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. There was an upward trend in maximum clone size with increasing fibrosis, inflammatory activity and with seroconversion from HBV e-antigen (HBeAg)-positive to HBeAg-negative, but these differences did not reach statistical significance. Maximum hepatocyte clone size did not differ between patients with and without a coexisting hepatocellular carcinoma. Thus, large hepatocyte clones containing integrated HBV DNA were detected during all stages of chronic HBV infection. Using laser microdissection, no significant difference in clone size was observed between foci of HBV surface antigen (HBsAg)-positive and HBsAg-negative hepatocytes, suggesting that expression of HBsAg is not a significant factor in clonal expansion. Laser microdissection also revealed that hepatocytes with normal-appearing histology make up a major fraction of the cells undergoing clonal expansion. Thus, preneoplasia does not appear to be a factor in the clonal expansion detected in our assays. Computer simulations suggest that the large hepatocyte clones are not produced by random hepatocyte turnover but have an as-yet-unknown selective advantage that drives increased clonal expansion in the HBV-infected liver.

Antiviral therapy with entecavir combined with post-exposure "prime-boost" vaccination eliminates duck hepatitis B virus-infected hepatocytes and prevents the development of persistent infection.

Short-term antiviral therapy with the nucleoside analogue entecavir (ETV), given at an early stage of duck hepatitis B virus (DHBV) infection, restricts virus spread and leads to clearance of DHBV-infected hepatocytes in approximately 50% of ETV-treated ducks, whereas widespread and persistent DHBV infection develops in 100% of untreated ducks. To increase the treatment response rate, ETV treatment was combined in the current study with a post-exposure "prime-boost" vaccination protocol. Four groups of 14-day-old ducks were inoculated intravenously with a dose of DHBV previously shown to induce persistent DHBV infection. One hour post-infection (p.i.), ducks were primed with DNA vaccines that expressed DHBV core (DHBc) and surface (pre-S/S and S) antigens (Groups A, B) or the DNA vector alone (Groups C, D). ETV (Groups A, C) or water (Groups B, D) was simultaneously administered by gavage and continued for 14 days. Ducks were boosted 7 days p.i. with recombinant fowlpoxvirus (rFPV) strains also expressing DHBc and pre-S/S antigens (Groups A, B) or the FPV-M3 vector (Groups C, D). DHBV-infected hepatocytes were observed in the liver of all ducks at day 4 p.i. with reduced numbers in the ETV-treated ducks. Ducks treated with ETV plus the control vectors showed restricted spread of DHBV infection during ETV treatment, but in 60% of cases, infection became widespread after ETV was stopped. In contrast, at 14 and 67 days p.i., 100% of ducks treated with ETV and "prime-boost" vaccination had no detectable DHBV-infected hepatocytes and had cleared the DHBV infection. These findings suggest that ETV treatment combined with post-exposure "prime-boost" vaccination induced immune responses that eliminated DHBV-infected hepatocytes and prevented the development of persistent DHBV infection.

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. Hepatocyte death, proliferation and curing are also important features of chronic infections, though the outcomes are different. In particular, immune selection due to persistent attack by antiviral CTL is thought to play a role in the emergence of hepatocytes infected with mutant strains of hepatitis B virus (HBV) (e.g. HBV e antigen-negative strains) and in the emergence of hepatocytes that appear refractory to HBV infection. In both instances, clonal expansion of subpopulations of hepatocytes may be inferred to have taken place. Interestingly, foci of altered hepatocytes and hepatocellular carcinomas (HCC) typically do no support virus replication. Thus, immune selection of hepatocytes by antiviral CTL, by inducing clonal expansion, may also play an important role in the progression to HCC. In this review, we discuss the evidence in support of roles for hepatocyte turnover in the resolution of transient and progression of chronic HBV infections.

A duck hepatitis B virus strain with a knockout mutation in the putative X ORF shows similar infectivity and in vivo growth characteristics to wild-type virus.

Hepadnaviruses including human hepatitis B virus (HBV) and duck hepatitis B virus (DHBV) express X proteins, HBx and DHBx, respectively. Both HBx and DHBx are transcriptional activators and modulate cellular signaling in in vitro assays. To test whether the DHBx protein plays a role in virus infection, we compared the in vivo infectivity and growth characteristics of a DHBV3 strain with a stop codon in the X-like ORF (DHBV3-X-K.O.) to those of the wild-type DHBV3 strain. Here we report that the two strains showed no significant difference in (i). their ability to induce infection that resulted in stable viraemia measured by serum surface antigen (DHBsAg) and DHBV DNA, and detection of viral proteins and replicative DNA intermediates in the liver; (ii). the rate of spread of infection in liver and extrahepatic sites after low-dose virus inoculation; and (iii). the ability to produce transient or persistent infection under balanced age/dose conditions designed to detect small differences between the strains. Thus, none of the infection parameters assayed were detectably affected by the X-ORF knockout mutation, raising the question whether DHBx expression plays a physiological role during in vivo infection with wild-type DHBV.

Structural and functional homology between duck and chicken interferon-gamma.

The Duck interferon gamma (DuIFN-gamma) cDNA was cloned from a phytohaemaglutinin-stimulated duck spleen cDNA library screened using a chicken IFN-gamma (ChIFN-gamma) cDNA probe. The DuIFN-gamma cDNA is 1392 nt long and shows 99% and 80% sequence identity with another cloned DuIFN-gamma cDNA, and with ChIFN-gamma cDNA, respectively. The cDNA contains a 495 bp ORF that encodes a putative 164 amino acid (AA) protein that shares 67% identity with ChIFN-gamma, but only 30-35% identity with mammalian IFN-gamma. The predicted three-dimensional (3D) structures of DuIFN-gamma and ChIFN-gamma are similar when analysed by comparative protein modelling. Culture supernatant collected from COS cells transfected with DuIFN-gamma cDNA was able to activate nitrite secretion from a chicken macrophage cell line (HD11) in a dose-dependent fashion. This activity could not be neutralised by an anti-ChIFN-gamma monoclonal antibody (Mab 85) that was able to neutralise the activity of ChIFN-gamma. Recombinant DuIFN-gamma (rDuIFN-gamma) protein was expressed in E. coli as an N-terminally His-tagged protein and was purified on a nickel affinity column. The eluted protein, which was detected as a approximately 18 kDa band with a purity of >90%, was also detected by Western blot using the anti-ChIFN-gamma monoclonal antibody (Mab 9.1). The rDuIFN-gamma was shown to activate nitrite secretion by HD11 cells in a dose-dependent fashion with a specific activity that was approximately 16-fold lower than a rChIFN-gamma control. Two rabbit antisera raised against rDuIFN-gamma were able to neutralise COS cell-expressed DuIFN-gamma activity; one of these also neutralised ChIFN-gamma activity. These findings indicate that DuIFN-gamma shares structural and functional identity with ChIFN-gamma, which is consistent with our previous results which demonstrated cross reactivity with other lymphokines from the two species.

Kinetics of hepadnavirus loss from the liver during inhibition of viral DNA synthesis.

Hepadnaviruses replicate by reverse transcription, which takes place in the cytoplasm of the infected hepatocyte. Viral RNAs, including the pregenome, are transcribed from a covalently closed circular (ccc) viral DNA that is found in the nucleus. Inhibitors of the viral reverse transcriptase can block new DNA synthesis but have no direct effect on the up to 50 or more copies of cccDNA that maintain the infected state. Thus, during antiviral therapy, the rates of loss of cccDNA, infected hepatocytes (1 or more molecules of cccDNA), and replicating DNAs may be quite different. In the present study, we asked how these losses compared when woodchucks chronically infected with woodchuck hepatitis virus were treated with L-FMAU [1-(2-fluoro-5-methyl-beta-L-arabinofuranosyl) uracil], an inhibitor of viral DNA synthesis. Viremia was suppressed for at least 8 months, after which drug-resistant virus began replicating to high titers. In addition, replicating viral DNAs were virtually absent from the liver after 6 weeks of treatment. In contrast, cccDNA declined more slowly, consistent with a half-life of approximately 33 to 50 days. The loss of cccDNA was comparable to that expected from the estimated death rate of hepatocytes in these woodchucks, suggesting that death of infected cells was one of the major routes for elimination of cccDNA. However, the decline in the actual number of infected hepatocytes lagged behind the decline in cccDNA, so that the average cccDNA copy number in infected cells dropped during the early phase of therapy. This observation was consistent with the possibility that some fraction of cccDNA was distributed to daughter cells in those infected hepatocytes that passed through mitosis.

Immune responses to duck hepatitis B virus infection.

The duck hepatitis B virus (DHBV) was the first hepatitis B virus identified from an avian host. It is a member of the Hepadnaviridae family of viruses. All members of this family display similar genomic organization and replication strategies and cause species-specific infections that result in either transient (acute) or persistent infection. Hepadnavirus infection occurs primarily in hepatocytes in the liver with release of infectious virions and non-infectious 'empty' surface antigen particles into the bloodstream. Hepadnavirus replication is non-cytopathic and immune responses to viral antigens are thought to be responsible for the liver damage seen in both transient and persistent infection and for the clearance of virus from infected cells. This has provided the basis for the use of vaccines and prophylactic treatments for individuals at high risk of human hepatitis B virus (HBV) infection. It follows that detailed understanding of the immune responses induced during transient and persistent infection may well facilitate the development of more effective approaches to immunotherapy in patients with persistent infection and may also provide a means of reducing the liver damage associated with this infection, without reducing the effectiveness of the immunity required to eliminate the virus. Immune responses to hepadnavirus infection have been studied primarily in humans, following natural infection with HBV, but studies have also been performed with the woodchuck hepatitis virus (WHV) and the DHBV models. This manuscript reviews the recent studies of immune responses to DHBV infection.

Characterisation of duck thrombocytes.

Duck thrombocytes were initially identified in peripheral blood mononuclear cells (PBMCs) purified from whole blood on Ficoll-Paque density gradients by examining stained smears of these cells. These thrombocytes could be readily purified from lymphocytes on the FACStar cell sorter by their increased side-scatter. They were similar to chicken thrombocytes in both appearance and function; they had a diameter of 4.5-6 microm and contained large vacuoles and were able to phagocytose carbon and Staphylococcus aureus. A monoclonal antibody (mAb) BA3 was generated which binds specifically to duck thrombocytes and has facilitated the characterisation of these cells which comprise up to 50-60 per cent of cells in Ficoll-Paque purified duck PBMCs.

Lamivudine therapy of WHV-infected woodchucks.

Hepatitis B viruses establish a chronic, productive, and noncytopathic infection of hepatocytes. Viral products are produced by transcription from multiple copies (5-50) of covalently closed circular (ccc) viral DNA. This cccDNA does not replicate, but can be replaced by DNA precursors that are synthesized in the cytoplasm. The present study was carried out to determine if long-term treatment with an inhibitor of viral DNA synthesis would lead to loss of virus products, including cccDNA, from the liver of woodchucks chronically infected with woodchuck hepatitis virus. Viral DNA synthesis was inhibited with the nucleoside analog, lamivudine (2'-deoxy-3'-thiacytidine). Lamivudine treatment produced a slow but progressive decline in viral titers in serum, to about 0.3% or less of the initial level. However, even after maintenance of drug therapy for 3-12 months, > 95% of the hepatocytes in most animals were still infected. Significant declines in the percentage of infected hepatocytes and of intrahepatic cccDNA levels were observed in only three woodchucks, two in the group receiving lamivudine and one in the placebo control group. Moreover, virus titers eventually rose in woodchucks receiving lamivudine, suggesting that drug-resistant viruses began to spread through the liver starting at least as early as 9-12 months of treatment. Three types of mutation that may be associated with drug resistance were found at this time, in a region upstream of the YMDD motif in the active site of the viral reverse transcriptase. The YMDD motif itself remained unchanged. Not unexpectedly, the lamivudine therapy did not have a impact on development of liver cancer.

Characterization of age- and dose-related outcomes of duck hepatitis B virus infection.

Experimental inoculation of naive ducks with duck hepatitis B virus (DHBV) can lead to one of three outcomes, namely, persistent viremia, transient infection with or without viremia, or no evidence of infection. The ability of individual ducks to resolve DHBV infection was found to be linked to the age of the duck at the time of inoculation and the dose of inoculated virus. (1) In recently hatched ducks inoculated intravenously (i.v.) with 4 x 10(4) DHBV DNA genomes, a switch from persistent viremia to transient antibody appearance was seen at an age of inoculation between 7 and 14 days. A 25-fold increase in the dose of virus (1 x 10(6) DHBV genomes) delayed this switch by 7 days. (2) When 4-month-old ducks were inoculated i.v. with different doses of virus, only those receiving the highest dose (2 x 10(11) DHBV genomes) showed viremia and extensive viral replication and histological changes in the liver; 2/3 ducks in this group had a transient infection, while the third duck had viral replication and histological changes in the liver that were still present at day 120 postinoculation (p.i.). In all ducks receiving lower doses (1 x 10(3), 1 x 10(6), 1 x 10(9) DHBV genomes) antibodies to viral surface and core antigens developed without detectable viral replication in the liver on days 6, 9, or 12 p.i. (3) When 10- to 16-month-old ducks were inoculated i.v. with 2 x 10(11) DHBV genomes, all showed extensive viral replication in hepatocytes and mild to moderate histological changes in the liver on days 4 or 6 p.i. In 4/5 ducks viremia was not detected, anti-surface antibodies were first detected on day 8 p.i., and viral DNA and antigen were cleared from the liver by days 35-47 p.i. The remaining duck became viremic with persistence of virus in the liver until at least day 46 p.i. The findings of the study are consistent with a model for noncytopathic viruses (R. M. Zinkernagel (1996) Science 271, 173-178).

Protective efficacy of DNA vaccines against duck hepatitis B virus infection.

The efficacy of DNA vaccines encoding the duck hepatitis B virus (DHBV) pre-S/S and S proteins were tested in Pekin ducks. Plasmid pcDNA I/Amp DNA containing the DHBV pre-S/S or S genes was injected intramuscularly three times, at 3-week intervals. All pre-S/S and S-vaccinated ducks developed total anti-DHBs and specific anti-S antibodies with similar titers reaching 1/10,000 to 1/50,000 and 1/2,500 to 1/4,000, respectively, after the third vaccination. However, following virus challenge, significant differences in the rate of virus removal from the bloodstream and the presence of virus replication in the liver were found between the groups. In three of four S-vaccinated ducks, 90% of the inoculum was removed between <5 and 15 min postchallenge (p.c.) and no virus replication was detected in the liver at 4 days p.c. In contrast, in all four pre-S/S-vaccinated ducks, 90% of the inoculum was removed between 60 and 90 min p.c. and DHBsAg was detected in 10 to 40% of hepatocytes. Anti-S serum abolished virus infectivity when preincubated with DHBV before inoculation into 1-day-old ducklings and primary duck hepatocyte cultures, while anti-pre-S/S serum showed very limited capacity to neutralize virus infectivity in these two systems. Thus, although both DNA vaccines induced high titers of anti-DHBs antibodies, anti-S antibodies induced by the S-DNA construct were highly effective in neutralizing virus infectivity while similar levels of anti-S induced by the pre-S/S-DNA construct conferred only very limited protection. This phenomenon requires further clarification, particularly in light of the development of newer HBV vaccines containing pre-S proteins and a possible discrepancy between anti-HBs titers and protective efficacy.

An homologous in vitro assay to detect lymphokines released by PHA-activated duck peripheral blood lymphocytes and spleen cells.

When stimulated with phytohaemagglutinin duck lymphocytes released lymphokines which were detected by their ability to maintain the proliferation of duck lymphoblasts using an in vitro assay similar to that previously developed with the mammalian system to measure IL-2. The inability of duck lymphokines to maintain the proliferation of mammalian lymphoblasts (mouse) indicated that there was no functional homology between duck and mammalian lymphokines. However, duck lymphokines did maintain the proliferation of chicken lymphoblasts indicating functional homology of these growth factors between these two species. The duck lymphokine maintenance assay is a simple and reliable test, and should be useful as an in vitro assay for the detection of factors released by antigen-specific lymphocytes when cultured in the presence of viral antigens.

Optimization of an in vitro assay which measures the proliferation of duck T lymphocytes from peripheral blood in response to stimulation with PHA and ConA.

The in vitro proliferative responses of duck PBMCs purified from Ficoll-Paque density gradients to the mitogens PHA and ConA show a great deal of duck-to-duck variation. Better responses were consistently obtained by using nylon wool fractionation to increase the proportion of duck T lymphocytes in PBMC preparations and then culturing these preparations with homologous monocytes, purified from PBMC preparations by their adherence properties. We have also established that the addition of homologous red blood cells enhances the in vitro proliferative responses of duck T lymphocytes, especially when limiting doses of PHA and ConA are used. Duck T lymphocytes showed greater and more consistent proliferation when cultures were incubated at 37 degrees C as compared to incubation at 41.6 degrees C. The improved consistency of higher proliferative responses with this assay should make it more suitable for detecting in vitro proliferative responses of antigen-specific T lymphocytes, as a measure of in vivo induced cell mediated immune responses.

Kinetics of duck hepatitis B virus infection following low dose virus inoculation: one virus DNA genome is infectious in neonatal ducks.

Using pooled serum from congenitally duck hepatitis B virus (DHBV)-infected ducks as inoculum, we examined the effect of virus dose on the incubation period of infection and on the patterns of spread of virus infection in the liver. The pooled serum inoculum contained 9.5 x 10(9) DHBV genomes per milliliter and had an infectivity titre (ID50) in newly hatched ducks of 1.5 x 10(10) per milliliter with a 95% confidence interval of 3.0 x 10(9) to 6.3 x 10(10) ID50/ml, indicating the equivalence between one DHBV genome and one infectious unit within the limits of the assays. The incubation period of infection was inversely related to the dose of inoculum and the onset of viraemia ranged from Day 6 with the highest dose to Day 14 or 29 with the lowest dose inoculum. To study the spread of virus infection from a low percentage of initially infected cells we inoculated newly hatched ducks intravenously with sufficient DHBV (1.5 x 10(3) ID50) to infect only approximately 0.0001% of total liver cells. DHBV infection first reached detectable levels on Day 4 postinoculation (p.i.) and was detected in approximately 0.035% of hepatocytes, most of which occurred as single cells or pairs of cells, indicating that a number of rounds of infection had occurred with the spread of virus both to adjoining cells, i.e., by cell-to-cell spread, and to cells located in other parts of the liver lobule. Despite some bird-to-bird variation in timing, the percentage of infected hepatocytes increased exponentially with a mean doubling time of 16 hr from Day 4 to Day 14 p.i., by which time replication was seen in > 95% of hepatocytes. This rapid dissemination from a small number of infected hepatocytes suggests that, in neonatal ducks, there are no major delays in virus replication within the liver, that any innate and adaptive defence mechanisms operating during the first 10 to 14 days of infection are insufficient to contain virus spread, and that even a small number of infected hepatocytes produce enough progeny to rapidly infect the remaining hepatocytes.

Identification of duck T lymphocytes using an anti-human T cell (CD3) antiserum.

Duck lymphocytes have not been classified into cells resembling B or T cells of mammals. Reagents used in the past to identify lymphocyte populations in other species have not been useful for this purpose and antibodies raised to duck immunoglobulin bind in high proportions to blood and organ lymphocytes of ducks as well as to their red blood cells. Here we report that a polyclonal rabbit antiserum reacting to the CD3 marker on human T cells has been used to identify duck T lymphocytes. These antibodies react with the intracytoplasmic portion of the human CD3 epsilon chain (amino acids 156-168), an epitope highly conserved between mammals. Immunohistochemical staining with this antiserum of sections of duck lymphoid organs and FACScan analysis of duck lymphoid cell suspensions identified a population of duck lymphocytes with a staining pattern similar to that seen for mammalian T cells. This anti-human CD3 immunoprecipitated a 23 kDa protein from a duck lymphoblast lysate: a size similar to the human CD3 epsilon chain. This is the first direct identification of duck T lymphocytes.

Woodchuck hepatitis virus infections: very rapid recovery after a prolonged viremia and infection of virtually every hepatocyte.

Earlier studies have suggested that transient hepadnavirus infections in mammals are associated with virus replication in a large fraction of hepatocytes. Although the viremia that occurred during transient infections in some individuals would presumably lead to virus replication in all hepatocytes, these studies did not reveal if this was the case. The question of the extent of hepatocyte infection was therefore reinvestigated because of the implications of the results for the mechanisms of virus clearance. Woodchucks were inoculated with woodchuck hepatitis virus, and the course of hepatic infection was determined. These studies indicated that essentially 100% of the hepatocytes became infected in the majority of woodchucks. In 7 of 10 woodchucks, the viral infection was then rapidly cleared from the liver, generally in less than 4 weeks. In another three woodchucks, though productive infection was just as rapidly cleared, viral covalently closed circular DNA remained for weeks to months after other indicators of virus infection had disappeared from the liver. Bromodeoxyuridine labeling and anti-proliferating cell nuclear antigen staining to detect hepatocytes passing through S phase indicated an increase in hepatocyte proliferation during the recovery phase of infection. The rate of cell division appeared to be sufficient to replace no more than 2 to 3% of the hepatocytes per day, at the times at which the biopsies were performed. Histopathologic evaluation of the biopsy samples did not provide evidence for a massive amount of liver regeneration. Models to explain virus clearance, with or without massive immune system-mediated destruction of infected hepatocytes, are reviewed.

Postnatal psychosis: a patient's experience with comments by the consulting psychiatrist.

The article describes a patient's experience of postnatal psychosis, a form of bipolar mood disorder which presents as the most severe form of psychological disorder suffered in the postnatal period. Accompanying the patient's description are comments from the consulting psychiatrist involved in the case and description of the three forms of postnatal mood change, i.e. the baby blues, postnatal psychosis and postnatal depression. The article is designed to assist in early recognition of postnatal psychosis, appreciation of the patient's altered state of mind, and encouraging acceptance of psychiatric treatment, all of which are essential for good management of postnatal mood disorders.

Inhibition of duck hepatitis B virus replication by hypericin.

Hypericin was found to be active against a member of the hepatitis B virus family, duck hepatitis B virus (DHBV). After a single 1 h incubation with hypericin, cells stably-transfected with a clone of DHBV stopped producing infectious virus for several days, though virus-like particles continued to be released into the culture medium. Characterization of these virions revealed a buoyant density characteristic of infectious virus preparations and lower than that of virus cores, suggesting that the particles were enveloped. Western blot analysis suggested, however, that the viral preS protein in surface antigen particles and, by inference, in virions, was present in covalently cross-linked aggregates. Evidence of a similar level of aggregation of the core subunit of virion nucleocapsids was not found, nor was there evidence of a similar high level of aggregation of cell-associated core and preS proteins. Hypericin was only slightly virucidal against DHBV and culture medium from treated cultures did not block initiation of infection when added to DHBV susceptible cultures prior to a challenge with infectious DHBV. Thus, the primary antiviral activity of hypericin against DHBV replication appears to be exerted at a late step in viral morphogenesis.

Rapid resolution of duck hepatitis B virus infections occurs after massive hepatocellular involvement.

A study was carried out to determine some of the factors that might distinguish transient from chronic hepadnavirus infection. First, to better characterize chronic infection, Pekin ducks, congenitally infected with the duck hepatitis B virus (DHBV), were used to assess age-dependent variations in viremia, percentage of DHBV-infected hepatocytes, and average levels of DNA replication intermediates in the cytoplasm and of covalently closed circular DNA in the nuclei of infected hepatocytes. Levels of viremia and viral DNA were found to peak at about the time of hatching but persisted at relatively constant levels in chronically infected birds up to 2 years of age. The percentage of infected hepatocytes was also constant, with DHBV replication in virtually 100% of hepatocytes in all birds. Next, we found that adolescent ducks inoculated intravenously with a large dose of DHBV also developed massive infection of hepatocytes with an early but low-level viremia, followed by rapid development of a neutralizing antibody response. No obvious quantitative or qualitative differences between transiently and chronically infected liver tissue were detected in the intracellular markers of viral replication examined. However, in the adolescent duck experiment, DHBV infection was rapidly cleared from the liver even when up to 80% of hepatocytes were initially infected. In all of these ducks, clearance of infection was accompanied by only a mild hepatitis, with no evidence that massive cell death contributed to the clearance. This finding suggested that mechanisms in addition to immune-mediated destruction of hepatocytes might make major contributions to clearance of infections, including physiological turnover of hepatocytes in the presence of a neutralizing antibody response and/or spontaneous loss of the capacity of hepatocytes to support virus replication.