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Background Pregnant women with chronic hepatitis B (CHB) exhibit unique clinical features in terms of postpartum immune system reconstitution and recovery from pregnancy-related changes. However, current studies focus primarily on the outcomes of maternal–infant transmission and postpartum hepatitis flares. We aimed to evaluate the profiles of hepatitis B core-related antigen (HBcrAg) and pregenomic RNA (pgRNA) in pregnant women with CHB. Methods This retrospective analysis included treatment-naïve pregnant women with CHB who were followed up regularly in an outpatient clinic from 2014 to 2021. Baseline HBcrAg and pgRNA levels were compared in patients with different disease phases. Changes in these parameters were examined in a subset of patients receiving antiviral prophylaxis. HBcrAg and pgRNA levels were measured before treatment, at 32 weeks of gestation, and postpartum. Results The final analysis included a total of 121 patients, 100 of whom were hepatitis B e antigen (HBeAg)–positive (96 and 4 in the immune-tolerant and -indeterminate phases, respectively) and 21 of whom were HBeAg-negative (6 and 15 in the immune-active and -inactive carrier phases, respectively). The HBeAg-negative group vs the HBeAg-positive group had lower levels of baseline HBcrAg (median [interquartile range {IQR}], 3.7 [3.0–5.9] vs 8.6 [8.4–8.7] log10 U/mL; P <.01) and pgRNA (median [IQR], 0.0 [0.0–2.5] vs 7.8 [7.6–8.1] log10 copies/mL; P <.01). The serum levels of HBcrAg and pgRNA were highest in immune-tolerant carriers and lowest in immune-inactive carriers. In HBeAg-positive patients, the correlation coefficients of HBcrAg and pgRNA with hepatitis B virus (HBV) DNA were 0.40 and 0.43, respectively; in HBeAg-negative patients, they were 0.53 and 0.51, respectively (all P <.05). The correlation coefficients with hepatitis B surface antigen (HBsAg) were 0.55 and 0.52 (P <.05) in HBeAg-positive patients, respectively, while in HBeAg-negative patients they were 0.42 and 0.37, respectively (P >.05). Among 96 patients receiving antiviral prophylaxis, we detected a rapid decrease in HBV DNA to an undetectable level during treatment but relatively stable levels of pgRNA and HBcrAg. Conclusions HBcrAg and pgRNA levels are lower in HBeAg-negative patients than in HBeAg-positive patients. These 2 markers are significantly associated with HBV DNA irrespective of HBeAg status, while they are significantly associated with HBsAg only in HBeAg-positive patients. [ABSTRACT FROM AUTHOR]

Background: Newer biomarkers of Hepatitis B virus (HBV) infection and treatment response have not been well-characterized in individuals with HBV/HIV coinfection. Methods: Pre-genomic RNA (pgRNA) and quantitative HBsAg (qHBsAg) were used to evaluate the associations with baseline characteristics. Participants included two separate groups – 236 with HBV/HIV coinfection enrolled in a cross-sectional cohort in Ghana and 47 from an HBV nucleoside/nucleotide treatment trial comparing tenofovir to adefovir in the United States. Results: In both cohorts, HBe antigenemia was highly associated with pgRNA and HBV DNA levels. In the treatment cohort, pre-treatment pgRNA serum concentration was 7.0 log10 U/mL, and mean qHBsAg was 201,297 IU/mL. The observed treatment-associated decrease in pgRNA was consistent with a biphasic decline curve that reached second-phase kinetics following treatment week 12. Changes from baseline were significantly correlated with changes in serum ALT (r = - 0.518; P = 0.023) but not with changes in HBV DNA (r = 0.132, P = NS). qHBsAg also correlated with ALT change (r = - 0.488, P = 0.034). Conclusion: pgRNA and qHBsAg represent newer biomarkers of HBV replication that may help monitor response and treatment outcomes. HBV pgRNA is highly associated with both HBeAg and ALT and may predict both active replication from the closed circular DNA (cccDNA) template as well as hepatic injury.

Abstract Background The co-occurrence of chronic hepatitis B (CHB) and metabolic dysfunction-associated fatty liver disease (MAFLD) has drawn considerable attention due to its impact on disease outcomes. This study aimed to investigate the association between hepatic steatosis and hepatitis B virus (HBV) and analyzed the influence of hepatic steatosis on hepatitis B virology in patients with CHB. Methods In this cross-sectional study, 272 patients infected with HBV who were treatment-naïve or had ceased antiviral treatment for > 6 months were categorized into the CHB group (n = 128) and CHB + MAFLD group (n = 144). Furthermore, based on whether HBV DNA was higher than 2000 IU/mL, patients were categorized into the high-level HBV DNA group (n = 129) and the low-level HBV DNA group (n = 143). The impact of hepatic steatosis on hepatitis B virology was analyzed within the CHB cohort. Multivariate logistic regression analysis was employed to identify independent factors influencing pre-genomic RNA (pgRNA) levels below the lower limit of detection (LLD) in patients with CHB. Results Among the 272 patients, compared with CHB group, HBV DNA levels (4.11 vs. 3.62 log10 IU/mL, P = 0.045), hepatitis B surface antigen (HBsAg) levels (3.52 vs. 3.20 log10 IU/mL, P = 0.008) and the hepatitis B e antigen (HBeAg) positive rate (33.6% vs. 22.2%, P = 0.036) were significantly decreased in the CHB + MAFLD group; In 143 low-level HBV DNA patients, the CHB + MAFLD group exhibited decreased levels of pgRNA and HBsAg compared to the CHB group. However, in 129 high-level HBV DNA patients, a more significant decrease was observed in pgRNA (3.85 vs 3.35 log10 copies/mL, P = 0.044) and HBsAg (3.85 vs 3.59 log10 IU/mL, P = 0.033); Spearman correlation analysis revealed a negative correlation between hepatic steatosis and pgRNA (r = − 0.529, P

Background: The co-occurrence of chronic hepatitis B (CHB) and metabolic dysfunction-associated fatty liver disease (MAFLD) has drawn considerable attention due to its impact on disease outcomes. This study aimed to investigate the association between hepatic steatosis and hepatitis B virus (HBV) and analyzed the influence of hepatic steatosis on hepatitis B virology in patients with CHB. Methods: In this cross-sectional study, 272 patients infected with HBV who were treatment-naïve or had ceased antiviral treatment for > 6 months were categorized into the CHB group (n = 128) and CHB + MAFLD group (n = 144). Furthermore, based on whether HBV DNA was higher than 2000 IU/mL, patients were categorized into the high-level HBV DNA group (n = 129) and the low-level HBV DNA group (n = 143). The impact of hepatic steatosis on hepatitis B virology was analyzed within the CHB cohort. Multivariate logistic regression analysis was employed to identify independent factors influencing pre-genomic RNA (pgRNA) levels below the lower limit of detection (LLD) in patients with CHB. Results: Among the 272 patients, compared with CHB group, HBV DNA levels (4.11 vs. 3.62 log10 IU/mL, P = 0.045), hepatitis B surface antigen (HBsAg) levels (3.52 vs. 3.20 log10 IU/mL, P = 0.008) and the hepatitis B e antigen (HBeAg) positive rate (33.6% vs. 22.2%, P = 0.036) were significantly decreased in the CHB + MAFLD group; In 143 low-level HBV DNA patients, the CHB + MAFLD group exhibited decreased levels of pgRNA and HBsAg compared to the CHB group. However, in 129 high-level HBV DNA patients, a more significant decrease was observed in pgRNA (3.85 vs 3.35 log10 copies/mL, P = 0.044) and HBsAg (3.85 vs 3.59 log10 IU/mL, P = 0.033); Spearman correlation analysis revealed a negative correlation between hepatic steatosis and pgRNA (r = − 0.529, P < 0.001), HBV DNA (r = − 0.456, P < 0.001), HBsAg (r = − 0.465, P < 0.001) and HBeAg (r = − 0.339, P < 0.001) levels; Multivariate logistic regression analysis identified HBV DNA (odds ratio [OR] = 0.283, P < 0.001), HBsAg (OR = 0.300, P < 0.001), and controlled attenuation parameter (CAP) values (OR = 1.013, P = 0.038) as independent factors influencing pgRNA levels below the LLD in patients with CHB. Conclusions: This study establishes a negative correlation between hepatic steatosis and hepatitis B virology, demonstrating decreased HBV expression in patients with CHB + MAFLD. [ABSTRACT FROM AUTHOR]

Hepatitis B virus (HBV) pregenomic RNA (pgRNA) is packaged into capsid where reverse transcription takes place to synthesize viral DNA genome, and the encapsidated pgRNA is the predominant species of serum HBV RNA in patients as a serological biomarker. In this study, by utilizing various conditional HBV replication and secretion systems, we analyzed the intracellular and extracellular capsid pgRNA and revealed that the 3′ terminus of capsid pgRNA is scatteredly distributed between DR2 and poly(A) tail, except that the viral polymerase priming-defective mutant Y63D retained the sequence upstream of 3′ DR1. Mechanistically, the heterogeneity of capsid RNA 3′ terminus is due to the endogenous viral RNaseH activity during reverse transcription and exogenous MNase digestion during capsid RNA isolation; cellular ribonucleases may also participate in this process as the Y63D pgRNA 3′ terminus in the immunoprecipitated capsid without prior MNase treatment remains truncated into 3′ DR1. The major pgRNA splicing variant 1 of 2.1 kb and the artificial 3′ DR1 and ε deletion mutants also possess a truncated 3′ end of capsid RNA, indicating that the underrepresentation of the 3′ end of encapsidated pgRNA is independent of pgRNA length or 3′ terminal sequences. Altogether, our study suggests that the 3′ region of HBV capsid pgRNA downstream of 3′ DR1 is likely positioned outside of the capsid or loosely encapsulated, and thus is ribonuclease accessible. Furthermore, the detailed features of capsid pgRNA 3′ terminus will shed light on HBV pgRNA encapsidation and reverse transcription and aid the development of better diagnostics of serum HBV RNA. IMPORTANCE The biogenesis and clinical application of serum HBV pgRNA have been a research hotspot in recent years. This study further characterized the heterogeneity of the 3′ terminus of capsid RNA by utilizing a variety of experimental systems conditionally supporting HBV genome replication and secretion, and reveal that the 3′ truncation of capsid pgRNA is catalyzed by cellular ribonuclease(s) and viral RNaseH at positions after and before 3′ DR1, respectively, indicating the 3′ DR1 as a boundary between the encapsidated portion of pgRNA for reverse transcription and the 3′ unprotected terminus, which is independent of pgRNA length and the 3′ terminal sequence. Thus, our study provides new insights into the mechanism of pgRNA encapsidation and reverse transcription, as well as the optimization of serum HBV RNA diagnostics. [ABSTRACT FROM AUTHOR]

ntroduction: Serum hepatitis B virus (HBV) RNA is a promising new biomarker to manage and predict clinical outcomes of chronic hepatitis B (CHB) infection. However, the HBV serum transcriptome within encapsidated particles, which is the biomarker analyte measured in serum, remains poorly characterized. This study aimed to evaluate serum HBV RNA transcript composition and proportionality by PCR-cDNA nanopore sequencing of samples from CHB patients having varied HBV genotype (gt, A to F) and HBeAg status. Methods: Longitudinal specimens from 3 individuals during and following pregnancy (approximately 7 months between time points) were also investigated. HBV RNA extracted from 16 serum samples obtained from 13 patients (73.3% female, 84.6% Asian) was sequenced and serum HBV RNA isoform detection and quantification were performed using three bioinformatic workflows; FLAIR, RATTLE, and a GraphMap-based workflow within the Galaxy application. A spikein RNA variant (SIRV) control mix was used to assess run quality and coverage. The proportionality of transcript isoforms was based on total HBV reads determined by each workflow. Results: All chosen isoform detection workflows showed high agreement in transcript proportionality and composition for most samples. HBV pregenomic RNA (pgRNA) was the most frequently observed transcript isoform (93.8% of patient samples), while other detected transcripts included pgRNA spliced variants, 3′ truncated variants and HBx mRNA, depending on the isoform detection method. Spliced variants of pgRNA were primarily observed in HBV gtB, C, E, or F-infected patients, with the Sp1 spliced variant detected most frequently. Twelve other pgRNA spliced variant transcripts were identified, including 3 previously unidentified transcripts, although spliced isoform identification was very dependent on the workflow used to analyze sequence data. Longitudinal sampling among pregnant and post-partum antiviral-treated individuals showed increasing proportions of 3′ truncated pgRNA variants over time. Conclusions: This study demonstrated long-read sequencing as a promising tool for the characterization of the serum HBV transcriptome. However, further studies are needed to better understand how serum HBV RNA isoform type and proportion are linked to CHB disease progression and antiviral treatment response [ABSTRACT FROM AUTHOR]

IntroductionSerum hepatitis B virus (HBV) RNA is a promising new biomarker to manage and predict clinical outcomes of chronic hepatitis B (CHB) infection. However, the HBV serum transcriptome within encapsidated particles, which is the biomarker analyte measured in serum, remains poorly characterized. This study aimed to evaluate serum HBV RNA transcript composition and proportionality by PCR-cDNA nanopore sequencing of samples from CHB patients having varied HBV genotype (gt, A to F) and HBeAg status.MethodsLongitudinal specimens from 3 individuals during and following pregnancy (approximately 7 months between time points) were also investigated. HBV RNA extracted from 16 serum samples obtained from 13 patients (73.3% female, 84.6% Asian) was sequenced and serum HBV RNA isoform detection and quantification were performed using three bioinformatic workflows; FLAIR, RATTLE, and a GraphMap-based workflow within the Galaxy application. A spike-in RNA variant (SIRV) control mix was used to assess run quality and coverage. The proportionality of transcript isoforms was based on total HBV reads determined by each workflow.ResultsAll chosen isoform detection workflows showed high agreement in transcript proportionality and composition for most samples. HBV pregenomic RNA (pgRNA) was the most frequently observed transcript isoform (93.8% of patient samples), while other detected transcripts included pgRNA spliced variants, 3′ truncated variants and HBx mRNA, depending on the isoform detection method. Spliced variants of pgRNA were primarily observed in HBV gtB, C, E, or F-infected patients, with the Sp1 spliced variant detected most frequently. Twelve other pgRNA spliced variant transcripts were identified, including 3 previously unidentified transcripts, although spliced isoform identification was very dependent on the workflow used to analyze sequence data. Longitudinal sampling among pregnant and post-partum antiviral-treated individuals showed increasing proportions of 3′ truncated pgRNA variants over time.ConclusionsThis study demonstrated long-read sequencing as a promising tool for the characterization of the serum HBV transcriptome. However, further studies are needed to better understand how serum HBV RNA isoform type and proportion are linked to CHB disease progression and antiviral treatment response.

Aim: Assess the in vitro effect of hepatitis B virus (HBV) genotype H (HBV/H) with the small surface HBV protein (HBs) C107R mutation on hepatitis B surface antigen (HBsAg) detection, TGFB1, CAT and IFNB1A expression, and the response to IFN-β-1a treatment. Methods: HBV/H wild-type and HBs C107R variant replicons were constructed and transfected into hepatic stellate cells and/or Huh7 that were later treated with IFN-β-1a. HBsAg, HBV-DNA, pgRNA, TGFB1, CAT and IFNB1A expression was analyzed. 3D HBs structure from wild-type and C107R were foreseen by AlphaFold protein predictor, and IFN-β-1a antiviral effect was evaluated. Results:C107R mutation did not impact viral replication, but HBsAg serologic detection was affected. Wild-type and C107R similarly modified gene expression and responded to IFN-β-1a. Conclusion:C107R disrupts the Cys107/Cys138 disulfide bond and impairs HBsAg detection. Independently of the mutation, there were changes in TGFB1, CAT and IFNB1A expression, and a medium response to IFN-β-1a treatment compared with genotype A and C. [ABSTRACT FROM AUTHOR]

Objective To evaluate the predictive efficacy of serum pregenomic RNA (pgRNA) level in HBeAg clearance for chronic hepatitis B (CHB) patients treated with polyethylene glycol interferon alfa (PEG-IFN-α). Methods Twenty-five CHB patients with HBeAg positive and quantitative < 50 S/CO treated in our outpatient department from November 2015 to July 2019 were recruited in this prospective study. They were treated with PEG-IFN-α alone or combined with nucleoside and nucleotide analogs (NAs) for 48 to 96 weeks. After treatment, the patients were followed up in the clinic. According to whether HBeAg was cleared during treatment or follow-up, the patients were divided into HBeAg clearance group (HBeAg quantitative < 1.00 COI) and HBeAg non-clearance group (HBeAg ≥1.00 COI). The baseline characteristics and dynamic changes of virological indexes were analyzed for every patient during treatment. The predictive value of virological markers to HBeAg clearance was evaluated by binary logistic regression analysis and receiver operating characteristic (ROC) curve. Results At the end of treatment, 13 patients (13/25, 52.00%) achieved HBeAg clearance. There were significant differences in the levels of serum pgRNA and HBcrAg between the 2 groups at baseline (P < 0.05); Baseline pgRNA was significantly correlated with HBcrAg (r=0.575, P=0.003) and HBeAg (r=0.496, P=0.012), and baseline HBcrAg was significantly correlated with HBeAg (r=0.888, P < 0.001) and HBsAg (r=0.423, P=0.035). During the treatment, the overall level of HBeAg in 25 patients showed a downward trend when compared with the baseline level, and the decline of HBeAg level was greater in the HBeAg clearance group than the HBeAg non-clearance group at different time points (P < 0.05). From baseline to 12 weeks, the levels of pgRNA and HBsAg were decreased significantly, and its decline of pgRNA was more significant in the HBeAg clearance group than the non-clearance group (P < 0.05). During the whole treatment process, the level of HBcrAg was in a slow downward trend, and a significant decrease was observed only at 12 to 24 week in the HBeAg clearance group (P < 0.05), not at other time points. ROC curve analysis showed that baseline pgRNA and HBcrAg were good indicators to predict HBeAg clearance. Multivariate binary logistic regression analysis indicated that baseline pgRNA level was an independent predictor of HBeAg clearance after treatment (P < 0.05), with an AUC of 0.763, cutoff value of 2.42 log10 copies/mL, a sensitivity of 69.23% and a specificity of 53.85%. Conclusion Pretreatment serum level of pgRNA shows good predictive value for the efficacy of PEG-IFN-α in CHB patients with low HBeAg level.

HIV and hepatitis B virus (HBV) coinfection is relatively common. Initiation of antiretroviral therapy (ART) in people with HIV (PWH) causes a progressive restoration of cell-mediated immune functions. In the presence of overt or occult coinfections, immune restoration might lead to immune reconstitution inflammatory syndrome (IRIS). Here, we describe the clinical, immunological, virological, and histological characterization of a case of HBV-related IRIS hepatitis in a PWH after ART initiation. A liver biopsy was performed during HBV-related IRIS hepatic flare, and liver samples were analyzed through immunohistochemistry and molecular techniques, with the assessment of intrahepatic HBV-DNA, covalently closed circular DNA, and HBV pregenomic RNA through a droplet digital polymerase chain reaction system. Immune activation and senescence were also longitudinally assessed. In this clinical case, the hepatic flare occurred 6 weeks after ART initiation with a therapeutic regimen including tenofovir alafenamide (TAF) and emtricitabine (FTC). The episode was self-limiting, characterized by hyperactivation of peripheral blood CD4+ and CD8+ T-lymphocytes, and resolved without ART discontinuation, leading to the achievement of HBsAg seroconversion (HBsAg-/HBsAb+) and HBV-DNA plasma undetectability. Notably, hyperactivation of the immune system plays a pivotal role in promoting the control of HBV replication, thus triggering the achievement of HBsAg seroconversion during treatment with TAF/FTC. [ABSTRACT FROM AUTHOR]

HBV nucleos(t)ide reverse transcriptase inhibitors (NrtIs) do not completely suppress HBV replication. Previous reports indicate persistent viremia during NrtI treatment despite HBV DNA being undetectable. HBV core inhibitors may enhance viral suppression when combined with NrtIs. This phase II trial (NCT03576066) evaluated the efficacy and safety of the investigational core inhibitor, vebicorvir (VBR), in virologically- suppressed patients on NrtIs. Non-cirrhotic, NrtI-suppressed patients with chronic HBV were randomised to VBR 300 mg once daily or matching placebo (PBO) for 24 weeks. Treatment was stratified by hepatitis B e antigen (HBeAg) status. The primary endpoint was change from Baseline in serum HBeAg or hepatitis B surface antigen (HBsAg) after 24 weeks. Of 73 patients enrolled, 47 were HBeAg positive and 26 were HBeAg negative. In HBeAg-positive and -negative patients, there were no differences in the change from Baseline at Week 24 for HBsAg or HBeAg. Using a novel, high-sensitivity assay to detect HBV DNA, a greater proportion of patients with detectable HBV DNA at Baseline achieved undetectable HBV DNA at Week 24 in the VBR+NrtI vs. PBO+NrtI group. In HBeAg-positive patients, a greater change from Baseline in HBV pregenomic (pg)RNA was observed at Week 24 with VBR+NrtI vs. PBO+NrtI. Treatment-emergent adverse events (TEAEs) in VBR+NrtI patients included upper respiratory tract infection, nausea, and pruritus. No serious adverse events, Grade 4 TEAEs, or deaths were reported. In this 24-week study, VBR+NrtI demonstrated a favourable safety and tolerability profile. While there were no significant changes in viral antigen levels, enhanced viral suppression was demonstrated by greater changes in DNA and pgRNA with the addition of VBR compared to NrtI alone. NCT03576066. Core inhibitors represent a novel approach for the treatment of chronic hepatitis B virus (HBV) infection, with mechanisms of action distinct from existing treatments. In this study, vebicorvir added to existing therapy reduced HBV replication to a greater extent than existing treatment and was generally safe and well tolerated. [Display omitted] • Complete suppression of HBV replication is essential for finite treatment regimens. • Vebicorvir (VBR) is a novel inhibitor of the HBV core protein. • VBR interferes with two additional steps in HBV replication than NrtIs. • Added to NrtI, VBR did not significantly change mean HBV antigens over 24 weeks. • Added to NrtI, VBR further reduced HBV DNA and pgRNA by high-sensitivity PCR assays. [ABSTRACT FROM AUTHOR]

Background: Over 296 million people worldwide are living with chronic Hepatitis B (CHB) infection who require monitoring of viral activity and disease progression. Serum HBV RNA is a promising new biomarker in CHB management. No standardized method for serum HBV RNA quantification has been established. Objectives: To develop and validate, both analytically and clinically, a 3′ RACE RT-qPCR assay for quantification of serum HBV RNA. Study design: The 3′ RACE RT-qPCR method was developed using published primers. The analytical limit of detection and quantification, linearity, inter- and intra-assay repeatability, and clinical specificity and sensitivity were evaluated using synthetic pre-genomic RNA and specimens from various patient populations. Intra- and inter-laboratory ring trials involving three laboratories were completed. Results: The 3′ RACE RT-qPCR assay dynamic range is 25 to 108 copies/µL of synthetic RNA. Theoretical and measured serum HBV RNA quantities from a serially diluted sample showed excellent linearity (R2=0.9795). The inter- and intra-assay repeatability were 94.91% and 95.12%, respectively. Clinical specificity and sensitivity were 100% and 90%, respectively, in treated patients. Inter-laboratory analysis demonstrated moderate to high agreement among participating laboratories (κ = 0.581 to 0.867). High agreement was also observed between both operators participating in the intra-laboratory evaluation (κ = 0.867). Conclusions: Our methodology for the quantification of serum HBV RNA is specific and repeatable and employs a biologically relevant RNA standard suitable for medium throughput laboratories. Method standardization is required to facilitate the comparison of studies and better understand the clinical role of this novel biomarker.

Objectives: To investigate the factors and virological significance of serum hepatitis B virus (HBV) pregenomic RNA (pgRNA) status after long-term antiviral therapy with nucleos(t)ide analogues (NAs) in patients with chronic hepatitis B (CHB). Methods: In total, 51 treatment-naïve patients with CHB were included in the study. Clinical data were collected at baseline, during 5 years and at year 10 of NA treatment. Serum HBV pgRNA status of 51 patients was determined at year 5. Results: At year 5, 45% of the patients remained positive for HBV pgRNA. There were significant differences in baseline hepatitis B e antigen (HBeAg) status, HBV DNA load and hepatitis B surface antigen (HBsAg) levels between patients testing positive and negative for HBV pgRNA at year 5. Serum HBV pgRNA status and serum HBV DNA load were correlated after 5 years of NA treatment (r = 0.347, P = 0.013). Being HBV pgRNA positive at year 5 was an independent risk factor for sustainedly undetectable HBV DNA after 10 years of NA treatment (odds ratio 13.638, 95% confidence interval 1.32–140.81; P = 0.028). Furthermore, HBV pgRNA status at year 5 was associated with HBV DNA re-appearance at year 10 (P = 0.009). Conclusions: HBV pgRNA status at year 5 can reveal HBV covalently closed circular DNA (cccDNA) activity, and HBV pgRNA positivity after long-term antiviral therapy may indicate higher transcriptional activity of HBV cccDNA. Long-term dynamic monitoring of HBV pgRNA should be considered.

Background: The existence of hepatic cancer stem cells (CSCs) contributes to chemotherapy resistance and cancer recurrence after treatment or surgery. However, very little is known about the hepatitis B virus (HBV) replication and its relationship with the stemness of hepatocellular carcinoma (HCC) in HBV-related HCC patients. Methods: We collected tumor tissues (T), matched adjacent non-tumor tissues (NT), and distal non-tumor tissues (FNT) from 55 HCC patients for analysis. Results: We found HBV DNA levels were higher in T samples than NT and FNT samples, but HBV pgRNA and total RNA expressed lower in T samples. HBV pgRNA and total RNA correlate to HBV DNA among the T, NT, and FNT samples. Further evidence for HBV replication in T samples was provided by HBV S, reverse transcriptase, and X genes sequencing, showing that HBV sequences and genotypes differed between T and matched NT and FNT samples. HBV pgRNA and total RNA showed more frequent significant correlations with CSC markers in NT samples in HBsAg-positive patients. The markers CD133 and OCT4 expressed higher in FNT samples, and HBV replication marker of pgRNA levels was significantly positively correlated to these two markers only in FNT samples. The detection of pgRNA and OCT4 in FNT was correlated to the recurrence of HCC in the resection of HCC patients. Analysis of HBV receptor, sodium taurocholate co-transporting polypeptide (NTCP), showed that NTCP was correlated negatively to CSC markers in T samples, except for the CD44. Conclusion: HBV replication may present in HCC with a weak transcriptomic signature. Moreover, the expression level of HBV pgRNA in distal non-tumor tissues is a sensitive marker for HBV replication and prognosis, which is associated with CSC-related markers especially with OCT4 in distal non-tumor tissues and recurrence of HCC in HBV-related HCC patients. [ABSTRACT FROM AUTHOR]

Serum hepatitis B virus pregenomic RNA (HBV pgRNA) is a potential biomarker that is correlated with covalently closed circular DNA. The long-term dynamics of HBV pgRNA in patients with chronic hepatitis B need to be explored. One hundred naïve nucleos(t)ide analog-treated patients with chronic hepatitis B were enrolled to analyze the dynamics of HBV pgRNA over 9 years. The positive rates of HBV pgRNAs declined gradually and showed biphasic kinetics. Serum HBV pgRNA levels in patients treated with entecavir became negative later than those treated with adefovir or lamivudine. Patients who remain positive for HBV pgRNA after 9 years of treatment may have higher viral transcription efficiencies. The reverse transcription efficiency of hepatitis B e-antigen (HBeAg)-positive patients was higher than that of HBeAg-negative patients at baseline and showed no difference after 24-week nucleos(t)ide analog treatment. The trajectory of serum HBV pgRNA-negative transformation differs in patients with different characteristics. Long-term dynamic monitoring of serum HBV pgRNA levels has significance in hepatitis B treatment.

BackgroundThe existence of hepatic cancer stem cells (CSCs) contributes to chemotherapy resistance and cancer recurrence after treatment or surgery. However, very little is known about the hepatitis B virus (HBV) replication and its relationship with the stemness of hepatocellular carcinoma (HCC) in HBV-related HCC patients.MethodsWe collected tumor tissues (T), matched adjacent non-tumor tissues (NT), and distal non-tumor tissues (FNT) from 55 HCC patients for analysis.ResultsWe found HBV DNA levels were higher in T samples than NT and FNT samples, but HBV pgRNA and total RNA expressed lower in T samples. HBV pgRNA and total RNA correlate to HBV DNA among the T, NT, and FNT samples. Further evidence for HBV replication in T samples was provided by HBV S, reverse transcriptase, and X genes sequencing, showing that HBV sequences and genotypes differed between T and matched NT and FNT samples. HBV pgRNA and total RNA showed more frequent significant correlations with CSC markers in NT samples in HBsAg-positive patients. The markers CD133 and OCT4 expressed higher in FNT samples, and HBV replication marker of pgRNA levels was significantly positively correlated to these two markers only in FNT samples. The detection of pgRNA and OCT4 in FNT was correlated to the recurrence of HCC in the resection of HCC patients. Analysis of HBV receptor, sodium taurocholate co-transporting polypeptide (NTCP), showed that NTCP was correlated negatively to CSC markers in T samples, except for the CD44.ConclusionHBV replication may present in HCC with a weak transcriptomic signature. Moreover, the expression level of HBV pgRNA in distal non-tumor tissues is a sensitive marker for HBV replication and prognosis, which is associated with CSC-related markers especially with OCT4 in distal non-tumor tissues and recurrence of HCC in HBV-related HCC patients.

Background: Newer biomarkers of Hepatitis B virus (HBV) infection and treatment response have not been well-characterized in individuals with HBV/HIV coinfection., Methods: Pre-genomic RNA (pgRNA) and quantitative HBsAg (qHBsAg) were used to evaluate the associations with baseline characteristics. Participants included two separate groups - 236 with HBV/HIV coinfection enrolled in a cross-sectional cohort in Ghana and 47 from an HBV nucleoside/nucleotide treatment trial comparing tenofovir to adefovir in the United States., Results: In both cohorts, HBe antigenemia was highly associated with pgRNA and HBV DNA levels. In the treatment cohort, pre-treatment pgRNA serum concentration was 7.0 log 10 U/mL, and mean qHBsAg was 201,297 IU/mL. The observed treatment-associated decrease in pgRNA was consistent with a biphasic decline curve that reached second-phase kinetics following treatment week 12. Changes from baseline were significantly correlated with changes in serum ALT (r = - 0.518; P = 0.023) but not with changes in HBV DNA (r = 0.132, P = NS). qHBsAg also correlated with ALT change (r = - 0.488, P = 0.034)., Conclusion: pgRNA and qHBsAg represent newer biomarkers of HBV replication that may help monitor response and treatment outcomes. HBV pgRNA is highly associated with both HBeAg and ALT and may predict both active replication from the closed circular DNA (cccDNA) template as well as hepatic injury., Competing Interests: MA, MS, and GC are employees and shareholders of Abbott Laboratories. All other authors have no conflicts of interest., (Copyright © 2024 Pathogens and Immunity.)

DDX17 is a member of the DEAD-box helicase family proteins involved in cellular RNA folding, splicing, and translation. It has been reported that DDX17 serves as a cofactor of host zinc finger antiviral protein (ZAP)-mediated retroviral RNA degradation and exerts direct antiviral function against Raft Valley fever virus through binding to specific stem-loop structures of viral RNA. Intriguingly, we have previously shown that ZAP inhibits hepatitis B virus (HBV) replication through promoting viral RNA decay, and the ZAP-responsive element (ZRE) of HBV pregenomic RNA (pgRNA) contains a stemloop structure, specifically epsilon, which serves as the packaging signal for pgRNA encapsidation. In this study, we demonstrated that the endogenous DDX17 is constitutively expressed in human hepatocyte-derived cells but dispensable for ZAP-mediated HBV RNA degradation. However, DDX17 was found to inhibit HBV replication primarily by reducing the level of cytoplasmic encapsidated pgRNA in a helicase-dependent manner. Immunofluorescence assay revealed that DDX17 could gain access to cytoplasm from nucleus in the presence of HBV RNA. In addition, RNA immunoprecipitation and electrophoretic mobility shift assays demonstrated that the enzymatically active DDX17 competes with HBV polymerase to bind to pgRNA at the 59 epsilon motif. In summary, our study suggests that DDX17 serves as an intrinsic host restriction factor against HBV through interfering with pgRNA encapsidation. [ABSTRACT FROM AUTHOR]

• After 5 years of antiviral treatment, 45.10% of patients remained positive for hepatitis B virus pregenomic RNA (HBV pgRNA). • Serum HBV pgRNA was correlated with serum HBV DNA after 5 years of antiviral treatment. • HBV pgRNA can reveal HBV covalently closed circular DNA (cccDNA) activity after 5 years of antiviral treatment. • Positive HBV pgRNA after 5 years of treatment may indicate higher cccDNA activity. To investigate the factors and virological significance of serum hepatitis B virus (HBV) pregenomic RNA (pgRNA) status after long-term antiviral therapy with nucleos(t)ide analogues (NAs) in patients with chronic hepatitis B (CHB). In total, 51 treatment-naïve patients with CHB were included in the study. Clinical data were collected at baseline, during 5 years and at year 10 of NA treatment. Serum HBV pgRNA status of 51 patients was determined at year 5. At year 5, 45% of the patients remained positive for HBV pgRNA. There were significant differences in baseline hepatitis B e antigen (HBeAg) status, HBV DNA load and hepatitis B surface antigen (HBsAg) levels between patients testing positive and negative for HBV pgRNA at year 5. Serum HBV pgRNA status and serum HBV DNA load were correlated after 5 years of NA treatment (r = 0.347, P = 0.013). Being HBV pgRNA positive at year 5 was an independent risk factor for sustainedly undetectable HBV DNA after 10 years of NA treatment (odds ratio 13.638, 95% confidence interval 1.32–140.81; P = 0.028). Furthermore, HBV pgRNA status at year 5 was associated with HBV DNA re-appearance at year 10 (P = 0.009). HBV pgRNA status at year 5 can reveal HBV covalently closed circular DNA (cccDNA) activity, and HBV pgRNA positivity after long-term antiviral therapy may indicate higher transcriptional activity of HBV cccDNA. Long-term dynamic monitoring of HBV pgRNA should be considered. [ABSTRACT FROM AUTHOR]

Background: Interferon alfa (IFN-α) has been proved effective in treating chronic hepatitis B (CHB), owing to its ability to suppress hepatitis B surface antigen and hepatitis B virus (HBV) covalently closed circular DNA. However, the underlying mechanisms are unclear.Methods: We investigated the antiviral activities of exosomes from responders and nonresponders to pegylated IFN-α (PegIFN-α) as well as the supernatants of IFN-α-treated macrophages derived from THP-1 (the human leukemia monocyte cell line). Then the expression profiles of exosomal microRNAs (miRNAs) were analyzed using miRNA sequencing. The luciferase reporter assay was used to locate the binding position of HBV genomic sequence targeted by the identified miRNA.Results: Exosomes from PegIFN-α-treated patients, particularly responders, as well as the supernatants of IFN-α-treated macrophages exhibited anti-HBV activities, as manifested by the suppression of hepatitis B surface antigen, hepatitis B e antigen, HBV DNA, and covalently closed circular DNA levels in HBV-related cell lines. PegIFN-α treatment up-regulated exosomal hsa-miR-193a-5p, hsa-miR-25-5p, and hsa-miR-574-5p, which could partially inhibit HBV replication and transcription, and hsa-miR-574-5p reduced pregenomic RNA and polymerase messenger RNA levels by binding to the 2750-2757 position of the HBV genomic sequence.Conclusions: Exosomes can transfer IFN-α-related miRNAs from macrophages to HBV-infected hepatocytes, and they exhibit antiviral activities against HBV replication and expression. [ABSTRACT FROM AUTHOR]

Chronic infection with hepatitis B virus (HBV) sometime induces lethal cirrhosis and hepatocellular carcinoma. Although nucleot(s)ide analogs are used as main treatment for HBV infection, the emergence of the drug‐resistant viruses has become a problem. To discover novel antivirals with low side effects and low risk of emergence of resistant viruses, screening for anti‐HBV compounds was performed with compound libraries of inhibitors targeting G‐protein‐coupled receptors (GPCRs). HepG2‐hNTCP C4 cells infected with HBV were treated with various GPCR inhibitors and harvested at 14 day postinfection for quantification of core protein in the first screening or relaxed circular DNA in the second screening. Finally, we identified a cannabinoid receptor 1 inhibitor, rimonabant, as a candidate showing anti‐HBV effect. In HepG2‐hNTCP C4 cells, treatment with rimonabant suppressed HBV propagation at the viral RNA transcription step but had no effect on entry or covalently closed circular DNA level. The values of half maximal inhibitory concentration, half maximal effective concentration, and selectivity index of rimonabant in primary human hepatocyte (PHH) are 2.77 μm, 40.4 μm, and 14.6, respectively. Transcriptome analysis of rimonabant‐treated primary hepatocytes by RNA sequencing revealed that the transcriptional activity of hepatocyte nuclear factor 4α (HNF4α), which is known to stimulate viral RNA synthesis, was depressed. By treatment of PHH with rimonabant, the expression level of HNF4α protein and the production of the messenger RNAs (mRNAs) of downstream factors promoted by HNF4α were reduced while the amount of HNF4α mRNA was not altered. These results suggest that treatment with rimonabant suppresses HBV propagation through the inhibition of HNF4α activity. [ABSTRACT FROM AUTHOR]

Aim. To define the role of DNA-methyltransferases of type 1 and type 3A in hepatitis B viral cycle. Materials and methods. Human hepatoma cells HepG2 with stable expression of 1.1-mer HBV genome were transfected with vectors encoding DNA-methyltransferase 1 (DNMT1), DNA-methyltransferase 3A (DNMT3A) or were co-transfected with these vectors. Total HBV DNA copy number, relative expression of pregenomic RNA (pgRNA), S-protein-encoding RNA (S-RNA) and cccDNA were analyzed by quantitative and semi-quantitative real-time PCR-analysis with TaqMan probes for assessment of DNMTs-mediated effects on HBV. Results. DNMT1 and DNMT3A suppress HBV transcription and replication, though to different magnitude. cccDNA pool is enlarged statistically significantly ≈2-fold (P

Objective: To explore the clinical changes in levels of the new clinical marker serum hepatitis B virus (HBV) pregenomic RNA (pgRNA) in patients with chronic hepatitis B (CHB) with long-term antiviral therapy. Methods: 100 CHB cases who were initially treated with nucleos(t)ide analogues (NAs) at Peking University First Hospital were included. The levels of alanine aminotransferase (ALT), HBV DNA, hepatitis B e-antigen (HBeAg), and hepatitis B surface antigen (HBsAg) during the follow-up period were measured. The TaqMan-based real-time quantitative PCR method was used to detect serum HBV pgRNA levels. The independent sample t -test and Mann-Whitney U test were used to compare continuous variables between groups, while Pearson's χ (2) test and Fisher's exact test were used to compare categorical variables. Results: HBV pgRNA levels decreased significantly in patients who developed virological responses at 48 weeks ( n = 54) during subsequent treatment compared to those who did not ( n = 46). The HBV pgRNA level was lower in HBeAg-positive patients than in HBeAg-negative patients ( P < 0.05 or P < 0.01). Patients with higher HBV DNA and HBeAg-positivity levels at baseline had a higher HBV pgRNA level following antiviral therapy. There was no statistically significant difference in HBV pgRNA levels in patients with different HBV pgRNA levels at baseline after antiviral therapy. There was no correlation between serum HBV pgRNA and HBsAg at baseline, but there was a correlation after long-term antiviral therapy, while there was a weak correlation between HBV pgRNA and HBsAg at the fifth and ninth years of antiviral therapy ( r = 0.262, P = 0.031; r = 0.288, P = 0.008). Conclusion: HBV pgRNA levels were higher with higher HBV activity in CHB patients with long-term antiviral therapy.

Even though an approved vaccine for hepatitis B virus (HBV) is available and widely used, over 257 million individuals worldwide are living with chronic hepatitis B (CHB) who require monitoring of treatment response, viral activity, and disease progression to reduce their risk of HBV-related liver disease. There is currently a lack of predictive markers to guide clinical management and to allow treatment cessation with reduced risk of viral reactivation. Novel HBV biomarkers are in development in an effort to improve the management of people living with CHB, to predict disease outcomes of CHB, and further understand the natural history of HBV. This review focuses on novel HBV biomarkers and their use in the clinical setting, including the description of and methodology for quantification of serum HBV RNA, hepatitis B core-related antigen (HBcrAg), quantitative hepatitis B surface antigen (qHBsAg), including ultrasensitive HBsAg detection, quantitative anti-hepatitis B core antigen (qAHBc), and detection of HBV nucleic acid-related antigen (HBV-NRAg). The utility of these biomarkers in treatment-naïve and treated CHB patients in several clinical situations is further discussed. Novel HBV biomarkers have been observed to provide critical clinical information and show promise for improving patient management and our understanding of the natural history of HBV.

Importance: The biogenesis and clinical application of serum HBV pgRNA have been a research hotspot in recent years. This study further characterized the heterogeneity of the 3' terminus of capsid RNA by utilizing a variety of experimental systems conditionally supporting HBV genome replication and secretion, and reveal that the 3' truncation of capsid pgRNA is catalyzed by cellular ribonuclease(s) and viral RNaseH at positions after and before 3' DR1, respectively, indicating the 3' DR1 as a boundary between the encapsidated portion of pgRNA for reverse transcription and the 3' unprotected terminus, which is independent of pgRNA length and the 3' terminal sequence. Thus, our study provides new insights into the mechanism of pgRNA encapsidation and reverse transcription, as well as the optimization of serum HBV RNA diagnostics., Competing Interests: The authors declare no conflict of interest.

Background & Aims The Hepatitis B Virus (HBV) may gain entry into non-liver cells but does not actively replicate in them. We investigated the possibility that these cells possess mechanisms that block HBV core promoter (HBVCP) transcription, specifically absent in liver cells, which together with other liver-specific mechanisms, such as sodium-taurocholate cotransporting polypeptide-mediated entry, enable liver cells to effectively produce HBV. Methods Liver and non-liver cell lines were screened for their capacity to activate the HBVCP and synthesize pre-genomic RNA (pgRNA). Transcription regulators differentially expressed between cells with active or inactive HBVCP were determined by human transcriptome array. Slug ( SNAI2 ) and SRY-related HMG box 7 (SOX7) transcriptional repressors were identified and shown to bind specifically to the HBVCP by electrophoretic mobility shift assay. The resultant inhibitory effect on HBVCP transcription was validated using luciferase reporter and assays for pgRNA, HBcAg and cccDNA accumulation in cells with HBV replicon and HBV infection models. To further confirm their specific activity, short peptide mimetics generated from Slug zinc-finger domains and SOX7 HMG-box were generated. Results The HBVCP was found to be active in liver and selected non-liver cells. These cells have low/negligible expression of Slug and SOX7, which inhibit HBVCP transcription specifically by binding at the pgRNA initiator site and competitively displacing hepatocyte nuclear factor 4α, respectively. Overexpression of Slug and/or SOX7 specifically reduced HBVCP transcription, significantly diminishing pgRNA synthesis, HBcAg and cccDNA accumulation in HBV-infected primary human hepatocytes. Similar results were obtained with Slug and SOX7 stapled peptides individually, which were even more potent in combination. Conclusions Slug and SOX7 are transcriptional repressors that bind specifically to the HBVCP. Their absence or weak expression in liver cells contribute to the favorable host environment for the active and efficient production of HBV. Lay summary Hepatitis B virus (HBV) replication occurs efficiently in human liver because of the specificity of viral uptake receptors and presence of numerous liver-enriched transcription activators. Herein, we show that the specific lack of transcriptional inhibitory mechanisms in liver cells also contribute to effective HBV production. HBV replication is kept low in non-liver cells as transcriptional repressors Slug and SRY-related HMG box 7 (SOX7) actively bind to the transcriptional initiator and displace transcription activators, respectively, within the HBV core promoter. [ABSTRACT FROM AUTHOR]

Hepatitis B virus (HBV) is the major cause of liver cirrhosis and hepatocellular carcinoma. After entering a hepatocyte, HBV forms a nuclear viral episome and produces pregenomic (pg) RNA with a stem–loop structure called an epsilon, which acts to signal encapsidation. We previously demonstrated that TGF-β upregulates activation-induced cytidine deaminase (AID) expression in hepatocytes, which in turn downregulates HBV transcripts by recruiting the RNA exosome complex. The molecular mechanism underlying AID-mediated HBV RNA reduction remains largely unclear. Here we used a pgRNA reporter system having a reporter gene within pgRNA to identify sis- and trans-acting elements in AID-mediated HBV RNA reduction. We found that the epsilon RNA and C-terminus of AID are required for AID-mediated HBV RNA reduction. Importantly, this reduction was reproduced in a hydrodynamic HBV transfection mouse model. The molecular mechanism of AID-mediated HBV RNA reduction is discussed. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are a class of small, single-stranded, noncoding, functional RNAs. Hepatitis B virus (HBV) is an enveloped DNA virus with virions and subviral forms of particles that lack a core. It was not known whether HBV encodes miRNAs. Here, we identified an HBV-encoded miRNA (called HBV-miR-3) by deep sequencing and Northern blotting. HBV-miR-3 is located at nucleotides (nt) 373 to 393 of the HBV genome and was generated from 3.5-kb, 2.4-kb, and 2.1-kb HBV in a classic miRNA biogenesis (Drosha-Dicer-dependent) manner. HBV-miR-3 was highly expressed in hepatoma cell lines with an integrated HBV genome and HBV+ hepatoma tumors. In patients with HBV infection, HBV-miR-3 was released into the circulation by exosomes and HBV virions, and HBV-miR-3 expression had a positive correlation with HBV titers in the sera of patients in the acute phase of HBV infection. More interestingly, we found that HBV-miR-3 represses HBsAg, HBeAg, and replication of HBV. HBV-miR-3 targets the unique site of the HBV 3.5-kb transcript to specifically reduce HBc protein expression, levels of pregenomic RNA (pgRNA), and HBV replication intermediate (HBV-RI) generation but does not affect the HBV DNA polymerase level, thus suppressing HBV virion production (replication). This may explain the low levels of HBV virion generation with abundant subviral particles lacking core during HBV replication, which may contribute to the development of persistent infection in patients. Taken together, our findings shed light on novel mechanisms by which HBV-encoded miRNA controls the process of self-replication by regulating HBV transcript during infection. [ABSTRACT FROM AUTHOR]

HBV cure rates remain low despite prolonged nucleos(t)ide (NrtI) therapy, likely due to persistent residual viral replication and an inability to eliminate covalently closed circular DNA (cccDNA). Therapies with novel mechanisms of action against hepatitis B virus (HBV) are being explored with the goal of achieving sustained off-treatment response and a functional cure without requiring lifelong therapy. Recent studies have indicated that serum HBV DNA levels (a biomarker for viral replication) combined with serum pregenomic RNA (pgRNA) levels (a surrogate for intrahepatic cccDNA transcriptional activity), may provide a better prediction for the risk of liver-related complications. Current HBV DNA assays, such as the COBAS AmpliPrep/COBAS TaqMan HBV test v2.0, quantitate HBV DNA down to 20 IU/mL, but are not able to monitor loss of residual virus in patients on NrtI therapy. There are no commercially available assays approved to detect serum/plasma HBV pgRNA levels. We have developed a multi-assay panel of highly sensitive nucleic acid assays designed to monitor levels of HBV DNA, pgRNA and total nucleic acids (TNA, composite DNA + pgRNA) in clinical specimens and to monitor changes during treatment with new antiviral combination regimens. • A quantitative assay was developed to detect HBV pgRNA in patient serum or plasma. • Semi-quantitative gel-based assays can monitor residual levels of HBV nucleic acids. • HBV pgRNA is correlated with other HBV serum/plasma biomarkers. [ABSTRACT FROM AUTHOR]

Background: Hepatitis B virus (HBV) DNA in serum of chronically infected patients declines by 3-4 log10 units at loss of HBe antigen (HBeAg) from serum. The mechanisms behind this decline, and the much smaller decline of surface antigen (HBsAg) levels, are still not well known. The aim of this study was to get a better understanding of this process by analysing both serum and intrahepatic markers of HBV replication. Methods: Levels of HBV DNA and HBsAg in serum, and covalently closed circular DNA (cccDNA), pregenomic RNA (pgRNA) and S-RNA and total intrahepatic HBV DNA (ihDNA) in liver biopsies from 84 chronically infected patients (16 positive and 68 negative for HBeAg) were analysed. Results: Lower HBV DNA levels within HBeAg-positive stage reflected lower levels of cccDNA and pgRNA with strong correlation. In HBeAg-negative patients, ihDNA levels were greater and HBV DNA levels in serum lower than expected from pgRNA levels. A lower HBV DNA/HBsAg ratio corresponded with lower pgRNA/cccDNA (p < 0.01) and higher S-RNA/cccDNA (p < 0.0001) ratios, suggesting that in HBeAg-negative patients transcription of pgRNA, but not of S-RNA, becomes suppressed. Conclusions: The marked reduction of HBV DNA in serum after loss of HBeAg appears to be due to combined reduction of cccDNA, pgRNA and yet unidentified mechanisms downstream of reverse transcription. Such mechanisms include faster clearance of circulating virus or blocked secretion of virions, the latter supported by the observed relative increase of ihDNA in HBeAg-negative patients. The smaller reduction of S-RNA than of pgRNA partly explains why HBsAg remain high in the HBeAg-negative stage, supporting the possibility of HBsAg synthesis from integrated HBV DNA. [ABSTRACT FROM AUTHOR]

Background: The initiation of hepatitis B virus (HBV) replication involves the formation of covalently closed circular DNA (cccDNA) and its transcription into pregenomic RNA (pgRNA) in hepatocyte nuclei. The regulatory mechanism of HBV replication by acetyltransferase is thus far not well understood, but human acetyltransferase has been reported as being involved in the regulation of HBV replication. Results: Depletion of KAT8 or HAT1 via RNA interference (RNAi) markedly down-regulated HBV-DNA and pgRNA levels in HepG2.2.15 cells, with KAT8 knockdown reducing both HBsAg and HBeAg more than HAT1 knockdown. Consistent with these observations, HBV replication regulators hepatocyte nuclear factor-4-á (HNF4á) and peroxisome proliferator-activated receptor gamma coactivator- (PPARGC-) 1-á were decreased following knockdown of HAT1 or KAT8. Conclusions: These data suggest that KAT8 or HAT1 regulate HBV replication and may be potential drug targets of anti-HBV therapy. [ABSTRACT FROM AUTHOR]

Activation-induced cytidine deaminase (AID) is essential for the somatic hypermutation (SHM) and class-switch recombination (CSR) of lg genes. The mechanism by which AID triggers SHM and (SR has been explained by two distinct models. In the DNA deamination model, AID converts cytidine bases in DNA into uridine. The uridine is recognized by the DNA repair system, which produces DNA strand breakages and point mutations. In the alternative model, RNA edi- ted by AID is responsible for triggering (SR and SHM. However, RNA deamination by AID has not been demonstrated. Here we found that C-to-T and 6-to-A mutations accumulated in hepatitis B virus (HBV) nudeocapsid DNA when AID was expressed in HBV-replicating hepatic cell lines. AID expression caused C-to-T mutations in the nucleocapsid DNA of RNase H-defective HBV. which does not produce plus-strand viral DNA. Furthermore, the RT-P(R products of nucleocapsid viral RNA from AID-expressing cells exhibited significant C-to-T mutations, whereas viral RNAs outside the nucleocapsid did not accumulate C-to-U mutations. Moreover, AID was packaged within the nucleocapsid by forming a ribonucleo-protein complex with HBV RNA and the HBV polymerase protein. The encapsidation of the AID protein with viral RNA and DNA provides an efficient environment for evaluating AID's RNA and DNA deamination activities. A bona fide RNA-editing enzyme, apolipoprotein B mRNA editing catalytic polypeptide 1, induced a similar level of C-to-U mutations in nucleocapsid RNA as AID. Taken together, the results indicate that AID can deaminate the nucleocapsid RNA of HBV. [ABSTRACT FROM AUTHOR]

In this report, the anti-hepatitis B virus (HBV) activity of dehydrocheilanthifoline (DHCH), a quaternary ammonium alkaloid isolated from the traditional Chinese medicine Corydalis saxicola Bunting (Papaveraceae), was determined in vitro. Following six days of treatment, DHCH efficiently suppressed the secretions of HBsAg and HBeAg in HepG2.2.15 cell cultures, with a half-maximal inhibitory concentration (IC50) of 15.84 and 17.12 μM, and with a therapeutic index (TI) of 7.32 and 6.77, respectively. Further studies revealed that DHCH reduced the levels of extracellular DNA, intracellular DNA and covalently closed circular DNA (cccDNA) of HBV in a dose-dependent and time-dependent manner, with IC50 values of 15.08, 7.62 and 8.25 μM, respectively after six days of treatment. In contrast, the level of viral pre-genomic RNA (pgRNA) increased 6.13-fold after treatment with DHCH. Together, it was demonstrated for the first time that DHCH could significantly inhibit the replication of HBV, which warrants further studies on the antiviral mechanisms of DHCH, and suggests that it may be a promising candidate in the therapy of HBV infection. [ABSTRACT FROM AUTHOR]

Hepatitis B virus (HBV) infection is one of the major global health problems, especially in economically under-developed or developing countries. HBV infection can lead to a number of clinical outcomes including chronic infection, cirrhosis and liver cancer. It ranks among the top 10 causes of death, being responsible for around 1 million deaths every year. Despite the availability of a highly efficient vaccine and potent antiviral agents, HBV infection still remains a significant clinical problem, particularly in those high endemicity areas where vaccination of large populations has not been possible due to economic reasons. Although HBV is among the smallest viruses in terms of virion and genome size, it has numerous unique features that make it completely distinct from other DNA viruses. It has a partially double stranded DNA with highly complex genome organization, life cycle and natural history. Remarkably distinct from other DNA viruses, it uses an RNA intermediate called pregenomic RNA (pgRNA) and reverse transcriptase for its genome replication. Genome replication is accomplished by a complex mechanism of primer shifting facilitated by direct repeat sequences encoded in the genome. Further, the genome has evolved in such a manner that every single nucleotide of the genome is used for either coding viral proteins or used as regulatory regions or both. Moreover, it utilizes internal in-frame translation initiation codons, as well as different reading frames from the same RNA to generate different proteins with diverse functions. HBV also shows considerable genetic variability which has been related with clinical outcomes, replication potential, therapeutic response etc. This review aims at reviewing fundamental events of the viral life cycle including viral replication, transcription and translation, from the molecular standpoint, as well as, highlights the clinical relevance of genetic variability of HBV. [ABSTRACT FROM AUTHOR]

Serum hepatitis B virus pregenomic RNA (HBV pgRNA) is a potential biomarker that is correlated with covalently closed circular DNA. The long-term dynamics of HBV pgRNA in patients with chronic hepatitis B need to be explored. One hundred naïve nucleos(t)ide analog-treated patients with chronic hepatitis B were enrolled to analyze the dynamics of HBV pgRNA over 9 years. The positive rates of HBV pgRNAs declined gradually and showed biphasic kinetics. Serum HBV pgRNA levels in patients treated with entecavir became negative later than those treated with adefovir or lamivudine. Patients who remain positive for HBV pgRNA after 9 years of treatment may have higher viral transcription efficiencies. The reverse transcription efficiency of hepatitis B e-antigen (HBeAg)-positive patients was higher than that of HBeAg-negative patients at baseline and showed no difference after 24-week nucleos(t)ide analog treatment. The trajectory of serum HBV pgRNA-negative transformation differs in patients with different characteristics. Long-term dynamic monitoring of serum HBV pgRNA levels has significance in hepatitis B treatment., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Pan, Tian, Xu, Luo, Tan, Han, Kang, Chen, Yang and Xu.)

Even though an approved vaccine for hepatitis B virus (HBV) is available and widely used, over 257 million individuals worldwide are living with chronic hepatitis B (CHB) who require monitoring of treatment response, viral activity, and disease progression to reduce their risk of HBV-related liver disease. There is currently a lack of predictive markers to guide clinical management and to allow treatment cessation with reduced risk of viral reactivation. Novel HBV biomarkers are in development in an effort to improve the management of people living with CHB, to predict disease outcomes of CHB, and further understand the natural history of HBV. This review focuses on novel HBV biomarkers and their use in the clinical setting, including the description of and methodology for quantification of serum HBV RNA, hepatitis B core-related antigen (HBcrAg), quantitative hepatitis B surface antigen (qHBsAg), including ultrasensitive HBsAg detection, quantitative anti-hepatitis B core antigen (qAHBc), and detection of HBV nucleic acid-related antigen (HBV-NRAg). The utility of these biomarkers in treatment-naïve and treated CHB patients in several clinical situations is further discussed. Novel HBV biomarkers have been observed to provide critical clinical information and show promise for improving patient management and our understanding of the natural history of HBV. [ABSTRACT FROM AUTHOR]

DDX17 is a member of the DEAD-box helicase family proteins involved in cellular RNA folding, splicing, and translation. It has been reported that DDX17 serves as a cofactor of host zinc finger antiviral protein (ZAP)-mediated retroviral RNA degradation and exerts direct antiviral function against Raft Valley fever virus through binding to specific stem-loop structures of viral RNA. Intriguingly, we have previously shown that ZAP inhibits hepatitis B virus (HBV) replication through promoting viral RNA decay, and the ZAP-responsive element (ZRE) of HBV pregenomic RNA (pgRNA) contains a stem-loop structure, specifically epsilon, which serves as the packaging signal for pgRNA encapsidation. In this study, we demonstrated that the endogenous DDX17 is constitutively expressed in human hepatocyte-derived cells but dispensable for ZAP-mediated HBV RNA degradation. However, DDX17 was found to inhibit HBV replication primarily by reducing the level of cytoplasmic encapsidated pgRNA in a helicase-dependent manner. Immunofluorescence assay revealed that DDX17 could gain access to cytoplasm from nucleus in the presence of HBV RNA. In addition, RNA immunoprecipitation and electrophoretic mobility shift assays demonstrated that the enzymatically active DDX17 competes with HBV polymerase to bind to pgRNA at the 5' epsilon motif. In summary, our study suggests that DDX17 serves as an intrinsic host restriction factor against HBV through interfering with pgRNA encapsidation. IMPORTANCE Hepatitis B virus (HBV) chronic infection, a long-studied but yet incurable disease, remains a major public health concern worldwide. Given that HBV replication cycle highly depends on host factors, deepening our understanding of the host-virus interaction is thus of great significance in the journey of finding a cure. In eukaryotic cells, RNA helicases of the DEAD box family are highly conserved enzymes involved in diverse processes of cellular RNA metabolism. Emerging data have shown that DDX17, a typical member of the DEAD box family, functions as an antiviral factor through interacting with viral RNA. In this study, we, for the first time, demonstrate that DDX17 inhibits HBV through blocking the formation of viral replication complex, which not only broadens the antiviral spectrum of DDX17 but also provides new insight into the molecular mechanism of DDX17-mediated virus-host interaction.

Hepatitis B virus (HBV) is a major human pathogen that causes liver diseases. The main HBV RNAs are unspliced transcripts that encode the key viral proteins. Recent studies have shown that some of the HBV spliced transcript isoforms are predictive of liver cancer, yet the roles of these spliced transcripts remain elusive. Furthermore, there are nine major HBV genotypes common in different regions of the world, these genotypes may express different spliced transcript isoforms. To systematically study the HBV splice variants, we transfected human hepatoma cells, Huh7, with four HBV genotypes (A2, B2, C2 and D3), followed by deep RNA-sequencing. We found that 13-28 % of HBV RNAs were splice variants, which were reproducibly detected across independent biological replicates. These comprised 6 novel and 10 previously identified splice variants. In particular, a novel, singly spliced transcript was detected in genotypes A2 and D3 at high levels. The biological relevance of these splice variants was supported by their identification in HBV-positive liver biopsy and serum samples, and in HBV-infected primary human hepatocytes. Interestingly the levels of HBV splice variants varied across the genotypes, but the spliced pregenomic RNA SP1 and SP9 were the two most abundant splice variants. Counterintuitively, these singly spliced SP1 and SP9 variants had a suboptimal 5' splice site, supporting the idea that splicing of HBV RNAs is tightly controlled by the viral post-transcriptional regulatory RNA element.