ICTV Virus Taxonomy Profile: Deltavirus.

Hepatitis delta virus, the only member of the only species in the genus Deltavirus, is a unique human pathogen. Its ~1.7 kb circular negative-sense RNA genome encodes a protein, hepatitis delta antigen, which occurs in two forms, small and large, both with unique functions. Hepatitis delta virus uses host RNA polymerase II to replicate via double rolling circle RNA synthesis. Newly synthesized linear RNAs are circularized after autocatalytic cleavage and ligation. Hepatitis delta virus requires the envelope of the helper virus, hepatitis B virus (family Hepadnaviridae), to produce infectious particles. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of Deltavirus which is available at www.ictv.global/report/deltavirus.

A novel toolbox for the in vitro assay of hepatitis D virus infection.

Hepatitis D virus (HDV) is a defective RNA virus that requires the presence of hepatitis B virus (HBV) for its life cycle. The in vitro HDV infection system is widely used as a surrogate model to study cellular infection with both viruses owing to its practical feasibility. However, previous methods for running this system were less efficient for high-throughput screening and large-scale studies. Here, we developed a novel method for the production of infectious HDV by adenoviral vector (AdV)-mediated transduction. We demonstrated that the AdV-based method yields 10-fold higher viral titers than the transient-transfection approach. The HDV-containing supernatant derived from AdV-infected Huh7 cells can be used as the inoculum in infectivity assays without requiring further concentration prior to use. Furthermore, we devloped a chemiluminescent immunoassay (HDV-CLEIA) to quantitatively determine intracellular HDAg with a dynamic range of 5-11,000 pg/mL. HDV-CLEIA can be used as an alternative approach to assess HDV infection. The advantages of our updated methodology were demonstrated through in vitro HDV infection of HepaRG cells and by evaluating the neutralization activity using antibodies that target various regions of the HBV/HDV envelope proteins. Together, the methods presented here comprise a novel toolbox of in vitro assays for studying HDV infection.

Hepatitis B and D viruses replication interference: Influence of hepatitis B genotype.

AIM: To study the hepatitis B virus (HBV) and hepatitis D virus (HDV) replication interferences in patients with chronic hepatitis delta infected with different HBV genotypes. METHODS: We conducted a transversal study including 68 chronic hepatitis delta (CHD) (37 HIV-positive) patients and a control group of 49 chronic hepatitis B (CHB) (22 HIV-positive) patients. In addition, a dynamic follow-up was performed in 16 CHD patients. In all the samples, the surface antigen of hepatitis B (HBsAg) serum titers were analyzed with the Monolisa HBsAg Ultra system (Bio-Rad), using as quantification standard a serial dilution curve of an international HBsAg standard. Serum HBV-DNA titers were analyzed using the Roche Cobas TaqMan (Roche, Barcelona, Spain), and the serum HDV-RNA using an in-house real-time qRT-PCR method, with TaqMan probes. HBV genotype was determined with the line immunoassay LiPA HBV genotyping system (Innogenetics, Ghent, Belgium). In those patients negative for LiPA assay, a nested PCR method of complete HBsAg coding region, followed by sequence analysis was applied. RESULTS: No differences in the HBV-DNA levels were found in CHB patients infected with different HBV genotypes. However, in CHD patients the HBV-DNA levels were lower in those infected with HBV-A than in those with HBV-D, both in HIV negative [median (IQR): 1.25 (1.00-1.35) vs 2.95 (2.07-3.93) log10 (copies/mL), P = 0.013] and HIV positive patients [2.63 (1.24-2.69) vs 7.25 (4.61-7.55) log10 (copies/mL), P < 0.001]. This was confirmed in the dynamic study of the HBV/HDV patients. These differences induce an under-estimation of HBV-A incidence in patients with CHD analyzed with LiPA assay. Finally, the HBsAg titers reflected no significant differences in CHD patients infected with HBV-A or D. CONCLUSION: Viral replication interference between HBV and HDV is HBV-genotype dependent, and more evident in patients infected with HBV-genotype A, than with HBV-D or E.

Hepatitis D virus infection, replication and cross-talk with the hepatitis B virus.

Viral hepatitis remains a worldwide public health problem. The hepatitis D virus (HDV) must either coinfect or superinfect with the hepatitis B virus (HBV). HDV contains a small RNA genome (approximately 1.7 kb) with a single open reading frame (ORF) and requires HBV supplying surface antigens (HBsAgs) to assemble a new HDV virion. During HDV replication, two isoforms of a delta antigen, a small delta antigen (SDAg) and a large delta antigen (LDAg), are produced from the same ORF of the HDV genome. The SDAg is required for HDV replication, whereas the interaction of LDAg with HBsAgs is crucial for packaging of HDV RNA. Various clinical outcomes of HBV/HDV dual infection have been reported, but the molecular interaction between HBV and HDV is poorly understood, especially regarding how HBV and HDV compete with HBsAgs for assembling virions. In this paper, we review the role of endoplasmic reticulum stress induced by HBsAgs and the molecular pathway involved in their promotion of LDAg nuclear export. Because the nuclear sublocalization and export of LDAg is regulated by posttranslational modifications (PTMs), including acetylation, phosphorylation, and isoprenylation, we also summarize the relationship among HBsAg-induced endoplasmic reticulum stress signaling, LDAg PTMs, and nuclear export mechanisms in this review.

Identification of a binding site for ASF/SF2 on an RNA fragment derived from the hepatitis delta virus genome.

The hepatitis delta virus (HDV) is a small (~1700 nucleotides) RNA pathogen which encodes only one open reading frame. Consequently, HDV is dependent on host proteins to replicate its RNA genome. Recently, we reported that ASF/SF2 binds directly and specifically to an HDV-derived RNA fragment which has RNA polymerase II promoter activity. Here, we localized the binding site of ASF/SF2 on the HDV RNA fragment by performing binding experiments using purified recombinant ASF/SF2 combined with deletion analysis and site-directed mutagenesis. In addition, we investigated the requirement of ASF/SF2 for HDV RNA replication using RNAi-mediated knock-down of ASF/SF2 in 293 cells replicating HDV RNA. Overall, our results indicate that ASF/SF2 binds to a purine-rich region distant from both the previously published initiation site of HDV mRNA transcription and binding site of RNAP II, and suggest that this protein is not involved in HDV replication in the cellular system used.

Squalamine as a broad-spectrum systemic antiviral agent with therapeutic potential.

Antiviral compounds that increase the resistance of host tissues represent an attractive class of therapeutic. Here, we show that squalamine, a compound previously isolated from the tissues of the dogfish shark (Squalus acanthias) and the sea lamprey (Petromyzon marinus), exhibits broad-spectrum antiviral activity against human pathogens, which were studied in vitro as well as in vivo. Both RNA- and DNA-enveloped viruses are shown to be susceptible. The proposed mechanism involves the capacity of squalamine, a cationic amphipathic sterol, to neutralize the negative electrostatic surface charge of intracellular membranes in a way that renders the cell less effective in supporting viral replication. Because squalamine can be readily synthesized and has a known safety profile in man, we believe its potential as a broad-spectrum human antiviral agent should be explored.

Hepatitis delta virus infection: open issues.

Hepatitis delta virus (HDV) consists of a circular single-stranded RNA genome which assembles two viral proteins and acquires a lipid envelope in which the hepatitis B surface antigens (HBsAg) are embedded. HDV does not encode its own polymerase, but exploits a cellular enzyme for its replication. A better understanding of the mechanisms of HDV replication mechanism would provide new insights for antiviral strategies. Based on genomic variability, eight major genotypes of HDV have been identified, which differ as much as 40% in the nucleotide sequence. The cloning of HDV-RNA has provided genetic probes for the measurement of HDV-RNA in serum and liver; the sensitivity of HDV-RNA detection improved significantly when the reverse transcriptase-polymerase chain reaction (PCR) technique was introduced. As no commercial test is standardized for viral load detection, home-made assays have been developed in the different referral centers, which may not be comparable. Quantification of HDV in serum by real-time PCR has been recently proposed in the management of chronically infected patients. No specific inhibitors of HDV are available at present and, in spite of the crucial relationship between HDV and HBV, drugs that block HBV have only a theoretical but no sound effect on HDV replication.

Hepatitis D virus: an update.

Hepatitis D virus (HDV) infection involves a distinct subgroup of individuals simultaneously infected with the hepatitis B virus (HBV) and characterized by an often severe chronic liver disease. HDV is a defective RNA agent needing the presence of HBV for its life cycle. HDV is present worldwide, but the distribution pattern is not uniform. Different strains are classified into eight genotypes represented in specific regions and associated with peculiar disease outcome. Two major specific patterns of infection can occur, i.e. co-infection with HDV and HBV or HDV superinfection of a chronic HBV carrier. Co-infection often leads to eradication of both agents, whereas superinfection mostly evolves to HDV chronicity. HDV-associated chronic liver disease (chronic hepatitis D) is characterized by necro-inflammation and relentless deposition of fibrosis, which may, over decades, result in the development of cirrhosis. HDV has a single-stranded, circular RNA genome. The virion is composed of an envelope, provided by the helper HBV and surrounding the RNA genome and the HDV antigen (HDAg). Replication occurs in the hepatocyte nucleus using cellular polymerases and via a rolling circle process, during which the RNA genome is copied into a full-length, complementary RNA. HDV infection can be diagnosed by the presence of antibodies directed against HDAg (anti-HD) and HDV RNA in serum. Treatment involves the administration of pegylated interferon-α and is effective in only about 20% of patients. Liver transplantation is indicated in case of liver failure.

Hepatitis delta virus proteins repress hepatitis B virus enhancers and activate the alpha/beta interferon-inducible MxA gene.

Co-infection and superinfection of hepatitis B virus (HBV) with hepatitis delta virus (HDV) leads to suppression of HBV replication both in patients and in animal and cellular models. The mechanisms behind this inhibition have not previously been explored fully. HBV replication is governed by four promoters and two enhancers, Enh1 and Enh2. Repression of these enhancers has been reported to be one of the main mechanisms of HBV inhibition. Moreover, in a previous study, it has been demonstrated that alpha interferon (IFN-alpha)-inducible MxA protein inhibits HBV replication. HDV encodes two proteins, p24 and p27. p27 was shown to activate several heterologous promoters, including HBV promoters. In an attempt to analyse the mechanisms of HBV inhibition by HDV, the question was raised whether HDV proteins could act directly by repressing HBV enhancers, and/or indirectly by activating the MxA gene. This issue was addressed in a co-transfection model in Huh-7 cells, using p24- or p27-expressing plasmids along with Enh1, Enh2, HBV and MxA promoter-luciferase constructs. Enh1 and Enh2 were strongly repressed, by 60 and 80 % and 40 and 60 %, by p24 and p27, respectively. In addition, p27 was responsible for threefold activation of the MxA promoter and potentiation of IFN-alpha on this promoter. MxA mRNA quantification and a virus yield reduction assay confirmed these results. In conclusion, this study shows that HDV proteins inhibit HBV replication by trans-repressing its enhancers and by trans-activating the IFN-alpha-inducible MxA gene.

Primary human hepatocytes are susceptible to infection by hepatitis delta virus assembled with envelope proteins of woodchuck hepatitis virus.

Hepatitis B virus (HBV) and hepatitis delta virus (HDV) share the HBV envelope proteins. When woodchucks chronically infected with woodchuck hepatitis virus (WHV) are superinfected with HDV, they produce HDV with a WHV envelope, wHDV. Several lines of evidence are provided that wHDV infects not only cultured primary woodchuck hepatocytes (PWH) but also primary human hepatocytes (PHH). Surprisingly, HBV-enveloped HDV (hHDV) and wHDV infected PHH with comparable efficiencies; however, hHDV did not infect PWH. The basis for these host range specificities was investigated using as inhibitors peptides bearing species-specific pre-S (where S is the small envelope protein) sequences. It was found that pre-S1 contributed to the ability of wHDV to infect both PHH and PWH. In addition, the inability of hHDV to infect PWH was not overcome using a chimeric form of hHDV containing WHV S protein, again supporting the essential role of pre-S1 in infection of target cells. One interpretation of these data is that host range specificity of HDV is determined entirely by pre-S1 and that the WHV and HBV pre-S1 proteins recognize different receptors on PHH.

A pilot study of 2 years of interferon treatment in patients with chronic delta hepatitis.

High dose interferon treatment for 1 year is the only established treatment for chronic hepatitis D, but it is associated with a high relapse rate after treatment discontinuation. In this study, patients were treated with 10 MU interferon alpha 2b, thrice weekly for 2 years. Twenty-three patients were recruited and 15 completed the 2-year treatment and 6 months follow-up periods. Treatment response was assessed biochemically [normal alanine aminotransferase (ALT)], virologically (undetectable hepatitis D virus RNA) and histologically (at least 2 point decrease in the Knodell score) at the end of treatment (EOT) and at the end of follow-up. Out of 15 patients who finished the 2-year treatment period, seven patients (47%) had a biochemical response but only two (13%) had a normal ALT after follow-up. ALT decreased from the baseline value of 143.1 +/- 121.7 (mean +/- SD) to 39.7 +/- 20.6 (P < 0.01) at EOT. Virological response was observed in six patients at EOT and in two patients at follow-up. Two patients lost hepatitis B surface antigen. Of the 12 patients with paired liver biopsies, a histological improvement was observed in eight patients. Interferon treatment leads to a complete or partial response in a substantial number of patients but 2 years of treatment does not appear to increase sustained response rates over 1 year treatment.

Myristoylation signal transfer from the large to the middle or the small HBV envelope protein leads to a loss of HDV particles infectivity.

A myristate linked to the N-terminus of the large hepatitis B virus (HBV) envelope protein was found to be required for infectivity of the hepatitis delta virus (HDV). Myristoylation of the large HBV envelope protein being known as indispensable for HBV infectivity, this result further demonstrates the similarities between the HBV and HDV entry pathways. In addition, the transfer of the N-myristoylation signal from the large to the middle or the small HBV envelope protein led in both cases to a loss of HDV infectivity. Hence, it is suggested that viral entry could depend on a physical link, or a spatial association, between the N-terminal receptor-binding polypeptide of the large protein and the myristoyl anchor linked to glycine-2.

Immunoadhesins containing pre-S domains of hepatitis B virus large envelope protein are secreted and inhibit virus infection.

Hepatitis B virus (HBV) replication produces three envelope proteins (L, M, and S) that have a common C terminus. L, the largest, contains a domain, pre-S1, not present on M. Similarly M contains a domain, pre-S2, not present on S. The pre-S1 region has important functions in the HBV life cycle. Thus, as an approach to studying these roles, the pre-S1 and/or pre-S2 sequences of HBV (serotype adw2, genotype A) were expressed as N-terminal fusions to the Fc domain of a rabbit immunoglobulin G chain. Such proteins, known as immunoadhesins (IA), were highly expressed following transfection of cultured cells and, when the pre-S1 region was present, >80% were secreted. The IA were myristoylated at a glycine penultimate to the N terminus, although mutation studies showed that this modification was not needed for secretion. As few as 30 amino acids from the N terminus of pre-S1 were both necessary and sufficient to drive secretion of IA. Even expression of pre-S1 plus pre-S2, in the absence of an immunoglobulin chain, led to efficient secretion. Overall, these studies demonstrate an unexpected ability of the N terminus of pre-S1 to promote protein secretion. In addition, some of these secreted IA, at nanomolar concentrations, inhibited infection of primary human hepatocytes either by hepatitis delta virus (HDV), a subviral agent that uses HBV envelope proteins, or HBV. These IA have potential to be part of antiviral therapies against chronic HDV and HBV, and may help understand the attachment and entry mechanisms used by these important human pathogens.

Role of N glycosylation of hepatitis B virus envelope proteins in morphogenesis and infectivity of hepatitis delta virus.

Hepatitis delta virus (HDV) particles are coated with the large (L), middle (M), and small (S) hepatitis B virus envelope proteins. In the present study, we constructed glycosylation-defective envelope protein mutants and evaluated their capacity to assist in the maturation of infectious HDV in vitro. We observed that the removal of N-linked carbohydrates on the S, M, and L proteins was tolerated for the assembly of subviral hepatitis B virus (HBV) particles but was partially inhibitory for the formation of HDV virions. However, when assayed on primary cultures of human hepatocytes, virions coated with S, M, and L proteins lacking N-linked glycans were infectious. Furthermore, in the absence of M, HDV particles coated with nonglycosylated S and L proteins retained infectivity. These results indicate that carbohydrates on the HBV envelope proteins are not essential for the in vitro infectivity of HDV.

Use of a prenylation inhibitor as a novel antiviral agent.

No specific therapy exists for hepatitis delta virus (HDV), which can cause severe liver disease. Molecular genetic studies have implicated the prenylation site of large delta antigen as a critical determinant of HDV particle assembly. We have established a cell culture model which produces HDV-like particles, and we show that delta antigen prenylation can be pharmacologically inhibited by the prenylation inhibitor BZA-5B. Furthermore, BZA-5B specifically abolishes particle production in a dose-dependent manner. These results demonstrate that the use of such a prenylation inhibitor-based antiviral therapy may be feasible and identify a novel class of potential antiviral agents.

Assembly of hepatitis delta virus-like empty particles in yeast.

Large delta antigen (L-HDAg) of hepatitis delta virus (HDV) and small-form hepatitis B surface antigen (HBsAg) of helper hepatitis B virus have previously been shown to be the minimum components for the assembly of HDV-like particles in mammalian cells. Extending from this finding, we coexpressed L-HDAg and small HBsAg in Saccharomyces cerevisiae to study their assembly in yeast cells. The assembly of virus particles from L-HDAg and HBsAg in yeast was demonstrated by their coexistence in the same isopycnic fractions and by the coimmunoprecipitation of L-HDAg with HBsAg using an antibody against HBsAg (anti-HBs). Furthermore, after purification by affinity chromatography with anti-HBs, HDV-like particles with size and morphology similar to those derived from mammalian cells could be visualized by electron microscopy. Mice immunized with yeast-derived HDV-like particles simultaneously acquired antibodies against HBsAg and HDAg, indicating that both viral proteins are antigenic. The results indicated that S. cerevisiae could serve as a host for the assembly of HDV-like empty particles. This system may be useful in investigating cellular processes involved in HDV assembly and in producing ample amount of HDV-like particles for structural and immunological studies.

Apparent helper-independent infection of woodchucks by hepatitis delta virus and subsequent rescue with woodchuck hepatitis virus.

Hepatitis delta virus (HDV) is a subviral agent of humans which is dependent upon hepatitis B virus as a helper for transmission. HDV can be experimentally transmitted to woodchucks by using woodchuck hepatitis virus (WHV) as the helper. We used this model system to study two types of HDV infections: those of animals already chronically infected with WHV and those of animals without any evidence of prior exposure to WHV. At 5 to 10 days after infection with HDV, liver biopsies of these two groups of animals indicated that around 1% of the hepatocytes were infected (HDV antigen positive). Moreover, similar amounts of replicative forms of HDV RNA were detected. In contrast, by 20 days postinfection, the two groups of animals were quite different in the extent of the HDV infection. The animals chronically infected with WHV showed spread of the infection within the liver and the release of high titers of HDV into the serum. In contrast, the animals not previously exposed to WHV showed a progressive reduction in liver involvement, and at no time up to 165 days postinfection could we detect HDV particles in the serum. However, if these animals were inoculated with a relatively high titer of WHV at either 7 or even 33 days after the HDV infection, HDV viremia was observed. Our data support the interpretation that in these animals, hepatocytes were initially infected in the absence of helper virus, HDV genome replication took place, and ultimately these replicating genomes were rescued by the secondary WHV infection. The observation that HDV can survive in the liver for at least 33 days in the absence of coinfecting helper virus may be relevant to the reemergence of HDV infection following liver transplantation.

HBV and HDV replication in experimental models: effect of interferon.

The use of HBV and HDV experimental models has significantly contributed to understand the viral life cycle and to systematically test antiviral effects of various drugs on a pre-clinical level. Similar replication strategies of related hepadna viruses permit the use of chimpanzees (Pan troglodytes), woodchucks (Marmota monax), ground and tree squirrels (Spermophilus beecheyi) or Pekin ducks (Anas domesticus) as appropriate animal models. Cell culture systems for in vitro infection or transfection using both primary cultures of human and non-human hepatocytes and non-hepatocytes and cell lines have recently been identified. The advantages and restrictions of these experimental models with respect to evaluation of interferon effects on viral and hepatocellular gene expression are discussed.

Hepatitis delta virus replication in vitro is not affected by interferon-alpha or -gamma despite intact cellular responses to interferon and dsRNA.

The hepatitis delta virus (HDV) genome consists of circular ssRNA which has extensive intramolecular complementarity and can form a dsRNA rod-like structure. If such RNA species were to exist in an unmasked form in cells, they would be expected to induce interferon (IFN) expression and activate two IFN-inducible dsRNA-dependent enzymes with anti-viral activity, namely the dsRNA-dependent protein kinase (PKR) and 2',5' oligoadenylate (2',5' A) synthetase. Since the virus replicates to high copy number for prolonged periods in infected cells it is apparently able to evade these antiviral mechanisms. The RNA genome may be masked and fail to induce or activate the antiviral response, or the virus may inhibit such a response. Treatment of a hepatoma cell line, Huh7, and a fibrosarcoma cell line, HT1080, stably transfected with a trimeric HDV cDNA construct, with IFN-alpha or IFN-gamma for up to seven days failed to influence the level of expression of genomic or antigenomic HDV RNA, or delta antigen (Ag). This is consistent with either failure of activation or inhibition of the IFN response. However the induction of several IFN-responsive genes, including PKR, 2',5' A synthetase and class I MHC is normal and cotransfection of a construct expressing delta Ag did not affect expression from an IFN-inducible chloramphenicol acetyltransferase construct. In addition, the activation of PKR is not inhibited in HDV-expressing cells and antiviral assays suggest that the ability of these cells to mount an antiviral response to at least two cytopathic viruses is unaffected. IFN-beta is inducible normally by dsRNA in cells transfected with the delta cDNA trimer. We conclude that HDV replication is not inhibited by IFN-alpha or IFN-gamma, even though the responses of cells expressing HDV RNA and antigen to IFN and dsRNA are intact.

Expression of hepatitis delta virus RNA deletions: cis and trans requirements for self-cleavage, ligation, and RNA packaging.

The hepatitis delta virus (HDV) genome is a circular, single-stranded, rod-shaped, 1.7-kb RNA that replicates via a rolling-circle mechanism. Viral ribozymes function to cleave replication intermediates which are then ligated to generate the circular product. HDV expresses two forms of a single protein, the small and large delta antigens (delta Ag-S and delta Ag-L), which associate with viral RNA in a ribonucleoprotein (RNP) structure. While delta Ag-S is required for RNA replication, delta Ag-L inhibits this process but promotes the assembly of the RNP into mature virions. In this study, we have expressed full-length and deleted HDV RNA inside cells to determine the minimal RNA sequences required for self-cleavage, ligation, RNP packaging, and virion assembly and to assess the role of either delta antigen in each of these processes. We report the following findings. (i) The cleavage and ligation reactions did not require either delta antigen and were not inhibited in their presence. (ii) delta Ag-L, in the absence of delta Ag-S, formed an RNP with HDV RNA which could be assembled into secreted virus-like particles. (iii) Full-length HDV RNAs were stabilized in the presence of either delta antigen and accumulated to much higher levels than in their absence. (iv) As few as 348 nucleotides of HDV RNA were competent for circle formation, RNP assembly, and incorporation into virus-like particles. (v) An HDV RNA incapable of folding into the rod-like structure was not packaged by delta Ag-L.