Large Hepatitis Delta Antigen Research Articles

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.

Sequence diversity of hepatitis D virus in Mongolia.

The Hepatitis Delta Virus (HDV) is a defective, single-stranded RNA virusoid encoding for a single protein, the Hepatitis Delta Antigen (HDAg), which requires the hepatitis B virus (HBV) envelope protein (HBsAg) for its transmission. Currently, hepatitis D is the most aggressive form of viral hepatitis and treatment options are limited. Worldwide 12 million people are chronically infected with HDV being at high risk for progression to cirrhosis and development of liver cancer. Although it is well established that Mongolia is the country with the highest prevalence of HDV infections, the information on the molecular epidemiology and factors contributing to HDV sequence diversity are largely unclear. The aim of the study was to characterize the sequence diversity of HDV in rural areas from Mongolia and to determine the extent of HLA class I-associated selection pressure. From the HepMongolia cohort from rural areas in Mongolia, 451 HBsAg-positive individuals were selected and anti-HDV, HDV-RNA and the sequence of the large HDAg was determined. For all individuals the HLA class I locus was genotyped. Residues under selection pressure in the presence of individual HLA class I types were identified with the recently published analysis tool HAMdetector. Of 431 HBsAg positive patients, 281 were anti-HDV positive (65%), and HDV-RNA could be detected in 207 of 281 (74%) of patients. The complete large HDAg was successfully sequenced from 131 samples. Phylogenetic analysis revealed that all Mongolian HDV isolates belong to genotype 1, however, they separate into several different clusters without clear regional association. In turn, from phylogeny there is strong evidence for recent local transmission events. Importantly, we found multiple residues with strong support for HLA class I-associated selection pressure consistent with a functional CD8+ T cell response directed against HDV. HDV isolates from Mongolia are highly diverse. The molecular epidemiology suggests circulation of multiple subtypes and provides evidence for ongoing recent transmissions.

Identification of a novel interaction site between the large hepatitis delta antigen and clathrin that regulates the assembly of genotype III hepatitis delta virus

BackgroundHepatitis delta virus (HDV), a satellite virus of hepatitis B virus (HBV), is a small, defective RNA virus strongly associated with the most severe form of hepatitis and progressive chronic liver disease and cirrhosis. Chronic hepatitis D, resulting from HBV/HDV coinfection, is considered to be the most severe form of viral hepatitis and affects 12–20 million people worldwide. Involved in the endocytosis and exocytosis of cellular and viral proteins, clathrin contributes to the pathogenesis and morphogenesis of HDV. Previously, we demonstrated that HDV-I and -II large hepatitis delta antigens (HDAg-L) possess a putative clathrin box that interacts with clathrin heavy chain (CHC) and supports HDV assembly.MethodsVirus assembly and vesicular trafficking of HDV virus-like particles (VLPs) were evaluated in Huh7 cells expressing HDV-I, -II and -III HDAg-L and hepatitis B surface antigen (HBsAg). To elucidate the interaction motif between HDAg-L and CHC, site-directed mutagenesis was performed to introduce mutations into HDAg-L and CHC and analyzed using coimmunoprecipitation or pull-down assays.ResultsComparable to HDV-I virus-like particles (VLPs), HDV-III VLPs were produced at a similar level and secreted into the medium via clathrin-mediated post-Golgi vesicular trafficking. Mutation at F27 or E33 of CHC abolished the binding of CHC to the C-terminus of HDV-III HDAg-L. Mutation at W207 of HDV-III HDAg-L inhibited its association with CHC and interfered with HDV-III VLP formation. We elucidated mechanism of the binding of HDV-III HDAg-L to CHC and confirmed the pivotal role of clathrin binding in the assembly of genotype III HDV.ConclusionsA novel W box which was identified at the C terminus of HDV-III HDAg-L is known to differ from the conventional clathrin box but also interacts with CHC. The novel W box of HDAg-L constitutes a new molecular target for anti-HDV-III therapeutics.

1H, 15N and 13C backbone and side chain solution NMR assignments of the truncated small hepatitis delta antigen Δ60-S-HDAg.

Hepatitis D virus (HDV) is a defective virus that relies on hepatitis B virus envelope proteins to complete its replication cycle. The HDV genome contains two isoforms of hepatitis delta antigen: the small and the large hepatitis delta antigens (S- and L-HDAg). Here we report the 1H, 13C and 15N backbone and side chain resonance assignments of an N-terminally truncated form of S-HDAg (SΔ60), which lacks the 1-60 oligomerization domain. We derived secondary structures based on NMR chemical shifts, which will be used in further structural and functional studies. We show that SΔ60 is partially disordered, and that the central structured part contains two well-defined α-helices of 22 and 17 residues, respectively. A temperature titration allowed to identify the residues involved in hydrogen bonds.

A Rapid Point-of-Care Test for the Serodiagnosis of Hepatitis Delta Virus Infection

Hepatitis Delta virus (HDV) is a satellite of the Hepatitis B virus (HBV) and causes severe liver disease. The estimated prevalence of 15–20 million infected people worldwide may be underestimated as international diagnostic guidelines are not routinely followed. Possible reasons for this include the limited awareness among healthcare providers, the requirement for costly equipment and specialized training, and a lack of access to reliable tests in regions with poor medical infrastructure. In this study, we developed an HDV rapid test for the detection of antibodies against the hepatitis delta antigen (anti-HDV) in serum and plasma. The test is based on a novel recombinant large hepatitis delta antigen that can detect anti-HDV in a concentration-dependent manner with pan-genotypic activity across all known HDV genotypes. We evaluated the performance of this test on a cohort of 474 patient samples and found that it has a sensitivity of 94.6% (314/332) and a specificity of 100% (142/142) when compared to a diagnostic gold-standard ELISA. It also works robustly for a broad range of anti-HDV titers. We anticipate this novel HDV rapid test to be an important tool for epidemiological studies and clinical diagnostics, especially in regions that currently lack access to reliable HDV testing.

Comprehensive Analysis of Hepatitis Delta Virus Assembly Determinants According to Genotypes: Lessons From a Study of 526 Hepatitis Delta Virus Clinical Strains.

Human hepatitis Delta virus (HDV) infection is associated to the most severe viral hepatic disease, including severe acute liver decompensation and progression to cirrhosis, and hepatocellular carcinoma. HDV is a satellite of hepatitis B virus (HBV) that requires the HBV envelope proteins for assembly of HDV virions. HDV and HBV exhibit a large genetic diversity that extends, respectively to eight (HDV-1 to -8) and to ten (HBV/A to/J) genotypes. Molecular determinants of HDV virion assembly consist of a C-terminal Proline-rich domain in the large Hepatitis Delta Antigen (HDAg) protein, also known as the Delta packaging domain (DPD) and of a Tryptophan-rich domain, the HDV matrix domain (HMD) in the C-terminal region of the HBV envelope proteins. In this study, we performed a systematic genotyping of HBV and HDV in a cohort 1,590 HDV-RNA-positive serum samples collected between 2001 to 2014, from patients originated from diverse parts of the world, thus reflecting a large genetic diversity. Among these samples, 526 HBV (HBV/A, B, C, D, E, and G) and HDV (HDV-1, 2, 3, and 5 to -8) genotype couples could be obtained. We provide results of a comprehensive analysis of the amino-acid sequence conservation within the HMD and structural and functional features of the DPD that may account for the yet optimal interactions between HDV and its helper HBV.

Future treatments for hepatitis delta virus infection.

Around 15-20 million people develop chronic hepatitis delta virus worldwide. Hepatitis delta virus (HDV) is a defective RNA virus requiring the presence of the hepatitis B virus surface antigen (HBsAg) to complete its life cycle. HDV infects hepatocytes using the hepatitis B virus (HBV) receptor, the sodium taurocholate cotransporting polypeptide (NTCP). The HDV genome is a circular single-stranded RNA which encodes for a single hepatitis delta antigen (HDAg) that exists in two forms (S-HDAg and L-HDAg), and its replication is mediated by the host RNA polymerases. The HBsAg-coated HDV virions contain a ribonucleoprotein (RNP) formed by the RNA genome packaged with small and large HDAg. Farnesylation of the L-HDAg is the limiting step for anchoring this RNP to HBsAg, and thus for assembling, secreting and propagating virion particles. There is an important risk of morbidity and mortality caused by end-stage liver disease and hepatocellular carcinoma with HDV and current treatment is pegylated-interferon (PEG-IFN) for 48weeks with no other options in patients who fail treatment. The ideal goal for HDV treatment is the clearance of HBsAg, but a reasonably achievable goal is a sustained HDV virological response (negative HDV RNA 6months after stopping treatment). New drug development must take into account the interaction of HBV and HDV. In this review, we will present the new insights in the HDV life cycle that have led to the development of novel classes of drugs and discuss antiviral approaches in phase II and III of development: bulevirtide (entry inhibitor), lonafarnib, (prenylation inhibitor) and REP 2139 (HBsAg release inhibitor).

Master of Disguise: Hepatitis Delta Virus Packaging and Spread Facilitated by Diverse Viral Envelope Proteins.

To better understand the origins of hepatitis delta virus (HDV), Perez-Vargaset al. sought to characterize the capability of HDV in utilizing envelope glycoproteins from viruses other than hepatitis B virus (HBV)(1). To this end, the authors compared the efficiency of HDV assembly and release in the presence of HBV surface antigens (HBsAg), vesicular stomatitis virus (VSV) G protein, or hepatitis C virus (HCV) E1 and E2 glycoproteins. The HDV particles generated with these respective glycoproteins were infectious to cells displaying appropriate receptorsin vitro. HDV incorporation of VSV-G or HCV-E1E2 wasassisted by farnesylation of the large hepatitis delta antigen (HDAg-L), a mechanism also seen withHBsAg. This article is protected by copyright. All rights reserved.

Structural Pattern Differences in Unbranched Rod-like RNA of Hepatitis Delta Virus affect RNA Editing.

Hepatitis delta virus (HDV) RNA forms an unbranched rod-like structure and complexes with the delta antigen (HDAg). Host ADAR1-catalyzed RNA editing at the amber/W site of the small HDAg leads to the production of the large HDAg, which inhibits replication and is required for virion assembly. For HDV genotype 1, amber/W editing is controlled by HDAg and the RNA structure immediate vicinity and downstream of the editing site. Here, the effects of 20 mutants carrying an increased length of consecutive base-pairing at various sites in HDV RNA on amber/W site editing were examined. All nine mutants carrying genomic regions that formed up to 15 consecutive base pairs, which is also the maximum length observed in 41 naturally occurring HDV genomes, showed normal editing rate. However, mutants carrying a 16 or 17 consecutive base-paired antigenomic segment located as far as 114 nt upstream could increase editing efficiency, possibly by interfering with HDAg binding. These data show for the first time that extended base-pairing upstream of the amber/W site could increase HDV RNA editing efficiency. Furthermore, it appears that the naturally occurring HDV RNA structures have been selected for suboptimal amber/W RNA editing, which favors the HDV replication cycle.

Recapitulation of HDV infection in a fully permissive hepatoma cell line allows efficient drug evaluation

Hepatitis delta virus (HDV) depends on the helper function of hepatitis B virus (HBV), which provides the envelope proteins for progeny virus secretion. Current infection-competent cell culture models do not support assembly and secretion of HDV. By stably transducing HepG2 cells with genes encoding the NTCP-receptor and the HBV envelope proteins we produce a cell line (HepNB2.7) that allows continuous secretion of infectious progeny HDV following primary infection. Evaluation of antiviral drugs shows that the entry inhibitor Myrcludex B (IC50: 1.4 nM) and interferon-α (IC50: 28 IU/ml, but max. 60–80% inhibition) interfere with primary infection. Lonafarnib inhibits virus secretion (IC50: 36 nM) but leads to a substantial intracellular accumulation of large hepatitis delta antigen and replicative intermediates, accompanied by the induction of innate immune responses. This work provides a cell line that supports the complete HDV replication cycle and presents a convenient tool for antiviral drug evaluation.

Genetic diversity of hepatitis D virus genotype‐1 in Europe allows classification into subtypes

Hepatitis delta virus (HDV) is an RNA virus which leads to both acute and chronic forms of hepatitis. At present, HDV isolates have been classified into eight major genotypes distributed over different geographical regions. Recent increase in HDV sequences in Europe and worldwide has enabled us to revisit the taxonomic classification of HDV. A total of 116 large hepatitis delta antigen (L-HDAg) nucleotide sequences and 13 full-length HDV genome sequences belonging to genotype-1 from our European cohort, as well as 621 L-HDAg nucleotide sequences belonging to genotype-1 to genotype-8 retrieved from the GenBank NCBI were included in this study. All 116 isolates of our cohort and 341 of 621 isolates (60%) account for genotype-1, while the remaining 40% of isolates were unevenly distributed across genotype-2 to genotype-8. Phylogenetic analysis of 98 L-HDAg sequences selected after elimination of redundant sequences of all 737 isolates was performed to identify plausible subtypes within HDV genotype-1. Pairwise genetic distances for L-HDAg sequences were calculated to estimate the inter-genotype and inter-subtype differences. The HDV genotype-1 isolates phylogenetically formed five distinct clusters (genotype 1a-1e), each of them corresponding to a distinct geographic region. Two distinct subtypes for HDV genotype-2 and -4 (ie -2a and -2b; -4a and -4b, respectively) could be identified based on isolate sequences from GenBank. The previously defined genotype-1 to genotype-8 have an inter-genotypic difference of ≥10%, while the newly defined subtypes of genotype-1, -2 and -4 show an inter-subtype difference of ≥3% to <10% from the average diversity. In addition, we identified unique amino acid residues, known as specificity-determining positions, amongst the proposed subtypes.

Amino Acid Substitutions within HLA-B*27-Restricted T Cell Epitopes Prevent Recognition by Hepatitis Delta Virus-Specific CD8+ T Cells.

Virus-specific CD8 T cell response seems to play a significant role in the outcome of hepatitis delta virus (HDV) infection. However, the HDV-specific T cell epitope repertoire and mechanisms of CD8 T cell failure in HDV infection have been poorly characterized. We therefore aimed to characterize HDV-specific CD8 T cell epitopes and the impacts of viral mutations on immune escape. In this study, we predicted peptide epitopes binding the most frequent human leukocyte antigen (HLA) types and assessed their HLA binding capacities. These epitopes were characterized in HDV-infected patients by intracellular gamma interferon (IFN-γ) staining. Sequence analysis of large hepatitis delta antigen (L-HDAg) and HLA typing were performed in 104 patients. The impacts of substitutions within epitopes on the CD8 T cell response were evaluated experimentally and by in silico studies. We identified two HLA-B*27-restricted CD8 T cell epitopes within L-HDAg. These novel epitopes are located in a relatively conserved region of L-HDAg. However, we detected molecular footprints within the epitopes in HLA-B*27-positive patients with chronic HDV infections. The variant peptides were not cross-recognized in HLA-B*27-positive patients with resolved HDV infections, indicating that the substitutions represent viral escape mutations. Molecular modeling of HLA-B*27 complexes with the L-HDAg epitope and its potential viral escape mutations indicated that the structural and electrostatic properties of the bound peptides differ considerably at the T cell receptor interface, which provides a possible molecular explanation for the escape mechanism. This viral escape from the HLA-B*27-restricted CD8 T cell response correlates with a chronic outcome of hepatitis D infection. T cell failure resulting from immune escape may contribute to the high chronicity rate in HDV infection.IMPORTANCE Hepatitis delta virus (HDV) causes severe chronic hepatitis, which affects 20 million people worldwide. Only a small number of patients are able to clear the virus, possibly mediated by a virus-specific T cell response. Here, we performed a systematic screen to define CD8 epitopes and investigated the role of CD8 T cells in the outcome of hepatitis delta and how they fail to eliminate HDV. Overall the number of epitopes identified was very low compared to other hepatotropic viruses. We identified, two HLA-B*27-restricted epitopes in patients with resolved infections. In HLA-B*27-positive patients with chronic HDV infections, however, we detected escape mutations within these identified epitopes that could lead to viral evasion of immune responses. These findings support evidence showing that HLA-B*27 is important for virus-specific CD8 T cell responses, similar to other viral infections. These results have implications for the clinical prognosis of HDV infection and for vaccine development.

Cellular Nuclear Export Factors TAP and Aly Are Required for HDAg-L-mediated Assembly of Hepatitis Delta Virus

Hepatitis delta virus (HDV) is a satellite virus of hepatitis B virus (HBV). HDV genome encodes two forms of hepatitis delta antigen (HDAg), small HDAg (HDAg-S), which is required for viral replication, and large HDAg (HDAg-L), which is essential for viral assembly. HDAg-L is identical to HDAg-S except that it bears a 19-amino acid extension at the C terminus. Both HDAgs contain a nuclear localization signal (NLS), but only HDAg-L contains a CRM1-independent nuclear export signal at its C terminus. The nuclear export activity of HDAg-L is important for HDV particle formation. However, the mechanisms of HDAg-L-mediated nuclear export of HDV ribonucleoprotein are not clear. In this study, the host cellular RNA export complex TAP-Aly was found to form a complex with HDAg-L, but not with an export-defective HDAg-L mutant, in which Pro205 was replaced by Ala. HDAg-L was found to colocalize with TAP and Aly in the nucleus. The C-terminal domain of HDAg-L was shown to directly interact with the N terminus of TAP, whereas an HDAg-L mutant lacking the NLS failed to interact with full-length TAP. In addition, small hairpin RNA-mediated down-regulation of TAP or Aly reduced nuclear export of HDAg-L and assembly of HDV virions. Furthermore, a peptide, TAT-HDAg-L(198–210), containing the 10-amino acid TAT peptide and HDAg-L(198–210), inhibited the interaction between HDAg-L and TAP and blocked HDV virion assembly and secretion. These data demonstrate that formation and release of HDV particles are mediated by TAP and Aly.

Evidence of an Exponential Decay Pattern of the Hepatitis Delta Virus Evolution Rate and Fluctuations in Quasispecies Complexity in Long-Term Studies of Chronic Delta Infection.

Chronic HDV infection can cause a severe form of viral hepatitis for which there is no specific treatment. Characterization of the hepatitis B or C viral quasispecies has provided insight into treatment failure and disease recurrence following liver transplantation, has proven useful to understand hepatitis B e antigen seroconversion, and has helped to predict whether hepatitis C infection will resolve or become chronic. It is likely that characterization of the hepatitis delta virus (HDV) quasispecies will ultimately have similar value for the management of this infection. This study sought to determine the RNA evolution rates in serum of chronic hepatitis delta (CHD) treatment-naïve patients, using next-generation sequencing methods. The region selected for study encompassed nucleotide positions 910 to 1270 of the genome and included the amber/W codon. Amber/W is a substrate of the editing process by the ADAR1 host enzyme and is essential for encoding the 2 delta antigens (HDAg). The amber codon encodes the small (unedited) HDAg form and the W codon the large (edited) HDAg form. The evolution rate was analyzed taking into account the time elapsed between samples, the percentage of unedited and edited genomes, and the complexity of the viral population. The longitudinal studies included 29 sequential samples from CHD patients followed up for a mean of 11.5 years. In total, 121,116 sequences were analyzed. The HDV evolution rate ranged from 9.5x10-3 to 1.2x10-3 substitutions/site/year and showed a negative correlation with the time elapsed between samples (p<0.05). An accumulation of transition-type changes was found to be responsible for higher evolution rates. The percentages of unedited and edited genomes and the quasispecies complexity showed no relationships with the evolution rate, but the fluctuations in the percentages of genomes and in complexity suggest continuous adaptation of HDV to the host conditions.

P180: Molecular signatures reveal a novel HLA‐B*15‐restricted CD8 T cell epitope on the large hepatitis delta antigen

Journal of Viral HepatitisVolume 22, Issue S2 p. 109-109 Poster PresentationFree Access P180: Molecular signatures reveal a novel HLA-B*15-restricted CD8 T cell epitope on the large hepatitis delta antigen First published: 25 June 2015 https://doi.org/10.1111/jvh.163_12425AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume22, IssueS2Special Issue: 15th International Symposium on Viral Hepatitis and Liver Diseases (ISVHLD) held from June 26th to 28th, 2015, in Berlin, GermanyJune 2015Pages 109-109 RelatedInformation

Molecular epidemiology of hepatitis delta virus in the Western Pacific region

BackgroundHepatitis delta virus (HDV) is a defective RNA virus requiring the presence of the hepatitis B virus (HBV) for the completion of its life cycle. Active replication of HDV can lead to severe hepatitis, and although present worldwide has an irregular geographical distribution, especially in the Asian Pacific region. ObjectivesThe aim of this study was to determine the prevalence and molecular epidemiology of HDV isolates in Oceania following the 1998 evaluation of the hepatitis B vaccine program. Study designSera collected from 184 hepatitis B surface antigen (HBsAg) positive Pacific Islanders living in Micronesia, Polynesia and Melanesia were tested for HDV RNA. ResultsTwenty of 54 patients with chronic hepatitis B (CHB) from Kiribati were positive for serum HDV RNA (37%), whilst sera from patients with CHB from Tonga (59), Fiji (42) and Vanuatu (29) were negative. The mean HDV RNA load for the 20 samples was 7.00log10copies/mL. Phylogenetic analysis revealed that the Kiribati HDV isolates were of genotype 1 and clustered with a previously published isolate from Nauru forming a distinct clade of Pacific HDV. All Micronesian isolates contained a serine at codon 202 of large hepatitis delta antigen (L-HDAg) demonstrating possible relatedness to strains of HDV-1 of African origin. ConclusionsThis study has confirmed endemic HDV infection in Micronesia and identified Kiribati as having amongst the highest prevalence for HDV viraemia in patients with CHB. Further investigations are ongoing into the origins of this unique HDV Pacific strain, and its inter-relationship with HBV.

Different antiviral effects induced by interferon or entecavir in HDV infected human-chimeric uPA/SCID mice determined by new intrahepatic qRT-PCR assay

Chronic co-infection with hepatitis B and Delta viruses (HBV, HDV) is associated with more severe liver damage compared to HBV mono-infection. Knowledge of the virological changes induced by the different antiviral treatments available is scant. Aim of our study was to investigate the antiviral effects induced by pegylated interferon alpha (peg-IFN-α) and HBV polymerase inhibitors on HDV productivity in human-chimeric uPA/SCID mice and by using a novel strand-specific qRT-PCR strategy enabling detection of all HDV RNA replicative intermediates in patient liver biopsies. Methods: Chronic HBV/HDV infected humanized mice were treated with peg-IFN-α (n = 3), entecavir (ETV) (n = 4) or remained untreated (n = 3). Viremia was monitored by qRT-PCR. Genomic and antigenomic HDV RNA species were determined in liver samples obtained from humanized mice and patients using a new qRT-PCR assay based on a magnetic bead separation strategy. HDAg expression levels were analysed by immunohistochemistry and immunoblotting. Results: Ratios of genomic to antigenomic HDV RNA detected in humanized mice were comparable to those detected in HDV replicative patients (3.9 – 73.0). 4 weeks of peg-IFN-α administration led to 2.0-log reduction of both HBV and HDV viremia and to 1.2-log reduction of circulating HBsAg. In contrast, 4-week ETV treatment did not alter HDV titres and HBsAg levels substantially (< 0.5 log reduction), while it provoked a clear HBV viremia decrease of 3.3-log. Consistent with the serological data, IFN induced a strong decline of both genomic and antigenomic intrahepatic HDV RNA loads, whereas HDV RNA levels remained unchanged in ETV treated mice. Peg-IFN-α strongly reduced the amount of HDAg-positive human hepatocytes. Immunoblotting indicated that both small and large HDAg decreased upon IFN administration, while intrahepatic HDAg contents remained unchanged in ETV treated mice compared to untreated control mice. Conclusions: ETV mediated suppression of HBV replication did not significantly affect HBsAg levels, HDV productivity and release, as documented by the maintenance of HDV viremia and intrahepatic loads. In contrast, 4-week peg-IFN-α treatment efficiently suppressed HDV productivity by lowering both intrahepatic levels of genomic and antigenomic HDV RNA and amounts of HDAg-positive cells. The antiviral effects induced by IFN on HDV infected hepatocytes in a system lacking adaptive immune responses suggest that by targeting HDV replication intrahepatic clearance could be achievable.

Persistent hepatitis D virus mono-infection in humanized mice is efficiently converted by hepatitis B virus to a productive co-infection

Clinical studies have shown that hepatitis delta virus (HDV) infection can persist for years and intrahepatic latency of the large delta antigen (HDAg) has been detected following liver transplantation. However, large HDAg arising via RNA-editing is associated with increasing amounts of non-infectious HDV quasi-species. This study investigated whether HDV could persist intrahepatically in the absence of HBV in vivo and whether infectious HDV could subsequently be released following HBV super-infection. Humanized mice were infected with HDV particles lacking HBV. To test for rescue of latent HDV infection 3 and 6 weeks HDV mono-infected mice were super-infected with HBV. Viral loads and cell toxicity were determined by qRT-PCR and immunohistochemistry. The presence of HDAg-positive human hepatocytes determined after 2, 3, and 6 weeks of HDV inoculation demonstrated establishment and maintenance of intrahepatic HDV mono-infection. Although intrahepatic amounts of large HDAg and edited HDV RNA forms increased over time in HDV mono-infected livers, HBV super-infection led to prompt viremia development (up to 10(8) HDV RNA and 10(7) HBV-DNA copies/ml) even after 6 weeks of latent mono-infection. Concurrently, the number of HDAg-positive human hepatocytes increased, demonstrating intrahepatic HDV spreading. The infectivity of the rescued HDV virions was verified by serial passage in naive chimeric mice. HDV mono-infection can persist intrahepatically for at least 6 weeks before being rescued by HBV. Conversion of a latent HDV infection to a productive HBV/HDV co-infection may contribute to HDV persistence even in patients with low HBV replication and in the setting of liver transplantation.

Hepatitis D Virus Isolates with Low Replication and Epithelial-Mesenchymal Transition-Inducing Activity Are Associated with Disease Remission

Clearance of hepatitis D virus (HDV) viremia leads to disease remission. Large hepatitis delta antigen (L-HDAg) has been reported to activate transforming growth factor β, which may induce epithelial-mesenchymal transition (EMT) and fibrogenesis. This study analyzed serum HDV RNA "quasispecies" in HDV-infected patients at two stages of infection: before and after alanine aminotransferase (ALT) elevations. Included in the study were four patients who went into remission after ALT elevation and three patients who did not go into remission and progressed to cirrhosis or hepatocellular carcinoma. Full-length HDV cDNA clones were obtained from the most abundant HDV RNA species at the pre- and post-ALT elevation stages. Using an in vitro model consisting of Huh-7 cells transfected with cloned HDV cDNAs, the pre- or post-ALT elevation dominant HDV RNA species were characterized for (i) their replication capacity by measuring HDV RNA and HDAg levels in transfected cells and (ii) their capacity to induce EMT by measuring the levels of the mesenchymal-cell-specific protein vimentin, the EMT regulators twist and snail, and the epithelial-cell-specific protein E-cadherin. Results show that in patients in remission, the post-ALT elevation dominant HDV RNA species had a lower replication capacity in vitro and lower EMT activity than their pre-ALT elevation counterparts. This was not true of patients who did not go into remission. The expression of L-HDAg, but not small HDAg, increased the expression of the EMT-related proteins. It is concluded that in chronically infected patients, HDV quasispecies with a low replication capacity and low EMT activity are associated with disease remission.

Multiple genomic sequences of hepatitis delta virus are associated with cDNA promoter activity and RNA double rolling-circle replication

To understand how DNA-dependent RNA polymerase II (pol II) recognizes hepatitis delta virus (HDV) RNA as a template, it is first necessary to identify the HDV sequence that acts as a promoter of pol II-initiated RNA synthesis. Therefore, we isolated the pol II-response element from HDV cDNA and examined the regulation by hepatitis delta antigens (HDAgs). Two HDV cDNA fragments containing bidirectional promoter activity were identified. One was located at nt 1582-1683 (transcription-promoter region 1, TR-P1) and the other at nt 1223-1363 (transcription-internal region 5, TR-I5). The promoter activities of these two regions were enhanced by HDAgs to differing degrees. Next, the role of these sequences in an HDV cDNA-free RNA replication system was characterized by site-directed mutagenesis. Our data showed that: (i) the AUG codon at the HDAg ORF of HDV RNA (nt 1599-1601) that mutates to UAG (amber stop codon) results in loss of dimeric but not monomeric HDV RNA synthesis. (ii) A 5 nt mutation of TR-P1 (P1-m5, nt 1670-1674) abolishes RNA replication completely. Two-nucleotide-mutated RNA (P1-m2, nt 1662-1663) is able to synthesize short RNAs but not monomeric HDV RNA. (iii) A mutation in 5 nt at the TR-I5 region (I5-m5, nt 1351-1355) also abolishes HDV replication. Mutants with 2 nt mutations (I5-m2, nt 1351-1352) or 3 nt mutations (I5-m3, nt 1353-1355) inhibit HDV dimeric but not monomeric RNA synthesis. Furthermore, large HDAg is expressed in cells transfected with I5-m3 and I5-m2 RNAs and that demonstrate the RNA-editing event in the monomeric HDV RNA. These results provide further understanding of the double rolling-circle mechanism in HDV RNA replication.