Genetic variability of hepatitis B virus in acute and in different phases of chronic infection in Brazil.

The selection pressure imposed by the host immune system impacts on hepatitis B virus (HBV) variability. This study evaluates HBV genetic diversity, nucleos(t)ide analogs resistance and HBsAg escape mutations in HBV patients under distinct selective pressures. One hundred and thirteen individuals in different phases of HBV infection were included: 13 HBeAg-positive chronic infection, 9 HBeAg-positive chronic hepatitis, 47 HBeAg-negative chronic infection (ENI), 29 HBeAg-negative chronic hepatitis (ENH) and 15 acute infected individuals. Samples were PCR amplified, sequenced and genetically analyzed for the overlapping POL/S genes. Most HBV carriers presented genotype A (84/113; 74.3%), subgenotype A1 (67/84; 79.7%), irrespective of group, followed by genotypes D (20/113; 17.7%), F (8/113; 7.1%) and E (1/113; 0.9%). Clinically relevant mutations in polymerase (tL180M/M204V) and in the Major Hydrophilic Region of HBsAg (sY100C, T118A/M, sM133T, sD144A and sG145R) were observed. Our findings, however, indicated that most polymorphic sites were located in the cytosolic loops (CYL1-2) and transmembrane domain 4 (TMD4) of HBsAg. Lower viral loads and higher HBV genetic diversity were observed in ENI and ENH groups (p < 0.001), suggesting that these groups are subjected to a higher selective pressure. Our results provide information on the molecular characteristics of HBV in a diverse clinical setting, and may guide future studies on the balance of HBV quasispecies at different stages of infection.

Frequency of HBsAg variants in occult hepatitis B virus infected patients and detection by ARCHITECT HBsAg quantitative.

Objective: To analyze the amino acid substitution caused by mutations in the major hydrophilic region (MHR) of the S-region genes in the serum samples of occult hepatitis B virus infection (OBI), and to explore the reasons for the missed detection of HBsAg. Method: The full-length gene of the S-region in hepatitis B virus(HBV) in the chronic hepatitis B virus(CHB)(10 samples) and OBI groups(42 samples) was amplified using a lab-developed, two-round PCR amplification technology. The PCR amplification products were sequenced/clone sequenced, and the nucleotide sequences of the S-region gene in HBV were compared to the respective genotype consensus sequence. Results: Only 20 of the 42 samples in the OBI group had the S-region genes successfully amplified, with the lowest HBV DNA load of 20.1IU/ml. As S-region genes in HBV, 68 cloned strains were sequenced. In the OBI and CHB groups MHR region, with a mutation rate of 3.21% (155/4828) and 0.70% (5/710). The genetic mutation rate was significantly higher in the OBI group than in the CHB group (P<0.05). The common mutation types in the MHR region were: I126T, L162R, K122E, C124R, and C147Y.Mutations at s122, s126, and s162 were associated with subgenotypes, most of which being C genotypes. The high-frequency mutation sites L162R and K122E found in this study have not been reported in previous literature. Conclusion: The results of this study confirmed that MHR mutations can cause the missed detection of HBsAg, giving rise to OBI.

Discovery and mechanistic study of Imperatorin that inhibits HBsAg expression and cccDNA transcription.

Chronic hepatitis B virus (HBV) infection remains a significant global health challenge due to its link to severe conditions like HBV-related cirrhosis and hepatocellular carcinoma (HCC). Although current treatments effectively reduce viral levels, they have limited impact on certain HBV elements, namely hepatitis B surface antigen (HBsAg) and covalently closed circular DNA (cccDNA). This highlights the urgent need for innovative pharmaceutical and biological interventions that can disrupt HBsAg production originating from cccDNA. In this study, we identified a natural furanocoumarin compound, Imperatorin, which markedly inhibited the expression of HBsAg from cccDNA, by screening a library of natural compounds derived from Chinese herbal medicines using ELISA assay and qRT-PCR. The pharmacodynamics study of Imperatorin was explored on HBV infected HepG2-NTCP/PHHs and HBV-infected humanized mouse model. Proteome analysis was performed on HBV infected HepG2-NTCP cells following Imperatorin treatment. Molecular docking and bio-layer interferometry (BLI) were used for finding the target of Imperatorin. Our findings demonstrated Imperatorin remarkably reduced the level of HBsAg, HBV RNAs, HBV DNA and transcriptional activity of cccDNA both in vitro and in vivo. Additionally, Imperatorin effectively restrained the actions of HBV promoters responsible for cccDNA transcription. Mechanistic study revealed that Imperatorin directly binds to ERK and subsequently interfering with the activation of CAMP response element-binding protein (CREB), a crucial transcriptional factor for HBV and has been demonstrated to bind to the PreS2/S and X promoter regions of HBV. Importantly, the absence of ERK could nullify the antiviral impact triggered by Imperatorin. Collectively, the natural compound Imperatorin may be an effective candidate agent for inhibiting HBsAg production and cccDNA transcription by impeding the activities of HBV promoters through ERK-CREB axis.

Effect of S-region mutations on HBsAg in HBsAg-negative HBV-infected patients.

BACKGROUND: Occult HBV infection (OBI) is a special form of hepatitis B virus (HBV) infection that may cause Liver cirrhosis and hepatocellular carcinoma, causing significant harm to patients. Given the insidious nature of OBI, it is usually not easy to be detected. Most of the samples currently studied are concentrated on blood donors, however, patients in this special state have not been fully studied. This project aimed to study the effect of HBV S region mutations on HBsAg in patients with clinical OBI. METHODS: Collect 107 HBsAg-/HBV DNA + blood samples from Beijing Youan Hospital, Capital Medical University from August 2022 to April 2023. Next, the successfully extracted and amplified HBV DNA S regions were sequenced. Construct mutant plasmids to verify the cell function of the high-frequency mutation sites and explore the possible molecular mechanism. RESULTS: Sixty-eight HBsAg-negative samples were sequenced, revealing high-frequency amino acid substitution sites in the HBV S protein, including immune escape mutations (i.e., sY100C、sK122R、sI126T、sT131P、and sS114T) and TMD (Transmembrane domain) region substitutions (i.e., sT5A、sG10D、sF20S、and sS3N). We constructed a portion of the mutant plasmids and found that sT5A, sF20S, sG10D, sS3N, sI68T, and sI126T single point mutations or combined mutations may decrease HBsAg expression or change the antigenicity of HBsAg leading to detection failure. CONCLUSIONS: HBsAg-negative patients may show various mutations and amino acid replacement sites at high frequency in the HBV S-region, and these mutations may lead to undetectable Hepatitis B surface antigen (HBsAg), HBsAg antigenic changes or secretion inhibition.

Characterization of mutations in hepatitis B virus DNA isolated from Japanese HBsAg-positive blood donors in 2021 and 2022.

Missense mutations in certain small envelope proteins diminish the efficacy of antibodies. Consequently, tracking the incidence and types of vaccine-escape mutations (VEMs) was crucial both before and after the introduction of universal hepatitis B vaccination in Japan in 2016. In this study, we isolated hepatitis B virus (HBV) DNA from 58 of 169 hepatitis B surface antigen (HBsAg)-positive blood samples from Japanese blood donors and determined the nucleotide sequence encoding the small envelope protein. DNA from six (10%) of the samples had VEMs, but no missense mutations, such as G145R, were detected. Complete HBV genome sequences were obtained from 29 of the 58 samples; the viral genotype was A1 in one (3%), A2 in three (10%), B1 in nine (31%), B2 in five (17%), B4 in one (3%), and C2 in 10 (34%) samples. Tenofovir-resistance mutations were detected in two (7%) samples. In addition, several core promoter mutations, such as 1762A>T and 1764G>A, and a precore nonsense mutation, 1986G>A, which are risk factors for HBV-related chronic liver disease, were detected. These findings provide a baseline for future research and highlight the importance of ongoing monitoring of VEMs and drug resistance mutations in HBV DNA from HBsAg-positive blood donors without HBV antibodies.

Mathematical Models of Early Hepatitis B Virus Dynamics in Humanized Mice.

Analyzing the impact of the adaptive immune response during acute hepatitis B virus (HBV) infection is essential for understanding disease progression and control. Here we developed mathematical models of HBV infection which either lack terms for adaptive immune responses, or assume adaptive immune responses in the form of cytolytic immune killing, non-cytolytic immune cure, or non-cytolytic-mediated block of viral production. We validated the model that does not include immune responses against temporal serum hepatitis B DNA (sHBV) and temporal serum hepatitis B surface-antigen (HBsAg) experimental data from mice engrafted with human hepatocytes (HEP). Moreover, we validated the immune models against sHBV and HBsAg experimental data from mice engrafted with HEP and human immune system (HEP/HIS). As expected, the model that does not include adaptive immune responses matches the observed high sHBV and HBsAg concentrations in all HEP mice. By contrast, while all immune response models predict reduction in sHBV and HBsAg concentrations in HEP/HIS mice, the Akaike Information Criterion cannot discriminate between non-cytolytic cure (resulting in a class of cells refractory to reinfection) and antiviral block functions (of up to 99 % viral production 1-3 weeks following peak viral load). We can, however, reject cytolytic killing, as it can only match the sHBV and HBsAg data when we predict unrealistic levels of hepatocyte loss.

Preclinical Antiviral and Safety Profiling of the HBV RNA Destabilizer AB-161.

HBV RNA destabilizers are a class of small-molecule compounds that target the noncanonical poly(A) RNA polymerases PAPD5 and PAPD7, resulting in HBV RNA degradation and the suppression of viral proteins including the hepatitis B surface antigen (HBsAg). AB-161 is a next-generation HBV RNA destabilizer with potent antiviral activity, inhibiting HBsAg expressed from cccDNA and integrated HBV DNA in HBV cell-based models. AB-161 exhibits broad HBV genotype coverage, maintains activity against variants resistant to nucleoside analogs, and shows additive effects on HBV replication when combined with other classes of HBV inhibitors. In AAV-HBV-transduced mice, the dose-dependent reduction of HBsAg correlated with concentrations of AB-161 in the liver reaching above its effective concentration mediating 90% inhibition (EC90), compared to concentrations in plasma which were substantially below its EC90, indicating that high liver exposure drives antiviral activities. In preclinical 13-week safety studies, minor non-adverse delays in sensory nerve conductance velocity were noted in the high-dose groups in rats and dogs. However, all nerve conduction metrics remained within physiologically normal ranges, with no neurobehavioral or histopathological findings. Despite the improved neurotoxicity profile, microscopic findings associated with male reproductive toxicity were detected in dogs, which subsequently led to the discontinuation of AB-161's clinical development.

The N-linked glycosylation modifications in the hepatitis B surface protein impact cellular autophagy, HBV replication, and HBV secretion.

N-linked glycosylation is a pivotal post-translational modification that significantly influences various aspects of protein biology. Autophagy, a critical cellular process, is instrumental in cell survival and maintenance. The hepatitis B virus (HBV) has evolved mechanisms to manipulate this process to ensure its survival within host cells. Significantly, post-translational N-linked glycosylation in the large surface protein of HBV (LHBs) influences virion assembly, infectivity, and immune evasion. This study investigated the role of N-linked glycosylation of LHBs in autophagy, and its subsequent effects on HBV replication and secretion. LHBs plasmids were constructed by incorporating single-, double-, and triple-mutated N-linked glycosylation sites through amino acid substitutions at N4, N112, and N309. In comparison to the wild-type LHBs, N-glycan mutants, including N309Q, N4-309Q, N112-309Q, and N4-112-309Q, induced autophagy gene expression and led to autophagosome accumulation in hepatoma cells. Acridine orange staining of cells expressing LHBs mutations revealed impaired lysosomal acidification, suggesting potential blockage of autophagic flux at later stages. Furthermore, N-glycan mutants increased the mRNA expression of HBV surface antigen (HBsAg). Notably, N309Q significantly elevated HBx oncogene level. The LHBs mutants, particularly N309Q and N112-309Q, significantly enhanced HBV replication, whereas N309Q, N4-309Q, and N4-112-309Q markedly increased HBV progeny secretion. Remarkably, our findings demonstrated that autophagy is indispensable for the impact of N-linked glycosylation mutations in LHBs on HBV secretion, as evidenced by experiments with a 3-methyladenine (3-MA) inhibitor. Our study provides pioneering insights into the interplay between N-linked glycosylation mutations in LHBs, host autophagy, and the HBV life cycle. Additionally, we offer a new clue for further investigation into carcinogenesis of hepatocellular carcinoma (HCC). These findings underscore the potential of targeting either N-linked glycosylation modifications or the autophagic pathway for the development of innovative therapies against HBV and/or HCC.

Juveniles, young adults, and infants with hepatitis B virus infection: A genomic study.

Integration of hepatitis B virus (HBV) DNA into the human genome is recognized as an oncogenic factor and a barrier to hepatitis B cure. In the study, biopsy liver tissues were collected from adolescents and young adults with acute HBV infection younger than or equal to 35 years of age and from HBV-infected infant patients younger than or equal to 6 months of age. A high-throughput sequencing method was used to detect HBV DNA integration. Totally, 12 adolescents, young adults, and 6 infants were included. Among the 12 patients with acute HBV infection, immunohistochemical staining of intrahepatic hepatitis B surface antigen for all displayed negative results, and no HBV DNA integrants in the hepatocyte DNA were confirmed. All infant patients had elevated levels of alanine aminotransferase and high levels of serum HBV DNA. Numerous gene sites of hepatocyte DNA were integrated by HBV DNA for each infant patient, ranging from 120 to 430 integration sites. The fragile histidine triad gene was the high-frequency integrated site in the intragenic region for infant patients. In conclusion, hepatocyte DNA is integrated by HBV DNA in babies with active hepatitis B but seems seldom affected among adolescents and young adults with acute HBV infection. Infantile hepatitis B should be taken seriously considering abundant HBV DNA integration events.

Multiscale modeling of HBV infection integrating intra- and intercellular viral propagation to analyze extracellular viral markers.

Chronic infection with hepatitis B virus (HBV) is caused by the persistence of closed circular DNA (cccDNA) in the nucleus of infected hepatocytes. Despite available therapeutic anti-HBV agents, eliminating the cccDNA remains challenging. Thus, quantifying and understanding the dynamics of cccDNA are essential for developing effective treatment strategies and new drugs. However, such study requires repeated liver biopsy to measure the intrahepatic cccDNA, which is basically not accepted because liver biopsy is potentially morbid and not common during hepatitis B treatment. We here aimed to develop a noninvasive method for quantifying cccDNA in the liver using surrogate markers in peripheral blood. We constructed a multiscale mathematical model that explicitly incorporates both intracellular and intercellular HBV infection processes. The model, based on age-structured partial differential equations, integrates experimental data from in vitro and in vivo investigations. By applying this model, we roughly predicted the amount and dynamics of intrahepatic cccDNA within a certain range using specific viral markers in serum samples, including HBV DNA, HBsAg, HBeAg, and HBcrAg. Our study represents a significant step towards advancing the understanding of chronic HBV infection. The noninvasive quantification of cccDNA using our proposed method holds promise for improving clinical analyses and treatment strategies. By comprehensively describing the interactions of all components involved in HBV infection, our multiscale mathematical model provides a valuable framework for further research and the development of targeted interventions.

HBsAg level defines different clinical phenotypes of HBeAg(-) chronic HBV infection related to HBV polymerase-specific CD8+ cell response quality.

Background: HBe-antigen(Ag)-negative chronic hepatitis B virus (HBV) infection is characterized by little liver fibrosis progression and vigorous HBV-multispecific CD8+ T-cell response. Aims: To assess whether HBsAg level could discriminate different HBeAg-negative chronic HBV infection subtypes with dissimilar quality of HBV-specific CD8+ T-cell response. Methods: We recruited 63 HBeAg-negative chronic HBV infection patients in which indirect markers of liver inflammation/fibrosis, portal pressure, viral load (VL), and HBV-specific CD8+ cell effector function were correlated with HBsAg level. Results: A positive linear trend between HBsAg level and APRI, liver stiffness (LS), liver transaminases, and HBV VL, and a negative correlation with platelet count were observed. Frequency of cases with HBV-specific CD8+ T-cell proliferation against at least two HBV epitopes was higher in HBsAg < 1,000 IU/ml group. CD8+ T-cell expansion after HBVpolymerase456-63-specific stimulation was impaired in HBsAg > 1,000 IU/ml group, while the response against HBVcore18-27 was preserved and response against envelope183-91 was nearly abolished, regardless of HBsAg level. Cases with preserved HBVpolymerase456-63 CD8+ cell response had lower LS/duration of infection and APRI/duration of infection rates. HBV-polymerase456-63-specific CD8+ T-cell proliferation intensity was negatively correlated with LS/years of infection ratio. Conclusion: HBsAg > 1,000 IU/ml HBeAg-negative chronic HBV infection group shows indirect data of higher degree of inflammation, liver stiffness, and fibrosis progression speed, which are related to an impaired HBV-polymerase-specific CD8+ T-cell response.

Distribution and Clinical Significance of Hepatitis B virus A1762T/G1764A Double Mutation in Chronic Hepatitis B Patients: A Cross-Sectional Study.

BACKGROUND: Chronic hepatitis B (CHB) is well-known as a major risk for liver cirrhosis and hepatocellular carcinoma (HCC). The A1762T/G1764A double mutation in the hepatitis B virus genome affects the production of HBe antigen and is established as a predictive marker for progression to HCC. Thus, this study aimed to investigate the prevalence and clinical significance of the mutation in Thai CHB patients. METHODS:  A cross-sectional study was conducted in 78 Thai CHB patients who were assessed for hepatitis B profiles, HBsAg, HBeAg and anti-HBeAg, transaminitis, liver fibrosis defined by FIB-4 (FIB-4) score and AST to platelet ratio index (APRI), alpha-fetoprotein (AFP) and active hepatitis B status. HBV A1762T/G1764A mutation was examined by SYBR Green I Real-time PCR. Chi-square and Mann-Whiney U tests were performed to determine the association between the mutation and variables. RESULTS: The prevalence of patients infected with the A1762T/G1764A mutation was 44.9%. The mutation was associated with HBeAg status (p=0.027) and HBsAg levels (p=0.008), transaminitis (p=0.011), and active hepatitis B (p=0.037), but not liver fibrosis markers, FIB-4 score and APRI, and AFP. Binary logistic regression identified the mutation as a predictive factor of active hepatitis B (OR 3.5, 95%CI, 1.1-11.3, p=0.037). Patients infected with the mutant exhibited significantly higher levels of HBsAg (p=0.011) and HBV viral load (p=0.047), but lower levels of HBeAg (p=0.12) than those infected with the wild-type HBV. CONCLUSION: The data indicate the high prevalence of the A1762T/G1764A mutation and its significant association with the severity of Thai CHB patients and the HBV mutation is proposed as a predictive marker of active hepatitis B status in CHB patients.

Characterization of a breakthrough vaccine escape strain associated with overt hepatitis B virus infection.

Hepatitis B virus (HBV) vaccine is composed of the purified hepatitis B surface antigen (HBsAg) that is produced by recombinant DNA technology. The neutralizing antibodies induced by vaccination target mainly the "a" determinant, aa124-147, of the outer viral envelope (HBsAg). In the present work, we demonstrate a case study for vaccinated patient that is infected with a vaccine escape HBV strain (Eg200). Characterization of the isolate Eg200 showed that it belongs to the genotype D and an uncommon sub-genotype in Egypt; D9. The DNA sequence encoding HBsAg was sequenced. Mutational analysis of the HBsAg showed a double mutation in the "a" determinant of this HBV isolate; T125M and P127T. However, such substitutions were found to be conserved to the detected serotype, ayw3, of Eg200 isolate. This case report indicates that continuous characterization of breakthrough vaccine escape strains of HBV is essential to develop the immunization strategies against HBV infection.

Comprehensive analysis of antigenic variations and genomic properties of hepatitis B virus in clinical samples in the mid-north east region of Bangladesh.

This investigation delineates an exhaustive analysis of the clinical, immunological, and genomic landscapes of hepatitis B virus (HBV) infection across a cohort of 22 verified patients. The demographic analysis unveiled a pronounced male bias (77.27%), with patient ages spanning 20 to 85 years and durations of illness ranging from 10 days to 4 years. Predominant clinical manifestations included fever, fatigue, anorexia, abdominal discomfort, and arthralgia, alongside observed co-morbidities such as chronic renal disorders and hepatocellular carcinoma. Antigenic profiling of the HBV envelope proteins elucidated significant heterogeneity among the infected subjects, particularly highlighted by discordances in the detection capabilities of small and large HBsAg assays, suggesting antigenic diversity. Quantitative assessment of viral loads unveiled a broad spectrum, accompanied by atypical HBeAg reactivity patterns, challenging the reliability of existing serological markers. Correlative studies between viral burden and antigenicity of the envelope proteins unearthed phenomena indicative of diagnostic evasion. Notably, samples demonstrating robust viral replication were paradoxically undetectable by the large HBsAg ELISA kit, advocating for more sophisticated diagnostic methodologies. Genotypic examination of three HBV isolates classified them as genotype D (D2), with phylogenetic alignment to strains from various global origins. Mutational profiling identified pivotal mutations within the basic core promoter and preS2/S1 regions, associated with an augmented risk of hepatocellular carcinoma. Further, mutations discerned in the small HBsAg and RT/overlap regions were recognized as contributors to vaccine and/or diagnostic escape mechanisms. In summation, this scholarly discourse elucidates the intricate interplay of clinical presentations, antigenic diversity, and genomic attributes in HBV infection, accentuating the imperative for ongoing investigative endeavors to refine diagnostic and therapeutic modalities.

PreS1BP mediates inhibition of Hepatitis B virus replication by promoting HBx protein degradation.

BACKGROUND: PreS1-binding protein (PreS1BP), recognized as a nucleolar protein and tumor suppressor, influences the replication of various viruses, including vesicular stomatitis virus (VSV) and herpes simplex virus type 1 (HSV-1). Its role in hepatitis B virus (HBV) replication and the underlying mechanisms, however, remain elusive. METHODS: We investigated PreS1BP expression levels in an HBV-replicating cell and animal model and analyzed the impact of its overexpression on viral replication metrics. HBV DNA, covalently closed circular DNA (cccDNA), hepatitis B surface antigen (HBsAg), hepatitis B core antigen (HBcAg), and HBV RNA levels were assessed in HBV-expressing stable cell lines under varying PreS1BP conditions. Furthermore, co-immunoprecipitation and ubiquitination assays were used to detect PreS1BP- hepatitis B virus X protein (HBx) interactions and HBx stability modulated by PreS1BP. RESULTS: Our study revealed a marked decrease in PreS1BP expression in the presence of active HBV replication. Functional assays showed that PreS1BP overexpression significantly inhibited HBV replication and transcription, evidenced by the reduction in HBV DNA, cccDNA, HBsAg, HBcAg, and HBV RNA levels. At the molecular level, PreS1BP facilitated the degradation of HBx in a dose-dependent fashion, whereas siRNA-mediated knockdown of PreS1BP led to an increase in HBx levels. Subsequent investigations uncovered that PreS1BP accelerated HBx protein degradation via K63-linked ubiquitination in a ubiquitin-proteasome system-dependent manner. Co-immunoprecipitation assays further established that PreS1BP enhances the recruitment of the proteasome 20S subunit alpha 3 (PSMA3) for interaction with HBx, thereby fostering its degradation. CONCLUSIONS: These findings unveil a previously unidentified mechanism wherein PreS1BP mediates HBx protein degradation through the ubiquitin-proteasome system, consequentially inhibiting HBV replication. This insight positions PreS1BP as a promising therapeutic target for future HBV interventions. Further studies are warranted to explore the clinical applicability of modulating PreS1BP in HBV therapy.

Cytokines Expression Compared to the Determinants of Cellular Apoptosis Prominently Attributes to the Deleterious Effects of 'A' Determinant Surface Gene Mutations in HBV Transfected Hepatoma Cell Line.

BACKGROUND: Previous studies have explored the role of AKT protein in anti-apoptotic/proliferative activities. However, there has been a lack of information regarding the role of Akt in association with cytokines expression in HBV-related (wild type HBV and HBV with mutations of 'a' determinant region) studies either in the case of HBV infection or in transfected hepatoma cells. The present study tries to determine the role of Akt and cytokines expression in the presence of small surface gene mutants in the hepatoma cell line. METHODS: Mutations of 'a' determinant region, viz. sA128V and sG145R, were created in wild-type pHBV1.3 by site-directed mutagenesis and transfected in hepatoma cell line. Secretory levels of HBsAg in the wild type as well as in both the mutants were analyzed by ELISA. Apoptotic analysis of transfected cells was studied by flow cytometry. Expression analysis of Akt and cytokines (TNF-alpha, IL-6, and IFN-gamma) was done by qPCR. RESULTS: The presence of significantly more alive cells in sG145R than sA128V transfected cells may be due to the up-regulation of the Akt gene expression. Cytokines expression was nearly similar between sA128V and wild-type pHBV1.3 transfected cells. Presence of sG145R showed dramatically high cytokines expression than sA128V and wild-type pHBV1.3. CONCLUSION: Cytokines expression predominantly contributes to the detrimental effects associated with the 'a' determinant region mutations particularly sG145R mutant. It may also be inferred that mechanisms associated with cellular apoptosis apparently do not play any major role to assign the 'a' determinant small surface gene mutation(s) for their pathological outcome.

Prevalence of hepatitis D virus infection in Central Italy has remained stable across the last 2 decades with dominance of subgenotypes 1 and characterized by elevated viral replication.

OBJECTIVES: Here we investigate Hepatitis D virus (HDV)-prevalence in Italy and its fluctuations over time and we provide an extensive characterization of HDV-infected patients. METHODS: The rate of HDV seroprevalence and HDV chronicity was assessed in 1579 hepatitis B surface antigen (HBsAg)+ patients collected from 2005 to 2022 in Central Italy. RESULTS: In total, 45.3% of HBsAg+ patients received HDV screening with an increasing temporal trend: 15.6% (2005-2010), 45.0% (2011-2014), 49.4% (2015-2018), 71.8% (2019-2022). By multivariable model, factors correlated with the lack of HDV screening were alanine-aminotransferase (ALT) less than two times of upper limit of normality (<2ULN) and previous time windows (P <0.002). Furthermore, 13.4% of HDV-screened patients resulted anti-HDV+ with a stable temporal trend. Among them, 80.8% had detectable HDV-ribonucleic acid (RNA) (median [IQR]:4.6 [3.6-5.6] log copies/ml) with altered ALT in 89.3% (median [IQR]:92 [62-177] U/L). Anti-HDV+ patients from Eastern/South-eastern Europe were younger than Italians (44 [37-54] vs 53 [47-62] years, P <0.0001), less frequently nucleos(t)ide analogs (NUC)-treated (58.5% vs 80%, P = 0.026) with higher HDV-RNA (4.8 [3.6-5.8] vs 3.9 [1.4-4.9] log copies/ml, P = 0.016) and HBsAg (9461 [4159-24,532] vs 4447 [737-13,336] IU/ml, P = 0.032). Phylogenetic analysis revealed the circulation of HDV subgenotype 1e (47.4%) and -1c (52.6%). Notably, subgenotype 1e correlated with higher ALT than 1c (168 [89-190] vs 58 [54-88] U/l, P = 0.015) despite comparable HDV-RNA. CONCLUSIONS: HDV-screening awareness is increasing over time even if some gaps persist to achieve HDV screening in all HBsAg+ patients. HDV prevalence in tertiary care centers tend to scarcely decline in native/non-native patients. Detection of subgenotypes, triggering variable inflammatory stimuli, supports the need to expand HDV molecular characterization.

Higher TP53BP2 expression is associated with HBsAg loss in peginterferon-α-treated patients with chronic hepatitis B.

BACKGROUND & AIMS: HBsAg loss is only observed in a small proportion of patients with chronic hepatitis B (CHB) who undergo interferon treatment. Investigating the host factors crucial for functional cure of CHB can aid in identifying individuals who would benefit from peginterferon-α (Peg-IFNα) therapy. METHODS: We conducted a genome-wide association study (GWAS) by enrolling 48 patients with CHB who achieved HBsAg loss after Peg-IFNα treatment and 47 patients who didn't. In the validation stage, we included 224 patients, of whom 90 had achieved HBsAg loss, to validate the identified significant single nucleotide polymorphisms. To verify the functional involvement of the candidate genes identified, we performed a series of in vitro and in vivo experiments. RESULTS: GWAS results indicated a significant association between the rs7519753 C allele and serum HBsAg loss in patients with CHB after Peg-IFNα treatment (p = 4.85 × 10-8, odds ratio = 14.47). This association was also observed in two independent validation cohorts. Expression quantitative trait locus analysis revealed higher hepatic TP53BP2 expression in individuals carrying the rs7519753 C allele (p = 2.90 × 10-6). RNA-sequencing of liver biopsies from patients with CHB after Peg-IFNα treatment revealed that hepatic TP53BP2 levels were significantly higher in the HBsAg loss group compared to the HBsAg persistence group (p = 0.035). In vitro and in vivo experiments demonstrated that loss of TP53BP2 decreased interferon-stimulated gene levels and the anti-HBV effect of IFN-α. Mechanistically, TP53BP2 was found to downregulate SOCS2, thereby facilitating JAK/STAT signaling. CONCLUSION: The rs7519753 C allele is associated with elevated hepatic TP53BP2 expression and an increased probability of serum HBsAg loss post-Peg-IFNα treatment in patients with CHB. TP53BP2 enhances the response of the hepatocyte to IFN-α by suppressing SOCS2 expression. IMPACT AND IMPLICATIONS: Chronic hepatitis B (CHB) remains a global public health issue. Although current antiviral therapies are more effective in halting disease progression, only a few patients achieve functional cure for hepatitis B with HBsAg loss, highlighting the urgent need for a cure for CHB. This study revealed that the rs7519753 C allele, which is associated with high expression of hepatic TP53BP2, significantly increases the likelihood of serum HBsAg loss in patients with CHB undergoing Peg-IFNα treatment. This finding not only provides a promising predictor for HBsAg loss but identifies a potential therapeutic target for Peg-IFNα treatment. We believe our results are of great interest to a wide range of stakeholders based on their potential clinical implications.

Spatial transcriptomics reveals a low extent of transcriptionally active hepatitis B virus integration in patients with HBsAg loss.

OBJECTIVE: Hepatitis B virus (HBV) can integrate into the chromosomes of infected hepatocytes, contributing to the production of hepatitis B surface antigen (HBsAg) and to hepatocarcinogenesis. In this study, we aimed to explore whether transcriptionally active HBV integration events spread throughout the liver tissue in different phases of chronic HBV infection, especially in patients with HBsAg loss. DESIGN: We constructed high-resolution spatial transcriptomes of liver biopsies containing 13 059 tissue spots from 18 patients with chronic HBV infection to analyse the occurrence and relative distribution of transcriptionally active viral integration events. Immunohistochemistry was performed to evaluate the expression of HBsAg and HBV core antigen. Intrahepatic covalently closed circular DNA (cccDNA) levels were quantified by real-time qPCR. RESULTS: Spatial transcriptome sequencing identified the presence of 13 154 virus-host chimeric reads in 7.86% (1026 of 13 059) of liver tissue spots in all patients, including three patients with HBsAg loss. These HBV integration sites were randomly distributed on chromosomes and can localise in host genes involved in hepatocarcinogenesis, such as ALB, CLU and APOB. Patients who were receiving or had received antiviral treatment had a significantly lower percentage of viral integration-containing spots and significantly fewer chimeric reads than treatment-naïve patients. Intrahepatic cccDNA levels correlated well with viral integration events. CONCLUSION: Transcriptionally active HBV integration occurred in chronically HBV-infected patients at different phases, including in patients with HBsAg loss. Antiviral treatment was associated with a decreased number and extent of transcriptionally active viral integrations, implying that early treatment intervention may further reduce the number of viral integration events.