Hepatitis B Virus DNA Integration and Clonal Expansion of Hepatocytes in the Chronically Infected Liver.

Human hepatitis B virus (HBV) can cause chronic, lifelong infection of the liver that may lead to persistent or episodic immune-mediated inflammation against virus-infected hepatocytes. This immune response results in elevated rates of killing of virus-infected hepatocytes, which may extend over many years or decades, lead to fibrosis and cirrhosis, and play a role in the high incidence of hepatocellular carcinoma (HCC) in HBV carriers. Immune-mediated inflammation appears to cause oxidative DNA damage to hepatocytes, which may also play a major role in hepatocarcinogenesis. An additional DNA damaging feature of chronic infections is random integration of HBV DNA into the chromosomal DNA of hepatocytes. While HBV DNA integration does not have a role in virus replication it may alter gene expression of the host cell. Indeed, most HCCs that arise in HBV carriers contain integrated HBV DNA and, in many, the integrant appears to have played a role in hepatocarcinogenesis. Clonal expansion of hepatocytes, which is a natural feature of liver biology, occurs because the hepatocyte population is self-renewing and therefore loses complexity due to random hepatocyte death and replacement by proliferation of surviving hepatocytes. This process may also represent a risk factor for the development of HCC. Interestingly, during chronic HBV infection, hepatocyte clones detected using integrated HBV DNA as lineage-specific markers, emerge that are larger than those expected to occur by random death and proliferation of hepatocytes. The emergence of these larger hepatocyte clones may reflect a survival advantage that could be explained by an ability to avoid the host immune response. While most of these larger hepatocyte clones are probably not preneoplastic, some may have already acquired preneoplastic changes. Thus, chronic inflammation in the HBV-infected liver may be responsible, at least in part, for both initiation of HCC via oxidative DNA damage and promotion of HCC via stimulation of hepatocyte proliferation through immune-mediated killing and compensatory division.

ICTV Virus Taxonomy Profile: Hepadnaviridae.

The family Hepadnaviridae comprises small enveloped viruses with a partially double-stranded DNA genome of 3.0-3.4 kb. All family members express three sets of proteins (preC/C, polymerase and preS/S) and replication involves reverse transcription within nucleocapsids in the cytoplasm of hepatocytes. Hepadnaviruses are hepatotropic and infections may be transient or persistent. There are five genera: Parahepadnavirus, Metahepadnavirus, Herpetohepadnavirus, Avihepadnavirus and Orthohepadnavirus. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Hepadnaviridae, which is available at ictv.global/report/hepadnaviridae.

ICTV Virus Taxonomy Profile: Deltavirus.

Hepatitis delta virus, the only member of the only species in the genus Deltavirus, is a unique human pathogen. Its ~1.7 kb circular negative-sense RNA genome encodes a protein, hepatitis delta antigen, which occurs in two forms, small and large, both with unique functions. Hepatitis delta virus uses host RNA polymerase II to replicate via double rolling circle RNA synthesis. Newly synthesized linear RNAs are circularized after autocatalytic cleavage and ligation. Hepatitis delta virus requires the envelope of the helper virus, hepatitis B virus (family Hepadnaviridae), to produce infectious particles. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of Deltavirus which is available at www.ictv.global/report/deltavirus.

Why, who and when to start treatment for chronic hepatitis B infection.

Chronic hepatitis B remains a major global health challenge due to morbidity and mortality from hepatocellular carcinoma and complications of liver cirrhosis. Current treatment regimens are non-curative and, once initiated, treatment is of indefinite duration for the majority. The decision to initiate treatment decisions is based on risk stratification. Advances in our understanding of the natural history of chronic hepatitis B have led to a paradigm shift in recommendations for treatment. Emerging non-invasive biomarkers of disease activity will further enhance disease stratification. In this review, we summarise the guidance from major international societies on treatment for chronic hepatitis B and explore some of the novel approaches to disease assessment.

Immune Tolerant Chronic Hepatitis B: The Unrecognized Risks.

Chronic infection with hepatitis B virus (HBV) progresses through multiple phases, including immune tolerant, immune active, immune control, and, in a subset of patients who achieve immune control, reactivation. The first, the immune tolerant phase, is considered to be prolonged in duration but essentially benign in nature, lacking long-term consequences, and thus not recommended for antiviral therapy. This review challenges the notion that the immune tolerant phase is truly benign and considers the possibility that events during this phase may contribute significantly to cirrhosis, hepatocellular carcinoma (HCC), and the premature death of 25% of HBV carriers worldwide. Thus, earlier treatment than recommended by current guidelines should be considered. Low therapeutic coverage exacerbated by restrictive treatment guidelines may facilitate disease progression in many patients but also increase the risk of neonatal and horizontal transmission from untreated mothers to their children. While a prophylactic vaccine exists, there are many areas worldwide where the treatment of adults and the delivery of an effective vaccination course to newborns present difficult challenges.

HBV DNA Integration and Clonal Hepatocyte Expansion in Chronic Hepatitis B Patients Considered Immune Tolerant.

BACKGROUND & AIMS: Chronic infection with hepatitis B virus (HBV) progresses through different phases. The first, called the immune-tolerant phase, has been associated with a lack of disease activity. We examined HBV-DNA integration, clonal hepatocyte expansion, HBV antigen expression, and HBV-specific immune responses in patients in the immune-tolerant phase to assess whether this designation is appropriate or if there is evidence of disease activity. METHODS: We studied HBV-DNA integration, clonal hepatocyte expansion, and expression of hepatitis B surface antigen and core antigen in liver tissues from 26 patients with chronic HBV infection (ages, 14-39 y); 9 patients were positive for hepatitis B e antigen (HBeAg) in the immune-tolerant phase and were matched for age with 10 HBeAg-positive patients with active disease and 7 HBeAg-negative patients with active disease. Peripheral blood samples were collected and HBV-specific T cells were quantified for each group. RESULTS: Detection of HBV antigens differed among groups. However, unexpectedly high numbers of HBV-DNA integrations, randomly distributed among chromosomes, were detected in all groups. Clonal hepatocyte expansion in patients considered immune tolerant also was greater than expected, potentially in response to hepatocyte turnover mediated by HBV-specific T cells, which were detected in peripheral blood cells from patients in all phases of infection. CONCLUSIONS: We measured HBV-specific T cells, HBV-DNA integration, and clonal hepatocyte expansion in different disease phases of young patients with chronic hepatitis B, with emphasis on the so-called immune-tolerant phase. A high level of HBV-DNA integration and clonal hepatocyte expansion in patients considered immune tolerant indicated that hepatocarcinogenesis could be underway-even in patients with early stage chronic HBV infection. Our findings do not support the concepts that this phase is devoid of markers of disease progression or that an immune response has not been initiated. We propose that this early phase be called a high-replication, low-inflammation stage. The timing of therapeutic interventions to minimize further genetic damage to the hepatocyte population should be reconsidered.

HBV replication, pathobiology and therapy: Unanswered questions.

This volume explores these and other issues of relevance to our understanding of the HBV life cycle and clinical management of chronic HBV infections. The ultimate goals of these studies is not just to obtain a more precise understanding of the HBV life cycle, but to also acquire an understanding that will lead to more effective treatments for an infection and pathogenic process that currently causes ∼500,000 to 1,000,000 deaths per year.

Animal models and the molecular biology of hepadnavirus infection.

Australian antigen, the envelope protein of hepatitis B virus (HBV), was discovered in 1967 as a prevalent serum antigen in hepatitis B patients. Early electron microscopy (EM) studies showed that this antigen was present in 22-nm particles in patient sera, which were believed to be incomplete virus. Complete virus, much less abundant than the 22-nm particles, was finally visualized in 1970. HBV was soon found to infect chimpanzees, gorillas, orangutans, gibbon apes, and, more recently, tree shrews (Tupaia belangeri) and cynomolgus macaques (Macaca fascicularis). This restricted host range placed limits on the kinds of studies that might be performed to better understand the biology and molecular biology of HBV and to develop antiviral therapies to treat chronic infections. About 10 years after the discovery of HBV, this problem was bypassed with the discovery of viruses related to HBV in woodchucks, ground squirrels, and ducks. Although unlikely animal models, their use revealed the key steps in hepadnavirus replication and in the host response to infection, including the fact that the viral nuclear episome is the ultimate target for immune clearance of transient infections and antiviral therapy of chronic infections. Studies with these and other animal models have also suggested interesting clues into the link between chronic HBV infection and hepatocellular carcinoma.

Molecular biology of hepatitis B virus infection.

Human hepatitis B virus (HBV) is the prototype of a family of small DNA viruses that productively infect hepatocytes, the major cell of the liver, and replicate by reverse transcription of a terminally redundant viral RNA, the pregenome. Upon infection, the circular, partially double-stranded virion DNA is converted in the nucleus to a covalently closed circular DNA (cccDNA) that assembles into a minichromosome, the template for viral mRNA synthesis. Infection of hepatocytes is non-cytopathic. Infection of the liver may be either transient (<6 months) or chronic and lifelong, depending on the ability of the host immune response to clear the infection. Chronic infections can cause immune-mediated liver damage progressing to cirrhosis and hepatocellular carcinoma (HCC). The mechanisms of carcinogenesis are unclear. Antiviral therapies with nucleoside analog inhibitors of viral DNA synthesis delay sequelae, but cannot cure HBV infections due to the persistence of cccDNA in hepatocytes.

Towards an HBV cure: state-of-the-art and unresolved questions--report of the ANRS workshop on HBV cure.

HBV infection is a major cause of liver cirrhosis and hepatocellular carcinoma. Although HBV infection can be efficiently prevented by vaccination, and treatments are available, to date there is no reliable cure for the >240 million individuals that are chronically infected worldwide. Current treatments can only achieve viral suppression, and lifelong therapy is needed in the majority of infected persons. In the framework of the French National Agency for Research on AIDS and Viral Hepatitis 'HBV Cure' programme, a scientific workshop was held in Paris in June 2014 to define the state-of-the-art and unanswered questions regarding HBV pathobiology, and to develop a concerted strategy towards an HBV cure. This review summarises our current understanding of HBV host-interactions leading to viral persistence, as well as the roadblocks to be overcome to ultimately address unmet medical needs in the treatment of chronic HBV infection.

Virology. Comment on "Specific and nonhepatotoxic degradation of nuclear hepatitis B virus cccDNA".

Lucifora et al. (Research Articles, 14 March 2014, p. 1221) report that the hepatitis B virus (HBV) transcriptional template, a long-lived covalently closed circular DNA (cccDNA) molecule, is degraded noncytolytically by agents that up-regulate APOBEC3A and 3B. If these results can be independently confirmed, they would represent a critical first step toward development of a cure for the 400 million patients who are chronically infected by HBV.

Duck hepatitis B virus covalently closed circular DNA appears to survive hepatocyte mitosis in the growing liver.

Nucleos(t)ide analogues that inhibit hepatitis B virus (HBV) DNA replication are typically used as monotherapy for chronically infected patients. Treatment with a nucleos(t)ide analogue eliminates most HBV DNA replication intermediates and produces a gradual decline in levels of covalently closed circular DNA (cccDNA), the template for viral RNA synthesis. It remains uncertain if levels of cccDNA decline primarily through hepatocyte death, or if loss also occurs during hepatocyte mitosis. To determine if cccDNA survives mitosis, growing ducklings infected with duck hepatitis B virus (DHBV) were treated with the nucleoside analogue, Entecavir. Viremia was suppressed at least 10(5)-fold, during a period when average liver mass increased 23-fold. Analysis of the data suggested that if cccDNA synthesis was completely inhibited, at least 49% of cccDNA survived hepatocyte mitosis. However, there was a large duck-to-duck variation in cccDNA levels, suggesting that low level cccDNA synthesis may contribute to this apparent survival through mitosis.

Identification of disubstituted sulfonamide compounds as specific inhibitors of hepatitis B virus covalently closed circular DNA formation.

Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) plays a central role in viral infection and persistence and is the basis for viral rebound after the cessation of therapy, as well as the elusiveness of a cure even after extended treatment. Therefore, there is an urgent need for the development of novel therapeutic agents that directly target cccDNA formation and maintenance. By employing an innovative cell-based cccDNA assay in which secreted HBV e antigen is a cccDNA-dependent surrogate, we screened an in-house small-molecule library consisting of 85,000 drug-like compounds. Two structurally related disubstituted sulfonamides (DSS), termed CCC-0975 and CCC-0346, emerged and were confirmed as inhibitors of cccDNA production, with low micromolar 50% effective concentrations (EC(50)s) in cell culture. Further mechanistic studies demonstrated that DSS compound treatment neither directly inhibited HBV DNA replication in cell culture nor reduced viral polymerase activity in the in vitro endogenous polymerase assay but synchronously reduced the levels of HBV cccDNA and its putative precursor, deproteinized relaxed circular DNA (DP-rcDNA). However, DSS compounds did not promote the intracellular decay of HBV DP-rcDNA and cccDNA, suggesting that the compounds interfere primarily with rcDNA conversion into cccDNA. In addition, we demonstrated that CCC-0975 was able to reduce cccDNA biosynthesis in duck HBV-infected primary duck hepatocytes. This is the first attempt, to our knowledge, to identify small molecules that target cccDNA formation, and DSS compounds thus potentially serve as proof-of-concept drug candidates for development into therapeutics to eliminate cccDNA from chronic HBV infection.

The persistence in the liver of residual duck hepatitis B virus covalently closed circular DNA is not dependent upon new viral DNA synthesis.

Residual hepatitis B virus (HBV) DNA can be detected following the resolution of acute HBV infection. Our previous work using duck hepatitis B virus (DHBV) infected ducks, indicated that ~80% of residual DHBV DNA in the liver is in the covalently closed circular DNA (cccDNA) form, suggesting that viral DNA synthesis is suppressed. The current study asked more directly if maintenance of residual DHBV cccDNA is dependent upon ongoing viral DNA synthesis. Ducks that recovered from acute DHBV infection were divided into 2 groups and treated with the antiviral drug, Entecavir (ETV), or placebo. No major differences in the stability of cccDNA or levels of residual cccDNA were observed in liver biopsy tissues taken 95 days apart from ETV treated and placebo control ducks. The data suggest that residual DHBV cccDNA is highly stable and present in a cell population with a rate of turnover similar to normal, uninfected hepatocytes.

Clonal expansion of normal-appearing human hepatocytes during chronic hepatitis B virus infection.

Chronic hepatitis B virus (HBV) infections are associated with persistent immune killing of infected hepatocytes. Hepatocytes constitute a largely self-renewing population. Thus, immune killing may exert selective pressure on the population, leading it to evolve in order to survive. A gradual course of hepatocyte evolution toward an HBV-resistant state is suggested by the substantial decline in the fraction of infected hepatocytes that occurs during the course of chronic infections. Consistent with hepatocyte evolution, clones of >1,000 hepatocytes develop postinfection in the noncirrhotic livers of chimpanzees chronically infected with HBV and of woodchucks infected with woodchuck hepatitis virus (W. S. Mason, A. R. Jilbert, and J. Summers, Proc. Natl. Acad. Sci. U. S. A. 102:1139-1144, 2005; W. S. Mason et al., J. Virol. 83:8396-8408, 2009). The present study was carried out to determine (i) if extensive clonal expansion of hepatocytes also occurred in human HBV carriers, particularly in the noncirrhotic liver, and (ii) if clonal expansion included normal-appearing hepatocytes, not just hepatocytes that appear premalignant. Host DNA extracted from fragments of noncancerous liver, collected during surgical resection of hepatocellular carcinoma (HCC), was analyzed by inverse PCR for randomly integrated HBV DNA as a marker of expanding hepatocyte lineages. This analysis detected extensive clonal expansion of hepatocytes, as previously found in chronically infected chimpanzees and woodchucks. Tissue sections were stained with hematoxylin and eosin (H&E), and DNA was extracted from the adjacent section for inverse PCR to detect integrated HBV DNA. This analysis revealed that clonal expansion can occur among normal-appearing human hepatocytes.

Detection of clonally expanded hepatocytes in chimpanzees with chronic hepatitis B virus infection.

During a hepadnavirus infection, viral DNA integrates at a low rate into random sites in the host DNA, producing unique virus-cell junctions detectable by inverse nested PCR (invPCR). These junctions serve as genetic markers of individual hepatocytes, providing a means to detect their subsequent proliferation into clones of two or more hepatocytes. A previous study suggested that the livers of 2.4-year-old woodchucks (Marmota monax) chronically infected with woodchuck hepatitis virus contained at least 100,000 clones of >1,000 hepatocytes (W. S. Mason, A. R. Jilbert, and J. Summers, Proc. Natl. Acad. Sci. USA 102:1139-1144, 2005). However, possible correlations between sites of viral-DNA integration and clonal expansion could not be explored because the woodchuck genome has not yet been sequenced. In order to further investigate this issue, we looked for similar clonal expansion of hepatocytes in the livers of chimpanzees chronically infected with hepatitis B virus (HBV). Liver samples for invPCR were collected from eight chimpanzees chronically infected with HBV for at least 20 years. Fifty clones ranging in size from approximately 35 to 10,000 hepatocytes were detected using invPCR in 32 liver biopsy fragments (approximately 1 mg) containing, in total, approximately 3 x 10(7) liver cells. Based on searching the analogous human genome, integration sites were found on all chromosomes except Y, approximately 30% in known or predicted genes. However, no obvious association between the extent of clonal expansion and the integration site was apparent. This suggests that the integration site per se is not responsible for the outgrowth of large clones of hepatocytes.

The amount of hepatocyte turnover that occurred during resolution of transient hepadnavirus infections was lower when virus replication was inhibited with entecavir.

Transient hepadnavirus infections can involve spread of virus to the entire hepatocyte population. In this situation hepatocytes present following recovery are derived from infected hepatocytes. During virus clearance antiviral cytokines are thought to block virus replication and formation of new covalently closed circular DNA (cccDNA), the viral transcriptional template. It remains unclear if existing cccDNA is eliminated noncytolytically or if hepatocyte death and proliferation, to compensate for killing of some of the infected hepatocytes, are needed to remove cccDNA from surviving infected hepatocytes. Interpreting the relationship between hepatocyte death and cccDNA elimination requires knowing both the amount of hepatocyte turnover and whether cccDNA synthesis is effectively blocked during the period of immune destruction of infected hepatocytes. We have addressed these questions by asking if treatment of woodchucks with the nucleoside analog inhibitor of viral DNA synthesis entecavir (ETV) reduced hepatocyte turnover during clearance of transient woodchuck hepatitis virus (WHV) infections. To estimate hepatocyte turnover, complexity analysis was carried out on virus-cell DNA junctions created by integration of WHV and present following recovery in the livers of WHV-infected control or ETV-treated woodchucks. We estimated that, on average, 2.2 to 4.8 times less hepatocyte turnover occurred during immune clearance in the ETV-treated woodchucks. Computer modeling of the complexity data suggests that mechanisms in addition to hepatocyte death were responsible for elimination of cccDNA during recovery from transient infections.