The distinct spatiotemporal evolutionary landscape of HBV and HDV largely determines the unique epidemic features of HDV globally.

Chronic infection of hepatitis B virus (HBV) and hepatitis D virus (HDV) causes the most severe form of viral hepatitis. Due to the dependence on HBV, HDV was deemed to co-evolve and co-migrate with HBV. However, we previously found that the naturally occurred HDV/HBV combinations do not always reflect the most efficient virological adaptation (Wang et al., 2021). Moreover, regions with heavy HBV burden do not always correlate with high HDV prevalence (e.g., East Asia), and vice versa (e.g., Central Asia). Herein, we systematically elucidated the spatiotemporal evolutionary landscape of HDV to understand the unique epidemic features of HDV. We found that the MRCA of HDV was from South America around the late 13th century, was globally dispersed mainly via Central Asia, and evolved into eight genotypes from the 19th to 20th century. In contrast, the MRCA of HBV was from Europe ∼23.7 thousand years ago (Kya), globally dispersed mainly via Africa and East Asia, and evolved into eight genotypes ∼1100 years ago. When HDV stepped in, all present-day HBV genotypes had already formed and its global genotypic distribution had stayed stable geographically. Nevertheless, regionalized HDV adapted to local HBV genotypes and human lineages, contributing to the global geographical separation of HDV genotypes. Additionally, a sharp increase in HDV infections was observed after the 20th century. In conclusion, HDV exhibited a distinct spatiotemporal distribution path compared with HBV. This unique evolutionary relationship largely fostered the unique epidemic features we observe nowadays. Moreover, HDV infections may continue to ramp up globally, thus more efforts are urgently needed to combat this disease.

Molecular determinants within the C-termini of L-HDAg that regulate hepatitis D virus replication and assembly.

Background & Aims: Hepatitis D virus (HDV) is the causative agent of chronic hepatitis delta, the most severe form of viral hepatitis. HDV encodes one protein, hepatitis delta antigen (HDAg), in two isoforms: S- and L-HDAg. They are identical in sequence except that L-HDAg contains an additional 19-20 amino acids at its C-terminus, which confer regulatory roles that are distinct from those of S-HDAg. Notably, these residues are divergent between different genotypes. We aimed to elucidate the molecular determinants within the C-termini that are essential for the regulatory role of L-HDAg in HDV replication and assembly. Methods: Northern blot, reverse-transcription quantitative PCR, and a newly established HDV trans-complementary system were used in this study. Results: C-termini of L-HDAg, albeit with high sequence variation among different genotypes, are interchangeable with respect to the trans-inhibitory function of L-HDAg and HDV assembly. The C-terminus of L-HDAg features a conserved prenylation CXXQ motif and is enriched with proline and hydrophobic residues. Abolishment of the CXXQ motif attenuated the inhibitory effect of L-HDAg on HDV replication. In contrast, the enrichment of proline and hydrophobic residues per se does not modify the trans-inhibitory function of L-HDAg. Nevertheless, these residues are essential for HDV assembly. Mechanistically, prolines and hydrophobic residues contribute to HDV assembly via a mode of action independent of the prenylated CXXQ motif. Conclusions: Within the C-terminus of L-HDAg, the CXXQ motif and the enrichment of proline and hydrophobic residues are all essential determinants of L-HDAg's regulatory roles in HDV replication and assembly. This intrinsic viral regulatory mechanism we elucidated deepens our understanding of the unique life cycle of HDV. Impact and implications: Hepatitis D virus (HDV) encodes one protein, hepatitis delta antigen (HDAg), in two isoforms: S- and L-HDAg. They are identical in sequence except that L-HDAg contains an additional 19-20 amino acids at its C-terminus. This C-terminal extension in L-HDAg confers regulatory roles in the HDV life cycle that are distinct from those of S-HDAg. Herein, we found that C-termini of L-HDAg, although with high sequence variation, are interchangeable among different HDV genotypes. Within the C-terminus of L-HDAg, the prenylation motif, and the enrichment of proline and hydrophobic residues are all essential determinants of L-HDAg's regulatory roles in HDV replication and assembly.

Drug repurposing screen identifies vidofludimus calcium and pyrazofurin as novel chemical entities for the development of hepatitis E interventions.

Hepatitis E virus (HEV) infection can cause severe complications and high mortality, particularly in pregnant women, organ transplant recipients, individuals with pre-existing liver disease and immunosuppressed patients. However, there are still unmet needs for treating chronic HEV infections. Herein, we screened a best-in-class drug repurposing library consisting of 262 drugs/compounds. Upon screening, we identified vidofludimus calcium and pyrazofurin as novel anti-HEV entities. Vidofludimus calcium is the next-generation dihydroorotate dehydrogenase (DHODH) inhibitor in the phase 3 pipeline to treat autoimmune diseases or SARS-CoV-2 infection. Pyrazofurin selectively targets uridine monophosphate synthetase (UMPS). Their anti-HEV effects were further investigated in a range of cell culture models and human liver organoids models with wild type HEV strains and ribavirin treatment failure-associated HEV strains. Encouragingly, both drugs exhibited a sizeable therapeutic window against HEV. For instance, the IC50 value of vidofludimus calcium is 4.6-7.6-fold lower than the current therapeutic doses in patients. Mechanistically, their anti-HEV mode of action depends on the blockage of pyrimidine synthesis. Notably, two drugs robustly inhibited ribavirin treatment failure-associated HEV mutants (Y1320H, G1634R). Their combination with IFN-α resulted in synergistic antiviral activity. In conclusion, we identified vidofludimus calcium and pyrazofurin as potent candidates for the treatment of HEV infections. Based on their antiviral potency, and also the favorable safety profile identified in clinical studies, our study supports the initiation of clinical studies to repurpose these drugs for treating chronic hepatitis E.

miR-26a exerts broad-spectrum antiviral effects via the enhancement of RIG-I-mediated type I interferon response by targeting USP15.

IMPORTANCE: miR-26a serves as a potent positive regulator of type I interferon (IFN) responses. By inhibiting USP15 expression, miR-26a promotes RIG-I K63-ubiquitination to enhance type I IFN responses, resulting in an active antiviral state against viruses. Being an intricate regulatory network, the activation of type I IFN responses could in turn suppress miR-26a expression to avoid the disordered activation that might result in the so-called "type I interferonopathy." The knowledge gained would be essential for the development of novel antiviral strategies against viral infection.