Cell culture-grown hepatitis C virus is infectious in vivo and can be recultured in vitro

Abstract

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...

High plasma level of nucleocapsid-free envelope glycoprotein-positive lipoproteins in hepatitis C patients

“…Infectious cell cultureproduced HCV (HCVcc) that can be propagated efficiently only in the human hepatoma cell line Huh7 has higher density than in vivo circulating viruses. 9 HCVcc are associated with apoE and apoC, but only marginally with apoB, in contrast to ex vivo-characterized LVPs. 10,11 Despite these differences, two sets of evidence further ascertain the role of lipoproteins in HCVcc assembly.…”

Entry and replication of recombinant hepatitis C viruses in cell culture

“…They are infectious in cell culture as well as in chimpanzee and humanized mice [5,54]. Methods to produce HCVcc have been published elsewhere [55,56].…”

“…As a consequence, HCV particles produced in Huh-7-derived cell lines do not fully resemble the lipoviroparticles circulating in infected patient serum, with possible implications for entry studies. In fact the much higher specific infectivity of HCV re-cultured from infected humanized mice compared to cell culture grown HCV is likely due to different particle properties of viruses grown in these environments [54]. HCV production in mice engrafted with a humanized liver [54], in PHH [91] or development of new cell lines fully competent for VLDL production is necessary to overcome this issue.…”

Experimental models for hepatitis C viral infection

“…27 Transfection of Huh7 and Huh7.5.1 cells with the in vitro-transcribed fulllength JFH-1 genome or a recombinant chimeric genome with another genotype 2a isolate, J6, resulted in the secretion of viral particles that were infectious in cultured cells, in chimeric mice, and in chimpanzees. [28][29][30][31] The infectivity of cells could be neutralized with antibodies against the HCV entry receptor CD81, antibodies against E2, or immunoglobulins from chronically infected patients. Importantly, the replication of cell-cultured HCV in this system was inhibited by IFN-␣ as well as by several HCVspecific antiviral compounds.…”