RNA Design Using incaRNAfbinv Demonstrated with the Identification of Functional RNA Motifs in Hepatitis Delta Virus.

Computational RNA design was introduced in the 1990s by Vienna's RNAinverse, which is a simple inverse RNA folding solver. Further developments and contemporary RNA design techniques, in addition to improved efficiency, offer more precise control over the designed sequences. incaRNAfbinv (incaRNAtion with RNA fragment-based inverse) is one such extension that builds upon RNAinverse and includes coarse-graining manipulations. The idea is that an RNA secondary structure can be decomposed to fragments of RNA motifs, and that a significant number of known natural RNA motifs exhibit a remarkable preservation in particular locations in a variety of genomes. This is taken into consideration by the ability of the user to select motifs that are known to be functional for a precise design, whilst the algorithm is more adaptable on other motifs. The latest version, incaRNAfbinv 2.0, is a free-to-use web-server which deploys the above methodology of fragment-based design. Its control over the decomposed RNA secondary structure motifs includes, among other advanced features, the insertion of constraints in a flexible manner. The resultant RNA designed sequences are ranked by their proximity to classical RNA design. Features and capabilities of incaRNAfbinv 2.0 are hereby illustrated with an example taken from hepatitis delta virus (HDV). The web-server is demonstrated in assisting to locate a known RNA motif that is responsible for HDV-3 RNA editing in more HDV genotypes than thought of before. This shows that computational RNA design by using inverse RNA folding is also a valuable strategy for locating functional RNA motifs in genomic data, in addition to artificially designing synthetic RNAs.

An hepatitis B and D virus infection model using human pluripotent stem cell-derived hepatocytes.

Current culture systems available for studying hepatitis D virus (HDV) are suboptimal. In this study, we demonstrate that hepatocyte-like cells (HLCs) derived from human pluripotent stem cells (hPSCs) are fully permissive to HDV infection across various tested genotypes. When co-infected with the helper hepatitis B virus (HBV) or transduced to express the HBV envelope protein HBsAg, HLCs effectively release infectious progeny virions. We also show that HBsAg-expressing HLCs support the extracellular spread of HDV, thus providing a valuable platform for testing available anti-HDV regimens. By challenging the cells along the differentiation with HDV infection, we have identified CD63 as a potential HDV co-entry factor that was rate-limiting for HDV infection in immature hepatocytes. Given their renewable source and the potential to derive hPSCs from individual patients, we propose HLCs as a promising model for investigating HDV biology. Our findings offer new insights into HDV infection and expand the repertoire of research tools available for the development of therapeutic interventions.

The Prevalence and Molecular Epidemiology of Hepatitis Delta Virus in Nigeria: The Results of a Nationwide Study.

Hepatitis delta virus (HDV) is a satellite of hepatitis B virus (HBV), which requires the HBV surface antigen (HBsAg) for its assembly and propagation. Although countries affected by HBV infection in Africa are well identified, data on HDV infection are still scarce, like in Nigeria, where HBV infection is endemic. In this study, we aimed to determine the prevalence of HDV infection and identify the circulating genotypes/strains in the country. A nationwide study was performed on 1281 HBsAg-positive samples collected from patients across eleven sites drawn from the six geopolitical zones in Nigeria. Anti-HDV antibody (HDV-Ab) screening and HDV-RNA viral load quantification were performed using a commercial ELISA assay and real-time RT-PCR kit, respectively. HDV genotyping was performed by the Sanger sequencing of amplicons from the so-called R0 region of the viral genome, followed by phylogenetic analyses. Of the 1281 HBsAg-positive samples, 61 (4.8%) were HDV-Ab positive, among which, 12 (19.7%) were HDV-RNA positive. Genotypes were obtained for nine of them: seven "African" HDV-1, one "Asian/European" HDV-1 and one HDV-6. This study shows that Nigeria is a country of low HDV prevalence where mainly "African" genotype-1 strains are circulating.

vHDvDB 2.0: Database and Group Comparison Server for Hepatitis Delta Virus.

The hepatitis delta virus (HDV) is a unique pathogen with significant global health implications, affecting individuals who are coinfected with the hepatitis B virus (HBV). HDV infection has profound clinical consequences, manifesting either as coinfection with HBV, resulting in acute hepatitis and potential liver failure, or as superinfection in chronic HBV cases, substantially increasing the risk of cirrhosis and hepatocellular carcinoma. Given the complex dynamics of HDV infection and the urgent need for advanced research tools, this article introduces vHDvDB 2.0, a comprehensive HDV full-length sequence database. This innovative platform integrates data preprocessing, secondary structure prediction, and epidemiological research tools. The primary goal of vHDvDB 2.0 is to consolidate HDV sequence data into a user-friendly repository, thereby facilitating access for researchers and enhancing the broader scientific understanding of HDV. The significance of this database lies in its potential to streamline HDV research by providing a centralized resource for analyzing viral sequences and exploring genotype-specific characteristics. It will also enable more in-depth research within the HDV sequence domains.

The diagnostic cascade for patients with hepatitis delta infection in France, 2018-2022: A cross-sectional study.

BACKGROUND AND AIMS: Chronic hepatitis D infection is the most severe form of viral hepatitis and can rapidly progress to cirrhosis or hepatocellular carcinoma. Despite recommendations for systematic screening of hepatitis B surface antigen (HBsAg)-positive individuals, data from real-world studies have reported a low frequency of hepatitis D (or delta) virus (HDV) screening. Our cross-sectional analysis evaluated the diagnostic cascade for hepatitis D infection in tertiary centres and described the characteristics of HDV-positive patients. METHODS: A total of 6772 individuals who tested HBsAg positive for the first time between 2018 and 2022 were retrospectively included. Demographic, clinical and laboratory data were analysed. RESULTS: A total of 5748 HBsAg-positive individuals (84.9%) were screened for HDV infection. The screening rate varied from 63% to 97% according to the screening strategy used in the centres including or not HDV reflex testing. The prevalence of HDV infection was 6.3%. HDV RNA levels were determined in 285 of the 364 (78.3%) HDV antibody screening-positive patients, and 167 (58.6%) had active HDV infection. 66.8% were males, with a mean age of 44.9 years. A total of 97.5% were born abroad, and 92.9% were HBeAg negative. At the time of diagnosis, HDV RNA levels were 6.0 Log UI/mL; 60.1% had alanine aminotransferase >40 U/L, and 56.3% had significant fibrosis (≥F2), including 41.6% with cirrhosis. The most common genotype was HDV-1 (75.4%). Coinfections were not uncommon: 7.4% were HIV positive, and 15.0% were HCV antibody positive. CONCLUSIONS: The present study highlights the need for increased screening and monitoring of HDV infection. Reflex testing helps to identify HDV-infected individuals.

Impact of Hepatitis Delta Virus Infection on the Selection of Hepatitis B Surface Antigen Mutations.

The interaction of multiple viruses in one host is thought to enhance the development of mutations. However, the impact of hepatitis D virus (HDV) positivity on the development of unique hepatitis B virus (HBV) mutations among people living with human immunodeficiency virus (HIV) (PLWH) remains poorly understood in African countries, including Botswana. We used HBV sequences generated from the Botswana Combination Prevention Project (BCPP), which is the largest pair-matched cluster-randomized HIV trial in Botswana. Only participants with available HBV sequences (n = 55) were included in our study ([HIV/HBV-positive (n = 50) and HIV/HBV/HDV-positive (n = 5)]. Geno2pheno was used to determine HBV genotypes, and HBV surface region sequences (all subgenotype A1) were aligned in AliView for mutational analysis, while the impact of mutations was assessed using Phyre2. Our results identified 182 common mutations between the two groups. In the HIV/HBV/HDV cohort, only three mutations (L95W, W156Q, C221Y) were classified as deleterious, with only L95W being the most frequent. In the HIV/HBV cohort, four mutations (W199R, C221A, C221S, W223G) were also classified as deleterious. Our results demonstrate the presence of unique HBV mutations among the HIV/HBV/HDV-positive cohort. Functional characterization of these mutations is recommended to determine their effect on HDV.

Interferon alpha induces a stronger antiviral effect than interferon lambda in HBV/HDV infected humanized mice.

Recent studies indicate that treatment of chronic hepatitis D virus (HDV) with either pegylated interferon (IFN)λ or pegylated IFNα monotherapy leads to a dramatic decline in HDV RNA. Herein, we investigated the innate antiviral efficacy of IFNλ and IFNα in humanized mice that lack an adaptive immune response. Humanized mice were either co-infected with hepatitis B virus (HBV) and HDV simultaneously, or HDV infection was performed subsequent to HBV infection (i.e., superinfected). After steady viral replication was achieved, mice received either IFNλ (n = 6) or IFNα (n = 7) for 12 (or 13) weeks. Pretreatment median levels of serum HBV DNA (8.8 [IQR:0.2] log IU/ml), HDV RNA (9.8 [0.5] log IU/ml), HBsAg (4.0 [0.4] log IU/ml) and human albumin, hAlb (6.9 [0.1] log ng/mL) were similar between mice treated with IFNα or IFNλ and between those coinfected versus superinfected. Compared to mice treated with IFNλ, mice treated with IFNα had a significantly greater decline in HBV, HDV, and HBsAg levels. In conclusion, IFNα induces stronger inhibition of HBV and HDV than IFNλ in humanized mice that lack an adaptive immune response. Further studies are needed to assess the respective role of the combined innate-and adaptive-immune systems in the treatment of HBV and HDV with IFNα and IFNλ.

[Chronic HBV and HDV infection]. / Chronische Hepatitis-B- und Hepatitis-D-Virusinfektion.

About 0,5% of the population in Germany has a chronic hepatitis B virus (HBV) infection. Untreated, chronic HBV infection can progress to liver cirrhosis and hepatocellular carcinoma (HCC). If diagnosed early, antiviral therapy can effectively prevent liver disease progression, but a cure is currently hardly achievable. About 5% of those chronically infected with HBV are also co-infected with the hepatitis D virus (HDV). HBV/HDV co-infection leads to liver cirrhosis in approximately 50% of patients within 5-10 years. Since 2020, the cell entry inhibitor bulevirtide is available as a specific therapy for HBV/HDV co-infection.

Hepatitis Delta Virus Surveillance Practice among Clinicians in Nigeria: A Cross-Sectional Survey.

BACKGROUND: The near total absence of routine Hepatitis Delta Virus (HDV) screening in many countries in sub-Saharan Africa is a major challenge to understanding the burden of HDV in the region. AIM: To evaluate Hepatitis Delta Virus screening practices and associated factors among clinicians in Nigeria. METHODS: A cross-sectional study was conducted in June-July 2022, in which a self-administered questionnaire that inquired about HDV awareness, screening practices, and treatment options was shared electronically with consenting clinicians practicing in Nigeria. At the end of the survey, data was analyzed using descriptive and inferential statistics. The level of significance was set at 0.05. RESULTS: At the end of the survey, 210 of the 213 responses retrieved from respondents were analyzed. The respondent's mean age was 38.60 ± 7.27 years with a male-to-female ratio of 1:2.5. They comprised 13.8% gastroenterologists and 86.2% respondents in other areas of clinical medicine. The study showed that 89.5% of the respondents knew that HDV infection occurs only in hepatitis B virus (HBV)-infected individuals. Most (91.4%) respondents do not screen for HDV in chronic HBV patients, mainly due to the non-availability of screening tools and lack of awareness of any screening test for HDV. Research interest was reported as the reason for screening among clinicians who had ever screened for HDV. Pegylated interferon was the main regimen used for treatment by 87.5% of respondents. About 2% did not know treatment options for HDV. A significant association between knowledge of HDV infection and area of specialty, as well as the nature of medical practice was noted (P = 0.008 and 0.013, respectively). CONCLUSION: The study showed a high level of awareness of HDV dependency on HBV, for natural infection to occur. However, it documented very minimal HDV screening in clinical settings and factors affecting screening among clinicians.

Assessing the diagnostic accuracy of serological tests for hepatitis delta virus diagnosis: a systematic review and meta-analysis.

Hepatitis Delta Virus (HDV), a satellite virus of Hepatitis B virus, exacerbates liver damage in affected individuals. Screening for HDV antibodies in HBsAg positive patients is recommended, but the diagnostic accuracy of serological tests remains uncertain. This review aimed to assess the diagnostic accuracy of serological tests for HDV. We searched PubMed, Web of Science, Cochrane Central Register of Controlled Trials, Scopus etc. for relevant studies. Studies measuring the sensitivity and specificity of serological HDV tests against PCR as a reference standard were included. Pooled sensitivity and specificity for each test method and sero-marker were calculated. The review included six studies with 11 study arms, evaluating ARCHITECT immunoassay, EIA, ELISA, QMAC, RIA, and Western Blot test methods targeting Anti-HDV IgG, Total anti-HDV and Anti-HDV IgM. Sensitivities for Anti-HDV IgG, Total Anti-HDV and Anti-HDV IgM, tests were 97.4%, 51.9%, and 62.0%, respectively, with specificities of 95.3%, 80.0%, and 85.0%. Our findings, with its limited number of studies, suggest that HDV serological tests, particularly those identifying Anti IgG exhibit high accuracy and can serve as effective screening tools for HDV.

Assessment of performance and comparison of three commercial HDV RNA detection assays: implications for diagnosis and treatment monitoring.

Objectives: Precise HDV-RNA detection and quantification are pivotal for diagnosis and monitoring of response to newly approved treatment. We evaluate the performance of three HDV RNA detection and quantification assays. Methods: Hepatitis Delta RT-PCR system kit, EurobioPlex HDV assay, and RoboGene HDV RNA Quantification kit 2.0 were used for testing 151 HBsAg-positive samples, 90 HDV-RNA negative and 61 HDV-RNA positive. We also evaluated serial dilutions of the WHO international standard for HDV, PEI 7657/12. All HDV-RNA positive samples were genotyped using a next-generation sequencing strategy. Results: Qualitative results indicated a 100% concordance between tests. Quantitative results correlated well, r2 = 0.703 (Vircell-vs-Eurobio), r2 = 0.833 (Vircell-vs-RoboGene), r2 = 0.835 (Robogene-vs-Eurobio). Bias index was 2.083 (Vircell-vs-Eurobio), -1.283 (Vircell-vs-RoboGene), and -3.36 (Robogene-vs-Eurobio). Using the WHO IS, Vircell overestimated the viral load by 0.98 log IU/mL, Eurobio by 1.46 log IU/mL, and RoboGene underestimated it by 0.98 log IU/mL. Fifty-nine samples were successfully genotyped (Genotype 1, n=52; Genotype 5, n=7; Genotype 6, n=1), with similar results for correlation and bias. Conclusion: This study underscores the necessity of using reliable HDV-RNA detection and quantification assays, as evidenced by the high concordance rates in qualitative detection and the observed variability in quantitative results. These findings highlight the importance of consistent assay use in clinical practice to ensure accurate diagnosis and effective treatment monitoring of HDV infection.

Method for Quantitative HDV RNA Detection: I, Manual Workflow (Serum and Liver Tissue) and II, Fully Automated High Throughput Workflow for Diagnostic Use.

The hepatitis delta virus (HDV) is a small RNA virus (1700 base pairs), which uses the surface proteins of the hepatitis B virus (HBV) as an envelope. Accurate and reliable quantitative detection of HDV RNA is central for scientific and translational clinical research or diagnostic purposes. However, HDV poses challenges for nucleic acid amplification techniques: (1) the circular genome displays high intramolecular base pairing; (2) high content of cytosine and guanine; and (3) enormous genomic diversity among the eight known HDV genotypes (GTs). Here, we provide step-by-step instructions for (A) a manual workflow to perform a quantitative HDV reverse transcription (RT)-PCR from serum and liver tissue and (B) a quantitative HDV RT-PCR assay with whole process control to be used for serum or plasma samples run on a fully automated system. Both assays target the conserved ribozyme region and demonstrate inclusivity for all eight HDV GTs. The choice of assay depends on the experimental needs and equipment availability. While the former is ideal for scientific research laboratories, the latter provides a useful tool in the field of translational research or diagnostics.

Prevalence of Hepatitis D in People Living with HIV: A National Cross-Sectional Pilot Study.

This study assesses the prevalence of hepatitis D virus (HDV) in people living with HIV (PLWHIV) in Greece. Given the compounding effects of HDV and hepatitis B (HBV) on liver disease progression, as well as the emergence of new therapeutic options such as bulevirtide, understanding regional disparities and the epidemiological impact of such co-infections is vital. A cross-sectional analysis was conducted utilizing 696 serum samples from PLWHIV attending five major university hospitals. The methodology included HDV antibody detection by ELISA and HDV RNA confirmation. Of the 30 HBsAg-positive samples analyzed, the study population was primarily male (93%), with a median age of 54 years. Participants had been on antiretroviral therapy for a median of 10 years, and the median CD4 count was 738 (539-1006) copies/mL. Additional serological findings revealed a 7% prevalence of hepatitis C virus (HCV) IgG antibodies and a 55% prevalence of hepatitis A virus (HAV) IgG antibodies. Seroreactivity for syphilis (RPR/VDRL/TPHA positive) was identified in 33% of the participants. The results indicated a low HDV prevalence, with only one individual (3%) testing positive for anti-HDV IgG antibodies and none for HDV RNA. This indicates a lower prevalence of HDV among PLWHIV with chronic HBV in Greece compared to global data.

Long-Term Treatment with Bulevirtide in Patients with Chronic Hepatitis D and Advanced Chronic Liver Disease.

Bulevirtide (BLV) is approved for the treatment of chronic hepatitis D (CHD). Because only limited long-term experience has been reported, we aimed to evaluate the efficacy and safety of BLV treatment in patients with advanced chronic liver disease (ACLD). We performed a retrospective analysis of patients with CHD who received BLV 2 mg/day for >12 months at a tertiary center. Virological response (VR) was defined as a reduction in hepatitis delta virus-ribonucleic acid (HDV-RNA) ≥2 log10 from baseline or HDV-RNA negativity and biochemical response (BR) as gender-specific normalization of transaminases. We identified 14 patients (9 men, 5 women; median age of 48 years; interquartile range (IQR) of 37-55), of whom 12 (86%) had suggested or assumed ACLD according to Baveno VI criteria. The median duration of BLV treatment was 26 months (IQR 17-27). During treatment, the mean HDV-RNA level decreased from log10 5.58 IU/ml to levels between log10 2.19 IU/ml and log10 3.19 IU/ml. HDV-RNA negativity was achieved in up to 63% after 24 months. VR and BR were 86% and 43% after 12 months, 90% and 60% after 18 months, 75% and 75% after 24 months, and 100% and 50% after 30 months, respectively. Two nonpersisting viral breakthroughs were observed after 24 months of treatment. The Child Pugh score and model of end-stage liver disease (MELD) scores remained stable or improved in 12 patients (86%). Only one patient developed hepatic decompensation after 24 months of treatment with ascites requiring large-volume paracentesis which was not associated with viral breakthrough, portal vein thrombosis, or hepatocellular carcinoma. Treatment with BLV beyond one year is effective and safe for patients with CHD and ACLD. Liver function remained stable or improved during treatment in the vast majority of patients, and only one case of hepatic decompensation occurred during a median follow-up of 26 months.

Seroprevalence of hepatitis D virus in Bulgarian outpatients with history of liver dysfunction.

The aim of this research was to define the prevalence of antibodies against hepatitis D virus (anti-HDV Ab) in a group of 26 outpatients with liver dysfunction in northeastern Bulgaria. Serum samples were obtained from April 2022 to December 2023 in the "Status" Medical Diagnostic Laboratory, Varna, Bulgaria. We found seroprevalence of anti-HDV Ab in 15.4% (CI: 4.3-34.8%) of the target population. Age and gender had no significant role in HDV seropositivity.

Prevalence and risk factors of transmission of hepatitis delta virus in pregnant women in the Center Region of Cameroon.

BACKGROUND: Hepatitis B virus (HBV) and hepatitis delta virus (HDV) co-infection has been described as the most severe form of viral hepatitis, and can be co-transmitted from mother-to-child. A seroprevalence of 4.0% of HDV infection was reported in pregnant women in Yaoundé, and 11.9% in the general population in Cameroon. Our objective was to describe the rate of HDV infection in HBsAg-positive pregnant women and to determine risk factors associated with mother-to-child transmission of HDV. MATERIALS AND METHODS: A cross-sectional, descriptive study was conducted from January 2019 to July 2022 among pregnant women attending antenatal contacts in seven health structures in the Centre Region of Cameroon. A consecutive sampling (non-probability sampling) was used to select only pregnant women of age over 21 years, who gave a written informed consent. Following an informed consent, an open-ended questionnaire was used for a Knowledge, Attitude and Practice (KAP) survey of these women, and their blood specimens collected and screened for HBsAg, anti-HIV and anti-HCV antibodies by rapid tests and ELISA. HBsAg-positive samples were further screened for HBeAg, anti-HDV, anti-HBs, and anti HBc antibodies by ELISA, and plasma HDV RNA load measured by RT-qPCR. RESULTS: Of 1992 pregnant women, a rate of 6.7% of HBsAg (133/1992) with highest rate in the rural areas, and 3.9% of hepatitis vaccination rate were recorded. Of 130, 42 (32.3%) were anti-HDV antibody-positive, and 47.6% had detectable HDV RNA viraemia. Of 44 anti-HDV-positive cases, 2 (4.5%) were co-infected with HBV and HCV, while 5 (11.4%) with HIV and HBV. Multiple pregnancies, the presence of tattoos and/or scarifications were significantly associated with the presence of anti-HDV antibodies. Of note, 80% of women with negative HBeAg and positive anti-HBe serological profile, had plasma HDV RNA load of more than log 3.25 (>10.000 copies/ml). CONCLUSION: These results show an intermediate rate of HDV infection among pregnant women with high level of HDV RNA viremia, which suggest an increased risk of vertical and horizontal co-transmission of HDV.