Detection and Genetic Characterization of Hepatitis B and D Viruses: A Multi-Site Cross-Sectional Study of People Who Use Illicit Drugs in the Amazon Region.

Hepatitis B (HBV) and delta (HDV) viruses are endemic in the Amazon region, but vaccine coverage against HBV is still limited. People who use illicit drugs (PWUDs) represent a high-risk group due to common risk behavior and socioeconomic factors that facilitate the acquisition and transmission of pathogens. The present study assessed the presence of HBV and HBV-HDV co-infection, identified viral sub-genotypes, and verified the occurrence of mutations in coding regions for HBsAg and part of the polymerase in HBV-infected PWUDs in municipalities of the Brazilian states of Amapá and Pará, in the Amazon region. In total, 1074 PWUDs provided blood samples and personal data in 30 municipalities of the Brazilian Amazon. HBV and HDV were detected by enzyme-linked immunosorbent assay and polymerase chain reaction. Viral genotypes were identified by nucleotide sequencing followed by phylogenetic analysis, whereas viral mutations were analyzed by specialized software. High rates of serological (32.2%) and molecular (7.2%) markers for HBV were detected, including cases of occult HBV infection (2.5%). Sub-genotypes A1, A2, D4, and F2a were most frequently found. Escape mutations due to vaccine and antiviral resistance were identified. Among PWUDs with HBV DNA, serological (19.5%) and molecular (11.7%) HDV markers were detected, such as HDV genotypes 1 and 3. These are worrying findings, presenting clear implications for urgent prevention and treatment needs for the carriers of these viruses.

Identification of Key Genes for Hepatitis Delta Virus-Related Hepatocellular Carcinoma by Bioinformatics Analysis.

BACKGROUND: It has been proposed that hepatitis delta virus (HDV) induces hepatic carcinogenesis by distinct molecular events compared with hepatocellular carcinoma (HCC) that is commonly induced by other hepatitis viruses. This study aimed to explore the underlying mechanism by identifying the key genes for HDV-HCC using bioinformatics analysis. METHODS: The GSE107170 dataset was downloaded and the differentially expressed genes (DEGs) were obtained by the online tool GEO2R. Gene otology (GO) functional analyses and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using R packages. The protein-protein interaction (PPI) network was constructed by Search Tool for the Retrieval of Interacting Genes/Proteins (STRING). Hub genes were selected by Cytoscape software according to degree algorithm. The hub genes were further validated in terms of expression and survival analysis based on public databases. RESULTS: A total of 93 commonly upregulated genes and 36 commonly downregulated genes were found. The top 5 upregulated hub genes were TFRC, ACTR2, ARPC1A, ARPC3, and ARPC2. The top 5 downregulated hub genes were CTNNB1, CCND1, CDKN1B, CDK4, and CDKN1A. In the validation analysis, the expressions of ARPC1A, ARPC3, and CDK4 were promoted in general liver cancer samples. Higher expressions of ARPC2 and CDK4 and lower expressions of CDKN1A, CCND1, and CDKN1B were associated with worse prognosis in general HCC patients. CONCLUSION: The present study identifies a series of key genes that may be involved in the carcinogenesis of HDV-HCC and used as prognostic factors.

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.

The genomic landscape of Mongolian hepatocellular carcinoma.

Mongolia has the highest incidence of hepatocellular carcinoma (HCC) in the world, but its causative factors and underlying tumor biology remain unknown. Here, we describe molecular characteristics of HCC from 76 Mongolian patients by whole-exome and transcriptome sequencing. We present a comprehensive analysis of mutational signatures, driver genes, and molecular subtypes of Mongolian HCC compared to 373 HCC patients of different races and ethnicities and diverse etiologies. Mongolian HCC consists of prognostic molecular subtypes similar to those found in patients from other areas of Asia, Europe, and North America, as well as other unique subtypes, suggesting the presence of distinct etiologies linked to Mongolian patients. In addition to common driver mutations (TP53, CTNNB1) frequently found in pan-cancer analysis, Mongolian HCC exhibits unique drivers (most notably GTF2IRD2B, PNRC2, and SPTA1), the latter of which is associated with hepatitis D viral infection. These results suggest the existence of new molecular mechanisms at play in Mongolian hepatocarcinogenesis.

Hepatitis delta virus interacts with splicing factor SF3B155 and alters pre-mRNA splicing of cell cycle control genes.

Hepatitis delta virus (HDV) is the agent responsible for the most severe form of human viral hepatitis. The HDV genome consists of a single-stranded circular RNA molecule that encodes for one single protein, the delta antigen. Given its simplicity, HDV must make use of several host cellular proteins to accomplish its life cycle processes, including transcription, replication, post-transcriptional, and post-translational modifications. Consequently, identification of the interactions established between HDV components and host proteins assumes a pivotal interest in the search of novel therapeutic targets. Here, we used the yeast three-hybrid system to screen a human liver cDNA library to identify host proteins that interact with the HDV genomic RNA. One of the identified proteins corresponded to the splicing factor SF3B155, a component of the U2snRNP complex that is essential for the early recognition of 3' splice sites in the pre-mRNAs of human genes. We show that the interaction between the HDV genomic RNA and SF3B155 occurs in vivo and that the expression of HDV promotes changes in splicing of human genes whose alternative splicing is SF3B155-dependent. We further show that expression of HDV triggers alterations in several constitutive and alternative splicing events in the tumor suppressor RBM5 transcript, with consequent reduction of its protein levels. This is the first description that HDV expression promotes changes in the splicing of human genes, and we suggest that the HDV-induced alternative splicing changes, through SF3B155 sequester, may contribute for the early progression to hepatocellular carcinoma characteristic of HDV-infected patients.

NTCP S267F variant associates with decreased susceptibility to HBV and HDV infection and decelerated progression of related liver diseases.

OBJECTIVES: To determine potential associations of the rs2296651 variant (c.800C>T, S267F) of NTCP with HBV and HBV plus concomitant HDV infection as well as with the progression of related liver diseases. METHODS: The S267F variant was genotyped by DNA sequencing in 620 HBV-infected patients and 214 healthy controls (HCs). Among the patients, 450 individuals were tested for HDV by a nested PCR assay. Logistic regression was applied to examine the association. RESULTS: The S267F variant was found more frequently among HCs (16%) compared to HBV-infected (6%) and HBV-HDV co-infected patients (3%) (HBV patients vs HC: OR=0.32, P=0.00002 and HDV patients vs. HC: OR=0.17, P=0.018). The frequency of S267F variant was inversely correlated with CHB, LC or HCC patients compared with HCs (OR=0.31, P=0.001; OR=0.32, P=0.013; OR=0.34, P=0.002, respectively). S267F variant was also associated with decreased risk of the development of advanced liver cirrhosis (LC) and hepatocellular carcinoma (HCC) (Child B and C vs. Child A, OR=0.26, adjusted P=0.016; BCLC B,C,D vs. BCLC A, OR=0.038, P=0.045, respectively). In addition, patients with the genotype CT had lower levels of AST, ALT, total and direct bilirubin as well as higher platelet counts, indicating an association with a more favorable clinical outcome. CONCLUSION: The NTCP S267F variant of the SLC10A1 gene exhibits protective effects against HBV and HDV infection and is associated with a reduced risk of developing to advanced stages of LC and HCC.

The functional role of sodium taurocholate cotransporting polypeptide NTCP in the life cycle of hepatitis B, C and D viruses.

Chronic hepatitis B, C and D virus (HBV, HCV and HDV) infections are a major cause of liver disease and cancer worldwide. Despite employing distinct replication strategies, the three viruses are exclusively hepatotropic, and therefore depend on hepatocyte-specific host factors. The sodium taurocholate co-transporting polypeptide (NTCP), a transmembrane protein highly expressed in human hepatocytes that mediates the transport of bile acids, plays a key role in HBV and HDV entry into hepatocytes. Recently, NTCP has been shown to modulate HCV infection of hepatocytes by regulating innate antiviral immune responses in the liver. Here, we review the current knowledge of the functional role and the molecular and cellular biology of NTCP in the life cycle of the three major hepatotropic viruses, highlight the impact of NTCP as an antiviral target and discuss future avenues of research.

High prevalence of hepatitis delta virus in Cameroon.

Hepatitis delta virus (HDV), a satellite virus of hepatitis B virus (HBV), infects an estimated 15-20 million people worldwide and confers a greater risk for accelerated progression to liver disease. However, limited HDV surveillance data are available in sub-Saharan Africa where HDV diversity is high. To determine the prevalence and diversity of HDV in Cameroon, serological and molecular characterization was performed on 1928 HBsAg positive specimens selected from retrospective viral surveillance studies conducted in Cameroon from 2010-2016. Samples were screened for HDV antibodies on the Abbott ARCHITECT instrument and for HDV RNA on the Abbott m2000 instrument by research assays. HDV positive specimens with sufficient viral load were selected for genomic sequencing. The seroprevalence of HDV in HBsAg positive samples from Cameroon was 46.73% [95% CI; 44.51-48.96%], with prevalence of active HDV infection being 34.2% [95% CI; 32.09-36.41%]. HDV genotypes 1, 6, 7 and 8 were identified amongst N = 211 sequences, including N = 145 genomes. HDV prevalence is high within the study cohort, indicating that a large portion of HBV infected individuals in Cameroon are at elevated risk for severe hepatitis and death. Collectively, these results emphasize the need for HBV vaccination and HDV testing in HBsAg positive patients in Cameroon.

HDV infection rates in northern Vietnam.

Hepatitis D caused by the hepatitis delta virus (HDV) is a serious health problem in many regions of the world. A total of 546 HBV-infected patients were enrolled from 2013 to 2015 and classified clinically into the subgroups of chronic hepatitis B (CHB, n = 191), liver cirrhosis (LC, n = 147) and hepatocellular carcinoma (HCC, n = 208). The patients were screened for HDV-RNA by nested PCR assays. HDV genotypes were assessed by direct sequencing, followed by phylogenetic analysis. HDV-RNA was identified in 13% (71/546) of HBV-infected patients. The highest HDV prevalence was found in the LC group (19.7%), followed by the HCC (12%) and CHB (8.9%) groups (P = 0.017). HDV/HBV coinfections were significantly associated with a rather unfavourable clinical outcome, in particular with LC development compared to HBV monoinfection. Phylogenetic analyses indicated that the genotype HDV1 was, with a prevalence of 91%, by far the most common genotype in Vietnam, followed by HDV2 with 9%. Other HDV genotypes were not observed. In accordance with previous data obtained a decade ago, our results confirm a continuing high prevalence of HDV infection in hepatitis B patients in northern Vietnam with the HDV1 genotype still being the predominant genotype. HDV nucleic acid testing to minimize the associated risk should be considered.

Amino Acid Substitutions within HLA-B*27-Restricted T Cell Epitopes Prevent Recognition by Hepatitis Delta Virus-Specific CD8+ T Cells.

Virus-specific CD8 T cell response seems to play a significant role in the outcome of hepatitis delta virus (HDV) infection. However, the HDV-specific T cell epitope repertoire and mechanisms of CD8 T cell failure in HDV infection have been poorly characterized. We therefore aimed to characterize HDV-specific CD8 T cell epitopes and the impacts of viral mutations on immune escape. In this study, we predicted peptide epitopes binding the most frequent human leukocyte antigen (HLA) types and assessed their HLA binding capacities. These epitopes were characterized in HDV-infected patients by intracellular gamma interferon (IFN-γ) staining. Sequence analysis of large hepatitis delta antigen (L-HDAg) and HLA typing were performed in 104 patients. The impacts of substitutions within epitopes on the CD8 T cell response were evaluated experimentally and by in silico studies. We identified two HLA-B*27-restricted CD8 T cell epitopes within L-HDAg. These novel epitopes are located in a relatively conserved region of L-HDAg. However, we detected molecular footprints within the epitopes in HLA-B*27-positive patients with chronic HDV infections. The variant peptides were not cross-recognized in HLA-B*27-positive patients with resolved HDV infections, indicating that the substitutions represent viral escape mutations. Molecular modeling of HLA-B*27 complexes with the L-HDAg epitope and its potential viral escape mutations indicated that the structural and electrostatic properties of the bound peptides differ considerably at the T cell receptor interface, which provides a possible molecular explanation for the escape mechanism. This viral escape from the HLA-B*27-restricted CD8 T cell response correlates with a chronic outcome of hepatitis D infection. T cell failure resulting from immune escape may contribute to the high chronicity rate in HDV infection.IMPORTANCE Hepatitis delta virus (HDV) causes severe chronic hepatitis, which affects 20 million people worldwide. Only a small number of patients are able to clear the virus, possibly mediated by a virus-specific T cell response. Here, we performed a systematic screen to define CD8 epitopes and investigated the role of CD8 T cells in the outcome of hepatitis delta and how they fail to eliminate HDV. Overall the number of epitopes identified was very low compared to other hepatotropic viruses. We identified, two HLA-B*27-restricted epitopes in patients with resolved infections. In HLA-B*27-positive patients with chronic HDV infections, however, we detected escape mutations within these identified epitopes that could lead to viral evasion of immune responses. These findings support evidence showing that HLA-B*27 is important for virus-specific CD8 T cell responses, similar to other viral infections. These results have implications for the clinical prognosis of HDV infection and for vaccine development.

The Hepatitis Delta Virus accumulation requires paraspeckle components and affects NEAT1 level and PSP1 localization.

The Hepatitis Delta Virus (HDV) relies mainly on host proteins for its replication. We previously identified that PSF and p54nrb associate with the HDV RNA genome during viral replication. Together with PSP1, these proteins are part of paraspeckles, which are subnuclear bodies nucleated by the long non-coding RNA NEAT1. In this work, we established the requirement for PSF, p54nrb and PSP1 in HDV replication using RNAi-mediated knockdown in HEK-293 cells replicating the HDV RNA genome. We determined that HDV replication induces the delocalization of PSP1 to cytoplasmic foci containing PABP and increases NEAT1 level causing an enlargement of NEAT1 foci. Overall, our data support a role for the main paraspeckles proteins in HDV life cycle and indicate that HDV replication causes a cellular stress and induces both a delocalization of the PSP1 to the cytoplasm and a disruption of paraspeckles.

Prevalence of Hepatitis B virus and Hepatitis D virus Coinfection in Western Burkina Faso and molecular characterization of the detected virus strains.

OBJECTIVES: In this study, we monitored the seroprevalence of HBV-HDV co-infection in different population groups in the Western part of Burkina Faso, and described the genetic diversity of the detected virus strains. METHODS: Between October 2013 and December 2014, venous blood samples were collected from different cohorts (blood donors, pregnant women, outpatients) in the western region of Burkina Faso. Samples were tested for HBsAg and total anti-HDV antibodies. Positive samples were further analysed for HBV-DNA and HDV-RNA. Genotyping of the detected virus strains was done by nucleotide sequencing and phylogenetic analyses. RESULTS: A total of 841 participants were included in this study. The mean age was 27.45 years (range: 7-89 years). HBsAg was found in 117 (13.9%) participants. Of the HBsAg positive samples, 4 (3.4%) were positive for total anti-HDV antibodies and negative for HDV RNA. Phylogenetic analyses based on the HBV complete genome (n=10) and S fragment sequences (n=35) showed that all strains belonged to genotype E. CONCLUSIONS: Our study showed a high HBsAg prevalence, but a low rate of HDV co-infection in HBsAg carriers from western Burkina Faso. The predominance of HBV genotype E in the country was confirmed. Our findings contribute to a better understanding of the burden of HBV and HDV infection in western Burkina Faso.

Genetic diversity of hepatitis B co-infection with hepatitis C, D and E viruses among Malaysian chronic hepatitis B patients.

BACKGROUND: Hepatitis B virus co-infection with other strains of viral hepatitis is associated with increased risk of liver cirrhosis and hepatic decompensation. OBJECTIVES: This is a prevalence study that assessed the genetic diversity of chronic hepatitis B patients and coinfection. METHODS: Chronic hepatitis B patients enrolled in this study were tested for antibodies of other hepatitis viruses using ELISA kits. Patient clinical profiles were collected and partial genes of HBV, HCV, and HEV were amplified, sequenced, and analyzed using phylogenetic analysis. The associations between variables were determined using the chi-squared test. RESULTS: Of the 82 patients recruited for this study, 53.7% were non-cirrhotic, 22.0% cirrhotic, 20.7% acute flare and 3.7% hepatocellular carcinoma. Majority (58%) of patients had a high level of ALT (≥34 U/L). Sequence analysis showed HBV (63.9%) belonged to genotype B, HEV belonged to genotype 4 while HCV belonged to genotype 3a and the genotypes were found to be significantly associated with the clinical stage of the patients (χ2=56.632; p<0.01). Similarly, Hepatitis B e antigen was also found to be significantly associated with the clinical stage of infection (χ2=51.952; p<0.01). CONCLUSION: This study revealed that genetic diversity was found to have a significant impact on the severity of infection.

A new class of hepatitis B and D virus entry inhibitors, proanthocyanidin and its analogs, that directly act on the viral large surface proteins.

Introduction of direct-acting antivirals against hepatitis C virus (HCV) has provided a revolutionary improvement in the treatment outcome. In contrast to HCV, however, the strategy for developing new antiviral agents against hepatitis B virus (HBV), especially viral-targeting compounds, is limited because HBV requires only four viral genes for its efficient replication/infection. Here, we identify an oligomeric flavonoid, proanthocyanidin (PAC) and its analogs, which inhibit HBV entry into host cells by targeting the HBV large surface protein (LHBs). Through cell-based chemical screening, PAC was identified to inhibit HBV infection with little cytotoxic effect. PAC prevented the attachment of the preS1 region in the LHBs to its cellular receptor, sodium taurocholate cotransporting polypeptide (NTCP). PAC was shown to target HBV particles and impair their infectivity, whereas it did not affect the NTCP-mediated bile acid transport activity. Chemical biological techniques demonstrated that PAC directly interacted with the region essential for receptor binding in the preS1 region in the LHBs protein. Importantly, PAC had a pan-genotypic anti-HBV activity and was also effective against a clinically relevant nucleoside analog-resistant HBV isolate. We further showed that PAC augmented the ability of a nucleoside analog, tenofovir, to interrupt HBV spread over time in primary human hepatocytes by cotreatment. Moreover, derivative analysis could identify small molecules that demonstrated more-potent anti-HBV activity over PAC. CONCLUSION: PAC and its analogs represent a new class of anti-HBV agents that directly target the preS1 region of the HBV large surface protein. These agents could contribute to the development of a potent, well-tolerated, and broadly active inhibitor of HBV infection. (Hepatology 2017;65:1104-1116).

Regulation of MAP4K4 gene expression by RNA interference through an engineered theophylline-dependent hepatitis delta virus ribozyme switch.

Riboswitches are functional non-coding RNA regulatory components that play an important role in the regulation of gene expression in diverse organisms. In particular, using riboswitches to modulate RNA interference (RNAi) enables temporal and spatial control of gene expression in mammalian cells. Herein, a ribozyme gene switch to activate RNAi was fabricated for the artificial regulation of versatile gene silencing through the interaction of an RNA aptamer with small molecules. The device comprised an allosteric HDV ribozyme with an embedded theophylline aptamer and a primary miRNA (pri-miRNA) to silence the MAP4K4 gene in hepatic (HepG2) cells, aiming to achieve dose-dependent control of the activation of RNAi, and then the regulation of the MAP4K4 gene by theophylline. Finally, we demonstrated the feasibility and applicability of utilizing HDV ribozyme switches to activate RNAi for regulating an endogenous gene in mammalian cells.

HDVDB: a data warehouse for hepatitis delta virus.

Hepatitis Delta Virus (HDV) is an RNA virus and causes delta hepatitis in humans. Although a lot of data is available for HDV, but retrieval of information is a complicated task. Current web database 'HDVDB' provides a comprehensive web-resource for HDV. The database is basically concerned with basic information about HDV and disease caused by this virus, genome structure, pathogenesis, epidemiology, symptoms and prevention, etc. Database also supplies sequence data and bibliographic information about HDV. A tool 'siHDV Predict' to design the effective siRNA molecule to control the activity of HDV, is also integrated in database. It is a user friendly information system available at public domain and provides annotated information about HDV for research scholars, scientists, pharma industry people for further study.

Ribozyme-independent replication of a defective hepatitis D virus RNA derived from hepatitis B/D patients receiving antiviral therapy.

Novel hepatitis D virus (HDV) RNA mutants carrying large fragment deletions were identified in the serum samples of two hepatitis B/D patients receiving antiviral therapy. Sequence analysis revealed that the deleted regions encompassed both ribozyme domains. The mutant persisted in the serum samples for at least 2 and 10 months, respectively in the two patients, raising the question of whether such mutants could replicate in the absence of ribozyme domains. Thirty anti-HDV antibody-positive serum samples derived from 17 patients receiving antiviral therapy were submitted for RT-PCR detection of HDV RNA deletion mutants. Large fragment HDV RNA deletions were found in 4 patients. Of them, two had liver biopsy samples available. Northern blot analysis revealed high molecular weight HDV RNA replication intermediates, genomic and anti-genomic senses, in the liver tissues. Transfection of an in vitro transcribed HDV RNA deletion mutant (1.28 kb) into Huh7 and HepG2 cells also resulted in generation of high molecular weight HDV RNA species in the hepatoma cells (>6.5 kb) with secretion of a 6.5 kb HDV RNA species into the medium. In conclusion, we discovered novel large fragment deletion mutants of HDV RNA in hepatitis B/D patients receiving antiviral therapy. Such mutants did not contain ribozyme domains but could replicate in the liver cells to generate high molecular weight but not unit-length HDV RNA.

Viral entry of hepatitis B and D viruses and bile salts transportation share common molecular determinants on sodium taurocholate cotransporting polypeptide.

UNLABELLED: The liver bile acids transporter sodium taurocholate cotransporting polypeptide (NTCP) is responsible for the majority of sodium-dependent bile salts uptake by hepatocytes. NTCP also functions as a cellular receptor for viral entry of hepatitis B virus (HBV) and hepatitis D virus (HDV) through a specific interaction between NTCP and the pre-S1 domain of HBV large envelope protein. However, it remains unknown if these two functions of NTCP are independent or if they interfere with each other. Here we show that binding of the pre-S1 domain to human NTCP blocks taurocholate uptake by the receptor; conversely, some bile acid substrates of NTCP inhibit HBV and HDV entry. Mutations of NTCP residues critical for bile salts binding severely impair viral infection by HDV and HBV; to a lesser extent, the residues important for sodium binding also inhibit viral infection. The mutation S267F, corresponding to a single nucleotide polymorphism (SNP) found in about 9% of the East Asian population, renders NTCP without either taurocholate transporting activity or the ability to support HBV or HDV infection in cell culture. These results demonstrate that molecular determinants critical for HBV and HDV entry overlap with that for bile salts uptake by NTCP, indicating that viral infection may interfere with the normal function of NTCP, and bile acids and their derivatives hold the potential for further development into antiviral drugs. IMPORTANCE: Human hepatitis B virus (HBV) and its satellite virus, hepatitis D virus (HDV), are important human pathogens. Available therapeutics against HBV are limited, and there is no drug that is clinically available for HDV infection. A liver bile acids transporter (sodium taurocholate cotransporting polypeptide [NTCP]) critical for maintaining homeostasis of bile acids serves as a functional receptor for HBV and HDV. We report here that the NTCP-binding lipopeptide that originates from the first 47 amino acids of the pre-S1 domain of the HBV L protein blocks taurocholate transport. Some bile salts dose dependently inhibit HBV and HDV infection mediated by NTCP; molecular determinants of NTCP critical for HBV and HDV entry overlap with that for bile acids transport. This work advances our understanding of NTCP-mediated HBV and HDV infection in relation to NTCP's physiological function. Our results also suggest that bile acids or their derivatives hold potential for development into novel drugs against HBV and HDV infection.

Proteomic changes in HEK-293 cells induced by hepatitis delta virus replication.

Hepatitis delta virus (HDV) infection greatly increases the risk of hepatocellular carcinoma in hepatitis B virus chronically infected patients. HDV is highly dependent on host factors for accomplishment of the replication cycle. However, these factors are largely unknown and the mechanisms involved in the pathogenicity of the virus still remain elusive. Here, we made use of the HEK-293 cell line, which was engineered in order to mimic HDV replication. Five different proteomes were analyzed and compared using a MS-based quantitative proteomics approach by (18)O/(16)O stable isotope labeling. About 3000 proteins were quantified and 89 found to be differentially expressed as a consequence HDV RNA replication. The down-regulation of p53 , HSPE, and ELAV as well as the up-regulation of Transportin 1 , EIF3D, and Cofilin 1 were validated by Western blot. A systems biology approach was additionally used to analyze altered pathways and networks. The G2/M DNA damage checkpoint and pyruvate metabolism were among the most affected pathways, and Cancer was the most likely disease associated to HDV replication. Western blot analysis allowed identifying 14-3-3 σ interactor as down-regulated protein acting in the G2/M cell cycle control checkpoint. This evidence supports deregulation of G2/M checkpoint as a possible mechanism involved in the promotion of HDV associated hepatocellular carcinoma. BIOLOGICAL SIGNIFICANCE: This manuscript provides a description of changes observed in the cellular proteome that arise as result of expression of the hepatitis delta virus (HDV) antigen as well as virus genome replication. Using a systems biology approach cancer was found to be the most probable disease associated with HDV replication. Additionally, results show that HDV alters the regulation of G2/M cell cycle control checkpoint. Taken together, our data provide new insights into probable mechanisms associated with the increased incidence of hepatocellular carcinoma observed in HDV infected patients.

Prevalence and clinical significance of hepatitis D virus co-infection in patients with chronic hepatitis B in Korea.

Hepatitis D virus (HDV) infection can cause severe acute and chronic liver disease in patients infected with hepatitis B virus (HBV). Despite the significant decline in the global HDV infection, it remains a major health concern in some countries. This study aimed to investigate the prevalence and clinical features of HDV co-infection in patients with chronic HBV infection in Korea, where HBV infection is endemic. Nine hundred forty patients [median age, 48 (18-94) years; men, 64.5%] infected chronically with HBV were enrolled consecutively. All patients who were positive for hepatitis B surface antigen (HBsAg) for at least 6 months and were tested for anti-HDV. A portion of the HDV delta antigen was amplified, sequenced, and subjected to molecular and phylogenetic analysis using sera from the patients who were anti-HDV positive. Clinical features and virologic markers were evaluated. Inactive HBsAg carriers, chronic hepatitis B, cirrhosis and hepatocellular carcinoma accounted for 29.5%, 44.7%, 17.9%, and 8.0%, respectively. Only three patients were positive for anti-HDV, corresponding to a 0.32% positive rate. All patients who were positive for anti-HDV were inactive HBsAg carriers. HDV RNA could be amplified by PCR from the sera of two patients. Phylogenetic analysis showed that both carried HDV genotype 1. In conclusion, the prevalence of HDV infection is very low (0.32%) in Korea. All HDVs were genotype 1 and detected in inactive HBsAg carriers. Therefore, HDV co-infection may not have a significant clinical impact in Korean patients with chronic HBV infection.