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Hepatitis B virus (HBV) is responsible for most of the viral hepatitis worldwide. HBV is a partially double stranded DNA virus that is composed of four main open reading frames (ORFs) encoding its important antigens, namely hepatitis B surface antigen (HBsAg), hepatitis B core antigen (HBcAg), HBV polymerase and hepatitis B X antigen (HBxAg). In this study, we report a successful method for the cloning and expression of HBcAg. The ORF of HBcAg was successfully amplified using polymerase chain reaction (PCR), cloned into the expression vector pRSET-B and transformed to Escherichia coli (E. coli) BL-21 (DE3) pLysS strain for protein expression. Successful expression of HBcAg was accomplished, in which an induced protein with a molecular weight of 24 kDa was obtained and confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. The produced HBcAg was successfully used for the diagnosis of HBV infected patient through detection of antibodies against HBcAg (anti-HBcAg) in the serum of the patient utilizing Western blotting. Overall, this study provides a simple, convenient and efficient protocol for the production of HBcAg that can be used as an important candidate to study the diagnosis and prognosis of HBV disease, as well as for understanding the epidemiological prevalence of HBV cases and production of anti-HBcAg., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2023 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.)

Abstract: The visualization of intrahepatic hepatitis B virus (HBV) DNA by in situ hybridization (ISH) has uncovered some interesting aspects of HBV life cycle at the single-cell level. In the current study, we intend to evaluate the reliability and robustness of this assay in the real-world clinical scenario and its relationship with currently available clinical biomarkers in chronic hepatitis B (CHB) patients.In this cross-sectional study, 94 CHB patients and 10 patients with non-HBV related liver diseases were enrolled. Liver biopsies and routine histopathology analysis were performed. Intrahepatic HBV DNA and viral antigens (HBsAg and HBcAg) were detected by ISH and immunohistochemistry (IHC), respectively. The basic biochemical and virological parameters such as alanine transaminase, serum HBV DNA, and serum HBsAg were measured.The HBV DNA-ISH assay showed 55.8% (53/94 cases) positive rate in CHB patients, no false positive was found in non-HBV related hepatitis. The IHC of HBsAg and HBcAg showed a positive rate of 94.7% (89/94 cases) and 19.5% (17/87 cases), respectively. Quantification of HBV DNA-ISH signal showed a significant correlation with serum HBV DNA (rs = 0.6223, P < .0001). In addition, the staining pattern of HBV DNA in situ in the context of collagen deposition informed the histopathological progression of chronic liver disease.The application of this ISH assay in evaluating intrahepatic viral replication in real-world CHB patients showed favorable performance. It can be a complementation to conventional liver histopathology examination and IHC detection of viral antigens. This methodology provides an intuitive assessment of virological and pathological state of CHB patients, and further supports clinical diagnosis and management., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2021 the Author(s). Published by Wolters Kluwer Health, Inc.)

Background: Most Chinese Blood Centers adopted mini pool (MP) nucleic acid testing (NAT) for HBV screening due to high cost of Individual donation (ID) NAT, and different proportions of MP-reactive but ID-non-reactive donations (MP+/ID-, defined as non-resolved donations) have been observed during daily donor screening process. Some of these non-resolved donations are occult HBV infections (OBIs), which pose potential risk of HBV transmission if they are not deferred. This study is aimed to further analyze these non-resolved donations., Methods: The non-resolved plasma samples were further analyzed by serological tests and various HBV DNA amplification assays including quantitative PCR (qPCR) and nested PCR amplifying the basic core and pre-core promoter regions (BCP/PC; 295 base pairs) and HBsAg (S) region (496 base pairs). Molecular characterizations of HBV DNA+ non-resolved samples were determined by sequencing analysis., Results: Of 17,226 MPs from 103,356 seronegative blood donations, 98 MPs were detected reactive for HBV. Fifty-six out of these 98 (57.1%) reactive MPs were resolved as HBV DNA+, but the remaining 42 pools (42.9%, 252 donations) were left non-resolved with a high rate (53.2%) of anti-HBc+. Surprisingly, among 42 non-resolved MPs, 17 contained one donation identified as OBIs by alternative NAT assays. Sequence analysis on HBV DNAs extracted from these OBI donations showed some key mutations in the S region that may lead to failure in HBsAg detection and vaccine escape., Conclusion: A total of 53.2% of the non-resolved donations were anti-HBc+, and OBIs were identified in 40.5% of these non-resolved pools. Therefore, non-resolved donations with anti-HBc+ might pose potential risk for HBV transmission. Our present analysis indicates that anti-HBc testing in non-resolved donations should be used to identify OBIs in order to further increase blood safety in China., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ye, Zhao, Li, Li, Zheng, Xiong, Zeng, Xu and Chen.)

We used HBV core antigen (HbcrAg), pre-genomic RNA (pg RNA) and other biomarkers to evaluate the therapeutic effect in HBV infected patients receiving anti-viral therapy. 127HBeAg-positive patients were enrolled: 35 patients received nucleotide therapy, 14 patients received interferon and 78 patients received combination therapy with both. HBcrAg, pg RNA and other biomarkers were detected at different time points, we defined the decreased titre of HBcrAg and HBeAg from baseline to 6 and baseline to 12 months as ∆HBcrAg and ∆HBeAg, which were used to predict HBeAg seroconversion. Furthermore, we used the time-dependent receiver operator curve of different markers to analyse HBeAg seroconversion. For HBeAg seroconversion: at 6 months, 0.75 log 10 U/mL of ∆HBcrAg and 1.47 log 10 PEI U/mL of ∆HBeAg showed maximum predictive value in receiver operator curve analysis (Youden's index values for area under the curve of 0.687 and 0.646, respectively). At 12 months, 2.05 log 10 U/mL of ∆HBcrAg and 1.92 log 10 PEI U/mL of ∆HBeAg showed improved prediction (maximum Youden's index values, with areas under the curve of 0.688 and 0.698, respectively).pg RNA was a better predictor of outcome due and the concentrations of 6.20 log 10 I U/mL of pg RNA and 8.0 log 10 U/mL of HBcrAg were cut-off values for response in a Kaplan-Meier curve analysis. Our results may be used to identify the pg RNA concentration in patients at baseline and ∆HBcrAg during therapy who are likely to achieve HBeAg seroconversion according to the cut-off value at different time points, thus helping to evaluate the therapeutic effect., (© 2020 John Wiley & Sons Ltd.)

Immunosuppression can lead to hepatitis B virus (HBV) reactivation in hepatitis B core antigen antibodies (anti-HBc) positive patients, especially those undergoing chemotherapy, although there is limited data on solid organ recipients, especially lung transplantation. Our aim was to analyze the risk of HBV reactivation and the potential impact of anti-HBc-positive status (both donors and recipients) on prognosis in a lung, kidney, and liver transplantation cohort.Retrospective analysis including data from all transplants in adults (2011-2012) in a tertiary hospital, with prospective HBV serology study to assess the risk of reactivation and its possible impact on survival.In total, 392 transplant recipients were included (196 kidney, 113 lung, 83 liver). Pre-transplantation anti-HBc screening was more frequent in liver recipients (P < .001) and donors (P < .001) than in kidney or lung. Fifty-five (14%) recipients were anti-HBc-positive and were not undergoing antiviral prophylaxis. Three (5.4%) cases of HBV reactivation occurred: 2 in pre-transplant anti-HBc-positive recipients and 1 with prior unknown anti-HBc status. All were HBeAg+ with HBV deoxyribonucleic acid (DNA) >10E8 IU/mL and only mild fibrosis. Baseline recipient anti-HBc positive status was the only factor associated with HBV reactivation. No reactivation cases occurred in lung or kidney recipients of anti-HBc positive grafts. Survival was lower in lung transplants, especially in human immunodeficiency virus-infected patients and those with prior immunosuppression.Anti-HBc positive status is a risk factor for HBV reactivation in solid organ recipients. Anti-HBc testing is highly recommended in solid-organ transplant recipients in order to identify those anti-HBc positive and therefore candidates for periodical hepatitis B surface antigen (HBsAg) and HBV DNA screening after transplant.

Background: Hepatitis B core-related antigen (HBcrAg) is a viral marker for the development of cirrhosis and hepatocellular carcinoma (HCC) in patients with chronic hepatitis B (CHB). However, the relationship between HBcrAg and HBV recurrence after liver transplantation (LT) is unclear., Aim: To investigate the correlation of serum HBcrAg level with HBV recurrence post-LT to evaluate the prognostic role of the pre-LT HBcrAg level., Methods: This retrospective cohort study enrolled 357 CHB patients who received LT for a median of 36.6 months. Univariate and multivariate analyses and time-dependent receiver operating characteristic (ROC) curves for markers associated with HBV recurrence were analysed., Results: 48 patients (13.4%) had HBV recurrence after LT. HBcrAg, detectable HBV DNA, HCC and HCC recurrence were associated with HBV recurrence. In a multivariate analysis, HBcrAg level was independently associated with HBV recurrence, and the relationship between HBcrAg level and incident HBV recurrence was significant and graded (HR: 3.17 per unit; 95% CI: 1.97-5.11; P for trend < .001). Additionally, HBcrAg level was superior to HBV DNA level in predicting HBV recurrence by time-dependent ROC analysis. Patients with an HBcrAg ≥ 5.0 log U/mL had a significantly higher 5-year cumulative recurrence rate than those with an HBcrAg < 5.0 log U/mL (37.6% vs 6%, P < .001); the adjusted hazard ratio was 5.27 (95% CI 2.47-11.25, P < .001)., Conclusion: An elevated serum HBcrAg level was independently associated with the risk of HBV recurrence in patients with CHB after LT., (© 2019 John Wiley & Sons Ltd.)

Background & Aims: The impact of hepatitis B core antibody (anti-HBc) positive liver grafts on survival and the risk of de novo hepatitis B virus (HBV) infection after liver transplantation (LT) remain controversial. Therefore, we aimed to analyze this risk and the associated outcomes in a large cohort of patients., Methods: This was a retrospective study that included all adults who underwent LT at Queen Mary Hospital, Hong Kong, between 2000 and 2015. Data were retrieved from a prospectively collected database. Antiviral monotherapy prophylaxis was given for patients receiving grafts from anti-HBc positive donors., Results: A total of 964 LTs were performed during the study period, with 416 (43.2%) anti-HBc positive and 548 (56.8%) anti-HBc negative donors. The median follow-up time was 7.8 years. Perioperative outcomes (hospital mortality, complications, primary nonfunction and delayed graft function) were similar between the 2 groups. The 1-, 5- and 10-year graft survival rates were comparable in anti-HBc positive (93.3%, 85.3% and 76.8%) and anti-HBc negative groups (92.5%, 82.9% and 78.4%, p = 0.944). The 1-, 5- and 10-year patient survival rates in anti-HBc positive group were 94.2%, 87% and 79% and were similar to the anti-HBc negative group (93.5%, 84% and 79.7%, p = 0.712). One-hundred and eight HBsAg negative recipients received anti-HBc positive grafts, of whom 64 received lamivudine and 44 entecavir monotherapy prophylaxis. The risk of de novo HBV was 3/108 (2.8%) and all occurred in the lamivudine era. There were 659 HBsAg-positive patients and 308 (46.7%) received anti-HBc positive grafts. The risk of HBV recurrence was similar between the 2 groups. Donor anti-HBc status did not impact on long-term patient and graft survival, or the risk of hepatocellular carcinoma recurrence after LT., Conclusions: De novo HBV was exceedingly rare especially with entecavir prophylaxis. Anti-HBc positive grafts did not impact on perioperative and long-term outcomes after transplant., Lay Summary: The risk of de novo hepatitis B infection after liver transplantation was rare when using hepatitis B core positive liver grafts with entecavir monotherapy prophylaxis. Hepatitis B core antibody status did not impact on perioperative and long-term outcomes after liver transplantation. This provides support for the clinical use of hepatitis B core positive liver grafts when required., (Copyright © 2019 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Hepatitis B virus (HBV) infects approximately 257 million individuals worldwide. Recent advances in the virology and diagnosis of HBV infection are summarized in this review article. A novel classification of the different phases of chronic HBV infection, proposed by the European Association for the Study of the Liver (EASL), is presented. New diagnostic and monitoring tools are now available, including rapid diagnostic tests for hepatitis B surface antigen (HBsAg) detection, HBsAg quantification assays, an HBV core-related antigen (HBcrAg) quantification test, and HBV RNA quantification testing. Their clinical utility under study is discussed in this review.

An electrochemical immunosensor devoted the core hepatitis B antibody detection, based on polytyramine (PTy) and carbon nanotubes (CNT) composite was developed. The antibody interactions with immobilized antigens were detected by reduction on the electron transfer from ionic species coming from reactive amine groups of PTy. The synthesis in acid medium of PTy-CNT composite favorite a great amount of NH 3 + ionic species, forming a nanocomposite with high catalytic activity on the electrode surface. As proof-of-concept, antibodies against the core hepatitis B virus were label-free and reagentless electrochemically detected by square wave voltammetry (SWV) through decrease on cathodic peaks. It was recently reported that hepatitis B core antigen antibodies (anti-HBc) is a powerful biomarker for hepatitis B virus (HBV) infection, being more specific than HBsAg due to the possibility of detecting the occult HBV infections. The nanostructured film was characterized by atomic force microscopy and electrochemical techniques. This immunosensor showed linear responses from 1.0 to 5.0 ng mL -1 and a limit of detection of 0.89 ng mL -1 anti-HBc. It was also tested in assays with negative and positive blood samples using 0.1 M KCl as electrolyte support on readings showing specific responses. This easy reagentless detection platform, showing a remarkable potential to development of bolder and simpler HBV assay for screening of blood bags, in attempting to circumvent point-of-care testing limitations., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Occupational health nurses (OHNs) need adequate knowledge to interpret the serology markers when screening workers for Hepatitis B virus.

Hepatitis B virus core antigen (HBcAg) is the major structural protein of hepatitis B virus (HBV). The presence of anti-HBcAg antibody in a blood serum indicates that a person has been exposed to HBV. This study demonstrated that the immobilization of HBcAg onto the gold nanoparticles-decorated reduced graphene oxide (rGO-en-AuNPs) nanocomposite could be used as an antigen-functionalized surface to sense the presence of anti-HBcAg. The modified rGO-en-AuNPs/HBcAg was then allowed to undergo impedimetric detection of anti-HBcAg with anti-estradiol antibody and bovine serum albumin as the interferences. Upon successful detection of anti-HBcAg in spiked buffer samples, impedimetric detection of the antibody was then further carried out in spiked human serum samples. The electrochemical response showed a linear relationship between electron transfer resistance and the concentration of anti-HBcAg ranging from 3.91ngmL -1 to 125.00ngmL -1 with lowest limit of detection (LOD) of 3.80ngmL -1 at 3σm -1 . This established method exhibits potential as a fast and convenient way to detect anti-HBcAg., (Copyright © 2018 Elsevier B.V. All rights reserved.)

BACKGROUND The aim of this study was to investigate the clinical characteristics of individuals with chronic hepatitis B virus (HBV) infection with persistent low levels of hepatitis B surface antigen (HBsAg) and to undertake a correlation analysis of the clinical characteristics. MATERIAL AND METHODS The study included 1,204 subjects with chronic HBV infection. Serum HBsAg, HBV envelope antigen (HBeAg), and HBV core antigen (HBcAg) levels were measured using the chemiluminescent microparticle immunoassay (CMIA) and the neutralization test. HBV DNA was measured using real-time fluorescence quantitative polymerase chain reaction (RT-FQ-PCR). RESULTS There were 1,023 subjects in the high-level HBsAg group (HBsAg level ≥10 IU/mL) and 181 subjects in the low-level HBsAg group (HBsAg level <10 IU/mL). In the low-level HBsAg group, the main serological pattern (93.37%) was HBsAg and HBeAg and HBcAg-positive (HBV-M2), and the asymptomatic carrier (ASC) status was 98.34%. The low-level HBsAg group had a lower HBV DNA-positive rate compared with the high-level HBsAg group (40.33% vs. 75.07%), with a normal distribution across all age groups (P>0.05). The low-level HBsAg group included an older age group. A low-level of HBsAg was positively correlated with a low level of replication of HBV DNA (r=0.452). CONCLUSIONS The findings of this study showed that individuals with chronic HBV infection and sustained low-levels of HBsAg were an older population and had a lower level of replicating HBV DNA when compared with individuals with high levels of HBsAg, and the majority (93.7%) were also HBsAg and HBeAg and HBcAg-positive.

To analyze the relationship between the factors related to the occurrence of HBV-GN and serum HBsAg-positive glomerulonephritis. A total of 56 patients were enrolled in the present study. All enrolled cases were divided into two groups according to whether HBsAg and/or HBcAg was present in renal kidney tissue: patients with Hepatitis B virus-associated nephritis (HBV-GN group, 30 cases) and patients with hepatitis B virus-combined nephritis (HBV-CG group, 26 cases). We sought to analyze the differences in clinical features and pathological characteristics in both groups. The rate of HBeAg positivity in the HBV-GN group was considerably increased in the HBV-CG group (P < 0.05), and the number of patients with HBsAg+HBeAg+HBcAb+ in the HBV-GN group was considerably increased in the HBV-CG group (21 cases vs 10 cases) (P < 0.05). Moreover, the number of MCD patients diagnosed by renal biopsy in the HBV-GN group was reduced compared with the HBV-CG group (1 case vs 7 cases) (P < 0.05). HBV infection and high-virus proliferation status were closely related with HBV-GN., (© 2017 Wiley Periodicals, Inc.)

Quantitative hepatitis B core antigen (anti-HBc) measurements could play an important role in evaluating therapeutic outcomes and optimizing the antiviral therapy of chronic hepatitis B infection. In this study, we have developed a simple and rapid fluorescence point-of-care test based on a lateral flow immunoassay (LFIA) method integrated with Eu (III) chelate microparticles to quantitatively determine anti-HBc concentrations in serum. This assay is based on a direct competitive immunoassay performed on lateral flow test strips with an assay time of 15 min. The Eu (III) chelate microparticle-based LFIA assay could quantitatively detect anti-HBc levels with a limit of detection of 0.31 IU mL -1 , and exhibited a wide linear range (0.63-640 IU mL -1 ). The intra- and inter-assay coefficients of variation for anti-HBc were both less than 10% and a satisfactory dilution test and accuracy were demonstrated. There were no statistically significant differences in sensitivity or specificity in serum samples between the Eu (III) chelate microparticle-based LFIA strips and the Abbott Architect kit. A simple, rapid and effective quantitative detection of anti-HBc was possible using the Eu (III) chelate microparticle-based LFIA strips. The strips will provide diagnostic value for clinical application.

Background: The natural history of HBV infection includes immune tolerance (IT), immune clearance (IC), HBeAg-negative inactive/quiescent carrier (ENQ), and HBeAg-negative hepatitis (ENH) phases. As the current biomarkers for discriminating the four phases still have some weaknesses, additional serological indicators are needed. Hepatitis B core-related antigen (HBcrAg) encoded with the precore/core gene contains denatured HBeAg, HBV core antigen (HBcAg), and a 22-KDa precore protein (p22cr) and has been demonstrated to have a close association with the natural history of hepatitis B infection. However, no specific cutoff values and diagnostic parameters have been identified to evaluate the diagnostic efficacy. This study aimed to clarify the distribution of HBcrAg levels and evaluate the diagnostic performance during the natural history of HBV infection in a Western Chinese population., Methods: In this study, 294 samples were collected from treatment-naive HBV infection patients in different phases (IT = 64; IC = 72; ENQ = 100, and ENH = 58). We detected the HBcrAg values and analyzed the relationship between HBcrAg and HBV DNA. HBsAg and other clinical parameters were quantitatively detected., Results: The HBcrAg levels of IT, IC, ENQ, and ENH were 9.30 log U/mL, 8.80 log U/mL, 3.00 log U/mL, and 5.10 log U/mL, respectively (p < 0.0001). Receiver operating characteristic curve analysis demonstrated that the areas under the curves (AUCs) for HBcrAg and quantitative HBsAg at cutoff values of 9.25 log U/mL and 4.355 log IU/mL for distinguishing the IT phase from the IC phase were 0.704 and 0.694, with a sensitivity of 53.13% and 79.69% and specificity of 76.39% and 59.72%, respectively. AUCs of HBcrAg and quantitative HBsAg at cutoff values of 4.15 log U/mL and 2.395 log IU/mL for discriminating between the ENQ and ENH phases were 0.931 and 0.653, with a sensitivity of 87.93% and 91.38% and specificity of 84.00% and 39.00%, respectively., Conclusions: HBcrAg levels varied significantly among the four natural phases of HBV infection and had higher predictive performance than quantitative HBsAg for distinguishing between ENQ-patients and ENH-patients and a similar performance as HBsAg for the discrimination between IT and IC phases, which indicated that HBcrAg could be a potential serological marker for HBV infection.

MicroRNAs (miRNAs) are a class of small, single-stranded, noncoding, functional RNAs. Hepatitis B virus (HBV) is an enveloped DNA virus with virions and subviral forms of particles that lack a core. It was not known whether HBV encodes miRNAs. Here, we identified an HBV-encoded miRNA (called HBV-miR-3) by deep sequencing and Northern blotting. HBV-miR-3 is located at nucleotides (nt) 373 to 393 of the HBV genome and was generated from 3.5-kb, 2.4-kb, and 2.1-kb HBV in a classic miRNA biogenesis (Drosha-Dicer-dependent) manner. HBV-miR-3 was highly expressed in hepatoma cell lines with an integrated HBV genome and HBV + hepatoma tumors. In patients with HBV infection, HBV-miR-3 was released into the circulation by exosomes and HBV virions, and HBV-miR-3 expression had a positive correlation with HBV titers in the sera of patients in the acute phase of HBV infection. More interestingly, we found that HBV-miR-3 represses HBsAg, HBeAg, and replication of HBV. HBV-miR-3 targets the unique site of the HBV 3.5-kb transcript to specifically reduce HBc protein expression, levels of pregenomic RNA (pgRNA), and HBV replication intermediate (HBV-RI) generation but does not affect the HBV DNA polymerase level, thus suppressing HBV virion production (replication). This may explain the low levels of HBV virion generation with abundant subviral particles lacking core during HBV replication, which may contribute to the development of persistent infection in patients. Taken together, our findings shed light on novel mechanisms by which HBV-encoded miRNA controls the process of self-replication by regulating HBV transcript during infection. IMPORTANCE Hepatitis B is a liver infection caused by the hepatitis B virus (HBV) that can become a long-term, chronic infection and lead to cirrhosis or liver cancer. HBV is a small DNA virus that belongs to the hepadnavirus family, with virions and subviral forms of particles that lack a core. MicroRNA (miRNA), a small (∼22-nt) noncoding RNA, was recently found to be an important regulator of gene expression. We found that HBV encodes miRNA (HBV-miR-3). More importantly, we revealed that HBV-miR-3 targets its transcripts to attenuate HBV replication. This may contribute to explaining how HBV infection leads to mild damage in liver cells and the subsequent establishment/maintenance of persistent infection. Our findings highlight a mechanism by which HBV-encoded miRNA controls the process of self-replication by regulating the virus itself during infection and might provide new biomarkers for diagnosis and treatment of hepatitis B., (Copyright © 2017 American Society for Microbiology.)

Chronic hepatitis B virus (HBV) infection is associated with lower prevalence of hyperlipidemia (HLP). However, occult HBV infection (OBI) in HLP patients has not yet been explored. OBI is defined as the presence of detectable HBV DNA in serum or liver tissue but undetectable HBV surface antigen in serum. In this study, 1,036 HLP patients and 1,134 replacement blood donor controls were recruited. Among them, 252 HLP patients and 255 blood donors with antibody to HBV core positive were selected and analyzed. HBV DNA was confirmed by nucleic acid testing assays, and nucleotide mutations were analyzed. OBI was detected in 9.5% (24/252) of HLP patients and 2.4% (6/255) of blood donors, respectively (P < 0.001). In HLP population, 41.7% of OBI and 13.6% of non-OBI carriers were associated with daily alcohol consuming > 30 g/day (P < 0.01), while in control population those rates were not statistically different between OBI and non-OBI carriers (P > 0.05). Viral load of OBI in HLP patients was higher than that of OBI in blood donors (P < 0.05), which was a positive correlation between total cholesterol and HBV viral load levels (r = 0.474 P = 0.019). HBV vaccination rate was found significantly lower in OBI HLP patients than that in non-OBI HLP patients (P < 0.01). Importantly, mutations were found in basic core promoter region of HBV among OBI HLP patients. In conclusion, the frequency of OBI is significantly higher in HLP patients, especially those patients with heavy daily alcohol consumption.

The presence of anti-hepatitis B virus (HBV) core antibody (anti-HBc) is considered a sensitive lifetime marker of HBV infection. Here, we examined this dogma by investigating the prevalence of hepatitis B viremia in anti-HBc negative complete vaccines in Taiwan.A total of 795 participants (1.7-20.0 years old) had completed HBV vaccination in infancy and were anti-HBc negative. Serum samples were available for 460 individuals with isolated anti-HBV surface antibodies (anti-HBs) (HBsAg-negative and anti-HBc negative) and for 245 individuals who tested negative for all 3 markers (triple seronegative). All samples were submitted for polymerase chain reaction (PCR) targeting both the preS/S and X/pre-C gene regions.Of the 460 participants with isolated anti-HBs, 26 (5.65%) were positive for HBV by 2-target PCR. Of the 245 triple seronegative samples, 12 (4.90%) were positive for HBV DNA. In the former group, the prevalence of viremia was significantly higher in individuals aged 6 to 10 years than in all other ages combined (11.82% vs 3.7%, P = 0.001). The anti-HBs titers were significantly lower in participants 6 to 10 years old than in all other ages combined (72.06 vs 99.64 mIU/mL, P = 0.038). In total, 7 (0.99%) subjects had quantifiable HBV DNA levels (280-18,820 IU/mL). Sequence analysis of the S gene revealed vaccine escape like mutations.Hepatitis B viremia can occur in completely vaccinated individuals who are negative for anti-HBc., Competing Interests: The authors declare no conflicts of interest.

Background: Hepatitis B virus (HBV) infection is strictly species and tissue specific, therefore none of the cell models established previously can reproduce the natural infection process of HBV in vitro. The aim of this study was to establish a new cell line that is susceptible to HBV and can support the replication of HBV., Methods: A hybrid cell line was established by fusing primary human hepatocytes with HepG2 cells. The hybrid cells were incubated with HBV-positive serum for 12 hours. HBV DNA was detected by quantitative fluorescence polymerase chain reaction (QF-PCR). HBsAg (surface antigen) and HBeAg (extracellular form of core antigen) were observed by electrochemiluminescence (ECL). HBcAg (core antigen) was detected by the indirect immunofluorescence technique. HBV covalently closed circular DNA (cccDNA) was analyzed by Southern blot hybridization and quantified using real-time PCR., Results: A new cell line was established and named HepCHLine-7. The extracellular HBV DNA was observed from Day 2 and the levels ranged from 9.80 (± 0.32) × 10(2) copies/mL to 3.12 (± 0.03) × 10(4) copies/mL. Intracellular HBV DNA was detected at Day 2 after infection and the levels ranged from 7.92 (± 1.08) × 10(3) copies/mL to 5.63 (± 0.11) × 10(5) copies/mL. HBsAg in the culture medium was detected from Day 4 to Day 20. HBeAg secretion was positive from Day 5 to Day 20. HBcAg constantly showed positive signals in approximately 20% (± 0.82%) of hybrid cells. Intracellular HBV cccDNA could be detected as early as 2 days postinfection and the highest level was 15.76 (± 0.26) copies/cell., Conclusion: HepCHLine-7 cells were susceptible to HBV and supported the replication of HBV. They are therefore suitable for studying the complete life cycle of HBV., (Copyright © 2014. Published by Elsevier B.V.)

The dysfunction of peripheral blood mononuclear cell (PBMC) plays an important role in hepatitis B virus (HBV) chronic infection and tolerance. This study is aimed to explore the changes of expression and distribution of Hepatitis B virus core antigen (HBcAg) in the PBMCs of patients infected with chronic hepatitis B virus by using confocal laser scanning microscopy (CLSM). The levels of HBcAg in PBMCs were correlated with the HBV load in serum, and the change of HBcAg distribution in different PBMC organelles may represent various HBV infection states. HBcAg was mainly distributed in the nuclei of PBMC in the cases of immune tolerance and no inflammatory activity. Taken together, our data suggest that the measurement of HBcAg and its distribution in PBMCs using CLSM may serve as an alternative approach to monitor HBV load and the immune states of HBV infection with ease of using and improved sensitivity., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Aim: To study the intrahepatic expression of hepatitis B surface antigen (HBsAg) and hepatitis B core antigen (HBcAg) in chronic hepatitis B patients with and without hepatocellular carcinoma., Methods: A total of 33 chronic hepatitis B patients (mean age of 40.3 ± 2.5 years), comprising of 14 HBeAg positive and 19 HBeAg negative patients; and 13 patients with hepatitis B virus related hepatocellular carcinoma (mean age of 49.6 ± 4.7 years), were included in our study. Immunohistochemical staining for HBcAg and HBsAg was done using standard streptavidin-biotin-immunoperoxidase technique on paraffin-embedded liver biopsies. The HBcAg and HBsAg staining distributions and patterns were described according to a modified classification system., Results: Compared to the HBeAg negative patients, the HBeAg positive patients were younger, had higher mean HBV DNA and alanine transaminases levels. All the HBeAg positive patients had intrahepatic HBcAg staining; predominantly with "diffuse" distribution (79%) and "mixed cytoplasmic/nuclear" pattern (79%). In comparison, only 5% of the HBeAg-negative patients had intrahepatic HBcAg staining. However, the intrahepatic HBsAg staining has wider distribution among the HBeAg negative patients, namely; majority of the HBeAg negative cases had "patchy" HBsAg distribution compared to "rare" distribution among the HBeAg positive cases. All but one patient with HCC were HBeAg negative with either undetectable HBV DNA or very low level of viremia. Intrahepatic HBcAg and HBsAg were seen in 13 (100%) and 10 (77%) of the HCC patients respectively. Interestingly, among the 9 HCC patients on anti-viral therapy with suppressed HBV DNA, HBcAg and HBsAg were detected in tumor tissues but not the adjacent liver in 4 (44%) and 1 (11%) patient respectively., Conclusion: Isolated intrahepatic HBcAg and HBsAg can be present in tumors of patients with suppressed HBV DNA on antiviral therapy; that may predispose them to cancer development.

Background & Aims: Previous studies predicted the prognosis of hepatocellular carcinoma (HCC) mainly based on tumour-related factors, whereas the impacts of hepatitis B virus (HBV)-related factors are usually ignored. The objective of this exploratory study was to investigate the prognostic role of hepatitis B core antibody (HBcAb) on post-operative survival and recurrence of HCC., Methods: A retrospective analysis of 3388 HBsAg positive (HBV-related) HCC patients treated by curative resection was performed. Multivariate analysis of independent prognostic factors was performed by Cox proportional hazards regression model., Results: HBcAb positivity was an independent prognostic factor for recurrence-free survival (RFS) of HBV-related patients (P < 0.001, HR: 1.723, 95% CI: 1.278-2.324), and the 1-, 3-, 5-year RFS rates for HBcAb-negative patients were significantly better than those of HBcAb-positive patients (92.5%, 72.1% and 65.9% vs 77.9%, 58.6% and 46.9%, P < 0.001). HBcAb-positive HCC was much bigger (P = 0.006), more often involved with vascular invasion (P = 0.001), elevated AFP (P = 0.001) and ALT (P = 0.046) levels, but less often involved with capsule formation (P = 0.034). Besides vascular invasion, tumour size, interferon-α treatment, AFP and GGT level, HBcAb positivity was an independent prognostic factor for early intrahepatic recurrence of HBV-related patients (P = 0.025, HR: 1.766, 95% CI: 1.073-2.907) and the majority of HBcAb-positive recurrence were early recurrence while most of HBcAb-negative recurrence were late recurrence (P = 0.004)., Conclusion: Positive HBcAb may represent a more invasive phenotype of HBV-related HCC, and is associated with a higher risk of early intrahepatic recurrence and poorer RFS of HBV-related patients after curative resection., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

A serologic response to hepatitis B virus (HBV) defined as 'anti-HBc alone' is commonly observed, but its significance remains unclear. This study aimed to define the relationship between 'anti-HBc alone' serostatus and HBV infection, including HBV-specific T- and B-cell memory responses. We enrolled 31 'anti-HBc alone' patients. Total HBV DNA and cccDNA were tested by nested polymerase chain reaction (PCR) analysis in liver samples from 22 'anti-HBc alone' patients vs controls (chronic or resolved HBV infection), followed by HBsAg/HBcAg immunohistochemical (IHC) staining. IFN-γ secretion by HBV-specific T cells was compared in individuals who were 'anti-HBc alone' (n = 27), resolved HBV (n = 21), chronic HBV (n = 24) and 12 healthy controls using enzyme-linked immunospot (ELISpot) assays. An HBsAg-IgG B-cell ELISpot assay was performed in 'anti-HBc alone' patients before and after one dose of recombinant HBsAg vaccine. The majority (23/31, 74.2%) of the 'anti-HBc alone' individuals were co-infected with HCV. Infrequent intrahepatic total HBV DNA (2/22, 9.1%) and cccDNA (1/22, 4.5%) were detected in biopsies; HBsAg and HBcAg IHC staining was negative. HBV-specific T-cell responses were similar between 'anti-HBc alone' individuals and HBV resolvers. Circulating HBV-memory B-cell responses were detected in all 'anti-HBc alone' individuals, consistent with an HBsAg-specific memory pool. After one HBV vaccine dose, increased anti-HBs antibody levels were observed, accompanied by an expansion of HBsAg-specific memory B cells (P = 0.0226). 'Anti-HBc alone' individuals showed HBV-specific T-cell and memory B-cell responses typical of previous viral exposure and protective memory, suggesting a resolved infection., (© 2015 John Wiley & Sons Ltd.)

Our report aimed to investigate the physiopathological patterns of liver lesions patients during hepatitis. After being diagnosed clinically as hepatopathy (n=9), the liver biopsy samples were prepared to paraffin-sections and further subjected to routine and immunohistochemical stains. Among these lesioned livers invaded by viruses, immunity-regulatory molecules, such as CD20, CD4 and CD8, were significantly expressed. In addition, antigens of the hepatitis B virus (HBsAg and HBcAg) were positively detected in non-dependent way of hepatitis intervention. Furthermore, the pro-fibrotic promotor of vimentin (Vim) was labelled immunologically in mesenchymal cells within virus-attacked livers. These observations provide the clinicopathologic evidences that there is a potential association between immunodeficiency and collagenation in hepatitis patients when liver injury development. It also manifests that regulation of intrahepatic immunologic response may inhibition of excessive collagen generation prior to further progressing to hepatofibrosis.

Background: Although higher levels of hepatitis B virus (HBV) replication in HIV-HBV co-infection may relate to liver disease progression, this has not been completely elucidated. We used expression of hepatitis B core antigen (HBcAg) in liver biopsies from HIV-HBV co-infected and HBV mono-infected patients as a marker for HBV replication, and related these findings to clinical and histological parameters., Methods: Data from 244 HBV patients were compared with 34 HIV-HBV patients. Liver biopsies were scored for inflammation, fibrosis, HBcAg, and hepatitis B surface antigen. Univariate and multivariate analyses were performed., Results: HBcAg, but not hepatitis B surface antigen, staining was stronger in HIV co-infected than in HBV mono-infected. Co-infected and HBV mono-infected had similar alanine aminotransferase, inflammatory and fibrosis scores, and hepatitis B e antigen status. HBcAg staining correlated with HIV after correcting for HBV DNA and hepatitis B e antigen. CD4 counts and HIV RNA level did not correlate with intensity of HBcAg staining. HBV DNA levels were higher in HIV co-infected and correlated with HBcAg staining., Conclusions: By looking at HBcAg as a reflection of HBV replication in HIV-HBV co-infected with controlled HIV, our findings suggest that these patients may have subtle immune function defects, which could lead to adverse liver disease outcomes.

There is a lack of knowledge regarding the effect of peginterferon (PEG-IFN) on the expression of intrahepatic hepatitis B core and surface antigen (HBcAg and HBsAg) in chronic hepatitis B (CHB) and its relation with response to therapy. Fifty-two HBeAg-positive and 67 HBeAg-negative CHB patients with paired liver biopsies taken at baseline and after 1 year of PEG-IFN therapy were studied. After PEG-IFN therapy, HBeAg-negative patients showed a significant reduction in both intrahepatic HBcAg (P = 0.04) and HBsAg expression (P < 0.001). In contrast, a reduction in intrahepatic HBcAg expression was not observed in HBeAg-positive patients, while a trend in reduction of intrahepatic HBsAg staining was found (P = 0.09). Post-treatment, 7 (13%) HBeAg-positive and 9 (14%) HBeAg-negative patients had no expression of intrahepatic HBsAg. Patients without any intrahepatic HBsAg expression post-treatment were more likely to achieve a combined response (HBeAg loss with hepatitis B virus (HBV) DNA <2000 IU/mL for HBeAg -positive and HBV DNA <2000 IU/mL and normal alanine aminotransferase for HBeAg-negative CHB): 71% vs 5% for HBeAg-positive (P < 0.001) and 60% vs 16% for HBeAg-negative patients (P = 0.004), respectively. Moreover, a more profound decline of serum HBsAg was observed in patients with absence of intrahepatic HBsAg staining (3.1 vs 0.4 log IU/mL, P < 0.001 and 1.7 vs 0.4 log IU/mL, P = 0.005 for HBeAg-positive and HBeAg-negative CHB, respectively). In conclusion, PEG-IFN reduces expression of intrahepatic HBsAg. Loss of HBsAg as assessed by immunohistochemistry from the liver predicts a sustained response and is reflected in a pronounced serum HBsAg decline., (© 2014 John Wiley & Sons Ltd.)

Background/aims: To confirm the relationship between hepatitis B recurrence and Hepatocellular carcinoma recurrence., Methodology: Data from 340 patients undergoing liver transplantation for HBV-related liver disease were retrospectively evaluated. Clinically relevant variables were analyzed using univariate models. Significant variables were subjected to multivariate logistic regression analysis to identify the independent predictors for HBV recurrence. Fifteen samples removed from HCC recurrence patients were stained for HBsAg and HBcAg., Results: The analyzed population included 283 male and 57 female patients. The mean age was 48.5±9.33 years and median follow-up was 47 months. Hepatitis B relapsed in 16 patients (4.7%). Univariate analysis indicated that HCC (P=0.022) and HCC recurrence (P=0.000) were associated to post transplantation HB. Multivariate analysis identified HCC recurrence as an independent risk factor for HB recurrence (hazard ratio: 23.262 (95% CI: 3.752, 144.216); P <0.001). Three of 15 metastatic lesions were positive for HBsAg and 1 lesion was positive for HBcAg. Conclusion: HCC recurrence is an independent risk factor for post transplantation recurrence of hepatitis.

Liver transplant in the us remains a successful life-saving procedure for patients with irreversible liver disease. In 2012, 6256 adult liver transplants were performed, and more than 65,000 people were living with a transplanted liver. The number of adults who registered on the liver transplant waiting list decreased for the first time since 2002; 10,143 candidates were added, compared with 10,359 in 2011. However, the median waiting time for active wait-listed adult candidates increased, as did the number of candidates removed from the list because they were too sick to undergo transplant. The overall deceased donor transplant rate decreased to 42.3 per 100 patient-years, and varied geographically from 18.9 to 228.0 per 100 patient-years. Graft survival continues to improve, especially for donation after circulatory death livers. The number of new active pediatric candidates added to the waiting list also decreased. Almost 75% of pediatric candidates listed in 2009 underwent transplant within 3 years; the 2012 rate of deceased donor transplants among active pediatric wait-listed candidates was 136 per 100 patient-years. Graft survival for deceased donor pediatric transplants was 92.8% at 30 days. Medicare paid for some or all of the care for more than 30% of liver transplants in 2010., (© Copyright 2013 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Background: Hepatocellular carcinoma (HCC) incidence is increasing in the United States. Hepatitis B virus (HBV) and hepatitis C virus (HCV) are major causes of HCC. Hepatitis infection in patients with HCC is generally diagnosed by serology, which is not always consistent with the presence of HBV and HCV in the liver. The relationship of liver viral status to serostatus in hepatocarcinogenesis is not fully understood., Methods: HBV and HCV were evaluated in formalin-fixed, paraffin-embedded liver tissue specimens in a retrospective study of 61 U.S. HCC cases of known serologic status. HBV DNA and HCV RNA were detected by PCR, reverse transcription PCR (RT-PCR), and pyrosequencing, and HBsAg and HBcAg were evaluated by immunohistochemistry., Results: Viral markers were detected in the liver tissue of 25 of 61 (41%) HCC cases. Tissue viral and serologic status were discordant in 27 (44%) cases, including those with apparent "occult" infection. Specifically, HBV DNA was detected in tissue of 4 of 39 (10%) serum HBsAg (-) cases, including 1 anti-HCV(+) case; and HCV RNA was detected in tissue of 3 of 42 (7%) anti-HCV seronegative cases, including two with serologic evidence of HBV., Conclusions: Viral hepatitis, including HBV-HCV coinfection, may be unrecognized in up to 17% of patients with HCC when based on serology alone. Further research is needed to understand the clinical significance of viral makers in liver tissue of patients with HCC in the absence of serologic indices., Impact: The contribution of HBV and HCV to the increasing incidence of HCC in the United States may be underestimated., (©2013 AACR.)

Introduction: Liver transplantation (OLT) is the treatment of choice for advanced hepatic disease. The growing gap between waiting list patients and the number of donations has led to acceptance of less than optimal donors. The aim of this study was to evaluate the 5-year experience with anti hepatitis B core antigen (HBc)-positive liver donors., Patients and Methods: All recipients of anti-HBc-positive grafts from January 2005 to December 2010 were evaluated annually after OLT for liver disease etiology, Model for End-Stage Liver Disease (MELD) score, and the presence of hepatocellular carcinoma (HCC) liver biopsy histology and serology for hepatitis B virus (HBsAg, anti-HBs, HBV-DNA), hepatitis C virus, and hepatitis D virus as well as antiviral prophylaxis to prevent de novo HBV., Results: Among the 249 OLT performed from January 2005 to December 2010, (9.3%) cases used grafts from anti-HBc-positive donors. Etiologics of liver disease among the recipients were HBV (n = 13; 32.5%), HCV (n = 13; 32.5%) or other causes (n = 14; 35%). In 20 of the 40 patients (50%), HCC was found in the explanted organ. Of 40 recipients of anti-HBc-positive grafts 11 died, and 7 (17.5%) required retransplantation. Various regimens were employed as post-transplantation antiviral prophylaxis: (l) Immune globulin (25.8%); (2) Oral antiviral drugs (9.7%); and (3) combined prophylaxis (51.6%) or no treatment (12.9%). No difference was observed in patient or graft survival in relation to the etiology of liver disease, the MELD score, or the presence of HCC at the time of OLT, except graft survival was significantly reduced among recipient who underwent transplantation for non-HBV or non-HCV liver diseases compared with those engrafted due to viral hepatitis (P = .0062). No difference was observed in histologic features (grading and staging) compared with the antiviral prophylactic therapy; the 2 patients (5%) who developed de novo HBV had not received prophylaxis after OLT., Conclusions: Matching anti-HBc-positive grafts to recipients without HBV infection before OLT, may be especially safe., (Copyright © 2013. Published by Elsevier Inc.)

Toll-like receptor 4 (TLR4) plays an important role in innate immunity. The aim of our study was to detect the expression of TLR4 in HBV-associated glomerulonephritis (HBV-GN) and explore the pathogenesis of TLR4 in inhibition of HBV replication in kidney. Immunohistochemical methods were used to detect the distribution of immunoglobulin, HBsAg, HBcAg and TLR4. Because TLR4 was mainly distributed in renal tubules and interstitial spaces, we used human proximal tubular epithelial cells (HK-2) as target cells, which were cultured with HBV-DNA-positive serum. Cells were divided into four groups: A, a normal control group; B, an HBV-induced group; C, an HBV and 10 μg/ml LPS (TLR4-stimulating factor) group; and D, an HBV and 5 μg/ml CLI095 (TLR4 inhibitor) group. The morphology of HK-2 cells was observed by microscopy, and the expression of α-SMA was examined by immunofluorescence before and after culturing with HBV-DNA-positive serum. MTT was used to detect HK2 proliferation. The expression of TLR4 protein was detected by immunofluorescence and western-blotting assays, HBsAg and HBeAg levels in supernatants were measured by ELISA, and the expression of HBV-DNA was measured by semi-quantitative reverse transcription-PCR (RT-PCR). The immunohistochemical staining for TLR4 in the normal control group was negative. The expression of TLR4 in HBV-GN was significantly higher than in the HBV-antigen-positive and -negative primary glomerulonephritis (PGN) groups and was mainly distributed in renal tubules and the interstitial area, where the distribution of HBsAg had similar intensity. The expression of TLR4 was significantly associated with renal tubular and interstitial lesions. In an in vitro study with prolonged infection with HBV-DNA-positive serum, more irregularly shaped cells and fewer HK-2 were observed. The expression of α-SMA in the cytoplasm of HK-2 was significantly increased after HBV infection. Immunofluorescence results showed that almost no expression of TLR4 in HK2 cells cultured with normal serum (group A), while the expression of TLR4 was increased after HBV infection (group B). With increasing LPS concentration, the rate of the proliferation of HK2 cells, the levels of HBV-DNA in the supernatant, and the expression of HBsAg and HBeAg decreased. TLR4 was mainly expressed in HBV-GN in renal tubules and interstitial spaces and was significantly associated with renal tubular and interstitial lesions. The stimulation of TLR4 not only inhibited HBV replication but also inevitably induced immune injury to cells. Therefore, we speculate that TLR4 may be involved in an immune inflammatory reaction by inhibiting HBV replication in HK2 cells, which could have an antiviral effect during HBV infection in the kidney.

This study was carried out to investigate the effect of fibronectin (FN) on primary cultured human fetal hepatocytes infected by hepatitis B virus (HBV) in vitro. Human fetal hepatocytes infected by HBV were cultured on culture dishes coated with FN in vitro. HBsAg and HBeAg in supernatants were detected by ELISA, HBcAg in nuclei by immunohistochemistry, HBV DNA by fluorescent quantitative PCR and covalently closed circular DNA (cccDNA) by nested PCR. The results were compared with cells cultured on dishes that were not coated with FN. Positivity for HBsAg and HBeAg in supernatants appeared from day 5 on the non-coated dishes, while positivity for HBsAg and HBeAg appeared from day 1 on the coated dishes. Positivity for HBcAg in the nuclei on the non‑coated dishes was displayed from day 2 with a positive rate of 15%, while positivity for HBcAg on the coated dishes was displayed from day 1 with a positive rate of >90%. HBV DNA in cells on the non-coated dishes was detected from day 4, while that on the coated dishes was detected from day 1. The non-coated dishes were positive for cccDNA from day 8, while the coated dishes were positive from day 2. FN coating is capable of accelerating HBV infection in primary cultured fetal hepatocytes in vitro, increasing the duration of HBV infection and the number of infected cells.

Background: To investigate the effect of HBV markers on HBV-GN., Methods: The immunohistochemistry was used to detect HBsAg and HBcAg in frozen sections of renal biopsy, the changes in HBV serum markers, renal functional parameters and clinical manifestations or symptoms were observed to analyze renal damage., Results: Using renal biopsy data from 329 cases, this study found that the most common pathological subtype in HBV-GN was mesangioproliferative glomerulonephritis (MsPGN) (24.9%, P <0.05), and 29.4% of patients who show serological HBsAg, HBeAg and anti-HBc positive developed membranoproliferative glomerulonephritis (MPGN) (P <0.05). The immunohistochemistry was used to detect HBsAg and HBcAg in frozen sections.50% of HBsAg and HBcAg deposits was observed in the glomeruli of MPGN patients, while 36.6% of HBsAg and 43.9% of HBcAg deposited in the glomeruli of MsPGN patients. The deposits of HBsAg and HBcAg in glomeruli were directly correlated with IgA, IgG, IgM and C3 deposits. In addition, cases with a moderate to severe decrease as reflected by the glomerular filtration rate (GFR) were predominantly patients with MPGN (31.6%, P <0.05) or MsPGN (21.1%, P <0.05). Patients who were serological HBsAg, HBeAg and anti-HBc positive or HBsAg, anti-HBe and anti-HBc positive mainly exhibited urine and renal parameter changes., Conclusion: Examination of HBV markers in serum and renal biopsy will be useful for clinicians to predict the renal damage in early stage when it is reversible in HBV-GN.

Aim: To analyze the presence of HBV DNA in blood donors admitted to blood banks with HBsAg negativity and isolated anti-HBc positivity., Methods: Sera samples of 2500 HBsAg negative donors were included in the study. HBsAg tests were assayed with VITROS analyzer. Anti-HBc total, anti-HBs, HBeAg, anti-HBe and anti-HBc IgM tests were manually studied with DIA PRO kit by employing ELISA method. HBV DNA test was evaluated with iQ5 Real Time PCR Detection System with real-time PCR method., Results: A total number of 401 (16.4%) HBsAg negative blood donors had anti-HBc positivity. Forty-five of 401 (1.8%) anti-HBc positive samples were anti-HBs negative. These 45 persons were evaluated for anti-HBc positivity. Thirty-six of 45 anti-HBc positive persons had only anti-HBc positivity, and other nine patients had Anti-HBe antibody. HBV DNA was detected in three (6.6 %) of 45 isolated Anti-HBc positive persons as in one of nine persons and two of 36 persons., Conclusion: The persons who have only HBs Ag negativity may not be appropriate to become blood donors. Anti-HBc test should be done for HBsAg negative persons. They should not be accepted as blood donors if Anti-HBc is found positive.

A structural determined heteropolytungstate, [K(4)(H(2)O)(8)Cl][K(4)(H(2)O)(4)PTi(2)W(10)O(40)]·NH(2)OH 1, has been synthesized and evaluated for in vitro antiviral activities against hepatitis B (HBV) and SARS virus. The identity and high purity of compound 1 were confirmed by elemental analysis, NMR, IR analysis and single-crystal X-ray diffraction. The compound 1, evaluated in HepG 2.2.15 cells expressing permanently HBV, significantly reduced the levels of HBV antigens and HBV DNA in a dose-dependent and time-dependent manner. EC(50) values were determined to be 54μM for HBeAg, 61μM for HBsAg and 2.66μM for supernatant HBV DNA, as compared to 1671, 1570, 169μM, respectively, for the commercially-available hepatitis B drug adefovir dipivoxil (ADV). Intracellular cccDNA, pgRNA and HBcAg were also found to be decreased by compound 1 in a concentration-dependent manner. Cytotoxicity results showed that compound 1 has low toxicity in HepG 2 cells with CC(50) value of 515.20μM. The results indicate that compound 1 can efficiently inhibit HBV replication in HepG 2.2.15 cells line in vitro. Additionally, compound 1 also shows high anti-SARS activity at an EC(50) of 7.08μM and toxicity with a CC(50) of 118.6μM against MDCK cells., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: Cellular immune responses seem to prevail in acute hepatitis, whereas chronically infected patients demonstrate generally suppressed cellular immune responses and significantly greater antibody responses., Aim: To study hepatitis B virus (HBV) specific T cell proliferative responses in HBV related liver diseases., Methods: We analyzed the T lymphocyte proliferative responses to the nonspecific mitogen phytohemagglutinin (PHA) and the HBV specific hepatitis B core antigen (HBcAg) by calculating T cell proliferation index in 10 acute viral hepatitis (AVH) patients, 19 chronic hepatitis B (CHB) patients, 10 HBV cirrhotics, 10 inactive carriers and 10 healthy controls using MTT assay., Results: The mean proliferation index (PI) to PHA was highest in healthy controls (133.2 +/- 58.1) and lowest in cirrhotics (44.1 +/- 46.9) with all other groups falling in between. On comparing the mean T cell responses to HBcAg, AVH patients had the highest mean response (186.48 +/- 116.37) followed by CHB (137.9 +/- 134.3), inactive carriers (63.2 +/- 41.2) and cirrhotics (55.5 +/- 42.7)., Conclusions: Patients with AVH had the highest T cell response to HBcAg, which probably explains the clearance of virus in these patients, in contrast to patients with cirrhosis who had the lowest T cell response.

Background and Aim: We investigated whether intrahepatic markers could predict response in chronic hepatitis B virus (HBV) patients treated with peg-interferon and adefovir for 48 weeks., Methods: Intrahepatic covalently closed circular DNA (cccDNA), total intrahepatic HBV DNA and the proportion of hepatitis B surface antigen (HBsAg) and hepatitis B core antigen (HBcAg) positive hepatocytes in 16 hepatitis B e antigen (HBeAg) positive and 24 HBeAg negative patients were measured at baseline and at end of treatment., Results: Baseline intrahepatic markers were not associated with sustained virological response (SVR) defined as HBV DNA < 2000 IU/mL and persistent normal alanine aminotransferase levels at the end of follow-up (week 72). At end of treatment, intrahepatic cccDNA and total intrahepatic HBV DNA in HBeAg positive patients were significantly lower in patients with HBeAg seroconversion (P = 0.016 and P = 0.010) with positive predictive values (PPV) for SVR of 80% and 80%, respectively. In HBeAg negative patients, intrahepatic cccDNA and total intrahepatic HBV DNA had declined significantly at end of treatment (P = 0.035 and P = 0.041) and corresponding PPV for SVR was 73% and 82%. In HBeAg positive patients, median proportion of HBcAg positive hepatocytes declined significantly (P = 0.002) at end of treatment. In HBeAg negative patients, the proportion of HBsAg positive hepatocytes had declined significantly at end of treatment (P = 0.0009). Using HBsAg ≤ 7.5% as a limit, PPV for SVR in HBeAg negative patients was 83%., Conclusions: At end of treatment in HBeAg positive patients, intrahepatic cccDNA and total intrahepatic HBV DNA were predictive for SVR. In HBeAg negative patients a proportion of < 7.5% HBsAg positive hepatocytes at end of treatment was a strong predictor for SVR., (© 2011 Journal of Gastroenterology and Hepatology Foundation and Blackwell Publishing Asia Pty Ltd.)

Objective: To explore the role of mother's peripheral blood mononuclear cell (PBMC) infected with hepatitis B virus (HBV) in intrauterine transmission., Methods: We have selected 60 cases of pregnant women with negative serum HBV DNA and positive PBMC HBV DNA from hospitalized patients. These women and their neonates acted as the experimental group. Twenty cases of pregnant women with HBV serum marker negative were selected. These women and their neonates served as the control group. Immunohistochemistry was employed to detect the expressions of HBsAg and HBcAg in cells of every placental layer and CD68 cells of placenta of the pregnant women whose neonates' PBMC HBV DNA was positive and/or whose neonates' serum HBV DNA positive., Results: In the experimental group, neonatal serum HBV DNA of only four cases were positive, only eight cases' neonatal PBMC HBV DNA were positive and four cases had HBV DNA positive in both neonatal serum and PBMC. The expressions of HBsAg and HBcAg were detected in CD68 cells of villous stroma and blood capillary in only eight cases of neonatal placenta with positive PBMC HBV DNA. HBV infection was found in cells of every layer in placenta in two of four cases with neonatal serum HBV DNA positive. The expressions of HBsAg and HBcAg were detected in trophoblastic cells, CD68 cells of villous stroma and blood capillary in two of four cases with HBV DNA positive in both neonatal serum and PBMC. In control group, no positive signals were detected in neonates and placenta., Conclusion: HBV-infected PBMC in pregnant women may lead to intrauterine infection.

Background/aims: Hepatitis B virus-associated glomerulonephritis (HBV-GN) is recognized as one of the major secondary nephropathies in HBV high-risk areas. To determine possible differences in the expression of HBV immune markers in tissues, we retrospectively examined HBV immune markers in the serum, renal tissues, and liver tissues in 132 HBV-GN children., Methods: All 132 patients had biopsy-proven HBV-GN including the presence of positive HBV antigens in the kidney. Serum-HBV immune markers were tested by an enzyme-linked immunosorbent assay. Renal and liver biopsies were done in 26 patients. All renal tissues were examined for HBV immune markers by immunofluorescence, and liver tissues were examined by immunohistochemistry., Results: Among the 132 patients, all showed varying degrees of kidney injury. Serum hepatitis B envelope antigen (HBeAg) was positive in 80 patients and negative in 52 patients. The positivity rate of Hepatitis B core antigen in renal tissue was statistically higher in serum HBeAg (-) than in serum HBeAg (+) patients (96.2% vs. 55.0%). Furthermore, there was no relationship between the presence of hepatitis B surface antigen and HBcAg in liver and renal tissue., Conclusion: HBV markers are not consistently present in serum, renal tissues, and liver tissues in children with HBV-GN.

In this study, the core antigen (HBcAg) gene region of hepatitis B virus (HBV) was transformed and expressed into an eukaryotic expression vector by recombinant DNA technology in order to obtain the protein used in anti-HBc tests which is being one of the most important marker for the serodiagnosis of HBV infections. For this purpose, HBV-DNA positive patient sera were used as the source of viral nucleic acids, and the primers coding HBcAg gene region have been designed. After the amplification of HBcAg gene region by polymerase chain reaction (PCR), the amplicons purified by Invisorb Spin Rapid PCR Kit" (Invitek, Germany), were cloned to pYES2.1 plasmid via the TOPO TA expression kit (Invitrogen, USA) and this plasmid was transformed to competent bacteria (TOPO 10F' Escherichia coli) by CaCl2 method. After competent bacteria were grown on LB (Lysogeny Broth) agar media supplemented with ampicillin, the plasmid "pYES2.1 + HBcAg" were isolated and transformed to Saccaromyces cerevisiae via the "S.c. EasyComp Transformation Kit" (Invitrogen, USA). Finally, the expression of HBcAg by the yeast was confirmed with the use of in house ELISA method. Since the diagnostic kits used in our country for hepatitis B serology are usually imported products, this creates a great economical burden. Thus, the experience and knowledge that builds up following such studies will help to produce our own diagnostic products using our equity.

Objective: To construct virus-like particles of hepatitis B core antigen, with HBsAg "a" epitope exposed on the surface., Methods: Hepatitis B surface antigen "a" epitope were inserted into the Hepatitis B core antigen, between the 78th (Asp) and the 79th (Pro) amino acids. The gene was synthesized after the codon optimized, then it was ligated to the express vector after been enzyme digest. The virus-like particles were observed by electron microscope and detected by ELISA after been expressed and purified. Immune the rabbits by the VLPs, then detect the antibody., Result: The virus-like particles were confirmed by electron microscope. Its antigenicity and immunogenicity were identified by ELISA., Conclusion: The prokaryotic express plasmid with the fusion gene has been constructed successfully. The virus-like particles have been expressed, purified and identified, which lays the foundation for its application in the further.

Objective: To investigate the inhibitory effect on HBV replication of antisense locked nucleic acid (LNA) targeting to both S and C genes in HBV transgenic mice., Methods: Thirty HBV transgenic mice were randomly divided into five groups (n = 6): glucose control group were treated with 5% glucose solution, liposome control group were treated with liposome alone, S group were treated with LNA targeting to S gene, C group were treated with LNA targeting to C gene, and dual-target group were treated with LNA targeting to both S and C genes. Antisense LNA was injected into mice via the tail vein. Serum HBsAg was quantified by TRFIA. Serum HBV DNA was quantified by real-time PCR. The expression of HBV C-mRNA in the liver was detected by RT-PCR. The expression of HBsAg and HBcAg in the liver was detected by immunohistochemistry. Serum ALB, ALT, BUN and CR were measured using an automatic biochemical analyzer. The effects of antisense LNA on mouse organs were investigated by HE staining., Results: 5 days after LNA injection, serum HBsAg levels in the dual-target group were reduced by 72.8%, and serum HBV DNA levels were decreased by 52.9%. These values were significantly higher than those in the control groups (all P < 0.05). No significant differences were noted in serum ALB, ALT, BUN and CR between the experiment groups and the control groups (all P > 0.05). The expression levels of HBsAg and HBcAg in the liver of dual-target group were significantly lower than those in the control groups. No significant histopathological abnormality was found in liver and kidney tissues in all groups., Conclusion: Antisense LNA targeting to both S and C genes can significantly inhibit HBV replication in transgenic mice.

Compared to HIV and hepatitis C virus, the residual infectious risk of hepatitis B virus (HBV) posed by blood products is about 10 times higher. In addition to HBsAg testing, screening for anti-HBc was recommended by the German Advisory Committee Blood in March 2005. Prevalence of anti-HBc in German blood donors was investigated at five test sites located in different geographic regions. In total, 12,000 blood donors were screened for anti-HBc by PRISM HBcore, and a statistically representative number of these were tested with Abbott Murex anti-HBc total, bioMérieux Hepanostika anti-HBc uniform, Bio-Rad Monolisa anti-HBc PLUS and Dade Behring Enzygnost anti-HBc. Anti-HBc repeat reactive samples were tested for anti-HBs, anti-HBe and HBV DNA by individual donation NAT. The mean prevalence of anti-HBc was 1.75% in donors that had not been tested for anti-HBc in the past. The percentage of anti-HBs in anti-HBc repeat reactive donors was 93.7%. Samples that were additionally reactive for anti-HBe were anti-HBc reactive in all tested assays. The sample to cut-off (S/Co) values for anti-HBc were lower (competitive assays) in samples that were also positive for anti-HBe, when compared to samples that were only anti-HBc reactive. Most commercially available anti-HBc assays provide sufficient sensitivity for routine screening purposes, and lacking specificity is no longer a serious issue for most of them. Assay differences were recognized for samples that were anti-HBc only reactive. The overall loss of 1.75% of positive testing donors can be significantly reduced to 0.45% by implementation of re-entry procedures for donors with an anti-HBs titre of over 100 IU/l and negative by sensitive ID-NAT.

Objective: To Screen an EIA kit that fits best for using in serological detection of anti-HBc in the national investigation of hepatitis B virus infection., Methods: Top 5 kinds of kits on the best seller list of China-made anti-HBc EIA kit were selected and each detect the anti-HBc panel (95 positive and 57 negative) 5 times. The co/s ratio of each test was recorded and transformed by Ig(co/s+1). Reliability indices ICC and CV, and validity indices AUC, pAUC and Se(FPR=e) were calculated and compared between each kit., Results: The indices of ICC and CV of the 5 kinds of kits arranged in the same order of A, B, D, C and E, from the best to the worst. And the differences between them were significantly important (Bootstrap method, P < 0.05), except for between kits B and D in CV index; The AUCs ranged from 0.991 to 0.997; For index pAUCs, differences were significant between kits B, D, C ,which had the biggest values, kits A and E which had the lowest ones. For index Se(FPR=e), significant differences were only seen between kits B and A, and B and E (Bootstrap method, P < 0.05)., Conclusion: The diagnostic ability of all the 5 kinds of kits arrange in an order of B, C, D, A and E, from the best to the worst.

The hepatitis B virus (HBV) is estimated to have infected about 350 million people worldwide, making it one of the most common human pathogens. Renal involvement is among its most common extra hepatic manifestations and usually manifests in the form of immune complex mediated glomerulopathy, such as membranous glomerulonephritis (MGN), membranoproliferative glomerulonephritis (MPGN), mesangioproliferative glomerulonephritis and immunoglobulin A (IgA) nephropathy. Occurrence of focal and segmental glomerular sclerosis (FSGS) with HBV infection is rare and only five cases have been reported earlier. We report two cases of hepatitis B associated FSGS. In both the cases, HBsAg was demonstrated in the renal tissue and both the cases showed response to treatment with lamivudine, thus indicating a possible causal association between the viral infection and occurrence of nephrotic syndrome.

Chronic hepatitis B virus (HBV) infection is a complex interaction between replicating noncytopathic virus and dysregulatory host antiviral immunity. Plasmacytoid dendritic cells (pDCs) contribute to innate antiviral immunity via secreting type I interferons. Toll-like receptor (TLR) 9 is involved in major pattern recognition receptors expressed in pDCs. The frequency of pDCs and TLR9 expression in peripheral blood mononuclear cells (PBMC) was determined, using flow cytometry. IFN-alpha production by PBMC was evaluated in vitro in the presence of cytidine phosphate guanosine (CpG) with/without pDCs. The correlation between TLR9, pDCs frequency and viral load was also evaluated. TLR9 expression in pDCs in chronic HBV patients was significantly ( approximately 50%) reduced, supported by approximately 70% reduction of TLR9 mRNA, in comparison to healthy controls, correlating with the impairment of IFN-alpha production in vitro. Furthermore, pDCs frequency in these patients was substantially reduced ( approximately 30%), inversely correlating with serum ALT levels and HBV viral load. HBsAg and HBcAg were detected by immunohistochemistry in pDCs in chronic HBV patients. We conclude that HBV infection results in reduced frequency of circulating pDCs and their functional impairment via inhibiting the expression of TLR9. These data may provide useful information in both basic research and clinical treatment of chronic HBV infection.

Objective: To examine the presence of hepatitis B surface antigen (HBsAg), hepatitis B core antigen (HBcAg), and hepatitis B virus (HBV) DNA in parotid tissues from patients with positive serum HBV markers., Methods: HBsAg and HBcAg were examined in parotid biopsy tissues from patients with suspected parotid tumor and positive serum HBV markers by immunocytochemistry, and HBV DNA was detected in parotid tissues by PCR., Results: Among the 22 patients with a parotid tumor, only one was pathologically confirmed as a neoplasm; all others were benign. HBsAg and HBcAg were present in parotid cells with positive rates of 45.5% (10/22) and 40.9% (9/22), respectively, with an overall positive rate of 54.5% (12/22). Of the 22 cases with serum markers of HBV infection, seven (31.8%) had both HBsAg and HBcAg in the parotid cells. HBV DNA was present in seven of the 12 samples in which hepatitis B antigen was detected (58.3%)., Conclusions: HBV in saliva might originate from the infected salivary glands and the infectious saliva could transmit HBV.

Background: a peculiar form of fulminant hepatitis known as Labrea hepatitis, probably related to hepatitis B and D, has been reported in Brazilian Amazon as early as the 1930s., Methods: we reviewed the postmortem liver biopsies of 9 patients with Labrea Hepatitis. Immunostaining for HBV and HDV antigens were performed., Results: we found several important characteristics in the liver tissues: 1) moderate hepatocellular necro-inflammation, 2) hepatocellular ballooning, 3) ballooned hepatocytes with fat droplets surrounding the nucleus (morula-like cells or spongiocytes), 4) mild to moderate necrosis and/or mild portoseptal fibrosis. Hepatitis B surface antigen (HBsAg) was identified in 7 of the 9 cases and was concentrated in the Morula-like cells. Hepatitis B core antigen (HBcAg) was present in 5 cases, mostly in the hepatocyte's nucleous. The hepatitis D virus antigen (HDV Ag) was present in 5 cases, mostly in the cytoplasm and concentrated in the Morula-like cells., Conclusion: labrea hepatitis is a fatal disease mostly affecting isolated communities in the Amazon. Evidence implicates HBV and HDV in the etiology of this disease, but this hypothesis needs to be confirmed with genotyping and sequencing research on HBV DNA and HDV RNA extracted from the liver and sera of these patients.

Background and Aim: The localization of hepatitis B virus (HBV) core antigen to the nucleus or cytoplasm of hepatocytes has biological implications for viral packaging and persistence. This study examined the relationship between the localization of hepatitis B virus antigens, histological activity, and viral titer in patients with chronic HBV infection., Methods: Liver biopsies from 110 patients with chronic HBV infection were studied. Ishak's scoring system was used for the histological analysis. The localization of hepatitis B surface antigen (HBsAg) and hepatitis B core antigen (HBcAg) and the percentage of hepatocytes stained positive by immunohistochemistry were correlated with viral titer, histological activity, and fibrosis indices using Spearman rank correlation., Results: In 88 hepatitis B e-antigen (HBeAg)-positive individuals, the nuclear localization of HBcAg correlated significantly with DNA titer (r = 0.435, P = 0.001) and negatively with fibrosis (r = -0.297, P = 0.005). The cytoplasmic localization correlated significantly with histological activity (r = 0.211, P = 0.049). In 22 HBeAg-negative individuals, the nuclear localization of HBcAg correlated significantly with histological activity (r = 0.625, P = 0.002), DNA titer (r = 0.651, P = 0.009), and fibrosis (r = 0.447, P = 0.042). The cytoplasmic localization correlated significantly with DNA titer (r = 0.524, P = 0.045) and fibrosis (r = 0.528, P = 0.012). There was no correlation of HBsAg expression with DNA titer, histological activity index, or fibrosis in both groups. HBeAg-positive patients presented at a younger age., Conclusion: In HBeAg-positive individuals, nuclear core antigen correlated with DNA titer, and cytoplasmic localization with histological activity, whereas in HBeAg-negative individuals, nuclear localization correlated with DNA titer, histological activity, and fibrosis, and cytoplasmic localization correlated with DNA titer and fibrosis, but not with histological activity. These observations suggest biological differences between HBeAg-positive and -negative disease.