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Hepatitis B virus (HBV) is a hepatotropic virus and an important human pathogen. There are an estimated 296 million people in the world that are chronically infected by this virus, and many of them will develop severe liver diseases including hepatitis, cirrhosis and hepatocellular carcinoma (HCC). HBV is a small DNA virus that replicates via the reverse transcription pathway. In this review, we summarize the molecular pathways that govern the replication of HBV and its interactions with host cells. We also discuss viral and non-viral factors that are associated with HBV-induced carcinogenesis and pathogenesis, as well as the role of host immune responses in HBV persistence and liver pathogenesis.

Hepatitis B virus (HBV) infection has a multi-dimensional effect on the host, which not only alters the dynamics of immune response but also persists in the hepatocytes to predispose oncogenic factors. The virus exists in multiple forms of which the nuclear localized covalently closed circular DNA (cccDNA) is the most stable and the primary reason for viral persistence even after clearance of surface antigen and viral DNA. The second reason is the existence of pregenomic RNA (pgRNA) containing virion particles. On the other hand, the integration of the viral genome in the host chromosome also leads to persistent production of viral proteins along with the chromosomal instabilities. The interferon treatment or administration of nucleot(s)ide analogs leads to reduction in the viral DNA load, but the pgRNA and surface antigen clearance are a slow process and complete loss of serological HBsAg is rare. The prolonged exposure of immune cells to the viral antigens, particularly HBs antigen, in the blood circulation results in T-cell exhaustion, which disrupts immune clearance of the virus and virus-infected cells. In addition, it predisposes immune-tolerant microenvironment, which facilitates the tumor progression. Thus cccDNA, pgRNA, and HBsAg along with the viral DNA could be the therapeutic targets in the early disease stages that may improve the quality of life of chronic hepatitis B patients by impeding the progression of the disease toward hepatocellular carcinoma.

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

Background Hydrodynamic injection (HI) of hepatitis B virus (HBV) mouse model is an useful tool for HBV related research in vivo. However, only 40% of C57/BL6 mice injected with 10 μg HBV genome contained plasmid (pAAV-HBV1.2), serum HBsAg more than 6 months and none of the BALB/c mice injected with 10 μg pAAV-HBV1.2 plasmid DNA, serum HBsAg positive more than 4 weeks in the previous study. Methods In this study, C57/BL6 and BALB/c mice were hydrodynamic injected with different doses of pAAV-HBV1.2 plasmid DNA. HBV related serum markers were detected by ELISA. ALT levels in the serum were measured using full automated biochemistry analyzer. HBcAg positive cells in the liver were detected by immunohistochemical staining. The mRNA levels of IRF3, ISGs including ISG15, OAS, PKR and immune factors including IFNγ, TNFα, TGFβ, IL-6, IL-10, PDL1 in liver of the mice were quantified by qRT-PCR. Results The results showed that the mice injected with 100 μg high-concentration or 1 μg low-concentration of pAAV-HBV1.2 plasmid DNA did not excert dominant influence on HBV persistence. In contrast, injection of 5 μg intermediate-dose of pAAV-HBV1.2 plasmid DNA led to significant prolonged HBsAg expression and HBV persistence in both C57/BL6 (80% of the mice with HBsAg positive more than 6 months) and BALB/c (60% of the mice with HBsAg positive more than 3 months) mice. IFNγ was significant up-regulated in liver of the mice injected with 1 μg or 100 μg pAAV-HBV1.2 plasmid DNA. TNFα was up-regulated significantly in liver of the mice injected with 100 μg pAAV-HBV1.2 plasmid DNA. Moreover, PDL1 was significant up-regulated in liver of the mice injected with 5 μg pAAV-HBV1.2 plasmid DNA. Conclusion In this paper we demonstrated that, in the HBV HI mouse model, the concentration of injected pAAV-HBV1.2 plasmid DNA contributes to the diverse kinetics of HBsAg and HBeAg in the serum as well as HBcAg expression level in the liver, which then determined the HBV persisternce, while the antiviral factors IFNγ, TNFα as well as immune negative regulatory factor PDL1 play important roles on HBV persistence.

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