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ILAR Journal V42(2) 2001
Animal Models of Hepatitis
Delta
Animal Models of Hepatitis Delta Virus Infection and Disease
John L. Gerin
| John L. Gerin, Ph.D., is Director of the Division of Molecular Virology and Immunology at Georgetown University Medical Center, Rockville, Maryland. |
Abstract
Hepatitis delta virus (HDV) is a defective RNA virus with similarities to unusual subviral pathogens of higher plants. It requires hepatitis B virus (HBV) for its replication/transmission, and HBV-infected humans are the only established host. HDV causes both severe acute hepatitis and rapidly progressive chronic disease in some individuals. The HDV life cycle involves remarkable features, such as ribozyme- mediated autocatalytic processes, Pol II-directed RNA synthesis from a single-stranded circular RNA template, and RNA editing. Much of our understanding of the nature of this pathogen derives from experimental studies in the chimpanzee model of HBV infection. The hepadnavirus-infected eastern woodchuck also is capable of supporting HDV replication and offers opportunities for the development of control strategies that might be applicable to human type D hepatitis.
Key Words: chimpanzee model; eastern woodchuck; hepatitis B surface antigens; hepatitis delta antigen; hepatitis delta virus; ribozyme; RNA editing; woodchuck hepatitis B virus
Introduction
The collection of viruses that comprise the viral hepatitides is taxonomically diverse, and each represents the prototype member of either a new genus or family of animal viruses. Even with this diversity, the agent known as the hepatitis delta virus (HDV1) is remarkable for its unique biological properties (Gerin et al. 2001; Modahl and Lai 2000). The discovery of this virus was due to the keen observations of an Italian gastroenterologist (Rizzetto et al.1977). The characterization of HDV, definition of its biological features, and role in chronic liver disease were gradually developed through a series of experimental studies in which animal models, especially the chimpanzee model of hepatitis B virus (HBV1) infection, played an essential part.
HDV: A Novel Animal Virus
HDV is approximately 36 nm in diameter and consists of a circular RNA genome and a nucleocapsid, hepatitis delta antigen (HDAg1), enveloped by a lipoprotein coat that consists of surface antigens (HBsAg1) contributed by a helper HBV. The HBsAg envelope protects the viral genome from the harsh extracellular environment and probably dictates the hepatocyte tropism. This dependence on HBV means that the HDV is defective and incapable of fully autonomous replication/transmission. The HDV genome (1.7 kb) is the smallest of the known human pathogens, and this limited coding capacity is reflected in the simple composition of the virus particle. The single viral gene product is encoded by the antigenomic RNA strand, thus HDV is a negative-stranded RNA virus. Its circular RNA genome and mode of replication resemble unusual subviral pathogens of higher plants; HDV is taxonomically the sole member of the Deltavirus genus (van Regenmortel et al. 2000).
The RNA genome contains several novel features that are essential for replication and distinguish HDV as a fascinating model for the study of RNA biology. Unique in animal virology, both the RNA genome and antigenome of HDV contain closely related RNA structural elements, termed ribozymes, which cleave the RNA at specific sites (Kuo et al.1988; Wu and Lai 1989). The ribozymes are essential to the double rolling circle mechanism of HDV replication (Branch and Robertson 1984) in which replication of genomic and antigenomic RNA from a circular template produces linear multimeric RNAs that are processed by autocatalytic cleavage at the ribozyme sites to yield linear RNAs of unit length. Unit length RNAs are then ligated to form circles, a step that may involve host enzymes (Reid and Lazinski 2000). Unlike other negative-stranded viruses, HDV does not encode a polymerase, and there is no known RNA-dependent RNA polymerase in humans, the only known natural host. Probably due to extensive intramolcular base-pairing in the HDV RNA (Kos et al. 1986; Wang et al. 1986), host DNA-dependent RNA polymerase, Pol II, may be recruited to recognize the viral RNA template (Gudima et al. 2000; Lo et al.1998; McNaughton et al. 1991; Modahl et al. 2000). Lastly, the viral nucleoprotein complex consists of two HDAg forms, a small protein (HDAg-S1) and a large protein (HDAg-L1). Both gene products are generated from the single open reading frame on the antigenomic RNA, and each has a different functional role in the virus life cycle. HDAg-S is required for HDV RNA replication, and HDAg-L, although it inhibits RNA synthesis, is necessary for virion formation. The virus manages the conflicting functions of these two proteins by the use of a host activity, known as RNA editing. During RNA replication, a proportion of antigenomic transcripts is edited by cellular double-stranded RNA adenosine deaminase (Casey and Gerin 1995; Polson et al. 1996, 1998) to yield, in the next round of replication, an mRNA in which the HDAg-S stop codon has been changed to a tryptophan codon. This process allows synthesis of the HDAg-L product, which is 19 amino acids longer than the HDAg-S form. As larger amounts of HDAg-L are produced, the mode of replication switches from RNA synthesis (promoted by HDAg-S) to virion packaging (promoted by HDAg-L).
Animal Models of Experimental Infection and Disease
Although hepatocyte cultures have been infected with HDV in vitro,replication is limited to single cycles involving a small proportion of cells. Cotransfection experiments with HDV and hepadnavirus cDNAs (Chang et al. 1991; Ryu et al. 1992; Sureau et al. 1992, 1993; Wu et al. 1991) have reproduced the complete replication cycle to include assembly and release and have greatly contributed to our current understanding of the molecular biology of this virus. Much of our knowledge of the natural history of HDV infection and disease, however, derives from studies in animal models. Due to the absolute dependence of HDV on HBV, the host range of HDV is limited to those species that support the replication of an hepadnavirus that is capable of providing the essential helper function, the hepatitis B surface antigen envelope. The relevant animal models in this regard are the chimpanzee and eastern woodchuck, natural hosts of the human HBV and woodchuck HBV (WHV1), respectively. Other possible animal models are discussed elsewhere in this volume (Tennant and Gerin 2001). These models either have not been evaluated for the ability to support HDV replication or have not done so in a sustainable fashion.
Chimpanzee
As described elsewhere in this volume, the chimpanzee has been an invaluable model of hepatitis B virus infection (Prince and Brotman 2001). This model was critical to progress in our understanding of HDV. When Rizzetto and colleagues first described delta antigen in the hepatocytes of patients with chronic type B hepatitis (Rizzetto et al.1977), it was considered to be a previously unrecognized HBV-specific antigen. Confirmation that it was a marker of a separate transmissible pathogen and elucidation of its unique properties came as a result of experimental infection of HBV-infected chimpanzees (Rizzetto et al.1980a,b). These studies revealed the unique nature of this agent and began to define the natural history of human infection and disease.
Biophysical and Biochemical Characterization of HDV
Experimental transmission of HDV in chimpanzees has permitted the biological amplification of HDV for both analytical and diagnostic purposes. HDV is difficult to study in human infection because the period of peak viremia in acute infection is relatively short and the levels of viremia in chronic infection are highly variable. In chimpanzee studies, frequent plasmaphereses taken during peak viremia provided large amounts of source material for the biophysical and biochemical characterization of HDV (Bonino et al. 1981, 1984; Rizzetto et al. 1980b). HDAg was shown to be an internal component of a virus-like particle that contained a small RNA (approximately 1.7 kb), and subsequent cloning of the genome and determination of its genomic organization and structural features were accomplished with material derived from the experimental infections (Denniston et al. 1986; Kos et al. 1986; Wang et al. 1986). Its size was determined by filtration using infectivity in chimpanzees as an endpoint (He et al. 1989). Source material from experimental infections permitted the development of serological and hybridization-based assays for the detection of HDV RNA, HDAg, and anti-HD in clinical samples (Bergmann and Gerin 1986; Denniston et al. 1986; Pohl et al. 1987; Rizzetto et al. 1980c).
Natural History of Infection and Disease
Chimpanzee studies demonstrated that HDV was defective and had an absolute requirement for HBV for its replication/transmission. Both coinfection, the simultaneous infection of a susceptible individual with HBV and HDV, and superinfection, HDV infection of an HBsAg-positive individual, have been documented (Purcell et al. 1987). Coinfection results in moderately severe hepatitis consisting of unimodal or bimodal elevation of serum enzyme markers of liver damage, alanine aminotransferase activity. In unimodal disease, both hepatitis B core antigen and HDAg are expressed in the infected hepatocytes at the same time, whereas in bimodal hepatitis, one or the other is expressed in the second episode. This order probably represents the relative concentrations of HBV and HDV in the source material. The coinfection pattern in chimpanzees results in a transient immunoglobulin M anti-HD response and a longer-lived immunoglobulin G anti-HD response. Superinfections result in more severe disease, and markers of HBV are suppressed during the acute HDV phase. More than 50% of superinfected chimpanzees develop markers of HDV persistence, although at low levels (Negro et al. 1988). Serial passage of HDV in the chimpanzee resulted in increasingly more severe acute hepatitis, remarkably more severe than that observed with other types of viral hepatitis in this model (Ponzetto et al. 1988). The endpoint titration of a standard inoculum in the passage series demonstrated the extremely high concentrations of HDV that occur in the acute phase: HDV had an infectious endpoint of 1011 infectious doses/mL of serum in contrast to an HBV endpoint of 106 infectious doses/mL (Ponzetto et al. 1987a). Although many features of human HDV infection are reproduced by experimental studies, chimpanzees usually do not develop the more progressive disease pattern that is generally seen in HDV worldwide. Possible explanations are that most HBV carrier animals used in the studies were hepatitis B e antigen negative at the outset, or the disease progression reflects the HDV-host interactions unique to the genotype of the standard inoculum, genotype I. Recent studies have revealed that most of the HBV carrier chimpanzees used in the HDV transmissions were infected with a chimpanzee genotype HBV (Hu et al. 2000), but it is not know whether HBV and HDV genotype interactions can influence the disease course. It does appear that the HDV genotype might influence disease severity in some settings (Casey and Gerin 1998; Casey et al. 1993).
Blood Product Safety
Many of the statements made by Prince and Brotman regarding the use of chimpanzees to assess the safety of blood products apply to the HDV setting, except that HBV-infected animals must be used due to the absolute requirement of HDV for the HBV envelope. Accordingly, the HBV-infected chimpanzee has been used to ensure the safety from HDV of HBsAg vaccines prepared from HBsAg-carrier plasma and the inactivation of clotting factor concentrates, which might be prepared from source material positive for HDV markers (Ponzetto et al.1986).
Eastern Woodchuck
The eastern woodchuck has been a valuable naturally occurring animal model of hepadnavirus infection and disease (Tennant and Gerin 2001) and has been used extensively in drug development for treatment of chronic type B hepatitis. Although there is no indication that HDV infection occurs in natural WHV infection of woodchucks, Ponzetto and colleagues were able to transmit HDV of human origin to WHV-carrier woodchucks, which caused acute or chronic hepatitis in a high proportion of animals (Ponzetto et al. 1984, 1987a). As in chimpanzees, the liver is the only organ involved despite WHV replication in extrahepatic tissues (Negro et al. 1989). HDV replication in woodhcucks depends on the presence of replicating WHV, and the HBsAg envelope of the source HDV is replaced by the underlying WHsAg envelope of the host animal (Ponzetto et al. 1984). There is evidence in this model that HDV might have the capacity to be latent for a short period of time and be expressed if the helper hepadnavirus arrives subsequently (Netter et al. 1994). A similar observation was made in the chimpanzee experimental model (Smedile et al. 1998), but whether these findings have epidemiological significance to the human situation remains unclear.
HDV has been passaged sequentially in woodchucks, yet still retains the capacity to infect chimpanzees with undimished virulence (Ponzetto et al. 1988). Because HDV prophylactic options for the large worldwide population of HBV carriers are limited, the WHV/woodchuck model has been used for the development of immunization strategies. There is some evidence that immunogens based on the HDAg sequence may modulate the course of experimental infection, although more work on that subject is needed (Gerin et al. 1994; Karayiannis et al. 1990, 1993). The model has not been widely applied to drug development of chronic HDV disease due to the rapid progression to endstage disease in the experimentally derived WHV carriers, which makes it difficult to develop HDV-carriers in a suitable time frame for experimental manipulation before loss to WHV-induced hepatocellular carcinoma. Current research efforts use WHV strains with disease progressions that are remarkably slower than the standard WHV inoculum and the development of standard infectious HDV inocula derived from transfections with cloned genotype-specific molecules (Casey and Gerin, unpublished data).
Certainly it is hoped that worldwide efforts for universal HBV immunization will eventually remove the need for specific control of this serious pathogen. It is first necessary to learn whether HDV is unique or, alternatively, whether other subviral human pathogens exist, possibly with relationships dependent on common human viruses. If we learn that such conditions exist, then animal models will surely provide insight into the potential for human disease and represent an important medical resource.
1Abbreviations us ed in this article: HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; HDAg, hepatitis delta antigen; HDAg-L, hepatitis delta antigen large protein; HDAg-S, hepatitis delta antigen small protein; HDV, hepatitis delta virus; WHV, woodchuck hepatitis B virus.
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