RESUMO
BACKGROUND AND AIMS: Accurate biomarkers to predict outcomes following discontinuation of nucleos(t)ide analogue (NA) therapy are needed. We evaluated serum hepatitis B core-related antigen (HBcrAg) level as a biomarker for predicting outcomes after NA discontinuation. METHODS: Patients with HBeAg-negative chronic hepatitis B (CHB) without cirrhosis were enrolled in a prospective trial evaluating clinical outcomes until 96 weeks after NA discontinuation. End of treatment (EOT) and off-treatment levels of serum HBcrAg, HBsAg, HBV RNA and HBV DNA were used to predict key clinical outcomes including hepatitis flare (ALT ≥5 × ULN and HBV DNA > 2000 IU/mL). The SCALE-B score was calculated for the purposes of model validation. RESULTS: HBcrAg was tested amongst 65 participants. The median age was 54 years, 54% were male and 83% were Asian. HBcrAg was detectable in 86% patients. HBcrAg level ≥4 log U/mL at EOT was predictive of hepatitis flare [8/10 (80%) vs. 17/55 (31%), p = .001]. The presence of either HBcrAg ≥4 log U/mL or detectable HBV RNA at EOT predicted for both biochemical relapse and hepatitis flare. The SCALE-B model at EOT predicted for virological relapse, biochemical relapse, hepatitis flare and HBsAg loss in this cohort. An increase in the serum HBcrAg level off-treatment was also associated with hepatitis flare. No participant with EOT HBcrAg level ≥4 log U/mL achieved HBsAg loss. CONCLUSIONS: High levels of serum HBcrAg predict for hepatitis flare after stopping NA therapy and low likelihood of HBsAg loss at week 96. People with high levels of serum HBcrAg are not suitable candidates for NA discontinuation.
RESUMO
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.
RESUMO
The early and mid-career researchers (EMCRs) of scientific communities represent the forefront of research and the future direction in which a field takes. The opinions of this key demographic are not commonly aggregated to audit fields and precisely demonstrate where challenges lie for the future. To address this, we initiated the inaugural International Emerging Researchers Workshop for the global Hepatitis B and Hepatitis D scientific community (75 individuals). The cohort was split into small discussion groups and the significant problems, challenges, and future directions were assessed. Here, we summarise the outcome of these discussions and outline the future directions suggested by the EMCR community. We show an effective approach to gauging and accumulating the ideas of EMCRs and provide a succinct summary of the significant gaps remaining in the Hepatitis B and Hepatitis D field.
Assuntos
Hepatite B , Hepatite D , Humanos , Hepatite B/virologia , Hepatite D/virologia , Pesquisa Biomédica , Pesquisadores , Vírus da Hepatite BRESUMO
Prior to nuclear export, the hepatitis B virus (HBV) pregenomic RNA may be spliced by the host cell spliceosome to form shorter RNA sequences known as splice variants. Due to deletions in the open reading frames, splice variants may encode novel fusion proteins. Although not essential for HBV replication, the role of splice variants and their novel fusion proteins largely remains unknown. Some splice variants and their encoded novel fusion proteins have been shown to impair or promote wild-type HBV replication in vitro, and although splice variants Sp3 and Sp9 are two of the most common splice variants identified to date, their in vitro replication phenotype and their impact on wild-type HBV replication are unclear. Here, we utilize greater than genome-length Sp3 and Sp9 constructs to investigate their replication phenotype in vitro, and their impact on wild-type HBV replication. We show that Sp3 and Sp9 were incapable of autonomous replication, which was rescued by providing the polymerase and core proteins in trans. Furthermore, we showed that Sp3 had no impact on wild-type HBV replication, whereas Sp9 strongly reduced wild-type HBV replication in co-transfection experiments. Knocking out Sp9 novel precore-surface and core-surface fusion protein partially restored replication, suggesting that these proteins contributed to suppression of wild-type HBV replication, providing further insights into factors regulating HBV replication in vitro. IMPORTANCE: The role of hepatitis B virus (HBV) splice variants in HBV replication and pathogenesis currently remains largely unknown. However, HBV splice variants have been associated with the development of hepatocellular carcinoma, suggesting a role in HBV pathogenesis. Several in vitro co-transfection studies have shown that different splice variants have varying impacts on wild-type HBV replication, perhaps contributing to viral persistence. Furthermore, all splice variants are predicted to produce novel fusion proteins. Sp1 hepatitis B splice protein contributes to liver disease progression and apoptosis; however, the function of other HBV splice variant novel fusion proteins remains largely unknown. We show that Sp9 markedly impairs HBV replication in a cell culture co-transfection model, mediated by expression of Sp9 novel fusion proteins. In contrast, Sp3 had no effect on wild-type HBV replication. Together, these studies provide further insights into viral factors contributing to regulation of HBV replication.
Assuntos
Hepatite B , Neoplasias Hepáticas , Isoformas de Proteínas , Proteínas Virais , Replicação Viral , Humanos , DNA Viral/genética , Hepatite B/virologia , Vírus da Hepatite B/genética , Vírus da Hepatite B/fisiologia , Fenótipo , Isoformas de Proteínas/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Carcinoma Hepatocelular/virologiaRESUMO
BACKGROUND: Chronic hepatitis B virus (HBV) infection is a major health problem for all Indigenous Australians. Post-2000, Hepatitis B surface antigen prevalence has decreased, although remaining four times higher among Indigenous compared with non-Indigenous people. AIMS: This study aimed to characterise the HBV from Indigenous populations in Queensland and the Torres Strait Islands. METHODS: Serum samples were collected, with consent, from people within Queensland Indigenous communities prior to 1990 as part of the Queensland Health vaccination programme. Ethics approval was subsequently obtained to further characterise the HBV from 93 of these stored samples. HBV DNA was extracted and genotype was obtained from 82 samples. HBV full genome sequencing was carried out for a subset of 14 samples. RESULTS: Seventy-eight samples were identified as genotype C (2 × C12, 3 × C13 and 73 × C14), one sample as genotype A (A2) and three samples as genotype D (1 × D2, 1 × D3 and 1 × D4). The HBV/C sequences identified were most closely related to sequences isolated from Papua New Guinea and Indonesia (Papua Province). CONCLUSIONS: The HBV isolated from the Torres Strait Islanders was notably different to the HBV/C4 strain isolated from Indigenous people of mainland northern Australia, with no evidence of recombination. This reflects the differences in culture and origin between Torres Strait Islanders and mainland Indigenous people.
Assuntos
Povos Aborígenes Australianos e Ilhéus do Estreito de Torres , Hepatite B Crônica , Humanos , Austrália/epidemiologia , Hepatite B Crônica/epidemiologia , Epidemiologia Molecular , Queensland/epidemiologiaRESUMO
BACKGROUND: The prevalence of chronic hepatitis B (CHB) in Aboriginal and Torres Strait Islander Australians in Far North Queensland (FNQ) is greater than twice that of the general Australian population. CHB is common in Torres Strait Islanders diagnosed with hepatocellular carcinoma (HCC) - and in Aboriginals with HCC living in the Northern Territory - however, Aboriginals diagnosed with HCC in FNQ very rarely have CHB. The explanation for this apparent disparity is uncertain. AIMS: To determine the HBV genotypes in the FNQ Aboriginal and Torres Strait Islander population and their correlation with clinical phenotype. METHODS: We determined the HBV genotype of Aboriginal and Torres Strait Islander Australians living with CHB in FNQ and correlated this with demographic and clinical findings. RESULTS: 134/197 (68%) enrolled individuals had a sufficient viral load for genotyping. All 40 people with HBV/D genotype had Aboriginal heritage, whereas 85/93 (91%) with HBV/C had Torres Strait Islander heritage (P < 0.0001). Individuals with HBV/D were younger than those with HBV/C (median (interquartile range) age: 43 (39-48) vs 53 (42-66) years, P = 0.0002). However, they were less likely to be HBeAg positive (1/40 (3%) vs 23/93 (25%), P = 0.001). All three HCCs developed in Torres Strait Islanders; two-thirds were infected with HBV/C14; genotyping was not possible in the other individual. All 10 diagnoses of cirrhosis occurred in Torres Strait Islanders, 6/10 were infected with HBV/C14, genotyping was not possible in the other four individuals. CONCLUSIONS: HBV genotypes in Aboriginal and Torres Strait Islander Australians in FNQ differ markedly, which could explain the significant differences in the clinical phenotype in the two populations and might be used to inform cost-effective CHB care in the region.
RESUMO
BACKGROUND AND AIMS: HBV RNA in peripheral blood reflects HBV cccDNA transcriptional activity and may predict clinical outcomes. The prospective Melbourne HBV-STOP trial studied nucleot(s)ide analog discontinuation in HBeAg-negative non-cirrhotic participants with long-term virological suppression. Ninety-six weeks after stopping treatment, the proportion of participants with virological relapse (HBV DNA > 2000 IU/mL), biochemical relapse (ALT > 2 × ULN and HBV DNA > 2000 IU/mL), or hepatitis flare (ALT > 5 × ULN and HBV DNA > 2000 IU/mL) was 89%, 58%, and 38%, respectively. We evaluated the ability of serum HBV RNA levels to predict these outcomes. APPROACH RESULTS: HBV RNA levels were measured using the Roche cobas 6800/8800 HBV RNA Investigational Assay. Sixty-five participants had baseline and longitudinal off-treatment specimens available for RNA testing. HBV RNA was detectable at baseline in 25% of participants and was associated with a higher risk of biochemical relapse (81% vs. 51%, p value 0.04) and hepatitis flare (63% vs. 31%, p value 0.04). Participants who had undetectable serum HBV RNA as well as HBsAg ≤ 100 IU/mL at baseline were less likely to experience virological relapse (4 of 9, 44%) than participants with detectable HBV RNA and HBsAg level > 100 IU/mL (15/15, 100%; p value 0.0009). Off-treatment levels of HBV RNA were correlated with HBV DNA and were associated with the risk of hepatitis flare. CONCLUSIONS: Serum HBV RNA may be a useful biomarker for guiding clinical decision-making before stopping nucleot(s)ide analog therapy. Baseline HBV RNA and HBsAg levels are associated with the risk of clinical relapse, hepatitis flare, and disease remission off-treatment.
Assuntos
Hepatite B Crônica , Nucleosídeos , Humanos , Antivirais/uso terapêutico , DNA Viral , Antígenos E da Hepatite B , Antígenos de Superfície da Hepatite B , Vírus da Hepatite B/genética , Hepatite B Crônica/tratamento farmacológico , Nucleosídeos/uso terapêutico , Estudos Prospectivos , RNA , Exacerbação dos SintomasRESUMO
BACKGROUNDS AND AIMS: We investigated associations between hepatitis B virus (HBV) genome-length haplotype number (HN) at baseline in subjects with HBeAg-positive chronic hepatitis B (CHB), and the likelihood of achieving functional cure during direct-acting antiviral therapy METHOD: We analysed 86 HBeAg-positive baseline samples from patients with HBV genotypes A and D who were enrolled in a Phase II trial of tenofovir disoproxil fumarate (TDF) to determine if HN was a biomarker of HBsAg loss during therapy. Findings were validated using baseline samples from 181 patients with HBV genotypes A and D from an independent clinical trial utilising TDF or tenofovir alafenamide therapy in HBeAg-positive CHB. RESULTS: In the HBeAg-positive test cohort, patients with genotypes A or D and ≤2 haplotypes had a minimum of 21-fold higher likelihood of achieving HBsAg loss on TDF. Baseline HN (p < 0.0001) was a stronger predictor of HBsAg loss on therapy than HBsAg titre (p = 0.03), HBeAg titre (p = 0.0002), or the presence of HBV basal core promoter (A1762T, p = 0.0379 and G1764A, p = 0.0176) or G1896A precore mutations (p = 0.0218). This finding was validated in the independent validation cohort. HN was statistically higher in patients with HBV genotypes B or C infection compared to genotypes A and D. CONCLUSION: Baseline HN ≤2 predicts which patients with HBV genotypes A or D will more likely progress to functional cure on current direct-acting antiviral therapy, with greater accuracy than current biomarkers including baseline HBsAg and HBeAg titre.
Assuntos
Hepatite B Crônica , Hepatite C Crônica , Humanos , Vírus da Hepatite B/genética , Hepatite B Crônica/diagnóstico , Hepatite B Crônica/tratamento farmacológico , Hepatite B Crônica/genética , Antígenos E da Hepatite B/genética , Antígenos de Superfície da Hepatite B/genética , Antivirais/uso terapêutico , Haplótipos , Hepatite C Crônica/tratamento farmacológico , Tenofovir/uso terapêutico , Genótipo , DNA Viral/genética , DNA Viral/análiseRESUMO
BACKGROUND: Chronic hepatitis B is a public health concern in Aboriginal communities in the Northern Territory of Australia with prevalence almost four times the non-Aboriginal population. Infection is suspected to mainly occur in early life, however, the mode of transmission and vaccine effectiveness is not known in this population. WHO has set a target for hepatitis B elimination by 2030; elimination in this disproportionately affected population in Australia will require understanding of the modes of transmission and vaccine effectiveness. METHODS: We conducted the study at four very remote Aboriginal communities. We approached mothers who had chronic hepatitis B and had given birth between 1988 and 2013 for consent. We obtained hepatitis B serology, immunisation and birth details from the medical record. If both mother and child had hepatitis B viral DNA detected, we performed viral whole genome sequencing. RESULTS: We approached 45 women for consent, of whom 23 agreed to participate. We included 20 mothers and 38 of their children. Of the 20 included mothers, 5 (25%) had children who were hepatitis B immune by exposure and 3 (15%) had children with evidence of chronic hepatitis B infection at the time of assessment. Hepatitis B immunoglobulin (HBIg) had been given at birth in 29/38 (76.3, 95% CI 59.8-88.6) children, and 26 children (68.4, 95% CI 51.3-82.5) were fully vaccinated. Of the 3 children who had chronic hepatitis B, all had received HBIg at birth and two were fully vaccinated. Of the 5 who were immune by exposure, 4 had received HBIg at birth and one was fully vaccinated. Whole genome sequencing revealed one episode of definite mother to child transmission. There was also one definite case of horizontal transmission. CONCLUSIONS: Chronic hepatitis B in this context is a sensitive issue, with a high proportion of women refusing consent. Although uncommon, there is ongoing transmission of hepatitis B to Aboriginal children in remote northern Australia despite vaccination, and this is likely occurring by both vertical and horizontal routes. Prevention will require ongoing investment to overcome the many barriers experienced by this population in accessing care.
Assuntos
Hepatite B Crônica , Hepatite B , Criança , Recém-Nascido , Pré-Escolar , Feminino , Humanos , Hepatite B Crônica/prevenção & controle , Vacinas contra Hepatite B , Transmissão Vertical de Doenças Infecciosas/prevenção & controle , Hepatite B/prevenção & controle , Northern Territory/epidemiologiaRESUMO
BACKGROUND: We evaluated the patterns of peripheral Toll-like receptor (TLR) signaling activity and the expression of TLRs and natural killer (NK) cell activation in a cohort of patients experiencing severe hepatitis flares after stopping nucleot(s)ide analogues (NAs) therapy. METHODS: Samples were collected longitudinally from patients with chronic hepatitis B who were enrolled in a prospective study of NA discontinuation. Patients experiencing hepatitis flares were compared with patients with normal alanine aminotransferase. Peripheral blood mononuclear cells (PBMCs) were stimulated with TLR ligands and cytokine secretion in the cell culture supernatant measured. Expression of TLR2/4, NKG2D, NKp46, and triggering receptor expressed on myeloid cells 1 (TREM-1) on monocytes, NK, and NK-T cells was measured. RESULTS: Seventeen patients with severe reactivation hepatitis flares were compared to 12 nonflare patients. Hepatitis flares were associated with increased activity of TLR2-8 and TLR9 signaling in PBMCs at the time of peak flare compared to baseline. Hepatitis flares were also associated with (1) upregulation of TLR2 and (2) TREM-1 receptor expression on NK. There were no differences at baseline between flare patients and nonflare patients. CONCLUSIONS: Hepatitis flares off NA therapy have a significant innate inflammatory response with upregulation of TLR signaling on peripheral monocytes and TLR2 and TREM-1 expression on NK cells. This implicates the innate immune system in the immunopathogenesis of hepatitis B flares.
Assuntos
Hepatite B Crônica , Células T Matadoras Naturais , Humanos , Vírus da Hepatite B , Receptor 2 Toll-Like , Receptor Gatilho 1 Expresso em Células Mieloides , Estudos Prospectivos , Receptores Toll-Like , Transdução de Sinais , Antivirais/uso terapêutico , Antígenos E da Hepatite BRESUMO
Chronic hepatitis B (CHB) is characterized by progression through different phases of hepatitis B virus (HBV) infection and disease. Although not necessary for HBV replication, there is increasing evidence that HBV splice variants are associated with liver disease progression and pathogenesis. However, there have been no studies till date on the frequency or diversity of splice variants for different HBV genotypes across the phases of CHB. Next generation sequencing data from 404 patient samples of HBV genotype A, B, C or D in Phase I, Phase II or Phase IV of CHB was analysed for HBV splice variants using an in house bioinformatics pipeline. HBV splice variants differed in frequency and type by genotype and phase of natural history. Splice variant Sp1 was the most frequently detected (206/404, 51% of patients), followed by Sp13 (151/404 37% of patients). The frequency of variants was generally highest in Phase II (123/165, 75% of patients), a phase typically associated with enhanced immune activation, followed by Phase I (69/99, 70% of patients). Splice variants were associated with reduced hepatitis B e antigen (HBeAg) levels and statistically reduced likelihood of achieving HBsAg loss (functional cure) in Phase II patients for Sp1 and Sp13 (p = .0014 and .0156, respectively). The frequency of HBV splice variants in patient serum differed markedly by HBV genotype and phase of CHB natural history. The increased levels of HBV splice variants detected in CHB phase II patients compared with the higher replicative Phase I in particular warrants further investigation.
Assuntos
Hepatite B Crônica , Hepatite B , DNA Viral/genética , Genótipo , Antígenos de Superfície da Hepatite B/genética , Antígenos E da Hepatite B , Vírus da Hepatite B/genética , HumanosRESUMO
BACKGROUND AND AIMS: Current guidelines recommend long-term nucleot(s)ide analogue (NA) therapy for patients with HBeAg-negative chronic hepatitis B (CHB). However, disease remission has been described after stopping NA therapy, as well as HBsAg loss. METHODS: We performed a prospective multi-centre cohort study of stopping NA therapy. Inclusion criteria were HBeAg-negative CHB, the absence of cirrhosis and HBVDNA
Assuntos
Antígenos de Superfície da Hepatite B , Hepatite B Crônica , Antivirais/uso terapêutico , Estudos de Coortes , DNA Viral , Feminino , Antígenos E da Hepatite B , Vírus da Hepatite B/genética , Hepatite B Crônica/diagnóstico , Hepatite B Crônica/tratamento farmacológico , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Resultado do TratamentoRESUMO
Areas with the highest burden of hepatitis B virus (HBV) infection are often low-middle-income countries with limited access to diagnosis due to isolation, affordability, and/or feasibility. Dried blood spots (DBSs) provide an alternative for remote areas where collection and transportation of serum is impractical. In this study, the application of DBS for serological and molecular detection of HBV and hepatitis D virus (HDV) was evaluated. Hepatitis B surface antigen was detected in 87 of 91 (95.6%) DBS. Seventeen of 21 (81%) had detectable HBeAg and 52 of 71 (73.2%) were anti-HBe positive. Anti-HD was detectable in 11 of 12 (91.6%) spiked control DBS after an initial failure to detect in patient DBS. HBV DNA was detected from 50 of 70 (71.4%) DBS with serum loads greater than 200 IU/mL in an in-house assay and 18 of 24 (75%) DBS with loads exceeding 389 IU/mL in a commercial assay. Using linear regression, HBV DNA loads from DBS were able to predict serum loads in 46 of 50 (92%) samples to within 1 log of actual serum load. HDV RNA was detected in 42 of 47 (89%) DBS with serum levels greater than 7200 IU/mL. DBSs are recommended for diagnosis of HBV, monitoring, and detection of high loads in pregnant women where peripheral blood testing remains unfeasible. Detection of HDV RNA from DBS may prove useful in endemic areas.
Assuntos
Teste em Amostras de Sangue Seco , Anticorpos Anti-Hepatite/análise , Hepatite B/diagnóstico , Hepatite D/diagnóstico , Antígenos de Superfície da Hepatite B/análise , Antígenos E da Hepatite B/análise , Humanos , Modelos LinearesRESUMO
Hepatitis delta virus (HDV) is a human pathogen, and the only known species in the genus Deltavirus. HDV is a satellite virus and depends on the hepatitis B virus (HBV) for packaging, release, and transmission. Extracellular HDV virions contain the genomic HDV RNA, a single-stranded negative-sense and covalently closed circular RNA molecule, which is associated with the HDV-encoded delta antigen forming a ribonucleoprotein complex, and enveloped by the HBV surface antigens. Replication occurs in the nucleus and is mediated by host enzymes and assisted by cis-acting ribozymes allowing the formation of monomer length molecules which are ligated by host ligases to form unbranched rod-like circles. Recently, meta-transcriptomic studies investigating various vertebrate and invertebrate samples identified RNA species with similarities to HDV RNA. The delta-like agents may be representatives of novel subviral agents or satellite viruses which share with HDV, the self-complementarity of the circular RNA genome, the ability to encode a protein, and the presence of ribozyme sequences. The widespread distribution of delta-like agents across different taxa with considerable phylogenetic distances may be instrumental in comprehending their evolutionary history by elucidating the transition from transcriptome to cellular circular RNAs to infectious subviral agents.
RESUMO
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.
Assuntos
Antivirais/farmacologia , Azocinas/farmacologia , Compostos Benzidrílicos/farmacologia , Genoma Viral , Vírus da Hepatite B/efeitos dos fármacos , Hepatite B/tratamento farmacológico , Hepatócitos/efeitos dos fármacos , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Fígado/efeitos dos fármacos , Tiazóis/farmacologia , Animais , Modelos Animais de Doenças , Células Hep G2 , Hepatite B/metabolismo , Hepatite B/patologia , Hepatite B/virologia , Vírus da Hepatite B/genética , Hepatócitos/metabolismo , Hepatócitos/patologia , Hepatócitos/virologia , Interações Hospedeiro-Patógeno , Humanos , Proteínas Inibidoras de Apoptose/metabolismo , Fígado/metabolismo , Fígado/patologia , Fígado/virologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Terapia de Alvo Molecular , Organoides , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
Recent interest in the origins and subsequent evolution of the hepatitis B virus (HBV) has strengthened with the discovery of ancient HBV sequences in fossilized remains of humans dating back to the Neolithic period around 7,000 years ago. Metagenomic analysis identified a number of African non-human primate HBV sequences in the oldest samples collected, indicating that human HBV may have at some stage, evolved in Africa following zoonotic transmissions from higher primates. Ancestral genotype A and D isolates were also discovered from the Bronze Age, not in Africa but rather Eurasia, implying a more complex evolutionary and migratory history for HBV than previously recognized. Most full-length ancient HBV sequences exhibited features of inter genotypic recombination, confirming the importance of recombination and the mutation rate of the error-prone viral replicase as drivers for successful HBV evolution. A model for the origin and evolution of HBV is proposed, which includes multiple cross-species transmissions and favors subsequent recombination events that result in a pathogen and can successfully transmit and cause persistent infection in the primate host.
RESUMO
Hepatitis delta virus (HDV) is considered a satellite virus that requires hepatitis B virus surface antigen for infectivity. HDV is endemic in some Pacific Island (PI) countries, including Kiribati and Nauru, with a unique genotype 1, "Pacific clade." The aims of this study were to determine the HDV genotypes in New Zealand and investigate the link of strains to other PI countries and the rest of the world through phylogenetics. Sequencing and phylogenetic analyses were performed on 16 HDV-positive serum samples from 14 individuals collected between 2009 and 2014 at Auckland Hospital. Thirteen of 14 strains were confirmed as genotype 1 and 1 was genotype 5. Eleven of the 13 genotype 1 strains clustered with the Pacific clade. These were isolated from subjects born in Samoa, Kiribati, Tuvalu, and Niue. Another genotype 1 strain isolated from a Maori health-care worker clustered most closely with a European strain. There was an African genotype 1 and genotype 5 from African-born subjects with HIV coinfection. This study supports the probable transmission of HDV Pacific clade around the PI from Micronesia to Polynesia. The data also confirm the need to screen hepatitis B surface antigen-positive individuals for HDV.
Assuntos
Hepatite B , Hepatite D , Genótipo , Hepatite B/epidemiologia , Hepatite D/epidemiologia , Vírus Delta da Hepatite/genética , Humanos , Nova Zelândia/epidemiologia , Ilhas do Pacífico/epidemiologia , FilogeniaRESUMO
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 13-28â% 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.