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

Abstract

HCV replicates in liver via an intermediate negative strand RNA. To study the relevance of HCV genome replication, quantitative strandspecific 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.

Hepatitis C virus double‐stranded RNA is the predominant form in human liver and in interferon‐treated cells

“…An established strand‐specific qRT/PCR assay was used to quantify HCV (−) strands in extracts of human liver and to determine the percentage of (−) strand RNA in double‐stranded form. This assay has several features that improve strand specificity, including an RT reaction temperature of 70°C, use of tagged RT primers, and post‐RT RNase H treatment . The assay was validated using (+) and (−) ssRNA in vitro transcripts (http://onlinelibrary.wiley.com/doi/10.1002/hep.28846/suppinfo) and was then applied to liver RNA extracts (Fig.…”

“…(B) Table delineating the ability of various assays to detect different forms of HCV RNA: free (1) strand, free (2) several features that improve strand specificity, including an RT reaction temperature of 708C, use of tagged RT primers, and post-RT RNase H treatment. (31) The assay was validated using (1) and (2) ssRNA in vitro transcripts (Supporting Fig. S3) and was then applied to liver RNA extracts (Fig.…”

Human cell types important for Hepatitis C Virus replication in vivo and in vitro. Old assertions and current evidence

“…Liver specimens had detectable amounts of negative strands by RT-PCR within 7 days after transplantation [94]. Levels of negative strand in the liver after transplants do not correlate with serum levels of HCV [94,95], and negative strands were more likely to be found in transplanted PBMC than in PBMC from individuals with chronic HCV infection [40,96]. …”

Rate-limiting pyrophosphate release by hepatitis C virus polymerase NS5B improves fidelity

“…The UTP:U misincorporation occurs readily, but the weak apparent binding affinity cannot be saturated at the low physiological concentration of UTP relative to ATP (570 µM and 3 mM, respectively (10)). Moreover, the ratio of (+) strand RNA to (-) strand RNA in vivo can reach up to 1000:1 depending on the cell type (15)(16)(17). Therefore, it would be more likely for the misincorporation necessary to form the S282T resistance variant to occur during (+) strand synthesis with an ATP:A misincorporation.…”