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Delta hepatitis: an update

      1. Introduction

      Twenty-five years ago Rizzetto et al. [
      • Rizzetto M.
      • Canese M.G.
      • Arico S.
      • Crivelli O.
      • Trepo C.G.
      • Bonino F.
      • et al.
      Immunofluorescence detection of new antigen-antibody system (delta/anti-delta) associated to hepatitis B virus in liver and in serum of HBsAg carriers.
      ], while examining liver biopsies from individuals infected with HBV, discovered by immunofluorescence a previously unrecognized nuclear antigen that was subsequently shown to be a specific marker of a novel human pathogen, HDV. The clinical association with HBV results from the fact that HDV is a defective virus that requires a helper function provided by HBV or other hepadnaviruses [
      • Rizzetto M.
      • Canese M.G.
      • Gerin J.L.
      • London W.T.
      • Sly D.L.
      • Purcell R.H.
      Transmission of the hepatitis B virus-associated delta antigen to chimpanzees.
      ]. Since the early studies, HDV has emerged as an important medical problem because it is highly pathogenic and causes a severe and rapidly progressive form of liver disease [
      • Rizzetto M.
      • Verme G.
      • Recchia S.
      • Bonino F.
      • Farci P.
      • Arico S.
      • et al.
      Chronic hepatitis in carriers of hepatitis B surface antigen, with intrahepatic expression of the delta antigen. An active and progressive disease unresponsive to immunosuppressive treatment.
      ]. The cloning and sequencing of the HDV genome in 1986 confirmed that HDV is unique in animal virology [
      • Wang K.S.
      • Choo Q.L.
      • Weiner A.J.
      • Ou J.H.
      • Najarian R.C.
      • Thayer R.M.
      • et al.
      Structure, sequence and expression of the hepatitis delta (δ) viral genome.
      ]. It is the first animal virus to possess a circular RNA genome, a finding that has only been seen in plant viruses [
      • Kos A.
      • Dijkema R.
      • Arnberg A.C.
      • van der Meide P.H.
      • Schellekens H.
      The hepatitis delta (δ) virus possesses a circular RNA.
      ]. Progress in molecular biology has provided the tools to further understand the unique virologic features of HDV, which continues to represent a major challenge to both virologists and clinicians.

      2. Hepatitis D virus

      2.1 Classification

      HDV does not resemble any known transmissible agent of animals, but it shares similarities with both viroids and virusoids of plants in terms of structural characteristics of the RNA genome and mode of viral replication [
      • Taylor J.M.
      Hepatitis delta virus and its replication.
      ]. The International Committee on Taxonomy of Viruses has proposed to classify HDV within the floating genus Deltavirus [
      • Murphy F.A.
      Virus Taxonomy.
      ].

      2.2 Structure and genome organization

      The virus is an enveloped, spherical particle with an average diameter of 36–43 nm [
      • He L.F.
      • Ford E.
      • Purcell R.H.
      • London W.T.
      • Phillips J.
      • Gerin J.L.
      The size of the hepatitis delta agent.
      ] containing in its interior a nucleocapsid of 19 nm in diameter [
      • Ryu W.S.
      • Netter H.J.
      • Bayer M.
      • Taylor J.
      Ribonucleoprotein complexes of hepatitis delta virus.
      ], which consists of an RNA genome and a single structural protein, hepatitis delta antigen (HDAg), that are encapsidated by the hepatitis B surface antigen (HBsAg). Such an antigen, which is the only function provided by the helper virus, is essential for HDV assembly and propagation [
      • Taylor J.M.
      Hepatitis delta virus and its replication.
      ]. There are two forms of HDAg, short (HDAg-S) and long (HDAg-L), that derive from the same open reading frame.
      The HDV genome consists of a single, minus-strand, circular RNA, approximately 1700 nucleotides in length, which represents the smallest known viral genome in the animal kingdom [
      • Wang K.S.
      • Choo Q.L.
      • Weiner A.J.
      • Ou J.H.
      • Najarian R.C.
      • Thayer R.M.
      • et al.
      Structure, sequence and expression of the hepatitis delta (δ) viral genome.
      ]. It possesses several distinctive features, some of which shared with plant virus RNAs, that make HDV unique relative to other animal RNA viruses [
      • Taylor J.M.
      Structure and replication of hepatitis delta virus.
      ]. The genome has a high degree, up to 70%, of intramolecular base-pairing that confers the potential to fold into an unbranched, rod-like structure [
      • Wang K.S.
      • Choo Q.L.
      • Weiner A.J.
      • Ou J.H.
      • Najarian R.C.
      • Thayer R.M.
      • et al.
      Structure, sequence and expression of the hepatitis delta (δ) viral genome.
      ]. Moreover, HDV shares with transmissible viroids and certain non-transmissible components of eukaryotic cells the capacity of its genomic and antigenomic RNAs to function as ribozymes capable of carrying out self-cleavage [
      • Sharmeen L.
      • Kuo M.Y.
      • Dinter-Gottlieb G.
      • Taylor J.
      • Antigenomic R.N.A.
      of human hepatitis delta virus can undergo self-cleavage.
      ] and self-ligation [
      • Sharmeen L.
      • Kuo M.Y.
      • Taylor J.
      • Self-ligating R.N.A.
      sequences on the antigenome of human hepatitis delta virus.
      ]. Although these autocatalytic RNA segments are functionally similar to the ‘hammerhead’ ribozymes described in plant viruses, their sequence and structure are quite different from those of other known ribozymes [
      • Perrotta A.T.
      • Shih I.
      • Been M.D.
      Imidazole rescue of a cytosine mutation in a self-cleaving ribozyme.
      ]. Another unique function described for the HDV ribozyme is general acid–base catalysis, which could allow RNA to catalyze other reactions, including peptide bond formation [
      • Nakano S.
      • Chadalavada D.M.
      • Bevilacqua P.C.
      General acid–base catalysis in the mechanism of a hepatitis delta virus ribozyme.
      ].

      2.3 Viral replication

      After HBsAg-mediated binding to a cellular receptor that is believed to be common to that of HBV, HDV penetrates into the hepatocyte and its genome is directed to the nucleoplasm, where genome replication occurs by the nuclear localization signal domain identified in the HDAg [
      • Taylor J.M.
      Structure and replication of hepatitis delta virus.
      ,
      • 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.
      ]. The mechanism of replication of the HDV genome is one of the most complex and fascinating aspects of the biology of this virus. HDV replicates according to a rolling-circle mechanism analogous to that proposed for viroids [
      • Branch A.D.
      • Robertson D.
      A replication cycle for viroids and other small infectious RNA's (Review).
      ]. Three major species of RNA accumulate in infected liver cells during HDV replication [
      • Taylor J.M.
      Hepatitis delta virus and its replication.
      ]: genomic RNA, its exact complement, antigenomic RNA, and a smaller form of polyadenylated RNA (0.8 kb) of antigenomic polarity, which is the messenger RNA (mRNA) containing the open reading frame (ORF) for the synthesis of HDAg. The HDV RNA genome is replicated by RNA-directed RNA synthesis operated by a host cell enzyme, RNA polymerase II. HDV has the unique ability to redirect such enzymes, which is normally DNA-dependent, to transcribe the viral RNA genome [
      • Taylor J.
      • Mason W.
      • Summers J.
      • Goldberg J.
      • Aldrich C.
      • Coates L.
      • et al.
      Replication of human hepatitis delta virus in primary cultures of woodchuck hepatocytes.
      ,
      • Fu T.B.
      • Taylor J.
      The RNAs of hepatitis delta virus are copied by RNA polymerase II in nuclear homogenates.
      ]. Initially, the genomic RNA serves as a template to synthesize multimeric linear transcripts of antigenomic polarity, which undergo autocatalytic cleavage and ligation to produce circular monomeric antigenomic RNA. This nascent RNA is also processed to produce the polyadenylated mRNA. The antigenomic RNA serves as a template for the replication of circular genomic RNA. An essential role in the replication of HDV is played by HDAg. It has been recently reported [
      • Yamaguchi Y.
      • Filipovska J.
      • Yano K.
      • Furuya A.
      • Inukai N.
      • Narita T.
      • et al.
      Stimulation of RNA polymerase II elongation by hepatitis delta antigen.
      ] that HDAg directly binds to RNA polymerase II and stimulates transcription by displacing a 66-kDa subunit of negative elongation factor (NELF) and promoting RNA polymerase II elongation.
      HDAg is an internal component of the nucleocapsid [
      • Bonino F.
      • Hoyer B.
      • Shih J.W.
      • Rizzetto M.
      • Purcell R.H.
      • Gerin J.L.
      Delta hepatitis agent: structural and antigenic properties of the delta-associated particle.
      ]. It contains several essential elements for the virus biology [
      • Lai M.M.
      The molecular biology of hepatitis delta virus (Review).
      ]: a coiled coil domain that enables HDAg dimerization; a nuclear localization signal that directs HDAg to the nucleus; a RNA-binding domain consisting of two arginine-rich regions; and an isoprenylation motif, unique to HDAg-L, at the C-terminus. HDAg-L differs from HDAg-S by the presence of 19 additional amino acids at the C-terminus [
      • Weiner A.J.
      • Choo Q.L.
      • Wang K.S.
      • Govindarajan S.
      • Redeker A.G.
      • Gerin J.L.
      • et al.
      A single antigenomic open reading frame of the hepatitis delta virus encodes the epitope(s) of both hepatitis delta antigen polypeptides p24 delta and p27 delta.
      ], which result from editing of the antigenomic RNA at position 1012 by a cellular enzyme, double-stranded RNA adenosine deaminase [
      • Polson A.G.
      • Bass B.L.
      • Casey J.L.
      RNA editing of hepatitis delta virus antigenome by dsRNA-adenosine deaminase.
      ]. This editing changes the UAG amber termination codon of the 195 amino acid. HDAg-S into UGG (encoding tryptophan), allowing for a C-terminal extension that leads to the synthesis of HDAg-L. HDAg-S is essential for viral replication, whereas HDAg-L acts as a dominant negative inhibitor of viral replication, but is required for virus assembly [
      • Lai M.M.
      The molecular biology of hepatitis delta virus (Review).
      ]. Evidence is accumulating to suggest that RNA editing plays a central role in the HDV replication cycle by acting as a biological regulator of the replication and assembly of HDV infectious virions [
      • Casey J.L.
      • Polson A.G.
      • Bass B.L.
      • Gerin J.L.
      Molecular biology of HDV: analysis of RNA editing and genotype variation.
      ].

      2.4 Viral heterogeneity

      Genetic variation is a hallmark of RNA viruses [
      • Holland J.
      • Spindler K.
      • Horodyski F.
      • Grabau E.
      • Nichol S.
      • VandePol S.
      Rapid evolution of RNA genomes (Review).
      ]. The first description of the genetic heterogeneity of HDV was reported in 1986 [
      • Wang K.S.
      • Choo Q.L.
      • Weiner A.J.
      • Ou J.H.
      • Najarian R.C.
      • Thayer R.M.
      • et al.
      Structure, sequence and expression of the hepatitis delta (δ) viral genome.
      ] when it was found that HDV circulates, within a single infected host, as a mixture of different, albeit closely related genomes. The distribution of these variants follows the model referred to as a quasispecies [
      • Eigen M.
      Self organization of matter and the evolution of biological macromolecules.
      ]. Subsequently, the wide application of refined molecular techniques has provided the tools to better characterize the degree of genetic heterogeneity of HDV, both within the same individual and between different individuals. The rate of HDV mutation has been calculated to be between 3×10−2 and 3×10−3 base substitutions per genome site per year [
      • Lee C.M.
      • Bih F.Y.
      • Chao Y.C.
      • Govindarajan S.
      • Lai M.M.
      Evolution of hepatitis delta virus RNA during chronic infection.
      ]. The genetic heterogeneity of HDV is not uniformly distributed over the entire viral genome [
      • Chao Y.C.
      • Chang M.F.
      • Gust I.
      • Lai M.M.
      Sequence conservation and divergence of hepatitis delta virus RNA.
      ], with the genomic and antigenomic cleavage domain and the RNA-binding domain of HDAg being the most conserved.
      Genetic analysis of HDV isolates collected worldwide has revealed that HDV has evolved into three major genotypes, designated I, II, and III [
      • Casey J.L.
      • Brown T.L.
      • Colan E.J.
      • Wignall F.S.
      • Gerin J.L.
      A genotype of hepatitis D virus that occurs in northern South America.
      ] that differ in their global distribution [
      • Casey J.L.
      • Polson A.G.
      • Bass B.L.
      • Gerin J.L.
      Molecular biology of HDV: analysis of RNA editing and genotype variation.
      ]. The sequence differences among these genotypes are significant; the most distantly related HDV isolates vary by as much as 40% over their entire genomic sequences and 35% for the amino acid sequence of HDAg [
      • Casey J.L.
      • Polson A.G.
      • Bass B.L.
      • Gerin J.L.
      Molecular biology of HDV: analysis of RNA editing and genotype variation.
      ]. Genotype I, the most common worldwide, is associated with a broad spectrum of pathogenicity. It is predominant in the United States, Europe and the Middle East. Recently, it has been associated with fulminant hepatitis in the city of Samara in Russia [
      • Flodgren E.
      • Bengtsson S.
      • Knutsson M.
      • Strebkova E.A.
      • Kidd A.H.
      • Alexeyev O.A.
      • et al.
      Recent high incidence of fulminant hepatitis in Samara, Russia: molecular analysis of prevailing hepatitis B and D virus strains.
      ]. Within genotype 1, two soubgroups, 1A and 1B, have been identified; 1A is predominant in Asia, 1B in the United States and both are common in the Mediterranean area. Genotype II, which seems to be associated with milder forms of liver disease, has been found in Japan and Taiwan, as well as more recently in the area of Yakutia in Russia [
      • Ivaniushina V.
      • Radjef N.
      • Alexeeva M.
      • Gault E.
      • Semenov S.
      • Salhi M.
      • et al.
      Hepatitis delta virus genotypes I and II cocirculate in an endemic area of Yakutia, Russia.
      ]. Genotype III, which has been identified only in northern South America, has been associated with outbreaks of severe and fulminant hepatitis [
      • Casey J.L.
      • Brown T.L.
      • Colan E.J.
      • Wignall F.S.
      • Gerin J.L.
      A genotype of hepatitis D virus that occurs in northern South America.
      ,
      • Casey J.L.
      • Niro G.A.
      • Engle R.E.
      • Vega A.
      • Gomez H.
      • McCarthy M.
      • et al.
      Hepatitis B virus (HBV)/hepatitis D virus (HDV) coinfection in outbreaks of acute hepatitis in the Peruvian Amazon basin: the roles of HDV genotype III and HBV genotype F.
      ,
      • Nakano T.
      • Shapiro C.N.
      • Hadler S.C.
      • Casey J.L.
      • Mizokami M.
      • Orito E.
      • et al.
      Characterization of hepatitis D virus genotype III among Yucpa Indians in Venezuela.
      ]. Interestingly, this genotype is linked with the coinfecting HBV genotype F [
      • Casey J.L.
      • Niro G.A.
      • Engle R.E.
      • Vega A.
      • Gomez H.
      • McCarthy M.
      • et al.
      Hepatitis B virus (HBV)/hepatitis D virus (HDV) coinfection in outbreaks of acute hepatitis in the Peruvian Amazon basin: the roles of HDV genotype III and HBV genotype F.
      ].

      3. Host range

      In nature, HDV infection has been found only in humans, whereas experimentally the host range of HDV is limited to those species that support the replication of HBV-related hepadnaviruses, capable of supplying a helper function to HDV, such as chimpanzees [
      • Rizzetto M.
      • Canese M.G.
      • Gerin J.L.
      • London W.T.
      • Sly D.L.
      • Purcell R.H.
      Transmission of the hepatitis B virus-associated delta antigen to chimpanzees.
      ], Eastern woodchucks [
      • Ponzetto A.
      • Cote P.J.
      • Popper H.
      • Hoyer B.H.
      • London W.T.
      • Ford E.C.
      • et al.
      Transmission of the hepatitis B virus-associated delta agent to the eastern woodchuck.
      ] and Pekin ducks [
      • Ponzetto A.
      • Rapicetta M.
      • Forzani B.
      • Smedile A.
      • Hele C.
      • Morace G.
      • et al.
      Hepatitis delta virus infection in Pekin ducks chronically infected by the duck hepatitis B virus.
      ]. In addition, different mouse models of HDV infection have been attempted, including mice injected either with serum from experimentally infected woodchucks [
      • Netter H.J.
      • Kajino K.
      • Taylor J.M.
      Experimental transmission of human hepatitis delta virus to the laboratory mouse.
      ] or with naked viral DNA or RNA sequences [
      • 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.
      ], as well as heterochimeric SCID mice engrafted with human hepatocytes [
      • Ohashi K.
      • Marion P.L.
      • Nakai H.
      • Meuse L.
      • Cullen J.M.
      • Bordier B.B.
      • et al.
      Sustained survival of human hepatocytes in mice: A model for in vivo infection with human hepatitis B and hepatitis delta viruses.
      ], but none of them have so far provided a reliable model of viral tropism and pathogenesis.

      4. Transmission and epidemiology

      Infection with HDV has a worldwide distribution, although there are considerable geographic differences that do not entirely mirror the prevalence of HBV infection [
      • Rizzetto M.
      • Hadziyannis S.
      • Hansson B.G.
      • Toukan A.
      • Gust I.
      Hepatitis delta virus infection in the world, epidemiological patterns and clinical expression.
      ]. In northern Europe and in the United States, where HDV is not endemic, the infection is mainly confined to intravevenous drug users [
      • Novick D.M.
      • Farci P.
      • Croxson T.S.
      • Taylor M.B.
      • Schneebaum C.W.
      • Lai M.E.
      • Hepatitis D.
      • et al.
      virus and human immunodeficiency virus antibodies in parenteral drug abusers who are hepatitis B surface antigen positive.
      ], whereas it has virtually disappeared in polytransfused subjects and hemophiliacs [
      • Rosina F.
      • Saracco G.
      • Rizzetto M.
      Risk of post-transfusion infection with the hepatitis delta virus. A multicenter study.
      ] as a result of universal blood screening for HBsAg and HBV vaccination. In areas where HDV is endemic in the general population, as in the Mediterranean basin, the inapparent parenteral route accounts for most cases of HDV transmission [
      • Bonino F.
      • Caporaso N.
      • Dentico P.
      • Marinucci G.
      • Valeri L.
      • Craxi A.
      • et al.
      Familiar clustering and spreading of hepatitis delta virus infection.
      ,
      • Sagnelli E.
      • Stroffolini T.
      • Ascione A.
      • Bonino F.
      • Chiaramonte M.
      • Colombo M.
      • et al.
      The epidemiology of hepatitis delta infection in Italy over the last 18 years.
      ,
      • Liaw Y.F.
      • Chiu K.W.
      • Chu C.M.
      • Sheen I.S.
      • Huang M.J.
      Heterosexual transmission of hepatitis delta virus in the general population of an area endemic for hepatitis B virus infection: a prospective study.
      ]. The advent of molecular epidemiology has provided new tools to further elucidate the modes and mechanisms of HDV transmission, confirming that HDV can be transmitted through sexual contacts [
      • Wu J.C.
      • Chen C.M.
      • Sheen I.J.
      • Lee S.D.
      • Tzeng H.M.
      • Choo K.B.
      Evidence of transmission of hepatitis D virus to spouses from sequence analysis of the viral genome.
      ], as well as among family members with a trend to form clusters [
      • Niro G.A.
      • Casey J.L.
      • Gravinese E.
      • Garrubba M.
      • Conoscitore P.
      • Sagnelli E.
      • et al.
      Intrafamilial transmission of hepatitis delta virus: molecular evidence.
      ].
      Evidence accrued in the last decade, however, is suggestive of a changing trend in the epidemiology of HDV. A decline in HDV prevalence in both acute [
      • Stroffolini T.
      • Ferrigno L.
      • Cialdea L.
      • Catapano R.
      • Palumbo F.
      • Novaco F.
      • et al.
      Incidence and risk factors of acute Delta hepatitis in Italy: results from a national surveillance system. SEIEVA Collaborating Group.
      ] and chronic hepatitis [
      • Sagnelli E.
      • Stroffolini T.
      • Ascione A.
      • Chiaramonte M.
      • Craxi A.
      • Giusti G.
      • et al.
      Decrease in HDV endemicity in Italy.
      ,
      • Hadziyannis S.J.
      Hepatitis delta: an overview.
      ,
      • Gaeta G.B.
      • Stroffolini T.
      • Chiaramonte M.
      • Ascione T.
      • Stornaiuolo G.
      • Lobello S.
      • et al.
      Chronic hepatitis D: a vanishing disease? An Italian multicenter study.
      ,
      • Navascues C.A.
      • Rodriguez M.
      • Sotorrio N.G.
      • Sala P.
      • Linares A.
      • Suarez A.
      • et al.
      Epidemiology of hepatitis D virus infection: changes in the last 14 years.
      ] has been observed in the Mediterranean area, most likely due to universal HBV vaccination, measures to control human immunodeficiency virus (HIV) and socioeconomic improvements, whereas new foci of HDV infection are emerging in other parts of the world. An outbreak of acute viral hepatitis with a high incidence of fulminant hepatitis, in which HDV accounted for 39% of the cases, has been reported between 1998 and 1999 in the city of Samara [
      • Flodgren E.
      • Bengtsson S.
      • Knutsson M.
      • Strebkova E.A.
      • Kidd A.H.
      • Alexeyev O.A.
      • et al.
      Recent high incidence of fulminant hepatitis in Samara, Russia: molecular analysis of prevailing hepatitis B and D virus strains.
      ], in southeastern Russia, which has remained largely isolated until 1991. This situation is reminiscent of that seen in Sweden in the early 1970s, as a result of the increased use of illicit parenteral drugs [
      • Hansson B.G.
      • Moestrup T.
      • Widell A.
      • Nordenfelt E.
      Infection with delta agent in Sweden: introduction of a new hepatitis agent.
      ]. The fulminant HDV strains from Samara were of genotype I and phylogenetically closest to Far Eastern and Eastern European strains. New foci of infection have also been identified in the island of Okinawa in Japan [
      • Sakugawa H.
      • Nakasone H.
      • Shokita H.
      • Nakayoshi T.
      • Kinjo F.
      • Saito A.
      • et al.
      Seroepidemiological study of hepatitis delta virus infection in Okinawa, Japan.
      ], in northern India [
      • Singh V.
      • Goenka M.K.
      • Bhasin D.K.
      • Kochhar R.
      • Singh K.
      A study of hepatitis delta virus infection in patients with acute and chronic liver disease from northern India.
      ] and in Albania [
      • Dalekos G.N.
      • Zervou E.
      • Karabini F.
      • Tsianos E.V.
      Prevalence of viral markers among refugees from southern Albania: increased incidence of infection with hepatitis A, B and D viruses.
      ]. South America, especially the subtropical area, remains an important potential reservoir for new outbreaks of HDV infection [
      • Manock S.R.
      • Kelley P.M.
      • Hyams K.C.
      • Douce R.
      • Smalligan R.D.
      • Watts D.M.
      • et al.
      An outbreak of fulminant hepatitis delta in the Waorani, an indigenous people of the Amazon basin of Ecuador.
      ].
      Overall, HDV infection is still a major public health concern. It has been estimated that approximately 5% of the global HBsAg carriers are also coinfected with HDV, leading to a total of 15 million persons infected with HDV worldwide. Thus, the absolute number of dire events on a global scale is still high and HDV remains a major cause of death and liver transplantation.

      5. Modes of infection and clinical expression

      In view of the absolute dependence of HDV on HBV coinfection, the modes of HDV infection are essentially two: simultaneous coinfection with HBV or superinfection of an HBsAg carrier [
      • Smedile A.
      • Rizzetto M.
      • Gerin J.L.
      Advances in hepatitis D virus biology and disease.
      ]. Persons with anti-HBs, being immune to HBV infection, are not susceptible to HDV infection.
      The clinical expression of acute hepatitis D acquired by confection with HBV may range from mild to severe, fulminant hepatitis [
      • Smedile A.
      • Rizzetto M.
      • Gerin J.L.
      Advances in hepatitis D virus biology and disease.
      ]. In most cases, it resembles a typical acute self-limited hepatitis that is clinically and histologically indistinguishable from the ordinary hepatitis B. The outcome is a complete recovery, as typically observed in acute type-B hepatitis, and in only 2% of cases it may progress to chronicity. Diagnosis is made by the concomitant appearance of markers of primary infection with HBV and HDV [
      • Smedile A.
      • Rizzetto M.
      • Gerin J.L.
      Advances in hepatitis D virus biology and disease.
      ].
      In the superinfection pattern, the preexisting HBV viremia provides the biological background for the full expression of the virulence of HDV [
      • Smedile A.
      • Rizzetto M.
      • Gerin J.L.
      Advances in hepatitis D virus biology and disease.
      ]. Clinically, this results in a severe acute hepatitis that may run a fulminant course [
      • Saracco G.
      • Macagno S.
      • Rosina F.
      • Rizzetto M.
      Serologic markers with fulminant hepatitis in persons positive for hepatitis B surface antigen. A worldwide epidemiologic and clinical survey.
      ]. It may present as an exacerbation of a preexisting HBV disease or as a new hepatitis in a previously asymptomatic HBsAg carrier. If the HBsAg state is unknown, it may be misdiagnosed as classical acute hepatitis B [
      • Farci P.
      • Smedile A.
      • Lavarini C.
      • Piantino P.
      • Crivelli O.
      • Caporaso N.
      • et al.
      Delta hepatitis in inapparent carriers of hepatitis B surface antigen. A disease simulating acute hepatitis B progressive to chronicity.
      ]. The correct diagnosis is suggested by a negative test for IgM anti-HBc and confirmed by the detection of HDV markers. Since the HBsAg carrier permits a continuous replication of HDV, the vast majority of HDV superinfected carriers develop progressive hepatitis (over 90% of the cases), whereas in a minority the superinfection may resolve [
      • Smedile A.
      • Ciancio A.
      • Rizzetto M.
      • Hepatitis D.
      Hepatitis D virus.
      ], with persistence of the original HBV infection or even clearance of HBsAg without seroconversion to anti-HBs [
      • Niro G.A.
      • Gravinese E.
      • Martini E.
      • Garrubba M.
      • Facciorusso D.
      • Conoscitore P.
      • et al.
      Clearance of hepatitis B surface antigen in chronic carriers of hepatitis delta antibodies.
      ].
      The setting of liver transplantation has permitted to recognize a third pattern of HDV infection, latent infection [
      • Ottobrelli A.
      • Marzano A.
      • Smedile A.
      • Recchia S.
      • Salizzoni M.
      • Cornu C.
      • et al.
      Patterns of hepatitis delta virus reinfection and disease in liver transplantation.
      ]. Within a few days after liver transplantation, HDV can establish infection despite low levels of HBV replication. This type of HDV infection remains latent and is associated with no signs of liver disease. However, when a shift to high levels of HBV replication occurs, the latent HDV infection is rapidly transformed into a florid hepatitis of the liver graft.

      6. Pathogenesis

      The pathogenesis of HDV-induced liver disease is still undefined. Although a direct cytopathic effect of the virus has been reported [
      • Cole S.M.
      • Gowans E.J.
      • Macnaughton T.B.
      • Hall P.D.
      • Burrell C.J.
      Direct evidence for cytotoxicity associated with expression of hepatitis delta virus antigen.
      ], this hypothesis is contradicted by the lack of liver injury observed in grafts expressing only HDAg [
      • Ottobrelli A.
      • Marzano A.
      • Smedile A.
      • Recchia S.
      • Salizzoni M.
      • Cornu C.
      • et al.
      Patterns of hepatitis delta virus reinfection and disease in liver transplantation.
      ,
      • Samuel D.
      • Zignego A.L.
      • Reynes M.
      • Feray C.
      • Arulnaden J.L.
      • David M.F.
      • et al.
      Long-term clinical and virological outcome after liver transplantation for cirrhosis caused by chronic delta hepatitis.
      ], as well as in hepatocytes from HDV-infected humans [
      • Verme G.
      • Amoroso P.
      • Lettieri G.
      • Pierri P.
      • David E.
      • Sessa F.
      • et al.
      A histological study of hepatitis delta virus liver disease.
      ], and transgenic mice [
      • Guilhot S.
      • Huang S.N.
      • Xia Y.P.
      • La Monica N.
      • Lai M.M.
      • Chisari F.V.
      Expression of the hepatitis delta virus large and small antigens in transgenic mice.
      ]. Of note, a deleterious effect of HDV replication on host cell proliferation has been documented using in vitro-transfected cells [
      • Wang D.
      • Pearlberg J.
      • Liu Y.T.
      • Ganem D.
      Deleterious effects of hepatitis delta virus replication on host cell proliferation.
      ]. A role of host immune mechanisms, such as specific CD4+ and CD8+ T lymphocytes [
      • Nisini R.
      • Paroli M.
      • Accapezzato D.
      • Bonino F.
      • Rosina F.
      • Santantonio T.
      • et al.
      Human CD4+ T-cell response to hepatitis delta virus: identification of multiple epitopes and characterization of T-helper cytokine profiles.
      ] in liver damage seems unlikely. Nevertheless, multiple types of autoimmune phenomena have been reported in chronic hepatitis D [
      • Smedile A.
      • Ciancio A.
      • Rizzetto M.
      • Hepatitis D.
      Hepatitis D virus.
      ], the most common being the presence of the LKM-3 autoantibody directed against uridine diphosphate glucuronosyl transferase [
      • Philipp T.
      • Durazzo M.
      • Trautwein C.
      • Alex B.
      • Straub P.
      • Lamb J.G.
      • et al.
      Recognition of uridine diphosphate glucuronosyl transferases by LKM-3 antibodies in chronic hepatitis D.
      ].
      The relationship between viral genotypes and clinical course of hepatitis D has represented an important area of investigation over the past few years. However, the critical question of whether genotypes play a differential role in the pathogenesis and severity of hepatitis D is still unsolved. Although the link of genotype III with outbreaks of fulminant hepatitis suggests an inherently higher pathogenicity, the association of genotype I with a wide spectrum of disease severity [
      • Niro G.A.
      • Smedile A.
      • Andriulli A.
      • Rizzetto M.
      • Gerin J.L.
      • Casey J.L.
      The predominance of hepatitis delta virus genotype I among chronically infected Italian patients.
      ] including fulminant hepatitis [
      • Flodgren E.
      • Bengtsson S.
      • Knutsson M.
      • Strebkova E.A.
      • Kidd A.H.
      • Alexeyev O.A.
      • et al.
      Recent high incidence of fulminant hepatitis in Samara, Russia: molecular analysis of prevailing hepatitis B and D virus strains.
      ] argues against a direct role of the genotype in the pathogenesis of the disease. Further studies are needed to elucidate whether there are major biological differences among genotypes in terms of efficiency of replication, packaging, infectivity, transmissibility and pathogenicity.

      7. Natural history

      The natural history of chronic HDV infection is characterized by a wide spectrum of clinical presentations. Since the earliest studies, HDV turned out to be a highly pathogenic virus causing a severe and rapidly progressive disease, with very infrequent spontaneous resolution [
      • Rizzetto M.
      • Verme G.
      • Recchia S.
      • Bonino F.
      • Farci P.
      • Arico S.
      • et al.
      Chronic hepatitis in carriers of hepatitis B surface antigen, with intrahepatic expression of the delta antigen. An active and progressive disease unresponsive to immunosuppressive treatment.
      ]. Cirrhosis was shown to develop in up to 70% of cases, among them in about 15% within 1–2 years of disease onset [
      • Saracco G.
      • Rosina F.
      • Brunetto M.R.
      • Amoroso P.
      • Caredda F.
      • Farci P.
      • et al.
      Rapidly progressive HBsAg-positive hepatitis in Italy. The role of hepatitis delta virus infection.
      ]. Although HDV cirrhosis, once established, may be a stable disease for many years [
      • Rosina F.
      • Conoscitore P.
      • Cuppone R.
      • Rocca G.
      • Giuliani A.
      • Cozzolongo R.
      • et al.
      Changing pattern of chronic hepatitis D in Southern Europe.
      ], coinfection with HDV was shown to significantly increase the risk of hepatocellular carcinoma (HCC) and death, during a median follow-up period of 6.6 years, in patients with compensated HBV cirrhosis [
      • Fattovich G.
      • Giustina G.
      • Christensen E.
      • Pantalena M.
      • Zagni I.
      • Realdi G.
      • et al.
      Influence of hepatitis delta virus infection on morbidity and mortality in compensated cirrhosis type B. The European Concerted Action on Viral Hepatitis (Eurohep).
      ]. HCC, previously considered a rare event in the course of HDV disease, developed in 42% of patients with HDV cirrhosis in Greece over 12 years of follow-up [
      • Hadziyannis S.J.
      Hepatitis delta: an overview.
      ]. The link of HDV with severe and progressive liver disease has been found at all ages, as suggested by the detection of markers of HDV infection in up to 40% of children with HBsAg positive cirrhosis [
      • Farci P.
      • Barbera C.
      • Navone C.
      • Bortolotti F.
      • Vajro P.
      • Caporaso N.
      • et al.
      Infection with the delta agent in children.
      ,
      • Maggiore G.
      • Hadchouel M.
      • Sessa F.
      • Vinci M.
      • Craxi A.
      • Marzani M.
      • et al.
      A retrospective study of the role of delta agent infection in children with HBsAg-positive chronic hepatitis.
      ,
      • Bortolotti F.
      • Di Marco V.
      • Vajro P.
      • Crivellaro C.
      • Zancan L.
      • Nebbia G.
      • et al.
      Long-term evolution of chronic delta hepatitis in children.
      ]. However, the current clinical scenario of HDV disease is changing because fresh and florid forms of chronic hepatitis D are becoming rare in the Mediterranean area, as a consequence of a dramatic decline in the prevalence of HDV infection over the past decade [
      • Rosina F.
      • Conoscitore P.
      • Cuppone R.
      • Rocca G.
      • Giuliani A.
      • Cozzolongo R.
      • et al.
      Changing pattern of chronic hepatitis D in Southern Europe.
      ].
      Although HDV is generally associated with a severe form of liver disease, a few studies performed in open populations in highly endemic areas, such as the island of Rhodes in Greece and the American Samoa [
      • Hadziyannis S.J.
      Hepatitis delta: an overview.
      ], have documented a high proportion of anti-HDV positive individuals without liver disease. Differences in disease outcome could be related to viral, host or still undefined enviromental factors. Among the viral factors, the interaction between HBV and HDV replication has been reevaluated in recent years by using highly sensitive PCR assays. Most patients with chronic HDV infection have antibodies to HBeAg and low levels of HBV DNA replication [
      • Hadziyannis S.J.
      • Sherman M.
      • Lieberman H.M.
      • Shafritz D.A.
      Liver disease activity and hepatitis B virus replication in chronic delta antigen-positive hepatitis B virus carriers.
      ,
      • Farci P.
      • Orgiana G.
      • Coiana A.
      • Peddis G.
      • Mandas A.
      • Lai M.E.
      • et al.
      Epidemiology of HDV infection in Sardinia, an island with a high endemicity for HBV: A multicenter study.
      ,
      • Sakugawa H.
      • Nakasone H.
      • Nakayoshi T.
      • Kawakami Y.
      • Yamashiro T.
      • Maeshiro T.
      • Hepatitis B.
      • et al.
      virus concentrations in serum determined by sensitive quantitative assays in patients with established chronic hepatitis delta virus infection.
      ]. Different from HDV-negative chronic hepatitis B, there is no correlation between serum HBV DNA and ALT levels in patients with chronic hepatitis D, suggesting that the liver damage in these patients is mainly caused by HDV [
      • Sakugawa H.
      • Nakasone H.
      • Nakayoshi T.
      • Kawakami Y.
      • Yamashiro T.
      • Maeshiro T.
      • Hepatitis B.
      • et al.
      virus concentrations in serum determined by sensitive quantitative assays in patients with established chronic hepatitis delta virus infection.
      ]. Similarly, HBV is suppressed in asymptomatic carriers of HDV [
      • Chen P.J.
      • Chen D.S.
      • Chen C.R.
      • Chen Y.Y.
      • Chen H.M.
      • Lai M.Y.
      • et al.
      Delta infection in asymptomatic carriers of hepatitis B surface antigen: low prevalence of delta activity and effective suppression of hepatitis B virus replication.
      ]. However, a pattern characterized by decreasing levels of HDV and reactivating HBV with moderately high ALT levels has been described during the course of chronic hepatitis D in Taiwan [
      • Wu J.C.
      • Chen T.Z.
      • Huang Y.S.
      • Yen F.S.
      • Ting L.T.
      • Sheng W.Y.
      • et al.
      Natural history of hepatitis D viral superinfection: significance of viremia detected by polymerase chain reaction.
      ]. In intravenous drug addicts, HDV infection is most often associated with HBeAg positivity and active HBV replication, a pattern that may increase the pathogenicity of HDV [
      • Smedile A.
      • Rosina F.
      • Saracco G.
      • Chiaberge E.
      • Lattore V.
      • Fabiano A.
      • Hepatitis B.
      • et al.
      virus replication modulates pathogenesis of hepatitis D virus in chronic hepatitis D.
      ].
      Another factor that may modify the natural history of chronic hepatitis D is coinfection with other viruses. In triple infection, several studies have shown that HDV plays a dominant role inhibiting both HBV and HCV [
      • Eyster M.E.
      • Sanders J.C.
      • Battegay M.
      • Di Bisceglie A.M.
      Suppression of hepatitis C virus (HCV) replication by hepatitis D virus (HDV) in HIV-infected hemophiliacs with chronic hepatitis B and C.
      ,
      • Sagnelli E.
      • Coppola N.
      • Scolastico C.
      • Filippini P.
      • Santantonio T.
      • Stroffolini T.
      • et al.
      Virologic and clinical expressions of reciprocal inhibitory effect of hepatitis B, C, and delta viruses in patients with chronic hepatitis.
      ,
      • Mathurin P.
      • Thibault V.
      • Kadidja K.
      • Ganne-Carrie N.
      • Moussalli J.
      • El Younsi M.
      • et al.
      Replication status and histological features of patients with triple (B, C, D) and dual (B, C) hepatic infections.
      ,
      • Jardi R.
      • Rodriguez F.
      • Buti M.
      • Costa X.
      • Cotrina M.
      • Galimany R.
      • et al.
      Role of hepatitis B, C, and D viruses in dual and triple infection: influence of viral genotypes and hepatitis B precore and basal core promoter mutations on viral replicative interference.
      ]. The course of chronic hepatitis D does not seem to be influenced by concomitant HIV infection [
      • Monno L.
      • Angarano G.
      • Santantonio T.
      • Milella M.
      • Carbonara S.
      • Fiore J.R.
      • et al.
      Lack of HBV and HDV replicative activity in HBsAg-positive intravenous drug addicts with immune deficiency due to HIV.
      ,
      • Pol S.
      • Wesenfelder L.
      • Dubois F.
      • Roingeard P.
      • Carnot F.
      • Driss F.
      • et al.
      Influence of human immunodeficiency virus infection on hepatitis delta virus superinfection in chronic HBsAg carriers.
      ,
      • Buti M.
      • Jardi R.
      • Allende H.
      • Cotrina M.
      • Rodriguez F.
      • Viladomiu L.
      • et al.
      Chronic delta hepatitis: is the prognosis worse when associated with hepatitis C virus and human immunodeficiency virus infections?.
      ], except in terms of a more elusive or absent immune response to HDV in subjects coinfected with HIV [
      • Roingeard P.
      • Dubois F.
      • Marcellin P.
      • Bernuau J.
      • Bonduelle S.
      • Benhamou J.P.
      • et al.
      Persistent delta antigenaemia in chronic delta hepatitis and its relation with human immunodeficiency virus infection.
      ].

      8. Diagnosis

      The advent of molecular techniques has provided highly sensitive tools to diagnose HDV infection. The detection of HDV RNA by polymerase chain reaction (PCR) is presently the most reliable diagnostic method. This molecular test has overcome the limitations of the direct detection of HDAg in serum by enzyme immunoassay or radioimmunoassay due to antigen sequestration in immune complexes with high-titered circulating antibodies [
      • Smedile A.
      • Ciancio A.
      • Rizzetto M.
      • Hepatitis D.
      Hepatitis D virus.
      ]. Its role is crucial not only in the early phase of infection, before antibody seroconversion, but also to investigate the molecular events during both acute and chronic hepatitis. PCR has also offered a sensitive tool for monitoring the efficacy of antiviral agents, since it can detect 10–100 copies of the viral genome in serum [
      • Smedile A.
      • Ciancio A.
      • Rizzetto M.
      • Hepatitis D.
      Hepatitis D virus.
      ]. Because of the genetic heterogeneity of HDV, primers from the most conserved region, the C-terminal half of the HDAg gene, are most useful in clinical practice [
      • Casey J.L.
      • Brown T.L.
      • Colan E.J.
      • Wignall F.S.
      • Gerin J.L.
      A genotype of hepatitis D virus that occurs in northern South America.
      ,
      • Niro G.A.
      • Smedile A.
      • Andriulli A.
      • Rizzetto M.
      • Gerin J.L.
      • Casey J.L.
      The predominance of hepatitis delta virus genotype I among chronically infected Italian patients.
      ]. The HDV genotype may be determined by restriction fragment length polymorphism analysis of PCR amplification products, by sequencing and, on liver biopsies, by immunohistochemical staining using genotype-specific anti-HD antibodies [
      • Hsu S.C.
      • Syu W.J.
      • Ting L.T.
      • Wu J.C.
      Immunohistochemical differentiation of hepatitis D virus genotypes.
      ].
      The diagnosis of HDV infection may also be indirect, based on the detection in serum of antibodies against HDAg (anti-HD) of the IgG and IgM classes [
      • Smedile A.
      • Rizzetto M.
      • Gerin J.L.
      Advances in hepatitis D virus biology and disease.
      ]. Testing for IgM anti-HD is crucial not only as a marker of primary HDV infection but also for its clinical relevance in the natural history of the disease [
      • Farci P.
      • Gerin J.L.
      • Aragona M.
      • Lindsey I.
      • Crivelli O.
      • Balestrieri A.
      • et al.
      Diagnostic and prognostic significance of the IgM antibody to the hepatitis delta virus.
      ]. As a rule, chronic hepatitis D is associated with high titers of both IgG and IgM anti-HD [
      • Manock S.R.
      • Kelley P.M.
      • Hyams K.C.
      • Douce R.
      • Smalligan R.D.
      • Watts D.M.
      • et al.
      An outbreak of fulminant hepatitis delta in the Waorani, an indigenous people of the Amazon basin of Ecuador.
      ], although the IgM are monomeric [7S] and not pentameric (19S) as in primary infection [
      • Macagno S.
      • Smedile A.
      • Caredda F.
      • Ottobrelli A.
      • Rizzetto M.
      Monomeric (7S) immunoglobulin M antibodies to hepatitis delta virus in hepatitis type D.
      ]. The decrease and disappearance of IgM anti-HD predicts impending resolution of chronic HDV disease, either spontaneous or induced by interferon (IFN) [
      • Borghesio E.
      • Rosina F.
      • Smedile A.
      • Lagget M.
      • Niro M.G.
      • Marinucci G.
      • et al.
      Serum immunoglobulin M antibody to hepatitis D as a surrogate marker of hepatitis D in interferon-treated patients and in patients who underwent liver transplantation.
      ].

      9. Treatment

      The serious nature of chronic hepatitis D and the uniqueness of the delta agent make this disease a difficult target for antiviral therapy [
      • Rizzetto M.
      • Rosina F.
      • Hepatitis D.
      Hepatitis D virus: treatment.
      ]. To date, only alfa IFN was shown to be beneficial, while other antiviral agents, including acyclovir [
      • Berk L.
      • de Man R.A.
      • Housset C.
      • Berthelot P.
      • Schalm S.W.
      Alpha lymphoblastoid interferon and acyclovir for chronic hepatitis delta.
      ], ribavirin [
      • Garripoli A.
      • Di Marco V.
      • Cozzolongo R.
      • Costa C.
      • Smedile A.
      • Fabiano A.
      • et al.
      Ribavirin treatment for chronic hepatitis D: a pilot study.
      ] and famciclovir [
      • Yurdaydin C.
      • Bozkaya H.
      • Gurel S.
      • Tillmann H.L.
      • Aslan N.
      • Okcu-Heper A.
      • et al.
      Famciclovir treatment of chronic delta hepatitis.
      ], failed to show any efficacy in chronic hepatitis D. Similarly, lamivudine, a nucleoside analog that potently inhibits HBV replication, has shown no effects on HDV replication nor on the disease activity [
      • Lau D.T.
      • Doo E.
      • Park Y.
      • Kleiner D.E.
      • Schmid P.
      • Kuhns M.C.
      • et al.
      Lamivudine for chronic delta hepatitis.
      ]. Experience with combination therapy with lamivudine and IFN is still limited, although preliminary data are not promising [
      • Wolters L.M.
      • van Nunen A.B.
      • Honkoop P.
      • Vossen A.C.
      • Niesters H.G.
      • Zondervan P.E.
      • et al.
      Lamivudine-high dose interferon combination therapy for chronic hepatitis B patients co-infected with the hepatitis D virus.
      ].
      Pilot studies as well as randomized controlled trials have shown that IFN-alpha can induce an amelioration, albeit transient, of chronic hepatitis D. However, its efficacy is related to the dose and duration of therapy. When an initial dose of 5 megaunits (MU) per m2 was given thrice weekly for 4 months followed by 3 MU per m2 for 8 additional months, the dose reduction was associated in most patients with a relapse in ALT levels, which were normal at the end of treatment in 25% of patients, but in only one patient at the end of the 12 months of follow-up [
      • Rosina F.
      • Pintus C.
      • Meschievitz C.
      • Rizzetto M.
      A randomized controlled trial of a 12-month course of recombinant human interferon-alpha in chronic delta (type D) hepatitis: a multicenter Italian study.
      ]. Higher doses (9 MU three times weekly) given for up to 1 year induced ALT normalization in 71% of patients at the end of treatment and in about 50% after 6 months of follow-up [
      • Farci P.
      • Mandas A.
      • Coiana A.
      • Lai M.E.
      • Desmet V.
      • Van Eyken P.
      • et al.
      Treatment of chronic hepatitis D with interferon alfa-2a.
      ]. This biochemical response correlated with an improvement in liver histology, but not with a loss of HDV viremia, as measured by PCR. The results of several studies confirmed that IFN has no or little effect on HDV replication [
      • Rizzetto M.
      • Rosina F.
      • Hepatitis D.
      Hepatitis D virus: treatment.
      ]. Clearance of HDV RNA, which is associated with a progressive decline in IgM anti-HD titer, was documented in less than 10% of treated patients. Individuals who clear HDV RNA may ultimately lose HBsAg as well [
      • Lau J.Y.
      • King R.
      • Tibbs C.J.
      • Catterall A.P.
      • Smith H.M.
      • Portmann B.C.
      • et al.
      Loss of HBsAg with interferon-alpha therapy in chronic hepatitis D virus infection.
      ,
      • Battegay M.
      • Simpson L.H.
      • Hoofnagle J.H.
      • Sallie R.
      • Di Bisceglie A.M.
      Elimination of hepatitis delta virus infection after loss of hepatitis B surface antigen in patients with chronic delta hepatitis.
      ]. In chronic hepatitis D there are no reliable features that can predict which patients will have long-term benefits from the use of IFN, although patients with a short disease duration are the most likely to respond [
      • Marzano A.
      • Ottobrelli A.
      • Spezia C.
      • Daziano E.
      • Soranzo M.L.
      • Rizzetto M.
      Treatment of early chronic delta hepatitis with lymphoblastoid alpha interferon: a pilot study.
      ], underscoring the importance of early treatment. In patients treated with a high dose and for a prolonged time, side effects are common and therefore close monitoring is essential for detecting major psychiatric and medical complications [
      • Rizzetto M.
      • Rosina F.
      • Hepatitis D.
      Hepatitis D virus: treatment.
      ,
      • Hadziyannis S.J.
      Use of alpha-interferon in the treatment of chronic delta hepatitis.
      ,
      • Gaudin J.L.
      • Faure P.
      • Godinot H.
      • Gerard F.
      • Trepo C.
      The French experience of treatment of chronic type D hepatitis with a 12-month course of interferon alpha-2B. Results of a randomized controlled trial.
      ].
      The efficacy of IFN in chronic hepatitis D has been primarily evaluated upon evidence on short-term outcomes [
      • Rizzetto M.
      • Rosina F.
      • Hepatitis D.
      Hepatitis D virus: treatment.
      ]. Recently, however, a long-term prospective study (up to 14 years) of patients treated with high doses of IFN (9 MU) for 1 year has provided important informations on the long-term effects of IFN [
      • Farci P.
      • Chessa L.
      • Peddis G.
      • Strazzera R.
      • Pascariello E.
      • Orgiana G.
      • et al.
      Influence of alpha interferon (IFN) on the natural history of chronic hepatitis D: dissociation of histologic and virologic response.
      ]. Remarkably, half the patients who had a biochemical response at the end of treatment still had normal ALT values after 14 years of follow-up. But the most striking finding of this study was the complete regression of liver fibrosis documented in some patients with persistent biochemical response and loss of IgM anti-HD, all of whom had an initial diagnosis of active cirrhosis that was confirmed in two subsequent biopsies performed at the end of treatment and 1 year thereafter. Thus, these data have challenged the belief that advanced liver fibrosis is irreversible. The long-term clinical and histologic improvement correlated with a significant and sustained decrease in the levels of HDV replication, leading in some patients to the clearance of HDV RNA and, eventually, of HBV. In another study, eradication of both HBV and HDV, associated with a complete reversion of liver fibrosis, was achieved in a single patient with active HDV cirrhosis treated with continuous therapy, 5 MU of IFN daily for up to 12 years [
      • Lau D.T.
      • Kleiner D.E.
      • Park Y.
      • Di Bisceglie A.M.
      • Hoofnagle J.H.
      Resolution of chronic delta hepatitis after 12 years of interferon alfa therapy.
      ].
      In conclusion, IFN-alpha is currently the only effective treatment for chronic hepatitis D. High doses (9 MU thrice weekly or 5 MU daily) for at least 1 year are required for achieving both short- and long-term effects on the disease outcome. In responders, therapy should be continued as long as possible, until eradication of HDV RNA and eventually loss of HBsAg. An individualized regimen in which the dose is titered according to tolerance and serum ALT levels is recommended. Despite the encouraging results, however, many problems—mainly related to the low rate of response and the high rate of relapse—remain to be solved. These limitations emphasize the need for improving the efficacy of IFN treatment, as well as for identifying innovative approaches and new antiviral agents that may benefit patients with chronic hepatitis D.
      Orthotopic liver transplantation is the only valid therapeutic option for end-stage HDV-related liver disease. The risk of HDV reinfection of the graft has been found to be lower than that of HBV reinfection and can be prevented by the continuous administration of anti-HBs immunoglobulins [
      • Samuel D.
      • Zignego A.L.
      • Reynes M.
      • Feray C.
      • Arulnaden J.L.
      • David M.F.
      • et al.
      Long-term clinical and virological outcome after liver transplantation for cirrhosis caused by chronic delta hepatitis.
      ,
      • Ciancio A.
      • Ottobrelli A.
      • Marzano A.
      • Olivero A.
      • Cerenzia M.T.
      • Smedile A.
      • et al.
      A long-term virological follow up in patients treated with orthotopic liver transplantation (OLT) for hepatitis delta virus (HDV).
      ].

      10. Prevention

      Because of the critical contribution of HBV to the life cycle of HDV, prevention of HDV infection can be successfully achieved by vaccination against HBV. To date no effective vaccine specific for HDV has been developed [
      • Ponzetto A.
      • Forzani B.
      • Forzani I.
      • D'Urso N.
      • Avanzini L.
      • Smedile A.
      • et al.
      Immunization with hepatitis delta antigen does not prevent superinfection with hepatitis delta virus in the woodchuck.
      ,
      • Karayiannis P.
      • Saldanha J.
      • Monjardino J.
      • Jackson A.
      • Luther S.
      • Thomas H.C.
      Immunization of woodchucks with hepatitis delta antigen expressed by recombinant vaccinia and baculoviruses, controls HDV superinfection.
      ,
      • Fiedler M.
      • Lu M.
      • Siegel F.
      • Whipple J.
      • Roggendorf M.
      Immunization of woodchucks (Marmota monax) with hepatitis delta virus DNA vaccine.
      ,
      • Huang Y.H.
      • Wu J.C.
      • Tao M.H.
      • Syu W.J.
      • Hsu S.C.
      • Chi W.K.
      • et al.
      DNA-Based immunization produces Th1 immune responses to hepatitis delta virus in a mouse model.
      ], which would represent the only means to eliminate the risk of HDV superinfection for over 300 million chronic carriers of HBsAg in the world.

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