Initiation of hepatitis delta virus (HDV) replication: HDV RNA encoding the large delta antigen cannot replicate.

Identification of the Functional Regions Required for Hepatitis D Virus Replication and Transcription by Linker-Scanning Mutagenesis of Viral Genome

Detection of hepatitis delta virus RNA by a nonradioactive in situ hybridization procedure

The intrahepatic distribution of hepatitis delta virus RNA was studied by in situ hybridization in 33 formalin-fixed, paraffin-embedded biopsies from 17 chronic hepatitis B virus carriers superinfected with hepatitis delta virus. The findings were correlated with the expression of the hepatitis delta antigen, the duration of the hepatitis delta virus infection and the eosinophilic degeneration of the hepatocytes. Intranuclear hepatitis delta virus RNA and antigen were found in 28 specimens, whereas the remaining five were negative for both markers. Hepatitis delta virus RNA and antigen were mostly found within the same cell. In 20 specimens, however, a variable number of hepatocytes showed the presence of hepatitis delta virus RNA alone. The percentage of these over the total number of infected cells was higher in the specimens taken within 1 year from the acute delta hepatitis, whereas their absence was invariably associated with a long-established hepatitis delta virus infection. Interestingly, the vast majority of hepatocytes undergoing eosinophilic degeneration, a change significantly associated with hepatitis delta virus infection, did not show the presence of either hepatitis delta virus RNA or the viral antigen, suggesting a lack of association, at the cellular level, between viral replication and cytopathological change. The specificity of the detection of hepatitis delta virus RNA was confirmed by negative findings in nine specimens from seven chronic hepatitis B virus carriers without evidence of past or current hepatitis delta virus infection. The loss in sensitivity due to the formalin fixation was estimated to be 50% of that obtained in frozen biopsies, as determined by counting autoradiographic grains over infected cells.(ABSTRACT TRUNCATED AT 250 WORDS)

The mechanism of regulation for the ribozyme activity of hepatitis delta virus (HDV) RNA in infected cells is unknown. Previously, we developed a direct assay capable of detecting the ribozyme activity of HDV dimer or trimer RNAs in vivo (K.-S. Jeng, A. Daniel, and M. M. C. Lai, J. Virol, 70:2403-2410, 1996). In this study, we used this method to examine the effects of hepatitis delta antigen (HDAg) on the ribozyme activities of HDV RNA in vivo. The HDV multimer cDNAs were cotransfected with plasmids encoding either HDV small delta antigen (SHDAg) or large delta antigen (LHDAg), and the self-cleavage of the primary transcripts from the HDV cDNA was analyzed at day 2 postransfection. The results were as follows. (i) Both HDAgs, particularly LHDAg, enhanced the self-cleavage activity of HDV RNA; however, HDAgs are not required for HDV RNA cleavage. (ii) HDAg could not restore the ribozyme activity of mutant HDV RNAs which have lost the ribozyme function. (iii) The enhancement of ribozyme activity by HDAg does not require HDV RNA replication. (iv) RNA-binding activity of HDAg is required for the enhancement of RNA cleavage. (v) The self-ligation activities of HDV ribozyme also were enhanced by HDAg. These results suggest that HDAg can regulate the cleavage and ligation of HDV RNA during the HDV life cycle.

Lacking an RNA-dependent RNA polymerase, hepatitis delta virus (HDV), which contains a circular RNA of 1.7 kilobases, is nonetheless able to replicate its RNA by use of cellular transcription machineries. Previously, we have shown that the replications of genomic- and antigenomic-strand HDV RNAs have different sensitivities to alpha-amanitin, suggesting that these two strands are synthesized in different transcription machineries in the cells, but the nature of these transcription machineries is not clear. In this study, we performed metabolic labeling and immunofluorescence staining of newly synthesized HDV RNA with bromouridine after HDV RNA transfection into hepatocytes and confirmed that HDV RNA synthesis had both alpha-amanitin-sensitive and -resistant components. The antigenomic RNA labeling was alpha-amanitin resistant and localized to the nucleolus. The genomic RNA labeling was alpha-amanitin sensitive and more diffusely localized in the nucleoplasm. Most of the genomic RNA labeling appeared to colocalize with the PML nuclear bodies. Furthermore, promyelocytic leukemia protein, RNA polymerase II (Pol II), and the Pol I-associated transcription factor SL1 could be precipitated together with hepatitis delta antigen, suggesting the association of HDV replication complex with the Pol I and Pol II transcription machineries. This conclusion was further confirmed by an in vitro replication assay. These findings provide additional evidence that HDV RNA synthesis occurs in the Pol I and Pol II transcription machineries, thus extending the capability of the cellular DNA-dependent RNA polymerases to utilizing RNA as templates.

The aim of this study was to evaluate whether patients with chronic hepatitis delta virus (HDV) infection treated with alpha interferon and subsequent loss of hepatitis B surface antigen (HBsAg) eliminate HDV. HDV RNA was detected in 26 of 28 patients with chronic delta hepatitis using the polymerase chain reaction. Seventeen patients in whom HDV RNA was detected were treated with alpha interferon; in 65%, HDV RNA remained detectable during treatment or reappeared after stopping therapy whereas in three patients HDV RNA remained absent (17.5%). HDV RNA became and remained undetectable in serum and liver of two of these three patients who lost HBsAg from serum and in one patient who was intermittently HBsAg negative during therapy. After loss of HBsAg, hepatitis B virus (HBV) DNA was still detectable in the liver, but not HBV RNA, indicating absent or very low HBV replication. Three patients were lost to follow up (17.5%). Two nontreated patients with chronic HDV infection also lost HBsAg during follow up; HDV RNA also became undetectable in their serum. Thus, HDV replication does not persist after the loss of HBsAg. Clearance of HBsAg may be a useful guide to when therapy can be stopped.

Hepatitis delta virus (HDV) replicates by RNA-dependent RNA synthesis according to a double rolling circle model. Also synthesized during replication is a 0.8-kb, polyadenylated mRNA encoding the hepatitis delta antigen (HDAg). It has been proposed that this mRNA species represents the initial product of HDV RNA replication; subsequent production of genomic-length HDV RNA relies on suppression of the HDV RNA polyadenylation signal by HDAg. However, this model was based on studies which required the use of an HDV cDNA copy to initiate HDV RNA replication in cell culture, thus introducing an artificial requirement for DNA-dependent RNA synthesis. We have now used an HDV cDNA-free RNA transfection system and a method that we developed to detect specifically the mRNA species transcribed from the HDV RNA template. We established that this polyadenylated mRNA is 0.8 kb in length and its 5' end begins at nucleotide 1631. Surprisingly, kinetic studies showed that this mRNA continued to be synthesized even late in the viral replication cycle and that the mRNA and the genomic-length RNA increased in parallel, even in the presence of HDAg. Thus, a switch from production of the HDAg mRNA to the full-length HDV RNA does not occur in this system, and suppression of the polyadenylation site by HDAg may not significantly regulate the synthesis of the HDAg mRNA, as previously proposed. These findings reveal novel insights into the mechanism of HDV RNA replication. A new model of HDV RNA replication and transcription is proposed.

To establish stable cell clones allowing continuous replication of hepatitis delta virus (HDV), Hep G2, a hepatoblastoma cell line containing no hepatitis B virus (HBV) DNA sequences, was transfected with a recombinant plasmid containing a tandem trimer of HDV cDNA (driven by the simian virus 40 late promoter) and a neomycin-resistance gene. After selection with the neomycin analogue G418, at least two of the resistant clones were shown to have intact delta antigen by specific immunoblotting, and the delta antigen was located in the cell nucleus by immunofluorescence. Transfected cloned viral DNAs were found to be integrated into cell chromosomes. Replication of the HDV genome was demonstrated by the presence of not only genomic and antigenomic HDV RNAs but also HDV RNAs in multimeric and circular forms. In addition, a 0.8-kilobase antigenomic RNA containing a poly(A) tail and encoding the delta-antigen open reading frame was documented. Continuous replication and transcription of the HDV genome was thus achieved in these transfected cell lines. The results confirmed that replication of HDV was unassisted by HBV. Stable passage of such cell lines strongly suggests that HDV lacks direct cytopathicity in hepatocytes. These clones should be useful in studying the details of the HDV life cycle and the relationship between HDV and its helper virus, HBV.

erosion.Pathologic diagnosis of Mycobacterium infection from bony specimens was recorded for 35 (92%) patients.For 29 (76%), diagnosis was conducted by molecular study, including 25 (66%) by the national reference mycobacterial laboratory.For 4 patients, diagnosis was confirmed by culture of M. bovis.Osteomyelitis/osteitis for 5 patients was considered BCG related according to pathologic diagnosis of Mycobacterium infection, BCG vaccination history, and lack of a history of contact with a person with tuberculosis.Thirty-two (84%) children underwent surgery (excision, debridement, open biopsy), 4 children received arthrotomy (3 ankle and knee joint), and 2 children underwent only aspiration biopsy.All patients received isoniazid and rifampin therapy; 33 patients also received pyrazinamide, and 6 received additional ethambutol therapy.Medications were adjusted after diagnoses changed from tuberculosis to BCG infection.Two patients had major sequelae, both involving the thoracic spine and causing severe kyphosis.Adverse reactions after BCG vaccination depend on the BCG dose, vaccine strain, vaccine administration method, injection technique, and recipient's underlying immune status (5).The vaccine strain and manufacturing process in Taiwan did not change during the study period.Findings were not associated with a specific batch of vaccine, inoculation age, underlying disease, or Salmonella spp.infection.Patients had no common birth place, hospital, or area of residence.We believe the increased number of cases resulted mainly from policy changes and laboratory facility improvements.A surgical approach to obtain a specimen is indicated.However, because medical treatment usually yields a good outcome (6), extensive debridement should be avoided.Although some patients with lower extremity involvement initially limped, most were able to walk well later.Vertebral involvement is rare.Unlike previously reported cases (7,8), both patients reported here who had vertebral involvement had sequelae.For young children with suspected vertebral tuberculosis but no tuberculosis contact history, a biopsy specimen for BCG studies is preferable to spondylectomy.Although no definite immunologic deficit was found in these BCG osteomyelitis/osteitis patients, 2 other compensated infants with disseminated BCG during the same period in Taiwan had identified immunodeficiency (9).Studies are ongoing by the Taiwan Centers for Disease Control to evaluate medical treatment duration, long-term outcomes, and more detailed immune genetic tests.

In hepatitis delta virus (HDV) infection, which is an important etiological cause of chronic liver disease, the relationship between serum quantitative HBsAg level and fibrosis and histological activity was investigated. Between 2014 and 2020, 98 patients with chronic HDV infection (53 noncirrhotic, 45 cirrhotic) participated in this prospectively designed study. Quantitative HBsAg levels of the patients were measured and their relationship with the stage of chronic liver disease was compared with histological activity index (HAI), fibrosis score and HDV RNA, model for end-stage liver disease score and other biochemical parameters. All patients were infected with genotype 1 (100%). HBeAg was positive in 8 (8.1%) of the patients. A correlation was found between quantitative HBsAg level and HDV RNA level in patients with both cirrhotic (r = 0.568; P < 0.001) and noncirrhotic (r = 0.644; P < 0.001) HDV infection. Alanine transaminase (P = 0.001; r = 0.495) and aspartate transaminase (P = 0.001; r = 0.511) levels correlated with quantitative HBsAg levels, more prominently in noncirrhotic patients. There was a correlation between quantitative HBsAg level and histological activity index (HAI) in patients with noncirrhotic HDV infection (P < 0.001; r = 0.664). In receiver operating characteristic analysis, both quantitative HBsAg (for cutoff: 1000; sensitivity 76%; specificity 17%; P = 0.335) and HDV RNA (for cutoff: 100000; sensitivity 2%; specificity 98%; P = 0.096) were not predictive markers for cirrhosis. Quantitative HBsAg level can be evaluated as an indicator of viral replication and histological activity in patients with chronic delta hepatitis without cirrhosis. We think that quantitative HBsAg level will be useful in the management of chronic HDV infection, especially in noncirrhotic patients.

Oral prenylation inhibition with lonafarnib in chronic hepatitis D infection: a proof-of-concept randomised, double-blind, placebo-controlled phase 2A trial

Previous studies have indicated that the replication of the RNA genome of hepatitis delta virus (HDV) involves redirection of RNA polymerase II (Pol II), a host enzyme that normally uses DNA as a template. However, there has been some controversy about whether in one part of this HDV RNA transcription, a polymerase other than Pol II is involved. The present study applied a recently described cell system (293-HDV) of tetracycline-inducible HDV RNA replication to provide new data regarding the involvement of host polymerases in HDV transcription. The data generated with a nuclear run-on assay demonstrated that synthesis not only of genomic RNA but also of its complement, the antigenome, could be inhibited by low concentrations of amanitin specific for Pol II transcription. Subsequent studies used immunoprecipitation and rate-zonal sedimentation of nuclear extracts together with double immunostaining of 293-HDV cells, in order to examine the associations between Pol II and HDV RNAs, as well as the small delta antigen, an HDV-encoded protein known to be essential for replication. Findings include evidence that HDV replication is somehow able to direct the available delta antigen to sites in the nucleoplasm, almost exclusively colocalized with Pol II in what others have described as transcription factories.

Hepatitis delta virus (HDV) superinfection of woodchuck chronic carriers of woodchuck hepatitis virus (WHV) results in acute and chronic disease. The different courses of disease mimicked the outcome of human HDV superinfection, making woodchucks valuable models for clinical studies of HDV. Ten of 11 woodchuck chronic carriers of WHV superinfected with HDV developed acute HDV infection with markers of viral replication in the serum and liver. One animal (DW128) had no serological markers of acute HDV infection. Nine of 11 (82%) superinfected animals developed chronic HDV infection. An unusual course of chronic HDV infection occurred in one woodchuck (DW128): no serum markers of acute or chronic HDV infection appeared but HDV RNA was detected in the liver, indicating that chronic HDV infection can occur without serological markers.

Hepatitis delta virus (HDV) is a satellite virus of the hepatitis B virus (HBV), with a single-stranded and rod-like circular RNA encoding only one protein, the hepatitis delta antigen (HDAg). Lacking its own replicase, the highly self-complementary HDV RNA hijacks host RNA polymerase II (Pol-II), eliciting a unique and incompletely understood RNA-templated transcriptional activity. Because transcription by Pol-II is regulated at multiple steps by various cyclin-dependent kinases (CDKs), we investigated whether CDKs contribute to HDV replication. Using selective compounds targeting transcriptional cyclin-dependent kinases (CDKs), we identified the Mediator kinase CDK8 and its paralog CDK19 as key cofactors for HDV replication. Loss of CDK8/19 activity by small-molecule inhibitor MSC2530818 or genetic knockouts completely prevents the establishment of HDV replication in multiple cell culture models and partially suppresses HDV RNA synthesis during the steady-state replication phase. Ectopic expression of the small HDAg, but not its methylation site R13 mutant, restored HDV replication in CDK8/19-deficient cells. Inactivation of CDK8/19 did not alter phosphorylation of small HDAg at Ser177, but was associated with reduced phosphorylation of the C-terminal domain of Pol-II, consistent with impaired transcriptional activity. Our findings reveal the essential role of CDK8/19 in mediating the transcriptional activity of Pol-II during HDV replication, which is partially counteracted by HDV-encoded small HDAg.

Hepatitis delta virus (HDV) RNA editing controls the formation of hepatitis-delta-antigen-S and -L and therefore indirectly regulates HDV replication. Editing is thought to be catalysed by the adenosine deaminase acting on RNA1 (ADAR1) of which two different forms exist, interferon (IFN)-alpha-inducible ADAR1-L and constitutively expressed ADAR1-S. ADAR1-L is hypothesized to be a part of the innate cellular immune system, responsible for deaminating adenosines in viral dsRNAs. We examined the influence of both forms on HDV RNA editing in IFN-alpha-stimulated and unstimulated hepatoma cells. For gene silencing, an antisense oligodeoxyribonucleotide against a common sequence of both forms of ADAR1 and another one specific for ADAR1-L alone were used. IFN-alpha treatment of host cells led to approximately twofold increase of RNA editing compared with unstimulated controls. If ADAR1-L expression was inhibited, this substantial increase in editing could no longer be observed. In unstimulated cells, ADAR1-L suppression had only minor effects on editing. Inhibition of both forms of ADAR1 simultaneously led to a substantial decrease of edited RNA independently of IFN-alpha-stimulation. In conclusion, the two forms of ADAR1 are responsible almost alone for HDV editing. In unstimulated cells, ADAR1-S is the main editing activity. The increase of edited RNA under IFN-alpha-stimulation is because of induction of ADAR1-L, showing for the first time that this IFN-inducible protein is involved in the base modification of replicating HDV RNA. Thus, induction of ADAR1-L may at least partially cause the antiviral effect of IFN-alpha in natural immune response to HDV as well as in case of therapeutic administration of IFN.