Hepatitis Delta Virus Biology Research Articles

Highlights from the 2023 International Meeting on the Molecular Biology of Hepatitis B virus.

Since its discovery in 1965, our understanding of the hepatitis B virus (HBV) replication cycle and host immune responses has increased markedly. In contrast, our knowledge of the molecular biology of hepatitis delta virus (HDV), which is associated with more severe liver disease, is less well understood. Despite the progress made, critical gaps remain in our knowledge of HBV and HDV replication and the mechanisms underlying viral persistence and evasion of host immunity. The International HBV Meeting is the leading annual scientific meeting for presenting the latest advances in HBV and HDV molecular virology, immunology, and epidemiology. In 2023, the annual scientific meeting was held in Kobe, Japan and this review summarises some of the advances presented at the Meeting and lists gaps in our knowledge that may facilitate the development of new therapies.

Deciphering the Role of Post-Translational Modifications and Cellular Location of Hepatitis Delta Virus (HDV) Antigens in HDV-Mediated Liver Damage in Mice.

Hepatitis D virus (HDV) infection represents the most severe form of chronic viral hepatitis. We have shown that the delivery of HDV replication-competent genomes to the hepatocytes using adeno-associated virus (AAV-HDV) as gene delivery vehicles offers a unique platform to investigate the molecular aspects of HDV and associated liver damage. For the purpose of this study, we generated HDV genomes modified by site-directed mutagenesis aimed to (i) prevent some post-translational modifications of HDV antigens (HDAgs) such as large-HDAg (L-HDAg) isoprenylation or short-HDAg (S-HDAg) phosphorylation; (ii) alter the localization of HDAgs within the subcellular compartments; and (iii) inhibit the right conformation of the delta ribozyme. First, the different HDV mutants were tested in vitro using plasmid-transfected Huh-7 cells and then in vivo in C57BL/6 mice using AAV vectors. We found that Ser177 phosphorylation and ribozymal activity are essential for HDV replication and HDAg expression. Mutations of the isoprenylation domain prevented the formation of infectious particles and increased cellular toxicity and liver damage. Furthermore, altering HDAg intracellular localization notably decreased viral replication, though liver damage remained unchanged versus normal HDAg distribution. In addition, a mutation in the nuclear export signal impaired the formation of infectious viral particles. These findings contribute valuable insights into the intricate mechanisms of HDV biology and have implications for therapeutic considerations.

Review article: emerging insights into the immunopathology, clinical and therapeutic aspects of hepatitis delta virus.

SummaryBackgroundHepatitis delta virus (HDV), which causes the most severe form of viral hepatitis, is an obligated hepatitis B (HBV) satellite virus that can either infect naïve subjects simultaneously with HBV (co‐infection), or chronically infect HBV carriers (super‐infection). An estimated 12 million people are infected by HDV worldwide.AimsTo summarise the most relevant aspects of the molecular biology of HDV, and to discuss the latest understanding of the induced pathology, interactions with the immune system, as well as both approved and investigational treatment options.MethodsReferences for this review were identified through searches of PubMed with the terms “HDV” “viral hepatitis” “co‐infection” and “super‐infection,” published between 1980 and October 2021ResultsThe limited access to the HDV‐infected liver has hampered the investigation of the intrahepatic compartment and our understanding of the mechanisms of HDV pathogenesis. In the absence of standardised and sensitive diagnostic tools, HDV is often underdiagnosed and owing to its strong dependence on host cellular factors, the development of direct antiviral agents has been challenging. New therapeutic agents targeting different steps of the viral cycle have recently been investigated, among which bulevirtide (which was conditionally approved by EMA in July 2020) and lonafarnib; both drugs having received orphan drug designation from both the EMA and FDA.ConclusionsThe HBV cure programme potentially offers a unique opportunity to enhance HDV treatment strategies. In addition, a more comprehensive analysis of the intrahepatic compartment is mandated to better understand any liver‐confined interaction of HDV with the host immune system.

AAV-HDV: An Attractive Platform for the In Vivo Study of HDV Biology and the Mechanism of Disease Pathogenesis.

Hepatitis delta virus (HDV) infection causes the most severe form of viral hepatitis, but little is known about the molecular mechanisms involved. We have recently developed an HDV mouse model based on the delivery of HDV replication-competent genomes using adeno-associated vectors (AAV), which developed a liver pathology very similar to the human disease and allowed us to perform mechanistic studies. We have generated different AAV-HDV mutants to eliminate the expression of HDV antigens (HDAgs), and we have characterized them both in vitro and in vivo. We confirmed that S-HDAg is essential for HDV replication and cannot be replaced by L-HDAg or host cellular proteins, and that L-HDAg is essential to produce the HDV infectious particle and inhibits its replication. We have also found that lack of L-HDAg resulted in the increase of S-HDAg expression levels and the exacerbation of liver damage, which was associated with an increment in liver inflammation but did not require T cells. Interestingly, early expression of L-HDAg significantly ameliorated the liver damage induced by the mutant expressing only S-HDAg. In summary, the use of AAV-HDV represents a very attractive platform to interrogate in vivo the role of viral components in the HDV life cycle and to better understand the mechanism of HDV-induced liver pathology.

COINFECTION BY HEPATITIS B AND HEPATITIS DELTA VIRUSES: MOLECULAR BIOLOGY OF PATHOGENS AND PROSPECTS OF PHARMACOTHERAPY

Hepatitis delta virus (HDV) is a defective virus and obligate companion of hepatitis B virus (HBV). The clinical significance of HDV and HBV coinfection is associated with more severe forms of acute or chronic viral hepatitis, a higher risk of developing liver cirrhosis and hepatocellular carcinoma compared to BGV-monoinfection. Current knowledge of the molecular biology of pathogens confirms the existence of a very complex relationship between the HBV, HDV, infected hepatocytes and cells of the immune system. Despite the fact that HBV viremia can now be controlled by using nucleoside analogues in the majority of patients, the problem of treating HDV and HBV coinfection is far from being resolved. Fundamental studies in recent years have led to significant progress in understanding the biology of HDV and provide the basis for the development and introduction into clinical practice highly effective antiviral drugs.

A new HDV mouse model identifies mitochondrial antiviral signaling protein (MAVS) as a key player in IFN-β induction

Studying hepatitis delta virus (HDV) and developing new treatments is hampered by the limited availability of small animal models. Herein, a description of a robust mouse model of HDV infection that mimics several important characteristics of the human disease is presented. HDV and hepatitis B virus (HBV) replication competent genomes were delivered to the mouse liver using adeno-associated viruses (AAV; AAV-HDV and AAV-HBV). Viral load, antigen expression and genomes were quantified at different time points after AAV injection. Furthermore, liver pathology, genome editing, and the activation of the innate immune response were evaluated. AAV-HDV infection initiated HDV replication in mouse hepatocytes. Genome editing was confirmed by the presence of small and large HDV antigens and sequencing. Viral replication was detected for 45days, even after the AAV-HDV vector had almost disappeared. In the presence of HBV, HDV infectious particles were detected in serum. Furthermore, as observed in patients, co-infection was associated with the reduction of HBV antigen expression and the onset of liver damage that included the alteration of genes involved in the development of liver pathologies. HDV replication induced a sustained type I interferon response, which was significantly reduced in immunodeficient mice and almost absent in mitochondrial antiviral signaling protein (MAVS)-deficient mice. The animal model described here reproduces important characteristics of human HDV infection and provides a valuable tool for characterizing the viral infection and for developing new treatments. Furthermore, MAVS was identified as a main player in HDV detection and adaptive immunity was found to be involved in the amplification of the innate immune response. Lay summary: Co-infection with hepatitis B and D virus (HBV and HDV, respectively) often causes a more severe disease condition than HBV alone. Gaining more insight into HDV and developing new treatments is hampered by limited availability of adequate immune competent small animal models and new ones are needed. Here, a mouse model of HDV infection is described, which mimics several important characteristics of the human disease, such as the initiation and maintenance of replication in murine hepatocytes, genome editing and, in the presence of HBV, generation of infectious particles. Lastly, the involvement of an adaptive immunity and the intracellular signaling molecule MAVS in mounting a strong and lasting innate response was shown. Thus, our model serves as a useful tool for the investigation of HDV biology and new treatments.

Hepatitis Delta Virus: A Peculiar Virus

The hepatitis delta virus (HDV) is distributed worldwide and related to the most severe form of viral hepatitis. HDV is a satellite RNA virus dependent on hepatitis B surface antigens to assemble its envelope and thus form new virions and propagate infection. HDV has a small 1.7 Kb genome making it the smallest known human virus. This deceivingly simple virus has unique biological features and many aspects of its life cycle remain elusive. The present review endeavors to gather the available information on HDV epidemiology and clinical features as well as HDV biology.

Interaction of Host Cellular Proteins with Components of the Hepatitis Delta Virus

The hepatitis delta virus (HDV) is the smallest known RNA pathogen capable of propagation in the human host and causes substantial global morbidity and mortality. Due to its small size and limited protein coding capacity, HDV is exquisitely reliant upon host cellular proteins to facilitate its transcription and replication. Remarkably, HDV does not encode an RNA-dependent RNA polymerase which is traditionally required to catalyze RNA-templated RNA synthesis. Furthermore, HDV lacks enzymes responsible for post-transcriptional and -translational modification, processes which are integral to the HDV life cycle. This review summarizes the known HDV-interacting proteins and discusses their significance in HDV biology.

The hepatitis delta virus RNA genome interacts with eEF1A1, p54nrb, hnRNP-L, GAPDH and ASF/SF2

Because of its extremely limited coding capacity, the hepatitis delta virus (HDV) takes over cellular machineries for its replication and propagation. Despite the functional importance of host factors in both HDV biology and pathogenicity, little is known about proteins that associate with its RNA genome. Here, we report the identification of several host proteins interacting with an RNA corresponding to the right terminal stem-loop domain of HDV genomic RNA, using mass spectrometry on a UV crosslinked ribonucleoprotein complex, RNA affinity chromatography, and screening of a library of purified RNA-binding proteins. Co-immunoprecipitation was used to confirm the interactions of eEF1A1, p54(nrb), hnRNP-L, GAPDH and ASF/SF2 with the right terminal stem-loop domain of HDV genomic RNA in vitro, and with both polarities of HDV RNA within HeLa cells. Our discovery that HDV RNA associates with RNA-processing pathways and translation machinery during its replication provides new insights into HDV biology and its pathogenicity.

The hepatitis delta virus RNA genome interacts with the human RNA polymerases I and III

The hepatitis delta virus (HDV) relies on human transcriptional machinery for its replication and transcription. Although the involvement of RNA polymerase II in HDV RNA biosynthesis is established, the contribution of additional polymerases remains uncertain. Here, we demonstrate the interaction of both RNA polymerase I and III with HDV RNA, both in vitro and in human cells. Binding of these polymerases occurs near the terminal stem-loop domains of both polarities of the HDV RNA genome. Based on interactions of HDV RNA with numerous host polymerases, our results suggest a higher level of complexity of HDV biology than previously envisioned.

Proteome analysis of a human liver carcinoma cell line stably expressing hepatitis delta virus ribonucleoproteins

Hepatitis delta virus (HDV) infects human hepatocytes already infected with the hepatitis B virus increasing about ten fold the risk of cirrhosis and fulminant hepatitis. The lack of an appropriate cell culture system capable of supporting virus replication has so far impaired the detailed investigation of the HDV biology including the identification of host factors involved in pathogenesis. Here, we made use of a HDV cDNA stably transfected cell line, Huh7-D12, in a proteomic approach to identify the changes in the protein expression profiles in human liver cells that arise as a consequence of HDV replication. Total protein extracts from Huh7-D12 cells and of the corresponding non transfected human liver carcinoma cell line, Huh7, were separated by 2-DE. Differentially expressed spots were identified by MALDI-TOF followed by database searching. We identified 23 differentially expressed proteins of which 15 were down regulated and 8 up regulated in Huh7-D12 cells. These proteins were found to be involved in different cellular pathways. The down regulation of the histone H1-binding protein and of triosephosphate isomerase was confirmed by Real time PCR, and the up regulation of the La protein and lamin A/C was validated by western blot.

Developments In Hepatitis Delta Research

The hepatitis delta virus (HDV) is the smallest human pathogen known so far. Several characteristics of the virus RNA genome resemble that of plant viroids. Since the hepatitis B virus surface antigens are part of the HDV envelope and are necessary for productive infection, HDV may be considered a satellite virus of HBV. Coinfection of the two viruses or superinfection with HDV in HBV carriers increases the risk of fulminant hepatitis and development of liver cirrhosis. At present, there is no specific treatment for hepatitis D. However, vaccination against HBV confers protection against coinfection with HDV. Although the increased rate of vaccination against HBV in developed countries reduced the prevalence of HDV, it is still a threat and remains endemic in many regions of the world. Here, we overview the epidemiology and treatment of hepatitis delta and report recent advances in the research of HDV biology. CLINICAL AND EPIDEMIOLOGICAL FEATURES The hepatitis delta virus (HDV) was discovered by the italian gastroenterologist Mario Rizzetto while studying liver biopsies of hepatitis B virus (HBV) infected patients (Rizzetto et al., 1977). Later, it was shown that HDV infects individuals previously infected with HBV, causing more severe hepatic lesions, and increasing the risk of fulminat hepatitis (Gorinvadarajan et al., 1984; Jacobson et al., 1985). Since the presence of HBV is necessary for the production of infectious HDV particles capable of propagating the infection, the HDV may be considered a HBV satellite virus (Rizzetto et al., 1980; Ponzetto et al, 1988). The clinical association between these two viruses is due to the fact that the genome of HDV does not encode for its own envelope proteins. The HDV envelope consists of HBV surface proteins (HBsAg; Smedile et al., 1994), and as a consequence HDV transmission occurs only in the presence of HBV. HDV replication seems to occur only in the liver, and all pathological abnormalities are limited to this organ. The lesions are similar to those observed during the course of other acute and chronic viral hepatitis. Often they consist of hepatocellular necrosis and inflamation. From the histological point of view, there are no significant differences between lesions caused by HDV and those caused by other hepatitis viruses. The clinical course of HDV infection may be variable. In general, the observed symptoms are more severe than those associated with other hepatitis viruses. The incubation period varies between 3 to 7 weeks. Following this period, the first symptoms can be detected, including fatigue, lethargy, and nausea. It is estimated that 60% to 70% of all hepatitis delta chronic patients will develop chirrosis. This percentage is about three times higher when compared to the percentage that is observed in hepatitis B patients (Rizzetto et al., 1983). The frequency of HDV associated fulminant hepatitis, the more severe form of the acute disease, is 10 times higher than the observed for other viral hepatitis. Fulminant hepatitis is usually associated with hepatic encephalopaties that, in the most severe cases are characterized by somnolence, abnormal behavior, and coma. The mortality associated with fulminant hepatitis is about 80%, independent of the treatment (Purcell and Gerin, 1990). Seroprevalence studies in individuals positive for HBsAg, show a non-uniform worldwide distribution of HDV (Ponzetto et al., 1985; see fig. 1). Developments In Hepatitis Delta Research

Hepatitis Delta Virus: The Molecular Basis of Laboratory Diagnosis

Infection with hepatitis delta virus (HDV), a satellite virus of hepatitis B virus (HBV), is associated with severe and sometimes fulminant hepatitis. The traditional methods for the diagnosis of HDV infection, such as detection of serum anti-HD antibodies, are sufficient for the clinical diagnosis of delta infection. However, such techniques lack the sensitivity and specificity required to more accurately characterize the nature of HDV infection and to assess the efficacy of therapies. Recent improvements in molecular techniques, such as HDV RNA hybridization and RT-PCR, have provided increased diagnostic precision and a more thorough understanding of the natural course of HDV infection. These advances have enhanced the clinician's ability to accurately evaluate the stage of HDV infection, response to therapy, and occurrence of reinfection after orthotopic liver transplant. This review focuses on the recent advances in the understanding of the molecular biology of HDV and in the laboratory diagnosis of HDV infection.

Review: hepatitis delta.

The causative agent of hepatitis delta virus (HDV) is the most unusual of all causative agents for all hepatitis viruses. Current knowledge of the molecular biology of HDV strengthens its proposed classification within the satellites, a family of subviral agents, some of which are pathogens of higher plants. Hepatitis delta virus is the only virus in the satellite family known to infect animal species, with hepatitis D having affected more than 10 million people worldwide who are also infected with its helper virus, HBV. Recently, the world map for hepatitis D appears somewhat modified, with decreasing HDV prevalence in certain areas and some new foci of HDV endemicity. Despite changing HDV prevalence, hepatitis. D, particularly the chronic form, is still an important health problem worldwide in terms of morbidity and mortality (mainly due to chronic liver disease, including hepatocellular carcinoma). Molecular studies have greatly advanced our understanding of the life cycle of HDV and of the function of its proteins. The new molecular information is of clinical relevance, with implications for the pathogenesis of liver damage, the diagnosis of HDV infection, for the natural course of the disease and, potentially, for therapy. Sensitive assays for HDV-RNA by polymerase chain reaction and sequencing techniques have clarified the patterns of HDV transmission and have confirmed the existence of unusual clinical forms of the virus and their relationship to replicative levels and genotypes of HDV. Prevention and treatment of hepatitis D are still in their infancy. However, liver transplantation for delta cirrhosis has proven far more successful than in any other viral form of cirrhosis, with few reinfections of the grafted liver, and has given important information on HDV biology and the pathogenesis of liver damage.

The molecular biology of hepatitis delta virus.

Hepatitis delta virus (HDV) contains a circular, viroid-like RNA genome, the only animal viral RNA of its kind. It possesses a ribozyme activity, which can autocatalytically cleave and ligate itself. The ribozyme has a unique structural requirement different from other known ribozymes. HDV RNA undergoes RNA-dependent RNA replication via a double rolling circle mechanism, which is probably mediated by cellular RNA polymerase II, utilizing modified cellular transcription machineries. HDV RNA encodes a single protein, hepatitis delta antigen, which is a nuclear, RNA-binding phosphoprotein and required for viral RNA replication. During replication, HDV RNA undergoes a specific RNA editing event to extend its open reading frame and produce a longer, isoprenylated delta antigen, which suppresses RNA replication and initiates viral particle assembly. Ribozyme, cell-mediated RNA-dependent RNA replication, and RNA editing are some of the unique properties and unresolved issues of the molecular biology of HDV.

Application of nucleic acid hybridization to the hepatitis delta virus research.

Cloning and sequencing of the genome of hepatitis delta virus (HDV) represented a remarkable progress in the knowledge of HDV infection and made available cDNA and RNA probes which can be successfully used to identify HDV-RNA in biological specimens. We review here the methods currently used for detecting HDV nucleic acids and report the most recent advances of the molecular biology of HDV. Finally, we examine the number of compelling reasons for the diagnostic laboratory to pursue this technology. Testing for HDV-RNA has become essential for the screening and the follow-up of both chronic HDV carriers, undergoing antiviral therapy and patients with terminal disease, eligible for liver transplant.