ABSTRACT Background & Aims The origin of hepatitis D virus (HDV) and related deltaviruses remains elusive. Contrarily, hepatitis B virus, HDV’s helper virus, and related hepadnaviruses display long-term association with primates. Current data suggest cross-order host shifts as a common mechanism in deltavirus ecology, but corroborative evidence is lacking. Here, we aimed to elucidate the genealogy of primate deltaviruses. Methods We screened 961 non-human primate (NHP) liver specimens obtained in Brazil between 2017-2023 for deltaviruses and hepadnaviruses. Complete deltaviral genomes were obtained via overlapping nested RT-PCR, cloned, expressed in vitro and analyzed via immunoblot and immunofluorescence analysis. Anti-deltaviral antibodies in NHP and vampire bats were detected via immunofluorescence analysis. Results We detected deltaviruses in two NHP. Complete deltaviral genomes exhibited common features including high self-complementarity, genomic and antigenomic ribozymes, and a delta antigen open reading frame. NHP deltaviruses were phylogenetically related to viruses found in common vampire bats. The NHP deltaviruses replicated in vitro without the expression of a large delta antigen. We did not detect anti-deltaviral antibodies in NHP sera (0/249), in contrast to sera from common vampire bats (7/112; 6.2%, 95% CI: 1.8-10.7), indicating viral circulation in bats. Ancestral state reconstruction suggested a bat origin of NHP deltaviruses. Targeted screening excluded a coinfecting hepadnavirus in the deltavirus-positive animals but led to the discovery of a hepadnavirus, corroborating a non-recent introduction of hepadnaviruses into the primate stem-lineage. Conclusions Our data are consistent with deltaviral cross-order host shifts and suggestive of the susceptibility of primates to reservoir-bound deltaviruses, lending credibility to a zoonotic origin of HDV.

Oncology patients receiving cytostatic therapy used to be at high risk of HBV infection when HBV screening measures were less reliable. Infections acquired under these conditions often persist, like those acquired perinatally or during early infancy. We studied the long-term clinical outcomes, viral characteristics, and virus-specific T-cell immunity of chronic HBV infection acquired during chemotherapy. We examined 16 chronically HBV-infected former paediatric oncology patients who were infected during cytostatic treatment in the 1980s. Patients underwent physical examination, laboratory liver function testing, non-invasive measurement of liver stiffness, and determination of HBV serology and DNA levels. If the material was sufficient, HBV sub-genotype, drug resistance and immune escape mutations, and mutations associated with HBeAg negativity were analysed. The frequency of HBV core-specific CD8+ T cells was measured after in vitro antigen-specific expansion. All but one patient were chronically infected with detectable HBsAg but were HBeAg-negative, mostly with low viraemia. Four patients were under ongoing effective antiviral therapy, and four required treatment initiation due to high viraemia or advanced liver disease. Hepatic effects were predominantly observed in highly viraemic patients. No drug resistance or immune escape mutations were observed. In two highly viraemic patients, basal core promoter and precore region mutations reducing HBeAg expression were identified. HBV core-specific CD8+ T cells were detected in all patients, but their frequency was low. In conclusion, more than 30 years after primary HBV infection was acquired during chemotherapy, the course of infection still resembles that of perinatally acquired infections.

Background Chronic liver diseases (CLD) leading to liver fibrosis and cirrhosis are a major cause of morbidity and mortality in sub-Saharan Africa and pose a significant burden on its health care systems. We aimed to elucidate the prevalence of fibrosis/cirrhosis in patients seeking health care in Kumasi, Ghana, and its underlying aetiologies.Methods In this cross-sectional study, we performed sonography, transient elastography as well as biochemical and virological analyses.Results Transient elastography indicated fibrosis/cirrhosis in 24.5% (113/461) of participants. Liver cirrhosis was significantly associated with known hepatitis B virus (HBV) infection, lack of formal education, hospitalisation, and male sex. Prevalence of active hepatitis B was significantly higher in patients with liver cirrhosis compared to controls (54.6% [30/55] vs. 17.1% [19/111]), as was anti-HBc (94.6% [52/55] vs. 80.2% [89/111]). CLD was mainly attributed to HBV (27.3%, 30/110), alcohol abuse (11.8%, 13/110), a combination of both (10.9%, 12/110), and metabolic dysfunction-associated steatotic liver disease (MASLD) (20%, 22/110). Antiviral treatment was indicated in 24 patients with active hepatitis B (number-needed-to-screen: 19.2). Hepatitis C and D viruses were of minor importance (2.7% [3/110] and 0.9% [1/110], respectively).Conclusions We found a high prevalence of CLD, predominantly caused by HBV, MASLD and alcohol. We confirmed the use of transient elastography as a non-invasive and easily applicable tool in resource-limited settings. Our findings underscore the need for systematic screening of hospitalised patients, especially men, in sub-Saharan Africa. Comprehensive screening, treatment, vaccination and prevention programs for HBV, as the leading cause of chronic liver disease, are warranted.

The myristoylated preS1 domain (myr-preS1) of the hepatitis B virus (HBV) large surface protein is essential for binding to the receptor protein, Na+/taurocholate co-transporting polypeptide (NTCP), and for the subsequent internalization of the virus-receptor complex. NTCP, which is expressed in hepatocytes, plays a physiological role in hepatic bile acid transport. Recent cryo-electron microscopy structures of the myr-preS1-NTCP complex were used to analyze virus-receptor interactions at the molecular level. Several interaction domains with high binding energies have been identified, including the interaction between myr-preS1 tryptophan 41 (W41) and the NTCP residues tyrosine 146 and phenylalanine 274 (Y146/F274), which are located at a considerable distance from the physiological bile acid binding sites of NTCP. The Y146A/F274A mutant of NTCP retained bile acid transport function but showed a reduced myr-preS1 binding. The W41G myr-preS1 mutant exhibited reduced binding to wild-type NTCP. The Y146A/F274A NTCP mutant did not support in vitro HBV infection, and the W41G myr-preS1 peptide was less effective in blocking infection compared with the wild-type myr-preS1 peptide. In conclusion, the myr-preS1-W41/NTCP-Y146/F274 interaction site, characterized by high binding energy, is essential for HBV entry into hepatocytes. Because this domain is spatially distinct from the bile acid binding and translocation sites of NTCP, it presents an attractive receptor target site for structure-based development of virus-selective HBV entry inhibitors that preserve the physiological bile acid transport function of NTCP. Significance Statement This study identified and validated a high energy interaction site between the hepatitis B virus and its receptor Na+/taurocholate co-transporting polypeptide that can be used for structure-based drug design of virus entry inhibitors.

BACKGROUND AND AIMS:Schistosomiasis is a neglected tropical disease affecting more than 250 million people worldwide. Eggs of the parasitic helminth S. mansoni cause major morbidity in the liver, spleen and intestine. Of note, egg-released soluble antigens (SEA) induce the transcription factor c-Jun in hepatocytes, promoting hepatocellular cell cycle activity, proliferation and apoptosis. In this study, we analysed the hepatic effect of pharmacological inhibition of c-Jun N-terminal kinase (JNK) after infection with S. mansoni. The JNK inhibitor SP600125 was chosen because it had no effect on schistosome viability. METHODS:Eight-week-old male mice were infected with 100 cercariae (♂ + ♀) and 6 weeks later treated with SP600125 via a subcutaneously implanted osmotic pump over 3 weeks. Hepatic damage, inflammation, fibrosis and metabolic aspects were analysed in liver and spleen tissue as well as in serum samples. RESULTS:JNK inhibitor-treated mice infected with S. mansoni showed a parasite-induced elevation of serum aminotransferases. Hepatic inflammation, the activation of hepatic stellate cells and metabolic exhaustion were observed in infected control mice. Additional SP600125 application almost doubled enhanced transaminases, hepatic cytokine expression, inflammation, necrosis, as well as HSC activation, and decreased glycogen stores to a minimum. CONCLUSIONS:Our findings suggest a protective role of JNK/c-Jun-signalling in hepatic inflammation, hepatic stellate cell activation, and metabolic exhaustion during S. mansoni infection.

Several bile acid (BA) transporters are involved in the enterohepatic BA circulation between the liver and gut, including the hepatic Na+/taurocholate cotransporting polypeptide (NTCP) and the intestinal apical sodium-dependent BA transporter (ASBT). Fluorescent BA derivatives are helpful to measure and visualize BA transport in vitro and in vivo. We used 4-nitrobenzo-2-oxa-1,3-diazole (NBD) as the labeling fluorophore and synthesized a series of 3-NBD-coupled BA. While 3α-NBD-taurocholic acid, 3β-NBD-taurocholic acid, 3α-NBD-glycocholic acid, and 3β-NBD-glycocholic acid showed significant transport rates for human NTCP, mouse mNtcp, and mouse mAsbt, human ASBT only showed reliable transport activity for 3α-NBD-glycocholic acid. In general, NBD coupling to the 3α-position proved superior to the 3β-position, and the NBD-BA with glycine conjugation exhibited the highest overall transport rates. None of the synthesized NBD-BA was transported by the organic anion transporting polypeptides OATP1B1 and OATP1B3. Overall, 3α-NBD-glycocholic acid is most appropriate for fluorescence-based transport assays to evaluate NTCP and ASBT inhibitors.

Background The African specific HBV genotype E (HBVgtE) is associated to a relative high viral replication and greater risk of hepatocellular carcinoma. However, studies concerning this genotype are still under-represented. This project aims at generating replication competent HBV DNA plasmid of genotype E to better understand biomarker production and pathogenesis by this genotype in vitro.

AimThis study aimed to determine the kinetics of occult hepatitis B virus infections (OBI) among people with HIV (PWH).MethodsThe study used archived plasma samples from longitudinal HIV natural history studies. We identified new OBI cases and assessed risk factors for OBI using Cox proportional hazards regression analysis.ResultsAt baseline, 8 of 382 [(2.1%) (95% CI: 1.06–4.1)] samples tested positive for hepatitis B surface antigen (HBsAg+). Of the 374 HBsAg-negative samples, 76 had sufficient sample volume for HBV DNA screening. OBI positivity (OBI+) at baseline was reported in 11 of 76 [14.7 95% CI (8.3–24.1)] HBsAg-negative (HBsAg−) participants. Baseline HBsAg-negative samples with sufficient follow-up samples (n = 90) were used for analysis of newly identified OBI cases. Participants contributed 129.74 person-years to the study and were followed for a median of 1.02 years (IQR: 1.00–2.00). Cumulatively, there were 34 newly identified OBI cases from the 90 participants, at the rate of 26.2/100 person-years (95% CI: 18.7–36.7). Newly identified OBI cases were more common among men than women (61.1% vs. 31.9%) and among participants with CD4+ T-cell counts ≤450 cells/mL (p-value = 0.02). Most of the newly identified OBI cases [55.9% (19/34)] were possible reactivations as they were previously HBV core antibody positive.ConclusionThere was a high rate of newly identified OBI among young PWH in Botswana, especially in men and in participants with lower CD4+ T-cell counts. OBI screening in PWH should be considered because of the risk of transmission, possible reactivation, and risk factors for the development of chronic liver disease, including hepatocellular carcinoma.

Background: Approximately 10–20% of subjects vaccinated with HBsAg-based hepatitis B virus (HBV) vaccines are non-responders. BM32 is a recombinant grass pollen allergy vaccine containing the HBV-derived preS surface antigen as an immunological carrier protein. PreS includes the binding site of HBV to its receptor on hepatocytes. We investigated whether immunological non-responsiveness to HBV after repeated HBsAg-based vaccinations could be overcome by immunization with VVX001 (i.e., alum-adsorbed BM325, a component of BM32). Methods: A subject failing to develop protective HBV-specific immunity after HBsAg-based vaccination received five monthly injections of 20 µg VVX001. PreS-specific antibody responses were measured by enzyme-linked immunosorbent assay (ELISA) and micro-array technology. Serum reactivity to subviral particles of different HBV genotypes was determined by sandwich ELISA. PreS-specific T cell responses were monitored by carboxyfluorescein diacetate succinimidyl ester (CFSE) staining and subsequent flow cytometry. HBV neutralization was assessed using cultured HBV-infected HepG2 cells. Results: Vaccination with VVX001 induced a strong and sustained preS-specific antibody response composed mainly of the IgG1 subclass. PreS-specific IgG antibodies were primarily directed to the N-terminal part of preS containing the sodium taurocholate co-transporting polypeptide (NTCP) attachment site. IgG reactivity to subviral particles as well as to the N-terminal preS-derived peptides was comparable for HBV genotypes A–H. A pronounced reactivity of CD3+CD4+ lymphocytes specific for preS after the complete injection course remaining up to one year after the last injection was found. Maximal HBV neutralization (98.4%) in vitro was achieved 1 month after the last injection, which correlated with the maximal IgG reactivity to the N-terminal part of preS. Conclusions: Our data suggest that VVX001 may be used as a preventive vaccination against HBV even in non-responders to HBsAg-based HBV vaccines.

Cellular entry of the hepatitis B and D viruses (HBV/HDV) requires binding of the viral surface polypeptide preS1 to the hepatobiliary transporter Na + -taurocholate co-transporting polypeptide (NTCP). This interaction can be blocked by bulevirtide (BLV, formerly Myrcludex B), a preS1 derivative and approved drug for treating HDV infection. Here, to elucidate the basis of this inhibitory function, we determined a cryo-EM structure of BLV-bound human NTCP. BLV forms two domains, a plug lodged in the bile salt transport tunnel of NTCP and a string that covers the receptor’s extracellular surface. The N-terminally attached myristoyl group of BLV interacts with the lipid-exposed surface of NTCP. Our structure reveals how BLV inhibits bile salt transport, rationalizes NTCP mutations that decrease the risk of HBV/HDV infection, and provides a basis for understanding the host specificity of HBV/HDV. Our results provide opportunities for structure-guided development of inhibitors that target HBV/HDV docking to NTCP.

The recent discovery of Hepatitis D (HDV)-like viruses across a wide range of taxa led to the establishment of the Kolmioviridae family. Recent studies suggest that kolmiovirids can be satellites of viruses other than Hepatitis B virus (HBV), challenging the strict HBV/HDV-association dogma. Studying whether kolmiovirids are able to replicate in any animal cell they enter is essential to assess their zoonotic potential. Here, we compared replication of three kolmiovirids: HDV, rodent (RDeV) and snake (SDeV) deltavirus in vitro and in vivo. We show that SDeV has the narrowest and RDeV the broadest host cell range. High resolution imaging of cells persistently replicating these viruses revealed nuclear viral hubs with a peculiar RNA-protein organization. Finally, in vivo hydrodynamic delivery of viral replicons showed that both HDV and RDeV, but not SDeV, efficiently replicate in mouse liver, forming massive nuclear viral hubs. Our comparative analysis lays the foundation for the discovery of specific host factors controlling Kolmioviridae host-shifting.