Cell Culture Systems for Hepatitis C Virus

Abstract

Due to the obligatory intracellular life style of viruses, cell culture systems for efficient viral propagation are crucial to obtain a detailed understanding of the virus host cell interaction. For hepatitis C virus (HCV) the development of permissive and authentic culture models has been and continues to be a challenging task. The first ef forts to culture HCV had limited success and range back to before the virus was molecularly cloned in 1989. Since then several major breakthroughs have gradually overcome limitations in culturing the virus and sequentially permitted analysis of viral RNA replication, cell entry and ultimately the complete replication cycle in cultured cells in 2005. Until today, basic and applied HCV research greatly benefit from these tremendous efforts which spurred multiple com plementary cell based model systems for distinct steps of the HCV replication cycle. When used in combination they now permit deep insights into the fascinating biology of HCV and its interplay with the host cell. In fact drug development has been much facilitated and our understanding of the molecular determinants of HCV replication has grown in parallel to these advances. Building on this ground work and further refining our cellular models to better mimic the ar chitecture, polarization and differentiation of natural hepatocytes should reveal novel unique aspects of HCV replication. Ultimately, models to culture primary HCV isolates across all genotypes may teach us important new lessons about viral functional adaptations that have evolved in exchange with its human host and that may ex plain the variable natural course of hepatitis C.

Characterization of nonprimate hepacivirus and construction of a functional molecular clone

“…For HCV, efforts to determine the complete genome sequence were pivotal given that the 3′-terminal highly conserved X tail remained undiscovered for 6 y after the discovery of HCV (26,27). This enabled the establishment of in vivo and in vitro assays for fundamental virologic studies, target identification and validation, and drug development (21,22,28).…”

“…Our efforts to coax NPHV replication in vitro have thus far proved unsuccessful. This should come as no surprise given the enormous difficulties encountered in attempts to establish robust culture systems for hepatitis viruses, including HBV and HCV (21,22). One major obstacle to establishing NPHV replication in culture is likely the cellular environment, in this case exacerbated by the lack of equine liver cell lines.…”

“…One major obstacle to establishing NPHV replication in culture is likely the cellular environment, in this case exacerbated by the lack of equine liver cell lines. For HCV, miR-122 supplementation was sufficient to promote replication in hepatic cells, with lower replication in nonhepatic cells (21,22). For NPHV, this strategy has been unsuccessful for the nonhepatic cell lines tested thus far.…”

“…Our attempt to establish NPHV infection in EFLCs was unsuccessful but it is possible that fetal cells at this early gestational age lack the proper hepatic phenotype. In future work, attempts should be made to evaluate later stage EFLCs, micropatterned cocultures of adult equine hepatocytes, hepatocyte-differentiated equine stem cells, or immortalized equine liver cells, similar to successful approaches for HCV (21,22).…”

“…To allow positive selection of replicating genomes, we constructed subgenomic replicons for NPHV (pNZP1-SGR), similar to those described for HCV (21,22) (Fig. 2D).…”