Quantitation of replication of the HCV genome in human livers with end‐stage cirrhosis by strand‐specific real‐time RT‐PCR assays: Methods and clinical relevance

HCV replicates in liver via an intermediate negative strand RNA. To study the relevance of HCV genome replication, quantitative strand‐specific HCV real‐time RT‐PCR assays were developed and applied to livers explanted because of end‐stage cirrhosis. The assays have broad ranges of determination and a high reproducibility and accuracy. Analysis of five different samples showed an even distribution of HCV genomes in four livers. Hepatic concentrations of positive (PS)‐ and negative (NS)‐strand RNA did correlate with each other, with PS/NS ratios ranging between 3 and 340. Hepatic concentrations of HCV‐PS or ‐NS RNA did not correlate with serum HCV‐RNA levels or with genotypes. A high HCV envelope‐2 protein expression correlated with a low NS concentration. HCV‐PS and ‐NS levels, E2 protein expression and genotype did not correlate with biochemical tests or with histological changes in the explanted liver, but the ratio NS/PS, a marker of viral replication, correlated with the severity of the recurrent post‐transplant hepatitis caused by HCV. This suggests the existence of an extra‐hepatic location of HCV with comparable viral replication rate being responsible for the infection of the newly transplanted liver. J. Med. Virol. 81:1569–1575, 2009. © 2009 Wiley‐Liss, Inc.