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Objective: Stereotactic radiofrequency pallidotomy has demonstrated improvement in motor fluctuations in patients with Parkinson's disease (PD), particularly levodopa (L-dopa)-induced dyskinesias. The authors aimed to determine whether or not unilateral pallidotomy with MR-guided focused ultrasound (MRgFUS) could safely improve Unified Dyskinesia Rating Scale (UDysRS; the primary outcome measure) scores over baseline scores in patients with PD., Methods: Twenty patients with PD and L-dopa responsiveness, asymmetrical motor signs, and motor fluctuations, including dyskinesias, participated in a 1-year multicenter open-label trial of unilateral MRgFUS ablation of the globus pallidus internus., Results: The sonication procedure was successfully completed in all 20 enrolled patients. MRgFUS-related adverse neurological events were generally mild and transient, including visual field deficit (n = 1), dysarthria (n = 4, 2 mild and 2 moderate), cognitive disturbance (n = 1), fine motor deficit (n = 2), and facial weakness (n = 1). Although 3 adverse events (AEs) were rated as severe (transient sonication-related pain in 2, nausea/vomiting in 1), no AE fulfilled US FDA criteria for a Serious Adverse Effect. Total UDysRS, the primary outcome measure, improved 59% after treatment (baseline mean score 36.1, 95% CI 4.88; at 3 months 14.2, 95% CI 5.72, p < 0.0001), which was sustained throughout the study (at 12 months 20.5, 95% CI 7.39, 43% improvement, p < 0.0001). The severity of motor signs on the treated side (Movement Disorder Society version of the United Parkinson's Disease Rating Scale [MDS-UPDRS] part III) in the "off" medication state also significantly improved (baseline mean score 20.0, 95% CI 2.4; at 3 months 10.6, 95% CI 1.86, 44.5% improvement, p < 0.0001; at 12 months 10.4, 95% CI 2.11, 45.2% improvement, p > 0.0001). The vast majority of patients showed a clinically meaningful level of improvement on the impairment component of the UDysRS or the motor component of the UPDRS, while 1 patient showed clinically meaningful worsening on the UPDRS at month 3., Conclusions: This study supports the feasibility and preliminary efficacy of MRgFUS pallidotomy in the treatment of patients with PD and motor fluctuations, including dyskinesias. These preliminary data support continued investigation, and a placebo-controlled, blinded trial is in progress. Clinical trial registration no.: NCT02263885 (clinicaltrials.gov).

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.

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.

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.

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.

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. We found that at least half of the foci of WHV core antigen-negative hepatocytes did not show clear morphological differences in either H&E or PAS (periodic acid Schiff) stained sections from surrounding hepatocytes, and were therefore not designated as FAH. In the second approach, we assayed core antigen-negative foci for the presence of fetuin B, a serum protein produced by normal hepatocytes, but not by neoplastic hepatocytes in hepatocellular carcinomas. Basophilic and amphophilic FAH had reduced levels of fetuin B compared to hepatocytes present in the surrounding liver; fetuin B staining was detected in clear cell FAH but the level could not be accurately assessed because of the displacement of fetuin B to the cell periphery by accumulated glycogen. The foci of morphologically normal WHV core antigen-negative hepatocytes had similar levels of fetuin B to that of the surrounding hepatocytes. The co-existence of at least four types of WHV core antigen-negative foci, including those with no obvious morphologic changes, raises the possibility that the different foci arise from distinct primary events. We hypothesize that a common event is loss of the ability to express WHV, allowing these hepatocytes to escape immune mediated cell death and to undergo clonal expansion to form distinct foci.

Synthesis of hepadnaviral DNA is dependent upon both the viral DNA polymerase and the viral core protein, the subunit of the nucleocapsids in which viral DNA synthesis takes place. In a study of natural isolates of duck hepatitis B virus (DHBV), we cloned full-length viral genomes from a puna teal. One of the clones failed to direct viral DNA replication in transfected cells, apparently as a result of a 3 nt inframe deletion of histidine 107 in the core protein. Histidine 107 is located in the center of a predicted helical region of the "insertion domain", a stretch of 45 amino acids which appears to be at the tip of a spike on the surface of the nucleocapsid. The mutation was introduced into a well-characterized strain of DHBV for further analysis. Core protein accumulated in cells transfected with the mutant DHBV but was partially degraded, suggesting that it was unstable. Assembled nucleocapsids were not detected by capsid gel electrophoresis. Interestingly, the mutant protein appeared to form chimeric nucleocapsids with wild-type core protein. The chimeric nucleocapsids supported viral DNA replication. These results suggest that the insertion domain of the spike may play a role either in assembly of stable nucleocapsids, possibly in formation of the dimer subunits, or in triggering nucleocapsid disintegration, required during initiation of new rounds of infection.

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. The Chiloe wigeon and puna teal hepatitis B viruses, the two new isolates most closely related to DHBV, also lacked an AUG start codon at the beginning of their X open reading frame (ORF). But as previously reported for the heron, Ross goose, and stork hepatitis B viruses, an AUG codon was found near the beginning of the X ORF of the mandarin duck, Orinoco, and ashy-headed sheldgoose viruses. In all of the new isolates, the X ORF ended with a stop codon at the same position. All of the cloned viruses replicated when transfected into the LMH line of chicken hepatoma cells. Significant differences between the new isolates and between these and previously reported isolates were detected in the pre-S domain of the viral envelope protein, which is believed to determine viral host range. Despite this, all of the new isolates were infectious for primary cultures of Pekin duck hepatocytes, and infectivity in young Pekin ducks was demonstrated for all but the ashy-headed sheldgoose isolate.

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. Before their use in vivo, expression of IFN-gamma and TNF-alpha from the adenovirus vectors was evaluated in vitro. Conditioned media from adenovirus-infected WC-3 cells was shown to inhibit WHV replication in baculovirus-transduced cells. Adenovirus super-infection of the liver in woodchucks led to declines in the percentage of hepatocytes with detectable core antigen and nucleic acids, and in levels of covalently closed circular DNA (cccDNA) and total WHV DNA, but a major long-term benefit of adenovirus super-infection during clevudine treatment was not demonstrated. Moreover, the effect took at least 2 weeks to develop suggesting that the declines in the percentage of WHV-infected cells, ccc, and total WHV DNA resulted from induction of the adaptive immune response by the adenovirus super-infection, and only indirectly from the expression of cytokines by the vectors.

We estimated the amount of hepatocyte turnover in the livers of three woodchucks undergoing clearance of a transient woodchuck hepatitis infection by determining the fate of integrated viral DNA as a genetic marker of the infected cell population. Integrated viral DNA was found to persist in liver tissue from recovered animals at essentially undiminished levels of 1 viral genome per 1,000-3,000 liver cells, suggesting that the hepatocytes in the recovered liver were derived primarily from the infected cell population. We determined the single and multicopy distribution of distinct viral cell junctions isolated from small pieces of liver after clearance of the infection to determine the cumulative amount of hepatocyte proliferation that had occurred during recovery. We estimated that proliferation was equivalent to a minimum of 0.7-1 complete random turnovers of the hepatocyte population of the liver. Our results indicated that during resolution of the transient infections a large fraction of the infected hepatocyte population was killed and replaced by hepatocyte cell division.

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.

Treatment of hepatitis B virus carriers with the nucleoside analog lamivudine suppresses virus replication. However, rather than completely eliminating the virus, long-term treatment often ends in the outgrowth of drug-resistant variants. Using woodchucks chronically infected with woodchuck hepatitis virus (WHV), we investigated the consequences of combining lamivudine treatment with immunotherapy mediated by an adenovirus superinfection. Eight infected woodchucks were treated with lamivudine and four were infected with approximately 10(13) particles of an adenovirus type 5 vector expressing beta-galactosidase. Serum samples and liver biopsies collected following the combination therapy revealed a 10- to 20-fold reduction in DNA replication intermediates in three of four woodchucks at 2 weeks after adenovirus infection. At the same time, covalently closed circular DNA (cccDNA) and viral mRNA levels both declined about two- to threefold in those woodchucks, while mRNA levels for gamma interferon and tumor necrosis factor alpha as well as for the T-cell markers CD4 and CD8 were elevated about twofold. Recovery from adenovirus infection was marked by elevation of sorbitol dehydrogenase, a marker for hepatocyte necrosis, as well as an 8- to 10-fold increase in expression of proliferating cell nuclear antigen, a marker for DNA synthesis, indicating significant hepatocyte turnover. The fact that replicative DNA levels declined more than cccDNA and mRNA levels following adenovirus infection suggests that the former decline either was cytokine induced or reflects instability of replicative DNA in regenerating hepatocytes. Virus titers in all four woodchucks were only transiently suppressed, suggesting that the effect of combination therapy is transient and, at least under the conditions used, does not cure chronic WHV infections.

Lamivudine [(-)-beta-L-2',3'-dideoxy-3'-thiacytidine] reduces woodchuck hepatitis virus (WHV) titers in the sera of chronically infected woodchucks by inhibiting viral DNA synthesis. However, after 6 to 12 months, WHV titers begin to increase toward pretreatment levels. Three WHV variants with mutations in the active site of the DNA polymerase gene are present at this time (W. S. Mason et al., Virology 245:18-32, 1998). We have asked if these mutant viruses were responsible for the lamivudine resistance and if their emergence caused an immediate rise in virus titers. Cell cultures studies implied that the mutants were resistant to lamivudine. Emergence of mutant WHV was not always associated, however, with an immediate rise in virus titers in the serum. One of the three types of mutant viruses became prominent in serum up to 7 months before titers in serum actually began to increase, at a time when wild-type virus was still predominant in the liver. The two other mutants did not show this behavior but were detected in serum and liver later, just at the time that virus titers began to rise. A factor linking all three mutants was that a similar duration of drug administration preceded the rise in titers, irrespective of which mutant ultimately prevailed. A simple explanation for these results is that the increase in virus titers following emergence of drug-resistant mutants can occur only as the preexisting wild-type virus is cleared from the hepatocyte population, allowing spread of the mutants. Thus, prolonged suppression of virus titers in the serum may sometimes be a measure of the stability of hepatocyte infection rather than of a successful therapeutic outcome.

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.

The template for synthesis of hepadnaviral RNAs is a covalently closed circular (ccc) DNA located in the nucleus of the infected hepatocyte. Hepatocytes are normally long-lived and nondividing, and antiviral therapies in chronically infected individuals face the problem of eliminating not only the replicative forms of viral DNA found in the cytoplasm but also the cccDNA from the nucleus. Because cccDNA does not replicate semiconservatively, it is not an obvious target for antiviral therapy. However, elimination of cccDNA might be facilitated if its half-life were short in comparison to the generation time of hepatocytes and if new cccDNA formation were effectively blocked. We have therefore measured cccDNA levels in woodchuck hepatocyte cultures following in vitro infection with woodchuck hepatitis virus and treatment with inhibitors of viral DNA synthesis. The viral reverse transcriptase inhibitors lamivudine (3TC) [(-)-beta-L-2',3'-dideoxy-3'-thiacytidine), FTC (5-fluoro-2',3'-dideoxy-3'-thiacytidine) and ddC (2',3'-dideoxycytidine) were added to the cultures beginning at 4 days postinfection. Treatment for up to 36 days with 3TC reduced the amount of cccDNA in the cultures not more than twofold compared to that of an untreated control. Treatment with ddC for 36 days and with FTC for 12 days resulted in effects similar to that of treatment with 3TC. Moreover, the declines in cccDNA appeared to reflect the loss of hepatocytes from the cultures rather than of cccDNA from hepatocytes. These results emphasize the important role of the longevity of the infected hepatocytes in the persistence of an infection.

Secondary amyloidosis is a common disease of water fowl and is characterized by the deposition of extracellular fibrils of amyloid A (AA) protein in the liver and certain other organs. Neither the normal role of serum amyloid A (SAA), a major acute phase response protein, nor the causes of secondary amyloidosis are well understood. To investigate a possible genetic contribution to disease susceptibility, we cloned and sequenced SAA cDNA derived from livers of domestic ducks. This revealed that the three C-terminal amino acids of SAA are removed during conversion to insoluble AA fibrils. Analysis of SAA cDNA sequences from several animals identified a distinct genetic dimorphism that may be relevant to susceptibility to secondary amyloid disease. The duck genome contained a single copy of the SAA gene that was expressed in liver and lung tissue of ducklings, even in the absence of induction of acute phase response. Genetic analysis of heterozygotes indicated that only one SAA allele is expressed in livers of adult birds. Immunofluorescence staining of livers from adult ducks displaying early symptoms of amyloidosis revealed what appear to be amyloid deposits within hepatocytes that are expressing unusually high amounts of SAA protein. This observation suggests that intracellular deposition of AA may represent an early event during development of secondary amyloidosis in older birds.

Duck hepatitis B virus (DHBV) DNA synthesis in congenitally infected ducks is inhibited by 2'-deoxycarbocyclic guanosine (2'-CDG). Three months of therapy reduces the number of infected hepatocytes at least 10-fold (W.S. Mason, J. Cullen, J. Saputelli, T.-T. Wu, C. Liu, W.T. London, E. Lustbader, P. Schaffer, A.P. O'Connell, I. Fourel, C.E. Aldrich, and A.R. Jilbert, Hepatology 19:393-411, 1994). The present study was performed to determine the kinetics of disappearance of infected hepatocytes and to evaluate the role of hepatocyte turnover in this process. Essentially all hepatocytes were infected before drug therapy. Oral treatment with 2'-CDG resulted in a prompt reduction in the number of infected hepatocytes. After 2 weeks, only 30 to 50% appeared to still be infected, and less than 10% were detectably infected after 5 weeks of therapy. To assess the possible role of hepatocyte turnover in these changes, 5-bromo-2'-deoxyuridine (BUdR) was administered 8 h before liver biopsy to label host DNA in hepatocytes passing through S phase, and stained nuclei were detected in tissue sections by using an antibody reactive to BUdR. The extent of nuclear labeling after 5 weeks was the same as that before therapy (ca. 1%). However, biopsies taken after 2 weeks of therapy showed a ca. 10-fold elevation in the number of nuclei labeled with BUdR. This result suggested that a rapid clearance of infected hepatocytes by 2'-CDG was caused not just by the inhibition of viral replication but also by an acceleration of the rate of hepatocyte turnover. To test this possibility further, antiviral therapy was carried out with another strong inhibitor of DHBV DNA synthesis, 5-fluoro-2',3'-dideoxy-3'-thiacytidine (524W), which did not accelerate hepatocyte turnover in ducks. 524W administration led to a strong inhibition of virus production but to a slower rate of decline in the number of infected hepatocytes, so that ca. 50% (and perhaps more) were still infected after 3 months of therapy. In addition, histopathologic evaluation of 2'-CDG-treated ducks revealed liver injury, especially at the start of therapy. No liver damage was observed during 524W therapy. These results imply that clearance of infected hepatocytes from the liver is correlated with hepatocyte turnover. Thus, in the absence of immune clearance or other sources for the accelerated elimination of infected hepatocytes, inhibitors of virus replication would have to be administered for a long period to substantially reduce the burden of infected hepatocytes in the liver.

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.

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.

We have examined the consequences on duck hepatitis B virus DNA synthesis of deleting the 5' and 3' copies of the 12 base sequence, DR1, from the viral pregenome. With the wild-type virus, reverse transcription initiates at nt 2537 within the 3' copy of DR1. When this sequence was deleted, initiation of reverse transcription was found at two other sites located closer to the 3' end of the pregenome (nt 2576 and nt 2644). The 3-base motif UUA was the only sequence common to these sites as well as the wild-type initiation site in DR1. Deletion of the 5' copy of DR1 did not alter minus strand synthesis, but led to aberrant priming of plus strand synthesis to generate predominantly linear rather than relaxed circular, double-stranded viral DNA, in agreement with the recent report by Loeb et al. (EMBO J. 10, 3533-3540, 1991). A mutant lacking only the 3' copy of DR1 rapidly converted to wild type in transfected cells. This apparently occurred as a consequence of conversion of newly synthesized relaxed circular to covalently closed circular (CCC) DNA, which might then serve as a template for the synthesis of wild-type viral RNAs. A mutant lacking only the 5' copy of DR1 did not exhibit this behavior. These results support the conclusion that amplified CCC DNA serves as transcriptional template.

Duck hepatitis B virus (DHBV) is produced in small amounts following transfection of human hepatoma or hepatoblastoma cell lines with cloned viral DNA. In a search for better hosts for DHBV replication, two avian liver cell lines were investigated. One of these cell lines, LMH, produced 5 to 10 times more DNA replicative intermediates and 10 to 20 times more infectious DHBV than did either of the two human cell lines, HuH-7 and Hep G2. Utilization of cell lines in genetic analyses of virus replication is often dependent upon obtaining efficient complementation between cotransfected viral genomes. We assayed transcomplementation of a viral polymerase (pol) gene mutant, which is rather inefficient in transfected human cells, and found that viral DNA synthesis was at least 20 times more efficient following cotransfection of LMH cells than in similarly transfected HuH-7 cells. Recombination, a potential interpretation problem in complementation assays, occurred at low levels in the cotransfected cultures but was substantially reduced or eliminated by creation of an LMH subline stably expressing the viral polymerase. This cell line, pol-7, supported the replication of DHBV pol mutants at ca. 10 to 15% of the level of virus replication obtained following transfection with wild-type viral DNA. By transcomplementation of a pol gene mutant in LMH cells, we were able to produce sufficient virus with the mutant genome to investigate the role of polymerase in covalently closed circular DNA amplification. Our results substantiate the hypothesis that covalently closed circular DNA is synthesized by the viral reverse transcriptase.

During the productive phase of chronic hepadnaviral infections, virion DNA synthesis occurs in the cytoplasm of the infected hepatocyte, but viral RNA is synthesized in the nucleus, apparently from a covalently closed, circular (CCC) viral DNA. J. Tuttleman, C. Pourcel, and J. Summers (1986a, Cell 47, 451-460) have shown that the intracellular levels of CCC DNA can increase during initiation of infection of duck hepatocytes in vitro with duck hepatitis B virus and during long term culture of infected duck hepatocytes in vitro. This amplification of CCC DNA occurs through the reverse transcription pathway. To distinguish between an entirely intracellular process of amplification and amplification due to multiple infections by extracellular virus in the virus producing cultures, suramin was added to the infected cultures to block superinfection. We found that CCC DNA amplification occurred at least as efficiently in the presence of suramin as in its absence. First, there was a net increase in the total amount of CCC DNA in the cultures both in the presence and in the absence of suramin. Second, synthesis of CCC DNA in the presence and absence of suramin was observed by density labeling of this viral DNA by growth of the cultures in medium containing BUdR. Amplification was also demonstrable in the presence of neutralizing duck antibodies. These results support the hypothesis of Tuttleman et al. (1986a) that CCC DNA amplification in chronically infected cultures and, by inference, the mechanism of persistent infection involves primarily intracellular regulatory mechanisms.

Primary cultures of woodchuck hepatocytes were demonstrated to be susceptible to in vitro infection by both woodchuck hepatitis virus and ground squirrel hepatitis virus, as evidenced by the appearance of DNA species characteristic of hepadnavirus replication. Initiation of infection by woodchuck hepatitis virus was blocked by the presence of suramin, polybrene, or dideoxycytidine. Viral CCC DNA, the putative template for viral RNA transcription, was detected at 2 days postinfection. Accumulation of intracellular intermediates in virion DNA synthesis was negligible until 7-10 days postinfection, but these DNA intermediates then increased dramatically in amount over the next few weeks. Results were obtained which suggested that the prolonged accumulation of intermediates in virion DNA synthesis was an intrinsic property of the infection of individual cells, and not the result of a slow spread of virus through the cultures.

A comparative study was made of sarcoma growth in 15I5 x 7(2) chickens infected in the wing web at 4 weeks of age with strains of subgroup B or G avian sarcoma viruses. Infection with sarcoma viruses of either subgroup B or G resulted in the formation of progressive wing web sarcomas at the site of inoculation. The survival times of the subgroup G virus-infected chickens were generally at least twice as great as the survival times of the subgroup B virus-infected chickens, which averaged 6-9 weeks postinoculation. At 5 weeks postinfection, a significantly higher titer of virus neutralizing antibody was detected in the subgroup G virus-infected chickens. Necropsy indicated that a high percentage of subgroup B virus-infected chickens exhibited fibrosarcomas at sites distal to the primary wing web sarcomas, whereas only a small percentage of subgroup G virus-infected chickens exhibited distal sarcomas. The results further indicated that the viral env gene is a determinant of the pattern of distal sarcoma formation.

Clone (cl.) 85 infection of 15I5 x 72 chickens at 4 weeks posthatch results in a lower frequency of distal sarcomas than does Prague (Pr)-B infection. As higher titers of virus-neutralizing antibody are generated in the cl.85-infected chickens, the possibility was addressed in the present study that the low frequency of cl.85-induced distal sarcomas is a direct consequence of the strong neutralizing response. Our observations indicate that ablation of humoral immunity by embryonic bursectomy does not serve to increase this frequency, implying that the antiviral humoral response is not required to limit formation of cl.85-induced distal sarcomas.

Suramin blocked in vitro infection by duck hepatitis B virus, a hepadnavirus, and Rous sarcoma virus, a retrovirus. Although suramin was able to inhibit the virus-encoded reverse transcriptase activities of these two viruses, this inhibition did not appear to account for the anti-viral effect of the drug. In particular, suramin was unable to block synthesis within cells of full-length viral DNAs when added subsequent to infection. The results are consistent with the hypothesis that suramin acted by blocking virus uptake or uncoating. As further support of this hypothesis, we found that suramin also blocked infection by hepatitis delta virus, an RNA virus that is not known to employ reverse transcriptase during the initiation of infection.

The plus strand of virion DNA of duck hepatitis B virus possessed, at its 5' terminus, a capped oligoribonucleotide 18 to 19 bases in length. This oligoribonucleotide had a unique 5' end, the heterogeneity in length reflecting two distinct junctions with plus-strand DNA that were 1 base apart. The sequence of the RNA differed from that predicted by the sequence of duck hepatitis B virus upstream of the 5' ends of plus-strand DNA but was identical to a downstream sequence corresponding to the 5' terminus of a major poly(A)+ viral RNA mapped by Büscher and co-workers (Cell 40:717-724, 1985). This RNA transcript is thought to serve as the template (i.e., the pregenome) for minus-strand synthesis via reverse transcription. The results suggest that the pregenome also donates a capped oligoribonucleotide that acts as the primer of plus-strand DNA synthesis, using the minus-strand DNA as template.

We characterized a number of important features of the structure of the cohesive overlap region of the DNA genome of duck hepatitis B virus. The 5'-terminal nucleotide of minus-strand DNA was localized to nucleotide 2537, a G residue within the 12-base repeat sequence DR1. This G residue was shown to be the site of a covalent linkage to a protein, consistent with speculation that this protein is the primer of minus-strand synthesis, which occurs by reverse transcription. The 3' terminus of the minus strand was heterogeneous, being mapped to nucleotides 2530 and 2531, indicating that the minus strand is terminally redundant by seven or eight bases and ends at the putative 5' end of the transcribed RNA template (pregenome) for reverse transcription. We previously demonstrated that the presumptive RNA primer of plus-strand synthesis remains attached to plus-strand DNA during virus maturation; moreover, the sequence of this primer suggested an origin from the 5' end of the pregenome (J.-M. Lien, C. E. Aldrich, and W. S. Mason, J. Virol. 57:229-236, 1986). We show here that over 75% of plus-strand primers are capped, further supporting the idea that these primers are uniquely derived from the 5' end of the pregenome. Finally, we found that seemingly mature duck hepatitis B virus genomes are incomplete by at least 12 bases, in that the 12-base repeat sequence DR2 is not copied into plus-strand DNA during virus maturation. Since DR2 in virion DNA is duplexed with the RNA primer of plus-strand synthesis, it is possible that the failure to make complete plus strands is due to an inability of the viral DNA polymerase to carry out a displacement of the bound RNA primer.