: Chronic HCV infection is a major threat to the public health. Current therapies have limited efficacy, but the search for more effective treatments is hampered by the lack of available animal models of HCV infection. The chimpanzee (Pan troglodytes) is the only animal species permissive for infection with this virus. This deficiency will be addressed by developing a small nonhuman primate model of hepatitis C involving the closely related, unclassified flavivirus GBV-B. GBV-B replicates to high titers, is hepatotropic, and causes liver disease in susceptible tamarins (Saquinus sp.). Since tamarins are more readily available than chimpanzees for such studies, GBV-B infection of these animals represents a potentially useful surrogate for studies of hepatitis C. However, although GBV-B among all animal viruses has the closest phylogenetic relationship to HCV, its proteins still share only approximately 25% identity at the amino acid level. Moreover, unlike HCV, GBV-B does not appear capable of establishing persistent infection in these animals. These features of GBV- B limit its usefulness. To overcome these limitations, the applicants will construct chimeric genome-length GBV-B cDNA clones in which specific functional domains of HCV are inserted in lieu of homologous GBV-B sequence. The hypothesis is that the close phylogenetic relationship between GBV-B and HCV will allow the rescue of viable chimeric viruses from these clones, and that these viruses will represent uniquely valuable resources to the research community since they will allow the in vivo evaluation of candidate inhibitors of critical HCV replication functions in a readily available and relatively inexpensive small, nonhuman primate species.
Under Aim 1, the investigators have constructed a fulllength, infectious cDNA copy of the GBV-B genome. The infectivity of RNA transcribed from this clone has been demonstrated following intrahepatic injection of the RNA in a susceptible tamarin.
In Aim 2, the investigators are constructing infectious chimeric flavivirus cDNAs containing the following HCV domains within a GBV-B background: the internal ribosome entry site (IRES), the major proteinase (NS3) with its cofactor molecule (NS4A), the RNA helicase (NS3) and the RNA dependent, RNA polymerase (NSSB).
In Aim 3, chimeras in which the structural proteins of GBV-B and HCV are placed within the genetic background of the alternate virus will be constructed. For both Aims 2 and 3, the applicants will assess the ability of RNAs transcribed from these chimeric cDNA clones to induce infection in tamarins following intrahepatic inoculation, and determine the extent to which the viruses rescued from these clones cause acute or chronic liver disease on subsequent passage in these nonhuman primates.
