Second, HDV prevalence has notdecreased in northern Europe. Prevalence in northernEurope and the USA was thought to be low and confinedto high-risk groups, such as intravenous drug-users;however, in London, UK, HDV prevalence in people withHBV increased from 26% in the 1980s to 85% in2005.72,73 In Germany, although the prevalence of anti-HDV antibody in those with HBV reduced from 186% in1992 to 68% in 1997, from 1999 onwards the rates haveincreased again to between 8% and 14%. 74HDV remainsprevalent in France.75The common factor in these threecountries is HDV infection in young individuals who

L-HDAg

Delta virion

Ribonucleoprotein

Hepatocytemembrane

receptor

L-HBsAg

S-HBsAg

M-HBsAg

S-HDAg

12 3

9

Nucleus

Genomic RNA

Endoplasmicreticulum

Golgi complex

Antigenomic RNAmRNA 6

7

68

4 4

5

Figure : Schematic representation of the delta virion and its replication cycle

(1) The virion attaches to the hepatocyte via an interaction between large-HBsAg and an uncharacterised membrane receptor in the host cell; (2) the virion enters the

cell and is uncoated; (3) the RNP is targeted to the nucleus; (4) genomic RNA is transcribed in the nucleus to form antigenomic RNA, which forms the template for

replication of new transcripts of the circular genome, and mRNA, which contains the open reading frame; (5) the mRNA is exported to the cytoplasm where it is

translated at the endoplasmic reticulum to form new molecules of hepatitis D antigen; (6) the new antigen molecules return to the nucleus where the small-HDAg

isoform supports further genome replication, and where both forms of hepatitis D antigen associate with new transcripts of genomic RNA to form new RNPs;

(7) RNPs are exported to the cytoplasm where large-HDAg facilitates association with HBV envelope proteins in the ER to form new virus particles; (8) these particles

bud through an intermediate compartment; (9) they are then exported from the hepatocyte via the trans-Golgi network to re-infect further cells.

RNP=ribonucleoprotein. mRNA=messenger RNA. HBV=hepatitis B virus. HBsAg=hepatitis B surface antigen. HDAg=hepatitis D antigen. ER=endoplasmic reticulum.


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have migrated from regions of high prevalence. Even inItaly the decline in HDV has now reached a plateau,76anda report has shown a high prevalence of HDV of 17% innon-EU citizens (mainly those from eastern Europe) whoare infected with HBV.77 Third, clustered outbreaks ofHDV superinfection continue to be reported, notably inVenezuela, Ecuador, Mongolia, and Greenland,7881whichare similar to those recorded in Samara (Russia), Okinawa(Japan), Central Africa, and the Amazon Basin in the1980s and 1990s.8285Thus, while outbreaks still occur andpopulation migration from endemic countries increases,the threat of HDV infection remains.

Modes of transmissionLike HBV, HDV is transmitted via the parenteral routethrough exposure to infected blood or body fluids, andtests in chimpanzees have shown that only a very small

inoculum is suffi cient to transmit infection.86 Thus,transmission rates remain high in intravenous drugusers. There is evidence for sexual transmission,87 andpeople with high-risk sexual activity are at increased riskfor infection.88Intrafamilial spread occurs and seems tobe common in regions of high prevalence, which isknown as inapparent parenteral transmission. Perinataltransmission of HDV is uncommon. Because of screeningof blood products, new infections in haemophiliacs, bloodtransfusion recipients, and patients receiving haemo-dialysis are no longer seen in developed countries.

Clinical expression and natural historyWith HBV and HDV co-infection, the fate of HDV isdetermined by the host response to HBV, which in more

than 95% of adults results in viral clearance. Acuteco-infection can be more severe than acute mono-infection with HBV, thereby resulting in acute liverfailure; however, disease expression is wide-ranging. Bycontrast, HDV superinfection of an individual withchronic HBV results in chronic HDV infection in mostpeople. In the remainder, replication of HDV stops, andthe natural history of the disease is that of the underlyingHBV; however, the residual liver disease might beadvanced. Important evidence from an Italian cohortshowed that 10% of patients with anti-HDV antibodiescleared HBsAg after a mean follow-up of 4 years,compared with 28% of those with HBV mono-infection.89The mechanism for increased rate of HBsAg loss afterclearance of HDV RNA is unknown, but an enhancedimmune response against HBV and HDV seemsplausible. Figure 3 shows the typical evolution of

serological and virological markers in co-infected patientsversus superinfected patients.

Superinfection can present as acute hepatitis in apreviously undiagnosed carrier of HBsAg, and is oftenmisdiagnosed as acute HBV or as worsening liverdisease due to chronic HBV.90 At initial histologicalassessment, patients with HDV superinfection areoften shown to have severe hepatitis with advancedfibrosis.9193 These patients undergo more rapidprogression to cirrhosis4,5 and increased hepaticdecompensation leading to death6,7 than do those withHBV infection alone. Despite the high rate ofprogression to cirrhosis, not all studies show anincreased rate of hepatocellular carcinoma, perhapsbecause of suppression of HBV replication by HDV.73

HighIntermediateLowVery low

Insufficient data

Genotype 58

Genotype 1

Genotype 1

Genotype 1/3

Genotype 1/2/4

Genotype 1

Figure : Worldwide prevalence of HDV and the geographic distribution of i ts genotypes

Two subtypes1A and 1Bhave been identified in HDV genotype 1. 1A is predominant in Asia and 1B in the USA. Both are common in the Mediterranean. HDV genotype 2 occurs in the Far East.

HDV genotype 3 occurs exclusively in the northern part of South America and is linked to HBV genotype F. Genotypes 58 have been identified primarily in patients from Africa. HDV=hepatitis D virus.


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In liver transplantation for HDV, hepatitis Bhyperimmune globulin (HBIg) leads to rapid reductionin HBsAg concentrations, and serum HDV RNAdeclines in parallel.94Graft re-infection is prevented bylong-term administration of HBIg, which prevents

HBV re-infection; hence, propagation of HDV cannotbe supported.95,96Originally, HDV was thought to persistas an isolated or latent infection after transplantation;however, more advanced and sensitive serologicalassays have shown that this latency does not occur.97Hence, the outcome of liver transplantation for HDV isvery good, with 5-year survival of more than 80%, andbetter than the outcome noted with transplantation forother causes of liver disease.95,98

Although early studies identified that HDV genotypeaffects the natural history of HDV, the more recentlyidentified genotypes 58, from Africa, are less wellcharacterised. A study from Taiwan99showed a lower rateof remission and more adverse outcomes in patients withgenotype 1 than in those with genotype 2. Patients with

genotype 4 often have mild liver disease,100but a variantof genotype 4 on the Miyako Island in Okinawa, Japan, isassociated with greater progression to cirrhosis thangenotype 4 is in Taiwan.101Genotype 3 has been linked tooutbreaks of severe, florid hepatitis (Labrea fever),

culminating in acute liver failure and death in theAmazon Basin of South America.44Outbreaks of severehepatitis due to genotype 1 have also taken place, butgenotype 1 is associated with a wide range of disease, 102thereby making the relation between genotype andnatural history diffi cult to interpret.

Although HBV genotype might also determine thenatural history of HDV, any effect is diffi cult to studybecause in many co-infected individuals the serum levelof HBV DNA is too low for genotype analysis. Nonetheless,Su and colleagues99showed a significantly lower remissionrate and more adverse outcomes in patients with HDVsuperinfection with HBV genotype C than with HBVgenotype B, in a population from Taiwan who were mostlyinfected with HDV genotypes 1 and 2. A study from Brazil

A

B

Time after exposure (weeks)

C

Anti-HBc IgGAnti-HBc IgMAnti-HD IgGAnti-HD IgM

HBsAgHDV RNAALT

Figure : Typical evolution of serological and virological markers in HDV infection

(A) Simultaneous co-infection with HBV and HDV, resulting in clearance of both viruses in almost all patients. (B) HDV superinfection of an HBV carrier with

self-limited outcome. The spontaneous clearance of HDV RNA might take years to occur (indicated by break on x axis) and, in few cases, can herald the loss of HBsAg

(not depicted). (C) HDV superinfection of a HBV carrier with chronic persistent viral replication; the more common outcome after superinfection. HDV=hepatitis

delta virus. ALT=alanine aminotransferase. HBV=hepatitis B virus. HBsAg=hepatitis B surface antigen.


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has suggested that HBV genotype determines the severity

of liver inflammation and the HDV viral load inindividuals with co-infection; however, the consequenceof this in relation to outcome is not clear.103

Patterns of viral dominanceCo-infection and superinfection with HDV suppressesHBV replication in patients and in model systems.About 7090% of patients with HDV co-infection areHBeAg negative, and most have low serum HBVDNA.73,104106Evidence has indicated that the small (p24)and large (p27) HDV proteins downregulate HBV repli-cation by repressing activity of the two HBV enhancerregions, and by transactivating the interferon-inducibleMxA gene, which inhibits HBV replication by reducing

the export of viral mRNA from the nucleus.107,108If HDVinfection is cleared either spontaneously or aftertreatment with interferon alfa, then HBV replicationcan reactivate.109

Many patients with HBV and HDV have serologicalevidence of exposure to hepatitis C virus (HCV)about30% in European cohorts.73,105 In those with tripleinfection, HDV is the dominant virus because it not onlysuppresses HBV replication, but also inhibits HCVreplication.110 Indeed, HCV RNA was detected in lessthan 19% of patients who were anti-HCV antibodypositive, HBsAg positive, and anti-HDV antibody posi-tive.73,105,110Most patients who are HCV RNA negative haveprobably cleared HCV infection, but further long-termfollow-up studies are needed, especially because patternsof viral dominance evolve over time in HBV and HCVco-infection.111A dynamic pattern of viral dominance alsoexists in HBV and HDV co-infection.112

PathogenesisThe mechanisms that determine whether an individualclears HDV spontaneously or becomes chronicallyinfected, and the processes that cause severe hepatitisand rapid progression of fibrosis, remain unclear.HDAg is not directly cytotoxic in human hepatocytes orin transgenic mice.113,114 Viral load of HDV was notassociated with severity of liver injury in a cohort of

patients in a clinical trial.106 However, evidence fromobservational cohort studies suggests that in the acutephase of HDV infection, HDV viraemia is associatedwith an increased level of alanine aminotransferase andsuppressed HBV. In the chronic phase, falling HDVRNA, reactivation of HBV, and moderate transaminitisculminate in a late phase that is characterised by eitherthe development of cirrhosis and hepatocellularcarcinoma due to replication of HDV or HBV, orremission with clearance of both viruses.115Hence, viralload of HDV and HBV fluctuates according to the stageof viral infection. Whether this fluctuation represents adirect relation with the pathogenesis of diseaseprogression remains unclear, and other factors shouldbe considered.

Host immune response

The host immune response is thought to have animportant role in viral clearance and liver injury. An earlystudy116 showed that the function of natural killer cellswas active in individuals who suppressed HDV RNAduring treatment with interferon, but further studiesinvestigating the innate immune response in thepathogenesis of HDV are needed. The adaptive immuneresponse in HDV infection is also poorly defined. In onestudy,117 weak HDAg-specific responses of CD4 T cellswere elicited in patients with inactive HDV infection(anti-HDV antibody positive but RNA negative), butabsent in those with chronic HDV infection. Furtherstudies118 have shown that chronic HDV infection isassociated with a response that is dominated by T-helper-2

(Th-2) cells, with a high frequency of T cells secretinginterleukin-10. Furthermore, after antiviral therapy,production of interleukin-10 that was HDV-peptidespecific remained higher in patients who did not respondto treatment than in those who did. Secretion ofinterferon-inducible protein-10 was, however, morefrequent in responders.119 These findings suggest thatHDV subverts the adaptive immune response away fromthe Th-1-biased CD4 and CD8 T-cell response that isneeded for viral clearance. Indeed, Huang andcolleagues120showed that the responses of HDV-specificCD8 cytotoxic T lymphocytes were detected in those withpast, but not active, infection.

CD4 T cells are essential to the antiviral immuneresponse because they help CD8 T cells and B cells bystimulating antigen presenting cells and cytokinesecretion. However, cytotoxic CD4 T cells that areperforin-positive might also have a role in directlykilling virus-infected cells. A higher frequency of thesecells were recorded in patients who were co-infectedwith HBV and HDV than with HBV or HCVmono-infection, and the frequency of perforin-positivecells was positively correlated with disease activity.These findings suggest that such perforin-positive cellsare implicated in the pathogenesis of HDV liverdisease.121These studies suggest an important role forthe adaptive immune response, but the precise

mechanisms are unknown.

Role of HDV genotype in pathogenesisEffi ciency of RNA editing is lower with genotype 2 thanwith genotype 1, but this difference is unlikely to accountfor the reduced disease activity.122 The sequence of theC-terminal moiety in large-HDAg varies widely betweengenotypes, with up to 74% divergence between the HDVpackaging signal domains of genotype 1 and other HDVgenotypes. The C-terminal sequence of the large antigenin genotype 1 results in better packaging ability than ingenotype 2, which interacts weakly with clathrin, leadingto less effi cient assembly of particles.123125 However, allHDV genotypes are able to bind clathrin to some degree,which lends support to the role of clathrin binding in the


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assembly of HDV particles.126 Which, if any, of thesemechanisms underpin the effect of genotype on thenatural history of HDV is unclear.

DiagnosisThe development of anti-HDV antibodies is universal inindividuals with HDV; therefore, every patient who isHBsAg positive should be tested for anti-HDV IgGantibodies, which persist even after the patient hascleared HDV infection. Although active HDV infectionwas diagnosed historically by the presence of anti-HDV IgM antibodies, it is now confirmed by the detectionof serum HDV RNA with sensitive real-time PCRassay.127 Covert HDV infection has not been reported;therefore, testing of HDV RNA in the absence of

anti-HDV antibodies is not indicated. Because of thevariability of the genome sequence, assays of HDV RNAmight produce false-negative results, and testing of anti-HDV IgM antibodies still has a role in patients whotest negative for HDV RNA, but have clinical featuresof HDV-related liver disease. International standard-isation of the HDV RNA assay is needed. Somelaboratories undertake quantitative assays, but serumconcentrations of HDV RNA do not correlate withdisease activity or stage of liver fibrosis.106 Serialquantification of HDV RNA is used to determine theresponse to antiviral treatment.128,129

HDV genotyping is usually only available in specialistcentres, but is gaining acceptance as a useful diagnostictest because patients with HDV genotype 1 are at ahigher risk of developing end-stage liver disease99 andhave a lower response to treatment with pegylatedinterferon than do patients with African genotypes(unpublished data). All patients with HDV also need

investigation for HCV and HIV because co-infectionwith these viruses is common.73,105,130The panel shows therelevance of serological and virological markers in HDV.Patients who test positive for serum HDV RNA shouldbe further investigated with liver biopsy to assess theseverity of liver disease. Studies have shown noassociation between levels of HDV RNA, HBsAg titre,values of liver tests, and the histological staging of liverdisease, thus reinforcing the pivotal part that liver biopsyplays in the assessment of HDV-related liver disease.Tests for non-invasively assessing the progression ofliver fibrosis, such as transient elastography, can beuseful for detecting advanced hepatic fibrosis, but havenot been validated in chronic HDV and therefore cannotyet be recommended. The aspartate aminotransferase to

HBsAg

Anti-HDV IgG AbTreat as per local HBV

guidelinesHCV/HIV Ab

Add ribavirin

+ve

-ve

>1 -ve

+ve

+ve

HCV RNA+ve HBV DNA>2000 IU/mL

Reactivation of HBV aftersuccessful control of HDV

HDV RNA

Liver biopsy

Consider PEG- IFN- for atleast 48 weeks

Consider addition of potentnucleos(t)ide analogue

Add potent nucleos(t)ideanalogue

Figure :Suggested algorithm for the investigation and management of a patient with HDV

HBsAg=hepatitis B surface antigen. HBV=hepatitis B virus. HCV=hepatitis C virus. HDV=hepatitis delta virus.

PEG-IFN-=pegylated interferon alfa. Ab=antibody.

Panel:Diagnostic markers in HDV infection and their

importance

Anti-HDV IgG antibody

Positive in all individuals exposed to HDV, and persists

long-term, even after viral clearance.

Anti-HDV IgM antibody

Positive in acute infection, negative in past infection but

persists in a large proportion of patients with chronic

infection. Sometimes used as surrogate marker for HDV

replication but not 100% sensitive or specific.

HDV RNA qualitative

Marker of HDV replication. Positive in all patients with

chronic infection. Negative in spontaneous or

treatment-induced viral clearance.

HDV RNA quantitative

Useful method to predict or monitor treatment response.

HBsAg qualitative

Must be positive for HDV infectivity.

HBsAg quantitative

Positively correlated with HDV RNA. Might be useful to

predict or monitor treatment response, since falling titre

heralds HBsAg loss, and hence HDV clearance.

HBeAg

Negative in about 85% of patients; associated with

detectable anti-HBe.

HBV DNA quantitative

Usually negative or low level because suppressed by HDV.

Might be increased, especially in patients with detectable

HBeAg. Can reactivate after spontaneous or

treatment-induced clearance of HDV.

ALT

Usually increased but does not correlate well with degree of

histological liver damage.

HDV=hepatitis delta virus. HBsAg=hepatitis B surface antigen. HBeAg=hepatitis B early

antigen. HBV=hepatitis B virus. ALT=alanine aminotransferase


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platelet ratio index was shown to have no use inpredicting the stage of fibrosis in patients with HDV.106The assessment of patients with HDV infection issummarised in figure 4.

TreatmentThe ideal endpoint of any treatment for HDV is not onlyclearance of HDV, but also of the helper virus HBV.Hence, a major challenge of defining the optimumtherapy is the added complexity of targeting two persistentviral infections. A meta-analysis of five studies oftreatment with recombinant interferon concluded thatsuch treatment was beneficial for HDV in terms of serumaminotransferase reduction, but the response was poorlysustained after discontinuation of treatment, and was notnecessarily associated with clearance of HDV RNA.There were better results with higher doses ofinterferonat least 5 MU daily or 9 MU thrice weekly for12 months, rather than for 6 months.131,132 High-doseinterferon induced delayed clearance of RNA, and

sometimes, loss of HBsAg with resultant improvementin histological inflammatory activity and stage offibrosis.132In a single-case study,133extended treatment for12 years led to clearance of HDV RNA and loss of HBsAgassociated with complete regression of fibrosis.

Consequently, some have advocated prolonging treatmentduration,47but studies have shown no additional benefitin giving treatment for 24 months rather than for12 months.134,135 The potential benefits of this approachshould be balanced with the side-effects, cost, andlogistical implications. Interferon-induced clearance ofHDV RNA has been associated with a fall in HBsAgconcentrations, and sometimes a loss of HBsAg, withabsence of HBsAg decline in patients who do not respondto treatment.128 However, the decrease in HBsAg oftenlags behind that of HDV RNA, and hence the mechanismbehind this finding is unclear. More data will emerge asquantification of HBsAg becomes more routinely used.

Investigators have examined the use of pegylatedinterferon for chronic HDV. Series from Italy,136France,109

Summary of therapy Regimen Number of

patients

Median age

(years)

Cirrhosis

(%)

Median baseline

HDV RNA

HDV

genotype

SVR

(%)

SBR (%)

Castelnau et al (2006)109

Open label pilot PEG monotherapy PEG-IFN2b; 15g/kg per week;

12 months

14 42 29 5* 1=86%

5=14%

43 57

Niro et al (2006)136

Multicentre randomised trial PEG vsPEG+rbv PEG-IFN2b; 15 g/kg per week;

72 weeks

+Rbv 800 mg per day for first

48 weeks

16

22

45

43

75

73

..

..

..

..

25

18

25

27

Erhardt et al (2006)137

Open label pilot PEG monotherapy PEG-IFN2b; 15 g/kg per week;

48 weeks

12 34 27 7* 1 or 2 17 17

Wedemeyer et al (2011)138

Multicentre randomised trial PEG+placebo vs

PEG+adv vsadv

PEG-IFN2a; 180 g per week;

48 weeks

+Adv 10 mg; once dailyAdv 10 mg; once daily 48 weeks

29

3130

38

4233

20

1424

6*

6*6*

1

11

31

260

45

3510

Yurdaydin et al (2008)139

Multicentre randomised trial LAM vsLAM+IFN vsIFN LAM 100 mg; once daily;

12 months

+IFN2a; 9 MU three times per

week for last 10 months

IFN2a; 9 MU three times per

week; 12 months

17

14

8

38

35

46

..

..

..

6*

6*

6*

..

..

..

12

36

50

24

21

50

Gunsar et al (2005)140

Single centre randomised trial IFN vsIFN+rbv IFN2a; 9 MU three times per

week 96 weeks

+Rbv 10001200 mg/day

10

21

39

38

30

24

..

..

..

..

20

20

235

235

Farci et al (2004)132

Single centre randomised trial High-dose IFN vs

low-dose IFN vsno Rx

IFN2a; 9 MU three times per

week 48 weeksIFN2a; 3 MU three times per

week 48 weeks

Untreated controls

14

14

13

35

35

38

71

64

61

6

6

6

1

1

1

0

0

0

50

7

8

SVR=sustained virological response (RNA negative 6 months after end of therapy). SBR=sustained biochemical response (normal aminotransferases 6 months after end of therapy). PEG=pegylated interferon.

IFN=interferon. Rbv=ribavirin. Adv=adefovir. LAM=lamivudine. Rx=prescription drug. *Log10copies per mL. Log10genome equivalents per mL.

Table:Summary of key trials of alpha-interferon for chronic hepatitis delta virus


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and Germany,137 have shown sustained virological

responses in 21% of 38 patients, 43% of 14 patients, and17% of 12 patients, respectively. In the HIDIT-1 trial, 138which randomised 90 patients from Germany, Turkey,and Greece, pegylated interferon led to a 28% sustainedvirological response; the addition of adefovir did notimprove virological response but did lead to increasedsuppression of HBsAg concentrations, whereas adefovirmonotherapy was ineffective.138Why response rates varybetween series is not clear, but might relate to baselineclinical, demographical, and virological characteristicsthat are heterogeneous. The table shows data for themain interferon trials.

The simplicity of the delta virus, particularly the lack ofviral polymerase, limits the targets for therapeutic

compounds. Investigators have studied nucleos(t)ideanalogues, which block the HBV polymerase, butlamivudine alone was not effective at reducingconcentrations of HDV RNA and did not increasesustained virological response when combined withinterferon.139,141144Famciclovir was also ineffective.145Riba-virin alone,146or combined with interferon140,147or pegylatedinterferon136 did not increase the virological response.Entecavir did not reduce HDV viraemia, but did reducealanine aminotransferase and HBV DNA in a smallsubset of patients who had low HDV RNA and high HBVDNA.148One report has shown HDV clearance associatedwith seroconversion of HBsAg in a man treated withpegylated interferon plus tenofovir and emtricitabine,who had high levels of both HBV DNA and HDV RNA.149In a long-term observational study150of 16 patients whowere co-infected with HIV, HBV, and HDV and on highlyactive antiretroviral therapy with anti-HBV activity,including tenofovir, a significant reduction in HDV RNAwas noted for a median of 61 years, and three patientsbecame RNA negative. The mechanism for this reductionis unclear, because it was not associated with acorresponding decrease in concentrations of HBsAg.Hence, treatment with nucleos(t)ide analogues is noteffective at reducing HDV replication, but might be usefulin patients with high concentrations of HBV replication,and might be of potential benefit when used long term by

gradually reducing HBsAg concentrations. The use ofolder analogues of nucleos(t)ides to induce HBVmutations is controversial. Lamivudine-induced muta-tions in the polymerase gene at rtM204V or rtM204I areassociated with changes in the overlapping envelope geneproducts, such as in sW196L/S, which inhibits secretionof HDV particles.151There is insuffi cient evidence to showwhether the failure to package and secrete HDV isbeneficial, or indeed whether the retention of HDV RNAwithin cells is harmful.

Baseline and treatment factors predicting the outcomeof interferon treatment are poorly defined. HDV RNA iscorrelated positively with HBsAg titre,106,128and baselinevalues of both predict response to therapy (unpublisheddata). HDV genotype 1 is associated with a reduced

response to pegylated interferon (unpublished data),

but whether this response relates to a property of theHDV sequence itself, or merely to the finding thatpatients with genotype 1 have increased viral loads, isunknown. By contrast with treatment response inpatients with chronic HCV, in which rates of sustainedvirological response to pegylated interferon aresubstantially reduced by the presence of underlyingcirrhosis, cirrhosis associated with HDV infection didnot obviously affect the response to pegylated inter-feron.136,137,152Baseline liver biochemical tests are also notassociated with treatment outcome.109

Although the kinetics of HDV RNA in therapy havebeen used to predict long-term virological outcome, dataare scarce.128,129 Castelnau and colleagues109 showed that

75% of patients achieving an end-of-treatment responsewere HDV RNA negative by month 6 of treatment,compared with no patients who did not respond totreatment; however, undetectable mid-treatment RNAdid not protect against relapse. The investigators alsonoted that HDV RNA values were lower at the end oftherapy than at baseline in some patients who did notrespond, which could justify extension of therapy in thisgroup. Yurdaydin and colleagues139 defined threevirological patterns of response to interferon: complete,in which RNA negativity at 6 months predicted sustainedvirological response; partial, in which incompletesuppression of RNA at 6 months predicted rebound flarein viral replication after discontinuation of therapy; andnon-response, in which viraemia persisted withoutdecline throughout treatment and follow-up. Livertransplantation is the only treatment option for patientswho have end-stage liver disease due to co-infection withHBV and HDV, and is appropriate for those with acuteliver failure that fulfils criteria for poor prognosis. 153Re-infection of the graft is reduced by long-termadministration of immunoglobulins against the HBsAg(HBIg) after transplantation,95,96 creating an HBsAg-negative environment in which HDV cannot survive.97The use of potent antivirals against HBV has furtherreduced the risk of HBV re-infection.

The accepted practice for treatment of chronic HDV is

subcutaneous injections of pegylated interferon everyweek for at least 48 weeks. This practice should beconsidered in patients with active replication of HDVRNA, and histological evidence of disease activity, andwith no contraindications to interferon therapy. We wouldadvocate an individualised approach to therapy in patientswho have not achieved clearance of HDV RNA by sensitivePCR-based assay at 48 weeks. Those who have persistent,high-level viraemia, with detectable anti-HDV IgMantibody, and ongoing transaminitis, are unlikely torespond to further therapy. By contrast, patients with afalling viral load, declining IgM antibody titre, andresolving transaminitis, or with progressive decline inHBsAg titre, might benefit from extending therapy to72 weeks, and perhaps beyond, if improvement is


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sustained and if tolerability is favourable. In patients with

a high concentration of HBV DNA, addition of a potentnucleos(t)ide analogue to inhibit HBV replication islogical; however, the long-term effectiveness has yet to bedefined. Potent nucleos(t)ide analogues are the treatmentchoice for patients in whom HBV replication is reactivatedafter successful control of HDV replication. Suchanalogues might also be used in patients with detectableserum HBV DNA who cannot be treated with pegylatedinterferon such as those with advanced liver disease.

Novel therapiesthe futureIncreased understanding of the molecular virology ofHDV, and the poor overall response to conventionalantiviral therapy, has driven the search for alternative

antiviral targets. Prenylation of large-HDAg is essentialfor viral assembly and secretion,33and in mouse modelsprenylation inhibitors inhibited the assembly andrelease of HDV, leading to rapid clearance of HDVRNA from serum.154 These compounds are in pre-clinical development. Other forms of post-translationalmodification of HDAg, such as acetylation, phos-phorylation, and methylation, might also prove fruitfulas targets for novel therapeutic compounds, althoughnone are yet in development. Studies showing thatmyristoylated synthetic peptides specific for theN-terminal region of the pre-S1 domain of HBsAg areable to inhibit viral attachment and hence HDVinfectivity, draw attention to an alternative therapeutictarget.14,17,155,156 As we learn more about cell-signallingpathways associated with the pathogenesis of HDV,further targets might be exposed.

Therapy for HDV deserves the resurgence of interestit has received. Treatment effectiveness remains unsatis-factory, and developments have lagged behind thoseachieved for the treatment of hepatitis B. The refinementof conventional antiviral regimens will continue withlarger collaborative trials, and drug development willhopefully follow the advances in HDV biology. Thesedevelopments remain necessary while worldwidemeasures to control the helper virus are inconsistent andthe prevalence of HDV does not decline in many areas,

resulting in a continuing and substantial health burden.Contributors

All authors contributed to the conception and writing of the Seminar.Sections were divided between SAH and PMH, and each authorundertook the relevant searches and wrote the assigned sections.SAH assembled the sections and all authors revised the final version.

Conflicts of interest

PMH has received payment for board membership from Roche;consultancy fees from Gilead, Bristol-Myers Squibb, and Phytopharm;and payment for participation in a speakers bureau from Gilead andBristol-Myers Squibb. HW has received payment for board membershipfrom Roche, Gilead, Bristol-Myers Squibb, and Schering Plough;consultancy fees from Roche and Novartis; grant support from Rocheand Gilead; payment for participation in a speakers bureau from Roche,Gilead, Bristol-Myers Squibb, Schering Plough, and Novartis; andpayment for developing educational presentations from Roche. SAH

declares that she has no conflicts of interest

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Hepatitis D review from the Lancet

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