We have demonstrated and/or confirmed that chimpanzees are susceptible to all 6 published HCV genotypes and important subtypes. For many of the genotypes, this is the first time the course of infection in chimpanzees has been described, to our knowledge. Overall, we found that the course of acute infection is similar in animals infected with the different HCV genotypes, with early appearance of viremia, relatively high peak genome titers, and development of acute hepatitis. It should be noted, however, that infection in 2 animals does not necessarily represent the typical course for a particular genotype or, for that matter, that particular strain. Also, we cannot rule out the possibility that the infection observed in animals after reverse titration could be influenced by exposure to apparently non-infectious virus challenges. However, we observed robust infections in both first- and second-passage animals, with no apparent difference. This is in agreement with data previously published by our group, in which it was found that chimpanzees inoculated with high doses (1,000,000 infectious doses of the H77 strain) of HCV had infections that were similar to those observed after inoculation with lower doses, only with a shorter incubation period [33
The chronicity rate of HCV in experimentally infected chimpanzees has varied in different studies, from as low as 33% to as high as 67% [9
]. In the present study, we found a chronicity rate of 56% among 16 experimentally infected chimpanzees. It is noteworthy that in the available samples taken from the same animals during the late chronic-phase infection (CH1410, CH1563, and CH1547), there was a clear indication of an increase in viral titers over time, compared with samples taken from the same animals during the late acute-phase infection ( and ), which suggests a relative loss of the host’s control of viremia [34
]. We observed that 5 of 7 animals with acute resolving infection did not seroconvert (4 animals) or had transient seroconversion (1 animal), whereas all 9 animals that developed a persistent infection seroconverted during the acute infection. Although it must be acknowledged that the humoral immune response of chimpanzees might differ from that of humans [35
], these data are in agreement with data from long-term follow-up studies of patients with transfusion-associated HCV infection [37
The virus pools characterized in the present study have been useful for a number of studies with chimpanzees. The chimpanzee is the only animal model in which to study protective immunity following resolved experimental infections or immunization with vaccine candidates. However, titrated challenge viruses previously were available only for genotype 1a and 1b strains. The current virus panel has made it possible to perform more extensive heterologous rechallenges. In a study by Prince et al [38
], in which chimpanzees with resolved genotype 1 infections were rechallenged with 100 CIDs of these respective genotype pools, it was found that heterologous challenge would lead to frequent viral persistence. In a recent study, we found that an animal with apparent sterilizing immunity against challenge with the homologous genotype 1a was not protected against acute infection with genotype 1b or 2a after challenge with 100 CIDs of the respective virus pools [39
]. In the testing of vaccine candidates, it is important to challenge with a virus dose that consistently infects the animal. Titrated virus pools of genotype 1a have been important for a number of vaccine studies with chimpanzees [40
]. The 1b pool presented here has been used in other vaccine studies [41
]. The virus-containing plasma pools could also be useful in the development of diagnostic assays, such as quantitative diagnostic assays or genotyping assays, and they have already been distributed to a number of investigators for these purposes.
Advances in HCV research have been hampered by a lack of readily available in vitro systems. The development of infectious HCV clones of strain H77 in 1997 paved the way for the development of such systems [43
]; genotype 1a (pHC-TN), 1b (pCV-J4L6S), and 2a (pJ6CF) infectious clones have since been developed from the virus pools characterized in the present study [26
]. A subgenomic replicon system for strain Con1 (genotype 1b) was developed in 1999 [45
]; however, this system currently is available only for genotype 1a, 1b, and 2a HCV strains. A retroviral pseudoparticle system bearing the HCV glycoproteins was developed in 2003 [46
], and the strains of our genotype pools have contributed to the development of this system for genotypes 1b, 2a, 3a, 4a, 5a, and 6a [30
]. In 2005, the JFH1 (genotype 2a)–based cell culture system was developed; this system provides robust HCV infection in Huh7.5 cells [10
]. The genotype pools presented here have served as the virus source for the development of JFH1-based intragenotypic and intergenotypic recombinants of genotypes 1b, 2a, 2b, 3a, 4a, 5a, and 6a, producing viruses with the genotype-specific core, E1, E2, p7, and NS2 in Huh7.5 cells [29
]. Thus, the virus genotype pools have contributed to the development of new experimental systems for HCV. The use of prototype strains in the development of such systems provides the added advantage of permitting the use of identical HCV strains in different experimental systems. For example, antibodies identified in vitro in the HCV pseudoparticle systems or in the JFH1-based cell culture systems could be used for passive immunoprophylaxis studies with chimpanzees in which a matched challenge virus from the genotype pools is used.
In the human liver–uPA-SCID mouse model, we observed robust infection with HCV strains that represent HCV genotypes 1–6, in most cases with peak HCV RNA titers that were 1log10
higher than those observed for the same strains in chimpanzees. Robust infection was seen in mice with different human liver grafts, but titration data indicated that HCV infectious doses that are 1–2 logs higher than those required to infect chimpanzees are required to infect human liver–chimeric mice. Plasma from infected mice with relatively high HCV RNA titers (often of >107
IU/mL; ) can be used to make additional antibody-free challenge pools of these HCV strains, as we reported for the H77C strain [16
]. The susceptibility of the human liver–chimeric mice to well-defined challenge viruses will permit more detailed studies of the cross-neutralization potential of antibody preparations. In addition, new drugs can be tested for therapeutic potential against the different genotypes [14
In summary, we have developed titrated challenge pools of genotypes 1–6 of HCV. These reference viruses are available to the scientific community and will contribute to studies of active and passive immunity against HCV. In addition, these challenge pools of genotypes 1–6 will permit controlled studies in vitro and in vivo, because different experimental systems have already been developed for these strains: for the HC-J6 (genotype 2a) strain, a consensus molecular clone that is infectious for chimpanzees, a retroviral pseudoparticle system that permits entry into Huh7 cells, and a JFH1-based intragenotypic recombinant that is infectious for Huh7 cells have been developed [28
]. We have demonstrated that infection of chimpanzees with all HCV genotypes resulted in relatively high HCV titers and liver inflammation, and that viruses from the challenge genotype pools readily infect human liver–chimeric mice, which will permit further studies with both animal models of HCV infection.