Combined small molecule and loss-of-function screen uncovers estrogen receptor alpha and CAD as host factors for HDV infection and antiviral targets.

OBJECTIVE: Hepatitis D virus (HDV) is a circular RNA virus coinfecting hepatocytes with hepatitis B virus. Chronic hepatitis D results in severe liver disease and an increased risk of liver cancer. Efficient therapeutic approaches against HDV are absent. DESIGN: Here, we combined an RNAi loss-of-function and small molecule screen to uncover host-dependency factors for HDV infection. RESULTS: Functional screening unravelled the hypoxia-inducible factor (HIF)-signalling and insulin-resistance pathways, RNA polymerase II, glycosaminoglycan biosynthesis and the pyrimidine metabolism as virus-hepatocyte dependency networks. Validation studies in primary human hepatocytes identified the carbamoyl-phosphatesynthetase 2, aspartate transcarbamylase and dihydroorotase (CAD) enzyme and estrogen receptor alpha (encoded by ESR1) as key host factors for HDV life cycle. Mechanistic studies revealed that the two host factors are required for viral replication. Inhibition studies using N-(phosphonoacetyl)-L-aspartic acid and fulvestrant, specific CAD and ESR1 inhibitors, respectively, uncovered their impact as antiviral targets. CONCLUSION: The discovery of HDV host-dependency factors elucidates the pathogenesis of viral disease biology and opens therapeutic strategies for HDV cure.

Mutations in Hepatitis D Virus Allow It to Escape Detection by CD8+ T Cells and Evolve at the Population Level.

BACKGROUND & AIMS: Hepatitis D virus (HDV) superinfection in patients with hepatitis B virus (HBV) is associated with rapid progression to liver cirrhosis and hepatocellular carcinoma. Treatment options are limited, and no vaccine is available. Although HDV-specific CD8+ T cells are thought to control the virus, little is known about which HDV epitopes are targeted by virus-specific CD8+ T cells or why these cells ultimately fail to control the infection. We aimed to define how HDV escapes the CD8+ T-cell-mediated response. METHODS: We collected plasma and DNA samples from 104 patients with chronic HDV and HBV infection at medical centers in Europe and the Middle East, sequenced HDV, typed human leukocyte antigen (HLA) class I alleles from patients, and searched for polymorphisms in HDV RNA associated with specific HLA class I alleles. We predicted epitopes in HDV that would be recognized by CD8+ T cells and corresponded with the identified virus polymorphisms in patients with resolved (n = 12) or chronic (n = 13) HDV infection. RESULTS: We identified 21 polymorphisms in HDV that were significantly associated with specific HLA class I alleles (P < .005). Five of these polymorphisms were found to correspond to epitopes in HDV that are recognized by CD8+ T cells; we confirmed that CD8+ T cells in culture targeted these HDV epitopes. HDV variant peptides were only partially cross-recognized by CD8+ T cells isolated from patients, indicating that the virus had escaped detection by these cells. These newly identified HDV epitopes were restricted by relatively infrequent HLA class I alleles, and they bound most frequently to HLA-B. In contrast, frequent HLA class I alleles were not associated with HDV sequence polymorphisms. CONCLUSIONS: We analyzed sequences of HDV RNA and HLA class I alleles that present epitope peptides to CD8+ T cells in patients with persistent HDV infection. We identified polymorphisms in the HDV proteome that associate with HLA class I alleles. Some variant peptides in epitopes from HDV were only partially recognized by CD8+ T cells isolated from patients; these could be mutations that allow HDV to escape the immune response, resulting in persistent infection. HDV escape from the immune response was associated with uncommon HLA class I alleles, indicating that HDV evolves, at the population level, to evade recognition by common HLA class I alleles.

Molecular Signature and Mechanisms of Hepatitis D Virus-Associated Hepatocellular Carcinoma.

There is limited data on the molecular mechanisms whereby hepatitis D virus (HDV) promotes liver cancer. Therefore, serum and liver specimens obtained at the time of liver transplantation from well-characterized patients with HDV-HCC (n = 5) and with non-HCC HDV cirrhosis (n = 7) were studied using an integrated genomic approach. Transcriptomic profiling was performed using laser capture-microdissected (LCM) malignant and nonmalignant hepatocytes, tumorous and nontumorous liver tissue from patients with HDV-HCC, and liver tissue from patients with non-HCC HDV cirrhosis. HDV-HCC was also compared with hepatitis B virus (HBV) HBV-HCC alone, and hepatitis C virus (HCV) HCV-HCC. HDV malignant hepatocytes were characterized by an enrichment of upregulated transcripts associated with pathways involved in cell-cycle/DNA replication, damage, and repair (Sonic Hedgehog, GADD45, DNA-damage-induced 14-3-3σ, cyclins and cell-cycle regulation, cell cycle: G2-M DNA-damage checkpoint regulation, and hereditary breast cancer). Moreover, a large network of genes identified functionally relate to DNA repair, cell cycle, mitotic apparatus, and cell division, including 4 cancer testis antigen genes, attesting to the critical role of genetic instability in this tumor. Besides being overexpressed, these genes were also strongly coregulated. Gene coregulation was high not only when compared with nonmalignant hepatocytes, but also to malignant hepatocytes from HBV-HCC alone or HCV-HCC. Activation and coregulation of genes critically associated with DNA replication, damage, and repair point to genetic instability as an important mechanism of HDV hepatocarcinogenesis. This specific HDV-HCC trait emerged also from the comparison of the molecular pathways identified for each hepatitis virus-associated HCC. Despite the dependence of HDV on HBV, these findings suggest that HDV and HBV promote carcinogenesis by distinct molecular mechanisms.Implications: This study identifies a molecular signature of HDV-associated hepatocellular carcinoma and suggests the potential for new biomarkers for early diagnostics. Mol Cancer Res; 16(9); 1406-19. ©2018 AACR.

Prevalence and molecular characterization of hepatitis D virus in Saudi Arabia: A single-center study.

BACKGROUND/AIMS: Hepatitis D virus (HDV) is a defective RNA virus that is dependent on hepatitis B surface antigen (HBsAg) for transmission and replication. HDV significance arises from the possibility of poor prognosis of hepatitis B virus (HBV) infection. In Saudi Arabia, HDV prevalence varied from 8 to 32% before the HBV vaccination program and ranged from 0 to 14.7% after the vaccination program was started. The last study, performed in 2004, showed a prevalence of 8.6% in hospital-based HBV cases and 3.3% in healthy donors. The aim of this study was to investigate the prevalence and molecular characterization of HDV in chronic hepatitis B (CHB) patients at the King Abdulaziz University Hospital in Jeddah, Saudi Arabia by molecular and serological techniques. To the best of our knowledge, this is the first study to detect HDV at the molecular level in Saudi Arabia. PATIENTS AND METHODS: The study included samples from 182 CHB patients from Jeddah; 13 samples with HBsAg negative were excluded. Samples were tested for HDV-Ab, viral RNA by reverse transcriptase-polymerase chain reaction (RT-PCR) in the HDV L-Ag region and sequence analysis. RESULTS: The mean age of the participants was 44.36 years; 75.1% of the participants were Saudi nationals, 58% were males. Nine samples were positive for HDV-Ab and four were borderline; all were subjected to RT-PCR amplification. Three of the positive HDV-Ab cases and 1 borderline case were positive by RT-PCR. All the positive cases had HBV genotype D, and the positive RT-PCR cases were positive for HBV DNA. One of the HDV viremic samples was of genotype 1 by sequencing. The prevalence of HDV in the study was 7.7%, which was lower in Saudis (6.3%) than in non-Saudis (11.9%). CONCLUSION: HDV coinfection does not seem to have an effect on the clinical status of the recruited CHB cases in this study. More studies are needed to investigate the genetic diversity in other areas such as the southern parts of the Kingdom.

Interleukin-28 gene polymorphisms may contribute to HBsAg persistence and the development of HBeAg-negative chronic hepatitis B.

BACKGROUND & AIMS: Aim of this study was to investigate whether a potential association exists between several single nucleotide polymorphisms (SNPs) of the IL-28B gene (rs12979860, rs1188122, rs8099917, rs8105790, rs12980275) and HBsAg persistence. Further, a potential effect on the development of HBeAg-negative CHB vs. inactive HBsAg carrier state was assessed in a genotype D HBV cohort. A cohort of chronic HDV patients was also used to see if they behave differently compared to chronic HBV patients. METHODS: This study was conducted in three main patient cohorts: Group 1 consisted of 482 patients with HBsAg persistence. Of them 143 were inactive carriers, 94 had HBeAg-positive chronic hepatitis B (CHB) and 245 had anti-HBe-positive CHB. Group 2 represents spontaneously recovered HBV patients; they were anti-HBs and anti-HBc positive. Group 3 consisted of 176 chronic hepatitis delta (CHD) patients with antidelta and HDV-RNA positivity. DNA sequencing was performed for genotyping. RESULTS: When patients with HBsAg persistence were compared with spontaneously recovered patients, a significant difference was observed for rs8105790 (P < 0.0001), rs12980275 (P < 0.02). Patients who had the CC/TC genotype for rs8105790 (P < 0.0001) and AA genotype for 1188122 (P < 0.02) were more likely to be inactive HBsAg carriers, when inactive HBsAg carriers were compared with HBeAg-negative CHB patients. Comparison of CHD patients vs. recovered HBV patients was parallel to that of HBV persistence vs. recovered HBV with similar significant differences in same SNPs. CONCLUSION: These results suggest that IL-28B polymorphisms may contribute to HBsAg persistence and the development of the inactive HBsAg carrier state.

Chronic HDV/HBV co-infection: predictors of disease stage---a case series of HDV-3 patients.

BACKGROUND & AIMS: Chronic HDV/HBV co-infection is perhaps the most intriguing amongst all viral hepatitis. Only few studies focus deeply on this topic, particularly with patients infected with HDV-3. This study aimed to identify predictors of advanced disease, examining a cross-sectional data of 64 patients. METHODS: Histological grading was used to characterize the disease stages and viral loads were tested as predictors of necroinflammatory activity and fibrosis. RESULTS: We identified three HDV/HBV co-infection patterns: patients with predominant HDV replication (56.3%), patients with similar viral loads of both viruses (40.6%), and patients with predominant HBV replication (3.1%). Mean HDV-RNA showed a positive trend regarding inflammatory activity and grade of fibrosis. HDV viral load correlated positively with serum levels of liver enzymes and inversely with platelets count. HBV viral load showed no correlation with any of the above parameters. Advanced fibrosis was associated with age, splenomegaly, and HDV viral load of more than 2 log10. Multiple logistic regression confirmed the independent effect of HDV viral predominance. Advanced necroinflammatory activity was independently associated with HDV viral load and splenomegaly. CONCLUSIONS: HDV may possibly play an important and direct role in the establishment of necroinflammatory activity and fibrosis. Data show an indigenous HDV genotype, HDV-3, similar to those described in the Amazon region.

Tumour necrosis factor-alpha, interleukin-10, interferon-gamma and vitamin D receptor gene polymorphisms in patients with chronic hepatitis delta.

No data exist to assess certain polymorphisms that have a potential effect on the immune response in patients with chronic hepatitis delta (CHD). The aim of this study was to investigate polymorphisms in 6 polymorphic sites: IL-10 -1082 (rs1800896), IL-10 -627 (rs1800872), IFN-γ +874 (rs62559044), TNF-α -308 (rs1800629), vitamin D receptor (VDR) FokI (rs2228570) and VDR TaqI (rs731236). The genotypes of 67 patients with CHD and 119 patients with chronic hepatitis B (CHB) were compared. In addition, 56 individuals with resolved hepatitis B virus (HBV) infection were used as a control group for patients with CHB. Polymorphisms in TNF-α, IL-10, and VDR genes were analysed using polymerase chain reaction/restriction fragment length polymorphism methods. The IFN-γ gene polymorphism was detected by allele-specific polymerase chain reaction (PCR). Patients with CDH were more likely to have advanced liver disease compared with patients with CHB (P < 0.0001). IL-10 -1082 and VDR TaqI polymorphisms showed significant differences between patients with CHD and CHB. The high secretory IL-10 -1082 genotype GG was less frequent in CHD compared with patients with CHB and resolved HBV (17.7%, 37.4% and 47.1%, respectively (P < 0.05 for CHD vs CHB and resolved HBV). The frequency of the high secretory VDR TaqI TT genotype was 86.6% in patients with CHD, 62.7% in patients with CHB and 62.5% in resolved HBV individuals (CHD vs CHB: P < 0.05). None of the polymorphisms analysed had an effect on HBV persistence. IL-10 -1082 and VDR TaqI polymorphisms may contribute to the more severe liver disease associated with CHD compared with CHB.

No impact of interleukin-28B polymorphisms on spontaneous or drug-induced hepatitis delta virus clearance.

BACKGROUND: rs12979860 and rs8099917 interleukin-28B polymorphisms are associated with spontaneous or interferon-alpha induced hepatitis C clearance, "CC" and "TT" genotypes (respectively) being the most favourable. There are no data on the influence of interleukin-28B polymorphisms on hepatitis delta clearance in hepatitis B/D co-infected patients. AIMS: The present study explores the potential influence of both rs12979860 and rs8099917 polymorphisms on delta infection outcome. METHODS: Retrospective-longitudinal study on 55 European patients observed for at least 4 years, selected from a cohort of 439 subjects positive for hepatitis delta antibodies and hepatitis B core antibodies. The rate of spontaneous and interferon induced delta-RNA clearance was compared in interleukin-28B rs12979860 "CC" vs "non CC", and in rs8099917 "TT" vs "non TT" genotypes. RESULTS: Prevalence of rs12979860C allele was 60%, consistent with the reported prevalence in Italy (67%, p=0.128). No significant differences in spontaneous clearance rate were observed between rs12979860 "CC" and "non CC" genotypes (13.3% vs 7.5%, respectively, p=0.60), and between rs8099917 "TT" and "non-TT" genotypes (11.1 vs 7.1%, respectively, p=0.67). No differences were observed for interferon-induced delta-RNA clearance either. CONCLUSIONS: Our data suggest that interleukin-28B polymorphisms might not influence hepatitis delta clearance rate in either natural history or interferon-alpha response.

Interferon-α for patients with chronic hepatitis delta: a systematic review of randomized clinical trials.

BACKGROUND: Hepatitis delta virus (HDV) infection therapy is unclear. This systematic analysis aimed to clarify the evidence on the efficacy of interferon (IFN)-α-based therapy in HDV. METHODS: We performed a systematic search on electronic databases including MEDLINE (1970 to January 2011), Web of Science, The Cochrane Library and ClinicalTrials.gov. Randomized clinical trials (RCTs) comparing IFN-α-based therapy with either another drug, placebo or no intervention were included. We excluded paediatric studies. We calculated relative risks (RRs) for comparison of treatment options on the primary outcome measure, which was defined as undetectable levels of HDV RNA and normal alanine aminotransferase at end of treatment (EOT; 1 year). RESULTS: Nine RCTs were included. Seven trials evaluated the treatment with IFN-α (n=132). The remaining two trials evaluated treatment with pegylated (PEG)-IFN-α (n=45). We found that 1-year treatment with high-dose IFN-α achieved better primary outcome rates than with PEG-IFN-α (RR=4.14, 95% CI 1.00, 17.14). Data for 1-year treatment with low-dose IFN-α compared with PEG-IFN-α were similar (RR=2.83, 95% CI 0.65, 12.40), as were low-dose IFN-α versus high-dose IFN-α (RR=0.68, 95% CI 0.31, 1.50). High-dose IFN-α and PEG-IFN-α reached similar HDV RNA suppression 24 weeks after EOT (RR=1.00, 95% CI 0.51, 1.97). None of the 55 patients assigned to no intervention obtained undetectable levels of HDV RNA and only one patient achieved normalization of alanine aminotransferase level. CONCLUSIONS: Based on available RCTs, 1-year high-dose IFN-α monotherapy appears to be more effective than PEG-IFN-α for treatment of HDV patients, with efficacy rates of approximately 30%. There is a lack of head-to-head comparisons. Combination therapies and longer treatment duration need to be investigated.

Quantitative longitudinal evaluations of hepatitis delta virus RNA and hepatitis B virus DNA shows a dynamic, complex replicative profile in chronic hepatitis B and D.

BACKGROUND & AIMS: This study presents a real-time reverse-transcription PCR (rt-RT-PCR) assay for hepatitis delta virus (HDV) RNA quantification, designed to clarify the interplay between HDV and hepatitis B virus (HBV) in chronic infection. METHODS: Serum HDV-RNA and HBV-DNA were analysed by rt-RT-PCR in a cross-sectional study of 37 untreated chronic HDV patients, 25 of whom were also longitudinally studied. RESULTS: In the cross-sectional study, both viruses were active in 15 (40.5%) patients and inactive in 4 (10.8%); HDV alone was active in 12 (32.4%) and HBV in 6 (16.2%). The longitudinal study showed seven replication profiles, with considerable fluctuating activity of one or both viruses, including alternating predominance. In 20% of cases, longitudinal HBV/HDV viral loads differed from cross-sectional results, indicating a risk of misinterpreting HBV/HDV interactions when assessing a single determination. Fluctuating HBV replication only increased in the presence of fluctuating HDV activity. HBsAg levels, stable in HBV single infection, fluctuated in HDV chronic infection. The results of both the cross-sectional and longitudinal study call into question the major suppressor effect of HDV over HBV, revealing an important role of HBV. CONCLUSIONS: Longitudinal evaluation of viremia shows a complex interaction between HBV and HDV and is essential to understand the pathophysiology of chronic HDV infection.

Virological and clinical implication of core promoter C1752/V1753 and T1764/G1766 mutations in hepatitis B virus genotype D infection in Mongolia.

BACKGROUND AND AIM: The aim of the present study was to reveal virological and clinical features of hepatitis B virus (HBV) genotype D infection. METHODS: One hundred and twenty-two Mongolian chronic liver disease (CLD) patients infected with HBV were subjected for serological HBV-markers screening and HBV-enzyme immunoassay (EIA) genotyping. Nucleotide sequences were analyzed for 48 HBV/D strains (23 isolated from hepatocellular carcinoma (HCC) and 25 from CLD patients). RESULTS: Prevalence of hepatitis B e antigen (HBeAg) positivity was low (25.9%) in young patients (< or =30 years old) indicating early HBeAg seroclearance in HBV/D carriers. The T1764/G1766 double mutation was the most common basal core promoter (BCP) mutation (29.2%) and was frequent in HBeAg-negative patients (39.3%). Patients harboring T1764/G1766 mutants exhibited lower HBV-DNA and HBV core antigen (HBcAg) levels than those with wild-type BCP strains (P = 0.024, 0.049, respectively). C1752 and/or V (not T) 1753 mutation was significantly prevalent in HCC patients (HCC vs CLD; 52.2% vs 20%, P = 0.033). T1762/A1764 mutation was detected in 75.0% of HCC patients with high viral load (> or =5 log copies/mL). Precore stop codon mutation A1896 was detected in (70.8%) of HBV/D-infected patients. CONCLUSIONS: In Mongolians infected with HBV/D, C1752 and/or V1753 mutation was associated with HCC.

Genetic changes in hepatitis delta virus from acutely and chronically infected woodchucks.

A woodchuck-derived hepatitis delta virus (HDV) inoculum was created by transfection of a genotype I HDV cDNA clone directly into the liver of a woodchuck that was chronically infected with woodchuck hepatitis virus. All woodchucks receiving this inoculum became positive for HDV RNA in serum, and 67% became chronically infected, similar to the rate of chronic HDV infection in humans. Analysis of HDV sequences obtained at 73 weeks postinfection indicated that changes had occurred at a rate of 0.5% per year; many of these modifications were consistent with editing by host RNA adenosine deaminase. The appearance of sequence changes, which were not evenly distributed on the genome, was correlated with the course of HDV infection. A limited number of modifications occurred in the consensus sequence of the viral genome that altered the sequence of the hepatitis delta antigen (HDAg). All chronically infected animals examined exhibited these changes 73 weeks following infection, but at earlier times, only one of the HDV carriers exhibited consensus sequence substitutions. On the other hand, sequence modifications in animals that eventually recovered from HDV infection were apparent after 27 weeks. The data are consistent with a model in which HDV sequence changes are selected by host immune responses. Chronic HDV infection in woodchucks may result from a delayed and weak immune response that is limited to a small number of epitopes on HDAg.

Genotypes and viremia of hepatitis B and D viruses are associated with outcomes of chronic hepatitis D patients.

BACKGROUND & AIMS: Genotypes and viremia of hepatitis D virus (HDV) and hepatitis B virus (HBV) may be associated with outcomes. This study evaluated the impact of viral genotypes and viremia on outcomes of dual HBV and HDV infection. METHODS: Viremia and viral genotypes were analyzed in 194 consecutive chronic hepatitis B patients with HDV superinfection and correlated with outcomes. RESULTS: The numbers of HBV genotype A, B, C, and nonclassified were 4, 57, 23, and 110, respectively. There were 51 genotype I HDV, 74 genotype II HDV, 8 genotype IV HDV, and 61 nonclassified HDV genotype. In a median follow-up of 135 months, 24 progressed to cirrhosis and 41 developed hepatocellular carcinoma. Patients infected with genotype I HDV had a lower remission rate (15.2% vs 40.2%; P = .007) and more adverse outcomes (cirrhosis, hepatocellular carcinoma, or mortality) (52.2% vs 25.0%; P= .005) than those with genotype II HDV. Patients infected with genotype C HBV had a lower remission rate (0 vs 32.1%; P = .005) and more adverse outcomes (70.0% vs 33.9%; P = .005) than those with genotype B HBV. The presence of HBV or HDV viremia was associated with lower remission rates compared with those negative for both (26.4% and 24.3% vs 69.2%; P < .001). In multivariate analysis, age, genotype C HBV, and genotype I HDV were independent factors associated with adverse outcomes. CONCLUSIONS: In chronic HBV and HDV dual infections, older age, genotype I HDV, and genotype C HBV correlated with adverse outcomes.