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.

Detection of Hepatocyte Clones Containing Integrated Hepatitis B Virus DNA Using Inverse Nested PCR.

Chronic hepatitis B virus (HBV) infection is a major cause of liver cirrhosis and hepatocellular carcinoma (HCC), leading to ~600,000 deaths per year worldwide. Many of the steps that occur during progression from the normal liver to cirrhosis and/or HCC are unknown. Integration of HBV DNA into random sites in the host cell genome occurs as a by-product of the HBV replication cycle and forms a unique junction between virus and cellular DNA.

Therapeutic Antiviral Effect of the Nucleic Acid Polymer REP 2055 against Persistent Duck Hepatitis B Virus Infection.

Previous studies have demonstrated that nucleic acid polymers (NAPs) have both entry and post-entry inhibitory activity against duck hepatitis B virus (DHBV) infection. The inhibitory activity exhibited by NAPs prevented DHBV infection of primary duck hepatocytes in vitro and protected ducks from DHBV infection in vivo and did not result from direct activation of the immune response. In the current study treatment of primary human hepatocytes with NAP REP 2055 did not induce expression of the TNF, IL6, IL10, IFNA4 or IFNB1 genes, confirming the lack of direct immunostimulation by REP 2055.

Conceptual models for the initiation of hepatitis B virus-associated hepatocellular carcinoma.

Although chronic hepatitis B virus (HBV) infection is a known risk factor for the development of hepatocellular carcinoma (HCC), the steps involved in the progression from normal liver to HCC are poorly understood. In this review, we apply five conceptual models, previously proposed by Vineis et al. to explain carcinogenesis in general, to explore the possible steps involved in the initiation and evolution of HBV-associated HCC.

Clonal expansion of hepatocytes with a selective advantage occurs during all stages of chronic hepatitis B virus infection.

Hepatocyte clone size was measured in liver samples of 21 patients in various stages of chronic hepatitis B virus (HBV) infection and from 21 to 76 years of age. Hepatocyte clones containing unique virus-cell DNA junctions formed by the integration of HBV DNA were detected using inverse nested PCR. The maximum hepatocyte clone size tended to increase with age, although there was considerable patient-to-patient variation in each age group.

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.

Nucleic acid polymers prevent the establishment of duck hepatitis B virus infection in vivo.

Nucleic acid polymers (NAPs) are novel, broad-spectrum antiviral compounds that use the sequence-independent properties of phosphorothioate oligonucleotides (PS-ONs) as amphipathic polymers to block amphipathic interactions involved in viral entry. Using the duck hepatitis B virus (DHBV) model of human hepatitis B virus infection, NAPs have been shown to have both entry and postentry antiviral activity against DHBV infection in vitro in primary duck hepatocytes (PDH). In the current study, various NAPs were assessed for their prophylactic activity in vivo against DHBV infection in ducks.

Nucleic acid polymers inhibit duck hepatitis B virus infection in vitro.

Nucleic acid polymers (NAPs) utilize the sequence-independent properties of phosphorothioate oligonucleotides (PS-ONs) to target protein interactions involved in viral replication. NAPs are broadly active against a diverse range of enveloped viruses that use type I entry mechanisms. The antiviral activity of NAPs against hepatitis B virus (HBV) infection was assessed in vitro in duck hepatitis B virus (DHBV)-infected primary duck hepatocytes (PDH).

The development of persistent duck hepatitis B virus infection can be prevented using antiviral therapy combined with DNA or recombinant fowlpoxvirus vaccines.

We recently reported the development of a successful post-exposure combination antiviral and "prime-boost" vaccination strategy using the duck hepatitis B virus (DHBV) model of human hepatitis B virus infection. The current study aimed to simplify the vaccination strategy and to test the post-exposure efficacy of combination therapy with the Bristol-Myers Squibb antiviral drug, entecavir (ETV) and either a single dose of DHBV DNA vaccines on day 0 post-infection (p.i.

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.

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.

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.

Pre-P is a secreted glycoprotein encoded as an N-terminal extension of the duck hepatitis B virus polymerase gene.

The duck hepatitis B virus (DHBV) pregenomic RNA is a bicistronic mRNA encoding the core and polymerase proteins. Thirteen AUGs (C2 to C14) and 10 stop codons (S1 to S10) are located between the C1 AUG for the core protein and the P1 AUG that initiates polymerase translation. We previously found that the translation of the DHBV polymerase is initiated by ribosomal shunting.

Immune selection during chronic hepadnavirus infection.

Purpose: Late-stage outcomes of chronic hepatitis B virus (HBV) infection, including fibrosis, cirrhosis, and hepatocellular carcinoma (HCC) result from persistent liver injury mediated by HBV antigen specific cytotoxic T lymphocytes (CTLs). Two other outcomes that often accompany chronic infection, the emergence of mutant viruses, including HBe-antigen negative (HBeAg (-)) HBV, and a reduction over time in the fraction of hepatocytes productively infected with HBV, may also result from persistent immune attack by antiviral CTLs. To gain insights into how these latter changes take place, we employed computer simulations of the chronically infected liver.

Hepatocyte turnover in transient and chronic hepadnavirus infections.

Hepatocyte turnover appears to be an important feature in the resolution of transient and progression of chronic hepadnavirus infections. Hepatocyte death, initiated through attack by antiviral cytotoxic T-lymphocytes (CTL), and compensatory hepatocyte proliferation, are both believed to be major contributing factors in the loss of virus DNA during immune resolution of transient infections. Noncytopathic curing of hepatocytes is also suggested to occur, though this mechanism does not prevent the death of large numbers of hepatocytes.

The liver of woodchucks chronically infected with the woodchuck hepatitis virus contains foci of virus core antigen-negative hepatocytes with both altered and normal morphology.

The livers of woodchucks chronically infected with woodchuck hepatitis virus (WHV) contain foci of morphologically altered hepatocytes (FAH) with "basophilic", "amphophilic" and "clear cell" phenotypes, which are possibly pre-neoplastic in nature. Interestingly, most fail to express detectable levels of WHV proteins and nucleic acids. We studied sections of WHV-infected liver tissue to determine if all foci of hepatocytes that failed to express detectable levels of WHV, as assessed by immunoperoxidase staining for WHV core antigen, could be classified morphologically as FAH.

DNA vaccines expressing the duck hepatitis B virus surface proteins lead to reduced numbers of infected hepatocytes and protect ducks against the development of chronic infection in a virus dose-dependent manner.

We tested the efficacy of DNA vaccines expressing the duck hepatitis B virus (DHBV) pre-surface (pre-S/S) and surface (S) proteins in modifying the outcome of infection in 14-day-old ducks. In two experiments, Pekin Aylesbury ducks were vaccinated on days 4 and 14 of age with plasmid DNA vaccines expressing either the DHBV pre-S/S or S proteins, or the control plasmid vector, pcDNA1.1Amp.

Vaccination of ducks with a whole-cell vaccine expressing duck hepatitis B virus core antigen elicits antiviral immune responses that enable rapid resolution of de novo infection.

As a first step in developing immuno-therapeutic vaccines for patients with chronic hepatitis B virus infection, we examined the ability of a whole-cell vaccine, expressing the duck hepatitis B virus (DHBV) core antigen (DHBcAg), to target infected cells leading to the resolution of de novo DHBV infections. Three separate experiments were performed. In each experiment, ducks were vaccinated at 7 and 14 days of age with primary duck embryonic fibroblasts (PDEF) that had been transfected 48 h earlier with plasmid DNA expressing DHBcAg with and without the addition of anti-DHBcAg (anti-DHBc) antibodies.

The competing roles of virus replication and hepatocyte death rates in the emergence of drug-resistant mutants: theoretical considerations.

Lamivudine therapy of individuals chronically infected with hepatitis B virus (HBV) may eventually fail due to the emergence of drug-resistant mutants. Nonetheless, the durability of the response generally exceeds 6-12 months. This durability appeared surprising in view of published evidence that the replication rate of drug-resistant mutants might be at least 10% of the replication rate of uninhibited wild-type virus.

Covalently closed circular DNA is the predominant form of duck hepatitis B virus DNA that persists following transient infection.

Residual hepatitis B virus (HBV) DNA can be detected in serum and liver after apparent recovery from transient infection. However, it is not known if this residual HBV DNA represents ongoing viral replication and antigen expression. In the current study, ducks inoculated with duck hepatitis B virus (DHBV) were monitored for residual DHBV DNA following recovery from transient infection until 9 months postinoculation (p.

Effect of antiviral treatment with entecavir on age- and dose-related outcomes of duck hepatitis B virus infection.

Entecavir (ETV), a potent inhibitor of the hepadnaviral polymerases, prevented the development of persistent infection when administered in the early stages of duck hepatitis B virus (DHBV) infection. In a preliminary experiment, ETV treatment commenced 24 h before infection showed no significant advantage over simultaneous ETV treatment and infection. In two further experiments 14-day-old ducks were inoculated with DHBV-positive serum containing 10(4), 10(6), 10(8), or 5 x 10(8) viral genomes (vge) and were treated orally with 1.

Identification and characterization of avihepadnaviruses isolated from exotic anseriformes maintained in captivity.

Five new hepadnaviruses were cloned from exotic ducks and geese, including the Chiloe wigeon, mandarin duck, puna teal, Orinoco sheldgoose, and ashy-headed sheldgoose. Sequence comparisons revealed that all but the mandarin duck viruses were closely related to existing isolates of duck hepatitis B virus (DHBV), while mandarin duck virus clones were closely related to Ross goose hepatitis B virus. Nonetheless, the S protein, core protein, and functional domains of the Pol protein were highly conserved in all of the new isolates.

Clonal expansion of hepatocytes during chronic woodchuck hepatitis virus infection.

Chronic hepadnavirus infections cause liver damage with ongoing death and regeneration of hepatocytes. In the present study we set out to quantify the extent of liver turnover by measuring the clonal proliferation of hepatocytes by using integrated viral DNA as a genetic marker for individual hepatocyte lineages. Liver tissue from woodchucks with chronic woodchuck hepatitis virus (WHV) infection was assayed for randomly integrated viral DNA by using inverse PCR.

Adenovirus-based gene therapy during clevudine treatment of woodchucks chronically infected with woodchuck hepatitis virus.

Interferon-alpha (IFN-alpha) is a potent suppressor of hepatitis B virus (HBV) replication in the HBV-transgenic mouse, depleting virus replication intermediates from infected hepatocytes via pathways mediated by interferon-gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha). It has also been hypothesized that cytokines induce curing of infected hepatocytes via non-cytolytic pathways during resolution of transient hepadnavirus infections. We have therefore evaluated therapy of chronic woodchuck hepatitis virus (WHV) infections using treatment with the nucleoside analog clevudine [L-FMAU; 1-(2-fluoro-5-methyl-b-L-arabinofuranosyl) uracil] and therapy with adenovirus vectors expressing INF-gamma, TNF-alpha, and beta-galactosidase.