Class A capsid assembly modulator apoptotic elimination of hepatocytes with high HBV core antigen level in vivo is dependent on de novo core protein translation.

Capsid assembly is critical in the hepatitis B virus (HBV) life cycle, mediated by the viral core protein. Capsid assembly is the target for new anti-viral therapeutics known as capsid assembly modulators (CAMs) of which the CAM-aberrant (CAM-A) class induces aberrant shaped core protein structures and leads to hepatocyte cell death. This study aimed to identify the mechanism of action of CAM-A modulators leading to HBV-infected hepatocyte elimination where CAM-A-mediated hepatitis B surface antigen (HBsAg) reduction was evaluated in a stable HBV replicating cell line and in AAV-HBV-transduced C57BL/6, C57BL/6 SCID, and HBV-infected chimeric mice with humanized livers. Results showed that in vivo treatment with CAM-A modulators induced pronounced reductions in hepatitis B e antigen (HBeAg) and HBsAg, associated with a transient alanine amino transferase (ALT) increase. Both HBsAg and HBeAg reductions and ALT increase were delayed in C57BL/6 SCID and chimeric mice, suggesting that adaptive immune responses may indirectly contribute. However, CD8+ T cell depletion in transduced wild-type mice did not impact antigen reduction, indicating that CD8+ T cell responses are not essential. Transient ALT elevation in AAV-HBV-transduced mice coincided with a transient increase in endoplasmic reticulum stress and apoptosis markers, followed by detection of a proliferation marker. Microarray data revealed antigen presentation pathway (major histocompatibility complex class I molecules) upregulation, overlapping with the apoptosis. Combination treatment with HBV-specific siRNA demonstrated that CAM-A-mediated HBsAg reduction is dependent on de novo core protein translation. To conclude, CAM-A treatment eradicates HBV-infected hepatocytes with high core protein levels through the induction of apoptosis, which can be a promising approach as part of a regimen to achieve functional cure. IMPORTANCE: Treatment with hepatitis B virus (HBV) capsid assembly modulators that induce the formation of aberrant HBV core protein structures (CAM-A) leads to programmed cell death, apoptosis, of HBV-infected hepatocytes and subsequent reduction of HBV antigens, which differentiates CAM-A from other CAMs. The effect is dependent on the de novo synthesis and high levels of core protein.

Oxygen-dependent histone lysine demethylase 4 restricts hepatitis B virus replication.

Mammalian cells have evolved strategies to regulate gene expression when oxygen is limited. Hypoxia-inducible factors (HIF) are the major transcriptional regulators of host gene expression. We previously reported that HIFs bind and activate hepatitis B virus (HBV) DNA transcription under low oxygen conditions; however, the global cellular response to low oxygen is mediated by a family of oxygenases that work in concert with HIFs. Recent studies have identified a role for chromatin modifiers in sensing cellular oxygen and orchestrating transcriptional responses, but their role in the HBV life cycle is as yet undefined. We demonstrated that histone lysine demethylase 4 (KDM4) can restrict HBV, and pharmacological or oxygen-mediated inhibition of the demethylase increases viral RNAs derived from both episomal and integrated copies of the viral genome. Sequencing studies demonstrated that KDM4 is a major regulator of the hepatic transcriptome, which defines hepatocellular permissivity to HBV infection. We propose a model where HBV exploits cellular oxygen sensors to replicate and persist in the liver. Understanding oxygen-dependent pathways that regulate HBV infection will facilitate the development of physiologically relevant cell-based models that support efficient HBV replication.

Fulminant hepatitis due to spontaneous reactivation of virus B in an immunocompetent patient

We present the case of a 52-year-old woman with a history of HBeAg-negative chronic hepatitis B virus (HBV) infection, viral load (VL) Z+<20,000U.l/ml with no evidence of liver fibrosis and, therefore, untreated. She presented to the emergency department with jaundice, epigastric pain, nausea, and vomiting. On admission, blood analysis revealed ALT 3982U/l, AST 3221U/l, Gamma-GT 80U/l, alkaline phosphatase 252U/l, LDH 960U/l, bilirrubin12.5mg/dl; no elevation of acute phase reactants, 141,000 platelets and coagulopathy with a prothrombin activity of 29%. Abdominal ultrasound showed no relevant findings. The serological profile revealed AgHBs+, anti-HBe+ y anti-HBc IgM+ and VL VHB>100 mills. Ul/ml, the remaining serology was negative and other causes of liver disease were ruled out. With the diagnosis of severe acute hepatitis (SAH) due to HBV reactivation (HBVR) treatment with entecavir was initiated. Given the analytical evolution (Table 1) and the appearance of encephalopathy grade I-II/IV, an urgent liver transplant was performed. The histological result of the explant was conclusive with intense interphase and lobular hepatitis with extensive areas of massive necrosis in both lobes, without hepatic fibrosis compatible with fulminant hepatitis (FH). (AU)

Novel non-HAP class A HBV capsid assembly modulators have distinct in vitro and in vivo profiles.

IMPORTANCE: Chronic hepatitis B is the most important cause of liver cancer worldwide and affects more than 290 million people. Current treatments are mostly suppressive and rarely lead to a cure. Therefore, there is a need for novel and curative drugs that target the host or the causative agent, hepatitis B virus itself. Capsid assembly modulators are an interesting class of antiviral molecules that may one day become part of curative treatment regimens for chronic hepatitis B. Here we explore the characteristics of a particularly interesting subclass of capsid assembly modulators. These so-called non-HAP CAM-As have intriguing properties in cell culture but also clear virus-infected cells from the mouse liver in a gradual and sustained way. We believe they represent a considerable improvement over previously reported molecules and may one day be part of curative treatment combinations for chronic hepatitis B.

Conditional replication and secretion of hepatitis B virus genome uncover the truncated 3' terminus of encapsidated viral pregenomic RNA.

IMPORTANCE: The biogenesis and clinical application of serum HBV pgRNA have been a research hotspot in recent years. This study further characterized the heterogeneity of the 3' terminus of capsid RNA by utilizing a variety of experimental systems conditionally supporting HBV genome replication and secretion, and reveal that the 3' truncation of capsid pgRNA is catalyzed by cellular ribonuclease(s) and viral RNaseH at positions after and before 3' DR1, respectively, indicating the 3' DR1 as a boundary between the encapsidated portion of pgRNA for reverse transcription and the 3' unprotected terminus, which is independent of pgRNA length and the 3' terminal sequence. Thus, our study provides new insights into the mechanism of pgRNA encapsidation and reverse transcription, as well as the optimization of serum HBV RNA diagnostics.

Conserved Lysine Residues of Hepatitis B Virus Core Protein Are Not Required for Covalently Closed Circular DNA Formation.

Hepatitis B virus (HBV) core protein (HBc), the building block of the viral capsid, plays a critical role throughout the HBV life cycle. There are two highly conserved lysine residues, namely, K7 and K96, on HBc, which have been proposed to function at various stages of viral replication, potentially through lysine-specific posttranslational modifications (PTMs). Here, we substituted K7 and K96 with alanine or arginine, which would also block potential PTMs on these two lysine residues, and tested the effects of these substitutions on HBV replication and infection. We found that the two lysine residues were dispensable for all intracellular steps of HBV replication. In particular, all mutants were competent to form the covalently closed circular DNA (cccDNA) via the intracellular amplification pathway, indicating that K7 and K96, or any PTMs of these residues, were not essential for nucleocapsid uncoating, a prerequisite for cccDNA formation. Furthermore, we found that K7A and K7R mutations did not affect de novo cccDNA formation and RNA transcription during infection, indicating that K7 or any PTMs of this residue were dispensable for HBV infection. In addition, we demonstrated that the HBc K7 coding sequence (AAA), as part of the HBV polyadenylation signal UAUAAA, was indispensable for viral RNA production, implicating this cis requirement at the RNA level, instead of any function of HBc-K7, likely constrains the identity of the 7th residue of HBc. In conclusion, our results provided novel insights regarding the roles of lysine residues on HBc, and their coding sequences, in the HBV life cycle. IMPORTANCE Hepatitis B virus (HBV) infection remains a public health burden that affects 296 million individuals worldwide. HBV core protein (HBc) is involved in almost all steps in the HBV life cycle. There are two conserved lysine residues on HBc. Here, we found that neither of them is essential for HBV intracellular replication, including the formation of covalently closed circular DNA (cccDNA), the molecular basis for establishing and sustaining the HBV infection. However, K96 is critical for virion morphogenesis, while the K7 coding sequence, but not HBc-K7 itself, is indispensable, as part of the RNA polyadenylation signal, for HBV RNA production from cccDNA. Our results provide novel insights regarding the role of the conserved lysine residues on HBc, and their coding sequences, in viral replication, and should facilitate the development of antiviral drugs against the HBV capsid protein.

Phytomedicines to Target Hepatitis B Virus DNA Replication: Current Limitations and Future Approaches.

Hepatitis B virus infection (HBV) is one of the most common causes of hepatitis, and may lead to cirrhosis or hepatocellular carcinoma. According to the World Health Organization (WHO), approximately 296 million people worldwide are carriers of the hepatitis B virus. Various nucleos(t)ide analogs, which specifically suppress viral replication, are the main treatment agents for HBV infection. However, the development of drug-resistant HBV strains due to viral genomic mutations in genes encoding the polymerase protein is a major obstacle to HBV treatment. In addition, adverse effects can occur in patients treated with nucleos(t)ide analogs. Thus, alternative anti-HBV drugs of plant origin are being investigated as they exhibit excellent safety profiles and have few or no side effects. In this study, phytomedicines/phytochemicals exerting significant inhibitory effects on HBV by interfering with its replication were reviewed based on different compound groups. In addition, the chemical structures of these compounds were developed. This will facilitate their commercial synthesis and further investigation of the molecular mechanisms underlying their effects. The limitations of compounds previously screened for their anti-HBV effect, as well as future approaches to anti-HBV research, have also been discussed.

Association of PD-L1 gene polymorphisms and circulating sPD-L1 levels with HBV infection susceptibility and related liver disease progression.

Soluble molecules of programmed death ligand 1 (sPD-L1) are known to modulate T-cell depletion, an important mechanism of hepatitis B virus (HBV) persistence and liver disease progression. In addition, PD-L1 polymorphisms in the 3'-UTR can influence PD-L1 expression and have been associated with cancer risk, although not definitively. The purpose of this study was to investigate the association of PD-L1 polymorphisms and circulating levels of sPD-L1 in HBV infection and live disease progression. In this study, five hundred fifty-one HBV infected patients of the three clinically well-defined subgroups chronic hepatitis B (CHB, n = 186), liver cirrhosis (LC, n = 142) and hepatocellular carcinoma (HCC, n = 223) and 240 healthy individuals (HC) were enrolled. PD-L1 polymorphisms (rs2297136 and rs4143815) were genotyped by in-house validated ARMS assays. Logistic regression models were applied in order to determine the association of PD-L1 polymorphisms with HBV infection as well as with progression of related liver diseases. Plasma sPD-L1 levels were quantified by ELISA assays. The PD-L1 rs2297136 AA genotype was associated with HBV infection susceptibility (HBV vs. HC: OR = 1.6; 95%CI = 1.1-2.3; p = 0.0087) and disease progression (LC vs. CHB: OR = 1.8; 95%CI = 1.1-2.9; p = 0.018). Whereas, the rs2297136 GG genotype was a protective factor for HCC development. Plasma sPD-L1 levels were significantly high in HBV patients (p < 0.0001) and higher in the LC followed by CHB and HCC groups. High sPD-L1 levels correlated with increased liver enzymes and with advanced liver disease progression (Child-pugh C > B > A, p < 0.0001) and BCLC classification (BCLC D > C > B > A, p = 0.031). We could, for the first time, conclude that PD-L1 rs2297136 polymorphism and plasma sPD-L1 protein levels associate with HBV infection and HBV-related liver disease progression.

Reactivation of hepatitis B virus infection in patients with rheumatoid arthritis receiving tofacitinib.

OBJECTIVES: The aim of this study was to investigate hepatitis B virus (HBV) reactivation in patients with rheumatoid arthritis (RA) receiving tofacitinib. METHOD: This was a retrospective study performed in a regional teaching hospital in southern Taiwan. During January 2017 and December 2020, patients with a clinician-confirmed diagnosis of RA using tofacitinib for at least 3 months were enrolled. Serum HBV DNA levels and serum alanine aminotransferase were followed up around every 3 to 6 months to assess HBV reactivation. RESULTS: A total of 98 patients with RA were enrolled, and eight were hepatitis B surface antigen positive (HBsAg+) (8.1%), 64 were HBsAg-negative (HBsAg-)/hepatitis B core antibody positive (HBcAb+) (65.3%). In the HBsAg+ patients, two patients received antiviral prophylaxis, and none of them had HBV reactivation or hepatitis flare-up. The HBV reactivation rate was 33.3% (2/6) in the HBsAg+ RA patient without antiviral prophylaxis. Among the HBsAg-/HBcAb+ patients, the HBV reactivation rate was 3.1% (2/64). The incidence rate of HBV reactivation was 153.8 per 1000 person-years for overall HBsAg+ patients and 250 per 1000 person-years after excluding patients receiving antiviral prophylaxis. The incidence rate was 11.2 per 1000 person-years for HBsAg-/HBcAb+ patients with RA receiving tofacitinib. CONCLUSION: Tofacitinib could induce HBV reactivation in both HBsAg+ and HBsAg-/HBcAb+ RA patients. HBsAg+ patients receiving tofacitinib have a high incidence rate of HBV reactivation, which could be prevented by antiviral prophylaxis. Although the risk of reactivation is low in HBsAg-/HBcAb+ patients, closely monitoring HBV DNA and alanine aminotransferase should be suggested.

Serum HBV pregenomic RNA exhibited opposite associations with NKdim and NKbright cell immunity in treatment-naïve chronic hepatitis B patients.

Hepatitis B virus (HBV) pregenomic RNA (pgRNA) is a new biomarker that reflects HBV replication, but its relationship with natural killer (NK) cell immunity in chronic hepatitis B (CHB) is unknown. We assessed serum HBV pgRNA levels in 323 CHB patients by reverse transcription-polymerase chain reaction, assessed cytokine production and activation and inhibitory markers of NK cells by flow cytometry, and measured serum cytokines by enzyme-linked immunosorbent assays (ELISAs). Among the different CHB phases, the serum HBV pgRNA level was highest in the immune-tolerant (IT) and immune-active (IA) phases. Regarding NK and NKdim cells, HBV pgRNA was negatively associated with frequencies, but positively associated with NKp44 and NKp46 expression (activation markers). Regarding NKbright cells, serum HBV pgRNA was positively associated with frequency and programmed cell death protein 1 (PD1) expression (inhibitory marker), but negatively associated with NKp44 and NKp46. Serum HBV pgRNA was not associated with NKp30 (activation marker) on NK cells or subsets. Lastly, serum HBV pgRNA was positively correlated with the levels of serum IL-7 and IL-12P40 (NK cell-promoting cytokines) and negatively correlated with serum prostaglandin E2 (PGE2) level (which negatively regulates NK cells). In conclusion, we found varied relationships between serum HBV pgRNA and NK cells and subsets, indicating that HBV pgRNA may play a complicated role in NK cell-related immunity, providing new information on HBV and host immunity.

Anti-HBV therapeutic potential of small molecule 3,5,6,7,3',4'-Hexamethoxyflavone in vitro and in vivo.

Current treatment methods for hepatitis B are mainly antiviral drug therapy and immunotherapy, in which antiviral drugs are mainly nucleoside analogue and interferon. They can significantly reduce the viral load but rarely achieve hepatitis B surface antigen (HBsAg) loss. 3,5,6,7,3',4'-Hexamethoxyflavone was screened out from a small molecule compound library for its lower cytotoxic effect and greater HBsAg inhibition activity. Meanwhile, we further performed experiments in HepG2.2.15, HepG2-NTCP cells, PHHs and HBV transgenic mouse model to evaluate the anti-HBV effects of 3,5,6,7,3',4'-Hexamethoxyflavone. Our study found that 3,5,6,7,3',4'-Hexamethoxyflavone can significantly reduce the level of HBV RNAs, HBV DNA and HBsAg, in addition, the activity of four HBV promoters and the ratio of total RNAs/cccDNA and 3.5 kb RNA/cccDNA were decreased by 3,5,6,7,3',4'-Hexamethoxyflavone. Mechanistically, we found HNF3α plays important roles in Hex mediated HBV transcription inhibition. Our study indicated Hex was a potential anti-HBV therapeutic drug.

The Functional and Antiviral Activity of Interferon Alpha-Inducible IFI6 Against Hepatitis B Virus Replication and Gene Expression.

Hepatitis B virus is an enveloped DNA virus, that infects more than three hundred and sixty million people worldwide and leads to severe chronic liver diseases. Interferon-alpha inducible protein 6 (IFI6) is an IFN-stimulated gene (ISG) whose expression is highly regulated by the stimulation of type I IFN-alpha that restricts various kinds of virus infections by targeting different stages of the viral life cycle. This study aims to investigate the antiviral activity of IFI6 against HBV replication and gene expression. The IFI6 was highly induced by the stimulation of IFN-α in hepatoma cells. The overexpression of IFI6 inhibited while knockdown of IFI6 elevated replication and gene expression of HBV in HepG2 cells. Further study determined that IFI6 inhibited HBV replication by reducing EnhII/Cp of the HBV without affecting liver enriched transcription factors that have significant importance in regulating HBV enhancer activity. Furthermore, deletion mutation of EnhII/Cp and CHIP analysis revealed 100 bps (1715-1815 nt) putative sites involved in IFI6 mediated inhibition of HBV. Detailed analysis with EMSA demonstrated that 1715-1770 nt of EnhII/Cp was specifically involved in binding with IFI6 and restricted EnhII/Cp promoter activity. Moreover, IFI6 was localized mainly inside the nucleus to involve in the anti-HBV activity of IFI6. In vivo analysis based on the hydrodynamic injection of IFI6 expression plasmid along with HBV revealed significant inhibition of HBV DNA replication and gene expression. Overall, our results suggested a novel mechanism of IFI6 mediated HBV regulation that could develop potential therapeutics for efficient HBV infection treatment.

Secondary prevention of hepatitis B virus-related hepatocellular carcinoma with current antiviral therapies.

Over the past decades, marked advancement has been made in the prevention and treatment of hepatitis B virus (HBV) infection. Due to highly effective antiviral therapies for chronic hepatitis B (CHB), long-term clinical outcomes in patients with CHB has also been dramatically improved. However, current antiviral therapies for CHB cannot completely abolish the risk of hepatocellular carcinoma (HCC). In addition, current treatment guidelines for CHB should be interpreted with caution given that HBV-associated hepatocarcinogenesis could be underway in patients who are not eligible for antiviral therapies by current guidelines. Therefore, efforts to reconcile treatment guidelines with recent clinical evidence should be made for reducing further development of HCC. In this article, we review the secondary prevention of HBV-related HCC with current antiviral therapies.

Updates on management strategies of hepatitis B virus reactivation in patients with resolved hepatitis virus B infection undergoing immunosuppressive therapy in rheumatology and the current situation in Niigata Rheumatic Center.

With the introduction of methotrexate, biological disease-modifying antirheumatic drugs (bDMARDs), and targeted synthetic DMARDs (tsDMARDs), the disease activity of patients with rheumatoid arthritis has been dramatically ameliorated. However, these drugs have strong immunosuppressive effects and can cause reactivation of hepatitis B virus (HBV) in patients with resolved HBV infection. Corticosteroids or immunosuppressants used for other connective tissue diseases or vasculitis also carry a risk of inducing reactivation of HBV. Therefore, every rheumatologist should know how to detect the resolved infection of HBV in patients with rheumatic diseases receiving immunosuppressive therapy and how to monitor it when resolved infection is revealed. Of note, the cut-off index was changed from 2.1 log copies/ml to 20 IU/ml (1.3 Log IU/ml) in 2017. Rheumatologists should start nucleic acid analog administration at reactivation of HBV while performing ongoing immunosuppressive therapy in order to prevent severe or fulminant hepatitis. A low titer of HBs antibody (Ab) or lack of HBs Ab is a risk factor of reactivation of HBV. However, the reactivation of HBV cannot be prevented by HBs Ab titers at baseline or changes overtime. Rheumatologists should recognize that every immunosuppressive therapy, regardless of the mode of action, has a potential risk of reactivation. To facilitate proper management of patients with HBV infection, collaboration between rheumatologists and hepatologists is strongly encouraged. Patients' education, systems for checking electronic medical charts, and multidisciplinary efforts are considered important for detecting HBV reactivation.

Hepatitis B Virus Screening and Real Life Data in Patients with Solid Tumor Receiving Chemotherapy.

BACKGROUND: Reactivation of the hepatitis B virus (HBV) either during or after chemotherapy may cause serious and sometimes fatal hepatitis. All patients undergoing chemotherapy should therefore be screened in terms of HBV before chemotherapy. The purpose of this research was to identify HBV screening rates in patients with solid cancer undergoing parenteral chemotherapy and to determine the outcomes of patients undergoing HBV screening. METHODS: Data for patients undergoing parenteral chemotherapy for solid cancer from January 1, 2012 to December 30, 2018 were retrieved from our electronic health record patient files in this retrospective study. Screening was defined as hepatitis B surface antigen (HBsAg) and/or hepatitis B core antibody (HBcAb) tests carried out within six months prior the first chemotherapy session. RESULTS: Four thousand fifty-eight (63%) of the 6440 patients who underwent parenteral chemotherapy were screened for HBsAg and/or HBcAb. The proportions of patients screened for HBsAg and HBcAb improved from 38.8% (2012) to 76.3% (2018), and from 0.2% (2012) to 43% (2018), respectively (P<0.001). The HBsAg and HBcAb positivity rates were 2.9% and 36.5%, respectively. Antiviral prophylaxis was started in 11.8% of HBsAg-negative/HBcAb-positive patients and 40.5% of HBsAg-positive patients. HBV reactivation did not occur in patients receiving antiviral prophylaxis, but was identified in 7.2% of HBsAg-positive patients and 0.6% of HBsAg-negative/HBcAb-positive patients without antiviral prophylaxis. CONCLUSION: Although HBV screening rates before chemotherapy are increasing among solid cancer patients, the rate of initiation of antiviral prophylaxis is still low. It is therefore important to raise awareness regarding HBV reactivation during/after chemotherapy.

Curcumin and Photobiomodulation in Chronic Viral Hepatitis and Hepatocellular Carcinoma.

Immune modulation is a very modern medical field for targeting viral infections. In the race to develop the best immune modulator against viruses, curcumin, as a natural product, is inexpensive, without side effects, and can stimulate very well certain areas of the human immune system. As a bright yellow component of turmeric spice, curcumin has been the subject of thousands of scientific and clinical studies in recent decades to prove its powerful antioxidant properties and anticancer effects. Curcumin has been shown to influence inter- and intracellular signaling pathways, with direct effects on gene expression of the antioxidant proteins and those that regulate the immunity. Experimental studies have shown that curcumin modulates several enzyme systems, reduces nitrosative stress, increases the antioxidant capacity, and decreases the lipid peroxidation, protecting against fatty liver pathogenesis and fibrotic changes. Hepatitis B virus (HBV) affects millions of people worldwide, having sometimes a dramatic evolution to chronic aggressive infection, cirrhosis, and hepatocellular carcinoma. All up-to-date treatments are limited, there is still a gap in the scientific knowledge, and a sterilization cure may not yet be possible with the removal of both covalently closed circular DNA (cccDNA) and the embedded HBV DNA. With a maximum light absorption at 420 nm, the cytotoxicity of curcumin as photosensitizer could be expanded by the intravenous blue laser blood irradiation (IVBLBI) or photobiomodulation in patients with chronic hepatitis B infection, Hepatitis B e-antigen (HBeAg)-positive, noncirrhotic, but nonresponsive to classical therapy. Photobiomodulation increases DNA repair by the biosynthesis of complex molecules with antioxidant properties, the outset of repairing enzyme systems and new phospholipids for regenerating the cell membranes. UltraBioavailable Curcumin and blue laser photobiomodulation could suppress the virus and control better the disease by reducing inflammation/fibrosis and stopping the progression of chronic hepatitis, reversing fibrosis, and diminishing the progression of cirrhosis, and decreasing the incidence of hepatocellular carcinoma. Photodynamic therapy with blue light and curcumin opens new avenues for the effective prevention and cure of chronic liver infections and hepatocellular carcinoma. Blue laser light and UltraBioavailable Curcumin could be a new valuable alternative for medical applications in chronic B viral hepatitis and hepatocarcinoma, saving millions of lives.

IL-18 variant increases risk of enhanced HBV DNA replication in chronic hepatitis.

BACKGROUND: The outcome ofhepatitis B (HBV) infection is influenced by immune responses and host genetics. Interleukin-18 (IL-18) is a determinant factor in controlling the balance of Th1/Th2 during antiviral response.Weexamine therole of two functional polymorphisms -607A/C and-137A/C inIL-18 gene with risk of chronic HBV infection. METHODS AND RESULTS: Genomic DNA isolates were obtained from 200 seropositive cases stratified according to their HBV DNA loads, and 200 blood donorsas a control population. Genotypes of the two polymorphisms were identified by ARMS-PCR method. The -607A allele, the-607AA and -607AC genotypes were associated with increased risk to develop chronic HBV infection (1.98, 5.11 and 3.5-fold risks, respectively). By contrast, the -137C minor allele and CG genotype had protected effects against chronic HBV infection. We found that -607A allele, -607AA and -607AC genotypes were significantly more frequent in patient's group with high HBV DNA levels compared to patient group with low HBV DNA level. Additionally, they were associated with increased 1.72, 6.04 and 3.28-fold risk of high HBV DNA replication. Patients carrying "-607A/-137 C" or "-607A/-137 G" haplotypes presented a high risk to develop chronic HBV infection (OR = 3.27; OR = 4.32, respectively). CONCLUSIONS: Taken together, our data suggest that theIL-18 -607A/C functional polymorphism was associated with susceptibility to enhanced replicative form of HBV DNA in chronic infection.

Intracellular Trafficking of HBV Particles.

The human hepatitis B virus (HBV), that is causative for more than 240 million cases of chronic liver inflammation (hepatitis), is an enveloped virus with a partially double-stranded DNA genome. After virion uptake by receptor-mediated endocytosis, the viral nucleocapsid is transported towards the nuclear pore complex. In the nuclear basket, the nucleocapsid disassembles. The viral genome that is covalently linked to the viral polymerase, which harbors a bipartite NLS, is imported into the nucleus. Here, the partially double-stranded DNA genome is converted in a minichromosome-like structure, the covalently closed circular DNA (cccDNA). The DNA virus HBV replicates via a pregenomic RNA (pgRNA)-intermediate that is reverse transcribed into DNA. HBV-infected cells release apart from the infectious viral parrticle two forms of non-infectious subviral particles (spheres and filaments), which are assembled by the surface proteins but lack any capsid and nucleic acid. In addition, naked capsids are released by HBV replicating cells. Infectious viral particles and filaments are released via multivesicular bodies; spheres are secreted by the classic constitutive secretory pathway. The release of naked capsids is still not fully understood, autophagosomal processes are discussed. This review describes intracellular trafficking pathways involved in virus entry, morphogenesis and release of (sub)viral particles.

Hepatitis B virus biology and life cycle.

Hepatitis B virus (HBV) specifically infects hepatocytes and causes severe liver diseases. The HBV life cycle is unique in that the genomic DNA (relaxed-circular partially double-stranded DNA: rcDNA) is converted to a molecular template DNA (covalently closed circular DNA: cccDNA) to amplify a viral RNA intermediate, which is then reverse-transcribed back to viral DNA. The highly stable characteristics of cccDNA result in chronic infection and a poor rate of cure. This complex life cycle of HBV offers a variety of targets to develop antiviral agents. We provide here an update on the current knowledge of HBV biology and its life cycle, which may help to identify new antiviral targets.

Synchronised infection identifies early rate-limiting steps in the hepatitis B virus life cycle.

Hepatitis B virus (HBV) is an enveloped DNA virus that contains a partially double-stranded relaxed circular (rc) DNA. Upon infection, rcDNA is delivered to the nucleus where it is repaired to covalently closed circular (ccc) DNA that serves as the transcription template for all viral RNAs. Our understanding of HBV particle entry dynamics and host pathways regulating intracellular virus trafficking and cccDNA formation is limited. The discovery of sodium taurocholate co-transporting peptide (NTCP) as the primary receptor allows studies on these early steps in viral life cycle. We employed a synchronised infection protocol to quantify HBV entry kinetics. HBV attachment to cells at 4°C is independent of NTCP, however, subsequent particle uptake is NTCP-dependent and reaches saturation at 12 h post-infection. HBV uptake is clathrin- and dynamin dependent with actin and tubulin playing a role in the first 6 h of infection. Cellular fractionation studies demonstrate HBV DNA in the nucleus within 6 h of infection and cccDNA was first detected at 24 h post-infection. Our studies show the majority (83%) of cell bound particles enter HepG2-NTCP cells, however, only a minority (<1%) of intracellular rcDNA was converted to cccDNA, highlighting this as a rate-limiting in establishing infection in vitro. This knowledge highlights the deficiencies in our in vitro cell culture systems and will inform the design and evaluation of physiologically relevant models that support efficient HBV replication.