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A cell culture system that allows the reproduction of the hepatitis B virus (HBV) life cycle is indispensable to exploring novel anti-HBV agents. To establish the screening system for anti-HBV agents, we exploited the high affinity and bright luminescence (HiBiT) tag and comprehensively explored the regions in the HBV genome where the HiBiT tag could be inserted. The plasmids for the HiBiT-tagged HBV molecular clones with a 1.38-fold HBV genome length were prepared. The HiBiT tag was inserted into five regions: preS1, preS2, hepatitis B e (HBe), hepatitis B X (HBx), and hepatitis B polymerase (HB pol). HiBiT-tagged HBVs were obtained by transfecting the prepared plasmids into sodium taurocholate cotransporting polypeptide-transduced HepG2 (HepG2/NTCP) cells, and their infectivity was evaluated in human primary hepatocytes and HepG2/NTCP cells. Among the evaluated viruses, the infection of HiBiT-tagged HBVs in the preS1 or the HB pol regions exhibited a time-dependent increase of the hepatitis B surface antigen (HBsAg) level after infection to HepG2/NTCP cells as well as human primary hepatocytes. Immunostaining of the hepatitis B core (HBc) antigen in infected cells confirmed these viruses are infectious to those cells. However, the time-dependent increase of the HiBiT signal was only detected after infection with the HiBiT-tagged HBV in the preS1 region. The inhibition of this HiBiT-tagged HBV infection in human primary hepatocytes and HepG2/NTCP cells by the preS1 peptide could be detected by measuring the HiBiT signal. The infection system with the HiBiT-tagged HBV in HepG2/NTCP cells facilitates easy, sensitive, and high-throughput screening of anti-HBV agents and will be a useful tool for assessing the viral life cycle and exploring antiviral agents., Importance: Hepatitis B virus (HBV) is the principal causative agent of chronic hepatitis. Despite the availability of vaccines in many countries, HBV infection has spread worldwide and caused chronic infection. In chronic hepatitis B patients, liver inflammation leads to cirrhosis, and the accumulation of viral genome integration into host chromosomes leads to the development of hepatocellular carcinoma. The currently available treatment strategy cannot expect the eradication of HBV. To explore novel anti-HBV agents, a cell culture system that can detect HBV infection easily is indispensable. In this study, we examined the regions in the HBV genome where the high affinity and bright luminescence (HiBiT) tag could be inserted and established an HBV infection system to monitor infection by measuring the HiBiT signal by infecting the HiBiT-tagged HBV in sodium taurocholate cotransporting polypeptide-transduced HepG2 (HepG2/NTCP) cells. This system can contribute to screening for novel anti-HBV agents., Competing Interests: The authors declare no conflict of interest.

Macaque restricts hepatitis B virus (HBV) infection because its receptor homologue, NTCP (mNTCP), cannot bind preS1 on viral surface. To reveal how mNTCP loses the viral receptor function, we here solve the cryo-electron microscopy structure of mNTCP. Superposing on the human NTCP (hNTCP)-preS1 complex structure shows that Arg158 of mNTCP causes steric clash to prevent preS1 from embedding onto the bile acid tunnel of NTCP. Cell-based mutation analysis confirms that only Gly158 permitted preS1 binding, in contrast to robust bile acid transport among mutations. As the second determinant, Asn86 on the extracellular surface of mNTCP shows less capacity to restrain preS1 from dynamic fluctuation than Lys86 of hNTCP, resulting in unstable preS1 binding. Additionally, presence of long-chain conjugated-bile acids in the tunnel induces steric hindrance with preS1 through their tailed-chain. This study presents structural basis in which multiple sites in mNTCP constitute a molecular barrier to strictly restrict HBV., (© 2024. The Author(s).)

Background: Occult hepatitis B infection (OBI) is characterized by the presence of hepatitis B virus (HBV) DNA at low levels in serum (<200 IU/mL) with a negative hepatitis B surface antigen (HBsAg) test. OBI remains a major challenge to blood safety, particularly in HBV-endemic regions like Cameroon, where HBV detection relies solely on HBsAg testing. This cross-sectional study aimed to investigate the actual incidence and genotype characteristics of OBI in Cameroonian blood donors., Methods: Between March and June 2023, samples were collected from 288 HBsAg-negative blood donors aged 18 to 55 years and analysed for antibodies against the HBV core (anti-HBc) and surface antigens (anti-HBs). Following DNA extraction from the serum samples, qualitative nested PCR and quantitative real-time PCR were used to detect HBV viral DNA and viral load respectively. For positive samples, sequencing of a fragment of the S gene was performed to identify the circulating HBV genotypes., Results: The findings revealed that 58% (n = 167/288) of blood donors tested positive for anti-HBc, 29% (n = 83/288) tested positive for anti-HBs, and 26% (n = 75/288) being positive for both anti-HBc and anti-HBs. Occult hepatitis was confirmed in 4.5% of the blood donors, all of whom belonged to either HBV genotypes A or E, which are predominant in Cameroon. The amino acid substitution sA184V associated with HBsAg detection failure in genotype E was observed in 70% of OBI sequences, and the HBsAg immune escape variants (sT131N and sS143L) implicated in OBI were also observed. The mutation rtN139K in the reverse transcriptase (RT) domain of the overlapping HBV polymerase (P) gene was present in 17% of OBI-positive sequences of genotype E, likely contributing to masking HBsAg secretion., Conclusion: The results suggest a considerable risk of transfusion-transmitted HBV in this region. Therefore, to ensure blood safety, nucleic acid testing (NAT) is recommended, as relying solely on HBsAg assays is insufficient to eliminate this risk., Competing Interests: The authors declare no conflict of interest., (Copyright: © 2024 Mbencho et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Chronic hepatitis B is a global health concern with a high risk of end-stage liver disease. Current standard-of-care agents have low cure rates, and new therapies are needed. Small interfering RNAs (siRNAs) that target viral RNAs fulfill a gap not addressed by standard-of-care agents and may contribute to a functional cure. Here, we describe the preclinical characterization of imdusiran (AB-729), a novel, pan-genotypic siRNA therapeutic that effectively reduces HBsAg, viral antigens, and viral replication in chronic hepatitis B patients and is currently in Phase 2 clinical studies. In hepatitis B virus (HBV) cell-based systems, imdusiran possessed pan-genotypic nanomolar potency and retained activity against HBV target site polymorphisms. Imdusiran was active against nucleos(t)ide analogue- and capsid assembly modulator-resistant HBV isolates, and combination with standard-of-care agents was additive. In an HBV adeno-associated virus mouse model, HBsAg was reduced up to 3.7 log 10 after a single imdusiran dose, with sustained suppression for 10 weeks. Imdusiran did not intrinsically stimulate cytokine release in healthy donor human whole blood, supportive of its mechanism of action as a direct acting RNA interference antiviral. Taken together, these data support imdusiran in combination treatment approaches toward chronic hepatitis B functional cure.

Current culture systems available for studying hepatitis D virus (HDV) are suboptimal. In this study, we demonstrate that hepatocyte-like cells (HLCs) derived from human pluripotent stem cells (hPSCs) are fully permissive to HDV infection across various tested genotypes. When co-infected with the helper hepatitis B virus (HBV) or transduced to express the HBV envelope protein HBsAg, HLCs effectively release infectious progeny virions. We also show that HBsAg-expressing HLCs support the extracellular spread of HDV, thus providing a valuable platform for testing available anti-HDV regimens. By challenging the cells along the differentiation with HDV infection, we have identified CD63 as a potential HDV co-entry factor that was rate-limiting for HDV infection in immature hepatocytes. Given their renewable source and the potential to derive hPSCs from individual patients, we propose HLCs as a promising model for investigating HDV biology. Our findings offer new insights into HDV infection and expand the repertoire of research tools available for the development of therapeutic interventions., (© 2024. The Author(s).)

Background: The hepatitis B e antigen (HBeAg)-negative infection Phase 3 is characterized by no or minimal signs of hepatic inflammation and the absence of hepatic fibrosis. However, underlying molecular mechanisms leading to this benign phenotype are poorly understood., Methods: Genotype A, B and D HBeAg-negative patient isolates with precore mutation G1896A from Phase 3 were analysed in comparison with respective HBeAg-positive rescue mutant and HBeAg-positive wild-type reference genomes regarding differences in viral replication, morphogenesis, infectivity and impact on NF-E2-related factor 2 (Nrf2)/antioxidant response element (ARE)-dependent gene expression and cellular kinome., Results: In comparison with reference genomes, the patient isolates are characterized by a lower intra- and extracellular hepatitis B surface antigen (HBsAg)-amount, and HBsAg-retention in the endoplasmic reticulum. Rescue of HBeAg expression increased HBsAg-amount but not its release. Expression of the isolated genomes is associated with a higher Nrf2/ARE-dependent gene expression as compared to reference genomes independent of HBeAg expression. Kinome analyses revealed a decreased activity of receptors involved in regulation of proliferative pathways for all patient isolates compared to the reference genomes. No specific conserved mutations could be found between all genomes from Phase 3., Conclusions: HBeAg-negative genomes from Phase 3 exhibit distinct molecular characteristics leading to lower HBsAg synthesis and release, enhanced oxidative stress protection and decreased activity of key kinases, triggering an antiproliferative stage, which might contribute to the lower probability of hepatocellular carcinoma. The observed differences cannot be associated with loss of HBeAg or specific mutations common to all analysed isolates, indicating the phenotype of Phase 3 derived genomes to be the result of a multifactorial process likely reflecting a conserved natural selection process., (© 2024 The Author(s). Liver International published by John Wiley & Sons Ltd.)

A substantial body of literature, including our own, points to a connection between hepatitis B virus (HBV) infection and the development of drug resistance in hepatocellular carcinoma (HCC), particularly against sorafenib. However, the influence of HBV on resistance to regorafenib, another therapeutic agent, has been less studied. In this study, we used the GEO database (GSE87630) and clinical samples to demonstrate that C-C motif chemokine receptor 9 (CCR9) was highly expressed in HBV-related HCC and predicted poor overall survival. Its overexpression correlated with HBsAg-positive HCC patients. Both univariate and multivariable Cox regression analysis elucidated CCR9 was an independent risk factor for poor overall survival in HCC patients. Our in vitro findings further revealed that HBV structural proteins, small HBV surface antigen (SHBs), triggered an upregulation of CCR9. Functional assays showed that SHBs enhanced HCC cell proliferation, migration, and invasion, increased ABCB1 and ABCC1 expression, and promoted regorafenib resistance via CCR9. Intriguingly, overexpression of HBV plasmid and an AAV-HBV mouse model both exhibited a significant elevation in global N6-methyladenosine (m6A) levels. Further investigations revealed that SHBs elevated these m6A levels, upregulated CCR9 and stabilized CCR9 mRNA through KIAA1429-mediated m6A modification, with sites 1373 and 1496 on CCR9 mRNA being critical for modification. In conclusion, SHBs promoted HCC progression and regorafenib resistance via KIAA1429-mediated m6A modification of CCR9. Our findings suggested that CCR9 could be a potential prognostic biomarker and a valuable molecular therapeutic target of regorafenib resistance in HBV-related HCC., (© 2024 Wiley Periodicals LLC.)

Hepatitis A and B are two crucial viral infections that still dramatically affect public health worldwide. Hepatitis A Virus (HAV) is the main cause of acute hepatitis, whereas Hepatitis B Virus (HBV) leads to the chronic form of the disease, possibly cirrhosis or liver failure. Therefore, vaccination has always been considered the most effective preventive method against pathogens. At this moment, we aimed at the immunoinformatic analysis of HAV-Viral Protein 1 (VP1) as the major capsid protein to come up with the most conserved immunogenic truncated protein to be fused by HBV surface antigen (HBs Ag) to achieve a bivalent vaccine against HAV and HBV using an AAY linker. Various computational approaches were employed to predict highly conserved regions and the most immunogenic B-cell and T-cell epitopes of HAV-VP1 capsid protein in both humans and BALB/c. Moreover, the predicted fusion protein was analyzed regarding primary and secondary structures and also homology validation. Afterward, the three-dimensional structure of vaccine constructs docked with various toll-like receptors (TLR) 2, 4 and 7. According to the bioinformatics tools, the region of 99-259 amino acids of VP1 was selected with high immunogenicity and conserved epitopes. T-cell epitope prediction showed that this region contains 32 antigenic peptides for Human leukocyte antigen (HLA) class I and 20 antigenic peptides in terms of HLA class II which are almost fully conserved in the Iranian population. The vaccine design includes 5 linear and 4 conformational B-cell lymphocyte (BCL) epitopes to induce humoral immune responses. The designed VP1-AAY-HBsAg fusion protein has the potency to be constructed and expressed to achieve a bivalent vaccine candidate, especially in the Iranian population. These findings led us to claim that the designed vaccine candidate provides potential pathways for creating an exploratory vaccine against Hepatitis A and Hepatitis B Viruses with high confidence for the identified strains., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Hepatitis B virus (HBV) expresses co-terminal large (L), middle (M), and small (S) envelope proteins containing preS1/preS2/S, preS2/S, and S domain alone, respectively. S and preS1 domains mediate sequential virion attachment to heparan sulfate proteoglycans and sodium taurocholate cotransporting polypeptide (NTCP), respectively, which can be blocked by anti-S and anti-preS1 antibodies. How anti-preS2 antibodies neutralize HBV infectivity remains enigmatic. The late stage of chronic HBV infection often selects for mutated preS2 translation initiation codon to prevent M protein expression, or in-frame preS2 deletions to shorten both L and M proteins. When introduced to infectious clone of genotype C or D, both M-minus mutations and most 5' preS2 deletions sustained virion production. Such mutant progeny viral particles were infectious in NTCP-reconstituted HepG2 cells. Neutralization experiments were performed on the genotype D clone. Although remaining susceptible to anti-preS1 and anti-S neutralizing antibodies, M-minus mutants were only partially neutralized by two anti-preS2 antibodies tested while preS2 deletion mutants were resistant. By infection experiments using viral particles with lost versus increased M protein expression, or a neutralization escaping preS2 deletion only present on L or M protein, we found that both full-length L and M proteins contributed to virus neutralization by the two anti-preS2 antibodies. Thus, immune escape could be a driving force for the selection of M-minus mutations, and especially preS2 deletions. The fact that both L and M proteins could mediate neutralization by anti-preS2 antibodies may shed light on the underlying molecular mechanism.IMPORTANCEThe large (L), middle (M), and small (S) envelope proteins of hepatitis B virus (HBV) contain preS1/preS2/S, preS2/S, and S domain alone, respectively. The discovery of heparan sulfate proteoglycans and sodium taurocholate cotransporting polypeptide (NTCP) as the low- and high-affinity HBV receptors could explain neutralizing potential of anti-S and anti-preS1 antibodies, respectively, but how anti-preS2 neutralizing antibodies work remains enigmatic. In this study, we found two M-minus mutants in the context of genotype D partially escaped two anti-preS2 neutralizing antibodies in NTCP-reconstituted HepG2 cells, while several naturally occurring preS2 deletion mutants escaped both antibodies. By point mutations to eliminate or enhance M protein expression, and by introducing preS2 deletion selectively to L or M protein, we found binding of anti-preS2 antibodies to both L and M proteins contributed to neutralization of wild-type HBV infectivity. Our finding may shed light on the possible mechanism(s) whereby anti-preS2 antibodies neutralize HBV infectivity., Competing Interests: The authors declare no conflict of interest.

This study aims to characterize the molecular profile of the hepatitis B virus (HBV) among socially vulnerable immigrants residing in Brazil to investigate the introduction of uncommon HBV strains into the country. Serum samples from 102 immigrants with positive serology for the HBV core antibody (anti-HBc) were tested for the presence of HBV DNA by PCR assays. Among these, 24 were also positive for the HBV surface antigen (HBsAg). The full or partial genome was sequenced to determine genotype by phylogenetic analysis. Participants were from Haiti (79.4%), Guinea-Bissau (11.8%), Venezuela (7.8%), and Colombia (1%). Of the 21 HBV DNA-positive samples, subgenotypes A1 (52.4%), A5 (28.6%), E (9.5%), F2 (4.8%), and F3 (4.8%) were identified. Among the 78 HBsAg-negative participants, four were positive for HBV DNA, resulting in an occult HBV infection rate of 5.1%. Phylogenetic analysis suggested that most strains were likely introduced to Brazil by migration. Importantly, 80% of A5 sequences had the A1762T/G1764A double mutation, linked to an increased risk of hepatocellular carcinoma development. In conclusion, this study is the first report of HBV subgenotype A5 in Brazil, shedding new light on the diversity of HBV strains circulating in the country. Understanding the genetic diversity of HBV in immigrant communities can lead to better prevention and control strategies, benefiting both immigrants and wider society.

Antigen presentation by HLA class II molecules to CD4 + T cells is an essential step for generating antibodies to hepatitis B antigens. In this study, we investigated the association between the HLA-DRB1 gene and the status of antibodies to hepatitis B surface and e antigens. Our results revealed a significant association between the status of anti-HBsAg and HLA-DRB1*04:03 (OR = 4.11, 95% CI = 1.50-10.84, p = 0.005, P adj.  = 0.05) as well as HLA-DRB1*15:01 (OR = 1.74, 95% CI = 1.20-2.50, p = 0.002, P adj.  = 0.045). MHC II binding predictions and in silico docking demonstrated strong binding affinity of HBsAg peptides to these two HLA-DRB1 molecules. Conversely, the status of anti-HBeAg was inversely associated with HLA-DRB1*14:54 (OR = 0.34, 95% CI = 0.18-0.64, p = 0.001, P adj.  = 0.011), and in silico analysis revealed weak binding affinity of HBeAg peptides to HLA-DRB1*14:54. In conclusion, these findings support the involvement of HLA-DRB1 in humoral immunity against HBV infection., (© 2024 Wiley Periodicals LLC.)

Spontaneous reactivation of the Hepatitis B virus (HBV) is rare in individuals with previously resolved infections. This report presents the case of a 71 year-old Japanese woman who experienced HBV reactivation without any prior immunosuppressive therapy or chemotherapy. Before the onset of liver injury, the patient was negative for hepatitis B surface antigen (HBsAg) but positive for hepatitis B surface antibody. She subsequently developed liver injury, with the reappearance of HBsAg and HBV DNA. The patient was successfully treated with tenofovir alafenamide, and prednisolone. Full-genome sequencing of HBV revealed subgenotype B1 without hepatitis B e-negative mutations in the precore and core promoter regions and 12 amino acid alterations in the pre-S1/S, P, and X genes. Notably, the S gene mutations D144A and K160N, which alter the antigenicity of HBsAg and potentially contribute to its reactivation, were identified. This case emphasizes the importance of vigilance for spontaneous reactivation of resolved HBV, highlighting the need for comprehensive genomic analysis to understand the associated virological intricacies., (© 2024. Japanese Society of Gastroenterology.)

Occult hepatitis B virus (HBV) infection (OBI) is a significant challenge for HBV prevention and control. We investigated the prevalence and surface (S) gene mutations of OBI among blood donors in Huzhou City, eastern China. The hepatitis B surface antigen (HBsAg) was routinely screened among 44,256 blood donors. HBV-DNA was detected using the Roche cobas®system. Serum samples that were HBsAg negative and HBV-DNA positive were selected, and the HBV S gene was amplified and sequenced. HBV genotype and S gene mutations were analyzed. The OBI rate in these blood donors was 0.070 % (31/44,256). Among the blood donors with OBI, only two cases (2/31, 6.5 %) were anti-HBc negative. The S gene sequences of 28 samples were successfully obtained, and we found that HBV genotype C (21/28, 70 %) was predominant among blood donors with OBI. Most S gene mutations were associated with OBI, and the high frequency mutations included N40S, G44E, Q51R/P, T113A/S,T118K/M, P120Q/S/T, and Y161F/S. Notably, amino acid substitutions at some sites differed from those reported previously, such as Y72F, G102V, P127L, Q129P, and S143T. Additionally, six novel mutations (S31I/N/R, P46L, S58C, C76Y, Y200F/C, and I208T) that may be associated with OBI were found. OBI was detected in a certain proportion of blood donors in Huzhou City. S gene mutations play an important role in OBI development. Further research is required to explore the functions of novel S gene mutants in OBI pathogenesis. The findings of this study may provide important insights to prevent HBV transmission through blood transfusions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that there are no competing interests (financial or non-financial) related to the content of the specific article. The authors further state that this article is a review article and that, for its compilation, research involving human participants and/or animals was not necessary., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background & Aims: New antiviral approaches that target multiple aspects of the HBV replication cycle to improve rates of functional cure are urgently required. HBV RNA represents a novel therapeutic target. Here, we programmed CRISPR-Cas13b endonuclease to specifically target the HBV pregenomic RNA and viral mRNAs in a novel approach to reduce HBV replication and protein expression., Methods: Cas13b CRISPR RNAs (crRNAs) were designed to target multiple regions of HBV pregenomic RNA. Mammalian cells transfected with replication competent wild-type HBV DNA of different genotypes, a HBV-expressing stable cell line, a HBV infection model and a hepatitis B surface antigen (HBsAg)-expressing stable cell line were transfected with PspCas13b-BFP (blue fluorescent protein) and crRNA plasmids, and the impact on HBV replication and protein expression was measured. Wild-type HBV DNA, PspCas13b-BFP and crRNA plasmids were simultaneously hydrodynamically injected into mice, and serum HBsAg was measured. PspCas13b mRNA and crRNA were also delivered to a HBsAg-expressing stable cell line via lipid nanoparticles and the impact on secreted HBsAg determined., Results: Our HBV-targeting crRNAs strongly suppressed HBV replication and protein expression in mammalian cells by up to 96% (p <0.0001). HBV protein expression was also reduced in a HBV-expressing stable cell line and in the HBV infection model. CRISPR-Cas13b crRNAs reduced HBsAg expression by 50% (p <0.0001) in vivo. Lipid nanoparticle-encapsulated PspCas13b mRNA reduced secreted HBsAg by 87% (p = 0.0168) in a HBsAg-expressing stable cell line., Conclusions: Together, these results show that CRISPR-Cas13b can be programmed to specifically target and degrade HBV RNAs to reduce HBV replication and protein expression, demonstrating its potential as a novel therapeutic option for chronic HBV infection., Impact and Implications: Owing to the limitations of current antiviral therapies for hepatitis B, there is an urgent need for new treatments that target multiple aspects of the HBV replication cycle to improve rates of functional cure. Here, we present CRISPR-Cas13b as a novel strategy to target HBV replication and protein expression, paving the way for its development as a potential new treatment option for patients living with chronic hepatitis B., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Hepatitis delta virus (HDV) is a satellite of hepatitis B virus (HBV), which requires the HBV surface antigen (HBsAg) for its assembly and propagation. Although countries affected by HBV infection in Africa are well identified, data on HDV infection are still scarce, like in Nigeria, where HBV infection is endemic. In this study, we aimed to determine the prevalence of HDV infection and identify the circulating genotypes/strains in the country. A nationwide study was performed on 1281 HBsAg-positive samples collected from patients across eleven sites drawn from the six geopolitical zones in Nigeria. Anti-HDV antibody (HDV-Ab) screening and HDV-RNA viral load quantification were performed using a commercial ELISA assay and real-time RT-PCR kit, respectively. HDV genotyping was performed by the Sanger sequencing of amplicons from the so-called R0 region of the viral genome, followed by phylogenetic analyses. Of the 1281 HBsAg-positive samples, 61 (4.8%) were HDV-Ab positive, among which, 12 (19.7%) were HDV-RNA positive. Genotypes were obtained for nine of them: seven "African" HDV-1, one "Asian/European" HDV-1 and one HDV-6. This study shows that Nigeria is a country of low HDV prevalence where mainly "African" genotype-1 strains are circulating.

The interaction of multiple viruses in one host is thought to enhance the development of mutations. However, the impact of hepatitis D virus (HDV) positivity on the development of unique hepatitis B virus (HBV) mutations among people living with human immunodeficiency virus (HIV) (PLWH) remains poorly understood in African countries, including Botswana. We used HBV sequences generated from the Botswana Combination Prevention Project (BCPP), which is the largest pair-matched cluster-randomized HIV trial in Botswana. Only participants with available HBV sequences ( n = 55) were included in our study ([HIV/HBV-positive ( n = 50) and HIV/HBV/HDV-positive ( n = 5)]. Geno2pheno was used to determine HBV genotypes, and HBV surface region sequences (all subgenotype A1) were aligned in AliView for mutational analysis, while the impact of mutations was assessed using Phyre2. Our results identified 182 common mutations between the two groups. In the HIV/HBV/HDV cohort, only three mutations ( L95W , W156Q , C221Y ) were classified as deleterious, with only L95W being the most frequent. In the HIV/HBV cohort, four mutations ( W199R , C221A , C221S , W223G ) were also classified as deleterious. Our results demonstrate the presence of unique HBV mutations among the HIV/HBV/HDV-positive cohort. Functional characterization of these mutations is recommended to determine their effect on HDV.

Hepatitis B virus (HBV) belongs to the genus Orthohepadnavirus, of Hepadnaviridae family, smallest human deoxyribonucleic acid (DNA) virus with 3200 bp in a partially double-stranded circular DNA. Globally, about 2 billion people are infected with over 65 million of the chronically infected residing in Africa. Ten HBV genotypes (A-J) have been reported across the globe. Based on the World Health Organization (WHO) African Regions including Kenya have high HBV prevalence rates yet the data on prevalence rates of the various HBV genotypes and their associated biomarkers is very scanty. A cross-sectional descriptive study with purposive sampling was conducted in which a census of patients with chronic Hepatitis B (CHB) with history >6-month were reviewed for eligibility. Demographics data was abstracted from patient files and blood samples drawn for genotyping, viral load using Rotor gene Q Polymerase Chain Reaction (PCR) equipment, Hepatitis B surface Antigen (HBsAg), Hepatitis B envelope antigen (HbeAg) and Hepatitis B core antibody (Anti-HBc) using Cobas e411 machine. Out of a total of 83 patients, 43 (52%) were eligible; males 29 (67.4%), females 14 (32.6%) with mean ages of 35.1±10.8 and 34.3±9.3 respectively. Genotypes A were 34(79.1%), B were 5(11.6%), C-D were 0 while E-J were 9(20.9%). All cases of genotype B were associated with co-infection of genotype A. Majority were HBeAg negative with HBV DNA >10 IU/ml (81.4% and 86.0% respectively) with distribution among all the genotypes. Across genotypes, viral load mean percentage comparisons were: A vs. A/B = 2600 (p = 0.09), A vs. E-J = 5260 (p = 0.09) and A/B vs. E-J = 200 (p = 0.28). The most prevalent genotype was A followed by mixed co-infection of genotype A/B. Genotype A was associated with HBV DNA viral loads > 10IU/ml and high rates of HBeAg negativity. Genotypes E-J were also detected though not characterized., Competing Interests: The authors have declared that no competing interests exist, (Copyright: © 2024 Gikunyu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Although a combination of immunoprophylaxis and antiviral therapy can effectively prevent mother-to-child transmission (MTCT) of hepatitis B virus (HBV), a considerable number of infants born to highly viremic mothers still develop occult HBV infection (OBI). To uncover the virological factor and risk predictor for OBI in infants, we found that the diversity and complexity of maternal HBV quasispecies in the case group were lower than those in the control group. Mutations with significant differences between the two groups were most enriched in the NTCPbd and PreC regions. Genetic distance at the amino-acid level of the PreC region, especially the combination of three amino-acid mutations in the PreC region, could strongly predict the risk of OBI in infants. HBV quasispecies in OBI infants were highly complex, and the non-synonymous substitutions were mainly found in the RT and HBsAg regions. The sK47E (rtQ55R) and sP49L mutations in OBI infants might contribute to OBI through inhibiting the production of HBV DNA and HBsAg, respectively. This study found the potential virological factors and risk predictors for OBI in infants born to highly viremic mothers, which might be helpful for controlling OBI in infants.

Occult hepatitis B virus infection (OBI) is characterized by the presence of HBV DNA in the absence of detectable HBsAg. OBI is an important risk factor for cirrhosis and hepatocellular carcinoma, but its pathogenesis has not been fully elucidated. Mutations in the HBV preS/S genes can lead to impaired secretion of either HBsAg or S-protein resulting in the accumulation of defective viruses or S protein in cells. In our previous work, the M133S mutation was present in the HBV S gene of maintenance hemodialysis (MHD) patients with OBI. In this study, we investigated the potential role of amino acid substitutions in S proteins in S protein production and secretion through the construction of mutant S gene plasmids, structural prediction, transcriptome sequencing analysis, and in vitro functional studies. Protein structure prediction showed that the S protein M133S mutant exhibited hydrophilic modifications, with greater aggregation and accumulation of the entire structure within the membrane phospholipid bilayer. Differential gene enrichment analysis of transcriptome sequencing data showed that differentially expressed genes were mainly concentrated in protein processing in the endoplasmic reticulum (ER). The expression of heat shock family proteins and ER chaperone molecules was significantly increased in the wild-type and mutant groups, whereas the expression of mitochondria-associated proteins was decreased. Immunofluorescence staining and protein blotting showed that the endoplasmic reticulum-associated protein PDI, the autophagy marker LC3, and the lysosome-associated protein LAMP2 co-localized with the S proteins in the wild-type and mutant strains, and their expression was increased. The mitochondria-associated TOMM20 protein was also co-expressed with the S protein, but expression was significantly reduced in the mutant. The M133S mutation in the S gene is expressed as a defective and misfolded protein that accumulates in the endoplasmic reticulum causing secretion-impaired endoplasmic reticulum stress, which in turn triggers mitochondrial autophagy and recruits lysosomes to fuse with the autophagosome, leading to mitochondrial clearance. This study preliminarily demonstrated that the mutation of M133S in the S gene can cause OBI and is associated with disease progression, providing a theoretical basis for the diagnosis and treatment of OBI., (© 2024. The Author(s).)

Despite the availability of a vaccine against hepatitis B virus (HBV), this infection still causes public health problems, particularly in susceptible populations. In Portugal, universal free vaccination started in 1994, and most HBV infections are diagnosed in immigrants from high-prevalence countries. Our aim was to assess the pattern of HBV genotypes/subgenotypes in samples collected between 2017 and 2021 from a convenience sample of 70 infected residents in Portugal. The HBV pol/HBsAg region was amplified and sequenced, allowing the analysis of RT sequences submitted to phylogenetic analysis and mutations assessment. A total of 37.1% of samples were from native Portuguese, aged 25-53 years (mean: 36.7 years), and the remaining samples were from individuals born outside of Portugal. A high diversity of HBV was identified: subgenotypes A1-A3 in 41.0% (16/39); D1, D3, and D4 in 30.7% (12/39); E in 23.1% (9/39); and F4 in 2.6% (1/39). Besides genotypes A and D, Portuguese were also infected with genotypes E and F, which are prevalent in Africa and South America, respectively. Resistance mutations in RT sequences were not found. The findings provide valuable insights for updating the HBV molecular epidemiology in Portugal. However, successful strategies to prevent and control the infection are still needed in the country, especially among susceptible and vulnerable populations.

Evolutionary changes in the hepatitis B virus (HBV) genome could reflect its adaptation to host-induced selective pressure. Leveraging paired human exome and ultra-deep HBV genome-sequencing data from 567 affected individuals with chronic hepatitis B, we comprehensively searched for the signatures of this evolutionary process by conducting "genome-to-genome" association tests between all human genetic variants and viral mutations. We identified significant associations between an East Asian-specific missense variant in the gene encoding the HBV entry receptor NTCP (rs2296651, NTCP S267F) and mutations within the receptor-binding region of HBV preS1. Through in silico modeling and in vitro preS1-NTCP binding assays, we observed that the associated HBV mutations are in proximity to the NTCP variant when bound and together partially increase binding affinity to NTCP S267F. Furthermore, we identified significant associations between HLA-A variation and viral mutations in HLA-A-restricted T cell epitopes. We used in silico binding prediction tools to evaluate the impact of the associated HBV mutations on HLA presentation and observed that mutations that result in weaker binding affinities to their cognate HLA alleles were enriched. Overall, our results suggest the emergence of HBV escape mutations that might alter the interaction between HBV PreS1 and its cellular receptor NTCP during viral entry into hepatocytes and confirm the role of HLA class I restriction in inducing HBV epitope variations., Competing Interests: Declaration of interests O.P, A.G., M.S., and V.S. are employees of Gilead Sciences Inc. O.N. is now an employee of SUN bioscience SA. S.R. is now an employee of Novartis AG. A. Scheck is now an employee of RIDGELINE Discovery GmbH. M.B. has received research funding from AB Altoimmune, Janssen, GSK, Gilead, and AbbVie. H.L.Y.C. is an advisor for Aligos, Arbutus, Gilead Sciences, Glaxo-Smith-Kline, Roche, Vaccitech, Vir Biotechnology, and Virion Therapeutics and a speaker for Echosens, Gilead Sciences, Roche, Mylan., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: In recent years, a gene-editing technology known as clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 has been developed and is progressively advancing into clinical trials. While current antiviral therapies are unable to eliminate the Hepatitis B virus (HBV), it stands as a prime target for the CRISPR/Cas9 technology. The objective of this study was to enhance the efficacy of CRISPR/Cas9 in suppressing HBV replication, lowering HBsAg and HBeAg levels, and eliminating covalently closed circular DNA (cccDNA)., Methods: To enhance the anti-HBV effectiveness of CRISPR/Cas9, our study delved into a dual-guide RNA (gRNA) strategy. After evaluating the antiviral activities of multiple gRNAs that effectively impeded HBV replication, we identified three specific gRNAs-namely 10, 4, and 21. These gRNAs were selected for their targeting of distinct yet conserved regions within the HBV genome., Results: In HBV-stable cell lines, namely HepAD38, and HBV infection models of HepG2-NTCP cells, our investigation revealed that the co-application of gRNA-10 with either gRNA-4 or gRNA-21 within the CRISPR/Cas9 system demonstrated heightened efficacy in impeding HBV replication, reducing the levels of HBsAg, HBeAg, and cccDNA levels, along with a more pronounced promotion of HBsAg clearance when compared to the use of a single gRNA., Conclusions: The CRISPR/Cas9 system employing dual gRNAs has proven highly effective in both suppressing HBV replication and facilitating HBsAg clearance. This promising outcome suggests that it holds potential to emerge as a novel approach for achieving the functional cure of patients with HBV infection.

Introduction: Human immunodeficiency virus (HIV) / hepatitis B virus (HBV) causes higher rates of liver disease compared to infection with just one virus. Co-infection can accelerate the progression to liver fibrosis or hepatocellular carcinoma and disturb the treatment response. APOBEC3G is a host defense factor which interferes with HIV-1 and HBV. We aimed to determine the prevalence of hepatitis B surface antigen (HBsAg) among HIV-infected patients and seronegative controls, and screen the HIV/HBV population for APOBEC3G variants rs8177832, rs35228531 and rs2294367, previously associated with HIV-1 infection susceptibility in Morocco., Methodology: A case control study was conducted on 404 individuals (204 HIV-infected and 200 eligible blood donors) from April to November 2021. HBsAg was measured on the Roche Cobas e411 automatic analyzer (Roche Diagnostics, Basel, Switzerland) and APOBEC3G polymorphisms were identified using the TaqMan genotyping allelic discrimination method. Fisher Exact test, odds ratio (OR) with 95% confidence interval (CI), and haplotype frequencies were calculated., Results: Of the 204 HIV-1 seropositive patients and 200 controls, 4.9% (95%CI: 2.38-8.83) and 2.50% (95% CI: 0.82-5.74) were HBsAg-positive respectively. There was a significant association between increasing age (> 40 years) and HBV infection among controls (p = 0.04). The distribution of genotypes and alleles frequencies of APOBEC3G variants was heterogenous and five different haplotypes with frequencies ≥ 5% were obtained, of which ACC (rs8177832, rs35228531, rs2294367) was the most prevalent., Conclusions: HBV co-infection is common among HIV-1 infected individuals in Morocco. Efforts should be made to prevent, treat and control HBV transmission in this population., Competing Interests: No Conflict of Interest is declared, (Copyright (c) 2024 Imane Belbacha, Rajae Bensghir, Kamal Filali Marhoum, Majda Laboudi, Khadija Hajoutt, Elmir ElHarti, Khalid Sadki, Hicham Oumzil.)

The human hepatitis delta virus (HDV) is a satellite RNA virus that depends on hepatitis B virus (HBV) surface proteins (HBsAg) to assemble into infectious virions targeting the same organ (liver) as HBV. Until recently, the evolutionary origin of HDV remained largely unknown. The application of bioinformatics on whole sequence databases lead to discoveries of HDV-like agents (DLA) and shed light on HDV's evolution, expanding our understanding of HDV biology. DLA were identified in heterogeneous groups of vertebrates and invertebrates, highlighting that the evolution of HDV, represented by eight distinct genotypes, is broader and more complex than previously foreseen. In this study, we focused on the characterization of three mammalian DLA discovered in woodchuck ( Marmota monax ), white-tailed deer ( Odocoileus virginianus ), and lesser dog-like bat ( Peropteryx macrotis ) in terms of replication, cell-type permissiveness, and spreading pathways. We generated replication-competent constructs expressing 1.1-fold over-length antigenomic RNA of each DLA. Replication was initiated by transfecting the cDNAs into human (HuH7, HeLa, HEK293T, A549) and non-human (Vero E6, CHO, PaKi, LMH) cell lines. Upon transfection and replication establishment, none of the DLA expressed a large delta antigen. A cell division-mediated viral amplification assay demonstrated the capability of non-human DLA to replicate and propagate in hepatic and non-hepatic tissues, without the requirement of envelope proteins from a helper virus. Remarkably L-HDAg but not S-HDAg from HDV can artificially mediate envelopment of WoDV and DeDV ribonucleoproteins (RNPs) by HBsAg to form infectious particles, as demonstrated by co-transfection of HuH7 cells with the respective DLA expression constructs and a plasmid encoding HBV envelope proteins. These chimeric viruses are sensitive to HDV entry inhibitors and allow synchronized infections for comparative replication studies. Our results provide a more detailed understanding of the molecular biology, evolution, and virus-host interaction of this unique group of animal viroid-like agents in relation to HDV.

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., (© 2024. The Author(s).)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 He, Liu, Jiang, Xu, Xiang and Lu.)

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., (© 2024 Wiley Periodicals LLC.)

This study explore the molecular mechanism of the synergistic effect of Chinese Yam polysaccharides and nucleoside analogues(NAs) on hepatitis B virus(HBV) resistance. Different concentrations of Chinese Yam polysaccharide and entecavir were ad-ded to HepG2.2.15 cells. After the cytotoxicity was detected by cell counting kit-8(CCK-8), the optimal concentration and time of the two drugs to inhibit HepG2.2.15 cells were screened out. They were divided into control group, Chinese Yam polysaccharide group, entecavir group and combination drug group(Chinese Yam polysaccharide + entecavir). The drugs were added to HepG2.2.15 cells, ELISA was used to detect the effects of each group of drugs on the secretion of hepatitis B virus surface antigen(HBsAg) and hepatitis B virus e antigen(HBeAg) in cell supernatant, probe quantitative real-time PCR(probe qRT-PCR) was used to detect the effects of drugs on HBV-DNA in HepG2.2.15 cells, and Western blot was used to detect the effects of each group of drugs on the expression of p38 MAPK, p-p38 MAPK, NTCP proteins in HepG2.2.15 cells. The qRT-PCR was used to detect the effect of drugs on the expression of p38 MAPK and NTCP mRNA in HepG2.2.15 cells. The results showed that compared with control group, the concentrations of HBeAg and HBsAg in Chinese Yam polysaccharide group, entecavir group and combination group decreased(P&lt;0.01 or P&lt;0.001), and both of them inhibited HBV-DNA in HepG2.2.15 cells(P&lt;0.01), and the HBV-DNA inhibition of HepG2.2.15 cells in the combination group was more obvious(P&lt;0.001), and the protein expression levels of p-p38 MAPK and NTCP were significantly decreased(P&lt;0.05 or P&lt;0.01), the mRNA expression level of p38 MAPK increased, and the mRNA expression level of NTCP decreased(P&lt;0.05 or P&lt;0.01). To sum up, Chinese Yam polysaccharide can reduce the expression of NTCP protein and mRNA through p38 MAPK signaling pathway and cooperate with entecavir in anti-HBV.

The concurrent seropositivity of HBsAg and anti-HBs has been described among patients with chronic hepatitis B (CHB), but its prevalence is variable. HBV S-gene mutations can affect the antigenicity of HBsAg. Patients with mutations in the 'α' determinant region of the S gene can develop severe HBV reactivation under immunosuppression. In this study at a tertiary liver center in the United States, we evaluated the frequency and virological characteristics of the HBsAg mutations among CHB patients with the presence of both HBsAg and anti-HBs. In this cohort, 45 (2.1%) of 2178 patients were identified to have a coexistence of HBsAg and anti-HBs, and 24 had available sera for the genome analysis of the Pre-S1, Pre-S2, and S regions. The frequency of mutations in the S gene was significantly higher among those older than 50 years (mean 8.5 vs. 5.4 mutations per subject, p = 0.03). Twelve patients (50%) had mutations in the 'α' determinant region of the S gene. Mutations at amino acid position 126 were most common in eight subjects. Three had a mutation at position 133. Only one patient had a mutation at position 145-the classic vaccine-escape mutation. Despite the universal HBV vaccination program, the vaccine-escape mutant is rare in our cohort of predominantly Asian 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., (© 2024. The Author(s).)

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., (© 2024. The Author(s).)

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., (© 2024. The Author(s).)

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., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

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., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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., Competing Interests: Declaration of competing interest The authors affirm no known financial conflicts or personal relationships that might have influenced the work presented in this paper., (Copyright © 2024. Published by Elsevier B.V.)

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., Competing Interests: NO authors have competing interests., (Copyright: © 2024 Tepjanta et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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 HBVpolymerase 456-63 -specific stimulation was impaired in HBsAg > 1,000 IU/ml group, while the response against HBVcore 18-27 was preserved and response against envelope 183-91 was nearly abolished, regardless of HBsAg level. Cases with preserved HBVpolymerase 456-63 CD8 + cell response had lower LS/duration of infection and APRI/duration of infection rates. HBV-polymerase 456-63 -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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Peña-Asensio, Calvo-Sánchez, Miquel-Plaza, Sanz-de-Villalobos, González-Praetorius, Delgado-Fernandez, Torralba and Larrubia.)

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Kitagawa et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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., (© 2024 Wiley Periodicals LLC.)

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., Competing Interests: Declaration of competing interest The authors declare that they have known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

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 (EC 90 ), compared to concentrations in plasma which were substantially below its EC 90 , 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.

Background and Aims: Hepatitis B virus X protein (HBx) play a key role in pathogenesis of HBV-induced hepatocellular carcinoma (HCC) by promoting epithelial to mesenchymal transition (EMT). In this study, we hypothesized that inhibition of HBx is an effective strategy to combat HCC., Methodology and Results: We designed and synthesized novel HBx gene specific single guide RNA (sgRNA) with CRISPR/Cas9 system and studied its in vitro effects on tumour properties of HepG2-2.15. Full length HBx gene was excised using HBx-CRISPR that resulted in significant knockdown of HBx expression in hepatoma cells. HBx-CRISPR also decreased levels of HBsAg and HBV cccDNA expression. A decreased expression of mesenchymal markers, proliferation and tumorigenic properties was observed in HBx-CRISPR treated cells as compared to controls in both two- and three- dimensional (2D and 3D) tumour models. Transcriptomics data showed that out of 1159 differentially expressed genes in HBx-CRISPR transfected cells as compared to controls, 70 genes were upregulated while 1089 genes associated with cell proliferation and EMT pathways were downregulated., Conclusion: Thus, targeting of HBx by CRISPR/Cas9 gene editing system reduces covalently closed circular DNA (cccDNA) levels, HBsAg production and mesenchymal characteristics of HBV-HCC cells. We envision inhibition of HBx by CRISPR as a novel therapeutic approach for HBV-induced HCC., (© 2023 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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.

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.

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., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest The authors have no competing interests to declare., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Despite good vaccine coverage and careful blood donor selection policies, hepatitis B virus (HBV) is still the most frequent viral infection among blood donors (BDs) in Italy, mostly in the occult form (OBI). We studied the virological features of OBI in BDs from South Italy by serology, molecular testing for HBV-DNA, and sequencing for HBV genotypes and mutations. One hundred and two samples from 95 BDs (22.1% first time, 87.9% regular, median age 57 years) positive for HBV-DNA and negative for HBsAg were retrospectively analyzed. HBV biomarkers were detected in 96.9% (anti-HBc in 44.2%, anti-HBc plus anti-HBs in 49.5%, anti-HBs alone in 3.2%). No risk factor was declared by 45.3% of donors. HBV-DNA levels were very low (median: 7 IU/mL). All samples harbored HBV genotype D and single or multiple mutations in the S gene were found in 28/36 sequences analyzed and in 75% of donors. Mutations were unrelated to gender, donor group or serological patterns. An HBsAg assay with enhanced sensitivity was positive in samples from seven donors (7.4%), two of which negative for HBV-DNA by real-time PCR. OBI still represents a risk for HBV transmission from blood donations; screening by highly sensitive serological and molecular assays is warranted.