Hepatitis B surface antigen levels and sequences of natural hepatitis B virus variants influence the assembly and secretion of hepatitis d virus.
ABSTRACT: Various domains of hepatitis B surface antigen (HBsAg) are essential for the assembly and secretion of hepatitis D virus (HDV). This study investigated the influences of the levels and sequences of HBsAg of naturally occurring HBV variants on the assembly and secretion of HDV. Six hepatitis B virus (HBV)-producing plasmids (three genotype B and three genotype C) and six HBsAg expression plasmids that expressed various HBsAg levels were constructed from the sera of HDV-infected patients. These plasmids were cotransfected with six expression plasmids of HDV of genotype 1, 2, or 4 into the Huh-7 hepatoma cell line. Serum HBsAg and HBV DNA levels were correlated with HDV RNA levels and outcomes of chronic hepatitis D (CHD) patients. The secretion of genotype 1, 2, or 4 HDV generally correlated with HBsAg levels but not with HBV genotypes or HBV DNA levels. Swapping and residue mutagenesis experiments of HBsAg-coding sequences revealed that the residue Pro-62 in the cytosolic domain-I affects the assembly and secretion of genotype 2 and 4 HDV and not those of genotype 1. The pre-S2 N-terminal deletion HBV mutant adversely affects secretion of the three HDV genotypes. In patients, serum HDV RNA levels correlated with HBsAg levels but not with HBV DNA levels. Viremia of HDV or HBV correlated with poor outcomes. In conclusion, the assembly and secretion of HDV were influenced by the amounts and sequences of HBsAg. For an effective treatment of CHD, reduction of HBsAg production in addition to the suppression of HBV and HDV replication might be crucial.
Project description:The nucleic acid polymer REP 2139 inhibits assembly/secretion of hepatitis B virus (HBV) subviral particles. Previously, REP 2139-Ca and pegylated interferon (pegIFN) in HBV/hepatitis delta virus (HDV) coinfection achieved high rates of HDV RNA and hepatitis B surface antigen (HBsAg) loss/seroconversion in the REP 301 study (NCT02233075). The REP 301-LTF study (NCT02876419) examined safety and efficacy during 3.5 years of follow-up. In the current study, participants completing therapy in the REP 301 study were followed for 3.5 years. Primary outcomes were safety and tolerability, and secondary outcomes were HDV functional cure (HDV RNA target not detected [TND], normal alanine aminotransferase [ALT]), HBV virologic control (HBV DNA ?2,000 IU/mL, normal ALT), HBV functional cure (HBV DNA TND; HBsAg <0.05 IU/mL, normal ALT), and HBsAg seroconversion. Supplemental analysis included high-sensitivity HBsAg (Abbott ARCHITECT HBsAg NEXT), HBV pregenomic RNA (pgRNA), HBsAg/hepatitis B surface antibody (anti-HBs) immune complexes (HBsAg ICs), and hepatitis B core-related antigen (HBcrAg). Asymptomatic grade 1-2 ALT elevations occurred in 2 participants accompanying viral rebound; no other safety or tolerability issues were observed. During therapy and follow-up, HBsAg reductions to <0.05 IU/mL were also <0.005 IU/mL. HBsAg ICs declined in 7 of 11 participants during REP 2139-Ca monotherapy and in 10 of 11 participants during follow-up. HDV functional cure persisted in 7 of 11 participants; HBV virologic control persisted in 3 and functional cure (with HBsAg seroconversion) persisted in 4 of these participants. Functional cure of HBV was accompanied by HBV pgRNA TND and HBcrAg <lower limit of quantitation. <i>Conclusion:</i> REP 2139-Ca + pegIFN is not associated with long-term safety or tolerability issues. The establishment of HDV functional cure and HBV virologic control/functional cure and HBsAg seroconversion are durable over 3.5 years and may reflect removal of integrated HBV DNA from the liver. Further investigation is warranted in larger studies.
Project description:<h4>Background</h4>Hepatitis D virus (HDV) infection is considered to cause more severe hepatitis than hepatitis B virus (HBV) monoinfection. With more than 9.5 million HBV-infected people, Vietnam will face an enormous health burden. The prevalence of HDV in Vietnamese HBsAg-positive patients is speculative. Therefore, we assessed the prevalence of HDV in Vietnamese patients, determined the HDV-genotype distribution and compared the findings with the clinical outcome.<h4>Methods</h4>266 sera of well-characterized HBsAg-positive patients in Northern Vietnam were analysed for the presence of HDV using newly developed HDV-specific RT-PCRs. Sequencing and phylogenetic analysis were performed for HDV-genotyping.<h4>Results</h4>The HDV-genome prevalence observed in the Vietnamese HBsAg-positive patients was high with 15.4% while patients with acute hepatitis showed 43.3%. Phylogenetic analysis demonstrated a predominance of HDV-genotype 1 clustering in an Asian clade while HDV-genotype 2 could be also detected. The serum aminotransferase levels (AST, ALT) as well as total and direct bilirubin were significantly elevated in HDV-positive individuals (p<0.05). HDV loads were mainly low (<300 to 4.108 HDV-copies/ml). Of note, higher HDV loads were mainly found in HBV-genotype mix samples in contrast to single HBV-infections. In HBV/HDV-coinfections, HBV loads were significantly higher in HBV-genotype C in comparison to HBV-genotype A samples (p<0.05).<h4>Conclusion</h4>HDV prevalence is high in Vietnamese individuals, especially in patients with acute hepatitis B. HDV replication activity showed a HBV-genotype dependency and could be associated with elevated liver parameters. Besides serological assays molecular tests are recommended for diagnosis of HDV. Finally, the high prevalence of HBV and HDV prompts the urgent need for HBV-vaccination coverage.
Project description:Hepatitis B virus (HBV) and hepatitis D virus (HDV) infections are major public health problems in sub-Saharan Africa. Whereas it is known that HBV infection is endemic in Nigeria, there is only little data about HDV prevalence available. Here, we assessed the HDV seroprevalence and determined the HDV and HBV genotypes distribution among HBsAg positive individuals in Southwestern Nigeria.This cross-sectional study involved 188 serum samples from HBsAg positive outpatients recruited at four tertiary hospitals in Southwestern Nigeria. Anti-HDV antibodies were detected by ELISA while HDV-RNA was detected by RT-PCR. Sequencing followed by phylogenetic analyses and HBV genotype-specific PCR were used to characterize HDV and HBV genotypes, respectively.Out of 188 HBsAg positive serum samples, 17 (9 %) showed detectable HDV-RNA. Anti-HDV antibodies test was possible from 103 samples and were observed in 4.9 % (5/103) patients. There was no significant difference in HDV prevalence between four main cities across the country. 64.7 % of HDV-RNA positive samples were from males and 35.3 % from females (P < 0.05). No significant associations were observed with regard to HDV seroprevalence and available demographic factors. Phylogenetic analyses demonstrated a predominance of HDV genotype 1 and HBV genotype E among the HDV-RNA/HBsAg positive patients.In conclusion, our study showed a high prevalence of HDV infection in HBsAg carriers and the predominance of HDV genotype 1 infection in Nigerian HBV endemic region. The findings contribute to a better understanding of the relevance of HDV/HBV co-infection and circulating genotypes.
Project description:Superinfection with hepatitis D virus (HDV) may increase the risk for hepatitis flares and chronic hepatic complications in patients with chronic hepatitis B virus (HBV) infection. This retrospective observational study aimed to examine the incidence of and factors associated with recent HDV superinfection among individuals coinfected with human immunodeficiency virus (HIV) and HBV.Anti-HDV immunoglobulin G (IgG) was sequentially determined in 375 HIV/HBV-coinfected patients to estimate the HDV incidence between 1992 and 2012. Plasma HDV and HBV loads and HBV surface antigen (HBsAg) levels were determined for the HDV seroconverters. A nested case-control study was conducted to identify the associated factors with HDV seroconversion. Phylogenetic analysis was performed using HDV sequences amplified from HDV seroconverters and HDV-seropositive patients at baseline.During 1762.4 person-years of follow-up [PYFU], 16 patients seroconverted for HDV, with an overall incidence rate of 9.07 per 1000 PYFU, which increased from 0 in 1992-2001, to 3.91 in 2002-2006, to 13.26 per 1000 PYFU in 2007-2012 (P < .05). Recent HDV infection was associated with elevated aminotransferase and bilirubin levels and elevated rapid plasma reagin titers. Of the 12 patients with HDV viremia, 2 were infected with genotype 2 and 10 with genotype 4. HBsAg levels remained elevated despite a significant decline of plasma HBV DNA load with combination antiretroviral therapy that contained lamivudine and/or tenofovir.Our findings show that the incidence of recent HDV infection in HIV/HBV-coinfected patients increased significantly from 1992-2001 to 2007-2011, and was associated with hepatitis flares and syphilis.
Project description:BACKGROUND:Characteristics of hepatitis B (HBV) and delta (HDV) coinfection in various geographical regions, including Israel, remain unclear. Here we studied HDV seroprevalence in Israel, assessed HDV/HBV viral loads, circulating genotypes and hepatitis delta antigen (HDAg) conservation. METHODS:Serological anti HDV IgG results from 8969 HBsAg positive individuals tested in 2010-2015 were retrospectively analyzed to determine HDV seroprevalence. In a cohort of HBV/HDV coinfected (n=58) and HBV monoinfected (n=27) patients, quantitative real-time PCR (qRT-PCR) and sequencing were performed to determine viral loads, genotypes and hepatitis delta antigen (HDAg) protein sequence. RESULTS:6.5% (587/8969) of the HBsAg positive patients were positive for anti HDV antibodies. HDV viral load was >2 log copies/ml higher than HBV viral load in most of the coinfected patients with detectable HDV RNA (86%, 50/58). HDV genotype 1 was identified in all patients, most of whom did not express HBV. While 66.6% (4/6) of the HBV/HDV co-expressing patients carried HBV-D2 only 18.5% (5/27) of the HBV monoinfections had HBV-D2 (p=0.03). Higher genetic variability in the HDAg protein sequence was associated with higher HDV viral load. CONCLUSIONS:The overall significant prevalence of HDV (6.5%) mandates HDV RNA testing for all coinfected patients. Patients positive for HDV RNA (characterized by low HBV DNA blood levels) carried HDV genotype 1. Taken together, the significant HDV seroprevalence and the lack of effective anti-HDV therapy, necessitates strict clinical surveillance especially in patients with higher HDV viral loads and increased viral evolution.
Project description:BACKGROUND:Patients coinfected with HBV and hepatitis D virus (HDV) have a greater risk of HCC and cirrhosis. The current study was undertaken to assess HDV genotype distribution and determine clinical characteristics of hepatitis delta virus (HDV) among HBsAg positive individuals in Shanghai. METHOD:This retrospective study involved 225 serum samples from HBsAg positive hospitalized patients from October 2010 to April 2013. HDV-specific RT-nested PCR was used to amplify HDV RNA. HDV genotypes were characterized by Next-generation sequencing (NGS), followed by phylogenetic analyses. HDV/HBV co-infected patients and HBV mono-infected patients were compared clinically and virologically. RESULTS:Out of the 225 HBsAg-positive serum samples with elevated transaminases, HDV-RNA was identified in 11 (4.9%) patients. The HBV loads in the HDV positive group were significantly lower than the HDV negative HBV-infected patients. The aminotransferase enzymes were significantly higher in HDV/HBV co-infected compared to HDV negative patients (P?<?0.05). Phylogenetic analyses indicated that HDV-2 genotype being the predominant genotype, other HDV genotypes were not observed. HDV/HBV patients were significantly associated with a rather unfavourable clinical outcome. CONCLUSION:In summary, the prevalence of HDV infection in patients with elevated transaminases is not low and the predominance of HDV genotype 2 infection in Shanghai. This finding helps us to better understand the correlation of HDV/HBV co-infection. Moreover, Next-generation sequencing (NGS) technologies provide a rapid, precise method for generating HDV genomes to define infecting genotypes.
Project description:Although hepatitis B virus (HBV) is hyperendemic and heterogeneous in its genetic diversity in Ethiopia, little is known about hepatitis D virus (HDV) circulating genotypes and molecular diversity.A total of 321 hepatitis B surface antigen (HBsAg) positives (125 HIV co-infected, 102 liver disease patients and 94 blood donors) were screened for anti-HDV antibody. The anti-HDV positive sera were subjected to Real time PCR for HDV-RNA confirmation. The non coding genome region (spanning from 467 to 834 nucleotides) commonly used for HDV genotyping as well as complete HDV genome were sequenced for genotyping and molecular analysis.The anti-HDV antibody was found to be 3.2% (3) in blood donors, 8.0% (10) in HIV co-infected individuals and 12.7% (13) in liver disease patients. None of the HIV co-infected patients who revealed HBV lamivudine (3TC) resistance at tyrosine-methionine/isoleucine-aspartate-aspartate (YM(I)DD) reverse transcriptase (RT) motif with concomitant vaccine escape gene mutants was positive for anti-HDV antibody. The HDV viremia rate was 33.3%, 30.0% and 23.1% in respect to the above study groups. All the six isolates sequenced were phylogenetically classified as HDV genotype 1 (HDV-1) and grouped into two monophyletic clusters. Amino acid (aa) residues analysis of clathrin heavy chain (CHC) domain and the isoprenylation signal site (Py) at 19 carboxyl (C)-terminal amino acids (aa 196-214) and the HDV RNA binding domain (aa 79-107) were highly conserved and showed a very little nucleotide variations. All the sequenced isolates showed serine at amino acid position 202. The RNA editing targets of the anti-genomic HDV RNA (nt1012) and its corresponding genomic RNA (nt 580) showed nucleotides A and C, respectively.The low seroprevalence and viraemic rates of HDV in particular during HIV-confection might be highly affected by HBV drug resistance selected HBsAg mutant variants in this setting, although HDV-1 sequences analysis revealed clade homogeneity and highly conserved structural and functional domains. Thus, the potential role of HBV drug resistance associated polymerase mutations and concomitant HBsAg protein variability on HDV viral assembly, secretion and infectivity needs further investigation.
Project description:Hepatitis B virus (HBV) and hepatitis delta virus (HDV) interplay was investigated by examining liver and serum samples from 21 coinfected and 22 HBV-monoinfected patients with chronic liver disease. Different real-time PCR assays were applied to evaluate intrahepatic amounts of HBV DNA, covalently closed circular DNA (cccDNA), pregenomic RNA (pgRNA), pre-S/S RNAs, and HDV RNA. Besides HBV DNA and HDV RNA levels, HBsAg concentrations in the sera were also determined. HDV-coinfected cases showed significantly lower median levels of serum HBV DNA (-5 log), intrahepatic relaxed-circular DNA (-2 log), and cccDNA (-2 log) than those of HBV-monoinfected cases. Interestingly, pgRNA and pre-S/S RNA amounts were significantly lower (both -1 log) in HDV-positive patients, whereas serum HBsAg concentrations were comparable between the two patient groups. Pre-S/S RNA and HBsAg amounts per cccDNA molecule were higher in HDV-positive patients (3-fold and 1 log, respectively), showing that HBV replication was reduced, whereas synthesis of envelope proteins was not specifically decreased. The ratios of cccDNA to intracellular total HBV DNA showed a larger proportion of cccDNA molecules in HDV-positive cases. For these patients, both intrahepatic and serum HDV RNA amounts were associated with cccDNA but not with HBsAg or HBV DNA levels. Finally, HBV genomes with large deletions in the basal core promoter/precore region were detected in 5/21 HDV-positive patients but in no HDV-negative patients and were associated with lower viremia levels. These findings provide significant information about the interference exerted by HDV on HBV replication and transcription activities in the human liver.
Project description:Hepatitis delta virus (HDV) coinfection will additionally aggravate the hepatitis B virus (HBV) burden in the coming decades, with an increase in HBV-related liver diseases. Between 2018 and 2019, a total of 205 HBV patients clinically characterized as chronic hepatitis B (CHB; <i>n</i> = 115), liver cirrhosis (LC; <i>n</i> = 21), and hepatocellular carcinoma (HCC; <i>n</i> = 69) were recruited. HBV surface antigen (HBsAg), antibodies against surface antigens (anti-HBs), and core antigens (anti-HBc) were determined by ELISA. The presence of hepatitis B viral DNA and hepatitis delta RNA was determined. Distinct HBV and HDV genotypes were phylogenetically reconstructed and vaccine escape mutations in the "a" determinant region of HBV were elucidated. All HBV patients were HbsAg positive, with 99% (<i>n</i> = 204) and 7% (<i>n</i> = 15) of them being positive for anti-HBc and anti-HBs, respectively. Anti-HBs positivity was higher among HCC (15%; <i>n</i> = 9) compared to CHB patients. The HBV-B genotype was predominant (65%; <i>n</i> = 134), followed by HBV-C (31%; <i>n</i> = 64), HBV-D, and HBV-G (3%; <i>n</i> = 7). HCC was observed frequently among young individuals with HBV-C genotypes. A low frequency (2%; <i>n</i> = 4) of vaccine escape mutations was observed. HBV-HDV coinfection was observed in 16% (<i>n</i> = 33) of patients with the predominant occurrence of the HDV-1 genotype. A significant association of genotypes with alanine aminotransferase (ALT) and aspartate aminotransferase (AST) enzyme levels was observed in HBV monoinfections. The prevalence of the HDV-1 genotype is high in Vietnam. No correlation was observed between HDV-HBV coinfections and disease progression when compared to HBV monoinfections.
Project description:The spreading of viral hepatitis among injecting drug users (IDU) is an emerging public health concern. This study explored the prevalence and the risks of hepatitis B virus (HBV), hepatitis C virus (HCV) and hepatitis D virus (HDV) among IDU-dominant prisoners in Taiwan. HBV surface antigen (HBsAg), antibodies to HCV (anti-HCV) and HDV (anti-HDV), viral load and HCV genotypes were measured in 1137(67.0%) of 1697 prisoners. 89.2% of participants were IDUs and none had HIV infection. The prevalence of HBsAg, anti-HCV, dual HBsAg/anti-HCV, HBsAg/anti-HDV, and triple HBsAg/anti-HCV/anti-HDV was 13.6%, 34.8%, 4.9%, 3.4%, and 2.8%, respectively. HBV viremia rate was significantly lower in HBV/HCV-coinfected than HBV mono-infected subjects (66.1% versus 89.9%, adjusted odds ratio/95% confidence intervals [aOR/CI] = 0.27/0.10-0.73). 47.5% anti-HCV-seropositive subjects (n = 396) were non-viremic, including 23.2% subjects were antivirals-induced. The predominant HCV genotypes were genotype 6(40.9%), 1a(24.0%) and 3(11.1%). HBsAg seropositivity was negatively correlated with HCV viremia among the treatment naïve HCV subjects (44.7% versus 72.4%, aOR/CI = 0.27/0.13-0.58). Anti-HCV seropositivity significantly increased the risk of anti-HDV-seropositivity among HBsAg carriers (57.1% versus 7.1%, aOR/CI = 15.73/6.04-40.96). In conclusion, IUDs remain as reservoirs for multiple hepatitis viruses infection among HIV-uninfected prisoners in Taiwan. HCV infection increased the risk of HDV infection but suppressed HBV replication in HBsAg carriers. An effective strategy is mandatory to control the epidemic in this high-risk group.