Lack of a small animal model of the human hepatitis C virus (HCV) has impeded development of antiviral therapies against this epidemic infection. By transplanting normal human hepatocytes into SCID mice carrying a plasminogen activator transgene (Alb-uPA), we generated mice with chimeric human livers. Homozygosity of Alb-uPA was associated with significantly higher levels of human hepatocyte engraftment, and these mice developed prolonged HCV infections with high viral titers after inoculation with infected human serum. Initial increases in total viral load were up to 1950-fold, with replication confirmed by detection of negative-strand viral RNA in transplanted livers. HCV viral proteins were localized to human hepatocyte nodules, and infection was serially passaged through three generations of mice confirming both synthesis and release of infectious viral particles. These chimeric mice represent the first murine model suitable for studying the human hepatitis C virus in vivo.

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Covalently closed circular DNA (cccDNA) is a crucial intermediate in the replication of hepadnaviruses. We inhibited the replication of duck hepatitis B virus in congenitally infected ducks with a combination of lamivudine and a dideoxyguanosine prodrug. Inhibition of viral replication should prevent renewal of the cccDNA pool, and its decay was measured in liver biopsy samples collected over a 5-month period. In three ducks, the cccDNA pools declined exponentially, with half-lives ranging from 35 to 57 days. In two others, the pools declined exponentially for about 70 days but then stabilized at about 6 copies/diploid genome. The selection of drug-resistant virus mutants is an unlikely explanation for this unexpected stabilization of cccDNA levels. Liver sections stained for the cell division marker PCNA showed that animals in which cccDNA loss was continuous had significantly greater numbers of PCNA-positive nuclei than did those animals in which cccDNA levels had plateaued.The hepadnaviruses include the human pathogen Hepatitis B virus (HBV) and animal viruses such as Duck hepatitis B virus (DHBV) and Woodchuck hepatitis B virus (WHV), which are important models for studying hepadnavirus biology. These viruses are characterized by small, circular, partially doublestranded DNA genomes. The minus strand of the viral DNA is covalently linked to protein at its 5Ј end and contains a nick at its 3Ј end. The plus strand is only partially complete (for reviews, see references 10 and 21). A crucial event in the replication of these viruses is the conversion of this form of the viral genome into a covalently closed circular DNA (cccDNA) form within the nucleus of a newly infected hepatocyte (15,17,26). cccDNA is the template for viral transcripts encoding structural proteins and the viral polymerase. It is also the template for the pregenomic RNA which is the precursor of the viral genome. Early in infection, the pool of nuclear cccDNA is amplified to about 20 molecules/cell (13,14,24). This is accomplished when an intracellular circuit of pregenomic RNA is encapsidated and converted to viral DNA in the cytoplasm and subsequently enters the nucleus and converts to cccDNA (29). Once a pool of cccDNA is established, capsids containing newly replicated viral DNA are enveloped and exported from the cell rather than entering the nucleus. This change in the fate of nucleocapsids is thought to be caused by rising levels of the pre-S envelope protein resulting from an increasing pool of cccDNA template (22, 23).Attempts to cure hepadnavirus infections by treatment with antiviral drugs suggest that the cccDNA pool is very stable. If cccDNA turnover were rapid, then inhibitors of the viral reverse transcriptase would prevent replenishment of the cccDNA pool, leading to its elimination after a short course of treatment. However, human HBV carriers treated with lamivudine for 3 months usually relapse quickly when treatment is discontinued, indicating that cccDNA persists (5). If treatment is continued for 1 to 2 years, drug-resist...

Superinfection exclusion is the phenomenon whereby a virus prevents the subsequent infection of an already infected host cell. The Pekin duck hepatitis B virus (DHBV) model was used to investigate superinfection exclusion in hepadnavirus infections. Superinfection exclusion was shown to occur both in vivo and in vitro with a genetically marked DHBV, DHBV-ClaI, which was unable to establish an infection in either DHBVinfected ducklings or DHBV-infected primary duck hepatocytes (PDHs). In addition, exclusion occurred in vivo even when the second virus had a replicative advantage. Superinfection exclusion appears to be restricted to DHBV, as adenovirus, herpes simplex virus type 1, and vesicular stomatitis virus were all capable of efficiently infecting DHBV-infected PDHs. Exclusion was dependent on gene expression by the original infecting virus, since UV-irradiated DHBV was unable to mediate the exclusion of DHBV-ClaI. Using recombinant adenoviruses expressing DHBV proteins, we determined that the large surface antigen mediated exclusion. The large surface antigen is known to cause down-regulation of a DHBV receptor, carboxypeptidase D (CPD). Receptor down-regulation is a mechanism of superinfection exclusion seen in other viral infections, and so it was investigated as a possible mechanism of DHBV-mediated exclusion. However, a mutant large surface antigen which did not down-regulate CPD was still capable of inhibiting DHBV infection of PDHs. In addition, exclusion of DHBV-ClaI did not correlate with a decrease in CPD levels. Finally, virus binding assays and confocal microscopy analysis of infected PDHs indicated that the block in infection occurs after internalization of the second virus. We suggest that superinfection exclusion may result from the role of the L surface antigen as a regulator of intracellular trafficking.Hepadnaviruses are a family of enveloped, hepatotropic viruses with small (3.0-to 3.2-kb), partially double-stranded DNA genomes (28). The family includes viruses infecting the woodchuck, ground squirrel, grey heron, snow goose, and Pekin duck (duck hepatitis B virus [DHBV]) as well as the medically important human hepatitis B virus (HBV).The virion is an icosahedral capsid made up of a core protein surrounded by a lipid bilayer that contains the viral envelope proteins. Contained within the capsid is the viral genome with the polymerase protein covalently attached to the 5Ј terminus of the minus strand. The hepadnavirus genome is organized into overlapping reading frames that encode the precore, core, polymerase, and surface proteins. The mammalian hepadnaviruses, as well as the majority of the avian hepadnaviruses, contain an additional open reading frame that encodes the X protein (10). Infection is initiated by the interaction of the virus with a receptor present on the surface of hepatocytes. Carboxypeptidase D (CPD) has been identified as a receptor for DHBV, although it appears that additional coreceptors are required (4,7,35). Following attachment, the virus enters the cell, likely by ...

Cytopathic effects are currently believed to contribute to hepatitis C virus (HCV)-induced liver injury and are readily observed in Huh7.5 cells infected with the JFH-1 HCV strain, manifesting as apoptosis highly correlated with growth arrest. Reactive oxygen species, which are induced by HCV infection, have recently emerged as activators of AMP-activated protein kinase. The net effect is ATP conservation via on/off switching of metabolic pathways that produce/consume ATP. Depending on the scenario, this can have either pro-survival or pro-apoptotic effects. We demonstrate reactive oxygen species-mediated activation of AMP-activated kinase in Huh7.5 cells during HCV (JFH-1)-induced growth arrest. Metabolic labeling experiments provided direct evidence that lipid synthesis is attenuated, and ␤-oxidation is enhanced in these cells. A striking increase in nuclear peroxisome proliferator-activated receptor ␣, which plays a dominant role in the expression of ␤-oxidation genes after ligand-induced activation, was also observed, and we provide evidence that peroxisome proliferator-activated receptor ␣ is constitutively activated in these cells. The combination of attenuated lipid synthesis and enhanced ␤-oxidation is not conducive to lipid accumulation, yet cellular lipids still accumulated during this stage of infection. Notably, the serum in the culture media was the only available source for polyunsaturated fatty acids, which were elevated (2-fold) in the infected cells, implicating altered lipid import/export pathways in these cells. This study also provided the first in vivo evidence for enhanced ␤-oxidation during HCV infection because HCV-infected SCID/Alb-uPA mice accumulated higher plasma ketones while fasting than did control mice. Overall, this study highlights the reprogramming of hepatocellular lipid metabolism and bioenergetics during HCV infection, which are predicted to impact both the HCV life cycle and pathogenesis.With ϳ200 million people infected worldwide, hepatitis C virus (HCV) 2 is a global health problem and a major cause of viral hepatitis. Persistent infection occurs in ϳ70% of infected patients leading to inflammation, insulin resistance, steatosis, fibrosis, and hepatocellular carcinoma (1). Current direct-acting antivirals are predicted to be a cure for most patients, but the high cost of this treatment means that the pathological consequences of persistent HCV infection will remain a concern.Although the pathology associated with chronic HCV infection was initially thought to be due to HCV-specific immune responses (2), the current opinion is that direct cytopathic effects in virally infected cells also contribute to HCV-associated liver injury (3, 4). The cellular mechanisms by which HCV replication might mediate liver injury are unclear, but there is no doubt that oxidative/nitrosative stress in HCV-infected cells plays an important role in the initiation and progression of liver damage (3, 5, 6). Oxidative/nitrosative stress essentially arises when the production of reactive oxygen (...