Markers Of Hepatitis B Virus Replication Research Articles

SARS-CoV-2 nsp13 suppresses hepatitis B virus replication by targeting cccDNA transcription.

To effectively combat hepatitis B virus (HBV), it is imperative to develop potent antiviral medications targeting covalently closed circular DNA (cccDNA). Our investigation aimed to assess the impact of SARS-CoV-2 nsp13 on HBV replication across diverse HBV models, confirming its ability to significantly reduce several HBV replication markers. Additionally, our identification of the interaction between nsp13 and SMC4 opens the door for further mechanistic exploration. This marks a paradigm shift in our approach to HBV antiviral therapy, introducing an entirely novel perspective. Our findings propose a novel strategy for developing anti-HBV drugs that specifically target HBV cccDNA.

Long-term Hepatitis B and Liver Outcomes Among Adults Taking Tenofovir-Containing Antiretroviral Therapy for HBV/HIV Coinfection in Zambia.

Long-term outcomes of tenofovir-containing antiretroviral therapy (ART) for hepatitis B virus (HBV)/human immunodeficiency virus (HIV) coinfection were evaluated in Zambia. A prospective cohort of adults with HIV and hepatitis B surface antigen (HBsAg)-positivity was enrolled at ART initiation. On tenofovir-containing ART, we ascertained HBV viral load (VL) non-suppression, alanine aminotransferase (ALT) elevation, serologic end-points, progression of liver fibrosis based on elastography, and hepatocellular carcinoma (HCC) incidence. We also described a subgroup (low HBV VL and no/minimal fibrosis at baseline) that, under current international guidelines, would not have been treated in the absence of their HIV infection. Among 289 participants at ART start, median age was 34 years, 40.1% were women, median CD4 count was 191 cells/mm3, 44.2% were hepatitis B e antigen-positive, and 28.4% had liver fibrosis/cirrhosis. Over median 5.91 years of ART, 13.6% developed HBV viral non-suppression, which was associated with advanced HIV disease. ALT elevation on ART was linked with HBV VL non-suppression. Regression of fibrosis and cirrhosis were common, progression to cirrhosis was absent, and no cases of HCC were ascertained. HBsAg seroclearance was 9.4% at 2 and 15.4% at 5 years, with higher rates among patients with low baseline HBV replication markers. Reassuring long-term liver outcomes were ascertained during tenofovir-based ART for HBV/HIV coinfection in Zambia. Higher than expected HBsAg seroclearance during ART underscores the need to include people with HIV in HBV cure research.

G-quadruplex in hepatitis B virus pregenomic RNA promotes its translation

Hepatitis B virus (HBV) is a hepatotropic DNA virus that has a very compact genome. Due to this genomic density, several distinct mechanisms are used to facilitate the viral life cycle. Recently, accumulating evidence show that G-quadruplex (G4) in different viruses play essential regulatory roles in key steps of the viral life cycle. Although G4 structures in the HBV genome have been reported, their function in HBV replication remains elusive. In this study, we treated an HBV replication-competent cell line and HBV-infected cells with the G4 structure stabilizer pyridostatin (PDS) and evaluated different HBV replication markers to better understand the role played by the G4. In both models, we found PDS had no effect on viral precore RNA (pcRNA) or pre-genomic RNA (pgRNA), but treatment did increase HBeAg/HBc ELISA reads and intracellular levels of viral core/capsid protein (HBc) in a dose-dependent manner, suggesting post-transcriptional regulation. To further dissect the mechanism of G4 involvement, we used invitro-synthesized HBV pcRNA and pgRNA. Interestingly, we found PDS treatment only enhanced HBc expression from pgRNA but not HBeAg expression from pcRNA. Our bioinformatic analysis and CD spectroscopy revealed that pgRNA harbors a conserved G4 structure. Finally, we introduced point mutations in pgRNA to disrupt its G4 structure and observed the resulting mutant failed to respond to PDS treatment and decreased HBc level in invitro translation assay. Taken together, our data demonstrate that HBV pgRNA contains a G4 structure that plays a vital role in the regulation of viral mRNA translation.

Antibiotic-induced gut bacteria depletion has no effect on HBV replication in HBV immune tolerance mouse model

Commensal microbiota is closely related to Hepatitis B virus (HBV) infection. Gut bacteria maturation accelerates HBV immune clearance in hydrodynamic injection (HDI) HBV mouse model. However, the effect of gut bacteria on HBV replication in recombinant adeno-associated virus (AAV)-HBV mouse model with immune tolerance remains obscure. We aim to investigate its role on HBV replication in AAV-HBV mouse model. C57BL/6 mice were administrated with broad-spectrum antibiotic mixtures (ABX) to deplete gut bacteria and intravenously injected with AAV-HBV to establish persistent HBV replication. Gut microbiota community was analyzed by fecal qPCR assay and 16S ribosomal RNA (rRNA) gene sequencing. HBV replication markers in blood and liver were determined by ELISA, qPCR assay and Western blot at indicated time points. Immune response in AAV-HBV mouse model was activated through HDI of HBV plasmid or poly(I:C) and then detected by quantifying the percentage of IFN-γ+/CD8+ T cells in the spleen via flow cytometry as well as the splenic IFN-γ mRNA level via qPCR assay. We found that antibiotic exposure remarkably decreased gut bacteria abundance and diversity. Antibiotic treatment failed to alter the levels of serological HBV antigens, intrahepatic HBV RNA transcripts and HBc protein in AAV-HBV mouse model, but contributed to HBsAg increase after breaking of immune tolerance. Overall, our data uncovered that antibiotic-induced gut bacteria depletion has no effect on HBV replication in immune tolerant AAV-HBV mouse model, providing new thoughts for elucidating the correlation between gut bacteria dysbiosis by antibiotic abuse and clinical chronic HBV infection.

A Novel Mouse Model Harboring Hepatitis B Virus Covalently Closed Circular DNA

Background and AimsThe persistence of viral covalently closed circular DNA (cccDNA) is the major obstacle for antiviral treatment against hepatitis B virus (HBV). Basic and translational studies are largely hampered due to the lack of feasible small animal models to support HBV cccDNA formation. The aim of this study is to establish a novel mouse model harboring cccDNA.MethodsAn adeno-associated virus (AAV) vector carrying a replication-deficient HBV1.04-fold genome (AAV-HBV1.04) was constructed. The linear HBV genome starts from nucleotide 403 and ends at 538, which results in the splitting of HBV surface and polymerase genes. Different HBV replication markers were evaluated for AAV-HBV1.04 plasmid–transfected cells, the AAV-HBV1.04 viral vector–transduced cells, and mice injected with the AAV-HBV1.04 viral vector.ResultsCompared with the previously reported AAV-HBV1.2 construct, direct transfection of AAV-HBV1.04 plasmid failed to produce hepatitis B surface antigen and progeny virus. Interestingly, AAV-HBV1.04 viral vector transduction could result in the formation of cccDNA and the production of all HBV replication markers in vitro and in vivo. The formation of cccDNA could be blocked by ATR (ataxia-telangiectasia and Rad3-related protein) inhibitors but not HBV reverse transcription inhibitor or capsid inhibitors. The AAV-HBV1.04 mouse supported long-term HBV replication and responded to antiviral treatments.ConclusionsThis AAV-HBV1.04 mouse model can support HBV cccDNA formation through ATR-mediated DNA damage response. The de novo formed cccDNA but not the parental AAV vector can lead to the production of hepatitis B surface antigen and HBV progeny. This model will provide a unique platform for studying HBV cccDNA and developing novel antivirals against HBV infection.

In Vivo Modelling of Hepatitis B Virus Subgenotype A1 Replication Using Adeno-Associated Viral Vectors

The paucity of animal models that simulate the replication of the hepatitis B virus (HBV) is an impediment to advancing new anti-viral treatments. The work reported here employed recombinant adeno-associated viruses (AAVs) to model HBV subgenotype A1 and subgenotype D3 replication in vitro and in vivo. Infection with subgenotype A1 is endemic to parts of sub-Saharan Africa, and it is associated with a high risk of hepatocellular carcinoma. Recombinant AAV serotype 2 (AAV2) and 8 (AAV8) vectors bearing greater-than-genome-length sequences of HBV DNA from subgenotype A1 and D3, were produced. Transduced liver-derived cultured cells produced HBV surface antigen and core antigen. Administration of AAV8 carrying HBV subgenotype A1 genome (AAV8-A1) to mice resulted in the sustained production of HBV replication markers over a six-month period, without elevated inflammatory cytokines, expression of interferon response genes or alanine transaminase activity. Markers of replication were generally higher in animals treated with subgenotype D3 genome-bearing AAVs than in those receiving the subgenotype A1-genome-bearing vectors. To validate the use of the AAV8-A1 murine model for anti-HBV drug development, the efficacy of anti-HBV artificial primary-microRNAs was assessed. Significant silencing of HBV markers was observed over a 6-month period after administering AAVs. These data indicate that AAVs conveniently and safely recapitulate the replication of different HBV subgenotypes, and the vectors may be used to assess antivirals’ potency.

Fast Differentiation of HepaRG Cells Allowing Hepatitis B and Delta Virus Infections.

HepaRG cells are liver bipotent progenitors acquiring hepatocytes features when differentiated in the presence of dimethylsulfoxide (DMSO). Differentiated HepaRG (dHepaRG) are considered the best surrogate model to primary human hepatocytes (PHH) and are susceptible to several hepatotropic viruses, including Hepatitis B Virus (HBV) and Hepatitis Delta Virus (HDV) infection. Despite these advantages, HepaRG cells are not widely used for the study of these two viruses because of their long differentiation process and their rather low and variable infection rates. Here, we tested the use of a cocktail of five chemicals (5C) combined or not with DMSO to accelerate the cells’ differentiation process. We found that NTCP-mediated HDV entry and replication are similar in HepaRG cells cultivated for only 1 week with 5C and DMSO or differentiated with the regular 4-week protocol. However, even though the NTCP-mediated HBV entry process seemed similar, cccDNA and subsequent HBV replication markers were lower in HepaRG cells cultivated for 1 week with 5C and DMSO compared to the regular differentiation protocol. In conclusion, we set up a new procedure allowing fast differentiation and efficient HDV-infection of HepaRG cells and identified differential culture conditions that may allow to decipher the mechanism behind the establishment of the HBV minichromosome.

Correlation of serum hepatitis B core-related antigen with hepatitis B virus total intrahepatic DNA and covalently closed circular-DNA viral load in HIV-hepatitis B coinfection.

To assess whether quantified hepatitis B core-related antigen (qHBcrAg) is a surrogate marker of intrahepatic replication in HIV and hepatitis B virus (HBV) coinfection. Cross-sectional study of 31 HIV-HBV-infected patients (total liver biopsies, n = 38) from a well defined cohort. Spearman's rank correlation coefficients were calculated between qHBcrAg and intrahepatic markers of HBV replication [total intrahepatic-DNA, covalently closed circular (ccc) DNA, cccDNA : total intrahepatic-DNA ratio]. At biopsy, 22 (71.0%) patients were hepatitis B 'e' antigen (HBeAg)-positive, 22 (71.0%) had detectable plasma HBV-DNA, and 17 (54.8%) were treated with tenofovir. Median levels (interquartile range) of intrahepatic markers were as follows: HBV cccDNA (n = 34), 0.26 copies/cell (0.4-2.89); total intrahepatic-DNA (n = 38), 2.38 copies/cell (0.58-207.9), and cccDNA : total intrahepatic-DNA ratio (n = 34), 0.05 (interquartile range = 0.01-0.12). There was a significantly strong correlation between qHBcrAg and cccDNA in all patients (Rho = 0.65, P < 0.001), while a moderate correlation was observed between qHBcrAg and total intrahepatic-DNA (Rho = 0.57, P < 0.001) or cccDNA : total intrahepatic-DNA ratio (Rho = -0.45, P = 0.01). Similar findings were observed for HBeAg-positive patients and those with detectable HBV-DNA, with the exception of qHBcrAg and cccDNA or cccDNA : total intrahepatic-DNA ratio. In contrast, no significant correlation between qHBcrAg and any intrahepatic marker was observed in HBeAg-negative patients or those with undetectable HBV-DNA. No significant difference was observed in median qHBcrAg levels across liver fibrosis stages (P = 0.5). qHBcrAg is a potential surrogate marker of cccDNA in HIV-HBV coinfected patients, yet might be less useful with undetectable serum HBV-DNA or HBeAg-negative status. Whether qHBcrAg provides further clinical utility compared with other serological markers remains debatable.

The association of vitamin D with hepatitis B virus replication: Bystander rather than offender

Low vitamin D is frequent in hepatitis B virus (HBV)-infected patients and several studies show an inverse association of serum vitamin D level with HBV viral load. However, the causal relationship remains unclear. HBV carriers receiving regular 6-month surveillance without current antiviral treatment or cirrhosis were invited to participate into this trial. The markers of HBV replication included serum HBV DNA and quantitative HBsAg (qHBsAg) levels. Those with undetectable HBV DNA or sufficient vitamin D levels, cancer or electrolyte imbalance were excluded. The eligible subjects were randomized to receive either vitamin D supplement 2000 IU per day for 2 months (vitamin D group) or none (control group). A total of 196 HBV carriers (93 males and 103 females; mean age 51.9±10.0 years) were screened. Of them, 28 patients had undetectable serum HBV DNA levels, which is defined as spontaneous viral clearance. The vitamin D levels were not different between patients with detectable HBV DNA and those without (p=0.18). After exclusion, 149 patients were randomized to two groups: 75 in vitamin D group and 74 in control group. After 2 months vitamin D supplement, the serum vitamin D levels were significantly higher in the vitamin D group than the control group (p<0.001). However, the serum qHBsAg and HBV DNA levels were comparable between these two groups. There is no causal relationship between vitamin D and HBV replication. The role of liver reserve on serum vitamin D levels in patients with chronic HBV infection needs further investigation.

Inhibition of replication of hepatitis B virus using transcriptional repressors that target the viral DNA

BackgroundChronic infection with hepatitis B virus (HBV) is a serious global health problem. Persistence of the virus occurs as a result of stability of the replication intermediate comprising covalently closed circular DNA (cccDNA). Development of drugs that are capable of disabling this cccDNA is vital.MethodsTo investigate an epigenetic approach to inactivating viral DNA, we engineered transcriptional repressors that comprise an HBV DNA-binding domain of transcription activator like effectors (TALEs) and a fused Krüppel Associated Box (KRAB). These repressor TALEs (rTALEs) targeted the viral surface open reading frame and were placed under transcription control of constitutively active or liver-specific promoters.ResultsEvaluation in cultured cells and following hydrodynamic injection of mice revealed that the rTALEs significantly inhibited production of markers of HBV replication without evidence of hepatotoxicity. Increased methylation of HBV DNA at CpG island II showed that the rTALEs caused intended epigenetic modification.ConclusionsEpigenetic modification of HBV DNA is a new and effective means of inactivating the virus in vivo. The approach has therapeutic potential and avoids potentially problematic unintended mutagenesis of gene editing.

Effect of hepatitis B virus (HBV) surface-gene variability on markers of replication during treated human immunodeficiency virus-HBV infection in Western Africa.

Replication markers exhibit substantial variation during chronic hepatitis B virus (HBV) infection, part of which can be explained by mutations on the surface (S) gene. We aimed to identify S-gene mutations possibly influencing the quantification of HBV replication markers (MUPIQH) in HBV genotype E infection, common to Western Africa. Seventy-three antiretroviral treatment (ART)-naïve human immunodeficiency virus (HIV)-HBV co-infected patients from Côte d'Ivoire, initiating anti-HBV-containing ART, had available HBV S-gene sequences. S-gene MUPIQHs were screened at ART initiation based on lower HBV-DNA or HBsAg quantification (qHBsAg) compared to wildtype. Their association with HBV virological response and qHBsAg slope during treatment was evaluated. Genotype E was predominant (95.9%). At ART initiation, median HBV-DNA was 7.27 log10 copies/mL (IQR = 5.26-8.33) and qHBsAg 4.08 log10 IU/mL (IQR = 3.49-4.61). Twelve S-gene MUPIQHs were identified among 21 patients (28.8%): sS140L (n = 4), sD144A (n = 1), sS167L (n = 2), sS174N (n = 6), sP178Q (n = 2), sG185L (n = 2), sW191L (n = 2), sP203Q/R (n = 2), sS204N/I/R/K/T/G (n = 7), sN207T (n = 2), sF212C (n = 1) and sV224A/Y (n = 7). MUPIQHs at positions s185+s191+s224 and s178+s204 were within highly covarying networks of S-gene mutations. Older age (P = 0.02), elevated transaminases (P = 0.03) and anti-hepatitis B "e" antibody-positive serology (P = 0.009) were significantly associated with prevalent MUPIQHs at ART initiation. During treatment, baseline MUPIQHs were not associated with time-to-undetectable HBV-DNA (P = 0.7) and qHBsAg levels decreased at similar rates between those with vs without MUPIQHs (P = 0.5). Several novel S-gene mutations were associated with reductions in replication markers among West African co-infected patients. These mutations, however, do not affect response during antiviral treatment. Their diagnostic and clinical consequences need clarification.

Viral Biomarkers in Chronic HBeAg Negative HBV Infection.

Viral biomarkers are important tools for monitoring chronic hepatitis B virus (HBV) hepatitis B early antigen (HBeAg) negative infection, both in its natural course as well as during and after treatment. The biomarkers consist of antibodies against viral epitopes, viral proteins, and molecular surrogate markers of the quantity and transcriptional activity of the stable episomal HBV covalently closed circular DNA (cccDNA) which is located in the nuclei of the infected hepatocytes. HBV deoxyribonucleic acid (DNA) or else viral load measurement in plasma or serum is a marker of HBV replication of major clinical importance. HBV DNA is used for staging and treatment monitoring as described in international scientific guidelines. Quantification of HBV antigens, mainly hepatitis B surface antigen (HBsAg) as well as Hepatitis B core related antigen (HBcrAg), play an important yet secondary role, especially in cases of low or undetectable HBV DNA and has been evaluated for the classification of the inactive carrier state, as a predictor of subsequent HBsAg clearance, treatment outcome, and development of hepatocellular carcinoma (HCC). The measurement of the replicative intermediate HBV RNA in serum is currently evaluated and may also prove to be a significant biomarker particularly in patients treated with nucleot(s)ide analogs. This review focuses on the viral biomarkers mentioned above and their role in HBV, HBeAg negative, infection.

Relative Abundance of Integrant-Derived Viral RNAs in Infected Tissues Harvested from Chronic Hepatitis B Virus Carriers.

Five matching sets of nonmalignant liver tissues and hepatocellular carcinoma (HCC) samples from individuals chronically infected with hepatitis B virus (HBV) were examined. The HBV genomic sequences were determined by using overlapping PCR amplicons covering the entire viral genome. Four pairs of tissues were infected with HBV genotype C, while one pair was infected with HBV genotype B. HBV replication markers were found in all tissues. In the majority of HCC samples, the levels of pregenomic/precore RNA (pgRNA) and covalently closed circular DNA (cccDNA) were lower than those in liver tissue counterparts. Regardless of the presence of HBV replication markers, (i) integrant-derived HBV RNAs (id-RNAs) were found in all tissues by reverse transcription-PCR (RT-PCR) analysis and were considerably abundant or predominant in 6/10 tissue samples (2 liver and 4 HCC samples), (ii) RNAs that were polyadenylated using the cryptic HBV polyadenylation signal and therefore could be produced by HBV replication or derived from integrated HBV DNA were found in 5/10 samples (3 liver and 2 HCC samples) and were considerably abundant species in 3/10 tissues (2 livers and 1 HCC), and (iii) cccDNA-transcribed RNAs polyadenylated near position 1931 were not abundant in 7/10 tissues (2 liver and 5 HCC samples) and were predominant in only two liver samples. Subsequent RNA sequencing analysis of selected liver/HCC samples also showed relative abundance of id-RNAs in most of the examined tissues. Our findings suggesting that id-RNAs could represent a significant source of HBV envelope proteins, which is independent of viral replication, are discussed in the context of the possible contribution of id-RNAs to the HBV life cycle.IMPORTANCE The relative abundance of integrant-derived HBV RNAs (id-RNAs) in chronically infected tissues suggest that id-RNAs coding for the envelope proteins may facilitate the production of a considerable fraction of surface antigens (HBsAg) in infected cells bearing HBV integrants. If the same cells support HBV replication, then a significant fraction of assembled HBV virions could bear id-RNA-derived HBsAg as a major component of their envelopes. Therefore, the infectivity of these HBV virions and their ability to facilitate virus cell-to-cell spread could be determined mainly by the properties of id-RNA-derived envelope proteins and not by the properties of replication-derived HBsAg. These interpretations suggest that id-RNAs may play a role in the maintenance of chronic HBV infection and therefore contribute to the HBV life cycle. Furthermore, the production of HBsAg from id-RNAs independently of viral replication may explain at least in part why treatment with interferon or nucleos(t)ides in most cases fails to achieve a loss of serum HBsAg.

Delta hepatitis: Toward improved diagnostics.

Delta hepatitis: Toward improved diagnostics.

Sustained Inhibition of HBV Replication InVivo after Systemic Injection of AAVs Encoding Artificial Antiviral Primary MicroRNAs.

Chronic infection with hepatitis B virus (HBV) remains a problem of global significance and improving available treatment is important to prevent life-threatening complications arising in persistently infected individuals. HBV is susceptible to silencing by exogenous artificial intermediates of the RNA interference (RNAi) pathway. However, toxicity of Pol III cassettes and short duration of silencing by effectors of the RNAi pathway may limit anti-HBV therapeutic utility. To advance RNAi-based HBV gene silencing, mono- and trimeric artificial primary microRNAs (pri-miRs) derived from pri-miR-31 were placed under control of the liver-specific modified murine transthyretin promoter. The sequences, which target the X sequence of HBV, were incorporated into recombinant hepatotropic self-complementary adeno-associated viruses (scAAVs). Systemic intravenous injection of the vectors into HBV transgenic mice at a dose of 1 × 1011 per animal effected significant suppression of markers of HBV replication for at least 32 weeks. The pri-miRs were processed according to the intended design, and intrahepatic antiviral guide sequences were detectable for 40 weeks after the injection. There was no evidence of toxicity, and innate immunostimulation was not detectable following the injections. This efficacy is an improvement on previously reported RNAi-based inhibition of HBV replication and is important to clinical translation of the technology.

Serum vitamin D level not correlated with hepatitis B virus replication in patients with chronic hepatitis B virus infection

SummaryBackgroundsVitamin D is an important immune modulator in chronic liver diseases. The study aims to investigate the relationship between vitamin D and hepatitis B virus replication.MethodsThis was a prospective study. Hepatitis B virus (HBV) carriers were enrolled from May to Oct 2014 in Taipei Tzu Chi Hospital. All have positive hepatitis B surface antigen (HBsAg) for more than 6 months. Serum alanine aminotransferase (ALT), quantitative HBsAg (qHBsAg), HBV DNA and 25(OH)D3 were measured in each participant.Results204 patients (male 103; mean age 47.86 ± 10.22 years) with chronic HBV infection (94.12% HBeAg‐negative) were enrolled. Of them, 13 (6.4%) had severe deficiency (25(OH)D3 &lt; 10 ng/mL), 176 (86.3%) insufficiency (25(OH)D3 ≥ 10 and &lt; 30 ng/mL) and 15 (7.4%) adequate (25(OH)D3 ≧ 30 ng/mL) vitamin D levels. In the regard of age and sex, there was no difference in vitamin D levels. Using linear regression analysis, there was no correlation between serum vitamin D and HBV DNA/qHBsAg levels. While serum vitamin D levels were categorized by quartiles, there was no association between vitamin D levels and HBV DNA/qHBsAg levels using univariate and multivariate analyses.ConclusionsIn this study, 92.6% of patients with chronic HBV infection have inadequate vitamin D levels. Serum vitamin D levels do not correlate with the markers of HBV replication including serum HBV DNA and qHBsAg levels.Copyright © 2017, The Gastroenterological Society of Taiwan, The Digestive Endoscopy Society of Taiwan and Taiwan Association for the Study of the Liver.

HDV RNA replication is associated with HBV repression and interferon-stimulated genes induction in super-infected hepatocytes

Hepatitis D virus (HDV) super-infection of Hepatitis B virus (HBV)-infected patients is the most aggressive form of viral hepatitis. HDV infection is not susceptible to direct anti-HBV drugs, and only suboptimal antiviral responses are obtained with interferon (IFN)-alpha-based therapy. To get insights on HDV replication and interplay with HBV in physiologically relevant hepatocytes, differentiated HepaRG (dHepaRG) cells, previously infected or not with HBV, were infected with HDV, and viral markers were extensively analyzed. Innate and IFN responses to HDV were monitored by measuring pro-inflammatory and interferon-stimulated gene (ISG) expression. Both mono- and super-infected dHepaRG cells supported a strong HDV intracellular replication, which was accompanied by a strong secretion of infectious HDV virions only in the super-infection setting and despite the low number of co-infected cells. Upon HDV super-infection, HBV replication markers including HBeAg, total HBV-DNA and pregenomic RNA were significantly decreased, confirming the interference of HDV on HBV. Yet, no decrease of circular covalently closed HBV DNA (cccDNA) and HBsAg levels was evidenced. At the peak of HDV-RNA accumulation and onset of interference on HBV replication, a strong type-I IFN response was observed, with interferon stimulated genes, RSAD2 (Viperin) and IFI78 (MxA) being highly induced. We established a cellular model to characterize in more detail the direct interference of HBV and HDV, and the indirect interplay between the two viruses via innate immune responses. This model will be instrumental to assess molecular and immunological mechanisms of this viral interference.

Quantification of serum HBV RNA allows differentiation of disease stages of hepatitis B virus (HBV) infections

Introduction: Inactive carriers of HBV infections are distinguished from individuals with active HBV infections by serum levels of HBV DNA. HBV RNA is another surrogate marker of HBV replication which can be quantified from serum. The aim of this study was to explore whether HBV RNA levels permits a better differentiation between the various stages of HBV infection. Methods: Serum samples from 12 patients with acute and 96 with chronic HBV infection (34 inactive HBsAg carrier with HBV DNA < 4 log10 copies/ml and ALT within normal ranges, 34 HBeAg negative and 28 HBeAg positive patients with chronic active hepatitis B (mean age 45.4 ± 15.6 (17 – 77) years, m/w = 73/35, mean ALT 8.7 ± 19.2 (0.22 – 100)µkat/ml, mean HBsAg 4.3 ± 0.9 (0.7 – 6.0) log10 ng/ml) were retrospectively analyzed. HBV DNA was measured by real-time PCR (limit of detection 2.6 log10 copies/ml). HBV-RNA levels were quantified based on a sensitive One-Step-RACE-PCR-Assay which was developed and validated (limit of detection 2.9 log10 copies/ml). Results: HBV RNA could be detected in 9 of 12 patients with acute HBV infection with a mean of 3.4 ± 1.6 (2.0 – 5.9) log10 copies/ml. HBV RNA was detectable in 29 out of 34 HBeAg negative patients and in all 28 patients with HBeAg positive chronic active HBV infection and showed a high correlation with HBV DNA levels (r = 0.93). Mean HBV DNA levels were significantly lower in HBeAg negative as compared to HBeAg positive patients (6.8 ± 1.3 (4.2 – 8.9) vs. 8.2 ± 1.4 (4.5 – 10.5) log10 copies/ml, p < 0.001), and so were mean HBV RNA levels (4.4 ± 1.5 (2.0 – 7.0) vs. 6.8 ± 1.2 (4.3 – 9.1) log10 copies/ml, p < 0.001). In contrast, HBV RNA was detectable in only in 2 out of 34 inactive HBsAg carriers (2.0 ± 0.2 (2.0 – 2.7) log10 copies/ml), while in 28 of those patients HBV DNA was still detectable with a mean of 3.1 ± 0.7 (2.0 – 4.6) log10 copies/ml. Conclusion: Serum levels of HBV RNA show a high correlation with HBV DNA levels in patients with active chronic HBV infection. However, in patients with inactive HBV infections, HBV RNA is less frequently detected than HBV DNA. It needs to be studied whether HBV RNA serum levels can identify patient inactive carrier status that have disease progression. Corresponding author: Krauel, Alexander E-Mail:Alexander.Krauel@medizin.uni-leipzig.de

Detection of Hepatitis B Virus Covalently Closed Circular DNA in the Plasma of Iranian HBeAg-Negative Patients With Chronic Hepatitis B.

Background:Covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) is a marker of HBV replication in the liver of patients infected with HBV.Objectives:This study aimed to investigate the association between the presence of cccDNA in the plasma samples of Iranian treatment-naive patients with chronic hepatitis B infection and HBV viral load and HBsAg levels.Patients and Methods:From April 2012 to May 2015, 106 treatment-naive patients with chronic hepatitis B infection were enrolled in this cross-sectional study. The HBsAg titer was measured by the Roche HBsAg II assay on the Cobas e411 system, and HBV DNA quantitation was performed using the COBAS TaqMan 48 kit. Real-time polymerase chain reaction was performed for the detection of HBV cccDNA.Results:The mean (SD) age of the patients was 41.1 ± 12.4 years (range, 20 - 62 years). From a total of 106 study participants, 67 (63.2%) were males. The HBV cccDNA was detected in plasma specimens in 19 (17.9%) out of the total 106 patients, and a significant relationship was found between the presence of cccDNA in plasma sample of males (23.9%) and females (7.7%) (P = 0.039). Also, a significant correlation was found between the presence of cccDNA in plasma sample of the patients and HBV viral load level (P < 0.0001) and HBsAg titer (P = 0.0043).Conclusions:This study showed that cccDNA can be detected in the plasma specimen of 17.9% of Iranian treatment-naive patients with chronic hepatitis B infection. Therefore, designing prospective studies focusing on the detection of cccDNA in these patients would provide more information.

Interferon-γ and Tumor Necrosis Factor-α Produced by T Cells Reduce the HBV Persistence Form, cccDNA, Without Cytolysis

Viral clearance involves immune cell cytolysis of infected cells. However, studies of hepatitis B virus (HBV) infection in chimpanzees have indicated that cytokines released by T cells also can promote viral clearance via noncytolytic processes. We investigated the noncytolytic mechanisms by which T cells eliminate HBV from infected hepatocytes. We performed a cytokine enzyme-linked immunosorbent assay of serum samples from patients with acute and chronic hepatitis B. Liver biopsy specimens were analyzed by in situ hybridization. HepG2-H1.3 cells, HBV-infected HepaRG cells, and primary human hepatocytes were incubated with interferon-γ (IFNγ) or tumor necrosis factor-α (TNF-α), or co-cultured with T cells. We measured markers of HBV replication, including the covalently closed circular DNA (cccDNA). Levels of IFNγ and TNF-α were increased in serum samples from patients with acute vs chronic hepatitis B and controls. In human hepatocytes with stably replicating HBV, as well as in HBV-infected primary human hepatocytes or HepaRG cells, IFNγ and TNF-α each induced deamination of cccDNA and interfered with its stability; their effects were additive. HBV-specific T cells, through secretion of IFNγ and TNF-α, inhibited HBV replication and reduced cccDNA in infected cells without the direct contact required for cytolysis. Blocking IFNγ and TNF-α after T-cell stimulation prevented the loss of cccDNA. Deprivation of cccDNA required activation of nuclear APOBEC3 deaminases by the cytokines. In liver biopsy specimens from patients with acute hepatitis B, but not chronic hepatitis B or controls, hepatocytes expressed APOBEC3A and APOBEC3B. IFNγ and TNF-α, produced by T cells, reduce levels of HBV cccDNA in hepatocytes by inducing deamination and subsequent cccDNA decay.