Challenge Pools of Hepatitis C Virus Genotypes 1–6 Prototype Strains: Replication Fitness and Pathogenicity in Chimpanzees and Human Liver-Chimeric Mouse Models

The most recent estimates suggest that 180 million people worldwide are infected with hepatitis C virus (HCV). In the United States, a total of ~3.2 million people are persistently infected with HCV, and >10,000 people die from HCV-related chronic liver disease each year. A continued increase in the number of HCV-infected patients with liver cirrhosis and hepatocellular carcinoma is expected during the next few decades, and HCV-associated end-stage liver disease is already the most common indication for liver transplantation in many Western countries. Thus, there is an urgent need for better drugs to treat this disease and for the development of a vaccine to prevent further spread. To promote such developments, it is important to define the pathogenesis of different HCV genotypes in available animal models and to develop well-characterized virus stocks that represent the various HCV variants that can be used in the development of new experimental in vitro systems and for in vivo studies of new drugs and passive and active immunization strategies. The HCV virion contains a positive-sense, single-stranded RNA genome that has a single long open reading frame (ORF) [1, 2]. Extensive genomic sequence analysis has demonstrated that HCV strains from around the world exhibit significant genetic heterogeneity, and on the basis of phylogenetic analysis of the ORF of representative isolates, HCV has been classified into 6 major genotypes (genotypes 1–6) and a number of subtypes (subtypes a, b, and so forth) [3–5]. Important differences in the geographic distribution of these genotypes exist, and recent studies have suggested important antigenic and serologic differences [2]. Furthermore, it is well established that the current standard therapy with interferon and ribavirin has a higher sustained virologic response rate in patients infected with genotypes 2 and 3, compared with patients infected with genotypes 1 and 4 [2]. A seventh major genotype has recently been identified in 3 Canadian and Belgian patients, who were presumably infected in Central Africa [6]. However, samples from these patients have not been readily available, and experimental infection with this new genotype could therefore not be included in the present study. The chimpanzee remains the only animal model that can be used for studies of the natural history of HCV and in challenge studies (eg, studies of immunogenicity and efficacy of HCV vaccine candidates) [7–9]. Despite the recent development of the strain JFH1 cell culture system, which permits virus propagation of a particular genotype 2a isolate in Huh7.5 cells [10], there is still no reproducible cell culture system based on the full-length sequence of other HCV genotype viruses. Thus, transmission to chimpanzees has been the only means to propagate experimentally HCV viruses of different strains. Urokinase-type plasminogen activator (uPA)–severe combined immunodeficient (SCID) mice engrafted with primary human hepatocytes (chimeric mice) are susceptible to infection with native HCV, and they produce infectious virions with a density similar to that observed in humans and experimentally infected chimpanzees [11–13]. This small animal model has been successfully used to assess the activity of antiviral compounds [14] and to evaluate protection and passive immunization studies of HCV [15–17], but because of the lack of an immune system, this model has not been useful for studies of HCV pathogenesis. Furthermore, this model has not permitted the generation of large quantities of reagents for studies of these specific genotypes. Our aim was to generate titrated challenge pools for the major HCV genotypes and important subtypes. Challenge viruses of genotypes 1–6 were characterized in chimpanzees and in human liver–chimeric mice.