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BackgroundHDV depends on HBsAg for its infectivity. HBsAg can derive from cccDNA and also from the integration of the so-called linear HBV-DNA in the genome of infected hepatocytes. Here, we elucidate the contribution of HBsAg production from linear HBV-DNA integration in sustaining HDV activity and its pathogenetic potential.Material and Methods70 liver biopsies from eAg-negative individuals (74% NUC-treated) were included: 35 with CHB and 35 with CHD. Droplet-digital PCR was used to quantify intrahepatic levels of cccDNA, pgRNA, HDV-RNA and HBs transcripts from cccDNA and from integrated HBV-DNA (Grudda, 2022). Next-generation sequencing by Illumina was applied to assess the integration of linear HBV-DNA in hepatocytes’ genome (in 22 CHB and 32 CHD).ResultsIndividuals with CHD and CHB had comparable age and NUC-treatment duration. CHD was characterized by lower cccDNA and pgRNA than CHB (median [IQR]: 1 [0.02–12] vs 24 [8–93] copies (cps)/1000 cells and 8 [1–147] vs 518 [57–3,894] cps/1000cells, P<0.0001 for both). In CHD, no correlation was observed between cccDNA and intrahepatic HDV-RNA, supporting that HDV replicative activity is not strictly related to the extent of HBV reservoir.At least 1 event of linear HBV-DNA integration was observed in 100% and 78.1% (25/32) of individuals with CHB and CHD (total number of unique HBV-integration events: 847 in CHB and 427 in CHD). Furthermore, in both CHB and CHD, a comparable production of HBs transcripts was observed, with >99% of them from integrated HBV-DNA (median [IQR] cps/1000cells: 12,776 [4,570–55,977] in CHB and 6,041 [323–29,446] in CHD).Among the 427 HBV-integration events observed in CHD, 180 involved coding regions of the hepatocytes’ genome, corresponding to a median (IQR) number of 5 (2–10) unique events per patient. Notably, the number of HBV-integration events in coding regions showed a positive correlation with the amount of integration-derived HBsAg transcripts and with serum HBsAg (Rho=0.54 and 0.64, P<0.01 for both). Even more, HBV-integration events were significantly more frequent in individuals with CHD characterized by higher serum HBsAg levels (median [IQR] number of unique HBV-integration events: 10 [7–16] in people with vs 2 [1–7] in people without serum HBsAg >4 logIU/ml; P=0.01).In 19/25 individuals with CHD characterized by >1 HBV-integration event, HBV-DNA integrants localised in human genes regulating cell proliferation. Among the 60 genes identified, 40 genes are already known to be specifically involved in hepatocarcinogenesis.ConclusionsHDV persistence is independent from the intrahepatic HBV reservoir and is sustained by HBsAg production from integrated HBV-DNA. Higher HBsAg levels (>4logIU/ml) can reflect an enrichment of HBV-DNA integration events in coding regions of hepatocytes’ genome.Localization of HBV integrants suggests that these events may potentially induce hepatocytes proliferation, paving the way for carcinogenesis.

BackgroundLimited information is available on the extent of HDV genetic diversification in ribozyme (critical for HDV replication) and HDAg domains (crucial for viral morphogenesis and containing cytotoxic T lymphocytes epitopes [CTLE]) and their correlation with virological and biochemical parameters.Methods103 individuals with chronic HDV infection were included. Full-length HDV genome sequences were obtained by Illumina (median [IQR] reads/seq: 62045[30460–91899]). Sub-genotypes 1 were defined by phylogenetic analysis. Amino acid (aa) residues were defined conserved if not mutated in 99% of sequences. HDAg domains and CTLE (N=18) were defined according to Pascarella 2010 and Kohsar 2021.ResultsIndividuals were mostly males with a median age of 54 (44–60) years, mainly from Eastern Europe (EE,51%) and Italy (IT,44.8%). Serum HDV-RNA and ALT were 5.6 (4.9–6.2)logIU/ml and 94 (65–152)U/L. Sub-genotypes 1c and 1e were the most prevalent (47.1% and 45.2%): 1c predominated in individuals from EE(77.6% vs 22.4%,P<0.001) while 1e in IT(77.5% vs 22.5%,P<0.001).HDV ribozyme was characterised by a high degree of genetic conservation. An opposite scenario was observed for HDAg in which the number of conserved aa in HDAg dropped to 36.7% (79/214), with 18.2% in coiled coil sequence (CCS), 27.3% RNA binding domain (RDB)2, 30.8% in RBD1, 33.3% in nuclear localization sequence (NLS), 40% in virus assembly signal (VAS) and 63.3% in RBD3. The degree of genetic conservation of CTLEs ranges from 11.1% in CTLEs 46–54 and 43–51 to 60.0% in 140–149.Notably, CTLE 170–179 from individuals with HDV-RNA >5logIU/ml (70% of conserved aa with vs 20% without HDV-RNA <5 logIU/ml, P=0.025) showed lower genetic conservation, suggesting that an enrichment of mutations in this CTLE can enhance viral replication.Finally, despite a comparable degree of genetic conservation between sub-genotypes 1e and 1c, they were characterized by divergent genetic pathways. In particular, sub-genotype 1c was significantly associated with the selection of 7 specific mutations (I16T/V, N22S, D47E, R112K, T180A, A202S, prevalence ranging from 26.5% to 44.9% vs 0% in 1e, P<0.001). Conversely, sub-genotype 1e was significantly associated with the selection of 6 specific mutations (D29E, D46E, K113R, R131K, M171L, I188V prevalence ranging from 23.9% to 43.5% vs 0% in 1c, P<0.009). Notably, in sub-genotype 1c, the co-presence of I16V/T+D47E+A202S correlated with ALT>3ULN (100% vs 27.5%, P=0.001)ConclusionSub-genotypes 1 are characterized by a conspicuous degree of genetic diversification in HDAg that has contributed to the selection of divergent genetic signatures. The enrichment of mutations in specific CTL epitopes could potentially hamper HDV recognition by immune response and in turn enhancing viral replication.Overall, the role of the high degree of genetic variability in affecting the proper HDV detection by the currently available diagnostic assays deserves further investigation.

Hepatocellular carcinoma (HCC) represents the second leading cause of cancer mortality worldwide and is mostly caused by hepatitis B virus (HBV) infection. HBV can induce HCC by an indirect mechanism of continuous necro-inflammation, contributing to hepatocyte damage and promoting cancer, as well as by viral intrinsic factors. Among them, the major contributors to the development of HBV-related HCC are represented by (i) HBV DNA integration in genes modulating cell proliferation, (ii) HBV pro-oncogenic proteins, such as HBx and HBs, and (iii) the accumulation of viral mutations, enhancing the tumorigenic features of HBV proteins. The currently available antiviral treatments, based on the usage of Nucleos(t)ide analogs (NUCs), substantially control HBV replication. However, even a successful NUC treatment does not completely abrogate HCC risk, since it rarely allows achievement of an HBV functional cure, the therapeutic end-point associated with HBsAg loss and more favorable liver outcomes. To date, novel therapeutic strategies based on innovative direct antivirals (nucleic acid polymers, small interfering RNAs, antisense oligonucleotides, covalently closed circular DNA (cccDNA) inhibitors, and capsid assembly modulators) and immune-therapeutics (therapeutic vaccines, checkpoint inhibitors, and Toll-like receptor agonists) are under evaluation in clinical trials. These approaches are showing promising data in terms of an HBV functional cure, thus representing novel strategies that could be beneficial for reducing the burden of HBV-related HCC. Lastly, further efforts in drug development are necessary to identify new compounds that could achieve a sterilizing HBV cure, implying the complete elimination of cccDNA and integrated HBV DNA, the only end-point that completely eradicates HBV and its related oncogenic risk.

Respiratory syncytial virus (RSV) is a leading cause of severe respiratory illness in infants, young children, as well as elderly and immunocompromised patients worldwide. RSV is classified into two major subtypes, RSV-A and RSV-B, and remains the most frequently detected pathogen in infants hospitalized with acute respiratory infections. Recent advances have brought both passive and active immunization strategies, including FDA-approved vaccines for older adults and pregnant women and new monoclonal antibodies (mAbs) for infant protection. Although significant progress has been made, the need remains for improved antiviral treatments, particularly for vulnerable infants and immunocompromised patients. Recent studies have identified multiple RSV mutations that confer resistance to current treatments. These mutations, detected in both in vitro studies and clinical isolates, often complicate therapeutic outcomes, underscoring the need for updated and effective management strategies. In this context, evaluating protein flexibility through tools like DisoMine provides insight into how specific mutations impact structural dynamics at binding sites, thus affecting ligand affinity. This review aims to synthesize these aspects, offering a comprehensive insight into ongoing efforts to counteract RSV and address the evolving challenge of drug resistance.

Subjective probabilities are important determinants of economic choice behaviour, and their elicitation is not trivial. Different methods are available in the literature. This paper compares three of them using an economic experiment with farmers, other experts (animal nutrition and production scientists, vets, as well as animal feed, dairy, and meat company representatives), and agricultural students: the frequency method (FM), the interval method (IM), and the quadratic scoring rule method (QSR). These methods vary in the degree of complexity and saliency of the incentive scheme. Elicited subjective probability distributions refer to methane emissions reductions that can be achieved by changing animal diets in livestock farming. The study investigates whether these methods produce consistent results across methods and participant groups. Subjective probability distributions do not significantly differ across methods and participant groups. Overall, these results support that the use of less complex methods such as the FM and IM may be preferable