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Hepatitis B Virus core protein (HBc) has multiple roles in the viral lifecycle: viral assembly, compartment for reverse transcription, intracellular trafficking, and nuclear functions. HBc displays assembly polymorphism - it can assemble into icosahedral capsid and aberrant non-capsid structures. It has been hypothesized that the assembly polymorphism is due to allosteric conformational changes of HBc dimer, the smallest assembly unit, however, the mechanism governing the polymorphic assembly of the HBc dimer is still elusive. By using the experimental antiviral drug BAY 41-4109, we successfully transformed the HBc assembly from icosahedral capsid to helical tube. Structural analyses of HBc dimers from helical tubes, T = 4 icosahedral capsid, and sheet-like HBc ensemble revealed differences within the inter-dimer interface. Disruption of the HBc inter-dimer interface may likely promote the various assembly forms of HBc. Our work provides new structural insights into the HBV assembly mechanism and strategic guide for anti-HBV drug design.

Chronic infection with hepatitis B virus (HBV) constitutes a major global public health threat, causing substantial disease burdens such as liver cirrhosis and hepatocellular carcinoma, thus representing high unmet medical needs. Currently available therapies are safe, well tolerated, and highly effective in decreasing viremia and improving measured clinical outcomes with low rates of antiviral resistance. However, long-term management remains a clinical challenge, mainly due to the slow kinetics of HBV surface antigen clearance. In this article, we review emerging antivirals directed at novel targets derived from mechanisms of viral cellular entry, viral replication, viral assembly, and the host immune response, leading to preclinical and clinical trials for possible future therapeutic intervention. The recent therapeutic advances in the development of all categories of HBV inhibitors may pave the way for regimens of finite duration that result in long-lasting control of chronic hepatitis B infection.

Background: HBV infection continues to be an important worldwide cause of morbidity and mortality. Patients with chronic hepatitis B can be successfully treated using nucleoside/nucleotide analogues. However, drug-resistant HBV mutants frequently arise, leading to treatment failure and progression to liver disease. Here, we report the effects of GLS4, a non-nucleosidic inhibitor that exhibits a novel and highly specific anti-HBV activity., Methods: The median inhibitory concentrations (IC(50)s) of GLS4 on HBV were measured by Southern blotting. HBV capsid and core protein levels were detected by immunoblotting. To determine the antiviral activity of GLS4 against adefovir dipivoxil (ADV)-resistant HBV mutants, HepG2 cells transiently transfected with PUC-HBV1.2 plasmids that contained one of three major ADV-resistant mutations (rtA181T, rtA181V and rtN236T) were treated with GLS4. Intracellular HBV replicative intermediates were detected by Southern blotting. The effect on the in vitro assembly of HBV capsid protein was examined using dynamic light scattering and electron microscopy., Results: The IC(50) of GLS4 was 0.012 μM, which is significantly lower than that of lamivudine (0.325 μM). Immunoblot analysis of HepG2.2.15 cells and transiently transfected HepG2 cells indicated that GLS4 treatment interfered with the formation of core particles (assembly). The ADV-resistant HBV mutant strains were also sensitive to GLS4. Upon examining the in vitro assembly of HBV core protein 149 by electron microscopy, increased aberrant particles were observed after GLS4 treatment., Conclusions: GLS4 is a new and unique potential anti-HBV agent that possesses a different mechanism of action than existing therapeutic drugs.

Background/aims: In chronic hepatitis B, hepatic progenitor cells (HPCs) activation and ductular reactions occurred in periportal and portal area. However, the association between hepatitis B virus (HBV) infection and HPCs activation remains unknown. We aim to investigate the expansion of HPCs in patients with end-stage chronic hepatitis B and its relationship to HBV infection., Methodology: Liver biopsy specimens from 16 cases of end-stage liver disease caused by chronic hepatitis B were studied. The quantities of serum HBV DNA were available in 13 patients. The number of HPCs and the area of ductular reactions were quantitively analyzed on the cytokeratin 7 (CK7)-stained sections. Double-staining combined either HBsAg or HBcAg with CK7 were performed to assess the histological relationship between HBV infection and HPCs activation., Results: All of the sections showed liver cirrhosis and severe inflammation (HAI ranged from 12 to 17). The number of HPCs correlated with the area of ductular reactions positively. Multivariate analysis showed that serum HBV DNA level was independently associated with HPCs activation and ductular reactions. Moreover, the expression area of HBsAg in liver tissue correlated with HPCs activation positively., Conclusions: In end-stage of chronic hepatitis B, the expansion of hepatic progenitor cells and ductular reactions were extensive. HBV infection may be involved in the proliferation of progenitor cells in cirrhotic environments.

The authors investigate the effects and mechanisms of the anti-hepatitis B virus (HBV) agent Bay 41-4109. HepG2.2.15 cells were used to investigate the antiviral effects of Bay 41-4109 by using real-time polymerase chain reaction (PCR), western blotting, and immunofluorescence. The C terminally truncated core protein was expressed and purified. Changes in hepatitis B capsid formation were assayed by dynamic light scattering and transmission electronic microscopy. Bay 41-4109 was found to be a highly selective and potent inhibitor of hepatitis B virus replication in HepG2.2.15 cells. This compound was equally effective at inhibiting HBV DNA release and the cytoplasmic HBcAg level, with 50% inhibitory concentrations of 32.6 and 132 nM, respectively. HBV DNA and HBcAg were inhibited in a dose-dependent manner, indicating that the anti-HBV mechanisms are associated with and dependent on the rate of HBcAg inhibition. Our results indicate that Bay 41-4109 treatment disassembled the core capsids and separated them into monomers or dimers, the form in which they could be further degraded into peptides. The core protein assembled in a misdirected manner cannot function effectively. Our results suggest that, based on its particular activities, Bay 41-4109 is a promising anti-HBV candidate.

Bispecific antibodies have immense potential for use in clinical applications. In the present study, a bispecific diabody against human red blood cells (RBCs) and hepatitis B virus surface antigen (HBsAg) was used to detect HBsAg in blood specimens. The bispecific diabody was constructed by crossing over the variable region of the heavy chains and the light chains of anti-RBC and anti-HBsAg antibodies with a short linker, SRGGGS. In enzyme-linked immunosorbent assays, this bispecific diabody showed specific binding to both RBCs and HBsAg. When this bispecific diabody was mixed with human blood specimens in the presence of HBsAg, the dual binding sites of the diabody caused agglutination of human RBCs. This diabody-mediated agglutination assay was then used to test 712 clinical blood specimens and showed 97.7% sensitivity and 100% specificity when the results were compared with those of the conventional immunoassay, which was used as a reference. This autologous RBC agglutination assay provides a simple approach for rapid screening for HBsAg in blood specimens.

Objective: To gain some insights into the critical events relating to HBV transcriptional regulation by comparing HBV replicative characteristics in different cell lines., Methods: Hepatic cell lines QSG-7701 and HepG2 were transfected with plasmid PUC18-HBV 1.2 by standard calcium phosphate precipitation method, and 1.0 microg pSEAP2-control vector was included in the transfection procedures to serve as an internal control monitoring the transfection efficiency. Hepatitis B surface antigen (HBsAg) in the medium was detected by ELISA method and HBV DNA was quantitated using fluorescent quantitative PCR. The intracellular HBsAg and HBcAg were detected with immunofluorescent staining. The gene expression profiles of QSG-7701 and HepG2 were compared using oligonucleotide microarray; partial differentially expressed genes were verified with quantitative RT-PCR., Results: In the medium of the cultured HepG2 cells, HBsAg and HBV DNA could be detected 6 days after the transfection, whereas in QSG-7701 cells, the HBsAg and HBV DNA could be detected for 2 weeks. The HBV DNA in the culture medium of QSG-7701 was about 50 times more than that of the HepG2 cells which were kept in 1 x 10(7)copies/ml(-3) x 10(7)copies/ml for 0 to 10 days after the transfection. On the 4th day after the transfection, 20% to 30% of the QSG-7701 cells were positive with HBsAg and HBcAg immunofluorescent staining. The gene microarray analysis showed that most transcription factors involved with HBV life cycle in QSG-7701 and HepG2 cells had similar levels, whereas some factors involved with HBV transcriptional regulation and core particle disassembly, such as interleukin-6 (R=5.1340), retinoid X receptor, alpha (R=5.1268), hepatic leukemia factor (R=3.2538), serine protease PRRS23 (R=2.8356), hepatitis B virus x interacting protein (R=0.4939), serine protease inhibitor Kazal type 1 (R=0.0740) and matrix metalloproteinase 3 (negative in QSG-7701) were all differentially expressed by HepG2 cells., Conclusion: Different than HepG2 cells, the QSG-7701 cells could support a high level and relatively stable HBV replication after HBV DNA transient transfection. The HBV core particles were probably recycled in the QSG-7701 cells. The differential gene expressions between QSG-7701 and HepG2 might explain the mechanism of the different HBV replication patterns. Hepatic cell line QSG-7701 might serve as a useful tool for HBV transcriptional regulation research.

Objective: To construct a series of recombinant replication competent plasmids of hepatitis B virus (HBV) full-length genome with varying mutations of precore and basic core promoter., Methods: The plasmid containing the entire HBV (1.2 copies) genome was used to construct objective plasmids. The objective competent vectors were constructed by molecular cloning and PCR-based site-directed mutagenesis in vitro and confirmed with sequence analysis. After introducing the plasmids into hepatocellular carcinoma cell line (Huh7) by calcium phosphate transfection, the culture supernatant was collected to detect HBsAg and HBV DNA to analyze viral replication and expression., Results: Ten competent vectors with varying precore and basic core promoter mutations were constructed. After transfecting into Huh7, the vectors replicated and secreted viral particles., Conclusions: Ten full-length competent HBV recombinants were obtained, which were harbouring varying mutations within precore and basic core promoter.

Objective: To investigate the correlation between impaired non-viral specific immune function of dendritic cell (DC) and viral clearance and cytotoxic T lymphocyte (CTL) response to HBV or HCV in patients with HBV and HCV coinfection., Methods: Twenty-five patients with HBV and HCV coinfection were investigated in this study. In 1994 and 2002, biochemical and virological markers and quantitative serum HBV DNA and HCV RNA levels were detected in these patients. According to the virus clearance status, these patients were divided into 4 groups: 14 patients with both HBV and HCV clearance (Group A), 6 patients with HCV clearance only (Group B), 3 patients with HBV clearance only (Group C), and 2 patients with persistent infection of HBV and HCV (Group D). Phenotypes and immune functions of monocyte-derived DCs were compared between these groups. 51Cr release assay were used to measure CTL response to epitopes derived from HBV, HCV or influenza virus (as positive control) in HLA-A2+ patients., Results: Impaired non-viral specific immune functions of DCs were observed in group B, C and D compared with group A and normal donors (Group N). These impaired functions included CD86 decreasing expression and lower capacity to stimulating allogenic T cells and uptaking antigen. The specific CTL response to HBV- and HCV-derived peptides could be induced in group A (12/12). The specific CTL response to HBV-derived peptides or to HCV-derived peptides could be induced in group C (3/3) or B (5/5), respectively. But the specific CTL response to both of two HBV-derived peptides or two HCV-derived peptides could not be induced in group C (0/3) or B (0/5), respectively. And no CTL response to HBV or HCV-derived peptides could be induced in groups D (0/1) and N (0/4)., Conclusion: 1. The results suggest that specific CTL response to HBV or HCV play a vital role in the viral clearance. 2. The DCs with impaired non-viral specific immune functions exist in chronic patients with HBV and/or HCV infection, but do not interfere with clearance and CTL response to HBV or HCV. It is reasonable to speculate that impaired functions of DCs result from viral infection.

Objective: To understand HBV serotypes and genotypes epidemiology in a northern city and a southern city in China., Methods: Using polymerase chain reaction (PCR) and direct sequencing of HBV DNA PCR products, the serotypes and genotypes of HBV in 530 from HBsAg positive samples. The enrolled patients were from Harbin, a northern city and Lianjiang, a southern city in China., Results: Comparison of the serotypes and genotypes of HBV between Harbin and Lianjiang showed that adrq+ was the most predominant hepatitis B virus serotype in both Harbin and Lianjiang (87.2% and 73.5%,respectively), adw2 was the next (12.0% and 25.7%, respectively); genotype C was the most frequent in Harbin and Lianjiang (87.8% and 73.2%, respectively), and genotype B was the next (12.2% and 26.1%, respectively) only 1 patient was infected by genotype D, and 1 patient was found to be co-infected by genotype B and C in Lianjiang., Conclusion: The results suggest that the percentage of HBV serotypes and genotypes between Harbin and Lianjiang was significantly different (P less than 0.001), but the main HBV serotype and genotype of the two cities were similar.

Objective: To investigate the relationship between different genotypes of hepatitis B virus (HBV) and the severity of liver diseases., Methods: The S nucleotide sequences of HBV strains isolated from plasma samples of 284 patients were detected and compared. Among them, 87 patients were HBV asymptomatic carriers (ASC), 157 chronic hepatitis B (CHB), 22 liver cirrhosis (LC), and 18 hepatocellular carcinoma (HCC)., Results: Genotypes B and C were predominant, with a 26.1% proportion and a 63.2% proportion respectively. The percentage of genotypes B and C in patients with ASC, CHB, LC, and HCC were significantly different (x(2)=15.09, P<0.001). Compared with genotype B, genotype C was more common in patients with CHB and HCC (59.6% vs 43.2%, chi(2)=10.87, P<0.001; 7.7% vs 1.4%, x(2)=7.41, P<0.001), but in patients with LC there was no different (7.7% vs 8.1%, chi(2)=1.29, P>0.05)., Conclusion: This study suggests that genotype B and C are predominant. And genotype C may induce more severe the liver inflammation than genotype B may do.

Abstract: Background and objectives: Host–virus interactions play a central role in determining the prognosis of hepatitis B virus infection. Multi-factors activated immune cells (MAICs) are autologous peripheral blood mononuclear cells activated with anti-CD3 monoclonal antibody, interleukin-2 and interferon-γ. The present pilot clinical trial was designed to determine whether adoptively transferred MAICs inhibit the replication of hepatitis B virus and is safe for patients. Study design: Fourteen patients with chronic hepatitis B were enrolled in the study. A total of (1.5–4.0)×109 peripheral blood mononuclear cells were isolated from each patient and activated by anti-CD3 monoclonal antibody, interleukin-2 and interferon-γ in vitro for 10 days to produce MAICs. Cell phenotypes and levels of cytokine secretion were determined during cell culture. Patients were followed up for 1 year after the transfusion of MAICs. Results: After 10 days of culture, peripheral blood mononuclear cells were expanded to a mean fold of 4.68±1.78 and activated effectively, as demonstrated by CD25 expression and cytokine secretion. Cell activation peaked on day 4. All patients accepted the MAICs transfusion with some slight adverse events, and fulminant hepatitis and bilirubinemia were not observed. Significant HBV inhibition was observed in 8 out of 14 patients, in which 5 patients achieved complete response (defined as that serum HBV DNA levels below the detection limit, occurrence of HBeAg seroconversion and normalization of ALT were observed together after MAICs transfusion and kept for at least 6 months) and 3 patients achieved partial response (defined as that serum HBV DNA levels below the detection limit, occurrence of HBeAg disappearance and normalization of ALT were observed together in 6 months after MAICs transfusion, but kept for less than 6 months). Conclusion: These findings strongly suggest that MAICs transfusion, which effectively inhibits the replication of hepatitis B virus, is safe for patients. [Copyright &y& Elsevier]