Hepatocellular Carcinoma and Hepatitis: Advanced Diagnosis and Management with a Focus on the Prevention of Hepatitis B-Related Hepatocellular Carcinoma.

Though the world-wide hepatitis B virus (HBV) vaccination program has been well completed for almost thirty years in many nations, almost HBV-related hepatocellular carcinoma (HCC) occurs in unvaccinated middle-aged and elderly adults. Apparently, treating 80% of qualified subjects could decrease HBV-related mortality by 65% in a short period. Nevertheless, globally, only 2.2% of CHB patients undergo antiviral therapy. The HBV markers related to HCC occurrence and prevention are as follows: the HCC risk is the highest at a baseline of HBV DNA of 6-7 log copies/mL, and it is the lowest at a baseline of an HBV DNA level of >8 log copies/mL and ≤4 log copies/mL (parabolic, and not linear pattern). The titer of an HBV core-related antigen (HBcrAg) reflecting the amount of HBV covalently closed circular DNA (ccc DNA) in the liver is related to HCC occurrence. The seroclearance of HBs antigen (HBsAg) is more crucial than HBV DNA negativity for the prevention of HCC. In terms of the secondary prevention of hepatitis B-related HCC involving antiviral therapies with nucleos(t)ide analogues (NAs), unsolved issues include the definition of the immune-tolerant phase; the optimal time for starting antiviral therapies with NAs; the limits of increased aminotransferase (ALT) levels as criteria for therapy in CHB patients; the normalization of ALT levels with NAs and the relation to the risk of HCC; and the relation between serum HBV levels and the risk of HCC. Moreover, the first-line therapy with NAs including entecavir (ETV), tenofovir disoproxil fumarate (TDF), and tenofovir alafenamide (TAF) remains to be clarified. Discussed here, therefore, are the recent findings of HBV markers related to HCC occurrence and prevention, unsolved issues, and the current secondary antiviral therapy for the prevention of HBV-related HCC.

Strain-specific responsiveness of hepatitis D virus to interferon-alpha treatment.

Background & Aims: Pegylated interferon alpha (pegIFNα) is commonly used for the treatment of people infected with HDV. However, its mode of action in HDV-infected cells remains elusive and only a minority of people respond to pegIFNα therapy. Herein, we aimed to assess the responsiveness of three different cloned HDV strains to pegIFNα. We used a previously cloned HDV genotype 1 strain (dubbed HDV-1a) that appeared insensitive to interferon-α in vitro, a new HDV strain (HDV-1p) we isolated from an individual achieving later sustained response to IFNα therapy, and one phylogenetically distant genotype 3 strain (HDV-3). Methods: PegIFNα was administered to human liver chimeric mice infected with HBV and the different HDV strains or to HBV/HDV infected human hepatocytes isolated from chimeric mice. Virological parameters and host responses were analysed by qPCR, sequencing, immunoblotting, RNA in situ hybridisation and immunofluorescence staining. Results: PegIFNα treatment efficiently reduced HDV RNA viraemia (∼2-log) and intrahepatic HDV markers both in mice infected with HBV/HDV-1p and HBV/HDV-3. In contrast, HDV parameters remained unaffected by pegIFNα treatment both in mice (up to 9 weeks) and in isolated cells infected with HBV/HDV-1a. Notably, HBV viraemia was efficiently lowered (∼2-log) and human interferon-stimulated genes similarly induced in all three HBV/HDV-infected mouse groups receiving pegIFNα. Genome sequencing revealed highly conserved ribozyme and L-hepatitis D antigen post-translational modification sites among all three isolates. Conclusions: Our comparative study indicates the ability of pegIFNα to lower HDV loads in stably infected human hepatocytes in vivo and the existence of complex virus-specific determinants of IFNα responsiveness. Impact and implications: Understanding factors counteracting HDV infections is paramount to develop curative therapies. We compared the responsiveness of three different cloned HDV strains to pegylated interferon alpha in chronically infected mice. The different responsiveness of these HDV isolates to treatment highlights a previously underestimated heterogeneity among HDV strains.

Evaluation and influencing factor analysis of the detection effect of hepatitis B virus surface antigen and treponema pallidum using rapid screening immunochromatography technology / 中国输血杂志

【Objective】 To evaluate the effectiveness of rapid initial screening using HBsAg and syphilis reagents of immunochromatography technology before blood donation, and explore the influencing factors. 【Methods】 The pre-donation screening of HBsAg and anti-TP and post-donation blood test results of blood donors in Yangzhou region from January 2020 to June 2023 were retrospectively analyzed. The HBsAg and anti-TP reactive samples by ELISA from January to June 2023 were, retested using colloidal gold immunochromatographic reagents, and the results were compared and analyzed. 【Results】 A total of 200 414 blood donors were screened, among which 781 were HBsAg and anti-TP positive, accounting for 0.39%. A total of 191 717 blood donors successfully donated blood, and 986 were HBsAg and anti-TP positive by ELISA, accounting for 0.51%. 62 HBsAg and 61 anti-TP reactive samples were retested using the initial screening reagent, with 24 HBsAg reactive samples and 26 anti-TP reactive samples, accounting for 38.71% and 42.62% respectively. 14 HBsAg and 6 anti-TP gray area samples were retested, but no reactivity was found.The reactivity rates of 9 samples with HBsAg detection S/CO values greater than 25.0 and 10 samples with anti-TP detection S/CO values greater than 15.0 were all 100%.There was a negative correlation between the reaction intensity (S/CO value) of reactive samples and interpretation time of initial screening reaction. 【Conclusion】 The rapid primary screening of hepatitis B and syphilis with immunochromatography technology among blood donors can effectively improve the quality of blood and the qualification rate of blood after collection. Through targeted training of primary screening staff, the quality of primary screening can be further improved, the rate of missed detection can be reduced, and costs can be saved, thus reducing the risk of transfusion transmitted infection and ensuring the health of blood donors.

Low seroprevalence of hepatitis delta virus co-infection in hepatitis B virus-infected blood donors in China: A multicenter study.

Hepatitis delta virus (HDV) coinfected with HBV causes severe viral hepatitis, however, the number of HDV infection may be underestimated. In the present study, we enrolled 1,141,331 blood donations, routinely tested for HBsAg and/or HBV DNA, from 21 blood establishments in China. 2,690 donors were HBsAg and/or HBV DNA positive after screening tests. After verification of HBsAg and HBV DNA, 1,490 samples were HBsAg confirmed-positive, including 1,459 HBV DNA-positive samples, and 825 samples were seronegative but HBV DNA positive. We first analyzed demographic characteristics of involved 2,690 donors with different HBV infection status and found the proportions of males, the older donors, workers and farmers were higher in HBsAg-/HBV DNA+ group. Then we evaluated specificity of HDV IgG and IgM antibody assays with 375 HBsAg and HBV DNA confirmed-negative samples, and 374 were tested negative using the two assays, respectively, suggesting a specificity of 99.73% for both assays (374/375, 95% Cl: 98.51-99.95%). Subsequently, we tested for HDV IgG and IgM of 2,315 HBsAg and/or HBV DNA confirmed-positive samples, and nine showed reactivity for IgG, while two were reactive for IgM. All these 11 reactive samples were tested again with another HDV pan-Ig and IgM testing assays and HDV RNA, and only one donor was identified as HDV IgG positive and HDV RNA negative, showing an HDV seroprevalence of 0.067% (95%CI: 0.012-0.38%) among HBsAg-positive blood donors in China. The positive donor was followed up for 2 years after the donation date, and decreased antibody titer of HDV IgG and HBsAg conversion were observed, and the infection status of the donor was HDV infection with recovery and occult hepatitis B virus infection with genotype C2. These results indicated a low seroprevalence of HDV infection among blood donors and a low risk of HDV transmission through blood transfusion in China.

Small Hepatitis B Virus Surface Antigen Promotes Hepatic Gluconeogenesis via Enhancing Glucagon/cAMP/Protein Kinase A/CREB Signaling.

Hepatitis B virus (HBV) is a major risk factor for serious liver diseases. The liver plays a unique role in controlling carbohydrate metabolism to maintain the glucose level within the normal range. Chronic HBV infection has been reported to associate with a high prevalence of diabetes. However, the detailed molecular mechanism underlying the potential association remains largely unknown. Here, we report that liver-targeted delivery of small HBV surface antigen (SHBs), the most abundant viral protein of HBV, could elevate blood glucose levels and impair glucose and insulin tolerance in mice by promoting hepatic gluconeogenesis. Hepatocytes with SHB expression also exhibited increased glucose production and expression of gluconeogenic genes glucose-6-phosphatase (G6pc) and phosphoenolpyruvate carboxykinase (PEPCK) in response to glucagon stimulation. Mechanistically, SHBs increased cellular levels of cyclic AMP (cAMP) and consequently activated protein kinase A (PKA) and its downstream effector cAMP-responsive element binding protein (CREB). SHBs-induced activation of CREB enhanced transcripts of gluconeogenic genes, thus promoting hepatic gluconeogenesis. The elevated cAMP level resulted from increased transcription activity and expression of adenylyl cyclase 1 (AC1) by SHBs through a binary E-box factor binding site (BEF). Taken together, we unveiled a novel pathogenic role and mechanism of SHBs in hepatic gluconeogenesis, and these results might highlight a potential target for preventive and therapeutic intervention in the development and progression of HBV-associated diabetes. IMPORTANCE Chronic HBV infection causes progressive liver damage and is found to be a risk factor for diabetes. However, the mechanism in the regulation of glucose metabolism by HBV remains to be established. In the current study, we demonstrate for the first time that the small hepatitis B virus surface antigen (SHBs) of HBV elevates AC1 transcription and expression to activate cAMP/PKA/CREB signaling and subsequently induces the expression of gluconeogenic genes and promotes hepatic gluconeogenesis both in vivo and in vitro. This study provides a direct link between HBV infection and diabetes and implicates that SHBs may represent a potential target for the treatment of HBV-induced metabolic disorders.

Multifaceted Defects in Monocytes in Different Phases of Chronic Hepatitis B Virus Infection: Lack of Restoration after Antiviral Therapy.

Monocytes play an important role in the control of microbial infection, but monocyte biology during chronic hepatitis B virus (HBV) infection (CHI) remains inadequately studied. We investigated the frequency, phenotype, and functions of monocyte subsets in different phases of CHI, namely, immune tolerance (IT), hepatitis B early antigen (HBeAg)-positive/HBeAg-negative chronic hepatitis B (EP-/EN-CHB, respectively), and inactive carrier (IC), identified factors responsible for their functional alterations, and determined the impact of antiviral therapy on these cells. Flow cytometric analysis indicated that HLA-DR+ CD14++ CD16- classical monocytes were significantly reduced while HLA-DR+ CD14++ CD16+ intermediate and HLA-DR+ CD14+ CD16++ nonclassical monocytes were expanded in IT and EP-/EN-CHB compared with those in IC and healthy controls (HC). In comparison to IC/HC, monocytes in IT and CHB exhibited diminished expression of Toll-like receptor 2 (TLR-2)/TLR-4/TLR-9 and cytokines interleukin-12 (IL-12)/tumor necrosis factor alpha (TNF-α)/IL-6 but produced higher levels of IL-10/transforming growth factor ß (TGF-ß). Further, monocytes in CHB/IT showed impaired phagocytosis and oxidative response relative to those in IC/HC. In vitro assays indicated that high titers of hepatitis B surface antigen (HBsAg) present in IT/CHB and of IL-4 in CHB triggered the functional defects in monocytes via induction of ß-catenin. Additionally, monocyte-derived M1 macrophages of CHB/IT produced fewer proinflammatory and more anti-inflammatory cytokines than those of IC/HC, while in CHB/IT, the monocytes skewed the differentiation of CD4+ T cells more toward regulatory T cells and a Th2 phenotype. Moreover, monocytes in CHB and IT overexpressed chemokine receptor CCR2, which coincided with increased intrahepatic accumulation of ß-catenin+ CD14+ cells. One year of tenofovir therapy failed to normalize monocyte functions or reduce serum HBsAg/IL-4 levels. Taken together, monocytes are functionally perturbed mostly in IT and EP-/EN-CHB phases. Targeting intramonocytic ß-catenin or reducing HBsAg/IL-4 levels might restore monocyte function and facilitate viral clearance. IMPORTANCE Chronic HBV infection (CHI) is a major cause of end-stage liver disease for which pharmacological treatments currently available are inadequate. Chronically HBV-infected patients fail to mount an efficient immune response to the virus, impeding viral clearance and recovery from hepatitis. Monocytes represent a central part of innate immunity, but a comprehensive understanding on monocyte involvement in CHI is still lacking. We here report a multitude of defects in monocytes in chronically HBV-infected patients that include alteration in subset distribution, Toll-like receptor expression, cytokine production, phagocytic activity, oxidative response, migratory ability, polarization of monocyte-derived macrophages, and monocyte-T-cell interaction. We demonstrated that high levels of hepatitis B virus surface antigen and IL-4 potentiate these defects in monocytes via ß-catenin induction while therapy with the nucleotide analog tenofovir fails to restore monocyte function. Our findings add to the continuing effort to devise new immunotherapeutic strategies that could reverse the immune defects in CHI.

Virological responses to tenofovir-alafenamide-containing antiretroviral therapy in people living with HIV co-infected with lamivudine-resistant or lamivudine-susceptible hepatitis B virus.

BACKGROUND: Data on the effectiveness of tenofovir alafenamide (TAF) against lamivudine-resistant (LAM-R) hepatitis B virus (HBV) among patients co-infected with human immunodeficiency virus (HIV) and HBV are limited. METHODS: Between April and December 2018, HIV-positive patients co-infected with LAM-R or lamivudine-susceptible (LAM-S) HBV who switched from tenofovir-disoproxil-fumarate-containing antiretroviral therapy (ART) to TAF-containing ART were followed for 96 weeks. Plasma HBV and HIV loads, HBV serological markers, and liver function before and after the switch were analysed. RESULTS: In total, 182 patients co-infected with HIV and HBV were included in this study: 45 with LAM-R HBV and 137 with LAM-S HBV. At baseline, 28.9% and 7.4% of patients in the LAM-R and LAM-S groups, respectively, tested positive for hepatitis B virus envelope antigen (HBeAg) (P<0.001), and the respective percentages of patients who had achieved plasma HBV DNA <20 IU/mL were 95.5% and 97.1%. At weeks 48 and 96, 100% and 94.9% of patients in the LAM-R group, respectively, and 97.1% and 95.6% of patients in the LAM-S group, respectively, maintained plasma HBV DNA <20 IU/mL. Lamivudine resistance of HBV and baseline hepatitis B virus surface antigen (HBsAg) level were associated with HBsAg decrement at week 96 at a degree of 0.25 log10 IU/mL [95% confidence interval (CI) 0.059-0.246] and 0.22 log10 IU/mL (per 1-log10IU/mL increase, 95% CI 0.018-0.101), respectively. At week 96, 2.2% (4/182) of patients had HBsAg loss; no patients in the LAM-R group and 25.0% (2/8) of patients in the LAM-S group had HBeAg seroconversion. CONCLUSIONS: Switching to TAF-containing regimens maintained high rates of HBV viral suppression in patients co-infected with either LAM-R or LAM-S HBV. The decrease in HBsAg was minimal, and HBsAg seroconversion occurred infrequently.

Nationwide retrospective study of hepatitis B virological response and liver stiffness improvement in 465 patients on nucleos(t)ide analogue.

BACKGROUND: Hepatitis B virus (HBV) nucleos(t)ide analog (NA) therapy reduces liver disease but requires prolonged therapy to achieve hepatitis B surface antigen (HBsAg) loss. There is limited North American real-world data using non-invasive tools for fibrosis assessment and few have compared 1st generation NA or lamivudine (LAM) to tenofovir disoproxil fumarate (TDF). AIM: To assess impact of NA on virological response and fibrosis regression using liver stiffness measurement (LSM) (i.e., FibroScan®). METHODS: Retrospective, observational cohort study from the Canadian HBV Network. Data collected included demographics, NA, HBV DNA, alanine aminotransferase (ALT), and LSM. Patients were HBV monoinfected patients, treatment naïve, and received 1 NA with minimum 1 year follow-up. RESULTS: In 465 (median 49 years, 37% female, 35% hepatitis B e antigen+ at baseline, 84% Asian, 6% White, and 9% Black). Percentage of 64 (n = 299) received TDF and 166 were LAM-treated with similar median duration of 3.9 and 3.7 years, respectively. The mean baseline LSM was 11.2 kPa (TDF) vs 8.3 kPa (LAM) (P = 0.003). At 5-year follow-up, the mean LSM was 7.0 kPa in TDF vs 6.7 kPa in LAM (P = 0.83). There was a significant difference in fibrosis regression between groups (i.e., mean -4.2 kPa change in TDF and -1.6 kPa in LAM, P < 0.05). The last available data on treatment showed that all had normal ALT, but more TDF patients were virologically suppressed (< 10 IU/mL) (n = 170/190, 89%) vs LAM-treated (n = 35/58, 60%) (P < 0.05). None cleared HBsAg. CONCLUSION: In this real-world North American study, approximately 5 years of NA achieves liver fibrosis regression rarely leads to HBsAg loss.

Dual role of neddylation in transcription of hepatitis B virus RNAs from cccDNA and production of viral surface antigen.

Background & Aims: HBV persistence is maintained by both an episomal covalently closed circular (ccc)DNA reservoir and genomic integration of HBV DNA fragments. While cccDNA transcription is regulated by Cullin4A-DDB1-HBx-mediated degradation of the SMC5/6 complex, HBsAg expression from integrants is largely SMC5/6 independent. Inhibiting neddylation of Cullin-RING ubiquitin ligases impairs degradation of substrates. Herein, we show that targeting neddylation pathway components by small-interfering (si)RNAs or the drug MLN4924 (pevonedistat) suppresses expression of HBV proteins from both cccDNA and integrants. Methods: An siRNA screen targeting secretory pathway regulators and neddylation genes was performed. Activity of MLN4924 was assessed in infection and integration models. Trans-complementation assays were used to study HBx function in cccDNA-driven expression. Results: siRNA screening uncovered neddylation pathway components (Nedd8, Ube2m) that promote HBsAg production post-transcriptionally. Likewise, MLN4924 inhibited production of HBsAg encoded by integrants and reduced intracellular HBsAg levels, independent of HBx. MLN4924 also profoundly inhibited cccDNA transcription in three infection models. Using the HBV inducible cell line HepAD38 as a model, we verified the dual action of MLN4924 on both cccDNA and integrants with sustained suppression of HBV markers during 42 days of treatment. Conclusions: Neddylation is required both for transcription of a cccDNA reservoir and for the genomic integration of viral DNA. Therefore, blocking neddylation might offer an attractive approach towards functional cure of chronic hepatitis B. Lay summary: Current treatments for chronic hepatitis B are rarely able to induce a functional cure. This is partly because of the presence of a pool of circular viral DNA in the host nucleus, as well as viral DNA fragments that are integrated into the host genome. Herein, we show that a host biological pathway called neddylation could play a key role in infection and viral DNA integration. Inhibiting this pathway could hold therapeutic promise for patients with chronic hepatitis B.

Laboratory Evaluation of Hepatitis C Virus Infection in Patients Undergoing Hemodialysis from North East India.

Introduction: Subjects undergoing hemodialysis have enhanced vulnerability to hepatitis C virus (HCV) infection due to invasive procedures and poor infection control practices. Early detection and treatment are essential to prevent cross-infection and mortality/morbidity. However, common use anti-HCV antibody tests lack the necessary accuracy, and alternative tests (e.g. core antigen detection kits) which are available need to be examined as a viable alternative. Method: A total of 270 continuous serum samples were collected from patients undergoing dialysis within 15 months of study period. Sequentially, multiple tests were performed - immunochromatography-based rapid test, third-generation ELISA i.e. (anti-HCV antibody detection), fourth-generation ELISA (HCV antigen-antibody combined detection assay), and HCV RNA quantitative real time polymerase chain reaction (qPCR) assay. Diagnostic parameters of serological kits were compared in terms of sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), accuracy, and so on. Statistical Package for the Social Sciences was used. Results: HCV-combined core antigen-antibody assays performed better than other serological assays in reference to the gold standard HCV RNA. This fourth-generation assay yielded a Kappa value of 0.947 compared with the value of 0.747 and 0.619 for anti-HCV ELISA and rapid detection test. Other parameters such as sensitivity, specificity, PPV, NPV, and so on were also better for fourth-generation ELISA compared with third-generation ELISA and other serological assays. HCV RNA was negative in 7.3% of anti-HCV-positive patients and was detected in 11.4% of anti-HCV ELISA-negative patients. In about 1.6% of HCV RNA-positive cases, fourth-generation ELISA was negative and had low HCV viral load (650 IU/ml and below). Fourth generation ELISA detected additional 7.4% HCV positive cases (compared to third generation kits) and upon cost effective analyis, additional cost to be bear for the better detection (by fourth generation kit) was found to be only INR 27 per 1% increased case detection. Conclusion: In resource scant setup, screening and follow-up of patients undergoing hemodialysis can be performed by fourth-generation HCV ELISA (antigen-antibody combined assay) instead of the current practice of anti-HCV antibody ELISA. Better yield in detection rate will compensate for slight addition to costs.

Control of occult hepatitis B virus infection.

Background: The diagnosis of hepatitis B virus (HBV) infection requires HBV DNA testing and serologic testing for detection of the surface antigen (HBsAg) and the hepatitis B core antibody (anti-HBc). There is a population of patients with occult HBV infection (OBI), which is not detected by HBsAg or HBV DNA quantification in blood, despite the presence of active replication in the liver. Scope: This document provides a definition of OBI and describes the diagnostic techniques currently used. It also addresses the detection of patients with risk factors and the need for screening for OBI in these patients. Summary: Correct diagnosis of OBI prevents HBV reactivation and transmission. Diagnosis of OBI is based on the detection of HBV DNA in patients with undetectable HBsAg in blood. Perspectives: A high number of patients with OBI may remain undiagnosed; therefore, screening for OBI in patients with factor risks is essential. For a correct diagnosis of OBI, it is necessary that new markers such as ultrasensitive HBsAg are incorporated, and a more comprehensive marker study is performed by including markers such as cccDNA.

Detection of peripheral blood T lymphocyte subpopulations among patients positive for both hepatitis B virus surface antigen and surface antibody / 中国热带医学

@#Abstract: Objective　To investigate the frequency of peripheral blood T cells among patients positive for both hepatitis B virus surface antigen (HBsAg) and surface antibody (HBsAb). Methods　 Thirty six patients with co-existence of HBsAg and HBsAb diagnosed were enrolled as the experimental group, who were admitted by Shanghai tenth people's hospital and Wuxi 9th people's hospital from 2014 to 2020. while 40 patients tested positive for HBsAg and negative for HBsAb served as controls, who were admitted by Wuxi 9th people's hospital. Flow cytometry was used to detect and compare the proportions of peripheral blood CD3+, CD4+ and CD8+ T cells between the experimental and control groups. In addition, the associations of serum HBsAb level with peripheral blood T cell proportions, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were examined among chronic hepatitis B patients with co-existence of HBsAg and HBsAb using Pearson correlation analysis. Results　The median age, gender distribution, mean ALT and AST concentrations, proportion of HBV DNA viral load>103 copies/mL, seroprevalence of HBV E antigen (HBeAg), seroprevalence of HBV E antibody (HBeAb), seroprevalence of HBV core antibody (HBcAb) were comparable between the experimental and control groups, and there were no significant difference in them (P>0.05). There were no significant difference between the experimental and control groups in terms of CD3+ T cell proportion [(71.83±1.50)% vs (72.75±1.47)%; t=0.66, P>0.05], CD4+ T cell proportion [(36.81±1.53)% vs (39.88±1.57)%; t=1.43, P>0.05] and CD8+ T cell proportion [(33.17±2.04)% vs (32.40±1.75)%; t=0.77, P>0.05]. Pearson correlation analysis revealed that the serum HBsAb level did not significantly correlate with peripheral blood CD3+ (r=0.026, P=0.65), CD4+ (r=‒0.08, P=0.16) and CD8+ T cell proportions (r=0.09, P=0.24), CD4+/CD8+ T proportion (r=‒0.005, P=0.35), serum ALT (r=0.04, P=0.56) and AST levels (r=0.002, P=0.69) among chronic hepatitis B patients with co-existence of HBsAg and HBsAb. Conclusions　There are no significant differences between HBsAg+/HBsAb+ and HBsAg+/HBsAb- CHB patients in terms of peripheral blood CD3+, CD4+ and CD8+ T cell proportions.

Silicon Nanowire Field-Effect Transistor as Label-Free Detection of Hepatitis B Virus Proteins with Opposite Net Charges.

The prevalence of hepatitis B virus (HBV) is a global healthcare threat, particularly chronic hepatitis B (CHB) that might lead to hepatocellular carcinoma (HCC) should not be neglected. Although many types of HBV diagnosis detection methods are available, some technical challenges, such as the high cost or lack of practical feasibility, need to be overcome. In this study, the polycrystalline silicon nanowire field-effect transistors (pSiNWFETs) were fabricated through commercial process technology and then chemically functionalized for sensing hepatitis B virus surface antigen (HBsAg) and hepatitis B virus X protein (HBx) at the femto-molar level. These two proteins have been suggested to be related to the HCC development, while the former is also the hallmark for HBV diagnosis, and the latter is an RNA-binding protein. Interestingly, these two proteins carried opposite net charges, which could serve as complementary candidates for evaluating the charge-based sensing mechanism in the pSiNWFET. The measurements on the threshold voltage shifts of pSiNWFETs showed a consistent correspondence to the polarity of the charges on the proteins studied. We believe that this report can pave the way towards developing an approachable tool for biomedical applications.

High prevalence of tryptophan-truncated S quasispecies in treatment-naïve chronic hepatitis B patients.

Hepatitis B virus surface antigen (HBsAg) encoded by the S gene is highly expressed during the replication cycle of hepatitis B virus (HBV). However, the frequent usage of tryptophan in HBsAg, which leads to a high cost of biosynthesis, is inconsistent with the high expression level of this protein. Tryptophan-truncated mutation of HBsAg, that is, a tryptophan to stop codon mutation resulting in truncated HBsAg, might help to maintain its high expression with lower biosynthetic cost. We aimed to investigate the prevalence of tryptophan-truncated S quasispecies in treatment-naïve patients with chronic hepatitis B (CHB) by applying CirSeq as well as a site-by-site algorithm developed by us to identify variants at extremely low frequencies in the carboxyl terminus of HBsAg. A total of 730 mutations were identified in 27 patients with CHB, varying from seven to 56 mutations per sample. The number of synonymous mutations was much higher than that of nonsynonymous mutations in the reverse transcriptase (RT) coding region and vice versa in the S coding region, implying that the evolutionary constraints on the RT and S genes might be different. We showed that 25 (92.6 %) of 27 patients had at least one S-truncated mutation, most of which were derived from tryptophan, indicating a high prevalence of tryptophan-truncated S mutations in treatment-naïve patients with CHB. In terms of the RT gene, 21 (77.8 %) patients had pre-existing drug-resistant mutations, while no truncated mutations were detected. Our findings that tryptophan-truncated S quasispecies and drug-resistant RT mutants were highly prevalent in treatment-naïve patients with CHB provide new insights into the composition of the HBV population, which might help optimize the treatment and management of patients with CHB.

D e novo synthesis of hepatitis B virus nucleocapsids is dispensable for the maintenance and transcriptional regulation of cccDNA.

BACKGROUND & AIMS: Chronic HBV infection cannot be cured by current therapeutics owing to their limited ability to reduce covalently closed circular (ccc)DNA levels in the livers of infected individuals. Therefore, greater understanding of the molecular determinants of cccDNA formation and persistence is required. One key issue is the extent to which de novo nucleocapsid-mediated replenishment (reimport) contributes to cccDNA levels in an infected hepatocyte. METHODS: We engineered an infectious HBV mutant with a genome encoding a stop codon at position T67 in the HBV core open reading frame (ΔHBc HBV). Importantly, ΔHBc HBV virions cannot initiate nucleocapsid synthesis upon infection. Long-term in vitro HBV infection markers were followed for up for 9 weeks in HepG2-NTCP cells (A3 clone) and HBV DNA was quantified using a newly-developed, highly-precise PCR assay (cccDNA inversion quantitative PCR). RESULTS: ΔHBc and wild-type (WT) HBV resulted in comparable expression of HBV surface antigen (HBsAg), which could be blocked using the entry inhibitor Myrcludex B, confirming bona fide infection via the receptor sodium taurocholate cotransporting polypeptide (NTCP). In primary human hepatocytes, Huh7-NTCP, HepG2-NTCP, and HepaRG-NTCP cells, comparable copy numbers of cccDNA were formed. cccDNA levels, transcription of viral RNA, and HBsAg secretion remained comparably stable in WT and ΔHBc HBV-infected cells for at least 9 weeks. CONCLUSIONS: Our results imply that de novo synthesised HBc plays a minor role in transcriptional regulation of cccDNA. Importantly, we show that initially-formed cccDNA is stable in hepatocytes without requiring continuous replenishment in in vitro infection systems and contribution from de novo DNA-containing nucleocapsids is not required. Thus, short-term therapeutic targeting of capsid-reimport is likely an inefficient strategy in eliminating cccDNA in chronically infected hepatocytes. LAY SUMMARY: The hepatitis B virus can maintain itself in the liver for a patient's lifetime, causing liver injury and cancer. We have clarified exactly how it maintains itself in an infected cell. This now means we have a better idea at how to target the virus and cure a chronic infection.

Havachoobe (Onosma dichroanthum Boiss) Root Extract Decreases the Hepatitis B Virus Surface Antigen Secretion in the PLC/PRF/5 Cell Line.

BACKGROUND: Many efforts are currently focused on functional treatment of the hepatitis B virus (HBV). This can be done by suppressing the secretion of HBV surface antigen (HBsAg). Scientific communities are very interested in natural products in that respect. OBJECTIVE: Use of root extract of Havachoobe (Onosma dichroanthum BoissI), a Northern Iranian native medical herb, for assessment of its anti-HBsAg secretion activity. METHODS: Havachoobe had been bought at a nearby apothecary store. Plant root extract was obtained using a hydroalcoholic process. Cytotoxic activity of the extract was examined on PLC/PRF/5 cells using MTT assay. ELISA has been used to measure HBsAg in the treated cell line supernatants. In addition, real-time PCR analysis was performed to evaluate the expression of HBsAg before and after treatment of Onosma in vitro. RESULTS: The results showed very low root extract cytotoxicity at concentrations under 8 µg/mL. Tissue culture infectious dose 50 was obtained at 63.78 µg/mL. In a dose-dependent and time-dependent manner, a significantly reduced HBsAg secretion was observed at a concentration of 8 ppm at 12 h post-treatment. The real-time PCR result showed relative decreased HBsAg expression at all doses at 12 h post-treatment time. DISCUSSION: In this study, we first reported anti-HBsAg activity on an Iranian herbal medicine. Havachoobe root extract was shown to be able to inhibit HBsAg in a dose-dependent and time-dependent manner. We find the extract exerts its inhibitory effect of HBsAg by targeting transcription of HBsAg.

Hepatitis B virus detected in paper currencies in a densely populated city of India: A plausible source of horizontal transmission?

BACKGROUND: The recent rise in the incidence of hepatitis B virus (HBV) infections in a densely populated city of eastern India ("mixing vessel" of people of varied socio-economic and immune status) prompted this study. Applying saliva on fingers for enumerating bank notes is a common practice in the Indian subcontinent. Paper notes may be a potential source of "horizontal" transmission of this virus, especially if there are cuts/bruises on the oral mucous membrane or skin. AIM: To investigate whether paper currencies could be a plausible mode of horizontal transmission of HBV infection. METHODS: Polymerase chain reactions (PCR) followed by nucleotide sequencing was done for the detection of HBV. Hepatitis B virus surface antigen enzyme-linked immunosorbent assay(HBsAg ELISA) was performed on all HBV deoxyribonucleic acid-positive samples to check the detectability of the virus. Atomic force microscopy (AFM) was carried out for visual confirmation of HBV particles in ultracentrifuged/immunoprecipitated samples from currency paper washings. RESULTS: HBV-specific PCRs on pellets obtained after ultracentrifugation/ immunoprecipitation of the currency paper washings detected potentially intact/viable HBV (genotype D2) in 7.14% of samples (n = 70). AFM gave the visual confirmation of HBV particles in ultracentrifuged/immunoprecipitated samples from currency paper washings. However, HBV isolates from the currency notes could not be detected by HBsAg ELISA. CONCLUSION: It is a common practice in the Indian subcontinent to count paper currencies by applying saliva on fingertips. Paper notes may be a potential source of "horizontal" transmission of this virus, especially if there are cuts/bruises on the oral mucous membrane or skin, but it was practically not possible to demonstrate experimentally such transmission. Detection of potentially intact/viable and "occult" HBV from currency poses potential risk of silent transmission of this virus among the general population.

Anti-HBc and hepatitis B virus DNA among HBsAg-negative blood donors from the main central blood bank units in Eastern Libya.

BACKGROUND: Post-transfusion hepatitis B virus (PTHB) infection is still a public health problem in the world. In many developed countries, nucleic acid testing (NAT) for detection of Hepatitis B Virus (HBV)-DNA has been implemented to enhance blood donation safety. In Libya, however, the testing for HBV infection is limited to the detection of HBV surface antigen (HBsAg) only. OBJECTIVES: To determine the frequency of anti-Hepatitis B core antibody (HBc) and HBV-DNA in HBsAg-negative, anti-HBc-positive blood donors in the main Central Blood Bank Units (CBBUs) in eastern Libya. METHODS: One thousand blood samples were obtained from healthy blood donors at the five main CBBUs in eastern Libya. The samples were screened for HBsAg and anti-HBc. Real-time polymerase chain reaction (PCR) was carried out to detect HBV-DNA in all anti-HBc-positive samples. RESULTS: A total of 94 (9.4%) donors were positive for anti-HBc. Of the 94 anti-HBc-positive samples, 9 samples (9.5%) tested positive for HBV-DNA by real-time PCR. CONCLUSION: The rate of anti-HBc among blood donors in this study (9.4%) was similar to that reported from other regions in the country. In the absence of advanced tests for the detection of HBV infection in blood donors, such as NAT, anti-HBc should be routinely tested for, at least for first-time donors.

Analysis of detection results of serum markers of four infectious diseases in 34 080 patients / 公共卫生与预防医学

Objective To investigate the positive rate of serum biomarkers of 4 infectious diseases including HBV, HCV, HIV, and TP in patients in Jinniu District People’s Hosptial of Chengdu. Methods The results of serum markers of the 4 infectious diseases in 34 080 patients detected in the Laboratory Department of Chengdu Jinniu District People's Hospital were analyzed retrospectively. Results Of these 34 080 patients, the positive rate of HIV antibody (anti-HIV1/2) was 0.32%, the positive rate of hepatitis B surface antigen (HBsAg) was 11.34%, the positive rate of hepatitis C antibody (anti-HCV) was 0.42%, and the positive rate of Treponema pallidum antibody (anti-TP) was 3.08%. The positive rates of anti-HIV1/2, HBsAg and anti-TP in males were higher than those in females (PsAg, and anti-HCV had the highest positive rate in the 30-59 age group, while anti-TP had the highest positive rate in the group older than 60 years old. Conclusion The positive detection rate of serum markers in four infectious diseases in patients in Chengdu Jinniu District People's Hospital before surgery, childbirth and blood transfusion was higher, and the male positive rate was higher than that of the female.

Affinity binding of aptamers to agarose with DNA tetrahedron for removal of hepatitis B virus surface antigen.

DNA nanostructures are effective intermediates for immobilizing biomolecules because of their high level of controllability, the flexibility to create desired nanostructures through precision "bottom-up" assembly and the convenience to building fine nanostructures. The purpose of this study was to demonstrate the potential use of aptamers bound to agarose through a DNA tetrahedral structure to make a novel type of immunosorbent for the removal of hepatitis B virus surface antigens. Our results show that the proposed immunosorbent exhibits favorable biocompatibility and specificity. Electrophoresis and confocal microscopy were used to confirm the formation of immunosorbents. The ARCHITECT HBsAg assay was performed to quantitatively measure hepatitis B surface antigen. The cytotoxic potential of the immunosorbent was determined by an in vitro viability assay using V79 lung fibroblasts, demonstrating that the immunosorbents are noncytotoxic. The high adsorption capacity of the novel DNA nanostructure-based adsorbents towards hepatitis B surface antigen indicated the potential application of these materials for the treatment of Hepatitis B infection.