Hepatitis B virus (HBV) DNA integration into the host cell genome is reportedly a major cause of liver cancer, and a source of hepatitis B surface antigen (HBsAg). High HBsAg levels can alter immune responses which therefore contributes to the progression of HBV-related disease. However, to what extent integration leads to the persistent circulating HBsAg is unclear. Here, we aimed to determine if the extent of HBV DNA integration is associated with the persistence of circulating HBsAg in people exposed to HBV. We established a digital droplet quantitative inverse PCR (dd-qinvPCR) method to quantify integrated HBV DNA in patients who had been exposed to HBV (anti-HBc positive and HBeAg-negative). Total DNA extracts from both liver resections (n = 32; 14 HBsAg-negative and 18 HBsAg-positive) and fine-needle aspirates (FNA, n = 10; 2 HBsAg-negative and 8 HBsAg-positive) were analysed. Using defined in vitro samples for assay establishment, we showed that dd-qinvPCR could detect integrations within an input of <80 cells. The frequency of integrated HBV DNA in those who had undergone HBsAg loss (n = 14, mean ± SD of 1.514 × 10-3 ± 1.839 × 10-3 integrations per cell) was on average 9-fold lower than those with active HBV infection (n = 18, 1.16 × 10-2 ± 1.76 × 10-2 integrations per cell; p = 0.0179). In conclusion, we have developed and validated a highly precise, sensitive and quantitative PCR-based method for the quantification of HBV integrations in clinical samples. Natural clearance of HBV is associated with fewer viral integrations. Future studies are needed to determine if dynamics of integrated HBV DNA can inform the development of curative therapies.

Hepatitis B virus (HBV) is a leading cause of chronic hepatitis and remains a significant global public health concern due to the lack of effective treatments. HBV replicates through reverse transcription within the viral capsid, making capsid assembly a promising antiviral target. However, no approved therapies currently target this process. In our previous study, we optimized the structure-activity relationship (SAR) of NVR 3-778 by modifying the A and B rings, leading to the identification of KR-26556 and Compound 3. In this study, we further synthesized derivatives to modify the C ring, resulting in the discovery of KR019 and KR026. These compounds exhibited over 170-fold higher selectivity than the reference compound while demonstrating potent antiviral activity in HBV-replicating cells. Mechanistic studies revealed that KR019 binds to the hydrophobic pocket at the core protein dimer-dimer interface, misdirecting capsid assembly into genome-free capsids and thereby inhibiting viral replication. Additionally, pharmacokinetic profiling confirmed favorable stability and safety. These findings highlight the strong antiviral potential of KR019 and KR026 and provide a foundation for further in vivo investigation.

To investigate the predictive value of serum hepatitis B virus (HBV) RNA on HBeAg seroconversion in treated chronic hepatitis B (CHB) patients.

Sixty-four HBeAg-positive CHB patients were selected. They were divided into HBeAg seroconversion group including 11 cases and HBeAg non-seroconversion group including 53 cases. HBV RNA levels and other laboratory results were measured at baseline and week 12, 24, 48, 72 during treatment in both groups. The predictive value of HBV RNA level for the seroconversion of HBeAg in patients treated for hepatitis B was analyzed.

Significant differences existed in serum HBV DNA and HBV RNA levels between the two groups at baseline while there was no significant difference in HBsAg. The correlation between HBV RNA and HBV DNA was significantly high ( r  = 0.707, P  < 0.05), while the correlation between HBV DNA and HBsAg ( r  = 0.474, P  < 0.05) or HBV RNA and HBsAg was poor ( r  = 0.372, P  < 0.05). Patients with younger age and higher HBV RNA levels at baseline and week 24 were less likely to have HBeAg seroconversion. HBV RNA was better than HBV DNA and HBsAg in predicting HBeAg seroconversion whether at baseline or week 12 and week 24. The area under the curve of HBV RNA level at 24th week was the highest, which was 0.942, and the cutoff value was 4.145 log 10 copies/ml.

HBV RNA level may be a suitable serum marker to predict whether HBeAg seroconversion can occur. CHB patients with serum HBV RNA level lower than 4.145 log 10 copies/ml at week 24 were more likely to achieve HBeAg seroconversion.

Viruses in the Hepadnaviridae family, including hepatitis B virus (HBV), replicate their double-stranded DNA (dsDNA) genomes through reverse transcription of an RNA intermediate, the pregenomic RNA (pgRNA), in the viral capsid within an infected cell. In the cell, capsids containing pgRNA, single-stranded DNA (ssDNA), and dsDNA are present. However, capsids containing dsDNA (referred to as mature genomes) are preferentially secreted in virions while only small amounts of capsids with pgRNA and ssDNA (referred to as immature genomes) are enveloped and secreted. The naturally occurring HBV core protein variant, CpF97L, is an exception; HBV CpF97L secretes high levels of ssDNA-containing virions in addition to dsDNA virions. We asked whether HBV CpF97L is capable of secreting pgRNA-containing virions as well. We found that HBV CpF97L secretes high levels of pgRNA-containing virions compared to wild-type (WT) HBV when reverse transcription was inhibited by entecavir or by the Y63F change in P protein. We detected pgRNA virions in Huh7 and HepG2 cell lines, indicating that RNA virion secretion was independent of the cell line used in virion propagation. More importantly, pgRNA virions were detected when dsDNA virions were synthesized as well. Our findings suggest that the capsids of CpF97L are constitutively matured, allowing for virions with immature genomes (ssDNA and pgRNA) to be secreted in addition to dsDNA virions.IMPORTANCEFinding a cure for hepatitis B is critical, as over 250 million people live with a hepatitis B virus (HBV) infection. HBV replicates through a series of nascent RNA and DNA intermediates in capsids, resulting in the secretion of a DNA virion to propagate the infection. HBV infections have been managed with nucleos(t)ide analogs (NAs), which terminate DNA synthesis during replication. During NA treatment, DNA levels plummet, RNA-containing capsids accumulate in infected cells and are secreted, albeit inefficiently, as virions. RNA virions in serum have therefore been proposed to be used as an indicator for covalently closed circular DNA (cccDNA) (HBV's minichromosome in hepatocytes) to determine patients who can be withdrawn from NAs without virological rebound. However, it is unknown if RNA virions are efficiently secreted by the frequent HBV variants that secrete high levels of ssDNA-containing virions, as these will lead to an erroneous overestimate of the cccDNA reservoir; hence, the need for our study.

Hepatitis B virus (HBV) exclusively infects hepatocytes and produces large amounts of subviral particles containing its surface antigen (HBsAg). T cell immunity is crucial for controlling and clearing HBV infection. However, the intercellular processes underlying HBsAg presentation to T cells are incompletely understood. Here, using preclinical mouse models, we show that, following HBsAg expression, the intrahepatic Batf3+XCR1+CCR7- conventional dendritic cell subset cDC1 presents HBsAg by MHC-I cross-dressing, driving CD8+ T cell response. Meanwhile, upon HBsAg access to lymphoid tissues, B cells acquire HBsAg directly in the follicles of lymphoid tissues and initiate CD4+ T cell responses sequentially in the follicular and interfollicular regions, guided by chemoattractant receptors CCR5 and EBI2, respectively. Finally, we identify ALCAM, LFA-1, and CD80 as key co-stimulatory signals essential for optimal T cell responses. Thus, these findings reveal the roadmap of non-canonical antigen presentation that drives T cell immunity against HBsAg, advancing novel therapeutic strategies for chronic HBV infection.

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 frequently occurs 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 the relative change of HBV RNA more accurately reflects the antiviral effectiveness of a CAM than the relative change in HBV DNA.

The covalently closed circular DNA (cccDNA) of the Hepatitis B virus (HBV) serves as a template for producing progeny viruses in virally infected hepatocytes. Promising cccDNA-targeting antiviral agents remain unavailable and unpredictable in the research and development pipelines, making sterile HBV elimination challenging at the current stage. The major challenge of discriminating trace amounts of cccDNA from the abundant HBV relaxed circular DNA (rcDNA), which is nearly identical to cccDNA in sequence, substantially discourages efforts to discover and directly screen cccDNA-targeting drugs. Therefore, an easy cccDNA cell culture system is required for high-throughput drug screening. In this study, we designed an HBV cccDNA self-generating stable cell culture system using a functional complementary concept and successfully generated an HBV cccDNA Gaussia luciferase reporter cell line in HepG2 and Huh7 cells. This design ensures that the Gluc signal is exclusively expressed upon cccDNA formation, allowing for the accurate and easy measurement of cccDNA levels via luminescent signals. Using this system, in conjunction with a firefly luciferase reporter to monitor cell activity, we screened 2074 drugs in the HepG2-HBV-cccDNA/Firefly cell line. Four compounds were selected for further experimentation and their anti-HBV effects were confirmed. Thus, this virus-free hepatitis B cccDNA cell culture system provides a valuable and convenient platform for the high-throughput screening of anti-HBV drugs.

Emerging evidence indicates that the treatment duration of pegylated interferon-α and HBsAb may predict relapse in chronic hepatitis B (CHB) patients who achieved clinical cure. However, the host immunological mechanisms contributing to clinical relapse remain poorly characterized.

This study aimed to identify the immunological factors associated with relapse in chronic hepatitis B (CHB) patients who achieved clinical cure based on pegylated interferon-alpha treatment.

CHB patients who achieved HBsAg loss after discontinuing pegylated interferon-alpha therapy were enrolled and followed up for at least 96 weeks. HBcrAg and immunological markers, including the proportion of follicular helper T (Tfh) cells were assessed by flow cytometry. Peripheral blood cytokine levels were measured using a Luminex assay. The primary outcome was the correlation between immunological markers with relapse at the end of treatment (EOT) and end of follow-up (EOF).

A total of 456 CHB patients were included. During the 96-week follow-up period, 37 patients (8.11%) experienced a relapse. Propensity score matching was performed at a 1:2 ratio, resulting in the inclusion of 37 relapsed (R) and 74 non-relapsed (NR) patients. The NR group exhibited higher proportions of Tfh cells than the R group in both EOT and EOF (EOT: 12.52% vs. 8.78%, p=0.008; EOF: 11.38% vs. 8.29%, p=0.008). A significantly greater proportion of CCR7loPD-1hi Tfh cells was observed in the NR group at the EOT (9.4% vs. 4.5%, p=0.009). The frequency of CCR7loPD-1hi Tfh cells was significantly and positively correlated with anti-HB levels in EOT (p=0.015, r=0.392). Serum levels (median, pg/mL) of CTLA-4 (EOT: 14.87 vs. 7.84; EOF: 14.87 vs. 7.97), PD-1 (EOT: 321.44 vs. 203.96; EOF: 288.45 vs. 166.04), and TIM-3 (EOT:4487 vs. 21254; EOF:2973 vs. 1768) were significantly higher in the R group than in the NR group at both EOT and EOF (p<0.05). At EOT, the HBcrAg level was significantly greater in the R than in the NR group (3.45±0.94 vs. 2.63±0.80 log10 U/mL, p=0.003).

Higher frequencies of CCR7loPD-1hi Tfh cells, lower levels of sCTLA-4 as well as sPD-1 in EOT were protective factors for relapse among CHB patients who experienced HBsAg loss after pegylated interferon-alpha therapy. CCR7loPD-1hiTfh cells produced insufficient IL-21 in patients who experienced recurrent disease, which resulted in a decrease in the ability of B cells to produce anti-HBs.

The Hepatitis B Virus (HBV) presents a formidable global health challenge, impacting hundreds of millions worldwide and imposing a considerable burden on healthcare systems. The elusive nature of the virus, with its ability to establish chronic infection and evade immune detection, and the absence of curative agents have prompted efforts to develop novel therapeutic approaches beyond current antiviral treatments. This review addresses the challenging concept of a functional cure for HBV, a state characterized by the suppression of HBV and HBsAg, mitigating disease progression and transmission without a complete cure. We provide an overview of HBV epidemiology and its clinical impact, followed by an exploration of the current treatment landscape and its limitations. The immunological basis of a functional cure is then discussed, exploring the intricate interplay between the virus and the host immune response. Emerging therapeutic approaches, such as RNA interference-based interventions, entry inhibitors, nucleic acid polymers, and therapeutic vaccines, are discussed with regard to their success in achieving a functional cure. Lastly, the review underscores the urgent need for innovative strategies to achieve a functional cure for HBV.

Steatotic liver disease (SLD) is prevalent among individuals with chronic hepatitis B virus (CHB), yet its impact on clinical outcomes remains controversial.

We used electronic health record data from 98 US healthcare-delivery systems to compare adults with (CHB-SLD) and without SLD (CHB-wo-SLD) from 2000 to 2024. We applied 1: 1 propensity score matching to balance cohorts by demographic and clinical characteristics. We further performed sensitivity analyses in the presence or absence of cirrhosis. We compared incidence rates (IR) and hazard ratios (HRs) of all-cause mortality, hepatocellular carcinoma (HCC), end-stage liver disease (ESLD) events, and detectable HBsAg and HBeAg as markers of seroclearance.

Among 124,932 individuals with CHB (12.43% CHB-SLD), there were 470,707 person-years of observations (median follow-up 2.95 years). Compared with CHB, individuals with CHB-SLD had a lower mortality risk (HR 0.44, 95% CI 0.40-0.48). Fibrosis risk was higher among those with CHB-SLD (vs CHB-wo-SLD) (HR 1.93, 95% CI 1.71-2.19); however, cirrhosis risk was comparable (HR 1.06, 95% CI 0.96-1.18) between cohorts, while HCC risk was lower in the CHB-SLD cohort (HR 0.83, 95% CI 0.70-0.96). The CHB-SLD cohort also had significantly reduced risks of ESLD events, including ascites, spontaneous bacterial peritonitis, variceal bleeding, hepatic encephalopathy, and hepatorenal syndrome (all p < 0.001). Additionally, detectable HBsAg and HBeAg IRs and HRs were lower among CHB-SLD compared to the CHB-wo-SLD cohort: 26.83 vs. 31.96 per 1,000 person-years (HR 0.80, 95% CI 0.73-0.87) and 8.52 vs. 11.36 per 1,000 person-years (HR 0.74, 95% CI 0.65-0.85), respectively. Sensitivity analyses stratified by cirrhosis status supported these findings.

CHB-SLD status was associated with more favorable outcomes, highlighting the complexity of CHB and SLD interactions.

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.

Hepatitis B virus (HBV) infection remains a significant global public health concern. Myricetin, a flavonoid compound widely distributed in natural plants, has demonstrated multiple biological functions in combating diseases such as cancer and inflammation. In this research, we explored the mechanism of myricetin against HBV replication. We employed various experiments such as ELISA, Southern Blot, Northern Blot, Western Blot, RT-qPCR, Dual luciferase reporter gene assay, ChIP, EMSA, IHC, Immunofluorescence, AAV infection, and isolation of primary human hepatocytes (PHH) in this study. Our results showed that myricetin significantly reduced the expression of HBV markers, including HBsAg, HBeAg and covalently closed circular DNA (cccDNA), in HepG2-NTCP cells and PHH. We further confirmed these findings using AAV-HBV cell and mouse models. Furthermore, we found that myricetin significantly downregulated HBV SP2 promoter activity. Mechanistically, myricetin reduced CEBPA expression, which in turn interfered with the binding of CEBPA to the HBV SP2 promoter, leading to an antiviral effect. In conclusion, myricetin exhibited promising antiviral activity against HBV, suggesting its potential for novel HBV treatment.

Chronic hepatitis B (CHB) is a major global health concern. This study aims to investigate the factors influencing hepatitis B surface antigen (HBsAg) clearance in CHB patients treated with pegylated interferon α-2b (Peg-IFNα-2b) for 48 weeks and to establish a predictive model.

This analysis is based on the "OASIS" project, a prospective real-world multicenter study in China. We included CHB patients who completed 48 weeks of Peg-IFNα-2b treatment. Patients were randomly assigned to a training set and a validation set in a ratio of approximately 4:1 by spss 26.0, and were divided into clearance and non-clearance groups based on HBsAg status at 48 weeks. Clinical data were analyzed using SPSS 26.0, employing chi-square tests for categorical data and Mann-Whitney U tests for continuous variables. Significant factors (p < 0.05) were incorporated into a binary logistic regression model to identify independent predictors of HBsAg clearance. The predictive model's performance was evaluated using ROC curve analysis.

We included 868 subjects, divided into the clearance group (187 cases) and the non-clearance group (681 cases). They were randomly assigned to a training set (702 cases) and a validation set (166 cases). Key predictors included female gender (OR = 1.879), lower baseline HBsAg levels (OR = 0.371), and cirrhosis (OR = 0.438). The final predictive model was: Logit(P) = 0.92 + Gender (Female) * 0.66 - HBsAg (log) * 0.96 - Cirrhosis * 0.88. ROC analysis showed an AUC of 0.80 for the training set and 0.82 for the validation set, indicating good predictive performance.

Gender, baseline HBsAg levels, and cirrhosis are significant predictors of HBsAg clearance in CHB patients after 48 weeks of Peg-IFNα-2b therapy. The developed predictive model demonstrates high accuracy and potential clinical utility.

Identifying hepatitis B virus (HBV) patients eligible for safe nucleos(t)ide analogues (NAs) discontinuation remains challenging. Discrepant data on combined HBV DNA and quantitative HBV surface antigen (qHBsAg) assessments are available. This study aimed to identify potential predictors for safe treatment discontinuation by evaluating clinical/virological outcomes in patients on long-term NA therapy.

A retrospective cohort of 139 chronic hepatitis B (CHB) patients - who consecutively started Entecavir or Tenofovir from 2007 to 2011 - was evaluated. The study population was selected based on anti-HBe positivity, absence of prior antiviral treatment, absence of non-HBV-related liver diseases or hepatocellular carcinoma (HCC), and long-term clinical/ultrasonographic/laboratory evaluations post-NA initiation. Serum samples collected before starting NA (T0) and over ten years (T1-T10) were tested for HBV DNA and qHBsAg.

Twenty-two/139 (15.8 %) CHB patients (12 chronic hepatitis, 10 cirrhosis) met the inclusion criteria. All patients showed a significant decrease in liver stiffness values in the ten years of follow-up (p = 0.001), and no hepatic decompensation occurred. Three/22 (13.6 %) patients developed HCC. Ten/22 patients (45.5 %; group-A) had fluctuating HBV DNA, while other 10/22 (45.5 %; group-B) showed undetectable HBV DNA for 5-9 years with more significant qHBsAg decline (p = 0.04) than group-A. Two/22 (9.1 %) patients showed a critical qHBsAg decline up to seroconversion together with undetectable HBV DNA.

Persistent undetectable HBV DNA levels correlate with qHBsAg reduction and the potential HBsAg seroclearance, suggesting that long-term HBV DNA monitoring in NA-treated CHB patients might help identify candidates for treatment discontinuation.

The RING finger protein 5 (RNF5), an E3 ubiquitin ligase, has demonstrated significant antiviral activity against various viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Kaposi's sarcoma-associated herpesvirus (KSHV). However, its role in hepatitis B virus (HBV) replication has not been previously studied. In this study, we demonstrate that RNF5 effectively inhibits HBV replication by promoting the degradation of the HBV Core protein through a Caspase-3-dependent pathway. We first show that RNF5 expression is upregulated in HBV-infected cells and patient samples, suggesting a role in the host's antiviral response. Subsequently, we investigate the mechanism by which RNF5 mediates its antiviral effect, finding that RNF5 targets the Core protein for degradation independently of its E3 ubiquitin ligase activity. The degradation of Core protein is mediated through a Caspase-3-dependent mechanism rather than the proteasomal pathway. Interestingly, RNF5's antiviral function does not rely on ubiquitination, indicating an alternative pathway involving apoptosis-related processes. These findings highlight the multifunctional role of RNF5 and suggest that targeting RNF5 could serve as a novel therapeutic approach to control HBV replication, providing new insights into the development of antiviral therapies against HBV.

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.

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.

Viruses are significant human pathogens responsible for pandemic outbreaks and seasonal epidemics. Viral infectious diseases impose a devastating global burden and have a profound impact on public health systems. During viral infections, alternative splicing (AS) plays a crucial role in regulating immune responses, altering the host's cellular environment, expanding viral genetic material, and facilitating viral replication. As research on AS in viral infections expands, it is crucial to consolidate data on virus-related splicing changes to improve our understanding of these viruses and associated diseases. To address this need, we created ASVirus (https://bddg.hznu.edu.cn/asvirus/), a comprehensive database of virus-associated AS events and their regulatory factors. ASVirus uniquely combines high-confidence, experimentally validated splicing data and investigates upstream regulatory mechanisms through a gene-splicing factor interaction network. Its user-friendly web interface offers detailed information into AS events from various viral families and the resulting mis-splicing in host genes, aiding the exploration of novel viral infection mechanisms and the identification of critical therapeutic targets for viral diseases.

The liver, as the primary metabolic organ, is susceptible to an array of factors that can harm liver cells and give rise to different liver diseases. Epigallocatechin gallate (EGCG), a natural compound found in green tea, exerts numerous beneficial effects on the human body. Notably, EGCG displays antioxidative, antibacterial, antiviral, anti-inflammatory, and anti-tumor properties. This review specifically highlights the pivotal role of EGCG in liver-related diseases, focusing on viral hepatitis, autoimmune hepatitis, fatty liver disease, and hepatocellular carcinoma. EGCG not only inhibits the entry and replication of hepatitis B and C viruses within hepatocytes, but also mitigates hepatocytic damage caused by hepatitis-induced inflammation. Furthermore, EGCG exhibits significant therapeutic potential against hepatocellular carcinoma. Combinatorial use of EGCG and anti-hepatocellular carcinoma drugs enhances the sensitivity of drug-resistant cancer cells to chemotherapeutic agents, leading to improved therapeutic outcomes. Thus, the combination of EGCG and anti-hepatocellular carcinoma drugs holds promise as an effective approach for treating drug-resistant hepatocellular carcinoma. In conclusion, EGCG possesses hepatoprotective properties against various forms of liver damage and emerges as a potential drug candidate for liver diseases.

Occult hepatitis B infection (OBI) is a serious public health issue. Although a number of effective hepatitis B vaccines are available, hepatitis B still poses a threat to global public health. Patients with OBI are usually asymptomatic, but there may be active HBV DNA present in their blood, leading to the risk of virus transmission during blood transfusions or organ transplantation, constituting a hazard to the health of recipients and increasing the risk of liver cirrhosis and liver cancer. Although China has progressed in the development of blood-screening technology, OBI is still a significant hidden danger to blood transfusion safety. Therefore, in blood screening and blood transfusion, strengthening the monitoring and management of OBI is crucial to ensure blood safety and protect public health.

Viral infections persist as a significant cause of morbidity and mortality worldwide. Conventional therapeutic approaches often fall short in fully eliminating viral infections, primarily due to the emergence of drug resistance. Natural killer (NK) cells, one of the important members of the innate immune system, possess potent immunosurveillance and cytotoxic functions, thereby playing a crucial role in the host's defense against viral infections. Chimeric antigen receptor (CAR)-NK cell therapy has been developed to redirect the cytotoxic function of NK cells specifically towards virus-infected cells, further enhancing their cytotoxic efficacy. In this manuscript, we review the role of NK cells in antiviral infections and explore the mechanisms by which viruses evade immune detection. Subsequently, we focus on the optimization strategies for CAR-NK cell therapy to address existing limitations. Furthermore, we discuss significant advancements in CAR-NK cell therapy targeting viral infections, including those caused by severe acute respiratory syndrome coronavirus 2, human immunodeficiency virus, hepatitis B virus, human cytomegalovirus, and Epstein-Barr virus.

Hepatitis B virus (HBV) infection is a well-documented independent risk factor for developing hepatocellular carcinoma (HCC). Consequently, extensive research has focused on elucidating the mechanisms by which HBV induces hepatocarcinogenesis. The majority of studies are dedicated to understanding how HBV DNA integration into the host genome, viral RNA expression, and the resulting protein transcripts affect cellular processes and promote the malignant transformation of hepatocytes. However, considering that most acute HBV infections are curable, immune suppression potentially contributes to the critical challenges in the treatment of chronic infections. Regulatory T cells (Tregs) are crucial in immune tolerance. Understanding the interplay of Tregs within the liver microenvironment following HBV infection could offer novel therapeutic approaches for treating HBV infections and preventing HBV-related HCC. Two viewpoints to targeting Tregs in the liver microenvironment include means of reducing their inhibitory function and decreasing Treg frequency. As these strategies may disrupt the immune balance and lead to autoimmune responses, careful and comprehensive profiling of the patient's immunological status and genetic factors is required to successfully employ this promising therapeutic approach.

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.

Covalently closed circular DNA (cccDNA) is a stable, episomal form of HBV DNA. cccDNA is a true marker for the intrahepatic events in controlled CHB infection. Quantifying cccDNA is critical for monitoring disease progression, and efficacy of anti-viral therapies.

To standardize the method, total HBV DNA was isolated from HepAD38 cells and digested with three exonuclease enzymes to remove linear and relaxed circular HBV DNA. Purified cccDNA quantification used ddPCR with specific primers. Treatment-naive chronic hepatitis B virus patients (nCHBV, n=36) with detectable HBV DNA and HBsAg were grouped by HBsAg levels: Group I (HBsAglo < 2000 IU/ml, n=11) and Group II (HBsAghi > 2000 IU/ml, n=25). cccDNA, HBV DNA and HBsAg were quantified in plasma and compared between groups. Correlation with clinical/histopathological features was done.

Non-digested 3.6^10⁶ tet-ve HepAD38 cells showed 316 copies/µl of total viral DNA. After digesting the linear, integrated, and relaxed circular DNA with triple enzymes, 15 copies/µl of cccDNA were detected. Similarly, after DNA digestion, HBsAglo patients showed a median of 8.5 copies/µl (IQR 2.75-9.75 copies/µl), and HBsAghi gave a median of 11 copies/µl (IQR 4-16 copies/µl) but with no significant difference between groups (p=0.093). Further, HBsAglo patients with low cccDNA copy numbers showed significantly higher fibrosis grades than HBsAghi (p=0.036).

We conclude that employing a combined approach utilizing three exonucleases, cccDNA-specific primers, and ddPCR enables the detection of cccDNA copies even in patients exhibiting low levels of HBsAg and HBV DNA. This integrated method offers additional validation as a surrogate diagnostic tool.

Infections caused by viruses as the smallest infectious agents, pose a major threat to global public health. Viral infections utilize different host mechanisms to facilitate their own propagation and pathogenesis. MicroRNAs (miRNAs), as small noncoding RNA molecules, play important regulatory roles in different diseases, including viral infections. They can promote or inhibit viral infection and have a pro-viral or antiviral role. Also, viral infections can modulate the expression of host miRNAs. Furthermore, viruses from different families evade the host immune response by producing their own miRNAs called viral miRNAs (v-miRNAs). Understanding the replication cycle of viruses and their relation with host miRNAs and v-miRNAs can help to find new treatments against viral infections. In this review, we aim to outline the structure, genome, and replication cycle of various viruses including hepatitis B, hepatitis C, influenza A virus, coronavirus, human immunodeficiency virus, human papillomavirus, herpes simplex virus, Epstein-Barr virus, Dengue virus, Zika virus, and Ebola virus. We also discuss the role of different host miRNAs and v-miRNAs and their role in the pathogenesis of these viral infections.

The advent of RNA interference (RNAi) technology through the use of short-interfering RNAs (siRNAs) represents a paradigm shift in the fight against viral infections. siRNAs, with their ability to directly target and silence specific posttranscriptional genes, offer a novel mechanism of action distinct from that of traditional pharmacotherapeutics. This review delves into the growing field of siRNA therapeutics against viral infections, highlighting their critical role in contemporary antiviral strategies. Importantly, this review will solely focus on the use of lipid nanoparticles (LNPs) as the ideal antiviral siRNA delivery agent for use in vivo. We discuss the challenges of siRNA delivery and how LNPs have emerged as a pivotal solution to enhance antiviral efficacy. Specifically, this review focuses on work that have preclinically tested LNP formulated siRNA on virus infection animal models. Since the COVID-19 pandemic, we have witnessed a resurgence in the field of RNA-based therapies, including siRNAs against viruses including, SARS-CoV-2. Notably, the critical importance of LNPs as the ideal carrier for precious 'RNA cargo' can no longer be ignored with the advent of mRNA-LNP based COVID-19 vaccines. siRNA-based therapeutics represents an emerging class of anti-infective drugs with a foreseeable future as suitable antiviral agents.

Hepatitis B virus (HBV) infections promote liver cancer initiation by inducing inflammation and cellular stress. Despite a primarily indirect effect on oncogenesis, HBV is associated with a recurrent genomic phenotype in hepatocellular carcinoma (HCC), suggesting that it impacts the biology of established HCC. Characterization of the interaction of HBV with host proteins and the mechanistic contributions of HBV to HCC initiation and maintenance could provide insights into HCC biology and uncover therapeutic vulnerabilities. In this study, we used affinity purification mass spectrometry to comprehensively map a network of 145 physical interactions between HBV and human proteins in HCC. A subset of the host factors targeted by HBV proteins were preferentially mutated in non-HBV-associated HCC, suggesting that their interaction with HBV influences HCC biology. HBV interacted with proteins involved in mRNA splicing, mitogenic signaling, and DNA repair, with the latter set interacting with the HBV oncoprotein X (HBx). HBx remodeled the PP2A phosphatase complex by excluding striatin regulatory subunits from the PP2A holoenzyme, and the HBx effects on PP2A caused Hippo kinase activation. In parallel, HBx activated mTOR complex 2, which can prevent YAP degradation. mTOR complex 2-mediated upregulation of YAP was observed in human HCC specimens and mouse HCC models and could be targeted with mTOR kinase inhibitors. Thus, HBV interaction with host proteins rewires HCC signaling rather than directly activating mitogenic pathways, providing an alternative paradigm for the cellular effects of a tumor-promoting virus. Significance: Integrative proteomic and genomic analysis of HBV/host interactions illuminated modifiers of hepatocellular carcinoma behavior and key signaling mechanisms in advanced disease, which suggested that HBV may have therapeutically actionable effects.

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.

We previously reported N-hydroxypyridinedione (HPD) compounds with mid-nanomolar efficacy and selectivity indexes around 300 against hepatitis B virus (HBV) replication. However, they lack pharmacological evaluation. Here, we report in vitro anti-HBV efficacy, cytotoxicity, and pharmacological characterization of 29 novel HPDs within seven subgroups. The best two compounds had EC50s of 61 and 190 nM and selectivity indexes of 526 and 1,071. Compounds with one halogen on the major R group were most effective and compounds with large ether R groups were most cytotoxic. Compounds were not cytotoxic in primary human hepatocytes. All compounds were freely soluble in pHs reflecting plasma (7.4) and the gastrointestinal tract (5 and 6.5). Almost all highly soluble compounds were passively permeable at pH 5.0 and 7.4. Only 2 of 11 compounds tested were likely to be effluxed by p-glycoprotein. The most potent HPDs inhibited HBV replication over human ribonuclease H1 activity by 10-fold. Four of 19 compounds inhibited CYP2D6 >50%, but their CYP2D6 IC50s were >8× higher than their anti-HBV EC50. No compound substantially inhibited CYP3A4. Thirteen of 15 compounds had human microsomal half-lives >30 min with medium to low rates of intrinsic clearance. Eleven of 12 compounds bound plasma proteins by ≥80%; however, effects against HBV replication for only one would likely be physiologically relevant. These results identify two lead candidate HPDs with pharmacological characteristics resembling commercially available drugs that are suitable for in vivo pharmacological and efficacy studies.

Hepatitis B virus (HBV) infection is a global health concern. The current sequential endpoints for the treatment of HBV infection include viral suppression, hepatitis B e antigen (HBeAg) seroconversion, functional cure, and covalently closed circular DNA (cccDNA) clearance. Serum hepatitis B core-related antigen (HBcrAg) is an emerging HBV marker comprising three components: HBeAg, hepatitis B core antigen, and p22cr. It responds well to the transcriptional activity of cccDNA in the patient's liver and is a promising alternative marker for serological testing. There is a strong correlation, and a decrease in its level corresponds to sustained viral suppression. In patients with chronic hepatitis B (CHB), serum HBcrAg levels are good predictors of HBeAg seroconversion (both spontaneous and after antiviral therapy), particularly in HBeAg-positive patients. Both low baseline HBcrAg levels and decreasing levels early in antiviral therapy favored HBsAg seroconversion, which may serve as a good surrogate option for treatment endpoints. In this review, we summarize the role of serum HBcrAg in the treatment of CHB. Therefore, long-term continuous monitoring of serum HBcrAg levels contributes to the clinical management of patients with CHB and optimizes the choice of treatment regimen, making it a promising marker for monitoring HBV cure.

Viruses frequently exploit the host's nucleocytoplasmic trafficking machinery to facilitate their replication and evade immune defenses. By encoding specialized proteins and other components, they strategically target host nuclear transport receptors (NTRs) and nucleoporins within the spiderweb-like inner channel of the nuclear pore complex (NPC), enabling efficient access to the host nucleus. This review explores the intricate mechanisms governing the nuclear import and export of viral components, with a focus on the interplay between viral factors and host determinants that are essential for these processes. Given the pivotal role of nucleocytoplasmic shuttling in the viral life cycle, we also examine therapeutic strategies aimed at disrupting the host's nuclear transport pathways. This includes evaluating the efficacy of pharmacological inhibitors in impairing viral replication and assessing their potential as antiviral treatments. Furthermore, we emphasize the need for continued research to develop targeted therapies that leverage vulnerabilities in nucleocytoplasmic trafficking. Emerging high-resolution techniques, such as advanced imaging and computational modeling, are transforming our understanding of the dynamic interactions between viruses and the NPC. These cutting-edge tools are driving progress in identifying novel therapeutic opportunities and uncovering deeper insights into viral pathogenesis. This review highlights the importance of these advancements in paving the way for innovative antiviral strategies.

The intricate link between cholesterol metabolism and host immune responses is well recognized, but the specific mechanisms by which cholesterol biosynthesis influences hepatitis B virus (HBV) replication remain unclear. In this study, we show that SREBP2, a key regulator of cholesterol metabolism, inhibits HBV replication by interacting directly with the HBx protein, thereby preventing its nuclear translocation. We also found that inhibiting the ER-to-Golgi transport of the SCAP-SREBP2 complex or blocking SREBP2 maturation significantly enhances HBV suppression. Notably, we demonstrate that the C-terminal domain (CTD) of SREBP2, rather than its N-terminal domain (NTD), mediates this inhibition by interacting with HBx and promoting its extracellular secretion, thus reducing nuclear HBx accumulation. These findings reveal a novel regulatory pathway that links cholesterol metabolism to HBV replication via SREBP2-mediated control of HBx localization. This insight provides a potential basis for new therapeutic strategies against HBV infection, addressing an important global health issue.

Hepatitis B virus (HBV) is a major global health issue, with an estimated 254 million people living with chronic HBV infection worldwide as of 2022. Chronic HBV infection is the leading cause of cirrhosis and liver cancer. Current treatment with nucleos(t)ide analogs is effective in the suppression of viral activity but generally requires lifelong treatment. They fail to eradicate the HBV viral reservoir, called covalently closed circular DNA (cccDNA), which replicates in the nucleus of liver cells. The cccDNA serves as the sole template for viral replication, as it generates the pregenomic RNA (pgRNA) necessary for producing new viral genomes. This stable form of viral DNA can reactivate the virus when treatment is stopped. HBV cccDNA is therefore one of the main challenges in curing chronic HBV infections. By targeting steps such as cccDNA formation, capsid assembly, or particle secretion, researchers continue to seek ways to interfere with HBV replication and to reduce its persistence, ultimately to eradicate HBV as a global health problem. This review provides an overview of what is currently known about cccDNA formation and biogenesis and the ongoing efforts to target and eradicate it to cure chronic HBV infections.

Chronic hepatitis B virus (HBV) infection can significantly increase the incidence of cirrhosis and liver cancer, and there is no curative treatment. The persistence of HBV covalently closed circular DNA (cccDNA) is the major obstacle of antiviral treatments. cccDNA is formed through repairing viral partially double-stranded relaxed circular DNA (rcDNA) by varies host factors. However, the detailed mechanisms are not well characterized. To dissect the biogenesis of cccDNA, we took advantage of an in vitro rcDNA repair system to precipitate host factors interacting with rcDNA and identified co-precipitated proteins by mass spectrometry. Results revealed the MRE11-RAD50-NBS1 (MRN) complex as a potential factor. Transiently or stably knockdown of MRE11, RAD50 or NBS1 in hepatocytes before HBV infection significantly decreased viral markers, including cccDNA, while reconstitution reversed the effect. Chromatin immunoprecipitation assay further validated the interaction of MRN complex and HBV DNA. However, MRN knockdown after HBV infection showed no effect on viral replication, which indicated that MRN complex inhibited the formation of cccDNA without affecting its stability or transcriptional activity. Interestingly, Mirin, a MRN complex inhibitor which can inhibit the exonuclease activity of MRE11 and MRN-dependent activation of ATM, but not ATM kinase inhibitor KU55933, could decrease cccDNA level. Likewise, the MRE11 endonuclease activity inhibitor PFM01 treatment decreased cccDNA. MRE11 nuclease assays indicated that rcDNA is a substrate of MRE11. Furthermore, the inhibition of ATR-CHK1 pathway, which is known to be involved in cccDNA formation, impaired the effect of MRN complex on cccDNA. Similarly, inhibition of MRE11 endonuclease activity mitigated the effect of ATR-CHK1 pathway on cccDNA. These findings indicate that MRN complex cooperates with ATR-CHK1 pathway to regulate the formation of HBV cccDNA. In summary, we identified host factors, specifically the MRN complex, regulating cccDNA formation during HBV infection. These findings provide insights into how HBV hijacks host enzymes to establish chronic infection and reveal new therapeutic opportunities.