Background and aim: Serum HBcrAg has been proposed as a useful biomarker in the management of HBV patients, however its role in patients with chronic hepatitis Delta (CHD) under treatment with Bulevirtide (BLV) is currently unknown.Aim: of the study was to investigate HBcrAg levels in CHD patients treated with BLV monotherapy.Methods: CHD patients treated with BLV 2 mg/day monotherapy in a single Center were enrolled. Serum HBcrAg levels were quantified by LUMIPULSE® G HBcrAg assay (Fujirebio Europe, LLOQ 3 Log10 U/mL). Virological (HDV-RNA undetectable or ≥2 Log10 decline vs. baseline), biochemical (ALT<40 U/L) and combined response (biochemical+virological) were assessed.Results: 405 serum samples were collected from 79 patients treated with BLV up to W144. At BLV start, median age was 55 (IQR 37-43) years, 53% males, 100% with cirrhosis (CPT-A score A in 87%), 100% NUC-treated, median ALT 97 (52-151) U/L, HBsAg 3.7 (3.4-3.9) Log10IU/mL, 92% HBeAg-negative, 90% HBV DNA undetectable, median HDV RNA 5.5 (4.2-6.2) Log10IU/mL. HBcrAg tested positive in 63 (80%) patients [4.0 (3.5-4.8) Log10 U/mL]. HBcrAg levels positively correlated with HBsAg (r=0.37, p=0.002) and HDV RNA levels (r=0.30, p=0.01), but not with ALT or HBV DNA. After 144 weeks of BLV monotherapy, rates of virological, biochemical, combined response and HDV RNA undetectability were 70%, 64%, 52% and 33%, respectively. HBcrAg levels significantly declined during therapy: -0.31 Log10 at week 48 (p=0.02), -0.63 Log10 at week 96 (p=0.003), and -0.62 Log10 at week 144 (p=0.01). During treatment, HBcrAg levels did not correlate with HDV RNA levels at single on-treatment timepoints but maintained a positive correlation with HBsAg levels over-time at all on-treatment timepoints (W48: r=0.35, p=0.007; W96: r=0.45, p=0.006; W144: r=0.50, p=0.01). Neither baseline nor on-treatment HBcrAg levels were associated with virological response or HDV RNA undetectability rates during therapy. Overall, HBcrAg levels became negative in 10 patients (week 48 in n=5, week 96 in n=3; week 144 in n=2): at the time of HBcrAg negativity, 7 had achieved a virological response (HDV RNA undetectable in 3), while 3 patients were suboptimal responders (<2 Log10 HDV RNA decline). Conversely, HBcrAg levels became positive during treatment in 5 patients who tested negative at baseline (<3.5 Log10 U/mL in all cases; n=4 at week 48, n=1 at week 144): at the time of HBcrAg positivity, 3 had achieved a virological response (HDV RNA undetectable in n=2), while 2 patients were suboptimal responders. During BLV monotherapy, no significant changes of HBsAg levels were observed.Conclusions: In CHD patients with cirrhosis treated with BLV monotherapy up to 144 weeks, baseline low HBcrAg levels were detectable in most patients and correlated with HBsAg and HDV RNA levels. During BLV, HBcrAg levels significantly declined and correlated with HBsAg levels, but not with HDV virological responses.

BACKGROUND:Quantitative hepatitis B surface antigen (qHBsAg) is a routine marker for monitoring patients with chronic hepatitis B (CHB) or Delta (CHD). New commercially available assays require independent evaluation in clinical laboratories before being adopted for routine use. This study assessed the performance of the new Siemens Atellica IM Quantitative HBsAg (QHBs) assay in patients with CHB or CHD. METHODS:Over a six-month period, all serum specimens from patients with CHB or CHD that were submitted to the laboratory for qHBsAg testing were prospectively analyzed using both the historically used Architect assay and the new Atellica assay. Furthermore, in a retrospective longitudinal analysis, we examined CHD patients who showed a reduction of at least 1 log10 UI/mL in HBsAg levels over the course of treatment. RESULTS:A total of 311 serum samples from 216 patients were prospectively evaluated. The Architect assay yielded slightly higher values than the Atellica assay (mean difference: +0.10 log₁₀ IU/mL). There was excellent agreement and correlation between the two assays (Spearman's Rs = 0.99; 95 % CI [0.9877-0.9922]). Three samples with low HBsAg concentrations (0.02-0.07 IU/mL) were detected by the Atellica assay but not by the Architect assay. In the longitudinal follow-up of five CHD patients (82 samples), both assays produced comparable results and could be used interchangeably without any potential clinical impact. CONCLUSION:The Atellica assay demonstrated equivalent performance to the Architect assay for qHBsAg monitoring in patients with CHB or CHD.

Background and Aims:Novel antiviral approaches capable of permanently inactivating the intrahepatic HBV DNA reservoir, the covalently closed circular DNA (cccDNA) and HBV DNA integrated into the host genome, are urgently needed. This study evaluated adenine base editing as a strategy to disrupt HBV replication by introducing mutations in the overlapping HBs/polymerase open reading frame (ORF). Methods:An adenine base editor (ABE) and 3 guide RNAs (gS1-gS3) were designed to introduce missense mutations within the HBs/polymerase ORF. ABE mRNA and individual gRNAs were co-transfected into HBV-infected HepG2-hNTCP cells and primary human hepatocytes. Antiviral efficacy was further assessed in HepG2.2.15 and PLC/PRF/5 cells harboring integrated HBV DNA. In vivo, lipid nanoparticles (LNP)-mediated delivery of ABE mRNA and gRNAs was evaluated in HBVcircle DNA-transduced mice and in HBV-infected human liver-chimeric mice. The impact of HBs editing on hepatitis D virus (HDV) release was assessed using PLC/PRF/5 and Huh7 cell-based HDV replication models. Results:Adenine base editing efficiently reduced HBsAg production and HBV replication in vitro by targeting both cccDNA and integrated HBV DNA. A single LNP injection of ABE-gS2 resulted in undetectable HBsAg in HBVcircle mice, while two injections achieved a 90% reduction in serum HBsAg in HBV-infected human liver chimeric mice. HBV DNA replication was also inhibited in vivo. Furthermore, HBs ORF base editing markedly suppressed HDV release in vitro. Conclusions:Adenine base editing of the HBs ORF effectively impairs HBV replication and HBsAg production in vitro and in vivo and concomitantly inhibits HDV release, highlighting its therapeutic potential.

Early diagnosis of Hepatitis C virus (HCV) infection is a cornerstone of elimination strategies. Fourth-generation antigen/antibody combination assays, such as Elecsys HCV Duo, promise to bridge the diagnostic gap left by antibody-only testing. In this real-world study, we assessed Elecsys HCV Duo performance using seroconversion panels and routine clinical samples, focusing on sensitivity, specificity, and positive predictive value (PPV) for active HCV detection, including challenging scenarios with low viral loads and HIV co-infections. The assay shortened the serological window by up to 203 days. When both antigen and antibody were detected, PPV reached 100%, enabling confident diagnosis without immediate RNA confirmation. The antigen component alone achieved a PPV of 97.6%, even in a low-prevalence setting (0.33%), better than previously reported assays. However, sensitivity for low viral loads (≤ 5 log10 IU/mL) was limited, with 95.5% of such cases missed. Elecsys HCV Duo offers a powerful tool for earlier detection and streamlined care, particularly in high-risk and resource-limited contexts. Yet, confirmatory RNA testing remains essential for low viral load cases. Broader validation is needed to confirm its utility in diverse populations, reinfection surveillance, and cost-effective screening strategies.

Hepatitis delta (HDV) infection affects 5% of hepatitis B (HBV)-positive patients and is associated with an increased risk of cirrhosis and hepatocellular carcinoma; however, it remains underdiagnosed. The first part of our Delta Describe study highlights the insufficient level of HDV screening among patients in metropolitan France. In this study, we report on their real-world management. Patients with at least one positive HDV RNA test performed in 2019 were identified through the major public and private laboratories in France. From January 2024 to July 2025, informed patients were interviewed, and physicians supplemented the collected data. A total of 547 patients were included, with a median age of 44 years; most originated from Africa or Eastern Europe. HIV and hepatitis C coinfections were reported in 15.2% and 4.6% of patients, respectively. Liver fibrosis was primarily assessed using FibroScan®. Most patients knew the year of their delta diagnosis, and 69.1% knew their fibrosis stage. Liver-related events occurred in 14.3% (67/468) of patients, mainly comprising portal hypertension (61.6%), liver failure (12.3%), and hepatocellular carcinoma (26%), and 45 patients (45/468) underwent liver transplantation. At the time of the survey, 47.1% of the patients reported undetectable HDV RNA; 40.6% (222/547) had currently or previously undergone BLV treatment. Among patients receiving ongoing treatment for HDV at the time of the survey, 84.8% were receiving nucleos(t)ide analogs (NUCs). In metropolitan France, HDV patients had access to specialized follow-up care and innovative therapies (bulevirtide), were mostly on NUCs, and demonstrated good disease awareness.

CAR T cell therapy has recently become a major treatment in hematology for lymphoma and myeloma patients. Following treatment, patients remain at risk of complications, including infections. Here, we report on a -81 year-old female patient who developed late HBV reactivation following CAR T cell.

Background & Aims:HBsAg loss improves clinical outcomes in persons with HIV and HBV coinfection. We aimed to evaluate if hepatitis B core-related antigen and circulating HBV RNA levels were associated with HBsAg loss in Euro-B, a multi-cohort collaboration including data from the Swiss HIV Cohort Study, EuroSIDA, and the French HIV/HBV cohort. Methods:We included persons with HIV, a positive HBsAg, and ≥6 months of follow-up on tenofovir-containing antiretroviral therapy. We evaluated quantitative HBsAg, HBV DNA, hepatitis B core-related antigen, and HBV RNA levels over time and assessed HBsAg loss (i.e. quantitative HBsAg <0.05 IU/ml) during tenofovir therapy. Results:Among 599 participants median age was 41 years (IQR 35-47), 18.4% were female and 47.3% HBeAg-positive. We observed HBsAg loss in 12.9% of participants after 2 years and in 18.2% during a median follow-up of 8.2 years (IQR 3.6-13.1). Individuals who were HBeAg-negative were more likely to have a negative hepatitis B core-related antigen and HBV RNA below the detection limit than participants who were HBeAg-positive. Quantitative HBsAg ≤1,000 IU/ml at baseline was the strongest predictor of HBsAg loss regardless of HBeAg status. Additionally, HBsAg loss was associated with lower baseline HBV RNA levels (odds ratio 0.66, 95% CI 0.49-0.88) and higher baseline HBV DNA levels in participants who were HBeAg-positive. Conclusions:In this European cohort of persons with HIV/HBV, 18% experienced HBsAg loss during tenofovir-containing antiretroviral therapy. In addition to low baseline quantitative HBsAg levels, HBV RNA may predict HBsAg loss in individuals who are HBeAg-positive. Impact and implications:The present study builds on a multi-cohort collaboration including persons with HIV/HBV from Europe. It provides estimates on the probability of HBsAg loss during long-term tenofovir-containing antiretroviral therapy and describes the potential of the novel biomarkers HBV RNA and hepatitis B core-related antigen as its predictors. The discrepancies regarding HBV RNA and HBcrAg levels before and during therapy observed between persons who were HBeAg-negative and HBeAg-positive with HIV/HBV may influence treatment decisions and the development of new treatment strategies. Clinical Trials Registration:The study is registered at ClinicalTrials.gov (NCT04984772).

Background & Aims Serum HBV RNA and hepatitis B core-related antigen (HBcrAg) levels have been proposed as useful biomarkers in the management of patients with HBV; however, their role in chronic hepatitis delta (CHD) is currently unknown. Methods Consecutive untreated patients with CHD were enrolled in a cross-sectional study in three EU centers. Clinical and virological characteristics were collected. Serum HBV RNA and HBcrAg levels were quantified by an automated real-time investigational assay (Cobas (R) 6800, Roche Diagnostics, Pleasanton, Ca, USA) and by LUMIPULSE (R) G HBcrAg assay (Fujirebio Europe), respectively. In 18 patients with available liver biopsies, intrahepatic analyses were performed. Results Overall, 240 patients with HDV were enrolled: median age 46 years, 62% male, 53% with cirrhosis, 57% nucleos(t)ide analogue treated, median ALT 70 U/L, median HBsAg 3.8 log(10) IU/ml, 88% HBeAg negative, and median HDV RNA 4.9 log(10) IU/ml. HBV RNA was positive (>10 copies/ml) in only 8% of patients (median 40 [13-82,000] copies/ml), whereas HBcrAg was >= 3 log(10) U/ml in 77% (median 4.2 [3.0-8.0] log(10) U/ml). By combining these biomarkers, three categories were identified: 23% double negative (HBV RNA/HBcrAg), 9% double positive (HBV RNA/HBcrAg) and 68% HBV RNA negative/HBcrAg positive. HBV RNA levels positively correlated with male sex and detectable HBV DNA, while positive HBcrAg correlated with higher HBsAg levels. Double-positive patients were younger, non-European, with elevated ALT and HDV RNA levels and detectable HBV DNA. Intrahepatic HDV RNA and HBV RNA were positive in most samples, while intrahepatic levels of covalently closed circular DNA were low. Conclusions In untreated CHD, most patients had undetectable HBV RNA but quantifiable HBcrAg ("divergent pattern") in the absence of HBeAg. Additional studies aiming to unravel the molecular mechanisms underlying these findings are warranted.

Objectives HIV, HBV, and HCV infections remain major public health concerns, particularly among vulnerable populations. Free-of-charge healthcare units provide essential care for individuals facing barriers to healthcare. However, systematic testing remains inconsistent. The DePASS initiative aimed to promote routine screening, assess infection seroprevalences, and identify barriers and facilitators to implementation. Methods This multicenter prospective observational study was conducted in 12 free-of-charge healthcare units across mainland France. Staff received training on HIV/HBV/HCV screening. Over a 9-month period, units offered HIV/HBV/HCV testing to all attendees, with HDV testing for HBV-positive individuals. Diagnoses and linkage to care were quantitatively assessed. A qualitative survey explored feasibility, acceptability, and perceived barriers among staff. Results Between December 2022 and May 2024, 6,602 individuals were tested. Seroprevalence was 1.76% for HIV, 2.32% for HCV, and 5.76% for HBV; 6.53% of HBV-positive individuals also tested positive for HDV. Among those with HIV, 90.4% were linked to care. Staff cited logistical constraints, limited time, and language barriers as major challenges. Conclusions Systematic HIV/HBV/HCV testing yielded diagnosis rates 7 to 9 times higher than those estimated in the general French population. Scaling up this model, combined with staff training and structural support, could substantially reduce undiagnosed infections in vulnerable people.

BackgroundCirculating HBV RNAs have been proposed as a biomarker that reflects the transcriptional activity of cccDNA and may help to evaluate HBV treatments activity. Different research assays have been proposed and, although 2 PCR-based research use only (RUO) investigational assays (IA) have been developed, the lack of standardized protocols represents an important limitation. Here we have designed and generated a stable clonal cell line producing an RNA-based standard for the calibration of PCR-based circulating HBV RNA assays.MethodsHBV RNA producing Huh7-derived stable cell lines were generated by transfecting pTriEX plasmids containing 1.1 length HBV DNA genomes carrying mutations in the catalytic site (YMAA mutation) and the TP-domain (Y63F) of the polymerase, and the ε-loop of the pgRNA (mutation A1G).ResultsThe clonal cell line (Huh7-3D29), carrying a double YMAA and Y63F mutation, displayed, and maintained over several passages in culture, a high RNA secretion phenotype with negligible residual secreted HBV DNA. Density gradient centrifugation showed that most of the secreted HBV RNA from Huh7-3D29 cells was detected in naked capsid and virions-like particles and only a minority in small extracellular vescicles (sEV). Nanopore sequencing of 5’RACE products shows that the majority of the Huh7-3D29 secreted HBV RNAs start at the 5' end of pgRNA and pgRNA derived spliced RNAs. Finally, Huh7-3D29 cells showed a high and up-scalable secreted RNA yield allowing 1,300 standard curves in 9 days from 1 flask.ConclusionWe generated a clonal cell line that produces high amounts of HBV RNAs with very low quantities of contaminating HBV DNAs, representing a stable source of RNA standard for HBV RNA assays calibration.Impact and ImplicationsSeveral investigational assays and 2 research use only (RUO) assays have been developed to detect and quantify circulating HBV RNAs, an emerging biomarker of cccDNA transcriptional activity and target engagement by new HBV treatments. The lack of a unique molecular standard for circulating HBV RNAs quantification represents an important limitation. Here we describe the generation of a stable clonal cell line producing and secreting an RNA-based standard containing all the HBV RNA species found in HBV patients’ sera (e.g., pgRNA, HBx transcripts). This new RNA standard will serve for the calibration of all PCR-based assays for circulating HBV RNA quantification to evaluate in a non-invasive manner the size of the transcriptionally active cccDNA pool and the activity of novel strategies aiming at curing HBV infection.

Background & AimsTranscription termination fine tunes gene expression and contributes to specify the function of RNAs in eukaryotic cells. Transcription termination of hepatitis B virus (HBV) is subjected to the recognition of the canonical polyadenylation signal (cPAS) common to all viral transcripts. The regulation of the usage of this cPAS and its impact on viral gene expression and replication is currently unknown.Approach & ResultsTo unravel the regulation of HBV transcript termination, we implemented a 3’ RACE-PCR assay coupled to single molecule sequencing both in in vitro infected hepatocytes and in chronically infected patients. The detection of a previously unidentified transcriptional readthrough indicated that the cPAS was not systematically recognized during HBV replication in vitro and in vivo. Gene expression downregulation experiments demonstrated a role for the RNA helicases DDX5 and DDX17 in promoting viral transcriptional readthrough, which was, in turn, associated to HBV RNA destabilization and decreased HBx protein expression. RNA and chromatin immunoprecipitation, together with mutation of cPAS sequence, suggested a direct role of DDX5 and DDX17 in functionally linking cPAS recognition to transcriptional readthrough, HBV RNA stability and replication.ConclusionsOur findings identify DDX5 and DDX17 as crucial determinants for HBV transcriptional fidelity and as host restriction factors for HBV replication.Impact and implicationsHepatitis B virus (HBV) covalently closed circular (ccc)DNA degradation or functional inactivation remains the holy grail to be attained to achieve HBV cure. Transcriptional fidelity is a cornerstone in gene expression regulation. Here, we demonstrate that two helicases, DDX5 and DDX17, inhibit the recognition of HBV polyadenylation signal and transcriptional termination, thus decreasing HBV RNA stability and acting as restriction factors for efficient cccDNA transcription and viral replication. The observation that DDX5 and DDX17 are downregulated in HBV chronically infected patients suggests a role for the helicases in HBV persistence in vivo. These results open new perspectives for researchers aiming at identifying new targets to neutralise cccDNA transcription.

Background and AimsIn France, Bulevirtide (BLV) was available in September 2019 through an early access program to treat patients with hepatitis Delta virus (HDV). The aim of this analysis was to evaluate the efficacy and safety of BLV in HIV patients with HDV coinfection.Patients and methodsPatients received BLV 2 mg +/- pegylated interferon (pegIFNα) according to the physician’s decision. The primary endpoint (per-protocol analysis) was the virological response rate at week 48, defined as the proportion of patients with undetectable serum HDV-RNA or HDV-RNA decline > 2 log10 IU/mL from baseline.ResultsCharacteristics of the 38 patients were as follow: 28 male, mean age 47.7 years, mean baseline HDV-RNA viral load 5.7 ± 1.2 log10 IU/ml. Median HIV viral load and mean CD4 count were 32 (30 - 65) cp/ml and 566 ± 307/mm3, respectively. Eight patients stopped treatment before week 48. At W48, 10 of 19 patients (52.6%) in the 2 mg BLV group, and 5 of 7 patients (71.4%) in the 2 mg BLV + pegIFNα group had reached virological response (no HDV-RNA available in 4 patients). At W48, 7/19 patients in the 2 mg BLV group, and 3/6 patients in the 2 mg BLV + pegIFNα group had combined response (virological response and normal ALT level).ConclusionAdults living with HIV coinfected with HDV can be treated by BLV with a virological response in more than 50% of patients. The combination of BLV and pegIFNα showed a strong virological response.IMPACT AND IMPLICATIONS• Bulevirtide is the only EMA-approved drug for HDV treatment and we showed that it can be used in adults living with HIV, with an overall good tolerability.• Bulevirtide induces a virologic response in more than 50% of patients suggesting that Bulevirtide should be considered as a first-line therapy in this specific population.• Bulevirtide in combination with pegIFNα could be used in patients without pegIFNα contra-indication.• No specific drug-drug interaction is reported.

Background We evaluated long-term trajectories of circulating hepatitis B virus (HBV) RNA and hepatitis B core-related antigen (HBcrAg) in persons with and without hepatitis B surface antigen (HBsAg) loss during tenofovir therapy in the Swiss HIV Cohort Study.Methods We included 29 persons with HIV with HBsAg loss and 29 matched persons with HIV without HBsAg loss. We compared HBV RNA and HBcrAg decline and assessed the cumulative proportions with undetectable HBV RNA and HBcrAg levels during tenofovir therapy using Kaplan-Meier estimates.Results HBsAg loss occurred after a median of 4 years (IQR, 1-8). All participants with HBsAg loss achieved suppressed HBV DNA and undetectable HBV RNA preceding undetectable quantitative HBsAg levels, whereas 79% achieved negative HBcrAg. In comparison, 79% of participants without HBsAg loss achieved undetectable HBV-RNA and 48% negative HBcrAg. After 2 years of tenofovir therapy, an HBV RNA decline >= 1 log10 copies/mL had 100% sensitivity and 36.4% specificity for HBsAg loss, whereas an HBcrAg decline >= 1 log10 U/mL had 91.0% sensitivity and 64.5% specificity.Conclusions HBV RNA suppression preceded undetectable quantitative HBsAg levels and had high sensitivity but low specificity for HBsAg loss during tenofovir therapy in persons with HIV. HBcrAg remained detectable in approximately 20% of persons with HBsAg loss and 50% of persons without HBsAg loss. In persons with HIV/hepatitis B virus (HBV) undergoing long-term tenofovir-containing antiretroviral therapy, HBV RNA suppression precedes hepatitis B surface antigen loss. Combining HBV RNA and hepatitis B core-related antigen decline during the first years of tenofovir therapy could improve predictions of hepatitis B surface antigen loss in novel HBV drug trials.

We present a drug design strategy based on structural knowledge of protein-protein interfaces selected through virus-host coevolution and translated into highly potential small molecules. This approach is grounded on Vinland, the most comprehensive atlas of virus-human protein-protein interactions with annotation of interacting domains. From this inspiration, we identified small viral protein domains responsible for interaction with human proteins. These peptides form a library of new chemical entities used to screen for replication modulators of several pathogens. As a proof of concept, a peptide from a KSHV protein, identified as an inhibitor of influenza virus replication, was translated into a small molecule series with low nanomolar antiviral activity. By targeting the NEET proteins, these molecules turn out to be of therapeutic interest in a nonalcoholic steatohepatitis mouse model with kidney lesions. This study provides a biomimetic framework to design original chemistries targeting cellular proteins, with indications going far beyond infectious diseases.

Objective A convenient, reproducible biomarker of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) transcriptional activity is lacking. We measured circulating HBV RNA (cirB-RNA) in untreated and nucleos(t)ide analogues (NUC) treated chronic hepatitis B (CHB) patients to define its correlation with intrahepatic viral markers and HBV core-related antigen (HBcrAg). Design Paired liver biopsy and serum samples were collected from 122 untreated and 30 NUC-treated CHB patients. We measured cirB-RNA, HBV DNA, hepatitis B surface antigen (HBsAg), HBcrAg and alanine aminotransferase levels. cirB-RNA was quantified using an investigational HBV RNA assay for use on the cobas 6800 system. The test detects a region spanning the HBV canonical polyadenylation site. cccDNA and 3.5 kb RNA in liver tissue were assessed by quantitative PCR and droplet digital PCR. Results cirB-RNA was detectable in 100% of HBeAg(+) chronic hepatitis (CH), 57% and 14% of HBeAg(−) CH and chronic infection untreated patients and 47% of NUC-treated patients. cirB-RNA undetectability was associated with lower intrahepatic cccDNA transcriptional activity, as well as serum HBcrAg, but no significant differences in HBsAg, in both untreated and treated patients. In untreated HBeAg(−) patients, cirB-RNA correlated with intrahepatic 3.5 kb RNA and cccDNA transcriptional activity, serum HBV DNA and HBcrAg, but not with HBsAg or total cccDNA levels. Combined undetectability of both cirB-RNA and HBcrAg detection in untreated HBeAg(−) patients identified a subgroup with the lowest levels of intrahepatic transcriptionally active cccDNA. Conclusion Our results support the usefulness of quantification of circulating HBV RNA expressed from cccDNA as an indicator of intrahepatic active viral reservoir in both untreated and NUC-treated CHB patients. Trial registration number NCT02602847 .

Hepatitis B virus (HBV) and hepatitis D virus (HDV) coinfection confers a greater risk for accelerated liver disease progression. Full-length characterization of HDV genome is necessary to understand pathogenesis and treatment response. However, owing to its high variability and tight structure, sequencing approaches remain challenging. Herein, we present a workflow to amplify, sequence, and analyze the whole HDV genome in a single fragment. Sequencing was based on the Oxford Nanopore Technologies long-read sequencing followed by a turnkey analysis pipeline (VIRiONT, VIRal in-house ONT sequencing analysis pipeline) that we developed and make available online for free. For the first time, HDV genome was successfully amplified and full-length sequenced in a single fragment, allowing accurate subtyping from 30 clinical samples. High variability of edition, a crucial step in viral life cycle, was found among samples (from 0% to 59%). Additionally, a new subtype of HDV genotype 1 was identified. We provide a complete workflow for assessment of HDV genome at full-length quasispecies resolution overcoming genome assembly issues and helping to identify modifications throughout the whole genome. This will help a better understanding of the impact of genotype/subtype, viral dynamics, and structural variants on HDV pathogenesis and treatment response.

Bulevirtide has been recently conditionally approved by the European Medicines Agency for the treatment of Chronic Hepatitis Delta, but the ideal duration of therapy is unknown. Here we describe the first case of cure of Hepatitis Delta following 3 years of Bulevirtide monotherapy in a patient with compensated cirrhosis and esophageal varices. During the 72-week off-Bulevirtide follow-up, virological and biochemical responses were maintained. In the off-therapy liver biopsy, intrahepatic HDV RNA and Hepatitis D antigen were undetectable, <1% hepatocytes were Hepatitis B surface antigen positive while hepatitis B core antigen was negative. Grading and staging improved compared to pre-treatment biopsy.