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Keywords:

  • cirrhosis;
  • delta hepatitis;
  • fulminant hepatitis;
  • hepatitis D virus

Abstract

  1. Top of page
  2. Abstract
  3. Epidemiology
  4. Clinical features and diagnosis
  5. Treatment
  6. Hepatitis D virus life cycle
  7. Mechanisms of hepatitis D virus disease
  8. Conclusions
  9. References

Hepatitis D virus (HDV) infection involves a distinct subgroup of individuals simultaneously infected with the hepatitis B virus (HBV) and characterized by an often severe chronic liver disease. HDV is a defective RNA agent needing the presence of HBV for its life cycle. HDV is present worldwide, but the distribution pattern is not uniform. Different strains are classified into eight genotypes represented in specific regions and associated with peculiar disease outcome. Two major specific patterns of infection can occur, i.e. co-infection with HDV and HBV or HDV superinfection of a chronic HBV carrier. Co-infection often leads to eradication of both agents, whereas superinfection mostly evolves to HDV chronicity. HDV-associated chronic liver disease (chronic hepatitis D) is characterized by necro-inflammation and relentless deposition of fibrosis, which may, over decades, result in the development of cirrhosis. HDV has a single-stranded, circular RNA genome. The virion is composed of an envelope, provided by the helper HBV and surrounding the RNA genome and the HDV antigen (HDAg). Replication occurs in the hepatocyte nucleus using cellular polymerases and via a rolling circle process, during which the RNA genome is copied into a full-length, complementary RNA. HDV infection can be diagnosed by the presence of antibodies directed against HDAg (anti-HD) and HDV RNA in serum. Treatment involves the administration of pegylated interferon-α and is effective in only about 20% of patients. Liver transplantation is indicated in case of liver failure.

The discovery of hepatitis D virus (HDV) dates back to the mid-1970s, and followed the detection of a novel nuclear antigen in patients with a severe form of chronic hepatitis B. The first report of this antigen, believed to be a hepatitis B antigen and called the delta antigen, was published in 1977 (1). Three years later, experiments in chimpanzees had already demonstrated that the hepatitis delta antigen (HDAg) was a structural component of a transmissible pathogen that required the hepatitis B virus (HBV) for its life cycle (2). The virion particle was shown to be composed of the HBV envelope proteins surrounding a ribonucleoprotein core-like structure comprising the HDAg and a molecule of RNA (3). The delta agent obtained the status of a distinct virus in 1983 with the official name of hepatitis delta virus. Nowadays, the term hepatitis D virus is preferred, even though ‘delta’ is still used. The uniqueness of this virus was confirmed in 1986, after cloning and sequencing of its genome (4). Thereafter, HDV obtained its own genus, the Deltavirus (5).

Epidemiology

  1. Top of page
  2. Abstract
  3. Epidemiology
  4. Clinical features and diagnosis
  5. Treatment
  6. Hepatitis D virus life cycle
  7. Mechanisms of hepatitis D virus disease
  8. Conclusions
  9. References

Overview

Hepatitis D virus concerns all age groups. The distribution pattern of this virus, investigated by seroprevalence studies of anti-HD in HBsAg-positive patients, is worldwide but not uniform (6). Regardless of the fact that HDV needs HBV for its life cycle, the distribution pattern of each virus is different. For example, 90% of HBV carriers are infected with both viruses in the Pacific Islands, whereas the rates decline to 8% in Italy and 5% in Japan (6). Current estimates suggest that 15–20 million people are infected with HDV. However, one should consider that these estimates are inaccurate and difficult to perform as systematic screening is not performed in HBV-infected individuals, especially if they present with normal liver enzymes (7). In addition, anti-HD may be lacking in immunodeficient patients and seroreversion may occur after resolution of the disease, rendering the diagnosis of past infections impossible. Main areas of prevalence are the Mediterranean basin, the Middle East, Central and Northern Asia, West and Central Africa, the Amazonian basin, Venezuela, Colombia and certain islands of the Pacific. The Far East is less concerned but HDV is nonetheless present in Taiwan, China and India (Fig. 1).

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

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The prevalence of HDV infection has significantly declined in some regions of the world such as Italy (8, 9), Spain (10), Taiwan (11) and Turkey (12), mainly because of vaccination campaigns against HBV, systematic screening of blood and blood products and of pregnant women, implementation of safety procedures against bloodborne infections among healthcare workers, switch to disposable syringes, improved socioeconomic conditions and the increased awareness of the general public on sexually transmissible agents that followed the acquired immunodeficiency syndrome scare (13).

The number of infected patients, however, stopped decreasing towards the end of the 1990s in Europe. The prevalence remained stable for example in London (7), in Hanover (14) and in Italy (13) and seemed to be increasing in France (15). This recrudescence in industrialized countries is mainly observed because of an increased immigration from Eastern Europe, Africa, the Middle East, Turkey and the ex-Soviet Union. Immigration from endemic regions is not the only cause: intravenous drug use, sexual practices and body modification procedures may also be involved. Moreover, HDV has emerged in new regions such as Russia (16), Northern India, Southern Albania, mainland China and some Pacific islands such as Okinawa.

Hepatitis D virus genotypes

The evolution rate of HDV RNA has been determined by longitudinal studies of the RNA quasispecies changes in chronic hepatitis D patients. This rate varies across a range comprised between 3 × 10−2 and 3 × 10−3 base substitutions per nucleotide per year (17). Highly conserved domains are located around the genomic and antigenomic RNA autocatalytic cleavage sites and the RNA-binding domain of HDAg (18, 19). HDV genomes of the different isolates present up to 39% heterogeneity. The different sequences have been classified into eight HDV genotypes (20). Except for genotype 1, which is represented worldwide, all other genotypes are mostly found in specific geographical areas. Genotype 2 prevails in Japan (21), Taiwan (19) and Russia (22), genotype 3 in the Amazonian region (23), genotype 4 in Japan (24, 25) and Taiwan (26) and genotypes 5–8 in Africa (20) (Fig. 1). Multiple genotypes infection can occur in patients at high risk of repeated exposure. A single genotype generally dominates and only 10% of the viral population is represented by the minor strain (27). Furthermore, chimeric forms of HDV RNA have been detected in Taiwanese patients infected with both genotypes 1 and 4 (28, 29).

The HDV genotype can be determined by restriction fragment length polymorphism analysis of polymerase chain reaction (PCR)-amplification products (30), by sequencing and by immunohistochemical staining of liver biopsies using genotype-specific antibodies (31).

Clinical features and diagnosis

  1. Top of page
  2. Abstract
  3. Epidemiology
  4. Clinical features and diagnosis
  5. Treatment
  6. Hepatitis D virus life cycle
  7. Mechanisms of hepatitis D virus disease
  8. Conclusions
  9. References

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-infection and superinfection. A third, minor pattern, the so-called helper-independent latent infection, has been reported in the liver transplant setting, and will be briefly discussed below.

Co-infection is a simultaneous infection with both viruses that leads to acute hepatitis B and D. From a clinical point of view, this is indistinguishable from acute hepatitis B (32), although it may be more severe and two peaks of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) may be observed. Because HBV is essential for HDV, the rate of progression to chronicity is the same as that of acute hepatitis B (<5%).

Superinfection is the HDV infection of an individual chronically infected with HBV. This way of infection causes severe acute hepatitis, which progresses to chronicity in almost all patients (up to 80%) (32). Once chronic HDV infection is established, it usually exacerbates the pre-existing liver disease due to HBV (33). HBV replication is, however, usually suppressed to low levels during the acute phase of HDV infection. This suppression becomes persistent in case of a chronic hepatitis D establishment (34, 35) (Table 1).

Table 1.   Co-infection and superinfection
 Co-infectionSuperinfection
  1. Comparison between clinical features of HDV infection in the two settings of co-infection and superinfection. Adapted from (183).

HBV infectionAcuteChronic
 OutcomeRecovery with seroclearanceUsually persistent infection
 Markers
  HBsAgPositive, early and transientPositive and persistent
  IgM anti-HBcPositiveNegative
  Anti-HBsPositive in recovery phaseNegative
HDV infectionAcuteAcute or chronic
 OutcomeRecovery with seroclearance (5% progress to chronicity)Usually persistent infection (80% progress to chronicity)
 Markers
  Serum HDAgEarly and short livedEarly and transient, undetectable later
  Liver HDAgPositive and transientPositive, may be negative at a late stage
  Serum HDV RNAPositive, early and transientPositive, early and persistent
Anti-HDVLate acute phase, low titreRapidly increasing, high titres
IgM anti-HDVPositive, transient pentamericRapidly increasing, high titres, monomeric

Helper-independent latent infection was initially reported to occur after liver transplantation (36). HBV infection of the grafted liver is usually prevented by administration of hepatitis B immunoglobulins. Hepatocytes may thus be infected with HDV alone. HDAg can be detected in the liver by immunohistochemistry before HBV recurrence, as the helper virus in only necessary for particle formation and not for viral replication (37). HDV viraemia (as determined by molecular hybridization) is only observed several months later on, when residual HBV evades neutralization, thus allowing for HDV rescue and cell-to-cell spread (36). This third pattern of infection has been revisited with the advent of more sensitive, reverse transcription (RT)-PCR-based techniques for detecting HDV RNA. Moreover, experiments on chimpanzees first infected with HDV and later challenged with HBV have shown a rescue of HDV when HBV inoculation was performed at day 7 but not at 1 month (38).

Route of hepatitis D virus transmission

The natural reservoir is man, even though chimpanzees infected with HBV and woodchucks infected with the woodchuck hepatitis virus can be infected by HDV. Infection with HDV is parenterally transmitted. In industrialized countries, high-risk populations include illicit drug users and people exposed to blood or blood products. HDV does not seem to be a typically sexually transmitted disease, as the frequency of infection in sexually promiscuous heterosexual or homosexual groups is lesser than that of HBV or HIV (39). In Taiwan, however, this route is the predominant way of transmission (40). In socially and economically disadvantaged populations, many infections occur by inapparent intrafamilial routes of transmission, facilitated by poor hygiene. Perinatal transmission of HDV is rare.

Markers and diagnosis

Hepatitis D virus induces innate and adaptive immune response in the infected host, which consist of immunoglobulin M (IgM) and IgG production (41). Therefore, in the serum, the three specific HDV markers are HDV RNA, HDAg and anti-HDV.

Hepatitis D virus RNA can be detected in serum by either molecular hybridization or RT-PCR. Hybridization assays have a detection limit of about 104–106 genomes/ml (42–44). This technique has been superseded by RT-PCR, which is more sensitive, with a detection limit of 10 genomes/ml (45–49). In liver samples, HDV RNA can be detected by in situ hybridization. This method is, however, not used in routine as it is very difficult and time-consuming. New automated assays are now being established to render possible the follow-up of viral RNA kinetic in the serum of infected patients during treatment (50, 51).

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

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

The diagnosis has of course to indicate whether there is an HDV infection, but it also has to distinguish among the three situations of infection: acute HBV/HDV co-infection, acute HDV superinfection of a chronic HBV carrier or HDV chronic infection.

As HDV is dependent on HBV, assessing the presence of HBsAg is necessary before investigating the other markers in order to establish the diagnosis.

Acute HBV/HVD co-infection is highlighted by the presence of a high titre of IgM anti-HBc, antibodies that disappear in chronic HBV infection. It bears otherwise the same characteristics as acute HDV superinfection. HDAg appears early but also disappears quickly. Repeated testing is necessary so that it does not elude detection (53). In immunodeficient patients, HDAg lasts longer as these people have a slow and weak immune response (54). HDV RNA is an early and sensitive marker of HDV replication in acute phase (43) and is present in 90% of the patients. In the setting of superinfection, the amount of HDV in the serum can reach 1012 RNA-containing particles per ml between 2 and 5 weeks post-inoculation, at the peak of acute infection. Anti-HD antibodies appear late but seroconversion allows one to establish diagnosis in the absence of other tests.

In chronic HDV infection, HDAg are complexed with anti-HD that are present at a high titre. HDAg are thus not detectable by ELISA but can be well visualized by immunoblot assay under denaturating conditions (55). Unfortunately, even though this technique is very sensitive (56), it is difficult to apply for routine detection, as it is time and labour consuming. The detection of the HDAg in the liver is only possible in about 50% of patients chronically infected for 10 years or more (49). HDV RNA is usually detectable in the serum. The titre of anti-HD antibodies of the IgG class is very high in chronic patients and may help distinguishing current from past infections. The persistence of anti-HD of the IgM class after the acute phase is characteristic of the progression to chronicity, at variance with other viral hepatitis infections (Table 1 and Fig. 2).

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

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To summarize, the first step towards establishing diagnosis is to test for anti-HD antibodies. Diagnosis can then be confirmed by immunohistochemical staining for HDAg in the liver or the detection of serum HDV RNA.

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

Natural history of the disease

Hepatitis D virus induces a usually severe form of hepatitis. However, the range of clinical manifestations is very broad as HDV infection can be associated with asymptomatic cases as well as with cases of fulminant hepatitis (57, 58).

Acute hepatitis occurs after an incubation time of 3–7 weeks. The preicteric phase is characterized by several non-specific symptoms such as fatigue, lethargy, anorexia or nausea and the appearance of biochemical markers, such as elevated serum ALT and AST activities. The icteric phase, which is not always observed, is characterized by elevated levels of serum bilirubin.

Fulminant viral hepatitis, which may occur especially in the setting of superinfection, is more frequent in hepatitis D than in hepatitis B alone (32). It is characterized by a massive hepatocyte necrosis that leads to liver failure and death in 80% of the patients, unless urgency liver transplantation is carried out.

The course of chronic hepatitis D is often more severe than other types of chronic hepatitis. Clinically, it may be asymptomatic or present with non-specific symptoms. The diagnosis is often fortuitous or may follow the appearance of late complications at the cirrhosis stage. ALT and AST levels are persistently elevated in most patients. Within 5–10 years, as many as 70–80% of chronic hepatitis D patients may develop cirrhosis (59, 60) and 15% within 1–2 years (61). Overall, the relative risk of developing cirrhosis during follow-up in patients co-infected with HBV and HDV seems two-fold compared with patients mono-infected with HBV (62). Cirrhosis due to HDV may remain stable for many years before progressing to liver failure or developing into hepatocellular carcinoma (HCC). Patients with HDV-associated cirrhosis have a probability of survival of 49 and 40% at 5 and 10 years respectively (63). The impact of HDV infection on the acceleration of HCC development in HBV-positive patients is controversial. A retrospective study on patients suffering from compensated, HBV-related cirrhosis in Western Europe, where HDV genotype 1 is predominant, has demonstrated a three-fold and two-fold risk increase, respectively, of developing HCC and of death in HDV patients compared with those mono-infected with HBV (64). A study in Taiwan, where genotype 2 prevails, has highlighted the fact that this specific genotype is less often associated with fulminant hepatitis or unfavourable long-term outcome than genotype 1 (30).

Factors influencing liver disease progression

Many factors can influence the outcome of chronic hepatitis D. A major one is the modality of infection with HBV (i.e. co-infection vs superinfection). Another one is the HDV genotype (23, 30). Indeed, infection with genotype 3, which is predominant in South America, induces a severe acute hepatitis with a high risk of liver failure (23, 65, 66). Another factor potentially involved in influencing disease outcome is the occurrence of specific HDAg species that have been reported in fulminant hepatitis (67). The HBV genotype is also responsible as it modulates the HDV viral load and correlates with adverse outcome (68, 69). Furthermore, high levels of HBV replication are associated with more severe liver damage also in the context of chronic hepatitis D (70).

Treatment

  1. Top of page
  2. Abstract
  3. Epidemiology
  4. Clinical features and diagnosis
  5. Treatment
  6. Hepatitis D virus life cycle
  7. Mechanisms of hepatitis D virus disease
  8. Conclusions
  9. References

Patients chronically infected with HBV may potentially be infected a second time with HDV after complete clearance of a first infection, although this phenomenon has been observed only in the chimpanzee experimental model (71) and not in the human infection. Ideally, then, the goal of treatment is to eradicate HDV together with HBV. HDV is considered eradicated when both HDV RNA in the serum and HDAg in the liver become persistently undetectable. However, it is only with HBsAg clearance that complete and definitive resolution is attained. Moreover, development of anti-HD antibodies will protect against re-infection. Viral clearance is accompanied with normalization of the ALT level, amelioration of liver necro-inflammation, while the progression of liver fibrosis stops.

Interferon-α and pegylated-interferon-α

Chronic hepatitis D is a difficult-to-treat disease. In most countries, the only approved treatment is still a high-dose, long-term administration of standard interferon-α (IFN-α), 9 millions units three times a week or 5 millions units daily for 12 months. Duration of therapy can be prolonged if HBsAg is not cleared and treatment well tolerated. Even though 50% of the patients present undetectable HDV RNA and sometimes normalization of ALT with high doses of IFN-α (72), HDV relapse is almost always observed after cessation of treatment, with a delay in a range of 2–6 months (72). Nevertheless, IFN-α treatment overall improves long-term clinical outcome and survival (73). Hepatic function and histology are improved. Interestingly, IFN-α did not show any antiviral effects against HDV in vitro, suggesting an indirect action potentially on the helper virus (74, 75) and/or on the host immune response. Unfortunately, IFN-α treatment is associated with several side effects such as neutropaenia, anaemia, fatigue and depression (76). Treatment is therefore contraindicated for a certain number of patients. In others, IFN-α dosing must be reduced while on treatment, with potential loss of efficacy.

Standard IFN-α has recently been replaced by its pegylated form (Peg-IFN-α), characterized by a longer half-life, a property that allows its weekly administration. Peg-IFN-α has demonstrated a better response to treatment in comparison with classical IFN-α. Non-responders to IFN-α may clear HDV RNA after a 6-month course with Peg-IFN-α (77). A stopping rule has been suggested in patients who show a less than 3 logs decrease in serum HDV RNA after 24 weeks of treatment: in such cases, in fact, the chances of long-term response are nil and cessation of treatment should be considered (78). However, Peg-IFN-α is still insufficient to cure the majority of chronic hepatitis D patients. In a prospective trial, only 21% of patients presented HDV RNA negativity and 26% presented a biochemical response (79). In three more trials, HDV RNA negativity occurred in 43% of patients (80), sustained response, defined by HDV RNA negativity and 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 therapy are cirrhosis and an HDV RNA level higher than 2.2 × 107 copies/ml (82, 83). In addition, the presence of anti-HD of the IgM class at the end of treatment is associated with treatment failure (82).

Other drug therapies

Alternative treatments have been tested, with limited results. The antivirals lamivudine, adefovir dipivoxil, famciclovir and entecavir, have been shown to have some efficacy against HBV but no efficacy against HDV either in monotherapy (84–88) or in combination with IFN-α (89, 90). Another antiviral, used in HCV treatment, the ribavirin, inhibits HDV replication in vitro (91, 92) but is ineffective in vivo even if associated with Peg-IFN-α (79).

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

Thymus-derived peptides, such as the thymic humoral factor-γ2 or the thymosine-α1, which have demonstrated some benefit in the treatment of HBV, alone or in combination with IFN-α, have been unhelpful in the treatment of HDV (94, 95).

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

Another new way proposed to treat HDV is the development of antisense nucleotides, small nucleic acid sequences that block the translation of the HDAg by specifically binding to the viral RNA. In in vitro studies, siRNA have demonstrated the ability to target the mRNA but not the genome and the antigenome, probably because these two molecules are located in the nucleus of the cell (98).

Liver transplantation

Liver transplantation is the management of choice in fulminant hepatitis D and end-stage chronic liver disease due to HDV. Patients undergoing liver transplantation receive also passive immunoprophylaxis against HBV reinfection with anti-HBs antibodies and administration of HBV polymerase inhibitors (99). This results in the complete clearance of both HBV and HDV in most patients after liver transplantation, with a survival rate at 5 years of almost 90% (100), better than what observed in patients mono-infected with HBV (38).

Hepatitis D virus life cycle

  1. Top of page
  2. Abstract
  3. Epidemiology
  4. Clinical features and diagnosis
  5. Treatment
  6. Hepatitis D virus life cycle
  7. Mechanisms of hepatitis D virus disease
  8. Conclusions
  9. References

Virion structure

Hepatitis D virus virions are roughly spherical particles (36–43 nm) (3, 101) containing a ribonucleic core-like structure surrounded by HBV envelope proteins and host lipids. The core-like structure (19 nm) is composed of one HDV genomic RNA complexed with about 70 molecules of HDAg in its large and small forms (102). HDAg form dimers through an antiparallel coiled coil. Dimers then interact to form octamers, which are arranged in 50 Å rings into a unique structure (103). The HDAg is thus not exposed at the virion outer surface. The envelope is composed of about 100 copies of 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 that of the Dane particle (55). It has also been demonstrated that S-HBsAg is sufficient for particle assembly, that L-HBsAg is necessary for infectivity (104) and that, finally, M-HBsAg is not essential for assembly or infectivity (105).

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

Hepatitis D virus RNA

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

The genome is a circular negative single-strand RNA composed of 1672–1697 nucleotides, depending on the strain (108). It is the smallest and the only circular RNA among animal viruses, this structural characteristic and mode of replication being otherwise only observed in plant viroids and virusoids. Because of the presence of 74% internal base pairing, this molecule has the ability to fold on itself as an unbranched, double-stranded, rod-like structure (4, 109). Nucleotide number one has been arbitrarily chosen using a unique Hind III restriction site (110). The genome contains a ribozyme domain spanning nucleotides 680–780 and a putative promoter site for the HDAg mRNA (111).

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

Finally, the mRNA directs the synthesis of HDAg. It is an 800-nucleotide linear RNA of the same polarity as the antigenome, which bears a 5′ cap structure and a 3′-poly(A) tail (115–117). The mRNA, at variance with the genome, is associated with polysomes during replication and can be translated in vitro (118). This molecule is unstable and is synthesized throughout replication (116) (Fig. 3).

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Figure 3.  Schematic representation of the antigenome, the genome and the HDV mRNA.

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Hepatitis D antigen

The only protein that is expressed by HDV is HDAg. A unique ORF, located on the antigenome, leads to the synthesis of two different forms of HDAg during the HDV cycle: S-HDAg (195 amino acids; 24 kDa) and L-HDAg, which has 19 additional amino acids at its C-terminus (214 amino acids; 27 kDa). The large form appears later, when some antigenomes are post-transcriptionnally edited by an enzyme, the adenosine deaminase acting on RNA-1 (ADAR-1) (119, 120). The adenosine at the amber/W site (amino acid position 1015) is deaminated into an inosine (UAG becomes UIG), and then paired to a cytosine during the next replication cycle (AUC becomes ACC). When this modified genome is transcribed into mRNA to produce an antigen, the stop codon (UAG) is replaced by a tryptophan (UGG) (118, 119, 121) (Fig. 4). The synthesis of L-HDAg then follows, reaching the next stop codon, which is located 19 codons downstream.

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Figure 4.  Editing of the HDV antigenomic RNA.

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S-HDAg is necessary for the initiation of genome replication (37), while the L-HDAg behaves as a dominant negative inhibitor (122, 123) and is essential for the assembly of the particle (106, 124).

HDAg binds to the RNA, an ability that is perhaps facilitated by the rod-like structure (125). HDAg may directly stimulate transcription elongation via the replacement of the negative elongation factor, a transcription repressor bound to the RNA polymerase II (RNAP II) (126).

In the absence of HBsAg, both L-HDAg and S-HDAg localize in the nuclei as they bear nuclear localization signals spanning amino acid residues 35–88 from the N-terminus (127, 128). In the presence of HBsAg, L-HDAg relocalizes to the cytoplasm (129). L-HDAg is indeed a nucleocytoplasmic shuttling protein as it also possesses a nuclear export signal in its C-terminus (130).

Several post-translational modifications of HDAg have been reported. L-HDAg contains a terminal CXXX box, which is a substrate for isoprenylation, a modification that enhances its replication inhibitory effect (131) and is necessary for viral particle formation. The prenylated L-HDAg is a lipophilic molecule that mediates the direct binding between the L-HDAg and the HBV envelope proteins (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 threonine residues, while L-HDAg is phosphorylated at serine residues only (134, 135). The major phosphorylation site is the serine 177. The protein kinase R has been shown to modulate HDV replication by phosphorylating S-HDAg (136). Phosphorylation of the serine 2 of S-HDAg increases HDV replication (137). HDAg phosphorylation is important for RNA replication, probably because it mediates its RNA-binding activity (137, 138).

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

Acetylation is another post-translational mechanism shown to modulate HDV replication as it regulates the nuclear localization of HDAg. A substitution of the lysine 72 by an alanine decreases the accumulation of viral RNA and induces an earlier appearance of L-HDAg (140).

Sumoylation is a newly described type of post-translational HDAg modification. S-HDAg is a small ubiquitin-like modifier 1 (SUMO1) target protein through multiple lysine residues. Experiments performed with S-HDAg fused to SUMO1 protein showed an increased HDV genomic RNA and mRNA synthesis, but no influence on antigenomic RNA synthesis (141) (Fig. 5).

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

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Virus attachment, entry, assembly and release

At of the time of writing, the receptor of HDV and its entry mechanism were unknown. Some information is nevertheless available. HDV and HBV share the same envelope proteins; we would thus expect the same or very similar mechanisms of attachment and entry for both viruses. However, HBV becomes non-infectious (at variance with HDV) when the virion is packaged with S-, M- and L-HBsAg lacking the N-linked carbohydrates (142). However, some similarities exist: residues 5–20 of the pre-S1 domain in the L-HBsAg are required for the entry of both viruses. In fact, entry can be inhibited by synthetic acylated (143) and myristylated (144, 145) peptides encompassing the first 50 amino acids of the pre-S1 domain. Entry appears to be preceded by attachment to the carbohydrate side chains of hepatocyte-associated heparan sulphate proteoglycans (146) as suramin inhibits in vitro HDV infection (147).

One of the classical routes of viral entry that could also be implicated is the clathrin-mediated endocytic pathway (129). Viruses such as the influenza virus (148), the reovirus (149) or the vesicular stomatitis virus (VSV) (150) are endocytosed together with their receptor into endosomes. HDAg has been identified as a clathrin adaptator-like protein as it specifically interacts with the clathrin heavy chain (CHC) (151). Clathrin is also implicated in the exocytosis mechanisms of some viruses such as the VSV or the HIV type 1. Interestingly, HDV assembly is reduced after CHC downregulation whereas HBV is not, suggesting that even though both viruses share the same envelope proteins, their assembly and release mechanisms are different (152). The assembly efficiency is, in addition, different between genotypes and correlate with the ability to interact with CHC.

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

Hepatitis D virus RNA replication

As HDV does not possess its own RNA polymerase, the virion uses the human transcriptional machinery for its replication. The implication of the RNAP II is now well established. RNAP II binds to the HDV genome and antigenome (154), the HDV replication is sensitive to α-amanitin (155) and the mRNA coding for the HDAg possesses a 5′ methylguanine cap and a poly(A) tail (115, 156). Resistance to α-amanitin, however, does not occur for the transcription of the antigenome, suggesting an involvement of RNAP I (157). Other evidences include the interaction between components of the SL1 transcription factor for RNAP I and S-HDAg (158), inhibition of the antigenome synthesis by anti-α-SL1 antibodies (158) and direct interaction between RNAP I and HDV RNA (159). Recent data suggest an implication of the third polymerase, the RNAP III, which also binds to both genomic and antigenomic HDV RNA (159).

Transcription occurs in the nucleus, where HDV RNA is brought into by the HDAg (160) and is activated by S-HDAg binding to HDV RNA. Replication occurs without any help coming from HBV and without any DNA intermediate. Replication proceeds via a double-rolling circle process, a mechanism proposed for the replication of plant viroids and small-satellite RNAs of some plant viruses (161). The transcription proceeds for more than one genome length, going twice through the ribozyme cleavage site. Then the antigenomic transcript undergoes an autocatalytic cleavage between nucleotides 688 and 689 via the antigenomic ribozyme, is folded into a rod-like structure and is ligated by cellular ligases (162) to form a circular template. Production of the genomic RNA from the antigenome occurs through the same mechanism; then the genomic RNA is either incorporated into new viral particles or used again as template for antigenomic RNA synthesis. The ratio between genomic and antigenomic RNA is asymmetric, as the genome is fifteen times more abundant than the antigenome (112). The mechanism of regulation is unknown but it has been reported that HDV RNA mutations could suppress genomic strand transcription, without affecting antigenomic strand synthesis, suggesting two separate means of regulation (163). The mRNA is transcribed from the genome, thus using the same template as for the antigenomic RNA. The transcription of these two elements nevertheless starts at different initiation sites (116), using different polymerases and in different subnuclear compartments. The antigenome is transcribed into the nucleolus, whereas the mRNA is transcribed into the nucleoplasm, like the genome (Fig. 6).

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

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Mechanisms of hepatitis D virus disease

  1. Top of page
  2. Abstract
  3. Epidemiology
  4. Clinical features and diagnosis
  5. Treatment
  6. Hepatitis D virus life cycle
  7. Mechanisms of hepatitis D virus disease
  8. Conclusions
  9. References

Hepatitis D virus only replicates in the liver. Pathologic changes are thus limited to this organ. Histologically, the liver presents cellular necrosis and inflammation. Even though some in vitro experiments have demonstrated a direct cytopathic effect of HDV (164, 165), other results and in vivo observations, such as the presence of inflammatory cells surrounding the infected hepatocytes (166) and the presence of various autoantibodies in the serum of patients, argue for immune-mediated liver damage.

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

Hepatitis D virus increases cell survival potential

Hepatitis D virus replication is associated with increased histone H3 acetylation within the clusterin promoter, resulting in an enhanced clusterin gene expression (167). Interestingly, this modification is the same as that associated with the expression of specific proteins of several oncogenic viruses, such as the adenovirus protein E1A (168, 169), the simian virus 40T antigen (170) and the E7 protein of the human papilloma virus (171). The increase of clusterin protein enhances the survival of cell infected with HDV (167). Clusterin has been reported to be upregulated in tumour cells and to play a significant role in tumourigenesis (172, 173). This protein could thus be implicated in the development of HCC in HDV-infected patients. However, conflicting results have been reported in the literature, such as inhibition of host cell proliferation (174), cell-cycle arrest (175) and cell death (165).

Hepatitis D virus inhibits IFN-α signalling

Hepatitis D virus, like many other viruses, seems to have developed an anti-IFN-α strategy. HDV directly inhibits the activation of the IFN-α signalling by interfering in the early steps of the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) signal transduction pathway. There is an inhibition of the tyrosine kinase 2 (Tyk2), STAT1 and STAT2 phosphorylation and a transcription impairment of several IFN-α-stimulated genes, such as the myxovirus resistance-A (MxA), the 2′,5′-oligoadenylate synthetase (2′,5′-OAS) and PKR in the presence of the virus (176). On the other hand, an upregulation of MxA transcription induced by the L-HDAg has been reported and suggested to account for the suppression of HBV replication (177).

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

Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation is implicated in inflammation processes and in cancer. L-HDAg has been shown to induce tumour necrosis factor-alpha (TNF-α)-induced NF-κB signalling, probably through the direct association with TNF receptor-associated factor 2 (TRAF2), a protein implicated in the early signal transduction events (178).

Changes in the cell proteome

Several studies have investigated the relationship among L-HDAg, S-HDAg, genomic RNA, antigenomic RNA or a combination of these elements and the proteome of the cell (179, 180). Proteins involved in pathways such as regulation of cell metabolism and energy pathways, nucleic acid and protein metabolism, transport, signal transduction, apoptosis and cell growth and maintenance show a modified expression profile. A small inhibitory RNA (siRNA) screening has also been performed to investigate the cellular proteins implicated in HDV replication. Cells stably expressing S-HDAg were transfected with siRNA before induction of viral replication (181). A direct interaction has been reported between a portion of the genome, described to act in vitro as an RNA promoter (111), and some cellular proteins. It should be pointed out that two of these proteins implicated in RNA processing or associated with the translation machinery: the eukaryotic translation elongation factor 1 alpha 1 (eEF1A1) and the glyceraldehyde-3-phosphate dehydrogenase, are considered and often used in research as housekeeping genes (182). Such observations suggest potential avenues of research in order to improve our understanding of the mechanisms of HDV replication and pathogenesis.

Conclusions

  1. Top of page
  2. Abstract
  3. Epidemiology
  4. Clinical features and diagnosis
  5. Treatment
  6. Hepatitis D virus life cycle
  7. Mechanisms of hepatitis D virus disease
  8. Conclusions
  9. References

Hepatitis D virus is an insufficiently characterized virus: After a decrease of its spread, largely because of the HBV vaccination campaigns and the increased awareness on bloodborne infections following the HIV scare, it still infects a steady proportion of HBV carriers worldwide. Thus, it is unfortunate that the interest in HDV research has faltered. The stable prevalence in Western countries, the frequent occurrence of severe outbreaks in diverse geographical areas, the severity of HDV-associated liver disease and the lack of efficient treatment should, however, encourage research. A better knowledge of the virus life cycle and pathogenesis will certainly help in identifying new approaches to treatment.

References

  1. Top of page
  2. Abstract
  3. Epidemiology
  4. Clinical features and diagnosis
  5. Treatment
  6. Hepatitis D virus life cycle
  7. Mechanisms of hepatitis D virus disease
  8. Conclusions
  9. References