Hepatitis D: Thirty years after
Authors: Mario Rizzetto
Journal: Journal of Hepatology (2009)

The key to the discovery of the Hepatitis D Virus (HDV) was the description in Turin, Italy in the mid-1970s of the delta antigen and antibody in carriers of the hepatitis B surface antigen. The new antigen was first thought to be a marker of the Hepatitis B Virus (HBV) and in view of its intricate true nature, it would have possibly died away as another odd antigenic subtype of HBV, like many that were described in the 1970s. Fortunately, instead, a collaboration started in 1978 between the Turin group, and the National Institute of Health and Georgetown University in the US. With American facilities and expertise this collaboration led just a year later, in 1979, to the unfolding of an unexpected and amazing chapter in virology. Experiments in chimpanzees demonstrated that the delta antigen was not a component of the HBV but of a separate defective virus requiring HBV for its infection; it was named the hepatitis D virus to conform to the nomenclature of hepatitis viruses and classified within the genus Deltavirus. The animal experiments were also seminal in proposing to future clinical interpretation, the paradigm of a pathogenic infection (hepatitis D), that could develop only in HBV-infected patients, was mainly transmitted by superinfection of HDV on chronic HBV carriers and had the ability to strongly inhibit the helper HBV. The discovery of the HDV has driven three directions of further research:(1)The understanding of the replicative and infectious mechanisms of the HDV.(2)The assessment of its epidemiological and medical impact.(3)The search for a therapy for chronic hepatitis D (CHD).This review summarizes the progress achieved in each field of research in the thirty years that have passed since the discovery of HDV. The key to the discovery of the Hepatitis D Virus (HDV) was the description in Turin, Italy in the mid-1970s of the delta antigen and antibody in carriers of the hepatitis B surface antigen. The new antigen was first thought to be a marker of the Hepatitis B Virus (HBV) and in view of its intricate true nature, it would have possibly died away as another odd antigenic subtype of HBV, like many that were described in the 1970s. Fortunately, instead, a collaboration started in 1978 between the Turin group, and the National Institute of Health and Georgetown University in the US. With American facilities and expertise this collaboration led just a year later, in 1979, to the unfolding of an unexpected and amazing chapter in virology. Experiments in chimpanzees demonstrated that the delta antigen was not a component of the HBV but of a separate defective virus requiring HBV for its infection; it was named the hepatitis D virus to conform to the nomenclature of hepatitis viruses and classified within the genus Deltavirus. The animal experiments were also seminal in proposing to future clinical interpretation, the paradigm of a pathogenic infection (hepatitis D), that could develop only in HBV-infected patients, was mainly transmitted by superinfection of HDV on chronic HBV carriers and had the ability to strongly inhibit the helper HBV. The discovery of the HDV has driven three directions of further research:(1)The understanding of the replicative and infectious mechanisms of the HDV.(2)The assessment of its epidemiological and medical impact.(3)The search for a therapy for chronic hepatitis D (CHD). This review summarizes the progress achieved in each field of research in the thirty years that have passed since the discovery of HDV. 1. Progress in HDV virologyThe genetic structure of the new virus appears rudimentary at first glance. The HD virion is a hybrid particle made up of the HD-Antigen (HD-Ag) and an RNA species enclosed within a HBsAg coat derived from HBV [1Rizzetto M. Canese M.G. Aricò S. Crivelli O. Trepo C. Bonino F. et al.Immunofluorescence detection of a new antigen-antibody system (delta/anti-delta) associated with hepatitis B virus in liver and in serum of HBsAg carriers.Gut. 1977; 18: 997-1003Crossref PubMed Scopus (658) Google Scholar, 2Rizzetto M. Hoyer B. Canese M.G. Shih J.W.K. Purcell R.H. Gerin J.L. Delta antigen: the association of delta antigen with hepatitis B surface antigen and ribonucleic acid in the serum of delta infected chimpanzees.Proc Natl Acad Sci New York. 1980; 77: 6124-6128Crossref PubMed Scopus (344) Google Scholar, 3Rizzetto M. Canese M.G. Gerin J.L. London W.T. Sly L.D. Purcell R.H. Transmission of the hepatitis B virus-associated delta antigen to chimpanzees.J Infect Dis. 1980; 141: 590-602Crossref PubMed Scopus (355) Google Scholar]. The virion contains a circular minute RNA genome of negative polarity, folding in native conditions into a nearly complementary rod-like structure. In the liver, the corresponding antigenomic circular strand of positive polarity contains only one open reading frame; this encodes the HD-Ag, the sole known protein of HDV, through the transcription of a 0.8 Kb messenger RNA. The very measure of the genome size indicated that HDV was not a conventional virus, as it was distinctly smaller than all known animal viruses [3Rizzetto M. Canese M.G. Gerin J.L. London W.T. Sly L.D. Purcell R.H. Transmission of the hepatitis B virus-associated delta antigen to chimpanzees.J Infect Dis. 1980; 141: 590-602Crossref PubMed Scopus (355) Google Scholar, 4Rizzetto M. Verme G. Delta hepatitis.J Hepatol. 1985; 1: 187-193Abstract Full Text PDF PubMed Scopus (82) Google Scholar]. Its size, and the folded RNA loop, were instead similar to the size and structure of the RNA viroids of higher plants; this first suggested that HDV may have originated from the plant rather than the animal world.A second unique feature of HDV was the recognition in 1989 that both the genomic and antigenomic strands contained a ribozyme, a RNA segment of less than 100 bases that retained the genetic information but was also able to self-cleave and self-ligate the circular HDV genome [[5]Branch A.D. Benenfeld B.J. Baroudy B.M. Wells F.V. Gerin J.L. Robertson H.D. An ultraviolet-sensitive RNA structural element in a viroid-like domain of the hepatitis delta virus.Science. 1989; 243: 649-652Crossref PubMed Scopus (60) Google Scholar]. The HDV ribozymes have been crystallized [[6]Ferré-D'Amaré A.R. Zhou K. Doudna J.A. Crystal structure of a hepatitis delta virus ribozyme.Nature. 1998; 395: 567-574Crossref PubMed Scopus (660) Google Scholar] and shown to catalyse cleavage of the HDV–RNA backbone with a rate of enhancement of 106- to 107-fold over the uncatalyzed rate. They use several catalytic strategies including the use of metal ions [[7]Been M.D. HDV ribozymes.Curr Top Microbiol Immunol. 2006; 307: 47-65Crossref PubMed Scopus (44) Google Scholar], intrinsic binding energy and acid-base catalysis under physiological conditions; the latter ability appears to be unique among ribozymes [[8]Nakano S. Chadalavada D.M. Bevilacqua P.C. General acid-base catalysis in the mechanism of a hepatitis delta virus.Science. 2000; 287: 1493-1497Crossref PubMed Scopus (362) Google Scholar]. Ribozymes are also present in viroids and this was taken as further evidence to support an origin of HDV [[9]Tsagris E.M. Martı´nez de Alba A.E. Gozmanova M. Kalantidis K. Viroids.Cell Microbiol. 2008; 10: 2168-2179Crossref PubMed Scopus (104) Google Scholar] from plants. However, viroids do not code for protein and the structure of the "hammerhead" ribozymes described in plant viruses is different from the HDV ribozymes, indicating that the catalytic domain of HDV–RNA represents a different ribozyme motif. More intriguing, the finding that the CPEB3 ribozyme, a conserved mammalian sequence residing in an intron of the CPEB3 gene, is structurally and biochemically related to the HDV ribozyme, suggests that HDV could derive from the human trascriptome and may have evolved in modern protein-dominated organisms rather than from an ancestral RNA world [[10]Salehi-Ashtiani K. Lupták A. Litovchick A. Szostak J.W. A genome wide search for ribozymes reveals an HDV-like sequence in the human CPEB3 gene.Science. 2006; 313: 1788-1792Crossref PubMed Scopus (211) Google Scholar]. Alternatively, human HDV may have evolved from a viroid-like satellite RNA of smaller original size, that acquired the RNA encoding the HD-Ag and the ribozyme through recombination with the human transcriptome [[11]Brazas R. Ganem D. A cellular homolog of hepatitis delta antigen: implications for viral replication and evolution.Science. 1996; 274: 90-94Crossref PubMed Scopus (106) Google Scholar].The dilemma confronting early research on HDV was how this virus could replicate; its minute genome coded only for the HD-Ag and could not possibly code for any of the complex enzymatic functions required for independent replication. It was intuitive that HDV exploited HBV and cellular functions, but what were these functions and what role did the HDV play in the replication process? Both questions have been answered in the last 30 years by the discovery of a surprisingly articulate sequence of cellular and viral interactions leading to HDV synthesis [12Taylor J.M. Structure and replication of hepatitis delta virus RNA.Curr Top Microbiol Immunol. 2006; 307: 1-23Crossref PubMed Scopus (67) Google Scholar, 13Lai M.M. RNA replication without RNA-dependent RNA polymerase: surprises from hepatitis delta virus.J Virol. 2005; 79: 7951-7958Crossref PubMed Scopus (106) Google Scholar].Crucial to the understanding of the respective role of HBV and host cells was the report by Dr. Fu and Dr. Taylor in 1993 [[14]Fu T.B. Taylor J. The RNAs of hepatitis delta virus are copied by RNA polymerase II in nuclear homogenates.J Virol. 1993; 67: 6965-6972Crossref PubMed Google Scholar] that hepatocytes nuclear extracts were able to replicate faithfully the whole genomic and antigenomic strand of HDV without any extraneous factor. The conclusion that HDV replication can proceed in host cells in the absence of HBV proteins was confirmed by in vitro and in vivo transfection experiments; the first have shown that cDNA constructs of HDV induce genome replication when transfected into cultured cells [[15]Kuo M.Y. Chao M. Taylor J. Initiation of replication of the human hepatitis delta virus genome from cloned DNA: role of delta antigen.J Virol. 1989; 63: 1945-1950Crossref PubMed Google Scholar], the second that DNA copies of the HDV–RNA and of the RNA itself injected into the tail vein of mice elicit typical HDV replication with a transient expression of HDV in several tissues of the rodent [[16]Chang J. Sigal L.J. Lerro A. Taylor J. Replication of the human hepatitis delta virus genome is initiated in mouse hepatocytes following intravenous injection of naked DNA or RNA sequences.J Virol. 2001; 75: 3469-3473Crossref PubMed Scopus (68) Google Scholar], besides the liver. Therefore, mammalian cells can autonomously support efficient HBV replication. Avian cells do not [[17]Liu Y.T. Brazas R. Ganem D. Efficient hepatitis delta virus RNA replication in avian cells requires a permissive factor(s) from mammalian cells.J Virol. 2001; 75: 7489-7493Crossref PubMed Scopus (5) Google Scholar]; however, the fusion of mammalian to avian cells rescues HDV synthesis, suggesting that avian cells lack factors permissive to HDV replication that are restored by mammalian cells.Clearly, HDV needs HBV only to borrow the HBsAg capsid whereby it attaches to hepatocytes and propagates infection [[18]Sureau C. The role of the HBV envelope proteins in the HDV replication cycle.Curr Top Microbiol Immunol. 2006; 307: 113-131Crossref PubMed Scopus (62) Google Scholar]. Within cells, HDV–RNA is associated with multiple copies of the HD proteins to assemble into a ribonucleoprotein complex, which is exported by the HBV envelope through the budding into the lumen of a pre-Golgi compartment before being secreted [[18]Sureau C. The role of the HBV envelope proteins in the HDV replication cycle.Curr Top Microbiol Immunol. 2006; 307: 113-131Crossref PubMed Scopus (62) Google Scholar]. Though the small HBsAg alone can package the HDV ribonucleoprotein and assemble into virions [[19]Wang C.J. Chen P.J. Wu J.C. Patel D. Chen D.S. Small-form hepatitis B surface antigen is sufficient to help in the assembly of hepatitis delta virus-like particles.J Virol. 1991; 65: 6630-6636PubMed Google Scholar], the large HBsAg protein bearing in S1 a receptor to cells, is required to confer infectivity [20Sureau C. Guerra B. Lanford R.E. Role of the large hepatitis B virus envelope protein in infectivity of the hepatitis delta virion.J Virol. 1993; 67: 366-372PubMed Google Scholar, 21Barrera A. Guerra B. Notvall L. Lanford R.E. Mapping of the hepatitis B virus pre-S1 domain involved in receptor recognition.J Virol. 2005; 79: 9786-9798Crossref PubMed Scopus (105) Google Scholar].The HBsAg coat is not exclusive, as HDV can coat also within the woodchuck hepatitis virus (WHV) surface antigen and in vivo HDV has been passed from chimpanzees to the rodent [[4]Rizzetto M. Verme G. Delta hepatitis.J Hepatol. 1985; 1: 187-193Abstract Full Text PDF PubMed Scopus (82) Google Scholar]. However, in apparent contradiction to the in vivo transmission of primate HDV to woodchucks, woodchuck-HDV was reported to infect cultured primary woodchuck hepatocytes as well as cultures of primary human hepatocytes, but human HDV could not infect woodchuck hepatocytes [[22]Gudima S. He Y. Chai N. Bruss V. Urban S. Mason W. et al.Primary human hepatocytes are susceptible to infection by hepatitis delta virus assembled with envelope proteins of woodchuck hepatitis virus.J Virol. 2008; 82: 7276-7283Crossref PubMed Scopus (28) Google Scholar]; the different host-range specificities seems determined by the different recognition of WHV and HBV Pre S1 proteins on human hepatocytes.Conventional RNA viruses undergo replication by a virus encoded RNA-dependent RNA-polymerase which replicates the viral genome; they cannot use cellular RNA-polymerases, as these accept only DNA templates. Hepatitis D virus does not possess any polymerase, therefore, the next enigma was how it replicated in the host. The explanation is that host RNA polymerase II (Pol II), is deceived by HDV and becomes redirected to read and copy its RNA. This was confirmed by transcriptional run-on experiments which have demonstrated inhibition of viral synthesis after the addition of low-dose amanitin [[23]Chang J. Nie X. Chang H.E. Han Z. Taylor J. Transcription of hepatitis delta virus RNA by RNA polymerase II.J Virol. 2008; 82: 1118-1127Crossref PubMed Scopus (75) Google Scholar], a toxin that selectively blocks the accumulation of RNA Pol II transcripts. Presumably the rod-like conformation of the viral RNA is recognized as double-stranded DNA for RNA Pol II binding. Immunoprecipitation experiments using blocking monoclonal antibodies to RNA Pol II [[24]Greco-Stewart V.S. Miron P. Abrahem A. Pelchat M. The human RNA polymerase II interacts with the terminal stem-loop regions of the hepatitis delta virus RNA genome.Virology. 2007; 357: 68-78Crossref PubMed Scopus (50) Google Scholar] have shown that human RNA Pol II binds directly to the terminal stem-loop regions of HDV–RNA and the interactions of the HD-Ag with the RNA Pol II clamp, a mobile structure that holds DNA and RNA in place, may facilitate the unusual RNA synthesis [[25]Yamaguchi Y. Mura T. Chanarat S. Okamoto S. Handa H. Hepatitis delta antigen binds to the clamp of RNA polymerase II and affects transcriptional fidelity.Genes Cells. 2007; 12: 863-875Crossref PubMed Scopus (40) Google Scholar]. Once redirected to copy HDV–RNA, RNA Pol II carries out unchecked its transcriptional activity, elongating a multimeric linear transcript of either the genome or the antigenome over the viral RNA circular template of the other; this elementary double-rolling circle mechanism of replication is unknown to animal viruses but operative in viroids [[13]Lai M.M. RNA replication without RNA-dependent RNA polymerase: surprises from hepatitis delta virus.J Virol. 2005; 79: 7951-7958Crossref PubMed Scopus (106) Google Scholar].The last step requires the rearrangement of the linear multimeric strand into genomic and antigenomic HDV–RNAs. This is where the HDV finally steps in; its ribozymes first cleave the redundant transcription product into a genomic/antigenomic size monomer and then ligate the linear monomer into the infectious circular form.The intricate RNA-cellular interactions are only part of the replicative story of HDV. Crucial and incredibly complex for such a small molecule are also the functions of the HD-Ag. The HD-Ag contains several functional domains [[26]Lazinski D.W. Taylor J.M. Relating structure to function in the hepatitis delta virus antigen.J Virol. 1993; 67: 2672-2680Crossref PubMed Google Scholar]; a coiled-coil domain facilitates protein–protein interactions [[27]Rozzelle Jr., J.E. Wang J.G. Wagner D.S. Erickson B.W. Lemon S.M. Self-association of a synthetic peptide from the N-terminus of the hepatitis delta virus protein into an immunoreactive alpha-helical multimer.Proc Natl Acad Sci USA. 1995; 92: 382-386Crossref PubMed Scopus (34) Google Scholar], a bipartite nuclear-localization signal drives the HD proteins to the nucleus [[28]Xia Y.P. Yeh C.T. Ou J.H. Lai M.M. Characterization of nuclear targeting signal of hepatitis delta antigen: nuclear transport as a protein complex.J Virol. 1992; 66: 914-921Crossref PubMed Google Scholar], an oligomerization domain and two arginine rich motifs support the binding to the RNA. It is edited by a cellular double-stranded adenine-deaminase into a small HD-Ag (SHD-Ag, 195 amino acid residues) and a large HD-Ag (LHD-Ag, 214 amino acid residues), the first promoting replication, the second virion assembly [[29]Casey J.L. RNA editing in hepatitis delta virus.Curr Top Microbiol Immunol. 2006; 307: 67-89Crossref PubMed Scopus (59) Google Scholar]. Many other functions of the HD-Ag isoforms have been recognized and added to the list in the last years [12Taylor J.M. Structure and replication of hepatitis delta virus RNA.Curr Top Microbiol Immunol. 2006; 307: 1-23Crossref PubMed Scopus (67) Google Scholar, 13Lai M.M. RNA replication without RNA-dependent RNA polymerase: surprises from hepatitis delta virus.J Virol. 2005; 79: 7951-7958Crossref PubMed Scopus (106) Google Scholar, 30Yamaguchi Y. Filipovska J. Yano K. Furuya A. Inukai N. Narita T. et al.Stimulation of RNA polymerase II elongation by hepatitis delta antigen.Science. 2001; 293: 124-127Crossref PubMed Scopus (127) Google Scholar].The dilemma is again how the two small HDV proteins can escort the HDV genome through different cellular compartments and become involved in the different steps of replication, transcription and formation of progeny virions. The answer is that the biological activities of the HD-Ag depend on a host of ordered protein–protein interactions regulated by post-translational modifications of the HD-Ag [[31]Huang W.H. Chen C.W. Wu H.L. Chen P.J. Post-translational modification of delta antigen of hepatitis D virus.Curr Top Microbiol Immunol. 2006; 307: 91-112Crossref PubMed Scopus (43) Google Scholar]; as a one gene–genome the HDV offers a unique opportunity to determine what these are and how they affect the functional roles of the HDV proteins.Phosphorylation [32Mu J.J. Wu H.L. Chiang B.L. Chang R.P. Chen D.S. Chen P.J. Characterization of the phosphorylated forms and the phosphorylated residues of hepatitis delta virus delta antigens.J Virol. 1999; 73: 10540-10545Crossref PubMed Google Scholar, 33Chen Y.S. Huang W.H. Hong S.Y. Tsay Y.G. Chen P.J. ERK1/2-mediated phosphorylation of small hepatitis delta antigen at serine 177 enhances hepatitis delta virus antigenomic RNA replication.J Virol. 2008; 82: 9345-9358Crossref PubMed Scopus (15) Google Scholar], acetylation [[34]Tseng C.H. Jeng K.S. Lai M.M. Transcription of subgenomic mRNA of hepatitis delta virus requires a modified hepatitis delta antigen that is distinct from antigenomic RNA synthesis.J Virol. 2008; 82: 9409-9416Crossref PubMed Scopus (20) Google Scholar], methylation [[35]Li Y.J. Stallcup M.R. Lai M.M. Hepatitis delta virus antigen is methylated at arginine residues, and methylation regulates subcellular localization and RNA replication.J Virol. 2004; 78: 13325-13334Crossref PubMed Scopus (68) Google Scholar], prenylation [[36]Glenn J.S. Watson J.A. Havel C.M. White J.M. Identification of a prenylation site in delta virus large antigen.Science. 1992; 256: 1331-1333Crossref PubMed Scopus (237) Google Scholar] and several other post translational modifications of the HD antigens [37Lee C.Z. Sheu J.C. Histone H1e interacts with small hepatitis delta antigen and affects hepatitis delta virus replication.Virology. 2008; 375: 197-204Crossref PubMed Scopus (10) Google Scholar, 38Huang W.H. Chen Y.S. Chen P.J. Nucleolar targeting of hepatitis delta antigen abolishes its ability to initiate viral antigenomic RNA replication.J Virol. 2008; 82: 692-699Crossref PubMed Scopus (22) Google Scholar, 39Huang W.H. Mai R.T. Lee Y.H. Transcription factor YY1 and its associated acetyltransferases CBP and p300 interact with hepatitis delta antigens and modulate hepatitis delta virus RNA replication.J Virol. 2008; 82: 7313-7324Crossref PubMed Scopus (26) Google Scholar] have been shown to orchestrate the life cycle of the virus. Different phosphorylation patterns of the small and large HD-Ag account for their distinct biological functions [[32]Mu J.J. Wu H.L. Chiang B.L. Chang R.P. Chen D.S. Chen P.J. Characterization of the phosphorylated forms and the phosphorylated residues of hepatitis delta virus delta antigens.J Virol. 1999; 73: 10540-10545Crossref PubMed Google Scholar] and phosphorilated SHD-Ag increases replication from antigenomic to genomic RNA [[33]Chen Y.S. Huang W.H. Hong S.Y. Tsay Y.G. Chen P.J. ERK1/2-mediated phosphorylation of small hepatitis delta antigen at serine 177 enhances hepatitis delta virus antigenomic RNA replication.J Virol. 2008; 82: 9345-9358Crossref PubMed Scopus (15) Google Scholar]. Acetylation and deacetylation of HD-Ag may provide a molecular switch for the synthesis of the different RNA species [[34]Tseng C.H. Jeng K.S. Lai M.M. Transcription of subgenomic mRNA of hepatitis delta virus requires a modified hepatitis delta antigen that is distinct from antigenomic RNA synthesis.J Virol. 2008; 82: 9409-9416Crossref PubMed Scopus (20) Google Scholar], and interactions with the multifunctional transcription factor YY1 and its associated acetyltransferase selectively modulates replication of the genomic and antigenomic forms [[39]Huang W.H. Mai R.T. Lee Y.H. Transcription factor YY1 and its associated acetyltransferases CBP and p300 interact with hepatitis delta antigens and modulate hepatitis delta virus RNA replication.J Virol. 2008; 82: 7313-7324Crossref PubMed Scopus (26) Google Scholar]. Methylation at arginine residues regulates the subcellular localization and also RNA replication [[35]Li Y.J. Stallcup M.R. Lai M.M. Hepatitis delta virus antigen is methylated at arginine residues, and methylation regulates subcellular localization and RNA replication.J Virol. 2004; 78: 13325-13334Crossref PubMed Scopus (68) Google Scholar]. Farnesylation of the LHD-Ag is essential to target this protein to cellular membranes containing the HBsAg in order to trigger viral assembly [[40]O'Malley B. Lazinski D.W. Roles of carboxyl-terminal and farnesylated residues in the functions of the large hepatitis delta antigen.J Virol. 2005; 79: 1142-1153Crossref PubMed Scopus (38) Google Scholar]. More intriguing, the participation of HDV proteins in the regulation of cellular gene expression could trigger HDV-related liver damage. It was reported that isoprenylation of LDH-Ag regulates Transforming-Growth Factor -β- induced signal-transduction and through this mechanism may induce liver fibrosis [[41]Choi S.H. Jeong S.H. Hwang S.B. Large hepatitis delta antigen modulates transforming growth factor-beta signaling cascades: implication of hepatitis delta virus-induced liver fibrosis.Gastroenterology. 2007; 132: 343-357Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar].2. Epidemiological and clinical impactIndirect antibody assays for the diagnosis of HDV became generally available at the beginning of the 1980s, prompting worldwide studies on the epidemiology and medical impact of the infection. Molecular assays for the direct measure of HDV–RNA in serum were developed but have not been made commercially available and are limited to specialized laboratories only; the current PCR assays are specific and have a sensitivity threshold of 10–100 copies of the HDV genome [42Smedile A. Niro M.G. Rizzetto M. Detection of serum HDV RNA by RT-PCR.in: NJ Methods in Molecular Medicine, vol. 95: Hepatitis B and D protocols, vol. 1. vol. 95. Humana Press Inc., Totowa2004: 85-93Google Scholar, 43Le Gal F. Gordien E. Affolabi D. Hanslik T. Alloui C. Dény P. et al.Quantification of hepatitis delta virus RNA in serum by consensus real-time PCR indicates different patterns of virological response to interferon therapy in chronically infected patients.J Clin Microbiol. 2005; 43: 2363-2369Crossref PubMed Scopus (100) Google Scholar], per ml of serum.Surveys in the 1980s showed that HDV is endemic worldwide, though with prevalences and patterns of infection varying in different areas; globally the number of HDV carriers was estimated at 15,000,000 at that time [[44]Rizzetto M. Ponzetto A. Forzani I. Hepatitis Delta Virus as a global health problem.Vaccine. 1990; 8: S10-S14Crossref PubMed Scopus (63) Google Scholar]. Medical scrutiny confirmed that CHD usually runs a severe and progressive course [[45]Rizzetto M. Verme G. Recchia S. Bonino F. Farci P. Aricò S. et al.Chronic HBsAg hepatitis with intrahepatic expression of the delta antigen. An active and progressive disease unresponsive to immunosuppressive treatment.Ann Intern Med. 1983; 98: 437-441Crossref PubMed Scopus (298) Google Scholar]. The prototype patient with CHD carried the HBsAg in blood, had elevated ALT, a liver biopsy exhibiting aggressive hepatitis but no markers of HBV replication; the discrepancy between a florid HBsAg-disease and the lack of HBV synthesis provided the best harbinger to the suspicion of an underlying HDV infection [[46]Smedile A. Rizzetto M. Gerin J.L. Advances in Hepatitis D Virus Biology and Disease.in: Progress in Liver Disease. vol. XII. Saunders Company, 1994: 157-175Google Scholar]. However, in epidemiological pockets as far away as the Greek island of Archangelos and Pacific Samoa, HDV caused no or insignificant disease, indicating that the course of HDV infections may span a clinical spectrum ranging from asymptomatic carriage of the virus to very severe disease [[47]Hadziyannis S.J. Hatzakis A. Papaioannou C. Anastassakos C. Vassiliadis E. Endemic hepatitis delta virus infection in a Greek community.Prog Clin Biol Res. 1987; 234: 181-202PubMed Google Scholar].A factor that may influence the course of disease is the genotype of the HDV. Currently this virus is divided into eight major genotypes differing as much as 40% in nucleotide sequence [[48]Dény P. Hepatitis delta virus genetic variability: from genotypes I, II, III to eight major clades?.Curr Top Microbiol Immunol. 2006; 307: 151-171Crossref PubMed Scopus (108) Google Scholar]. Genotype I is the most frequent worldwide and has variable pathogenicity. In a study from Taiwan CHD patients infected with genotype I HDV had a lower remission rate and more adverse outcomes [[49]Su C.W. Huang Y.H. Huo T.I. Shih H.H. Sheen I.J. Chen S.W. et al.Genotypes and viremia of hepatitis B and D viruses are associated with outcomes of chronic hepatitis D patients.Gastroenterology. 2006; 130: 1625-1635Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar] than those with genotype II HDV. Genotypes II and IV were found in East Asia causing relatively mild disease [[50]Wu J.C. Functional and clinical significance of hepatitis D virus genotype II infection.Curr Top Microbiol Immunol. 2006; 307: 173-186Crossref PubMed Scopus (35) Google Scholar]; however, a genotype II b variant isolated from HDV patients from the Miyako Island, Okinawa (II b-M) was associated with greater progression to cirrhosis then the II b genotype prevalent in Taiwan [[51]Watanabe H. Nagayama K. Enomoto N. Chinzei R. Yamashiro T. Izumi N. et al.Chronic hepatitis delta virus infection with genotype II b variant is correlated with progressive liver disease.J Gen Virol. 2003; 84: 3275-3289Crossref PubMed Scopus (48) Google Scholar]. Genotype III was associated with the genotype F of HBV and fulminant hepatitis in South America [[46]Smedile A. Rizzetto M. Gerin J.L. Advances in Hepatitis D Virus Biology and Disease.in: Progress in Liver Disease. vol. XII. Saunders Company, 1994: 157-175Google Scholar]. While the genotype of HBV does not seem to affect the interaction of HBsAg with HDV, the genotype of HDV may influence the efficiency of assembly with the HBsAg into virions. Aminoacid sequence variations in HBsAg expressed by naturally occurring variants of HBV diminished the assembly efficiency of genotype II and IV of HDV but not of genotype I [[52]Shih H.H. Jeng K.S. Syu W.J. Huang Y.H. Su C.W. Peng W.L. et al.Hepatitis B surface antigen levels and sequences of natural hepatitis B virus variants influence the assembly and secretion of hepatitis D virus.J Virol. 2008; 82: 2250-2264Crossref PubMed Scopus (27) Google Scholar]; the more efficient interactions between genotype I of HDV and HBV, might play a role for the wide distribution of HDV I in the world.Since the 1990s the circulation of HDV has declined significantly in Europe, consistent with the hypothesis that the virus was discovered at the time of a major epidemic that has been brought under control by now [[53]Hadziyannis S.J. Decreasing prevalence of hepatitis D virus infection