More than 1% of the world's population is chronically infected with hepatitis C virus (HCV). HCV infection can result in acute hepatitis, chronic hepatitis, and cirrhosis, which is strongly associated with development of hepatocellular carcinoma. Genetic studies of HCV replication have been hampered by lack of a bona fide infectious molecular clone. Full-length functional clones of HCV complementary DNA were constructed. RNA transcripts from the clones were found to be infectious and to cause disease in chimpanzees after direct intrahepatic inoculation. This work defines the structure of a functional HCV genome RNA and proves that HCV alone is sufficient to cause disease.

Responses in three chimpanzees were compared following challenge with a clonal hepatitis C virus (HCV) contained in plasma from an animal that had received infectious RNA transcripts. Two of the chimpanzees (Ch1552 and ChX0186) had recovered from a previous infection with HCV, while the third (Ch1605) was a naïve animal. All animals were challenged by reverse titration with decreasing dilutions of plasma and became serum RNA positive following challenge. Ch1605 displayed a typical disease profile for a chimpanzee. We observed increasing levels of serum RNA from week 1 postinoculation (p.i.), reaching a peak of 10 6 copies/ml at week 9 p.i., and alanine aminotransferase (ALT) elevations and seroconversion to HCV antibodies at week 10 p.i. In contrast, both Ch1552 and ChX0186 exhibited much shorter periods of viremia (4 weeks), low serum RNA levels (peak, 10 3 copies/ml), and minimal ALT elevations. A comparison of intrahepatic cytokine levels in Ch1552 and Ch1605 showed greater and earlier gamma interferon (IFN-␥) and tumor necrosis factor alpha responses in the previously infected animal, responses that were 30-fold greater than baseline responses at week 4 p.i. for IFN-␥ in Ch1552 compared to 12-fold in Ch1605 at week 10 p.i. These data indicate (i) that clonal HCV generated from an infectious RNA transcript will lead to a typical HCV infection in naïve chimpanzees, (ii) that there are memory immune responses in recovered chimpanzees that control HCV infection upon rechallenge, and (iii) that these responses seem to be T-cell mediated, as none of the animals had detectable antibody against the HCV envelope glycoproteins. These observations have encouraging implications for the development of a vaccine for HCV.

To study determinants of clinical outcome following HCV infection, viral kinetics, immune events, and intrahepatic cytokine markers were compared in 10 naive chimpanzees. Four of the animals cleared HCV; 6 developed persistent infections. All animals developed similar acute infections with increasing viremia from 1 to 2 weeks, followed by alanine aminotransferase (ALT) elevations and seroconversion. This viremia pattern consisted of a biphasic increase, a rapid slope (mean doubling time [t 2 ] ‫؍‬ 0.5 days) followed by a slower slope after the second week (t 2 ‫؍‬ 7.5 days). This slowing of virus replication correlated in all animals with increased intrahepatic 25 oligoadenylate synthetase 1 (2OAS-1) messenger RNA (mRNA) levels and was independent of disease outcome. An effective control of virus replication was observed following increases in intrahepatic interferon ␥ (IFN-␥) mRNA and ALT levels. Although this control was associated in all animals with a 2-log decrease in virus titer, the timing occurred approximately 2 weeks later in the chronic group (P < .05). Additionally, while cleared infections were characterized by a continual decrease in virus titer, the titers in the persistent infections reached a steady state level of 10 4 to 10 5 RNA copies/mL. This inability of the immune response to sustain viral clearance in the persistent infections was associated with a reduced intrahepatic CD3e and monocyte-induced protein 1␣ (MIP-1␣) mRNA induction. In conclusion, these data indicate that, regardless of outcome, chimpanzees generate responses that control HCV replication during the early and late acute phase. However, the pathogenesis of HCV may be determined by a more rapid onset of the induced response and the cell population that migrates to the liver. (HEPATOLOGY 2004;39:1709 -1720.) H uman infections with hepatitis C virus (HCV) frequently become persistent, leading to chronic hepatitis, cirrhosis, and the development of hepatocellular carcinoma. 1 Persistent infection occurs in up to 85% of patients 2 and between 30% and 60% of chimpanzees, 3 the only animal model for this virus. The spontaneous clearance of primary HCV infections has been associated with strong, multispecific T-cell responses, 4 -6 and rechallenge experiments in chimpanzees have indicated that memory T-cells rather than antibody to surface antigens are pivotal in rapidly controlling the virus. 7,8 Consequently, persistence of the virus is thought to be the result of poor, dysfunctional, or ineffective T-cell responses that are unable to control viral replication. 9,10 However, the acute phase of HCV infection is not well documented because the virus causes a mild or inapparent acute infection in humans, and most diagnoses are made during the chronic stage, many years after the events determining the outcome of infection have occurred. In this study, 10 naïve, MHC class I-diverse chimpanzees were inoculated with virus or RNA encoding the same clonal HCV sequence containing no detectable quasispecies diversity. The monoclonal virus us...