In situ distribution of hepatitis C virus replicative-intermediate RNA in hepatic tissue and its correlation with liver disease

Liver failure from chronic hepatitis C is the leading indication for liver transplantation in the United States. However, the pathogenesis of liver in jury resulting from chronic hepatitis C virus (HCV) infection is not well u nderstood. To examine the relationship between HCV replication in liver tis sue and hepatocellular injury, a strand-specific in situ hybridization proc edure was developed, The sensitivity and specificity of digoxigenin-labeled riboprobes were optimized by analyzing Northern blots and cell lines expre ssing HCV RNAs. For the current study, both genomic (sense) and replicative -intermediate (antisense) HCV RNAs were detected and quantified in 8 of 8 l iver tissue specimens from infected patients versus 0 of 11 liver tissue sp ecimens from noninfected controls. The distribution pattern for HCV replica tive-intermediate RNA in liver was different from that for HCV genomic RNA. HCV genomic RNA was variably distributed throughout infected livers and wa s located primarily in the cytoplasm of hepatocytes, with some signal in fi broblasts and/or macrophages in the surrounding fibroconnective tissue. How ever, HCV replicative-intermediate RNA showed a more focal pattern of distr ibution and was exclusively localized in the cytoplasm of hepatocytes. Ther e was no significant relationship between the distribution pattern for HCV genomic RNA and any indices of hepatocellular injury, However, a highly sig nificant correlation was observed between the percentage of cells staining positive for replicative-intermediate RNA and the degree of hepatic inflamm atory activity (P, < 0.0001), Furthermore, the ratio of cells staining posi tive for HCV replicative-intermediate versus genomic RNA correlated with th e histological severity of liver injury (P, 0.0065), supporting the hypothe sis that active replication of HCV in liver tissue may be a significant det erminant of hepatocellular injury.