Hepatitis B virus (HBV) is a global health problem with over 250 million people affected worldwide. Nucleos(t)ide analogues remain the standard of care and suppress production of progeny virions; however, they have limited effect on the viral transcriptome and long-term treatment is associated with off-target toxicities. Promising results are emerging from clinical trials and several drug classes have been evaluated, including capsid assembly modulators and RNA interfering agents. Whilst peripheral biomarkers are used to monitor responses and define treatment endpoints, they fail to reflect the full reservoir of infected hepatocytes. Given these limitations, consideration should be given to the merits of sampling liver tissue, especially in the context of clinical trials. In this review article, we will discuss methods for profiling HBV in liver tissue and their value to the HBV cure programme.

Liver cancer is the third leading cause of death globally, with hepatitis B virus (HBV) infection being identified as the primary risk factor for its development. The occurrence of HBV-related hepatocellular carcinoma (HCC) is attributed to various mechanisms, such as chronic inflammation and liver cell regeneration induced by the cytotoxic immune response triggered by the virus, abnormal activation of oncogenes arising from HBV DNA insertion mutations, and epigenetic alterations mediated by viral oncoproteins. The envelope protein of the HBV virus, known as hepatitis B surface antigen (HBsAg), is a key indicator of increased risk for developing HCC in HBsAg-positive individuals. The HBsAg seroclearance status is found to be associated with recurrence in HCC patients undergoing hepatectomy. Additional evidence indicates that HBsAg is essential to the entire process of tumor development, from initiation to advancement, and acts as an oncoprotein involved in accelerating tumor progression. This review comprehensively analyzes the extensive effects and internal mechanisms of HBsAg during the various stages of the initiation and progression of HCC. Furthermore, it highlights the importance and potential applications of HBsAg in the realms of HCC early diagnosis and personalized therapeutic interventions. An in-depth understanding of the molecular mechanism of HBsAg in the occurrence and development of HCC is provided, which is expected to develop more precise and efficient strategies for the prevention and management of HCC in the future.

HBsAg can be derived from intrahepatic cccDNA and integrated HBV DNA (iDNA). We examined the iDNA from liver tissues of 24 HBeAg(+) and 32 HBeAg(-) treatment-naive CHB participants.

Liver tissues were obtained from the North American Hepatitis B Research Network (HBRN). For cccDNA analysis, DNA was heat-denatured and digested by plasmid-safe ATP-dependent DNase to remove rcDNA and iDNA prior to qPCR. For iDNA detection, total DNA was subjected to HBV hybridization-targeted next generation sequencing (HBV-NGS) assay. The HBV-host junction sequences were identified by ChimericSeq. Comparison of HBV cccDNA and iDNA with serum and intrahepatic virological parameters were assessed.

Intrahepatic cccDNA, serum HBV DNA, HBV RNA, HBcrAg and qHBsAg were higher among the HBeAg(+) participants. Among the HBeAg(+) samples, 87% had positive intrahepatic HBcAg staining compared to 13% of HBeAg(-) samples (p<0.0001). HBsAg staining, in contrast, was present in over 85% of both HBeAg(+) and (-) livers. 23 (95.8%) HBeAg(+) participants had ≤50% iDNA of total HBV DNA whereas 25 (78.1%) HBeAg(-) participants had >50% iDNA in their livers. The iDNA junction-breakpoint distributions for the HBeAg(+) group were random with 15.9% localized to the DR2-DR1 region. In contrast, 52.4% of the iDNA were clustered at DR2-DR1 region among the HBeAg(-) participants. Microhomology-mediated end joining (MMEJ) patterns of dslDNA HBV integration was more frequent in HBeAg (+) livers.

Serum RNA and HBcrAg reflect the intrahepatic cccDNA concentrations. HBeAg(-) CHB participants had high levels of intrahepatic iDNA and HBsAg despite lower cccDNA levels suggesting that iDNA is the primary source of HBsAg in HBeAg(-) CHB.

Nucleic acid-based therapeutics have become a key pillar of the 'third wave' of modern medicine, following the eras of small molecule inhibitors and antibody drugs. Their rapid progress is heavily dependent on delivery technologies, with the development of N-acetylgalactosamine (GalNAc) conjugates marking a breakthrough in targeting liver diseases. This technology has gained significant attention for its role in addressing chronic conditions like chronic hepatitis B (CHB) and nonalcoholic steatohepatitis (NASH), which are challenging to treat with conventional methods.

This review explores the origins, mechanisms, and advantages of GalNAc-siRNA delivery systems, highlighting their ability to target hepatocytes via the asialoglycoprotein receptor (ASGPR). The literature reviewed covers preclinical and clinical advancements, particularly in CHB and NASH. Key developments in stabilization chemistry and conjugation technologies are examined, emphasizing their impact on enhancing therapeutic efficacy and patient compliance.

GalNAc-siRNA technology represents a transformative advancement in RNA interference (RNAi) therapies, addressing unmet needs in liver-targeted diseases. While significant progress has been made, challenges remain, including restricted targeting scope and scalability concerns. Continued innovation is expected to expand applications, improve delivery efficiency, and overcome limitations, establishing GalNAc-siRNA as a cornerstone for future nucleic acid-based treatments.

Hepatitis B virus (HBV) relaxed circular DNA (rcDNA) possesses an 8-9 nucleotide-long terminal redundancy (TR, or r) on the negative (-) strand DNA derived from the reverse transcription of viral pregenomic RNA (pgRNA). It remains unclear whether the TR forms a 5' or 3' flap structure on HBV rcDNA and which TR copy is removed during covalently closed circular DNA (cccDNA) formation. To address these questions, a mutant HBV cell line HepDES-C1822G was established with a C1822G mutation in the pgRNA coding sequence, altering the sequence of 3' TR of (-) strand DNA while the 5' TR remained wild type (wt). The production of HBV rcDNA and cccDNA in HepDES-C1822G cells was comparable to wt levels. Next-generation sequencing (NGS) analysis revealed that the positive (+) strand DNA of rcDNA and both strands of cccDNA predominantly carried the wt nt1822 residue, indicating that the 5' TR of (-) strand DNA serves as the template during rcDNA replication, forming a duplex with the (+) strand DNA, while the 3' TR forms a flap-like structure, which is subsequently removed during cccDNA formation. In a survey of known cellular flap endonucleases using a loss-of-function study, we found that the 3' flap endonuclease Mus81 plays a critical role in cccDNA formation in wild-type HBV replicating cells, alongside the 5' flap endonuclease FEN1. Additionally, we have mapped the potential Mus81 and FEN1 cleavage sites within the TR of nuclear DP-rcDNA by RACE-NGS analyses. The overlapping function between Mus81 and FEN1 in cccDNA formation indicates that the putative 5' and 3' flap formed by TR are dynamically interchangeable on rcDNA precursor. These findings shed light on HBV rcDNA structure and cccDNA formation mechanisms, contributing to our understanding of HBV replication cycle.

Hepatitis B virus (HBV) generates a double-stranded linear DNA (dslDNA) byproduct during replication. This dslDNA can undergo intermolecular and intramolecular nonhomologous end-joining (NHEJ) recombination, resulting in viral integration and dslDNA derived covalently closed circular DNAs (dsl-cccDNAs), respectively. The insertions and deletions (INDELs) at the end-joining site around the direct repeat (DR) 1 motif have been used to differentiate dsl-cccDNA from the authentic cccDNA. The frequency and characteristics of dsl-cccDNA in chronic hepatitis B (CHB) patients remain unclear.

HBV-targeted next-generation sequencing (NGS) was used to identify 32 dsl-cccDNA positive candidates, 22 HBeAg(+) and 10 HBeAg(-), from 56 liver biopsies of antiviral treatment-naive CHB patients for dsl-cccDNA confirmation and characterization by cccDNA-PCR NGS. INDELs within the DR2-1 region (nt 1600-1840) of the cccDNA were analyzed.

Various clonally expanded, heterogenous ∼22-nt deletions in the X-gene were detected in all 32 samples, which are likely quasi-species from the authentic cccDNA. We, therefore, defined dsl-cccDNA only by the presence of INDELs clustered at the DR1 surrounding region (nt 1800-1840). The percentage of dsl-cccDNA in total cccDNA was higher among HBeAg(+) compared to HBeAg(-) samples [11.32 (3.24-26.94)% vs. 7.72 (2.16-28.23)%, p=0.01]. The diversity of dsl-cccDNA species correlated with cccDNA levels (log-transformed; r=0.82, p<0.001), HBeAg(+) CHB (p<0.001), and serum HBV DNA (p<0.001).

dsl-cccDNA is more prevalent and heterogenous among the HBeAg(+) CHB subjects, which is likely due to the active viral cccDNA transcription and reverse transcription at the HBeAg(+) phase. The existence of replication-defective dsl-cccDNA may facilitate immune evasion and HBV integration, and complicate HBV pathogenesis. The potential impact of dsl-cccDNA in HBV therapeutic response deserves further assessment.

The hepatitis B virus surface antigen's (HBsAg) 'a' determinant comprises a sequence of amino acid residues located in the major hydrophilic region of the S protein, whose exchanges are closely associated with compromising the antigenicity and immunogenicity of that antigen. The HBsAg is generally present in the bloodstream of individuals with acute or chronic hepatitis B virus (HBV) infection. It is classically known as the HBV infection marker, and is therefore the first marker to be investigated in the laboratory in the clinical hypothesis of infection by this agent. One of the factors that compromises the HBsAg detection in the bloodstream by the assays adopted in serological screening in both clinical contexts is the loss of S protein antigenicity. This can occur due to mutations that emerge in the HBV genome regions that encode the S protein, especially for its immunodominant region - the 'a' determinant. These mutations can induce exchanges of amino acid residues in the S protein's primary structure, altering its tertiary structure and the antigenic conformation, which may not be recognized by anti-HBs antibodies, compromising the infection diagnosis. In addition, these exchanges can render ineffective the anti-HBs antibodies action acquired by vaccination, compromise the effectiveness of the chronically HBV infected patient's treatment, and also the HBsAg immunogenicity, by promoting its retention within the cell. In this review, the residues exchange that alter the S protein's structure is revisited, as well as the mechanisms that lead to the HBsAg antigenicity loss, and the clinical, laboratory and epidemiological consequences of this phenomenon.

Chronic hepatitis B virus (HBV) infection is a leading cause of liver cirrhosis and hepatocellular carcinoma, representing a global health problem for which a functional cure is difficult to achieve. The HBV core protein (HBc) is essential for multiple steps in the viral life cycle. It is the building block of the nucleocapsid in which viral DNA reverse transcription occurs, and its mediation role in viral-host cell interactions is critical to HBV infection persistence. However, systematic studies targeting HBc-interacting proteins remain lacking. Here, we combined HBc with the APEX2 to systematically identify HBc-related host proteins in living cells. Using functional screening, we confirmed that proteasome activator subunit 1 (PSME1) is a potent HBV-associated host factor. PSME1 expression was up-regulated upon HBV infection, and the protein level of HBc decreased after PSME1 knockdown. Mechanistically, the interaction between PSME1 and HBc inhibited the degradation of HBc by the 26S proteasome, thereby improving the stability of the HBc protein. Furthermore, PSME1 silencing inhibits HBV transcription in the HBV infection system. Our findings reveal an important mechanism by which PSME1 regulates HBc proteins and may facilitate the development of new antiviral therapies targeting PSME1 function.

Hepatitis B virus is one of the leading causes behind the neoplastic transformation of liver tissue and associated mortality. Despite the availability of many therapies and vaccines, the pathogenic landscape of the virus remains elusive; urging the development of novel strategies based on the fundamental infectious and transformative modalities of the virus-host interactome. Ubiquitination is a widely observed post-translational modification of several proteins, which either regulates the proteins' turnover or impacts their functionalities. In recent years, ample amount of literature has accumulated regarding the ubiquitination dynamics of the HBV proteins as well as the host proteins during HBV infection and carcinogenesis; with direct and detailed characterization of the involvement of HBV in these processes. Interestingly, while many of these ubiquitination events restrict HBV life cycle and carcinogenesis, several others promote the emergence of hepatocarcinoma by putting the virus in an advantageous position. This review sums up the snowballing literature on ubiquitination-mediated regulation of the host-HBV crosstalk, with special emphasis on its influence on the establishment and progression of hepatocellular carcinoma on a molecular level. With the advent of cutting-edge ubiquitination-targeted therapeutic approaches, the findings emanating from this review may potentiate the identification of novel anti-HBV targets for the formulation of novel anticancer strategies to control the HBV-induced hepato-carcinogenic process on a global scale.

Hepatitis B virus (HBV) is a globally prevalent human DNA virus responsible for over 250 million cases of chronic liver infections, leading to conditions such as liver inflammation, cirrhosis and hepatocellular carcinoma (HCC). Sodium taurocholate co-transporting polypeptide (NTCP) is a transmembrane protein highly expressed in human hepatocytes and functions as a bile acid (BA) transporter. NTCP has been identified as the receptor that HBV and its satellite virus, hepatitis delta virus (HDV), use to enter hepatocytes. HBV entry into hepatocytes is tightly regulated by various signaling pathways, and NTCP plays an important role as the initial stage of HBV infection. NTCP acts as an initiation signal, causing metabolic changes in hepatocytes and facilitating the entry of HBV into hepatocytes. Thus, a comprehensive understanding of NTCP's role is crucial. In this review, we will examine the regulatory mechanisms governing HBV pre-S1 binding to liver membrane NTCP, the role of NTCP in HBV internalization, and the transcriptional and translational regulation of NTCP expression. Additionally, we will discuss clinical drugs targeting NTCP, including combination therapies involving NTCP inhibitors, and consider the safety of NTCP as a therapeutic target.

Cryo-electron microscopy and tomography have allowed us to unveil the remarkable structure of icosahedral viruses. However, in the past few years, the idea that these viruses must have perfectly symmetric virions, but in some cases, it might not be true. This has opened the door to challenging paradigms in structural virology and raised new questions about the biological implications of "unusual" or "defective" symmetries and structures. Also, the continual improvement of these technologies, coupled with more rigorous sample purification protocols, improvements in data processing, and the use of artificial intelligence, has allowed solving the structure of sub-viral particles in highly heterogeneous samples and finding novel symmetries or structural defects. In this review, I initially analyzed the case of the symmetry and composition of hepatitis B virus-produced spherical sub-viral particles. Then, I focused on Alphaviruses as an example of "imperfect" icosahedrons and analyzed how structural biology has changed our understanding of the Alphavirus assembly and some biological implications arising from these discoveries.

Chronic hepatitis B virus infections are a significant cause of liver cirrhosis and cancer. Our research reveals that HBV infection leads to a marked increase in m6A modification of Foxp4 mRNA, resulting in enhanced stability of the mRNA and a subsequent increase in Foxp4 mRNA levels. Analysis of biopsy samples from chronic HBV patients demonstrated consistent upregulation of m6A-modified Foxp4 mRNA levels alongside increased Foxp4 mRNA levels. Functionally, Foxp4 was found to promote proliferation, migration, and invasion of hepatocellular carcinoma (HCC) cells in laboratory settings. Additionally, HBV gene expression was shown to activate the PI3K/AKT pathway by modulating Foxp4 mRNA stability in HCC cells. This study provides valuable insights into the underlying mechanisms of HBV infection and its potential implications for cancer development.

Hepatitis B virus (HBV) causes hepatitis B (HB) and distinct HBV genotypes can lead to different prognoses. However, HBV genotyping is rarely done in clinics, because the traditional method by PCR-based DNA sequencing is impractical for clinical diagnosis with tedious process and low success rate. Herein, we have established an ELISA-based genotyping method to quickly determine the HBV genotypes of HB patients in China. First, two commercial antibodies, 16D12 and 6H3 specific for HBV genotypes B and C respectively, are chosen as capture antibodies, since these two genotypes dominate in China. Then two home-made genotype-specific antibodies, B19 and C04, are used as the detection antibodies for genotypes B and C in sandwiched ELISA. The ELISA kit shows high sensitivity (> 95%) and specificity (> 95%) in detecting genotypes B and C of Chinese HB patients. Moreover, the ELISA kit has demonstrated higher success rate (98.7%) than PCR-based DNA sequencing (93.5%) and a commercial PCR-based genotyping kit (92.2%) for sera with HBV DNA ≥ 1000 IU/mL and HBsAg ≥ 250 IU/mL. Such an advantage is more obvious for the sera with HBV DNA < 1000 IU/mL. The kappa analysis between the ELISA and PCR-based DNA sequencing results exhibits a kappa of 0.836, indicating a good correlation.

Hepatitis B virus (HBV) exploits the endosomal sorting complexes required for transport (ESCRT)/multivesicular body (MVB) pathway for virion budding. In addition to enveloped virions, HBV-replicating cells nonlytically release non-enveloped (naked) capsids independent of the integral ESCRT machinery, but the exact secretory mechanism remains elusive. Here, we provide more detailed information about the existence and characteristics of naked capsid, as well as the viral and host regulations of naked capsid egress. HBV capsid/core protein has two highly conserved Lysine residues (K7/K96) that potentially undergo various types of posttranslational modifications for subsequent biological events. Mutagenesis study revealed that the K96 residue is critical for naked capsid egress, and the intracellular egress-competent capsids are associated with ubiquitinated host proteins. Consistent with a previous report, the ESCRT-III-binding protein Alix and its Bro1 domain are required for naked capsid secretion through binding to intracellular capsid, and we further found that the ubiquitinated Alix binds to wild type capsid but not K96R mutant. Moreover, screening of NEDD4 E3 ubiquitin ligase family members revealed that AIP4 stimulates the release of naked capsid, which relies on AIP4 protein integrity and E3 ligase activity. We further demonstrated that AIP4 interacts with Alix and promotes its ubiquitination, and AIP4 is essential for Alix-mediated naked capsid secretion. However, the Bro1 domain of Alix is non-ubiquitinated, indicating that Alix ubiquitination is not absolutely required for AIP4-induced naked capsid secretion. Taken together, our study sheds new light on the mechanism of HBV naked capsid egress in viral life cycle.

Virus-like particles (VLPs) have untapped potential for packaging and delivery of macromolecular cargo. To be a broadly useful platform, there needs to be a strategy for attaching macromolecules to the inside or the outside of the VLP with minimal modification of the platform or cargo. Here, we repurpose antiviral compounds that bind to hepatitis B virus (HBV) capsids to create a chemical tag to noncovalently attach cargo to the VLP. Our tag consists of a capsid assembly modulator, HAP13, connected to a linker terminating in maleimide. Our cargo is a green fluorescent protein (GFP) with a single addressable cysteine, a feature that can be engineered in many proteins. The HAP-GFP construct maintained HAP's intrinsic ability to bind HBV capsids and accelerate assembly. We investigated the capacity of HAP-GFP to coassemble with HBV capsid protein and bind to preassembled capsids. HAP-GFP binding was concentration-dependent, sensitive to capsid stability, and dependent on linker length. Long linkers had the greatest activity to bind capsids, while short linkers impeded assembly and damaged intact capsids. In coassembly reactions, >20 HAP-GFP molecules were presented on the outside and inside of the capsid, concentrating the cargo by more than 100-fold compared to bulk solution. We also tested an HAP-GFP with a cleavable linker so that external GFP molecules could be removed, resulting in exclusive internal packaging. These results demonstrate a generalizable strategy for attaching cargo to a VLP, supporting development of HBV as a modular VLP platform.

Chronic hepatitis B virus (HBV) infection is strongly associated with increased risk of liver cancer and cirrhosis. While existing treatments effectively inhibit the HBV life cycle, viral rebound occurs rapidly following treatment interruption. Consequently, functional cure rates of chronic HBV infection remain low and there is increased interest in a novel treatment modality, capsid assembly modulators (CAMs). Here, we develop a multiscale mathematical model of CAM treatment in chronic HBV infection. By fitting the model to participant data from a phase I trial of the first-generation CAM vebicorvir, we estimate the drug's dose-dependent effectiveness and identify the physiological mechanisms that drive the observed biphasic decline in HBV DNA and RNA, and mechanistic differences between HBeAg-positive and negative infection. Finally, we demonstrate analytically and numerically that HBV RNA is more sensitive than HBV DNA to increases in CAM effectiveness.

While certain human hepatitis B virus-targeting nucleoside analogs (NAs) serve as crucial anti-HBV drugs, HBV yet remains to be a major global health threat. E-CFCP is a 4'-modified and fluoromethylenated NA that exhibits potent antiviral activity against both wild-type and drug-resistant HBVs but less potent against human immunodeficiency virus type-1 (HIV-1). Here, we show that HIV-1 with HBV-associated amino acid substitutions introduced into the RT's dNTP-binding site (N-site) is highly susceptible to E-CFCP. We determined the X-ray structures of HBV-associated HIV-1 RT mutants complexed with DNA:E-CFCP-triphosphate (E-CFCP-TP). The structures revealed that exocyclic fluoromethylene pushes the Met184 sidechain backward, and the resultant enlarged hydrophobic pocket accommodates both the fluoromethylene and 4'-cyano moiety of E-CFCP. Structural comparison with the DNA:dGTP/entecavir-triphosphate complex also indicated that the cyclopentene moiety of the bound E-CFCP-TP is slightly skewed and deviated. This positioning partly corresponds to that of the bound dNTP observed in the HIV-1 RT mutant with drug-resistant mutations F160M/M184V, resulting in the attenuation of the structural effects of F160M/M184V substitutions. These results expand our knowledge of the interactions between NAs and the RT N-site and should help further design antiviral NAs against both HIV-1 and HBV.

Whether hepatitis B virus (HBV) infection of women prior to pregnancy can influence risk of congenital malformations in offspring remains controversial. We assessed the association between them by considering congenital malformations in the aggregate as well as risk of organs systems using a large national sample of Chinese women.

We performed a record-linkage cohort study of women who participated in National Free Preconception Health Examination Project, between January 1, 2010, and December 31, 2019 for whom data on congenital malformations in their offspring were available from the National Population-Based Birth Defects Surveillance Network. A total of 498,968 linked records were obtained, of which 127,371 were excluded because HBV status before pregnancy was unknown, the records involved multiple pregnancies, or pre-pregnancy examinations were conducted after conception. Based on pre-pregnancy status, mothers were assigned to two categories of HBsAg- or HBsAg+ and, in certain analyses, to three categories of HBsAg-, HBsAg+/HBeAg- or HBsAg+/HBeAg+. Potential associations of serological status with risk of congenital malformations, considered separately or in aggregate, were explored using multilevel logistic regression. Factors that might influence such associations were also explored.

Among the 371,597 women analyzed, 21,482 (5.78%) were HBsAg+ before pregnancy, and 8333 (2.24%) had a fetus or child diagnosed with congenital malformations, composed of 7744 HBsAg- women and 589 HBsAg+ women. HBsAg+ status was associated with increased risk of congenital malformations in the aggregate (OR 1.14, 95% CI 1.03-1.25) and of cardiovascular malformations specifically (OR 1.18, 95% CI 1.03-1.35). HBsAg+/HBeAg- status was associated with significantly higher risk of cardiovascular malformations (OR 1.19, 95% CI 1.01-1.39) as well as reproductive malformations (OR 1.51, 95% CI 1.02-2.23). Associations between HBsAg+ status before pregnancy and risk of congenital malformations was modified by alanine aminotransferase activity (P interaction < 0.05).

Prepregnancy HBV infection might be associated with fetal malformations. This association needs further investigation to confirm whether it is a causal association, and assess whether antiviral therapy of women with HBsAg+ planning to conceive might reduce the risk of fetal malformations.

The National Health Commission of the People's Republic of China, China; Science and Technology Department of Sichuan Province, China; and the Ministry of Science and Technology of the People's Republic of China.

In-cell self-assembly of natural viral capsids is an event that can be visualized under transmission electron microscopy (TEM) observations. By mimicking the self-assembly of natural viral capsids, various artificial protein- and peptide-based nanocages were developed; however, few studies have reported the in-cell self-assembly of such nanocages. Our group developed a β-Annulus peptide that can form a nanocage called artificial viral capsid in vitro, but in-cell self-assembly of the capsid has not been achieved. Here, we designed an artificial viral capsid decorated with a fluorescent protein, StayGold, to visualize in-cell self-assembly. Fluorescence anisotropy measurements and fluorescence resonance energy transfer imaging, in addition to TEM observations of the cells and super-resolution microscopy, revealed that StayGold-conjugated β-Annulus peptides self-assembled into the StayGold-decorated artificial viral capsid in a cell. Using these techniques, we achieved the in-cell self-assembly of an artificial viral capsid.

With the increasing momentum and success of monoclonal antibody therapy in conventional medical practices, there is a revived emphasis on the development of monoclonal antibodies targeting the hepatitis B surface antigen (anti-HBs) for the treatment of chronic hepatitis B (HBV) and hepatitis D (HDV). Combination therapies of anti-HBs monoclonal antibodies, and novel anti-HBV compounds and immunomodulatory drugs presenting a promising avenue to enhanced therapeutic outcomes in HBV/HDV cure regimens. In this review, we will cover the role of antibodies in the protection and clearance of HBV infection, the association of anti-HBV surface antigen antibodies (anti-HBs) in protection against HBV and how antibody effector functions, beyond neutralization, are likely necessary. Lastly, we will review clinical data from previous and ongoing clinical trials of passive antibody therapy to provide a state-of-the-are perspective on passive antibody therapies in combinations with additional novel agents.

Tripartite motif (TRIM) proteins, comprising a family of over 100 members with conserved motifs, exhibit diverse biological functions. Several TRIM proteins influence viral infections through direct antiviral mechanisms or by regulating host antiviral innate immune responses. To identify TRIM proteins modulating hepatitis B virus (HBV) replication, we assessed 45 human TRIMs in HBV-transfected HepG2 cells. Our study revealed that ectopic expression of 12 TRIM proteins significantly reduced HBV RNA and subsequent capsid-associated DNA levels. Notably, TRIM65 uniquely downregulated viral pregenomic (pg) RNA in an HBV-promoter-specific manner, suggesting a targeted antiviral effect. Mechanistically, TRIM65 inhibited HBV replication primarily at the transcriptional level via its E3 ubiquitin ligase activity and intact B-box domain. Though HNF4α emerged as a potential TRIM65 substrate, disrupting its binding site on the HBV genome did not completely abolish TRIM65's antiviral effect. In addition, neither HBx expression nor cellular MAVS signaling was essential to TRIM65-mediated regulation of HBV transcription. Furthermore, CRISPR-mediated knock-out of TRIM65 in the HepG2-NTCP cells boosted HBV infection, validating its endogenous role. These findings underscore TRIM proteins' capacity to inhibit HBV transcription and highlight TRIM65's pivotal role in this process.

The key to a curative treatment of hepatitis B virus (HBV) infection is the eradication of the intranuclear episomal covalently closed circular DNA (cccDNA), the stable persistence reservoir of HBV. Currently, established therapies can only limit HBV replication but fail to tackle the cccDNA. Thus, novel therapeutic approaches toward curative treatment are urgently needed. Recent publications indicated a strong association between the HBV core protein SUMOylation and the association with promyelocytic leukemia nuclear bodies (PML-NBs) on relaxed circular DNA to cccDNA conversion. We propose that interference with the cellular SUMOylation system and PML-NB integrity using arsenic trioxide provides a useful tool in the treatment of HBV infection. Our study showed a significant reduction in HBV-infected cells, core protein levels, HBV mRNA, and total DNA. Additionally, a reduction, albeit to a limited extent, of HBV cccDNA could be observed. Furthermore, this interference was also applied for the treatment of an established HBV infection, characterized by a stably present nuclear pool of cccDNA. Arsenic trioxide (ATO) treatment not only changed the amount of expressed HBV core protein but also induced a distinct relocalization to an extranuclear phenotype during infection. Moreover, ATO treatment resulted in the redistribution of transfected HBV core protein away from PML-NBs, a phenotype similar to that previously observed with SUMOylation-deficient HBV core. Taken together, these findings revealed the inhibition of HBV replication by ATO treatment during several steps of the viral replication cycle, including viral entry into the nucleus as well as cccDNA formation and maintenance. We propose ATO as a novel prospective treatment option for further pre-clinical and clinical studies against HBV infection.

The main challenge for the achievement of a functional cure for hepatitis B virus (HBV) is the covalently closed circular DNA (cccDNA), the highly stable persistence reservoir of HBV, which is maintained by further rounds of infection with newly generated progeny viruses or by intracellular recycling of mature nucleocapsids. Eradication of the cccDNA is considered to be the holy grail for HBV curative treatment; however, current therapeutic approaches fail to directly tackle this HBV persistence reservoir. The molecular effect of arsenic trioxide (ATO) on HBV infection, protein expression, and cccDNA formation and maintenance, however, has not been characterized and understood until now. In this study, we reveal ATO treatment as a novel and innovative therapeutic approach against HBV infections, repressing viral gene expression and replication as well as the stable cccDNA pool at low micromolar concentrations by affecting the cellular function of promyelocytic leukemia nuclear bodies.

Hepatitis B and C viruses (HBV and HCV) are significant causes of chronic liver diseases, with approximately 350 million infections globally. To accelerate the finding of effective treatment options, we introduce HBCVTr, a novel ligand-based drug design (LBDD) method for predicting the inhibitory activity of small molecules against HBV and HCV. HBCVTr employs a hybrid model consisting of double encoders of transformers and a deep neural network to learn the relationship between small molecules' simplified molecular-input line-entry system (SMILES) and their antiviral activity against HBV or HCV. The prediction accuracy of HBCVTr has surpassed baseline machine learning models and existing methods, with R-squared values of 0.641 and 0.721 for the HBV and HCV test sets, respectively. The trained models were successfully applied to virtual screening against 10 million compounds within 240 h, leading to the discovery of the top novel inhibitor candidates, including IJN04 for HBV and IJN12 and IJN19 for HCV. Molecular docking and dynamics simulations identified IJN04, IJN12, and IJN19 target proteins as the HBV core antigen, HCV NS5B RNA-dependent RNA polymerase, and HCV NS3/4A serine protease, respectively. Overall, HBCVTr offers a new and rapid drug discovery and development screening method targeting HBV and HCV.

The Hepatitis B Virus (HBV) ribonuclease H (RNase H) although promising remains an unexploited therapeutic target. HBV RNase H inhibition causes premature termination of viral minus-polarity DNA strands, prevents the synthesis of the viral positive-polarity DNA strand, and causes accumulation of RNA:DNA heteroduplexes within viral capsids. As part of our ongoing research to develop more potent anti-HBV RNase H inhibitors, we designed, synthesized and analyzed a library of 18 novel compounds (17 N-hydroyxpyridinedione (HPD) imine derivatives and 1 barbituric acid analogue) as potential leads for HBV treatment development. In cell assays, fourteen HPDs showed significant anti-HBV activity with EC50s from 1.1 to 2.5 μM and selectivity indices (SI) of up to 58. Three of them exhibited more than 3-fold improvement in the SI over the best previous HPD imine (SI = 13). To gain insight to the interaction between the tested compounds and the active site of HBV RNase H, docking experiments were undertaken. In almost all binding poses, the novel HPDs coordinated both active site Mg2+ ions via their oxygen trident. Furthermore, the novel HPDs displayed high cell permeability and solubility as well as good drug-like properties. These results reveal that HPD imines can be significantly active and selective HBV inhibitors, and that the HPD scaffold merits further development towards anti-HBV agents.

N6-methyladenosine (m6A) RNA methylation has recently emerged as a significant co-transcriptional modification involved in regulating various RNA functions. It plays a vital function in numerous biological processes. Enzymes referred to as m6A methyltransferases, such as the methyltransferaselike (METTL) 3-METTL14-Wilms tumor 1 (WT1)-associated protein (WTAP) complex, are responsible for adding m6A modifications, while m6A demethylases, including fat mass and obesity-associated protein (FTO) and alkB homolog 5 (ALKBH5), can remove m6A methylation. The functions of m6A-methylated RNA are regulated through the recognition and interaction of m6A reader proteins. Recent research has shown that m6A methylation takes place at multiple sites within hepatitis B virus (HBV) RNAs, and the location of these modifications can differentially impact the HBV infection. The addition of m6A modifications to HBV RNA can influence its stability and translation, thereby affecting viral replication and pathogenesis. Furthermore, HBV infection can also alter the m6A modification pattern of host RNA, indicating the virus's ability to manipulate host cellular processes, including m6A modification. This manipulation aids in establishing chronic infection, promoting liver disease, and contributing to pathogenesis. A comprehensive understanding of the functional roles of m6A modification during HBV infection is crucial for developing innovative approaches to combat HBV-mediated liver disease. In this review, we explore the functions of m6A modification in HBV replication and its impact on the development of liver disease.

Chronic hepatitis B (CHB) virus is the most common risk factor for developing liver malignancy. Autophagy is an essential element in human cell maintenance. Several studies have demonstrated that autophagy plays a vital role in liver cancer at different stages. In this systematic review, we intend to investigate the role of polymorphism and mutations of autophagy-related genes (ATGs) in the pathogenesis and carcinogenesis of the hepatitis B virus (HBV).

The search was conducted in online databases (Web of Science, PubMed, and Scopus) using Viruses, Infections, Polymorphism, Autophagy, and ATG. The study was conducted based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses criteria.

The primary search results led to 422 studies. By screening and eligibility evaluation, only four studies were relevant. The most important polymorphisms in hepatocellular carcinoma were rs2241880 in ATG16L1, rs77859116, rs510432, and rs548234 in ATG5. Furthermore, some polymorphisms are associated with an increased risk of HBV infection including, rs2241880 in ATG16L1 and rs6568431 in ATG5.

The current study highlights the importance of rs2241880 in ATG16L1 and rs77859116, rs510432, and rs548234 in ATG5 for HBV-induced HCC. Additionally, some mutations in ATG16L1 and ATG5 were important in risk of HBV infection. The study highlights the gap of knowledge in the field of ATG polymorphisms in HBV infection and HBV-induced HCC.

What is the impact of male hepatitis B virus (HBV) infection on sperm quality, embryonic development, and assisted reproductive outcomes?

Male HBV infection did not affect assisted reproductive outcomes, but HBV is capable of impairing human sperm and embryo formation in the early stages following fertilization.

HBV is found in germ cells and early embryos of patients with HBV. HBV may impair human sperm function via increasing reactive oxygen species.

We conducted a retrospective cohort study of 1581 infertile couples, including 496 male patients clinically confirmed to have hepatitis B infection, and a laboratory study of effects of HBV proteins on early embryos, using human embryonic stem cells (hESCs), human sperm, and golden hamster oocytes.

In total, 1581 infertile couples (24-40 years of age) who were admitted to a reproductive medicine center to undergo ART for the first time from January 2019 to November 2021 were selected as the study subjects. The case group was composed of 469 couples with hepatitis B surface antigen (HBsAg)-seropositive men and seronegative women (368 for IVF and 101 for ICSI treatment). The negative control group was composed of 1112 couples where both men and women were seronegative for hepatitis B antigen. We divided these couples into three comparison groups (IVF/ICSI, IVF, and ICSI). IVF of human sperm and hamster oocytes was used to evaluate the influence of the HBV HBs protein on formation of 2-cell embryos. Mitochondrial membrane potential (MMP) of hESCs was assayed via a fluorescence intensity system. Immunofluorescence staining of the phosphorylated histone H2A.X was applied to identify DNA damage to hESCs caused by the HBV X (HBx) protein.

Sperm concentration, total sperm number, and sperm with normal morphology were decreased in the couples with HBV-infected males in couples who were undergoing IVF/ICSI (male HBV(+) vs control: 469 vs 1112 individuals; sperm number, P < 0.01; normal sperm morphology, P < 0.01), IVF (368 vs 792; sperm number, P < 0.01; normal sperm morphology, P ≤ 0.05), and ICSI (101 vs 306; sperm number, P < 0.01; normal sperm morphology, P < 0.001). There was no significant difference in the number of embryo cleavages, blastocyst formation, biochemical pregnancy rate, clinical pregnancy rate, and live-birth rate between case and control groups. The 2PN fertilization rate in IVF/ICSI (P < 0.01) and ICSI (P < 0.05) couples, and the number of 2PN-fertilized oocytes in IVF (P < 0.001) couples were lower in couples with male HBV infection compared to control couples. HBV HBs protein reduced the MMP of human sperm and decreased 2-cell embryo formation in IVF of human sperm and zona-free-hamster oocyte. A reduction in fluorescence intensity and immunofluorescence staining of phosphorylated histone H2A.X indicated that HBx caused MMP impairment and DNA damage in human early embryonic cells, respectively.

N/A.

HBV can be examined in samples of sperm or discarded IVF early embryos from HBsAg-seropositive men and seronegative women. The hESC model in vitro may not fully mimic the natural embryos in vivo.

This study furthers our understanding of the influence of male HBV infection on embryonic development. Our results suggest that a semen-washing process may be necessary for male patients with HBV undergoing ART to minimize the potential negative effects of HBV infection on the early embryo.

This work was funded by grants from the National Natural Science Foundation of China, grant numbers 81870432 and 81570567 to X.Z., 81571994 to P.S., and 81950410640, the Natural Science Foundation of Guangdong Province, China (No. 2023A1515010660 to X.Z.), and the Li Ka Shing Shantou University Foundation (Grant No. L11112008). The authors have no conflicts of interest.

Viruses are vehicles to exchange genetic information and proteins between cells and organisms by infecting their target cells either cell-free, or depending on cell-cell contacts. Several viruses like certain retroviruses or herpesviruses transmit by both mechanisms. However, viruses have also evolved the properties to exchange proteins between cells independent of viral particle formation. This exchange of viral proteins can be directed to target cells prior to infection to interfere with restriction factors and intrinsic immunity, thus, making the target cell prone to infection. However, also bystander cells, e.g. immune cell populations, can be targeted by viral proteins to dampen antiviral responses. Mechanistically, viruses exploit several routes of cell-cell communication to exchange viral proteins like the formation of extracellular vesicles or the formation of long-distance connections like tunneling nanotubes. Although it is known that viral nucleic acids can be transferred between cells as well, this chapter concentrates on viral proteins of human pathogenic viruses covering all Baltimore classes and summarizes our current knowledge on intercellular transport of viral proteins between cells.

Hepatocellular carcinoma (HCC) remains one of the predominant causes of cancer-related mortality across the globe. It is attributed to obesity, excessive alcohol consumption, smoking, and infection by the hepatitis virus. Early diagnosis of HCC is essential, and local treatments such as surgical excision and percutaneous ablation are effective. Palliative systemic therapy, primarily with the tyrosine kinase inhibitor Sorafenib, is used in advanced cases. However, the prognosis for advanced HCC remains poor. This Review additionally describes the pathophysiological mechanisms of HCC, which include aberrant molecular signaling, genomic instability, persistent inflammation, and the paradoxical position of the immune system in promoting and suppressing HCC. The paper concludes by discussing the growing body of research on the relationship between mitochondria and HCC, suggesting that mitochondrial dysfunction may contribute to the progression of HCC. This Review focuses on immunological interactions between different mechanisms of HCC progression, including obesity, viral infection, and alcohol consumption.

Hepatitis B virus (HBV) infection is the leading cause of chronic liver disease worldwide. HBV-infected patients are at a lifetime risk of developing liver cirrhosis and hepatocellular carcinoma (HCC). Today, pegylated interferon (Peg-IFN) and nucleos(t)ide analogs (NAs) are used in the treatment of patients with chronic hepatitis B (CHB). Both treatment options have limitations. Despite effective viral suppression, NAs have little effect on covalently closed circular DNA (cccDNA), the stable episomal form of the HBV genome in hepatocytes. Therefore, the cure rate with NAs is low, and long-term treatment is required. Although the cure rate is better with Peg-IFN, it is difficult to tolerate due to drug side effects. Therefore, new treatment options are needed in the treatment of HBV infection. We can group new treatments under two headings: those that interfere with the viral life cycle and spread and those that modulate the immune response. Clinical studies show that combinations of treatments that directly target the viral life cycle and treatments that regulate the host immune system will be among the important treatment strategies in the future. As new direct-acting antiviral (DAA) and immunomodulatory therapies continue to emerge and evolve, functional cures in HBV treatment may be an achievable goal.

Hepatitis B virus (HBV) infection is closely related to viral hepatitis, liver cirrhosis, and hepatocellular carcinoma. HBV infection can reprogram metabolism processes of the host cells including glucose metabolism. The aberrant glucose metabolism may aid in viral infection and immune escape and may contribute to liver associated pathology. In this review, we discussed the interplay between HBV infection and glucose metabolism, which may provide new insights into HBV infection and pathology, novel intervention targets for HBV-related diseases.

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.

Chronic hepatitis B virus (HBV) infection is one of the leading causes of hepatocellular carcinoma (HCC). The HBV genome is prone to mutate and several variants are closely related to the malignant transformation of liver disease. G1896A mutation (G to A mutation at nucleotide 1896) is one of the most frequently observed mutations in the precore region of HBV, which prevents HBeAg expression and is strongly associated with HCC. However, the mechanisms by which this mutation causes HCC are unclear. Here, we explored the function and molecular mechanisms of the G1896A mutation during HBV-associated HCC. G1896A mutation remarkably enhanced the HBV replication in vitro. Moreover, it increased tumor formation and inhibited apoptosis of hepatoma cells, and decreased the sensitivity of HCC to sorafenib. Mechanistically, the G1896A mutation could activate ERK/MAPK pathway to enhanced sorafenib resistance in HCC cells and augmented cell survival and growth. Collectively, our study demonstrates for the first time that the G1896A mutation has a dual regulatory role in exacerbating HCC severity and sheds some light on the treatment of G1896A mutation-associated HCC patients.

Globally, a chronic-hepatitis B virus (HBV) infection is the leading cause of hepatocellular carcinoma (HCC). The transcription factor hypoxia-inducible factor 1 (HIF1) is often elevated in HCC, including HBV-associated HCC. Previous studies have suggested that the expression of the HIF1 subunit, HIF1α, is elevated in HBV-infected hepatocytes; however, whether HIF1 activity affects the HBV lifecycle has not been fully explored. We used a liver-derived cell line and ex vivo cultured primary hepatocytes as models to determine how HIF1 affects the HBV lifecycle. We observed that HIF1 elevates HBV RNA transcript levels, core protein levels, core protein localization to the cytoplasm, and HBV genome replication. Attenuating the transcription activity of HIF1 blocked HIF1-mediated effects on the HBV lifecycle. Our studies show that HIF1 regulates various stages of the HBV lifecycle in hepatocytes and could be a therapeutic target for blocking HBV replication and the development of HBV-associated diseases.

Infection with hepatitis B virus (HBV) is a main risk factor for hepatocellular carcinoma (HCC). Extracellular vesicles, such as exosomes, play an important role in tumor development and metastasis, including regulation of HBV-related HCC. In this study, we have characterized exosome microRNA and proteins released in vitro from hepatitis B virus (HBV)-related HCC cell lines SNU-423 and SNU-182 and immortalized normal hepatocyte cell lines (THLE2 and THLE3) using microRNA sequencing and mass spectrometry. Bioinformatics, including functional enrichment and network analysis, combined with survival analysis using data related to HCC in The Cancer Genome Atlas (TCGA) database, were applied to examine the prognostic significance of the results. More than 40 microRNAs and 200 proteins were significantly dysregulated (p < 0.05) in the exosomes released from HCC cells in comparison with the normal liver cells. The functional analysis of the differentially expressed exosomal miRNAs (i.e., mir-483, mir-133a, mir-34a, mir-155, mir-183, mir-182), their predicted targets, and exosomal differentially expressed proteins (i.e., POSTN, STAM, EXOC8, SNX9, COL1A2, IDH1, FN1) showed correlation with pathways associated with HBV, virus activity and invasion, exosome formation and adhesion, and exogenous protein binding. The results from this study may help in our understanding of the role of HBV infection in the development of HCC and in the development of new targets for treatment or non-invasive predictive biomarkers of HCC.

Human viral oncogenesis is a complex phenomenon and a major contributor to the global cancer burden. Several recent findings revealed cellular and molecular pathways that promote the development and initiation of malignancy when viruses cause an infection. Even, antiviral treatment has become an approach to eliminate the viral infections and prevent the activation of oncogenesis. Therefore, for a better understanding, the molecular pathogenesis of various oncogenic viruses like, hepatitis virus, human immunodeficiency viral (HIV), human papillomavirus (HPV), herpes simplex virus (HSV), and Epstein-Barr virus (EBV), could be explored, especially, to expand many potent antivirals that may escalate the apoptosis of infected malignant cells while sparing normal and healthy ones. Moreover, contemporary therapies, such as engineered antibodies antiviral agents targeting signaling pathways and cell biomarkers, could inhibit viral oncogenesis. This review elaborates the recent advancements in both natural and synthetic antivirals to control viral oncogenesis. The study also highlights the challenges and future perspectives of using antivirals in viral oncogenesis.

Viral hepatitis is a global health care challenge due to its worldwide distribution, chronic persistence, complications, and high prevalence with unchecked conditions in areas like sub-Saharan Africa. A high proportion of asymptomatic infections allows serious complications and poses infection risk to destination populations. This study aimed to determine the prevalence of both HBV and HCV among 3248 migrants from different parts of sub-Saharan Africa newly arrived at Kufra, Libya, a remote agricultural North African city. All these migrants were required by the Libyan authorities to undergo a complete medical check-up for different purposes such as joining new jobs, and obtaining licenses for trade and commerce. UAT sera from 3248 migrants, aged 18-53 years, attending the Al Kufra city hospital from January 01 to December 31, 2019, were screened for HBsAg and anti-HCV antibody by rapid tests and positive samples were further tested by ELISA method. The results showed that 761/3248 (23.4%) of the migrants were positive for HBV and 1014/3248 (31.2%) were positive for HCV. Migrants from sub-Saharan Africa carry high rates of HBV and HCV infection. This suggests the importance of increased attention to actions to deal with findings among positive migrants, and for awareness about risks of transmission to the local population. Study results indicate the value of routine migrant monitoring, the need for awareness in destination country health authorities, and the potential for impact on migrant destination populations.

According to the World Health Organization (WHO), an estimated 296 million people are chronically infected with hepatitis B virus (HBV). Approximately 15-25% of these people develop complications such as advanced chronic liver diseases (ACLDs). Mortality due to HBV-related complications accounted for an estimated 882,000 deaths in 2019. Potent preventive vaccines have already restricted new HBV infections, and several drugs are available to treat chronic HBV infections. However, the positive impacts of these drugs have been recorded in only a few patients with chronic HBV infection. These drugs do not show long-term efficacy and cannot halt the progression to complications. Thus, more effective and evidence-based therapeutic strategies need to be urgently developed for patients with chronic HBV infection. CHB is a pathological entity induced by HBV that progresses due to impaired host immunity. This indicates the inherent limitations of antiviral-drug-based monotherapy for treating patients with chronic HBV infection. Additionally, commercially available antiviral drugs are not available to patients in developing and resource-constrained countries, posing a challenge to achieving the following WHO goal: "Elimination of Hepatitis by 2030". As such, this review aimed to provide insights regarding evidence-based and effective management strategies for chronic HBV infection.

The burden of chronic hepatitis B virus (HBV) results in almost a million deaths per year. The most common treatment for chronic hepatitis B infection is long-term nucleoside analogs (NUC) or one-year interferon-alpha (pegylated or non-pegylated) therapy before or after NUC therapy. Unfortunately, these therapies rarely result in HBV functional cure because they do not eradicate HBV from the nucleus of the hepatocytes, where the covalently closed circular DNA (cccDNA) is formed and/or where the integrated HBV DNA persists in the host genome. Hence, the search continues for novel antiviral therapies that target different steps of the HBV replication cycle to cure chronically infected HBV individuals and eliminate HBV from the liver reservoirs.

The authors focus on capsid assembly modulators (CAMs). These molecules are unique because they impact not only one but several steps of HBV viral replication, including capsid assembly, capsid trafficking into the nucleus, reverse transcription, pre-genomic RNA (pgRNA), and polymerase protein co-packaging.

Mono- or combination therapy, including CAMs with other HBV drugs, may potentially eliminate hepatitis B infections. Nevertheless, more data on their potential effect on HBV elimination is needed, especially when used daily for 6-12 months.

Covalently closed circular (ccc) DNA is the template for hepatitis B virus (HBV) replication. The lack of small animal models for characterizing chronic HBV infection has hampered research progress in HBV pathogenesis and drug development. Here, we generated a spatiotemporally controlled recombinant cccDNA (rcccDNA) mouse model by combining Cre/loxP-mediated DNA recombination with the liver-specific "Tet-on/Cre" system. The mouse model harbors three transgenes: a single copy of the HBV genome (integrated at the Rosa26 locus, RHBV), H11-albumin-rtTA (spatiotemporal conditional module), and (tetO)₇-Cre (tetracycline response element), and is named as RHTC mouse. By supplying the RHTC mice with doxycycline (DOX)-containing drinking water for two days, the animals generate rcccDNA in hepatocytes, and the rcccDNA supports active HBV gene expression and can maintain HBV viremia persistence for over 60 weeks. Persistent HBV gene expression induces intrahepatic inflammation, fibrosis, and dysplastic pathology, which closely mirrors the disease progression in clinical patients. Bepirovirsen, an antisense oligonucleotide (ASO) targeting all HBV RNA species, showed dose-dependent antiviral effects in the RHTC mouse model. The spatiotemporally controlled rcccDNA mouse is convenient and reliable, providing versatile small animal model for studying cccDNA-centric HBV biology as well as evaluating antiviral therapeutics.