High levels of subgenomic HCV plasma RNA in immunosilent infections

Abstract

A genetic analysis of hepatitis C virus (HCV) in rare blood donors who remained HCV seronegative despite long-term high-level viremia revealed the chronic presence of HCV genomes with large in frame deletions in their structural genes. Full-length HCV genomes were only detected as minority variants. In one immunodeficiency virus (HIV) co-infected donor the truncated HCV genome transiently decreased in frequency concomitant with delayed seroconversion and re-emerged following partial seroreversion. The long-term production of heavily truncated HCV genomes in vivo suggests that these viruses retained the necessary elements for RNA replication while the deleted structural functions necessary for their spread in vivo was provided in trans by wild-type helper virus in co-infected cells. The absence of immunological pressure and a high viral load may therefore promote the emergence of truncated HCV subgenomic replicons in vivo.

Fig. 1. Virological and serological follow-up for subjects TN9, TN168 and TN78

Plasma HCV viral loads (VL) are expressed as HCV RNA/ml (left y axis). Antibody titers, determined by EIA 3.0, are expressed as signal over cut off ratios (S/CO, right y axis). Time points selected for amplification by RT-nPCR are indicated by shaded squares within the VL data points, the first and last being the baseline and exit time points respectively. Durations of anti-HIV and anti-HCV treatments are indicated for TN168.

Fig. 2. Detection of truncated HCV genomes

The amplified HCV PCR products corresponding to the 5′ half of the genome were separated by agarose gel electrophoresis. Full-length and deletion containing amplicons are labeled by white arrows (A and B). C: Amplification of the 5′ half genomes of subject TN78 and of regular seroconvertors. Time points are as follows: B, baseline; i-vii, intermediate; E, exit. MW: molecular weight marker in nucleotides. The 5′ amplicons selected for sequencing are highlighted by arrows.

Fig. 3. Genome organization of the deletion mutants from subjects TN9 and TN168

A: genomic organization of the HCV reference strain. B: Full HCV genome amplification and sequencing strategy diagram showing the location of the nPCR primers and sequencing primers. C: positions of the truncations observed in subjects TN9 and TN168.