This application proposes to examine the biological significance of liver-specific hepatitis C viral (HCV) quasispecies variants. HCV is a positive single-stranded blood-borne RNA virus which infects humans and may cause chronic liver injury including hepatitis, cirrhosis and hepatocellular carcinoma through chronic infection. The liver is the major site of replication of HCV although HCV RNA is also found in the blood of infected individuals. The genome of HCV varies considerably in nucleotide sequence from isolate to isolate, allowing HCV to be divided into genotypes and subtypes. However, multiple isolates from a single infected individual may also have considerable variability in nucleotide and amino acid sequence, particularly within the hypervariable region (HVR1) of the envelope gene. HRV1 is a sequence of approximately 27 amino acids at the likely N terminus of the HCV E2 glycoprotein. Variations in HRV1 provide markers for identification and tracing of HCV quasispecies variants. Quasispecies arise because of the high error frequency associated with viral RNA replication and immune pressure appears to be a factor in their selection. Preliminary experiments conducted by the applicants have shown that a greater number of HCV quasispecies variants are found within the liver compared to serum, even allowing for differences in viral load and that some liver-specific variants appear to be present. The applicants hypothesize that an excess of HCV quasispecies variants are continually being produced by errors in replication of HCV RNA within hepatocytes, some of which are cleared from serum by neutralizing antibodies, leaving behind in the circulation those variants which have escaped immune clearance. The significance of these differences will be examined through cloning and sequencing quasispecies variants present in both serum and liver. Changes in HCV quasispecies over time will be sought by comparing nucleotide sequence in HRV1 region before and after liver transplantation. The presence of possible neutralizing antibodies in serum directed against liver-specific quasispecies variants will be sought by enzyme-linked immunoassays based on peptides synthesized according to nucleotide sequences of the various cloned quasispecies. In addition, immunoreactivity to the whole of the E1 and E2 proteins cloned and expressed in mammalian cells will be determined. Differences in presence, titer and reactivity of antibodies against quasispecies will be compared to determine whether neutralizing antibodies account for clearance of some quasispecies from serum but not liver. These studies have great potential significance for understanding of viral clearance, development of persistent infection and for the development of a vaccine against HCV.
