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BACKGROUND AND AIMS The hepatitis B core-related antigen (HBcrAg), a composite antigen of precore/core gene including classical hepatitis B core protein (HBc) and HBeAg and, additionally, the precore-related antigen PreC, retaining the N-terminal signal peptide, has emerged as a surrogate marker to monitor the intrahepatic HBV covalently closed circular DNA (cccDNA) and to define meaningful treatment endpoints. APPROACH AND RESULTS Here, we found that the woodchuck hepatitis virus (WHV) precore/core gene products (i.e., WHV core-related antigen [WHcrAg]) include the WHV core protein and WHV e antigen (WHeAg) as well as the WHV PreC protein (WPreC) in infected woodchucks. Unlike in HBV infection, WHeAg and WPreC proteins were N-glycosylated, and no significant amounts of WHV empty virions were detected in WHV-infected woodchuck serum. WHeAg was the predominant form of WHcrAg, and a positive correlation was found between the serum WHeAg and intrahepatic cccDNA. Both WHeAg and WPreC antigens displayed heterogeneous proteolytic processing at their C-termini, resulting in multiple species. Analysis of the kinetics of each component of the precore/core-related antigen, along with serum viral DNA and surface antigens, in HBV-infected chimpanzees and WHV-infected woodchucks revealed multiple distinct phases of viral decline during natural resolution and in response to antiviral treatments. A positive correlation was found between HBc and intrahepatic cccDNA but not between HBeAg or HBcrAg and cccDNA in HBV-infected chimpanzees, suggesting that HBc can be a better marker for intrahepatic cccDNA. CONCLUSIONS In conclusion, careful monitoring of each component of HBcrAg along with other classical markers will help understand intrahepatic viral activities to elucidate natural resolution mechanisms as well as guide antiviral development.

The global burden of hepatitis B virus (HBV) is enormous, with 257 million persons chronically infected, resulting in more than 880,000 deaths per year worldwide. HBV exists as nine different genotypes, which differ in disease progression, natural history and response to therapy. HBV is an ancient virus, with the latest reports greatly expanding the host range of the Hepadnaviridae (to include fish and reptiles) and casting new light on the origins and evolution of this viral family. Although there is an effective preventive vaccine, there is no cure for chronic hepatitis B, largely owing to the persistence of a viral minichromosome that is not targeted by current therapies. HBV persistence is also facilitated through aberrant host immune responses, possibly due to the diverse intra-host viral populations that can respond to host-mounted and therapeutic selection pressures. This Review summarizes current knowledge on the influence of HBV diversity on disease progression and treatment response and the potential effect on new HBV therapies in the pipeline. The mechanisms by which HBV diversity can occur both within the individual host and at a population level are also discussed.

Background & Aims HBV consists of 9 major genotypes (A to I), 1 minor strain (designated J) and multiple subtypes, which may be associated with different clinical characteristics. As only cell lines expressing genotype D3 have been established, herein, we aimed to establish stable cell lines producing high-titer cell culture-generated HBV (HBVcc) of different genotypes and to explore their infectivity, virological features and responses to treatment. Methods Stable cell lines producing high titers of HBV genotype A2, B2, C1, E, F1b and H were generated by transfecting plasmids containing a replication-competent 1.3x length HBV genome and an antibiotic marker into HepG2 cells that can support HBV replication. Clones with the highest levels of HBV DNA and/or HBeAg were selected and expanded for large-scale purification of HBVcc. HBVcc of different genotypes were tested in cells and a humanized chimeric mouse model. Results HBVcc genotypes were infectious in mouse-passaged primary human hepatocytes (PXB cells) and responded differently to human interferon (IFN)-α with variable kinetics of reduction in HBV DNA, HBeAg and HBsAg. HBVcc of all genotypes were infectious in humanized chimeric mice but with variable kinetics of viremia and viral antigen production. Treatment of infected mice with human IFN-α resulted in modest and variable reductions of viremia and viral antigenemia. HBVcc passaged in humanized chimeric mice (HBVmp) infected PXB cells much more efficiently than that of the original HBVcc viral stock. Conclusions Herein, we generated stable cell lines producing HBV of various genotypes that are infectious in vitro and in vivo. We observe genotype-associated variations in viral antigen production, infection kinetics and responses to human IFN-α treatment in these models. Lay summary Stable cell lines producing high-titer cell culture-generated hepatitis B virus (HBV) of various genotypes were established. HBV genotypes showed stable infectivity in both in vitro and in vivo models, which are valuable tools for antiviral development.

Background & aims We aim to capture the economic impact of a potential cure for chronic hepatitis B infection (CHB) in three countries (USA, China and Australia) with different health systems and epidemics to estimate the threshold drug prices below which a CHB cure would be cost-saving and/or highly cost-effective. Methods We simulated patients' hepatitis B progression, under three scenarios: current long-term suppressive antiviral therapy, functional cure defined as sustained undetectable HBsAg and HBV DNA, and partial cure defined as sustained undetectable HBV DNA only after a finite, 48-week treatment. Results Compared with current long-term antiviral therapy, a 30% effective functional cure among patients with and without cirrhosis in the USA, China and Australia would yield 17.50, 17.32 and 20.42 QALYs per patient, and 20.61, 20.42 and 20.62 QALYs per patient respectively. In financial terms, for CHB patients with and without cirrhosis, this would be cost-saving at a one-time treatment cost under US$11 944 and US$6694, respectively, in the USA, US$1744 and US$1001 in China, and US$12 063 and US$10 983 in Australia. Conclusion We show that in purely economic terms, a CHB cure will be highly cost-effective even if effective in only 30% of treated patients. The threshold price for cure is largely determined by the current antiviral drug costs, since it will replace a daily antiviral pill that is inexpensive and effective, although not curative. The likely need for combination therapies to achieve cure will also present cost challenges. While cost-effectiveness is important, it cannot be the only consideration, as cure will provide many benefits in addition to reduced liver disease and HCC, including eliminating the need for a long-term daily pill and reducing stigma often associated with chronic viral infection.

A major unmet clinical need is a therapeutic capable of removing hepatitis B virus (HBV) genome from the liver of infected individuals to reduce their risk of developing liver cancer. A strategy to deliver such a therapy could utilize the ability to target and promote apoptosis of infected hepatocytes. Presently there is no clinically relevant strategy that has been shown to effectively remove persistent episomal covalently closed circular HBV DNA (cccDNA) from the nucleus of hepatocytes. We used linearized single genome length HBV DNA of various genotypes to establish a cccDNA-like reservoir in immunocompetent mice and showed that clinical-stage orally administered drugs that antagonize the function of cellular inhibitor of apoptosis proteins can eliminate HBV replication and episomal HBV genome in the liver. Primary human liver organoid models were used to confirm the clinical relevance of these results. This study underscores a clinically tenable strategy for the potential elimination of chronic HBV reservoirs in patients.

Current therapies rarely cure chronic hepatitis B virus (HBV) infection due to the persistence of the viral episome, the covalently closed circular DNA (cccDNA), in hepatocytes. The hepatitis B virus core-related antigen (HBcrAg), a mixture of the viral precore/core gene products, has emerged as one potential marker to monitor the levels and activities of intrahepatic cccDNA. In this study, a comprehensive characterization of precore/core gene products revealed that HBcrAg components included the classical hepatitis B virus core antigen (HBc) and e antigen (HBeAg) and, additionally, the precore-related antigen, PreC, retaining the N-terminal signal peptide. Both HBeAg and PreC antigens displayed heterogeneous proteolytic processing at their C termini resulting in multiple species, which varied with viral genotypes. HBeAg was the predominant form of HBcrAg in HBeAg-positive patients. Positive correlations were found between HBcrAg and PreC, between HBcrAg and HBeAg, and between PreC and HBeAg but not between HBcrAg and HBc. Serum HBeAg and PreC shared similar buoyant density and size distributions, and both displayed density and size heterogeneity. HBc, but not HBeAg or PreC antigen, was found as the main component of capsids in DNA-containing or empty virions. Neither HBeAg nor PreC protein was able to form capsids in cells or in vitro under physiological conditions. In conclusion, our study provides important new quantitative information on levels of each component of precore/core gene products as well as their biochemical and biophysical characteristics, implying that each component may have distinct functions and applications in reflecting intrahepatic viral activities. IMPORTANCE Chronic hepatitis B virus (HBV) infection afflicts approximately 257 million people, who are at high risk of progressing to chronic liver diseases, including fibrosis, cirrhosis, and hepatocellular carcinoma. Current therapies rarely achieve cure of HBV infection due to the persistence of the HBV episome, the covalently closed circular DNA (cccDNA), in the nuclei of infected hepatocytes. Peripheral markers of cccDNA levels and transcriptional activities are urgently required to guide antiviral therapy and drug development. Serum hepatitis B core-related antigen (HBcrAg) is one such emerging peripheral marker. We have characterized the components of HBcrAg in HBV-infected patients as well as in cell cultures. Our results provide important new quantitative information on levels of each HBcrAg component, as well as their biochemical and biophysical characteristics. Our findings suggest that each HBcrAg component may have distinct functions and applications in reflecting intrahepatic viral activities.

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

Viperin is an interferon-inducible protein that is pivotal for eliciting an effective immune response against an array of diverse viral pathogens. Here we describe a mechanism of viperin's broad antiviral activity by demonstrating the protein's ability to synergistically enhance the innate immune dsDNA signaling pathway to limit viral infection. Viperin co-localized with the key signaling molecules of the innate immune dsDNA sensing pathway, STING and TBK1; binding directly to STING and inducing enhanced K63-linked polyubiquitination of TBK1. Subsequent analysis identified viperin's necessity to bind the cytosolic iron-sulfur assembly component 2A, to prolong its enhancement of the type-I interferon response to aberrant dsDNA. Here we show that viperin facilitates the formation of a signaling enhanceosome, to coordinate efficient signal transduction following activation of the dsDNA signaling pathway, which results in an enhanced antiviral state. We also provide evidence for viperin's radical SAM enzymatic activity to self-limit its immunomodulatory functions. These data further define viperin's role as a positive regulator of innate immune signaling, offering a mechanism of viperin's broad antiviral capacity.

In HIV-hepatitis B virus (HBV) co-infection, adverse liver outcomes including liver fibrosis occur at higher frequency than in HBV-mono-infection, even following antiretroviral therapy (ART) that suppresses both HIV and HBV replication. To determine whether liver disease was associated with intrahepatic or circulating markers of inflammation or burden of HIV or HBV, liver biopsies and blood were collected from HIV-HBV co-infected individuals (n = 39) living in Bangkok, Thailand and naïve to ART. Transient elastography (TE) was performed. Intrahepatic and circulating markers of inflammation and microbial translocation were quantified by ELISA and bead arrays and HIV and HBV infection quantified by PCR. Liver fibrosis (measured by both transient elastography and liver biopsy) was statistically significantly associated with intrahepatic mRNA for CXCL10 and CXCR3 using linear and logistic regression analyses adjusted for CD4 T-cell count. There was no evidence of a relationship between liver fibrosis and circulating HBV DNA, qHBsAg, plasma HIV RNA or circulating cell-associated HIV RNA or DNA. Using immunohistochemistry of liver biopsies from this cohort, intrahepatic CXCL10 was detected in hepatocytes associated with inflammatory liver infiltrates in the portal tracts. In an in vitro model, we infected an HBV-infected hepatocyte cell line with HIV, followed by interferon-γ stimulation. HBV-infected cells lines produced significantly more CXCL10 than uninfected cells lines and this significantly increased in the presence of an increasing multiplicity of HIV infection. Conclusion: Enhanced production of CXCL10 following co-infection of hepatocytes with both HIV and HBV may contribute to accelerated liver disease in the setting of HIV-HBV co-infection., Author summary Chronic hepatitis B infection is common in people living with HIV due to shared routes of transmission. Liver disease including fibrosis and cirrhosis is accelerated in those with HIV-hepatitis B coinfection, and although outcomes are improved with antiviral treatment, increased morbidity and mortality are still seen for unknown reasons. We recruited people living with HIV-hepatitis B coinfection who had not been treated and assessed liver fibrosis using non-invasive means (transient elastography or ‘Fibroscan’) and liver biopsy. We measured multiple markers of HIV and HBV replication and inflammation in the liver as well as circulating in the blood to assess their relationship to liver fibrosis. We found that the inflammatory cytokine CXCL10 and its receptor CXCR3 in the liver were strongly associated with fibrosis. CXCL10 was localised to hepatocytes on fluorescent microscopy. We then infected a hepatocyte cell line with HIV and HBV and found that production of CXCL10 was greatly increased in the presence of both viruses. We conclude that increased CXCL10 in hepatocytes, as a result of co-infection of hepatocytes with HIV and HBV, may be an important new target for treatments to reduce liver disease in people living with HIV and HBV and should be further investigated.

Background & Aims HBsAg-specific antibody responses are difficult to detect during chronic hepatitis B infection (CHB) and are often overlooked. The aim of this study was to examine whether anti-HBs may be involved in functional cure (FC) by profiling anti-HBs responses in patients with CHB using a panel of specific assays. Methods Longitudinal serum samples were obtained from 25 patients with CHB who were infected with HBV genotype A and were undergoing nucleos(t)ide analogue (NA) treatment: 14 achieved FC while 11 remained infected (non-FC). Anti-HBs immune complexes (HBsAg-IC), FcγRIIIa dimer binding, epitope specificity and neutralisation efficacy were measured. Results HBsAg-IC peaks were detected prior to HBsAg loss in 10/14 FC patients. These HBsAg-IC peaks overlapped with either an alanine aminotransferase (ALT) flare (8/10 patients), or a rise in ALT (2/10 patients). HBsAg-IC peaks were detected in 7/11 non-FC patients, but were not associated with an ALT flare. FCγRIIIa binding was detected in 9/14 FC patients, independent from detection of overlapping HBsAg-IC/ALT peaks. FC patients had stable HBsAg epitope occupancy across the study, whereas non-FC patients had a reduction in HBsAg epitope occupancy within the first 12–24 weeks of NA treatment. Convalescent sera from FC patients recognised more HBsAg epitopes and neutralised HBV infection more potently than anti-HBs derived from vaccinees. Neutralisation potency appeared to increase post-HBsAg loss in 4/5 FC patients examined. Conclusions Using these assays, we confirm that anti-HBs responses are present and fluctuate over time in this cohort of patients with HBeAg+ CHB, who were infected with HBV genotype A and treated with NAs. Key anti-HBs profiles associated with either FC or failure to achieve FC were also identified, suggesting a role for anti-HBs responses in FC. Lay summary Using a panel of assays to characterise hepatitis B surface antibody (anti-HBs) responses in a group of patients with chronic hepatitis B, we identified anti-HBs profiles associated with either functional cure, or failure to achieve functional cure. Functional cure was associated with immune complex peaks which overlapped with alanine aminotransferase flares. Conversely, in those who did not achieve functional cure, immune complex peaks were present, but were not associated with alanine aminotransferase flares, and a decline in anti-HBs diversity was observed early during treatment.

Hepatitis B virus (HBV) is a major human pathogen that causes liver diseases. The main HBV RNAs are unspliced transcripts that encode the key viral proteins. Recent studies show that some of the HBV spliced transcript isoforms are predictive of liver cancer, yet the roles of these spliced transcripts remain elusive. Furthermore, a total of 9 major HBV genotypes were isolated from discrete geographical regions of the world, it is likely that these genotypes may express a broad variety of spliced transcript isoforms. To systematically study the HBV splice variants, we transfected the human hepatoma cells Huh7 with 4 HBV genotypes (A2, B2, C2, and D3), followed by deep RNA-sequencing. We found that 12-25% of HBV RNAs were splice variants, which were reproducibly detected across independent biological replicates. This accounted for a total of 6 novel and 6 previously identified splice variants. In particular, 2 highly abundant novel splice variants, in which we called the putative splice variants 1 and 5 (pSP1 and pSP5), were specifically expressed at high levels in genotypes D3 and B2, respectively. In general, the HBV splicing profiles varied across the genotypes except for the known spliced pgRNAs SP1 and SP9, which were present in all 4 major genotypes. Counterintuitively, these singly spliced SP1 and SP9 had a suboptimal 5′ splice site, suggesting that splicing of HBV RNAs is tightly controlled by the viral post-transcriptional regulatory RNA element.IMPORTANCEHBV infection affects over 257 million people worldwide. HBV is a major cause of liver diseases including cancer and there is no cure. Some HBV RNAs are spliced variants and their roles are largely unclear, although some splice variants have been previously found to be associated with liver cancer. HBV exists as 9 genotypes worldwide with marked differences in replicative capacity and disease sequelae. Whether HBV splice variants vary for the different genotypes is yet to be investigated in depth. Here we sequenced RNAs from 4 major HBV genotypes using a cell culture system. We found 6 new and 6 previously known splice variants across these genotypes. Some novel splice variants were present at high levels, suggesting they could be functionally important. Interestingly, although HBV has adapted to human hosts for over 50,000 years, the most frequently spliced location shared little flanking sequence similarity with that of humans.

BACKGROUND AND AIMS: We conducted haplotype analysis of complete hepatitis B virus (HBV) genomes following deep sequencing from 368 patients across multiple phases of chronic hepatitis B (CHB) infection from four major genotypes (A-D), analyzing 4,110 haplotypes to identify viral variants associated with treatment outcome and disease progression. APPROACH AND RESULTS: Between 18.2% and 41.8% of nucleotides and between 5.9% and 34.3% of amino acids were 100% conserved in all genotypes and phases examined, depending on the region analyzed. Hepatitis B e antigen (HBeAg) loss by week 192 was associated with different haplotype populations at baseline. Haplotype populations differed across the HBV genome and CHB history, this being most pronounced in the precore/core gene. Mean number of haplotypes (frequency) per patient was higher in immune-active, HBeAg-positive chronic hepatitis phase 2 (11.8) and HBeAg-negative chronic hepatitis phase 4 (16.2) compared to subjects in the "immune-tolerant," HBeAg-positive chronic infection phase 1 (4.3, P< 0.0001). Haplotype frequency was lowest in genotype B (6.2, P< 0.0001) compared to the other genotypes (A = 11.8, C = 11.8, D = 13.6). Haplotype genetic diversity increased over the course of CHB history, being lowest in phase 1, increasing in phase 2, and highest in phase 4 in all genotypes except genotype C. HBeAg loss by week 192 of tenofovir therapy was associated with different haplotype populations at baseline. CONCLUSIONS: Despite a degree of HBV haplotype diversity and heterogeneity across the phases of CHB natural history, highly conserved sequences in key genes and regulatory regions were identified in multiple HBV genotypes that should be further investigated as targets for antiviral therapies and predictors of treatment response.

Hepatitis B virus (HBV) is a diverse, partially double-stranded DNA virus, with 9 genotypes (A-I), and a putative 10th genotype (J), thus far characterised. Given the broadening interest in HBV sequencing, there is an increasing requirement for a consistent, unified approach to HBV genotype and subgenotype classification. We set out to generate an updated resource of reference sequences using the diversity of all genomic-length HBV sequences available in public databases. We collated and aligned genomic-length HBV sequences from public databases and used maximum-likelihood phylogenetic analysis to identify genotype clusters. Within each genotype, we examined the phylogenetic support for currently defined subgenotypes, as well as identifying well-supported clades and deriving reference sequences for them. An alignment of these reference sequences and maximum-likelihood phylogenetic trees of the sequences are provided to simplify classification. Based on the phylogenies generated, we present a comprehensive set of HBV reference sequences at the genotype and subgenotype level.

Nucleos(t)ide analouges (NUC) treatment prevents progression of liver fibrosis in subjects with chronic hepatitis B (CHB). However, risk for hepatocellular carcinoma (HCC) persists despite viral suppression. Specific HBV variants have been associated with adverse outcomes, including HCC, however the frequency of these variants during the seemingly benign immunotolerant (IT) phase is unknown. Next generation sequencing and detailed virological characterization on a cohort of treatment-naive IT subjects was performed to determine the frequency of clinically relevant viral variants. Samples from 97 subjects (genotype B/C 55%/45%, median HBV-DNA 8.5 log10 IU/mL, median HBsAg 4.8 log10IU/mL, median HBeAg 3.6 log10 PEIU/mL) were analysed. Despite subjects being in the IT phase, clinically relevant HBV variants were common at baseline, particularly in the basal core promoter (BCP, overlaps the hepatitis B X (HBx) gene), precore, and PreS regions. BCP/HBx variants were independently associated with lower baseline HBeAg, HBsAg and HBV-DNA titres. Precore variants were independently associated with higher baseline ALT. Increased viral diversity was associated with increased age and lower HBV DNA, HBsAg and HBeAg levels. Low level (

Migration from sub-Saharan Africa is contributing to the rising incidence of chronic hepatitis B (CHB) infection and its complications in Australia. African CHB is associated with unique genotypes, such as E and A1, which are associated with reduced vaccine efficacy and early-onset hepatocellular carcinoma, respectively, although the prevalence of these genotypes outside Africa is poorly described. Treatment-naïve children of African origin with CHB were recruited at the Royal Children's Hospital Melbourne. Population-based sequencing of the complete HBV genome, or the clinically relevant basal core promoter (BCP)/precore (PC) region, was performed, and the HBV genotype/subgenotype assigned by phylogenetic analysis. HBV was characterized in serum from 67 children, median age 12.5 years. HBV genotype E was most frequent (70 %), with genotype D [25 %; subgenotypes D6 (formerly D7)/D3/D2)] and subgenotype A1 (5 %) also being identified. Despite their young age, over 50 % of the children were HBeAg-negative and had seroconverted to anti-HBe, with this being associated with canonical BCP/PC mutations in the majority of cases. The profile of HBV in African children living in Australia was characterized by early HBeAg seroconversion and infection with HBV variants associated with poor clinical outcome, as well as genotypes previously associated with reduced vaccine efficacy or rapid progression to liver cancer. These findings have important ramifications for patient monitoring and treatment guidelines in the Australian paediatric setting.

Hepatitis B virus (HBV) from 76 adult immigrants in Australia from Myanmar was characterized to determine the prevalence of different HBV genotypes and subgenotypes. A mutational analysis was then performed to determine the presence of clinically significant mutations and correlate them to clinical outcomes. Initial genotyping revealed 68 patients with genotype C (89.5%) and eight patients with genotype B (10.5%). Phylogenetic analysis revealed the large majority of the genotype C infections were of subgenotype C1 (67/68). Sequencing of the HBV polymerase gene (and overlapping surface gene) revealed no mutations associated with antiviral resistance. HBV surface gene mutations were detected in 10 patients with subgenotype C1. HBV BCP/PC sequencing was obtained for 71/76 (93%) patients. BCP and/or PC mutations were identified in 57/71 (80%) of PCR positive patients. Treatment had been commenced for 15/76 (18%) patients, a further 26 untreated patients were in a stage of disease where HBV treatment would be considered standard of care. It was identified that genotype C1 is the predominant sub-genotype in this population. Genotype C is known to be associated with increased risk of development of HCC. This highlights the need for screening for HCC given the potential for the development of liver cancer. It was also identified that people with HBV were potentially not receiving optimal therapy in a timely fashion.

Hepatitis B virus infection in Africa is characterised by distinct genotypes with observed differences in natural history and clinical outcomes. Replication-competent cDNA clones of African genotypes were generated from patient-derived sequences identified in African children with chronic hepatitis B infection living in Australia: A1 (wild-type and basal core promotor (BCP) mutant), D2, D6, and E, comparing the replication phenotype to an established D3 cDNA clone in a transient transfection cell culture model. All clones replicated efficiently although less than the European D3 reference clone, and demonstrated marked differences in replication capacity, highest for subgenotypes A1 and D2. The BCP mutation increased the replication levels of the A1 subgenotype compared to wild-type. Intracellular and secreted surface antigen and HBeAg protein expression also varied across genotypes. We observed differences in functional activity in the upstream regulatory region across the genotypes that may contribute to the replication and protein differences observed.

Hepatitis B virus (HBV) is the prototype member of the family Hepadnaviridae and replicates via episomal copies of a covalently closed circular DNA (cccDNA) genome of approximately 3.2 kb. The chromatinization of this small viral genome, with overlapping open reading frames and regulatory elements, suggests an important role for epigenetic pathways to regulate HBV transcription. However, the host pathways that regulate HBV transcription and the temporal nature of promoter usage in infected cells are not well understood, in part due to the compact genome structure and overlapping open reading frames. To address this we developed a simple and cost-effective PCR assay to quantify the major viral RNAs and validated this technique using current state-of-art de novo HBV infection model systems. Our PCR method is three orders of magnitude more sensitive than Northern blot and requires relatively small amounts of starting material, making this an attractive tool for assessing HBV transcription.

Viperin is an interferon-inducible protein that is pivotal for eliciting an effective immune response against an array of diverse viral pathogens. Here we describe a mechanism of viperin’s broad antiviral activity by demonstrating the protein’s ability to synergistically enhance the innate immune dsDNA signalling pathway to limit viral infection. Viperin co-localised with the key signalling molecules of the innate immune dsDNA sensing pathway, STING and TBK1; binding directly to STING and inducing enhanced K63-linked polyubiquitination of TBK1. Subsequent analysis identified viperin’s necessity to bind the cytosolic iron-sulphur assembly component 2A, to prolong its enhancement of the type-I interferon response to aberrant dsDNA. Here we show that viperin facilitates the formation of a signalling enhanceosome, to coordinate efficient signal transduction following activation of the dsDNA signalling pathway; which results in an enhanced antiviral state. We also provide evidence for viperin’s radical SAM enzymatic activity to self-limit its immunomodulatory functions. This data further defines viperin’s role as a positive regulator of innate immune signalling, offering a mechanism of viperin’s broad antiviral capacity.

Chronic hepatitis B (CHB) is prevalent worldwide. The infectious agent, hepatitis B virus (HBV), replicates via an RNA intermediate and is error prone, leading to the rapid generation of closely related but not identical viral variants, including those that can escape host immune responses and antiviral treatments. The complexity of CHB can be further enhanced by the presence of HBV variants with large deletions in the genome generated via splicing (spHBV variants). Although spHBV variants are incapable of autonomous replication, their replication is rescued by wild-type HBV. spHBV variants have been shown to enhance wild-type virus replication, and their prevalence increases with liver disease progression. Single-molecule deep sequencing was performed on whole HBV genomes extracted from samples, including the liver explant, longitudinally collected from a subject with CHB over a 15-year period after liver transplantation. By employing novel bioinformatics methods, this analysis showed that the dynamics of the viral population across a period of changing treatment regimens was complex. The spHBV variants detected in the liver explant remained present posttransplantation, and a highly diverse novel spHBV population as well as variants with multiple deletions in the pre-S genes emerged. The identification of novel mutations outside the HBV reverse transcriptase gene that co-occurred with known drug resistance-associated mutations highlights the relevance of using full-genome deep sequencing and supports the hypothesis that drug resistance involves interactions across the full length of the HBV genome. IMPORTANCE Single-molecule sequencing allowed the characterization, in unprecedented detail, of the evolution of HBV populations and offered unique insights into the dynamics of defective and spHBV variants following liver transplantation and complex treatment regimens. This analysis also showed the rapid adaptation of HBV populations to treatment regimens with evolving drug resistance phenotypes and evidence of purifying selection across the whole genome. Finally, the new open-source bioinformatics tools with the capacity to easily identify potential spliced variants from deep sequencing data are freely available.

Chronic infection with hepatitis B virus (HBV) greatly increases the risk for liver cirrhosis and hepatocellular carcinoma (HCC). HBV isolates worldwide can be divided into ten genotypes. Moreover, the immune clearance phase selects for mutations in different parts of the viral genome. The outcome of HBV infection is shaped by the complex interplay of the mode of transmission, host genetic factors, viral genotype and adaptive mutations, as well as environmental factors. Core promoter mutations and mutations abolishing hepatitis B e antigen (HBeAg) expression have been implicated in acute liver failure, while genotypes B, C, subgenotype A1, core promoter mutations, preS deletions, C-terminal truncation of envelope proteins, and spliced pregenomic RNA are associated with HCC development. Our efforts to treat and prevent HBV infection are hampered by the emergence of drug resistant mutants and vaccine escape mutants. This paper provides an overview of the HBV life cycle, followed by review of HBV genotypes and mutants in terms of their biological properties and clinical significance.

International audience; Chronic HBV infection results in \\textgreater1 million deaths per year from cirrhosis and liver cancer. No known cure for chronic HBV exists, due in part to the continued presence of transcriptionally active DNA in the nucleus that is not directly targeted by current antiviral therapies. A coordinated approach is urgently needed to advance an HBV cure worldwide, such as those established in the HIV field. We propose the establishment of an International Coalition to Eliminate Hepatitis B Virus (ICE-HBV) to facilitate the formation of international working groups on HBV virology, immunology, innovative tools and clinical trials: to promote awareness and education as well as to drive changes in government policy and ensure funds are channelled to HBV cure research and drug development. With the ICE-HBV in place, it should be possible to enable a HBV cure within the next decade

Persistent hepatitis B virus (HBV) infection is established by the formation of an intranuclear pool of covalently closed circular DNA (cccDNA) in the liver. Very little is known about the intrahepatic distribution of HBV cccDNA in infected patients, particularly at the single-cell level. Here, we established a highly sensitive and specific ISH assay for the detection of HBV RNA, DNA, and cccDNA. The specificity of our cccDNA probe set was confirmed by its strict intranuclear signal and by a series of Southern blot analyses. Use of our in situ assay in conjunction with IHC or immunofluorescence uncovered a surprisingly mosaic distribution of viral antigens and nucleic acids. Most strikingly, a mutually exclusive pattern was found between HBV surface antigen-positive (HBsA-positive) and HBV DNA- and cccDNA-positive cells. A longitudinal observation of patients over a 1-year period of adeforvir therapy confirmed the persistence of a nuclear reservoir of viral DNA, although cytoplasmic DNA was effectively depleted in these individuals. In conclusion, our method for detecting viral nucleic acids, including cccDNA, with single-cell resolution provides a means for monitoring intrahepatic virological events in chronic HBV infection. More important, our observations unravel the complexity of the HBV life cycle in vivo.

Hepatitis B virus (HBV) exists as 9 major genotypes and multiple subtypes, many of which exhibit differences in pathogenicity and treatment response. Genotype H identified in Central America is associated with low incidence of liver disease and HCC, but higher incidence of occult HBV (low level HBV DNA positivity, HBsAg negative). The replication phenotype of genotype H associated with less severe forms of liver disease is unknown. We hypothesized that the reduced pathogenesis associated with this genotype may be due to by lower rates of viral replication and/or secretion compared to other characterised strains. We used transient transfection and infection cell culture models to characterise the replication phenotype, compared to our D3 reference strain. Genotype H exhibited reduced viral replication and altered envelope protein expression compared to genotype D, with functional studies showing that low replication was in part likely due to sequence differences in the major transcriptional regulatory region.

Background Alanine aminotransferase (ALT) flares during NA therapy are uncommon but occur. Evaluation of ALT flares during nucleos(t)ide analogue (NA) therapy is important as new immunomodulatory therapies for hepatitis B virus (HBV) are developed. We evaluated the association between ALT flares and HBsAg loss during long-term therapy for genotype A CHB. Methods This analysis included genotype A subjects from a phase III study of tenofovir vs adefovir in HBeAg-positive HBV. ALT flare was defined as (i) a rise in ALT >2x ULN from normal ALT levels; or (ii) a rise in ALT >2x baseline ALT level. HBsAg response at week 384 was recorded as one of HBsAg loss vs HBsAg decline (≥1 log10 IU/mL decline) vs non-response. The primary analysis evaluated the association between ALT flare and HBsAg response. Results 54 subjects were included. 23/54 (43%) subjects experienced an on-treatment ALT flare. 45% achieved an HBsAg reduction ≥1 log10 IU/mL, and of these 67% achieved HBsAg loss at a median of 102 weeks [IQR: 64-156]. Flare was associated with HBsAg decline vs non-response (67% vs 23%, P = .002), and were more common in subjects who achieved HBsAg loss vs non-response (56% vs 23%), P = .049). There was a median delay of 56 weeks [IQR: 40-80] between a flare and HBsAg loss. Conclusion In genotype A subjects undergoing long-term NA therapy, ALT flares predict for HBsAg response. The delay between ALT flare and HBsAg loss has implications for clinical trial design for early phase development of immunomodulatory strategies aiming for HBsAg loss.

ABSTRACT HBV causes persistent infection in approximately 300 million people and is associated with up to 2 million deaths annually. While the mechanisms by which HBV establishes and maintains infection are yet to be fully elucidated, there is mounting evidence that HBV infection in humans upregulates a range of innate immune responses and HBV has in turn has evolved mechanisms to suppress these responses. One such mechanism may be the hepatitis B e antigen (HBeAg), a soluble secreted protein which is also a major driver of adaptive immune responses. In this review, we review the literature on HBeAg-mediated regulation of innate immune responses and show that this regulation may extend beyond hepatocytes to other cell types such as NK cells which play an important role in viral clearance. Although further studies using new infection models are required, taken together these findings suggest that the HBeAg is an important regulator of the host response to infection and should not be overlooked in efforts to identify novel therapeutic targets against HBV.

Hepatitis B virus (HBV) infection can result in a spectrum of outcomes from immune-mediated control to disease progression, cirrhosis, and liver cancer. The host molecular pathways that influence and contribute to these outcomes need to be defined. Using an immunocompetent mouse model of chronic HBV infection, we identified some of the host cellular and molecular factors that impact on infection outcomes. Here, we show that cellular inhibitor of apoptosis proteins (cIAPs) attenuate TNF signaling during hepatitis B infection, and they restrict the death of infected hepatocytes, thus allowing viral persistence. Animals with a liver-specific cIAP1 and total cIAP2 deficiency efficiently control HBV infection compared with WT mice. This phenotype was partly recapitulated in mice that were deficient in cIAP2 alone. These results indicate that antagonizing the function of cIAPs may promote the clearance of HBV infection.

Interferon-α (IFN-α) is used to treat chronic hepatitis B virus (HBV) infection, but only 20%-40% of patients respond well. Clinical observations have suggested that HBV genotype is associated with the response to IFN therapy; however, its role in viral responsiveness to IFN in HBV-infected hepatocytes remains unclear. Here, we produced infectious virions of HBV genotypes A to D to infect three well-recognized cell-culture-based HBV infection systems, including primary human hepatocytes (PHH), differentiated HepaRG (dHepaRG), and HepG2-NTCP cells to quantitatively compare the antiviral effect of IFN-α on HBV across genotypes and cell models. The efficacy of IFN-α against HBV in hepatocytes was generally similar across genotypes A2, B5, C2, and D3; however, it was significantly different among the infection models given that the half maximal inhibitory concentration value of IFN-α for inhibition of viral DNA replication in PHH (20 U/mL) and dHepaRG cells were much lower than that in HepG2-NTCP cells (500 U/mL). Notably, even in PHH, IFN-α did not reduce HBV covalently closed circular DNA at the concentrations for which viral antigens and DNA replication intermediates were strongly reduced. The three cell-culture models exhibited differential cellular response to IFN-α. The genes reported to be associated with responsiveness to IFN-α in patients were robustly induced in PHH while weakly induced in HepG2-NTCP cells upon IFN-α treatment. Reduction or promotion of IFN response in PHH or HepG2-NTCP cells significantly attenuated or improved the inhibitory capacity of IFN-α on HBV replication, respectively.In the cell-culture-based HBV infection models, the sensitivity of HBV to IFN-α in hepatocytes is determined more by the cell-intrinsic IFN response than by viral genotype, and improvement of the IFN response in HepG2-NTCP cells promotes the efficacy of IFN-α against HBV. (Hepatology 2018;67:1237-1252).

Recent scientific advances, including the development of curative therapies for HCV and the establishment of global cure initiatives for HIV, have led to international calls seeking a cure for chronic infection with HBV.1 ,2 Over 240 million people live with chronic HBV, resulting in up to 780 000 deaths annually due to hepatic fibrosis/cirrhosis and hepatocellular carcinoma (HCC).3 ,4 Persons chronically infected with HBV who do not receive treatment have a lifelong risk of developing HCC, the third most common cause of disease globally.5–7 Along with scaled-up approaches to preventing and treating chronic HBV infection, having a safe and effective cure for HBV infection promises to minimise the global burden of HBV-related morbidity and mortality and reduce the economic and other burdens of lifelong treatment. Nevertheless, as HBV cure research proceeds, it is critical to anticipate and address the associated ethical issues to best protect the rights, interests and welfare of those who participate in the research as well as those who are or will become chronically infected with HBV. In this paper, after describing briefly the rationale for work aimed at achieving HBV cure, we delineate some of the key ethical issues that are especially salient for HBV cure research: (1) risks of interventions; (2) outcome measures, monitoring and modelling; (3) selection of study population; (4) language and consent; and (5) fairness. HBV infects hepatocytes and can establish a long-lived persistent reservoir through unintegrated covalently closed circular (ccc)DNA (and to a lesser extent integrated HBV DNA) that can continually produce HBV DNA and viral proteins, particularly hepatitis B surface antigen (HBsAg).8 Following the administration of nucleos(t)ide reverse transcriptase inhibitors or interferon α, the main strategies for treating chronic HBV infection,9 ,10 HBV DNA declines to often undetectable levels and liver inflammation improves, …

International audience; In 2017, the order Mononegavirales was expanded by the inclusion of a total of 69 novel species. Five new rhabdovirus genera and one new nyamivirus genus were established to harbor 41 of these species, whereas the remaining new species were assigned to already established genera. Furthermore, non-Latinized binomial species names replaced all paramyxovirus and pneumovirus species names, thereby accomplishing application of binomial species names throughout the entire order. This article presents the updated taxonomy of the order Mononegavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV).

The mechanisms by which hepatitis B virus (HBV) establishes and maintains chronic hepatitis B infection (CHB) are poorly defined. Innate immune responses play an important role in reducing HBV replication and pathogenesis. HBV has developed numerous mechanisms to escape these responses, including the production of the secreted hepatitis B e antigen (HBeAg), which has been shown to regulate antiviral toll-like receptor (TLR) and interleukin-1 (IL-1) signaling. IL-18 is a related cytokine that inhibits HBV replication in hepatoma cell lines and in the liver through the induction of gamma interferon (IFN-γ) by NK cells and T cells. We hypothesized that HBV or HBV proteins inhibit IFN-γ expression by NK cells as an accessory immunomodulatory function. We show that HBeAg protein inhibits the NF-κB pathway and thereby downregulates NK cell IFN-γ expression. Additionally, IFN-γ expression was significantly inhibited by exposure to serum from individuals with HBeAg-positive but not HBeAg-negative chronic HBV infection. Further, we show that the HBeAg protein suppresses IL-18-mediated NF-κB signaling in NK and hepatoma cells via modulation of the NF-κB pathway. Together, these findings show that the HBeAg inhibits IL-18 signaling and IFN-γ expression, which may play an important role in the establishment and/or maintenance of persistent HBV infection. IMPORTANCE It is becoming increasingly apparent that NK cells play a role in the establishment and/or maintenance of chronic hepatitis B infection. The secreted HBeAg is an important regulator of innate and adaptive immune responses. We now show that the HBeAg downregulates NK cell-mediated IFN-γ production and IL-18 signaling, which may contribute to the establishment of infection and/or viral persistence. Our findings build on previous studies showing that the HBeAg also suppresses the TLR and IL-1 signaling pathways, suggesting that this viral protein is a key regulator of antiviral innate immune responses.

Over 257 million people live with chronic hepatitis B virus (HBV) infection and there is no known cure. The effective preventative vaccine has no impact on existing infection. Despite the existence of drugs which efficiently suppress viral replication, treatment is usually life-long and finite therapies that cure HBV infection are urgently required. However, even if such therapies were available today, it is unlikely they would reach all of those who need it most, due to chronic hepatitis B (CHB) being largely undiagnosed across the globe and to the dire need for health systems promoting access to therapy. Considerable challenges to developing and implementing an effective HBV cure remain. Nonetheless, important advances towards a cure are being made, both in the development of a multitude of new therapeutic agents currently undergoing clinical trials, and through the establishment of a new global initiative dedicated to an HBV cure, ICE-HBV, that is working together with existing organisations to fast-track an HBV cure available to all.

Hepatitis B virus (HBV) infection continues to represent a significant health threat, affecting over 400 million people worldwide. Historically, the diagnosis and treatment of chronic hepatitis B (CHB) relied in detection of the hepatitis B surface antigen (HBsAg) and more recently realtime polymerase chain reaction (PCR) analysis. The advent of novel technologies and equipment for the identification and staging of the different stages of HBV infection has resulted in dramatic changes to patient monitoring and management. Through the use of rapid, quantitative HBsAg immunoassays, it is now possible to predict the likelihood of patient response to treatment as well as the clinical course of disease. Ultradeep sequencing technologies (also known as next-generation sequencing) overcome many of the traditional limitations associated with population-based sequencing approaches, and have provided significant insight into the viral response to therapeutic intervention and the molecular pathogenesis of CHB. The authors discuss recent developments in the molecular diagnosis of HBV infection, as well as potential advantages and caveats resultant of this rapid progression of technology.

Hepatitis B virus (HBV) from 40 adult African immigrants in Australia was characterized to determine the prevalence of different HBV genotypes and subgenotypes. A mutational analysis was then performed to determine the presence of clinically significant mutations and correlate them to clinical outcomes. Initial sequencing analysis revealed 13 with genotype A (32.5%), 13 with genotype D (32.5%), and 14 with genotype E (35%). Serology showed that 37 were HBeAg negative. Phylogenetic analysis identified a high prevalence (25%) of HBV subgenotype A1 in our cohort, a subgenotype which has been associated with more aggressive clinical disease. BCP/PC sequencing was obtained for 38 patients. BCP and/or PC mutations were identified in 36/38 (95%). The median viral load of all patients was 2995 IU/mL and most of the pathology results were within the normal range. Only one patient had an increased APRI score of 1.1 suggestive of cirrhosis. We present novel information on the HBV genotypes amongst the African population in Australia along with clinical correlates. The high prevalence of A1 subgenotype in this population supports the current Australian recommendation to commence hepatocellular carcinoma screening in Africans with chronic HBV from 20 years old.

The hepatitis B virus (HBV) exists as 9 major genotypes (A to I), one minor strain (designated J) and multiple subtypes. Marked differences in HBV natural history, disease progression and treatment response are exhibited by many of these genotypes and subtypes. For example, HBV genotype C is associated with later hepatitis B e antigen (HBeAg) seroconversion and high rates of liver cancer compared to other HBV genotypes, whereas genotype A2 is rarely associated with HBeAg-negative disease or liver cancer. The reasons for these and other differences in HBV natural history are yet to be determined but could in part be due to sequence differences in the HBV genome that alter replicative capacity and/or gene expression. Direct comparative studies on HBV replication and protein expression have been limited to date due largely to the absence of infectious HBV cDNA clones for each of the HBV genotypes present in the same genetic arrangement. We have produced replication-competent infectious cDNA clones of the most common subtypes of genotypes A to D, namely, A2, B2, C2, D3, and the minor strain J, and compared their HBV replication phenotype using transient-transfection models. We identified striking differences in HBV replicative capacity as well as HBeAg and surface (HBsAg) protein expression across genotypes, which may in part be due to sequence variability in regulatory regions of the HBV genome. Functional analysis showed that sequence differences in the major upstream regulatory region across genotypes impacted promoter activity. IMPORTANCE There have been very few studies directly comparing the replication phenotype of different HBV genotypes, for which there are marked differences in natural history and disease progression worldwide. We have generated replication-competent 1.3-mer cDNA clones of the major genotypes A2, B2, C2, and D3, as well as a recently identified strain J, and identified striking differences in replicative capacity and protein expression that may contribute to some of the observed differences in HBV natural history observed globally.

It has been over 50 years since the discovery of hepatitis B virus (HBV), yet 240 million people worldwide live with chronic HBV, resulting in up to 800000 deaths per year. A cure is yet to be achieved, due largely to a viral nuclear reservoir of transcriptionally active covalently closed circular DNA (cccDNA). While current antiviral therapies are effective at reducing viral replication, they have no impact on the existing cccDNA reservoir. Identifying mechanisms to either eliminate (complete cure) or inactivate (functional cure) HBV cccDNA are a major focus of HBV research worldwide. This review discusses recent advances in efforts to eliminate and/or regulate cccDNA, as well as future directions that may be considered in efforts to cure chronic HBV.

Chronic hepatitis B affects over 300 million people globally, and although there is an effective preventative vaccine, there is no cure. The recent discovery of the NTCP entry receptor means that infection models now permit investigation of drugs that target all stages of HBV life cycle. This is important as existing treatment strategies reduce the viral burden but rarely prevent expression of viral antigens associated with disease progression. It is likely this will only be achieved through the destruction or silencing of cccDNA in the hepatocyte nucleus. In this chapter we describe limitations and advantages of current and emerging treatment and cure strategies for chronic hepatitis B. We also discuss future drug targets with the hepatitis B life cycle that we believe are worthy of investigation. Together, these approaches should speed development of new therapeutic regimens that may lead to HBV cure in the foreseeable future.