D espite decades of research, the processes that govern liver development and regeneration are only partially understood. A good understanding of the mechanisms that play a role in these processes is important because it may lead to new treatments of human liver diseases. Several in vivo experimental conditions have been used to study liver regeneration and repair and the interaction of different cell types in these processes. These include partial hepatectomy, administration of toxic compounds, or a combination of both, and transgenic expression of certain proteins. 1,2 In contrast to in vitro experiments, these approaches raise fewer questions concerning the influence of artificial matrices on the function and behavior of hepatocytes and other liver cell types.Although these procedures have generated useful information on rodent liver regeneration, stem cell activation, and other processes, extrapolating these findings to the Abbreviations: uPA, urokinase plasminogen activator; HBV, hepatitis B virus; HCV, hepatitis C virus; HBsAg, hepatitis B surface antigen; HBeAg, hepatitis B e antigen; PAS, periodic-acid-Schiff. From the

Hepatitis C virus (HCV) is a major cause of chronic liver disease, frequently progressing to cirrhosis and increased risk of hepatocellular carcinoma. Current therapies are inadequate and progress in the field has been hampered by the lack of efficient HCV culture systems. By using a recently described HCV genotype 2a infectious clone that replicates and produces infectious virus in cell culture (HCVcc), we report here that HCVcc strain FL-J6͞JFH can establish long-term infections in chimpanzees and in mice containing human liver grafts. Importantly, virus recovered from these animals was highly infectious in cell culture, demonstrating efficient ex vivo culture of HCV. The improved infectivity of animal-derived HCV correlated with virions of a lower average buoyant density than HCVcc, suggesting that physical association with low-density factors influences viral infectivity. These results greatly extend the utility of the HCVcc genetic system to allow the complete in vitro and in vivo dissection of the HCV life cycle.animal model ͉ pathogenesis ͉ reverse genetics ͉ viral hepatitis A major limitation in hepatitis C virus (HCV) research has been the lack of virus culture systems. After identification of the viral genome in 1989 (1), early efforts focused on understanding the structure and function of individual viral gene products. HCV is an enveloped, positive-strand RNA virus classified in the family Flaviviridae (2). The 9.6-kb ssRNA genome encodes three structural (virion-associated) and seven nonstructural (intracellular) genes within a single ORF.The first functional cDNA clones of HCV were constructed in 1997, allowing chimpanzees to be infected after intrahepatic transfection with recombinant viral RNA (3, 4). Unfortunately, these infectious genomes failed to replicate in cell culture. By engineering HCV replicons to express a drug-selectable gene, it became possible to select for HCV RNA replication in cell culture (5). However, efficient replication required cell cultureadaptive mutations in the viral RNA (6). Moreover, only the intracellular aspects of HCV replication were modeled by these systems. For unknown reasons, cell culture-adaptive mutations can inhibit virion production in culture (T. Pietschmann and R. Bartenschlager, personal communication) and attenuate RNA infectivity in vivo (7).Recent progress in the field has come from the identification of JFH-1, a genotype 2a subgenomic replicon that does not require adaptive mutations for efficient RNA replication in culture (8). Based on this sequence, we constructed a chimeric JFH-1 genome containing the core to nonstructural protein 2 (NS2) region of HCV strain J6. This genome, FL-J6͞JFH, replicated and produced high levels of infectious virus in cell culture (HCVcc) (9), allowing us to study new aspects of the viral life cycle in tissue culture (9, 10). Similarly, full-length JFH-1 clones produced HCVcc, albeit with delayed kinetics of virus release (11, 12). HCVcc strain JFH-1 was able to transiently infect a chimpanzee, although replication le...

We developed a cell culture system and characterized HEV particles; we identified 3 ORF2 capsid proteins (ORF2i, ORF2g, and ORFc). These findings will advance our understanding of the HEV life cycle and improve diagnosis.

Hepatitis C virus (HCV) is a major cause of liver cirrhosis and hepatocellular carcinoma. Current antiviral therapy fails to clear infection in a substantial proportion of cases. Drug development is focused on nonstructural proteins required for RNA replication. Individuals undergoing orthotopic liver transplantation face rapid, universal reinfection of the graft. Therefore, antiviral strategies targeting the early stages of infection are urgently needed for the prevention of HCV infection. In this study, we identified the polyphenol, epigallocatechin-3-gallate (EGCG), as an inhibitor of HCV entry. Green tea catechins, such as EGCG and its derivatives, epigallocatechin (EGC), epicatechin gallate (ECG), and epicatechin (EC), have been previously found to exert antiviral and antioncogenic properties. EGCG had no effect on HCV RNA replication, assembly, or release of progeny virions. However, it potently inhibited Cell-culture-derived HCV (HCVcc) entry into hepatoma cell lines as well as primary human hepatocytes. The effect was independent of the HCV genotype, and both infection of cells by extracellular virions and cell-to-cell spread were blocked. Pretreatment of cells with EGCG before HCV inoculation did not reduce HCV infection, whereas the application of EGCG during inoculation strongly inhibited HCV infectivity. Moreover, treatment with EGCG directly during inoculation strongly inhibited HCV infectivity. Expression levels of all known HCV (co-)receptors were unaltered by EGCG. Finally, we showed that EGCG inhibits viral attachment to the cell, thus disrupting the initial step of HCV cell entry. Conclusion: The green tea molecule, EGCG, potently inhibits HCV entry and could be part of an antiviral strategy aimed at the prevention of HCV reinfection after liver transplantation.

Hypervariable region 1 (HVR1) of hepatitis C virus (HCV) E2 envelope glycoprotein has been implicated in virus neutralization and persistence. We deleted HVR1 from JFH1-based HCV recombinants expressing Core/E1/E2/p7/NS2 of genotypes 1 to 6, previously found to grow efficiently in human hepatoma Huh7.5 cells. The 2a ⌬HVR1 , 5a ⌬HVR1 , and 6a ⌬HVR1 Core-NS2 recombinants retained viability in Huh7.5 cells, whereas 1a ⌬HVR1 , 1b ⌬HVR1 , 2b ⌬HVR1 , 3a ⌬HVR1 , and 4a ⌬HVR1 recombinants were severely attenuated. However, except for recombinant 4a ⌬HVR1 , viruses eventually spread, and reverse genetics studies revealed adaptive envelope mutations that rescued the infectivity of 1a ⌬HVR1 , 1b ⌬HVR1 , 2b ⌬HVR1 , and 3a ⌬HVR1 recombinants. Thus, HVR1 might have distinct functional roles for different HCV isolates. Ultracentrifugation studies showed that deletion of HVR1 did not alter HCV RNA density distribution, whereas infectious particle density changed from a range of 1.0 to 1.1 g/ml to a single peak at ϳ1.1 g/ml, suggesting that HVR1 was critical for low-density HCV particle infectivity. Using chronic-phase HCV patient sera, we found three distinct neutralization profiles for the original viruses with these genotypes. In contrast, all HVR1-deleted viruses were highly sensitive with similar neutralization profiles. In vivo relevance for the role of HVR1 in protecting HCV from neutralization was demonstrated by ex vivo neutralization of 2a and 2a ⌬HVR1 produced in human liver chimeric mice. Due to the high density and neutralization susceptibility of HVR1-deleted viruses, we investigated whether a correlation existed between density and neutralization susceptibility for the original viruses with genotypes 1 to 6. Only the 2a virus displayed such a correlation. Our findings indicate that HVR1 of HCV shields important conserved neutralization epitopes with implications for viral persistence, immunotherapy, and vaccine development.