j.1478-3231.2010.02320.x

REVIEW ARTICLE

Hepatitis D virus: an updateStephanie Pascarella1 and Francesco Negro2,3

1 Department of Pathology and Immunology, University of Geneva, Geneva, Switzerland

2 Division of Clinical Pathology, University Hospital, Geneva, Switzerland

3 Division of Gastroenterology and Hepatology, University Hospital, Geneva, Switzerland

Keywords

cirrhosis delta hepatitis fulminant hepatitis

hepatitis D virus

Correspondence

Prof Francesco Negro, Divisions of Clinical

Pathology and of Gastroenterology and

Hepatology, University Hospital, 4 rue Gabrielle-

Perret-Gentil, 1211 Geneva 14, Switzerland

Tel: 141 22 3795 800

Fax: 141 22 3729 366

e-mail: [email protected]

Received 20 May 2010

Accepted 6 July 2010

DOI:10.1111/j.1478-3231.2010.02320.x

AbstractHepatitis D virus (HDV) infection involves a distinct subgroup of individualssimultaneously infected with the hepatitis B virus (HBV) and characterized byan often severe chronic liver disease. HDV is a defective RNA agent needingthe presence of HBV for its life cycle. HDV is present worldwide, but thedistribution pattern is not uniform. Different strains are classified into eightgenotypes represented in specific regions and associated with peculiar diseaseoutcome. Two major specific patterns of infection can occur, i.e. co-infectionwith HDV and HBV or HDV superinfection of a chronic HBV carrier.Co-infection often leads to eradication of both agents, whereas superinfectionmostly evolves to HDV chronicity. HDV-associated chronic liver disease(chronic hepatitis D) is characterized by necro-inflammation and relentlessdeposition of fibrosis, which may, over decades, result in the developmentof cirrhosis. HDV has a single-stranded, circular RNA genome. The virionis composed of an envelope, provided by the helper HBV and surroundingthe RNA genome and the HDV antigen (HDAg). Replication occurs in thehepatocyte nucleus using cellular polymerases and via a rolling circleprocess, during which the RNA genome is copied into a full-length, comple-mentary RNA. HDV infection can be diagnosed by the presence of antibodiesdirected against HDAg (anti-HD) and HDV RNA in serum. Treatmentinvolves the administration of pegylated interferon-a and is effective inonly about 20% of patients. Liver transplantation is indicated in case of liverfailure.

The discovery of hepatitis D virus (HDV) dates back tothe mid-1970s, and followed the detection of a novelnuclear antigen in patients with a severe form of chronichepatitis B. The first report of this antigen, believed to bea hepatitis B antigen and called the delta antigen, waspublished in 1977 (1). Three years later, experiments inchimpanzees had already demonstrated that the hepatitisdelta antigen (HDAg) was a structural component of atransmissible pathogen that required the hepatitis B virus(HBV) for its life cycle (2). The virion particle was shownto be composed of the HBVenvelope proteins surround-ing a ribonucleoprotein core-like structure comprisingthe HDAg and a molecule of RNA (3). The delta agentobtained the status of a distinct virus in 1983 with theofficial name of hepatitis delta virus. Nowadays, the termhepatitis D virus is preferred, even though delta isstill used. The uniqueness of this virus was confirmedin 1986, after cloning and sequencing of its genome(4). Thereafter, HDV obtained its own genus, theDeltavirus (5).

Epidemiology

Overview

Hepatitis D virus concerns all age groups. The distribu-tion pattern of this virus, investigated by seroprevalencestudies of anti-HD in HBsAg-positive patients, is world-wide but not uniform (6). Regardless of the fact thatHDV needs HBV for its life cycle, the distribution patternof each virus is different. For example, 90% of HBVcarriers are infected with both viruses in the PacificIslands, whereas the rates decline to 8% in Italy and 5%in Japan (6). Current estimates suggest that 1520million people are infected with HDV. However, oneshould consider that these estimates are inaccurate anddifficult to perform as systematic screening is not per-formed in HBV-infected individuals, especially if theypresent with normal liver enzymes (7). In addition, anti-HD may be lacking in immunodeficient patients andseroreversion may occur after resolution of the disease,rendering the diagnosis of past infections impossible.

Liver International (2010)c 2010 John Wiley & Sons A/S 7

Liver International ISSN 1478-3223

Main areas of prevalence are the Mediterranean basin,the Middle East, Central and Northern Asia, West andCentral Africa, the Amazonian basin, Venezuela, Colom-bia and certain islands of the Pacific. The Far East is lessconcerned but HDV is nonetheless present in Taiwan,China and India (Fig. 1).

The prevalence of HDV infection has significantlydeclined in some regions of the world such as Italy (8,9), Spain (10), Taiwan (11) and Turkey (12), mainlybecause of vaccination campaigns against HBV, systema-tic screening of blood and blood products and ofpregnant women, implementation of safety proceduresagainst bloodborne infections among healthcare workers,switch to disposable syringes, improved socioeconomicconditions and the increased awareness of the generalpublic on sexually transmissible agents that followed theacquired immunodeficiency syndrome scare (13).

The number of infected patients, however, stoppeddecreasing towards the end of the 1990s in Europe. Theprevalence remained stable for example in London (7), inHanover (14) and in Italy (13) and seemed to beincreasing in France (15). This recrudescence in indus-trialized countries is mainly observed because of anincreased immigration from Eastern Europe, Africa, theMiddle East, Turkey and the ex-Soviet Union. Immigra-tion from endemic regions is not the only cause: intrave-nous drug use, sexual practices and body modificationprocedures may also be involved. Moreover, HDV hasemerged in new regions such as Russia (16), NorthernIndia, Southern Albania, mainland China and somePacific islands such as Okinawa.

Hepatitis D virus genotypes

The evolution rate of HDV RNA has been determined bylongitudinal studies of the RNA quasispecies changes inchronic hepatitis D patients. This rate varies across a

range comprised between 3 102 and 3 103 basesubstitutions per nucleotide per year (17). Highly con-served domains are located around the genomic andantigenomic RNA autocatalytic cleavage sites and theRNA-binding domain of HDAg (18, 19). HDV genomesof the different isolates present up to 39% heterogeneity.The different sequences have been classified into eightHDV genotypes (20). Except for genotype 1, which isrepresented worldwide, all other genotypes are mostlyfound in specific geographical areas. Genotype 2 prevailsin Japan (21), Taiwan (19) and Russia (22), genotype 3 inthe Amazonian region (23), genotype 4 in Japan (24, 25)and Taiwan (26) and genotypes 58 in Africa (20)(Fig. 1). Multiple genotypes infection can occur inpatients at high risk of repeated exposure. A singlegenotype generally dominates and only 10% of the viralpopulation is represented by the minor strain (27).Furthermore, chimeric forms of HDV RNA have beendetected in Taiwanese patients infected with both geno-types 1 and 4 (28, 29).

The HDV genotype can be determined by restrictionfragment length polymorphism analysis of polymerasechain reaction (PCR)-amplification products (30), bysequencing and by immunohistochemical staining ofliver biopsies using genotype-specific antibodies (31).

Clinical features and diagnosis

Patterns of hepatitis D virus infection

As HBV is essential for HDV virion assembly and release,HDV infection is always associated with HBV infection.Two major patterns of infection can occur: co-infectionand superinfection. A third, minor pattern, the so-calledhelper-independent latent infection, has been reported inthe liver transplant setting, and will be briefly discussedbelow.

Fig. 1. Schematic representation of the main areas of HDV distribution in the world. Bold numbers represent the predominant HDV genotypefor the mentioned area. Adapted from (184) and (185).

Liver International (2010)8 c 2010 John Wiley & Sons A/S

Hepatitis D virus: an update Pascarella and Negro

Co-infection is a simultaneous infection with bothviruses that leads to acute hepatitis B and D. From aclinical point of view, this is indistinguishable from acutehepatitis B (32), although it may be more severe and twopeaks of serum alanine aminotransferase (ALT) andaspartate aminotransferase (AST) may be observed.Because HBV is essential for HDV, the rate of progressionto chronicity is the same as that of acute hepatitis B(o5%).

Superinfection is the HDV infection of an individualchronically infected with HBV. This way of infectioncauses severe acute hepatitis, which progresses to chroni-city in almost all patients (up to 80%) (32). Once chronicHDV infection is established, it usually exacerbates thepre-existing liver disease due to HBV (33). HBV replica-tion is, however, usually suppressed to low levels duringthe acute phase of HDV infection. This suppressionbecomes persistent in case of a chronic hepatitis Destablishment (34, 35) (Table 1).

Helper-independent latent infection was initially re-ported to occur after liver transplantation (36). HBVinfection of the grafted liver is usually preventedby administration of hepatitis B immunoglobulins.Hepatocytes may thus be infected with HDV alone.HDAg can be detected in the liver by immunohistochem-istry before HBV recurrence, as the helper virus in onlynecessary for particle formation and not for viral replica-tion (37). HDV viraemia (as determined by molecular

hybridization) is only observed several months later on,when residual HBV evades neutralization, thus allowingfor HDV rescue and cell-to-cell spread (36). This thirdpattern of infection has been revisited with the advent ofmore sensitive, reverse transcription (RT)-PCR-basedtechniques for detecting HDV RNA. Moreover, experi-ments on chimpanzees first infected with HDV and laterchallenged with HBV have shown a rescue of HDV whenHBV inoculation was performed at day 7 but not at 1month (38).

Route of hepatitis D virus transmission

The natural reservoir is man, even though chimpanzeesinfected with HBV and woodchucks infected with thewoodchuck hepatitis virus can be infected by HDV.Infection with HDV is parenterally transmitted. In in-dustrialized countries, high-risk populations includeillicit drug users and people exposed to blood or bloodproducts. HDV does not seem to be a typically sexuallytransmitted disease, as the frequency of infection insexually promiscuous heterosexual or homosexualgroups is lesser than that of HBVor HIV (39). In Taiwan,however, this route is the predominant way of transmis-sion (40). In socially and economically disadvantagedpopulations, many infections occur by inapparent intra-familial routes of transmission, facilitated by poor hy-giene. Perinatal transmission of HDV is rare.

Markers and diagnosis

Hepatitis D virus induces innate and adaptive immuneresponse in the infected host, which consist of immuno-globulin M (IgM) and IgG production (41). Therefore, inthe serum, the three specific HDV markers are HDVRNA, HDAg and anti-HDV.

Hepatitis D virus RNA can be detected in serum byeither molecular hybridization or RT-PCR. Hybridiza-tion assays have a detection limit of about 104106 gen-omes/ml (4244). This technique has been superseded byRT-PCR, which is more sensitive, with a detection limitof 10 genomes/ml (4549). In liver samples, HDV RNAcan be detected by in situ hybridization. This method is,however, not used in routine as it is very difficult andtime-consuming. New automated assays are now beingestablished to render possible the follow-up of viral RNAkinetic in the serum of infected patients during treatment(50, 51).

Serum HDAg can be detected by two different meth-ods, namely the enzyme-linked immunosorbent assay(ELISA) (52) and the radioimmunoassay (RIA). Theseassays are not available in the US for clinical diagnosis.HDAg can be detected by immunofluorescence or im-munohistochemical staining of liver biopsies.

As HDAg, serum anti-HDV IgM and IgG antibodiescan be detected by ELISA or RIA.

The diagnosis has of course to indicate whether thereis an HDV infection, but it also has to distinguish among

Table 1. Co-infection and superinfection

Co-infection Superinfection

HBV infection Acute ChronicOutcome Recovery with

seroclearanceUsually persistentinfection

MarkersHBsAg Positive, early and

transientPositive and persistent

IgM anti-HBc Positive NegativeAnti-HBs Positive in

recovery phaseNegative

HDV infection Acute Acute or chronicOutcome Recovery with

seroclearance(5% progress tochronicity)

Usually persistentinfection (80%progress to chronicity)

MarkersSerum HDAg Early and short

livedEarly and transient,undetectable later

Liver HDAg Positive andtransient

Positive, may benegative at a late stage

Serum HDVRNA

Positive, early andtransient

Positive, early andpersistent

Anti-HDV Late acute phase,low titre

Rapidly increasing,high titres

IgM anti-HDV Positive, transientpentameric

Rapidly increasing,high titres, monomeric

Comparison between clinical features of HDV infection in the two

settings of co-infection and superinfection. Adapted from (183).


Pascarella and Negro Hepatitis D virus: an update

the three situations of infection: acute HBV/HDV co-infection, acute HDV superinfection of a chronic HBVcarrier or HDV chronic infection.

As HDV is dependent on HBV, assessing the presenceof HBsAg is necessary before investigating the othermarkers in order to establish the diagnosis.

Acute HBV/HVD co-infection is highlighted by thepresence of a high titre of IgM anti-HBc, antibodies thatdisappear in chronic HBV infection. It bears otherwisethe same characteristics as acute HDV superinfection.HDAg appears early but also disappears quickly. Re-peated testing is necessary so that it does not eludedetection (53). In immunodeficient patients, HDAg lastslonger as these people have a slow and weak immuneresponse (54). HDV RNA is an early and sensitivemarker of HDV replication in acute phase (43) and ispresent in 90% of the patients. In the setting ofsuperinfection, the amount of HDV in the serum canreach 1012 RNA-containing particles per ml between 2and 5 weeks post-inoculation, at the peak of acuteinfection. Anti-HD antibodies appear late but serocon-version allows one to establish diagnosis in the absence ofother tests.

In chronic HDV infection, HDAg are complexed withanti-HD that are present at a high titre. HDAg are thusnot detectable by ELISA but can be well visualized byimmunoblot assay under denaturating conditions (55).Unfortunately, even though this technique is very sensi-tive (56), it is difficult to apply for routine detection, as itis time and labour consuming. The detection of theHDAg in the liver is only possible in about 50% ofpatients chronically infected for 10 years or more (49).

HDV RNA is usually detectable in the serum. The titre ofanti-HD antibodies of the IgG class is very high inchronic patients and may help distinguishing currentfrom past infections. The persistence of anti-HD of theIgM class after the acute phase is characteristic of theprogression to chronicity, at variance with other viralhepatitis infections (Table 1 and Fig. 2).

To summarize, the first step towards establishing diag-nosis is to test for anti-HD antibodies. Diagnosis can thenbe confirmed by immunohistochemical staining forHDAg in the liver or the detection of serum HDV RNA.

If HDV infection is confirmed, the next step is toevaluate liver grading and staging to determine whetherthe patient will benefit from a potential treatment.

Natural history of the disease

Hepatitis D virus induces a usually severe form ofhepatitis. However, the range of clinical manifestationsis very broad as HDV infection can be associated withasymptomatic cases as well as with cases of fulminanthepatitis (57, 58).

Acute hepatitis occurs after an incubation time of37 weeks. The preicteric phase is characterized byseveral non-specific symptoms such as fatigue, lethargy,anorexia or nausea and the appearance of biochemicalmarkers, such as elevated serum ALT and AST activities.The icteric phase, which is not always observed, ischaracterized by elevated levels of serum bilirubin.

Fulminant viral hepatitis, which may occur especiallyin the setting of superinfection, is more frequent inhepatitis D than in hepatitis B alone (32). It is

Fig. 2. Serologic pattern of type D hepatitis. Expression level of antigen, DNA or RNA, IgM and IgG for both HDV and HBV and ALT.



characterized by a massive hepatocyte necrosis that leadsto liver failure and death in 80% of the patients, unlessurgency liver transplantation is carried out.

The course of chronic hepatitis D is often more severethan other types of chronic hepatitis. Clinically, it may beasymptomatic or present with non-specific symptoms.The diagnosis is often fortuitous or may follow theappearance of late complications at the cirrhosis stage.ALT and AST levels are persistently elevated in mostpatients. Within 510 years, as many as 7080% ofchronic hepatitis D patients may develop cirrhosis (59,60) and 15% within 12 years (61). Overall, the relativerisk of developing cirrhosis during follow-up in patientsco-infected with HBV and HDV seems two-fold com-pared with patients mono-infected with HBV (62).Cirrhosis due to HDV may remain stable for many yearsbefore progressing to liver failure or developing intohepatocellular carcinoma (HCC). Patients with HDV-associated cirrhosis have a probability of survival of 49and 40% at 5 and 10 years respectively (63). The impactof HDV infection on the acceleration of HCC develop-ment in HBV-positive patients is controversial. A retro-spective study on patients suffering from compensated,HBV-related cirrhosis in Western Europe, where HDVgenotype 1 is predominant, has demonstrated a three-fold and two-fold risk increase, respectively, of develop-ing HCC and of death in HDV patients compared withthose mono-infected with HBV (64). A study in Taiwan,where genotype 2 prevails, has highlighted the fact thatthis specific genotype is less often associated with fulmi-nant hepatitis or unfavourable long-term outcome thangenotype 1 (30).

Factors influencing liver disease progression

Many factors can influence the outcome of chronichepatitis D. A major one is the modality of infection withHBV (i.e. co-infection vs superinfection). Another one isthe HDV genotype (23, 30). Indeed, infection withgenotype 3, which is predominant in South America,induces a severe acute hepatitis with a high risk ofliver failure (23, 65, 66). Another factor potentiallyinvolved in influencing disease outcome is the occur-rence of specific HDAg species that have been reported infulminant hepatitis (67). The HBV genotype is alsoresponsible as it modulates the HDV viral load andcorrelates with adverse outcome (68, 69). Furthermore,high levels of HBV replication are associated with moresevere liver damage also in the context of chronichepatitis D (70).

Treatment

Patients chronically infected with HBV may potentiallybe infected a second time with HDV after completeclearance of a first infection, although this phenomenonhas been observed only in the chimpanzee experimentalmodel (71) and not in the human infection. Ideally, then,

the goal of treatment is to eradicate HDV together withHBV. HDV is considered eradicated when both HDVRNA in the serum and HDAg in the liver becomepersistently undetectable. However, it is only with HBsAgclearance that complete and definitive resolution isattained. Moreover, development of anti-HD antibodieswill protect against re-infection. Viral clearance is accom-panied with normalization of the ALT level, ameliorationof liver necro-inflammation, while the progression ofliver fibrosis stops.

Interferon-a and pegylated-interferon-a

Chronic hepatitis D is a difficult-to-treat disease. In mostcountries, the only approved treatment is still a high-dose,long-term administration of standard interferon-a (IFN-a), 9 millions units three times a week or 5 millions unitsdaily for 12 months. Duration of therapy can be prolongedif HBsAg is not cleared and treatment well tolerated. Eventhough 50% of the patients present undetectable HDVRNA and sometimes normalization of ALT with highdoses of IFN-a (72), HDV relapse is almost alwaysobserved after cessation of treatment, with a delay in arange of 26 months (72). Nevertheless, IFN-a treatmentoverall improves long-term clinical outcome and survival(73). Hepatic function and histology are improved. Inter-estingly, IFN-a did not show any antiviral effects againstHDV in vitro, suggesting an indirect action potentially onthe helper virus (74, 75) and/or on the host immuneresponse. Unfortunately, IFN-a treatment is associatedwith several side effects such as neutropaenia, anaemia,fatigue and depression (76). Treatment is therefore contra-indicated for a certain number of patients. In others, IFN-a dosing must be reduced while on treatment, withpotential loss of efficacy.

Standard IFN-a has recently been replaced by itspegylated form (Peg-IFN-a), characterized by a longerhalf-life, a property that allows its weekly administration.Peg-IFN-a has demonstrated a better response to treat-ment in comparison with classical IFN-a. Non-respondersto IFN-a may clear HDV RNA after a 6-month coursewith Peg-IFN-a (77). A stopping rule has been suggestedin patients who show a less than 3 logs decrease in serumHDV RNA after 24 weeks of treatment: in such cases, infact, the chances of long-term response are nil andcessation of treatment should be considered (78). How-ever, Peg-IFN-a is still insufficient to cure the majority ofchronic hepatitis D patients. In a prospective trial, only21% of patients presented HDV RNA negativity and 26%presented a biochemical response (79). In three moretrials, HDV RNA negativity occurred in 43% of patients(80), sustained response, defined by HDV RNA negativityand normalization of ALT, occurred in 17% of patients(78) and 39% of patients achieved the primary endpoint,including three patients who lost HBsAg for up to 6 years(81). Major pretreatment predictors of response to ther-apy are cirrhosis and an HDV RNA level higher than2.2 107 copies/ml (82, 83). In addition, the presence of



anti-HD of the IgM class at the end of treatment isassociated with treatment failure (82).

Other drug therapies

Alternative treatments have been tested, with limitedresults. The antivirals lamivudine, adefovir dipivoxil,famciclovir and entecavir, have been shown to havesome efficacy against HBV but no efficacy against HDVeither in monotherapy (8488) or in combination withIFN-a (89, 90). Another antiviral, used in HCV treat-ment, the ribavirin, inhibits HDV replication in vitro(91, 92) but is ineffective in vivo even if associated withPeg-IFN-a (79).

Immunomodulatory drugs such as corticosteroids orlemivasole have not showed any beneficial effects (59, 93).

Thymus-derived peptides, such as the thymic humoralfactor-g2 or the thymosine-a1, which have demonstratedsome benefit in the treatment of HBV, alone or incombination with IFN-a, have been unhelpful in thetreatment of HDV (94, 95).

Isoprenylation inhibitors have been shown to reducevirus assembly in mouse, suggesting a potential utility forthe patients, but have not yet been tested in clinical trials(96, 97).

Another new way proposed to treat HDV is thedevelopment of antisense nucleotides, small nucleic acidsequences that block the translation of the HDAg byspecifically binding to the viral RNA. In in vitro studies,siRNA have demonstrated the ability to target the mRNAbut not the genome and the antigenome, probablybecause these two molecules are located in the nucleusof the cell (98).

Liver transplantation

Liver transplantation is the management of choice infulminant hepatitis D and end-stage chronic liver diseasedue to HDV. Patients undergoing liver transplantationreceive also passive immunoprophylaxis against HBVreinfection with anti-HBs antibodies and administrationof HBV polymerase inhibitors (99). This results in thecomplete clearance of both HBV and HDV in mostpatients after liver transplantation, with a survival rateat 5 years of almost 90% (100), better than what observedin patients mono-infected with HBV (38).

Hepatitis D virus life cycle

Virion structure

Hepatitis D virus virions are roughly spherical particles(3643 nm) (3, 101) containing a ribonucleic core-likestructure surrounded by HBVenvelope proteins and hostlipids. The core-like structure (19 nm) is composedof one HDV genomic RNA complexed with about70 molecules of HDAg in its large and small forms(102). HDAg form dimers through an antiparallelcoiled coil. Dimers then interact to form octamers, which

are arranged in 50 A rings into a unique structure (103).The HDAg is thus not exposed at the virion outer surface.The envelope is composed of about 100 copiesof the three envelope proteins of HBV: the small HbsAg(S-HBsAg), the middle (M-HBsAg) and the large(L-HBsAg). The relative proportion of these proteins,95:5:1, is more similar to that found in the 22 nm,HBsAg-positive empty particles of HBV than thatof the Dane particle (55). It has also been demonstratedthat S-HBsAg is sufficient for particle assembly, thatL-HBsAg is necessary for infectivity (104) and that,finally, M-HBsAg is not essential for assembly or infec-tivity (105).

L-HDAg (but not S-HDAg) and HBsAg are necessaryand sufficient to form particles, which obviously arenot infectious unless also HDV RNA is present (106).S-HDAg increases packaging efficiency (106, 107). Theantigenomic strand has never been found in the viralparticle.

Hepatitis D virus RNA

Three different RNAs accumulate during HDV replica-tion: the genome (300 000 copies), the antigenome(30 000 copies) and the mRNA (600 copies).

The genome is a circular negative single-strand RNAcomposed of 16721697 nucleotides, depending on thestrain (108). It is the smallest and the only circular RNAamong animal viruses, this structural characteristic andmode of replication being otherwise only observed inplant viroids and virusoids. Because of the presence of74% internal base pairing, this molecule has the ability tofold on itself as an unbranched, double-stranded, rod-like structure (4, 109). Nucleotide number one has beenarbitrarily chosen using a unique Hind III restriction site(110). The genome contains a ribozyme domain span-ning nucleotides 680780 and a putative promoter sitefor the HDAg mRNA (111).

The antigenome is the exact complement of thegenome, as replication occurs through RNA-directedRNA synthesis without any DNA intermediates (112).The antigenome contains a unique open reading frame(ORF) coding for the HDAg and, as the genome, aribozyme domain (113, 114).

Finally, the mRNA directs the synthesis of HDAg. It isan 800-nucleotide linear RNA of the same polarity as theantigenome, which bears a 50 cap structure and a 30-poly(A) tail (115117). The mRNA, at variance with thegenome, is associated with polysomes during replicationand can be translated in vitro (118). This molecule isunstable and is synthesized throughout replication (116)(Fig. 3).

Hepatitis D antigen

The only protein that is expressed by HDV is HDAg. Aunique ORF, located on the antigenome, leads to thesynthesis of two different forms of HDAg during the



HDV cycle: S-HDAg (195 amino acids; 24 kDa) andL-HDAg, which has 19 additional amino acids at itsC-terminus (214 amino acids; 27 kDa). The large formappears later, when some antigenomes are post-tran-scriptionnally edited by an enzyme, the adenosine dea-minase acting on RNA-1 (ADAR-1) (119, 120). Theadenosine at the amber/W site (amino acid position1015) is deaminated into an inosine (UAG becomesUIG), and then paired to a cytosine during the nextreplication cycle (AUC becomes ACC). When this mod-ified genome is transcribed into mRNA to produce anantigen, the stop codon (UAG) is replaced by a trypto-phan (UGG) (118, 119, 121) (Fig. 4). The synthesisof L-HDAg then follows, reaching the next stop codon,which is located 19 codons downstream.

S-HDAg is necessary for the initiation of genomereplication (37), while the L-HDAg behaves as a domi-nant negative inhibitor (122, 123) and is essential for theassembly of the particle (106, 124).

HDAg binds to the RNA, an ability that is perhapsfacilitated by the rod-like structure (125). HDAg maydirectly stimulate transcription elongation via the repla-cement of the negative elongation factor, a transcriptionrepressor bound to the RNA polymerase II (RNAP II)(126).

In the absence of HBsAg, both L-HDAg and S-HDAglocalize in the nuclei as they bear nuclear localizationsignals spanning amino acid residues 3588 from theN-terminus (127, 128). In the presence of HBsAg,L-HDAg relocalizes to the cytoplasm (129). L-HDAgis indeed a nucleocytoplasmic shuttling protein asit also possesses a nuclear export signal in its C-terminus(130).

Several post-translational modifications of HDAg havebeen reported. L-HDAg contains a terminal CXXX box,

which is a substrate for isoprenylation, a modificationthat enhances its replication inhibitory effect (131) and isnecessary for viral particle formation. The prenylatedL-HDAg is a lipophilic molecule that mediates the directbinding between the L-HDAg and the HBV envelopeproteins (132).

Both HDAg forms are phosphorylated, although L-HDAg is six times more phosphorylated than S-HDAg(133). S-HDAg is phosphorylated at serine and threonineresidues, while L-HDAg is phosphorylated at serineresidues only (134, 135). The major phosphorylation siteis the serine 177. The protein kinase R has been shown tomodulate HDV replication by phosphorylating S-HDAg(136). Phosphorylation of the serine 2 of S-HDAgincreases HDVreplication (137). HDAg phosphorylationis important for RNA replication, probably because itmediates its RNA-binding activity (137, 138).

The methylation of S-HDAg, which is observed in vitroand in vivo, is located in the RNA-binding domain.Mutations of this domain or use of a methylationinhibitor result in an inhibition of HDV RNA replica-tion: HDAg loses the ability to form speckled structuresin the nucleus and localizes in the cytoplasm (139).

Acetylation is another post-translational mechanismshown to modulate HDV replication as it regulates thenuclear localization of HDAg. A substitution of thelysine 72 by an alanine decreases the accumulation ofviral RNA and induces an earlier appearance of L-HDAg(140).

Sumoylation is a newly described type of post-transla-tional HDAg modification. S-HDAg is a small ubiquitin-like modifier 1 (SUMO1) target protein through multiplelysine residues. Experiments performed with S-HDAgfused to SUMO1 protein showed an increased HDVgenomic RNA and mRNA synthesis, but no influence onantigenomic RNA synthesis (141) (Fig. 5).

Virus attachment, entry, assembly and release

At of the time of writing, the receptor of HDV and itsentry mechanism were unknown. Some information is

Fig. 3. Schematic representation of the antigenome, the genomeand the HDV mRNA.

Fig. 4. Editing of the HDV antigenomic RNA.



nevertheless available. HDV and HBV share the sameenvelope proteins; we would thus expect the same or verysimilar mechanisms of attachment and entry for bothviruses. However, HBV becomes non-infectious (at var-iance with HDV) when the virion is packaged with S-,M- and L-HBsAg lacking the N-linked carbohydrates(142). However, some similarities exist: residues 520 ofthe pre-S1 domain in the L-HBsAg are required for theentry of both viruses. In fact, entry can be inhibited bysynthetic acylated (143) and myristylated (144, 145)peptides encompassing the first 50 amino acids of thepre-S1 domain. Entry appears to be preceded by attach-ment to the carbohydrate side chains of hepatocyte-associated heparan sulphate proteoglycans (146) assuramin inhibits in vitro HDV infection (147).

One of the classical routes of viral entry that could alsobe implicated is the clathrin-mediated endocytic pathway(129). Viruses such as the influenza virus (148), thereovirus (149) or the vesicular stomatitis virus (VSV)(150) are endocytosed together with their receptor intoendosomes. HDAg has been identified as a clathrinadaptator-like protein as it specifically interacts withthe clathrin heavy chain (CHC) (151). Clathrin is alsoimplicated in the exocytosis mechanisms of some virusessuch as the VSV or the HIV type 1. Interestingly, HDVassembly is reduced after CHC downregulation whereasHBV is not, suggesting that even though both viruses sharethe same envelope proteins, their assembly and releasemechanisms are different (152). The assembly efficiency is,in addition, different between genotypes and correlatewith the ability to interact with CHC.

Hepatitis D virus genotype 1 has an assembly effi-ciency that is higher than genotypes 2 and 3. A recentwork has pointed out the implication of the amino acidin position 205 in virion release. Substitution of theproline by an arginine or an alanine in genotype 1significantly decreased the secretion of viral particles.The reverse substitution in genotypes 2 or 3 increased theassembly efficiency of HDAg (153).

Hepatitis D virus RNA replication

As HDV does not possess its own RNA polymerase, thevirion uses the human transcriptional machinery for its

replication. The implication of the RNAP II is now wellestablished. RNAP II binds to the HDV genome andantigenome (154), the HDV replication is sensitive to a-amanitin (155) and the mRNA coding for the HDAgpossesses a 50 methylguanine cap and a poly(A) tail (115,156). Resistance to a-amanitin, however, does not occurfor the transcription of the antigenome, suggesting aninvolvement of RNAP I (157). Other evidences includethe interaction between components of the SL1 tran-scription factor for RNAP I and S-HDAg (158), inhibi-tion of the antigenome synthesis by anti-a-SL1antibodies (158) and direct interaction between RNAP Iand HDV RNA (159). Recent data suggest an implicationof the third polymerase, the RNAP III, which also bindsto both genomic and antigenomic HDV RNA (159).

Transcription occurs in the nucleus, where HDV RNAis brought into by the HDAg (160) and is activated by S-HDAg binding to HDV RNA. Replication occurs withoutany help coming from HBV and without any DNAintermediate. Replication proceeds via a double-rollingcircle process, a mechanism proposed for the replicationof plant viroids and small-satellite RNAs of some plantviruses (161). The transcription proceeds for more thanone genome length, going twice through the ribozymecleavage site. Then the antigenomic transcript undergoesan autocatalytic cleavage between nucleotides 688 and689 via the antigenomic ribozyme, is folded into a rod-like structure and is ligated by cellular ligases (162) toform a circular template. Production of the genomicRNA from the antigenome occurs through the samemechanism; then the genomic RNA is either incorpo-rated into new viral particles or used again as templatefor antigenomic RNA synthesis. The ratio between geno-mic and antigenomic RNA is asymmetric, as the genomeis fifteen times more abundant than the antigenome(112). The mechanism of regulation is unknown but ithas been reported that HDV RNA mutations couldsuppress genomic strand transcription, without affectingantigenomic strand synthesis, suggesting two separatemeans of regulation (163). The mRNA is transcribedfrom the genome, thus using the same template as for theantigenomic RNA. The transcription of these two ele-ments nevertheless starts at different initiation sites(116), using different polymerases and in different

Fig. 5. Localization of HDAg functional domains: RNA-binding domain, coiled-coil sequence, nuclear localization sequence and virus assemblysignal. Sites of post-translational modifications: phosphorylation (P), methylation (M), acetylation (A) and isoprenylation (I). Adapted from(186).



subnuclear compartments. The antigenome is tran-scribed into the nucleolus, whereas the mRNA is tran-scribed into the nucleoplasm, like the genome (Fig. 6).

Mechanisms of hepatitis D virus disease

Hepatitis D virus only replicates in the liver. Pathologicchanges are thus limited to this organ. Histologically, theliver presents cellular necrosis and inflammation. Eventhough some in vitro experiments have demonstrated adirect cytopathic effect of HDV (164, 165), other resultsand in vivo observations, such as the presence of inflam-matory cells surrounding the infected hepatocytes (166)and the presence of various autoantibodies in the serumof patients, argue for immune-mediated liver damage.

As HBV replication is strongly repressed in the pre-sence of HDV, liver damage is believed to be induced byHDV infection rather than by HBV.

Hepatitis D virus increases cell survival potential

Hepatitis D virus replication is associated with increasedhistone H3 acetylation within the clusterin promoter,resulting in an enhanced clusterin gene expression (167).Interestingly, this modification is the same as thatassociated with the expression of specific proteins ofseveral oncogenic viruses, such as the adenovirus proteinE1A (168, 169), the simian virus 40T antigen (170) andthe E7 protein of the human papilloma virus (171). Theincrease of clusterin protein enhances the survival of cellinfected with HDV (167). Clusterin has been reported tobe upregulated in tumour cells and to play a significantrole in tumourigenesis (172, 173). This protein couldthus be implicated in the development of HCC in HDV-infected patients. However, conflicting results have beenreported in the literature, such as inhibition of host cellproliferation (174), cell-cycle arrest (175) and cell death(165).

Hepatitis D virus inhibits IFN-a signalling

Hepatitis D virus, like many other viruses, seems to havedeveloped an anti-IFN-a strategy. HDV directly inhibitsthe activation of the IFN-a signalling by interfering in theearly steps of the Janus kinase (JAK)/signal transducersand activators of transcription (STAT) signal transduc-tion pathway. There is an inhibition of the tyrosinekinase 2 (Tyk2), STAT1 and STAT2 phosphorylation anda transcription impairment of several IFN-a-stimulatedgenes, such as the myxovirus resistance-A (MxA), the20,50-oligoadenylate synthetase (20,50-OAS) and PKR inthe presence of the virus (176). On the other hand,an upregulation of MxA transcription induced by theL-HDAg has been reported and suggested to account forthe suppression of HBV replication (177).

Hepatitis D virus-L antigen sensitizes to tumour necrosisfactor-a-induced nuclear factor kappa-light-chain-enhancer of activated B cells signalling

Nuclear factor kappa-light-chain-enhancer of activated Bcells (NF-kB) activation is implicated in inflammationprocesses and in cancer. L-HDAg has been shown toinduce tumour necrosis factor-alpha (TNF-a)-inducedNF-kB signalling, probably through the direct associa-tion with TNF receptor-associated factor 2 (TRAF2), aprotein implicated in the early signal transduction events(178).

Changes in the cell proteome

Several studies have investigated the relationship amongL-HDAg, S-HDAg, genomic RNA, antigenomic RNAor a combination of these elements and the proteome ofthe cell (179, 180). Proteins involved in pathways suchas regulation of cell metabolism and energy path-ways, nucleic acid and protein metabolism, transport,signal transduction, apoptosis and cell growth and

Fig. 6. Double-rolling circle replication of HDV and localization of the different replication events.



maintenance show a modified expression profile. A smallinhibitory RNA (siRNA) screening has also been per-formed to investigate the cellular proteins implicated inHDV replication. Cells stably expressing S-HDAg weretransfected with siRNA before induction of viral replica-tion (181). A direct interaction has been reported be-tween a portion of the genome, described to act in vitroas an RNA promoter (111), and some cellular proteins. Itshould be pointed out that two of these proteins im-plicated in RNA processing or associated with thetranslation machinery: the eukaryotic translation elonga-tion factor 1 alpha 1 (eEF1A1) and the glyceraldehyde-3-phosphate dehydrogenase, are considered and often usedin research as housekeeping genes (182). Such observa-tions suggest potential avenues of research in order toimprove our understanding of the mechanisms of HDVreplication and pathogenesis.

Conclusions

Hepatitis D virus is an insufficiently characterized virus:After a decrease of its spread, largely because of the HBVvaccination campaigns and the increased awareness onbloodborne infections following the HIV scare, it stillinfects a steady proportion of HBV carriers worldwide.Thus, it is unfortunate that the interest in HDV researchhas faltered. The stable prevalence in Western countries,the frequent occurrence of severe outbreaks in diversegeographical areas, the severity of HDV-associated liverdisease and the lack of efficient treatment should, how-ever, encourage research. A better knowledge of the viruslife cycle and pathogenesis will certainly help in identify-ing new approaches to treatment.

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