Hepatitis C is one of the most widespread infectious diseases in the world. About 180 million people are infected with hepatitis C virus (HCV) worldwide with a yearly incidence of 3-4 million.
While the acute phase of infection is mostly asymptomatic, the majority of acutely infected individuals develops chronic hepatitis and is at increased risk of developing liver cirrhosis and hepatocellular carcinoma.
Thus, HCV infection is a major contributor to end-stage liver disease and in developed countries to liver transplantation.
HCV is a small, enveloped virus classified as a member of the Flaviviridae family. Its genome consists of a 9.6 kb single stranded RNA of positive polarity composed of 5′ and 3′ untranslated regions (UTR) and one long open reading frame (ORF) encoding a polyprotein, which is co- and post-translationally cleaved and thus yields the structural (Core, E1, E2), p7 and nonstructural (NS2, NS3, NS4A, NS4B, NS5A, NS5B) proteins.
HCV isolates from around the world exhibit significant genetic heterogeneity. At least 7 major HCV genotypes (genotypes 1-7) have been identified, which differ by 31-33% at the nucleotide level and deduced amino acid level.
In addition, there are numerous subtypes (a, b, c, etc.), which differ by 20-25% on the nucleotide and deduced amino acid level.
Since its discovery in 1989, research on HCV has been hampered by the lack of appropriate cell culture systems allowing for research on the complete viral life cycle as well as new therapeutics and vaccines.
In 2001, a genotype 2a isolate (JFH1) was described, which subsequently was found to yield high RNA titers in the replicon system without adaptive mutations.
A major breakthrough occurred in 2005, when formation of infectious viral particles was reported after transfection of RNA transcripts from the JFH1 full-length consensus cDNA clone into Huh7 cells.
At the same time, it was demonstrated that the intragenotypic 2a/2a recombinant genome (J6/JFH1), in which the structural genes (Core, E1, E2), p7 and NS2 of JFH1 were replaced by the respective genes of clone J6CF, produced infectious viral particles in Huh7.5 cells (a cell line derived from bulk Huh7 cells) with an accelerated kinetic.
Cell culture derived J6/JFH viruses were apparently fully viable in vivo.
Despite the importance of the described cell culture systems they represent only a single isolate (genotype 2a) of HCV.
It is important to develop cell culture systems for representative strains of other HCV isolates, subtypes and genotypes, since neutralizing antibodies are not expected to cross-neutralize all genotypes and new specific antiviral compounds have differential efficiencies against different isolates, subtypes and genotypes.
To date, only the JFH1 (genotype 2a) clone could autonomously replicate and release infectious virus in cultured human hepatoma cells, Huh7 and Huh7.5; its efficient growth depended on mutations.
A JFH1 chimera with the 5′UTR-NS2 region from another genotype 2a strain cDNA clone, J6CF, had enhanced infectivity.
Besides, an H77 (genotype 1a) clone containing replicon-derived mutations was shown to produce infectious virus particles.
To facilitate HCV research and obtain basic knowledge for better and individualized treatment, the present inventors have aimed at developing culture systems for other HCV patient isolates.
Hence, improved and alternative HCV genomes of all genotypes, which are capable of expressing said virus when transfected into cells and are capable of infectivity in vivo, would be advantageous.