Structure and sequence at an RNA template 5' end influence insertion of transgenes by an R2 retrotransposon protein.

R2 non-long terminal repeat retrotransposons insert site-specifically into ribosomal RNA genes (rDNA) in a broad range of multicellular eukaryotes. R2-encoded proteins can be leveraged to mediate transgene insertion at 28S rDNA loci in cultured human cells. This strategy, Precise RNA-mediated INsertion of Transgenes (PRINT), relies on co-delivery of an mRNA encoding R2 protein and an RNA template encoding a transgene cassette of choice. Here we demonstrate that the PRINT RNA template 5' module, which as a complementary DNA 3' end will generate the transgene 5' junction with rDNA, influences the efficiency and mechanism of gene insertion. Iterative design and testing identified optimal 5' modules consisting of a hepatitis delta virus-like ribozyme fold with high thermodynamic stability, suggesting that RNA template degradation from its 5' end may limit transgene insertion efficiency. We also demonstrate that transgene 5' junction formation can be either precise, formed by annealing the 3' end of first-strand complementary DNA with the upstream target site, or imprecise, by end-joining, but this difference in junction formation mechanism is not a major determinant of insertion efficiency. Sequence characterization of imprecise end-joining events indicates surprisingly minimal reliance on microhomology. Our findings expand current understanding of the role of R2 retrotransposon transcript sequence and structure, and especially the 5' ribozyme fold, for retrotransposon mobility and RNA-templated gene synthesis in cells.

Growing Awareness: Limited Testing and Screening Bias for Hepatitis Delta Virus in Utah 2000-2021.

BACKGROUND: This study assessed the epidemiology of hepatitis delta virus (HDV) within the University of Utah UHealth health care system (2000-2021). METHODS: Analysis of HDV/HBV testing, diagnostic codes, liver enzymes, and comorbidities was performed. RESULTS: Among the 1962 HBV patients, only 22.2% underwent HDV testing, revealing an 8.3% positivity rate for HDV coinfections. This study observed a consistent increase in HBV and HDV cases, with higher HDV detection rates linked to increased testing. Limited HDV testing and potential screening biases were evident. DISCUSSION: Improved HDV testing and surveillance are crucial for early detection and implementation of targeted therapies.

Enrichment reveals extensive integration of hepatitis B virus DNA in hepatitis delta-infected patients.

INTRODUCTION: Hepatitis B virus (HBV) DNA may become integrated into the human genome of infected human hepatocytes. Expression of integrations can produce the surface antigen (HBsAg) that is required for synthesis of hepatitis D virus (HDV) particles and the abundant subviral particles in the blood of HBV- and HDV-infected subjects. Knowledge about the extent and variation of HBV integrations and impact on chronic HDV is still limited. METHODS: We investigated 50 pieces of liver explant tissue from five patients with hepatitis D-induced cirrhosis, using a deep sequencing strategy targeting HBV RNA. RESULTS: We found that integrations were abundant and highly expressed, however with large variation in number of integration derived (HBV/human chimeric) reads, both between and within patients. The median number of unique integrations for each patient correlated with serum levels of both HBsAg. Still, most of the HBV reads represented a few predominant integrations. CONCLUSIONS: The results suggest that HBV DNA integrates in a large proportion of hepatocytes, and that the HBsAg output from these integrations vary >100-fold depending on clone size and expression rate. A small part of the integrations seems to determine the serum levels of HBsAg and HDV RNA in HBV/HDV co-infected patients with liver cirrhosis.

Adjusted estimate of the prevalence of hepatitis delta virus in 25 countries and territories.

BACKGROUND & AIMS: Hepatitis delta virus (HDV) is a satellite RNA virus that requires the hepatitis B virus (HBV) for assembly and propagation. Individuals infected with HDV progress to advanced liver disease faster than HBV-monoinfected individuals. Recent studies have estimated the global prevalence of anti-HDV antibodies among the HBV-infected population to be 5-15%. This study aimed to better understand HDV prevalence at the population level in 25 countries/territories. METHODS: We conducted a literature review to determine the prevalence of anti-HDV and HDV RNA in hepatitis B surface antigen (HBsAg)-positive individuals in 25 countries/territories. Virtual meetings were held with experts from each setting to discuss the findings and collect unpublished data. Data were weighted for patient segments and regional heterogeneity to estimate the prevalence in the HBV-infected population. The findings were then combined with The Polaris Observatory HBV data to estimate the anti-HDV and HDV RNA prevalence in each country/territory at the population level. RESULTS: After adjusting for geographical distribution, disease stage and special populations, the anti-HDV prevalence among the HBsAg+ population changed from the literature estimate in 19 countries. The highest anti-HDV prevalence was 60.1% in Mongolia. Once adjusted for the size of the HBsAg+ population and HDV RNA positivity rate, China had the highest absolute number of HDV RNA+ cases. CONCLUSIONS: We found substantially lower HDV prevalence than previously reported, as prior meta-analyses primarily focused on studies conducted in groups/regions that have a higher probability of HBV infection: tertiary care centers, specific risk groups or geographical regions. There is large uncertainty in HDV prevalence estimates. The implementation of reflex testing would improve estimates, while also allowing earlier linkage to care for HDV RNA+ individuals. The logistical and economic burden of reflex testing on the health system would be limited, as only HBsAg+ cases would be screened. IMPACT AND IMPLICATIONS: There is a great deal of uncertainty surrounding the prevalence of hepatitis delta virus among people living with hepatitis B virus at the population level. In this study, we aimed to better understand the burden in 25 countries and territories, to refine techniques that can be used in future analyses. We found a lower prevalence in the majority of places studied than had been previously reported. These data can help inform policy makers on the need to screen people living with hepatitis B virus to find those coinfected with hepatitis delta virus and at high risk of progression, while also highlighting the pitfalls that other researchers have often fallen into.

Diversification of mammalian deltaviruses by host shifting.

Hepatitis delta virus (HDV) is an unusual RNA agent that replicates using host machinery but exploits hepatitis B virus (HBV) to mobilize its spread within and between hosts. In doing so, HDV enhances the virulence of HBV. How this seemingly improbable hyperparasitic lifestyle emerged is unknown, but it underpins the likelihood that HDV and related deltaviruses may alter other host-virus interactions. Here, we show that deltaviruses diversify by transmitting between mammalian species. Among 96,695 RNA sequence datasets, deltaviruses infected bats, rodents, and an artiodactyl from the Americas but were absent from geographically overrepresented Old World representatives of each mammalian order, suggesting a relatively recent diversification within the Americas. Consistent with diversification by host shifting, both bat and rodent-infecting deltaviruses were paraphyletic, and coevolutionary modeling rejected cospeciation with mammalian hosts. In addition, a 2-y field study showed common vampire bats in Peru were infected by two divergent deltaviruses, indicating multiple introductions to a single host species. One vampire bat-associated deltavirus was detected in the saliva of up to 35% of individuals, formed phylogeographically compartmentalized clades, and infected a sympatric bat, illustrating horizontal transmission within and between species on ecological timescales. Consistent absence of HBV-like viruses in two deltavirus-infected bat species indicated acquisitions of novel viral associations during the divergence of bat and human-infecting deltaviruses. Our analyses support an American zoonotic origin of HDV and reveal prospects for future cross-species emergence of deltaviruses. Given their peculiar life history, deltavirus host shifts will have different constraints and disease outcomes compared to ordinary animal pathogens.

Hepatitis D virus interferes with hepatitis B virus RNA production via interferon-dependent and -independent mechanisms.

BACKGROUND & AIMS: Chronic coinfection with HBV and HDV leads to the most aggressive form of chronic viral hepatitis. Herein, we aimed to elucidate the molecular mechanisms underlying the widely reported observation that HDV interferes with HBV in most coinfected patients. METHODS: Patient liver tissues, primary human hepatocytes, HepaRG cells and human liver chimeric mice were used to analyze the effect of HDV on HBV using virological and RNA-sequencing analyses, as well as RNA synthesis, stability and association assays. RESULTS: Transcriptomic analyses in cell culture and mouse models of coinfection enabled us to define an HDV-induced signature, mainly composed of interferon (IFN)-stimulated genes (ISGs). We also provide evidence that ISGs are upregulated in chronically HDV/HBV-coinfected patients but not in cells that only express HDV antigen (HDAg). Inhibition of the hepatocyte IFN response partially rescued the levels of HBV parameters. We observed less HBV RNA synthesis upon HDV infection or HDV protein expression. Additionally, HDV infection or expression of HDAg alone specifically accelerated the decay of HBV RNA, and HDAg was associated with HBV RNAs. On the contrary, HDAg expression did not affect other viruses such as HCV or SARS-CoV-2. CONCLUSIONS: Our data indicate that HDV interferes with HBV through both IFN-dependent and IFN-independent mechanisms. Specifically, we uncover a new viral interference mechanism in which proteins of a satellite virus affect the RNA production of its helper virus. Exploiting these findings could pave the way to the development of new therapeutic strategies against HBV. IMPACT AND IMPLICATIONS: Although the molecular mechanisms remained unexplored, it has long been known that despite its dependency, HDV decreases HBV viremia in patients. Herein, using in vitro and in vivo models, we showed that HDV interferes with HBV through both IFN-dependent and IFN-independent mechanisms affecting HBV RNA metabolism, and we defined the HDV-induced modulation signature. The mechanisms we uncovered could pave the way for the development of new therapeutic strategies against HBV by mimicking and/or increasing the effect of HDAg on HBV RNA. Additionally, the HDV-induced modulation signature could potentially be correlated with responsiveness to IFN-α treatment, thereby helping to guide management of HBV/HDV-coinfected patients.

Potent broadly neutralizing antibody VIR-3434 controls hepatitis B and D virus infection and reduces HBsAg in humanized mice.

BACKGROUND & AIMS: Chronic hepatitis B is a global public health problem, and coinfection with hepatitis delta virus (HDV) worsens disease outcome. Here, we describe a hepatitis B virus (HBV) surface antigen (HBsAg)-targeting monoclonal antibody (mAb) with the potential to treat chronic hepatitis B and chronic hepatitis D. METHODS: HBsAg-specific mAbs were isolated from memory B cells of HBV vaccinated individuals. In vitro neutralization was determined against HBV and HDV enveloped with HBsAg representing eight HBV genotypes. Human liver-chimeric mice were treated twice weekly with a candidate mAb starting 3 weeks post HBV inoculation (spreading phase) or during stable HBV or HBV/HDV coinfection (chronic phase). RESULTS: From a panel of human anti-HBs mAbs, VIR-3434 was selected and engineered for pre-clinical development. VIR-3434 targets a conserved, conformational epitope within the antigenic loop of HBsAg and neutralized HBV and HDV infection with higher potency than hepatitis B immunoglobulins in vitro. Neutralization was pan-genotypic against strains representative of HBV genotypes A-H. In the spreading phase of HBV infection in human liver-chimeric mice, a parental mAb of VIR-3434 (HBC34) prevented HBV dissemination and the increase in intrahepatic HBV RNA and covalently closed circular DNA. In the chronic phase of HBV infection or co-infection with HDV, HBC34 treatment decreased circulating HBsAg by >1 log and HDV RNA by >2 logs. CONCLUSIONS: The potently neutralizing anti-HBs mAb VIR-3434 reduces circulating HBsAg and HBV/HDV viremia in human liver-chimeric mice. VIR-3434 is currently in clinical development for treatment of patients with chronic hepatitis B or D. IMPACT AND IMPLICATIONS: Chronic infection with hepatitis B virus and co-infection with hepatitis D virus place approximately 290 million individuals worldwide at risk of severe liver disease and cancer. Available treatments result in low rates of functional cure or require lifelong therapy that does not eliminate the risk of liver disease. We isolated and characterized a potent human antibody that neutralizes hepatitis B and D viruses and reduces infection in a mouse model. This antibody could provide a new treatment for patients with chronic hepatitis B and D.

Hepatitis Delta Virus Genome RNA Synthesis Initiates at Position 1646 with a Nontemplated Guanosine.

Hepatitis delta virus (HDV) is a significant human pathogen that causes acute and chronic liver disease; there is no licensed therapy. HDV is a circular negative-sense single-stranded RNA (ssRNA) virus that produces three RNAs in infected cells, genome, antigenome, and mRNA; the latter encodes hepatitis delta antigen (HDAg), the viral protein. These RNAs are synthesized by host DNA-dependent RNA polymerase acting as an RNA-dependent RNA polymerase. Although HDV genome RNA accumulates to high levels in infected cells, the mechanism by which this process occurs remains poorly understood. For example, the nature of the 5' end of the genome, including the synthesis start site and its chemical composition, is not known. Analysis of this process has been challenging because the initiation site is part of an unstable precursor in the rolling-circle mechanism by which HDV genome RNA is synthesized. In this study, circular HDV antigenome RNAs synthesized in vitro were used to directly initiate HDV genome RNA synthesis in transfected cells, thus enabling the detection of the 5' end of the genome RNA. The 5' end of this RNA is capped, as expected for a polymerase II product. Initiation begins at position 1646 on the genome, which is located near the loop end proximal to the start site for HDAg mRNA synthesis. Unexpectedly, synthesis begins with a guanosine that is not conventionally templated by the HDV RNA. IMPORTANCE Hepatitis delta virus (HDV) is a unique virus that causes severe liver disease. It uses host RNA polymerase II to copy its circular RNA genome in a unique and poorly understood process. Although the virus RNA accumulates to high levels within infected cells, it is not known how the synthesis of the viral RNA begins or even where on the genome synthesis starts. Here, we identify the start site for the initiation of HDV genome RNA synthesis as position 1646, which is at one end of the closed-hairpin-like structure of the viral RNA. The 5' end of the RNA is capped, as expected for polymerase II products. However, RNA synthesis begins with a guanosine that is not present in the genome. Thus, although HDV uses polymerase II to synthesize the viral genome, some details of the initiation process are different. These differences could be important for successfully targeting virus replication.

N6-Methyladenine Modification of Hepatitis Delta Virus Regulates Its Virion Assembly by Recruiting YTHDF1.

Hepatitis delta virus (HDV) is a defective satellite virus that uses hepatitis B virus (HBV) envelope proteins to form its virions and infect hepatocytes via the HBV receptors. Concomitant HDV/HBV infection continues to be a major health problem, with at least 25 million people chronically infected worldwide. N6-methyladenine (m6A) modification of cellular and viral RNAs is the most prevalent internal modification that occurs cotranscriptionally, and this modification regulates various biological processes. We have previously described a wider range of functional roles of m6A methylation of HBV RNAs, including its imminent regulatory role in the encapsidation of pregenomic RNA. In this study, we present evidence that m6A methylation also plays an important role in the HDV life cycle. Using the methylated RNA immunoprecipitation (MeRIP) assay, we identified that the intracellular HDV genome and antigenome are m6A methylated in HDV- and HBV-coinfected primary human hepatocytes and HepG2 cell expressing sodium taurocholate cotransporting polypeptide (NTCP), while the extracellular HDV genome is not m6A methylated. We observed that HDV genome and delta antigen levels are significantly decreased in the absence of METTL3/14, while the extracellular HDV genome levels are increased by depletion of METTL3/14. Importantly, YTHDF1, an m6A reader protein, interacts with the m6A-methylated HDV genome and inhibits the interaction between the HDV genome and antigens. Thus, m6A of the HDV genome negatively regulates virion production by inhibiting the interaction of the HDV genome with delta antigens through the recruitment of YTHDF1. This is the first study that provides insight into the functional roles of m6A in the HDV life cycle. IMPORTANCE The functional roles of N6-methyladenine (m6A) modifications in the HBV life cycle have been recently highlighted. Here, we investigated the functional role of m6A modification in the HDV life cycle. HDV is a subviral agent of HBV, as it uses HBV envelope proteins to form its virions. We found that m6A methylation also occurs in the intracellular HDV genome and antigenome but not in the extracellular HDV genome. The m6A modification of the HDV genome recruits m6A reader protein (YTHDF1) onto the viral genome. The association of YTHDF1 with the HDV genome abrogates the interaction of delta antigens with the HDV genome and inhibits virion assembly. This study describes the unique effects of m6A on regulation of the HDV life cycle.

Prevalence and determinants of hepatitis delta virus infection among HIV/hepatitis B-coinfected adults in care in the United States.

Hepatitis delta virus (HDV) infection increases the risk of liver complications compared to hepatitis B virus (HBV) alone, particularly among persons with human immunodeficiency virus (HIV). However, no studies have evaluated the prevalence or determinants of HDV infection among people with HIV/HBV in the US. We performed a cross-sectional study among adults with HIV/HBV coinfection receiving care at eight sites within the Center for AIDS Research Network of Integrated Clinical Systems (CNICS) between 1996 and 2019. Among patients with available serum/plasma specimens, we selected the first specimen on or after their initial HBV qualifying test. All samples were tested for HDV IgG antibody and HDV RNA. Multivariable log-binomial generalized linear models were used to estimate prevalence ratios (PRs) with 95% CIs of HDV IgG antibody-positivity associated with determinants of interest (age, injection drug use [IDU], high-risk sexual behaviour). Among 597 adults with HIV/HBV coinfection in CNICS and available serum/plasma samples (median age, 43 years; 89.9% male; 52.8% Black; 42.4% White), 24/597 (4.0%; 95% CI, 2.4%-5.6%) were HDV IgG antibody-positive, and 10/596 (1.7%; 95% CI, 0.6%-2.7%) had detectable HDV RNA. In multivariable analysis, IDU was associated with exposure to HDV infection (adjusted PR = 2.50; 95% CI, 1.09-5.74). In conclusion, among a sample of adults with HIV/HBV coinfection in care in the US, 4.0% were HDV IgG antibody-positive, among whom 41.7% had detectable HDV RNA. History of IDU was associated with exposure to HDV infection. These findings emphasize the importance of HDV testing among persons with HIV/HBV coinfection, especially those with a history of IDU.

Silent HDV epidemics culminates in high levels of liver cirrhosis in endemic region despite 20 years of HBV vaccination.

The hepatitis delta virus (HDV) is believed to be a vanishing infection in countries with successful hepatitis B virus (HBV) vaccination programs. We assessed the current status of HDV infection in Tuva, a region of the Russia that has been highly endemic for HBV. The proportion of HDV-infected patients among HBsAg-positive patients in the regional registry in 2020 was 32.7% (786/2401). An analysis of the medical records of 514 HDV patients demonstrated that 37.5% (193/514) had liver cirrhosis at the first doctor's visit, and 7.4% of patients lived in families where another family member had HDV. All HDV patients were infected with genotype HDV-1, 94.5% had HBV genotype D, and 5.5% had genotype A. A serosurvey conducted among 1170 healthy volunteers showed that the average detection rate of HBsAg with anti-HDV was 1.0% (95% CI: 0.57-1.81%). No anti-HDV positive samples were detected in participants aged under 30 years. The HBsAg/anti-HDV positivity rate peaked at 7.4% in patients aged 50-59 years, which was significantly higher than in a similar age cohort surveyed in 2008 (1.6%, p < .0001). A Bayesian analysis showed that HDV circulation in Tuva resulted from two waves of introduction, the first in the 1810s (95% HPD: 1741-1834) from Central Asia, and the second in the 1960s (95% HPD: 1953-1979) from Russia. HBV has a much longer history of circulation in Tuva with the MRCA for the predominant genotype HBV-D dated to 972 (95% HPD: 535-1253) for subtype D1, 1274 (95% HPD: 936-1384) for D2, and 1173 (95% HPD: 1005-1618) for D3. A SkyGrid reconstruction of population dynamics showed an increase in the intensity of HDV spread in recent decades. This situation shows the need for HDV screening and prevention measures among people living with HBV.

Does hepatitis delta virus have a preference for hepatitis B virus genotype? A systematic review of the literature.

Hepatitis delta virus (HDV) is a deficient virus that requires the surface proteins of Hepatitis B virus (HBV) to complete its replication. HDV is thus only found in those already infected with HBV (~5% worldwide). There are eight different HDV genotypes (1-8) and 10 HBV genotypes (A-J), each having fairly distinct geographic distributions. While their pairings may be coincidental based on epidemiological occurrence, some evidence exists regarding possible virologic basis for genotype dominance and patterns. Here we sought to determine which HBV genotype is most often linked with active HDV infection and speculate on whether this may represent a viral 'preference'. Electronic databases with OVID Medline were comprehensively searched for studies published between 1977 and 2022 indexing the word 'genotype' and all permutations of 'HDV' (hepatitis D virus, hepatitis delta, etc.). Primary studies of patient samples reporting genotype data for either or both of HDV and HBV were tabulated. The initial search revealed 419 articles and was narrowed to 133 studies reporting genotype data for either or both HBV and HDV. We limited our search to cases with detectable HDV RNA. These represented over 5800 samples from over 70 countries. Of these, 1947 samples had paired genotype data for both viruses. The most common pairing was HDV-1 with HBV-D, but it remains unclear whether this represents a viral 'preference' or mere co-endemicity of the two viruses. Determining if there is a virologic link between HBV and HDV genotypes may have important implications for emerging HDV and HBV research.

Improved hepatitis delta virus genome characterization by single molecule full-length genome sequencing combined with VIRiONT pipeline.

Hepatitis B virus (HBV) and hepatitis D virus (HDV) coinfection confers a greater risk for accelerated liver disease progression. Full-length characterization of HDV genome is necessary to understand pathogenesis and treatment response. However, owing to its high variability and tight structure, sequencing approaches remain challenging. Herein, we present a workflow to amplify, sequence, and analyze the whole HDV genome in a single fragment. Sequencing was based on the Oxford Nanopore Technologies long-read sequencing followed by a turnkey analysis pipeline (VIRiONT, VIRal in-house ONT sequencing analysis pipeline) that we developed and make available online for free. For the first time, HDV genome was successfully amplified and full-length sequenced in a single fragment, allowing accurate subtyping from 30 clinical samples. High variability of edition, a crucial step in viral life cycle, was found among samples (from 0% to 59%). Additionally, a new subtype of HDV genotype 1 was identified. We provide a complete workflow for assessment of HDV genome at full-length quasispecies resolution overcoming genome assembly issues and helping to identify modifications throughout the whole genome. This will help a better understanding of the impact of genotype/subtype, viral dynamics, and structural variants on HDV pathogenesis and treatment response.

Diagnosis and Management of Hepatitis Delta Virus Infection.

Hepatitis D virus (HDV) depends on hepatitis B virus (HBV) to enter and exit hepatocytes and to replicate. Despite this dependency, HDV can cause severe liver disease. HDV accelerates liver fibrosis, increases the risk of hepatocellular carcinoma, and hastens hepatic decompensation compared to chronic HBV monoinfection. The Chronic Liver Disease Foundation (CLDF) formed an expert panel to publish updated guidelines on the testing, diagnosis, and management of hepatitis delta virus. The panel group performed network data review on the transmission, epidemiology, natural history, and disease sequelae of acute and chronic HDV infection. Based on current available evidence, we provide recommendations for screening, testing, diagnosis, and treatment of hepatitis D infection and review upcoming novel agents that may expand treatment options. The CLDF recommends universal HDV screening for all patients who are Hepatitis B surface antigen-positive. Initial screening should be with an assay to detect antibodies generated against HDV (anti-HDV). Patients who are positive for anti-HDV IgG antibodies should then undergo quantitative HDV RNA testing. We also provide an algorithm that describes CLDF recommendations on the screening, diagnosis, testing, and initial management of Hepatitis D infection.

Hepatitis Delta virus in migrants: The challenge of elimination (ANRS CO22 HEPATHER cohort).

Novel treatments for hepatitis Delta virus (HDV) infection provide promising opportunities to treat patients with hepatitis B virus (HBV) and HDV co-infection. However, current clinical trials on HDV treatment rarely explore patients' barriers to treatments. In Europe, HDV infection mostly affects young migrants from HDV-endemic areas who experience early liver-related mortality. Migrants are more likely to face multiple situations of statutory and socioeconomic insecurity and structural barriers than non-migrants. These obstacles may impact their quality of life and can (i) lead them to give secondary importance to certain HDV care options, (ii) delay treatment initiation and (iii) affect their adherence and commitment to care. Preliminary results from the ANRS CO22 HEPATHER cohort show that the majority (61.6%) of HBV-HDV co-infected migrants live in poverty. Moreover, half were diagnosed and a quarter of those who initiated HBV treatment had been in France for no more than two years, a period when language skills are often still poor and when knowledge of the health and administrative system may be lacking. We advocate for increased social science research, in particular qualitative studies, to investigate the effects that multiple forms of precarity (weak access to social rights, language barriers, housing insecurity, unexpected expenditures and other difficulties) may have on HDV screening opportunities, follow-up, and treatment pathways in migrants. This will help adapt communication and care around viral hepatitis, as well as inform and orient medical services and public health actors about the difficulties that migrants encounter.

Different evolutionary dynamics of hepatitis B virus genotypes A and D, and hepatitis D virus genotypes 1 and 2 in an endemic area of Yakutia, Russia.

BACKGROUND: The geographic distribution of the hepatitis B virus (HBV) and the hepatitis D virus (HDV) genotypes is uneven. We reconstructed the temporal evolution of HBV and HDV in Yakutia, one of the regions of Russia most affected by HBV and HDV, in an attempt to understand the possible mechanisms that led to unusual for Russia pattern of viral genotypes and to identify current distribution trends. METHODS: HBV and HDV genotypes were determined in sera collected in 2018-2019 in Yakutia from randomly selected 140 patients with HBV monoinfection and 59 patients with HBV/HDV. Total 86 HBV and 88 HDV genomic sequences isolated in Yakutia between 1997 and 2019 were subjected to phylodynamic and philogeographic Bayesian analysis using BEAST v1.10.4 software package. Bayesian SkyGrid reconstruction and Birth-Death Skyline analysis were applied to estimate HBV and HDV population dynamics. RESULTS: Currently, HBV-A and HDV-D genotypes are prevalent in Yakutia, in both monoinfected and HDV-coinfected patients. Bayesian analysis has shown that the high prevalence of HBV-A in Yakutia, which is not typical for Russia, initially emerged after the genotype was introduced from Eastern Europe in the fifteenth century (around 600 (95% HPD: 50-715) years ago). The acute hepatitis B epidemics in the 1990s in Yakutia were largely associated with this particular genotype, as indicated by temporal changes in HBV-A population dynamics. HBV-D had a longer history in Yakutia and demonstrated stable population dynamics, indicating ongoing viral circulation despite vaccination. No correlation between HBV and HDV genotypes was observed for coinfected patients in Yakutia (r = - 0.016069332). HDV-2b circulates in Russia in Yakutia only and resulted from a single wave of introduction from Central Asia 135 years ago (95% HPD: 60-350 years), while HDV-1 strains resulted from multiple introductions from Europe, the Middle East, Central Asia, and different parts of Russia starting 180 years ago (95% HPD: 150-210 years) and continuing to the present day. The population dynamics of HDV-1 and HDV-2 show no signs of decline despite 20 years of HBV vaccination. The Birth-Death Skyline analysis showed an increase in the viral population in recent years for both HDV genotypes, indicating ongoing HDV epidemics. CONCLUSIONS: Taken together, these data call for strict control of HBV vaccination quality and coverage, and implementation of HBV and HDV screening programs in Yakutia.

Signature of chronic hepatitis B virus infection in nails and hair.

BACKGROUND: Hepatitis B virus (HBV) is detected in extrahepatic tissues of individuals with HBV infection. Whether nails and hair contain HBV has been unknown. METHODS: We examined two patient groups: those with chronic HBV infection alone (n = 71), and those with both chronic HBV and hepatitis delta virus (HDV) infections (n = 15). HBV DNA in the patients' fingernails and hair were measured by real-time PCR. Hepatitis B surface antigen (HBsAg) of fingernails was evaluated by an enzyme immunoassay. HDV RNA in fingernails was measured by real-time PCR. Immunochemical staining was performed on nails. We used chimeric mice with humanized livers to evaluate the infectivity of nails. RESULTS: Of the 71 pairs of HBV-alone nail and hair samples, 70 (99%) nail and 60 (85%) hair samples were positive for ß-actin DNA. Of those 70 nail samples, 65 (93%) were HBV DNA-positive. Of the 60 hair samples, 49 (82%) were HBV DNA-positive. The serum HBV DNA level of the nail HBV DNA-positive patients was significantly higher than that of the nail HBV DNA-negative patients (p < 0.001). The hair HBV DNA-positive patients' serum HBV DNA level was significantly higher compared to the hair HBV DNA-negative patients (p < 0.001). The nail HBV DNA level was significantly higher than the hair HBV DNA level (p < 0.001). The nails and hair HBV DNA levels were correlated (r = 0.325, p < 0.05). A phylogenetic tree analysis of the complete genome sequence of HBV isolated from nails and hair identified the infection source. Of the 64 nail samples, 38 (59%) were HBsAg-positive. All 15 pairs of chronic HBV/HDV infection nail and hair samples were ß-actin DNA-positive. However, nail HBV DNA was detected in two patients (13%). None of the 15 patients were positive for hair HBV DNA. Nail HDV RNA was detected in three patients (20%). Of the 15 patients, eight (53%) were nail HBsAg-positive. HBsAg and hepatitis delta (HD) antigen were detected in the nails by immunochemical staining. Chimeric mice were not infected with PBS containing HBsAg and HBV DNA elucidated from nails. CONCLUSIONS: Nails and hair were the reservoir of HBV DNA. Moreover, nails can contain HBsAg, HDV RNA, and HD antigen.

Hepatitis Delta Virus Alters the Autophagy Process To Promote Its Genome Replication.

A substantial number of viruses have been demonstrated to subvert autophagy to promote their own replication. Recent publications have reported the proviral effect of autophagy induction on hepatitis B virus (HBV) replication. Hepatitis delta virus (HDV) is a defective virus and an occasional obligate satellite of HBV. However, no previous work has studied the relationship between autophagy and HDV. In this article, we analyze the impact of HBV and HDV replication on autophagy as well as the involvement of the autophagy machinery in the HDV life cycle when produced alone and in combination with HBV. We prove that HBxAg and HBsAg can induce early steps of autophagy but ultimately block flux. It is worth noting that the two isoforms of the HDV protein, the small HDAg (S-HDAg) and large HDAg (L-HDAg) isoforms, can also efficiently promote autophagosome accumulation and disturb autophagic flux. Using CRISPR-Cas9 technology to generate specific knockouts, we demonstrate that the autophagy machinery, specifically the proteins implicated in the elongation step (ATG7, ATG5, and LC3), is important for the release of HBV without affecting the level of intracellular HBV genomes. Surprisingly, the knockout of ATG5 and ATG7 decreased the intracellular HDV RNA level in both Huh7 and HepG2.2.15 cells without an additional effect on HDV secretion. Therefore, we conclude that HBV and HDV have evolved to utilize the autophagy machinery so as to assist at different steps of their life cycle.IMPORTANCE Hepatitis delta virus is a defective RNA virus that requires hepatitis B virus envelope proteins (HBsAg) to fulfill its life cycle. Thus, HDV can only infect individuals at the same time as HBV (coinfection) or superinfect individuals who are already chronic carriers of HBV. The presence of HDV in the liver accelerates the progression of infection to fibrosis and to hepatic cancer. Since current treatments against HBV are ineffective against HDV, it is of paramount importance to study the interaction between HBV, HDV, and host factors. This will help unravel new targets whereby a therapy that is capable of simultaneously impeding both viruses could be developed. In this research paper, we evidence that the autophagy machinery promotes the replication of HBV and HDV at different steps of their life cycle. Notwithstanding their contribution to HBV release, autophagy proteins seem to assist HDV intracellular replication but not its secretion.

Hepatitis delta virus propagation enabled by hepatitis C virus-Scientifically intriguing, but is it relevant to clinical practice?

In vitro cell culture experiments and animal models have demonstrated that hepatitis delta virus (HDV) can theoretically propagate being enveloped by human pathogenic viruses other than hepatitis B virus (HBV), namely hepatitis C virus (HCV) and dengue virus. However, the clinical relevance of these findings and whether HDV replication occurs in real-world hepatitis B surface antigen (HBsAg)-negative HCV patient cohorts remain unknown. To this aim, we analysed 323 HCV-RNA-positive and HBsAg-negative sera for the presence of HDV-RNA and anti-HDV antibodies (anti-HDV). All 323 (100%) samples were negative for HDV-RNA. Interestingly, 8/316 samples tested positive for anti-HDV. The HBV serology of these eight patients showed a positive result for HBV core antibodies (anti-HBc) indicating a seroconversion of an acute HBV infection in the past. None of the anti-HBc-negative patients were positive for anti-HDV. Our results indicate a distinctly low probability of replicative HDV infection in HCV mono-infected patients in Germany. Current German clinical guidelines rightly recommend performing HDV screening only in HBsAg-positive patients. However, larger studies on this subject should be performed in regions that are endemic for chronic HBV/HDV as well as HCV infections.

Molecular Phylogenetics of Hepatitis D Virus in New Zealand and the Implications for Pacific Island Countries.

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.