Hepatitis C virus molecular clones and their replication capacity in vivo and in cell culture
Section snippets
The hepatitis C virus
Primary infections with HCV are often asymptomatic or associated with only mild and rather non-specific symptoms. Nevertheless, HCV infections are by no means harmless because this virus has a high propensity to establish persistence which can last for decades, eventually life-long. Persistently infected patients have a high risk of contracting liver diseases, with the most severe manifestations being liver cirrhosis and hepatocellular carcinoma (Lauer and Walker, 2001). Given about 180 million
HCV replication cycle
Like almost all other positive-strand RNA viruses, HCV replication takes place in the cytoplasm of the host cell, which is primarily the hepatocyte. HCV particles bind to the host cell via a specific interaction of the viral glycoproteins with a yet unknown cellular receptor complex. So far, several receptor candidates have been identified, including CD81 (Pileri et al., 1998), SR-B1 (Scarselli et al., 2002), and the C-type lectins DC-SIGNR, L-SIGN and ASGPR (Gardner et al., 2003, Pohlmann et
Molecular cloning of functional HCV genomes: the consensus genome concept
To enable studies of the viral life cycle, it is imperative to have a cell culture system that allows the reproducible generation of high-titre virus stocks. Most often such a system is based on the inoculation of permissive host cells with virus present in a biological sample but this approach is limited by the fact that manipulations of the viral genome are hardly possible. Such manipulations, in particular mutagenesis, are, however, highly instructive and have provided invaluable insights
Genotype 1 isolates and the replicon system
Building on this important achievement, several additional HCV consensus genomes of other isolates were constructed and shown to be infectious in vivo (Table 1). However, despite intensive efforts reproducible and efficient replication of the genomes available up to that time could not be achieved in cell culture. In the light of the numerous reports indicating that HCV could be propagated in primary cell cultures and immortalized cell lines, but in all cases with very low efficiency (for
Molecular HCV clones supporting production of infectious virus in cell culture
With the intention of producing infectious HCV particles in cell culture, several groups constructed genomic replicons that have an analogous bicistronic design like subgenomic replicons but contain the complete HCV open reading frame downstream of the EMCV IRES (Fig. 1B). However, initial attempts to establish stable replication of such genomic replicons (also referred to as selectable full-length genomes) were not successful. Only when adaptive mutations were introduced, and in one case a
Conclusions and perspectives
Molecular HCV clones are and will continue to be a very important tool to study the complexity of the full viral replication cycle as well as HCV-host cell interaction in molecular detail. The past few years have seen tremendous progress in this field that started with the first establishment of subgenomic replicons and culminated recently in the development of an efficient and robust system for production of infectious virus particles in cell culture. Key for this success is the molecular
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2018, Antiviral ResearchCitation Excerpt :Re-challenge studies proved that animals were not necessarily protected against re-infection with the identical virus from a monoclonal infection (Bukh et al., 2008), a discovery with major implications for antiviral strategies based on vaccine candidates. Many of the in vivo infectious consensus clones of HCV were extensively tested in vitro, primarily in hepatoma cells (Bukh, 2016; Sakai et al., 2007; Gottwein et al., 2010; Bartenschlager and Sparacio, 2007) and invariably found non-viable. Moreover, most generated consensus clones regardless of whether they have been proven to be infectious in vivo, do not propagate in cell culture.
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2016, Journal of HepatologyCitation Excerpt :However, they facilitated development of in vitro systems for HCV, since subsequent studies could be performed with genomes known to have all genetic elements required for infection [82,97]. This research resulted in the development of HCV replicons in 1999 and in a true infectious culture system in 2005 [97,98]. Despite the inability to culture patient viruses, detailed characterization of the infectious genome of hepatitis C virus (HCV) resulted in the development of recombinant systems in vivo (infectious clones) and in vitro (replicons, pseudoviruses, and infectious cultures in human hepatoma cell lines).
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