HCV 5`UTR sequences remain unchanged at serum and PBMC during IFN-based therapy in HIV-coinfected Patients

FEDERICO BOLCIC; NATALIA LAUFER; FRANCO MORETTI; RITA REYNOSO; JORGE QUARLERI

Lisboa, Portugal

Workshop; 5th International Workshop on HIV and Hepatitis Co-infection; 2009

Background: The hepatitis C virus (HCV)RNA contains a well-defined structure of about 340 nucleotides in its 5´-untranslated region (UTR), called the internal ribosome-entry site (IRES). The IRES encompasses most of the 5´-UTR of the HCV RNA and is highly conserved compared with the rest of the viral genome. This conservation indicates that it plays an essential role in the viral life cycle, and allows the use of viral 5´-UTR for diagnostics and genotype classification. Extrahepatic sites capable of supporting HCV replication have been suggested. Peripheral blood mononuclear cells (PBMC) can act as HCV reservoir and keep different genotypic variations. The current HCV antiviral treatment is based on peg-interferon plus ribavirin. Controversial results were observed in the variation of 5´UTR during HCV treatment. The aim of this study was to analyze the effect of peg-IFN + ribavirin therapy in the HCV 5´UTR genomic sequence in serum and PBMC compartments in HIV-coinfected patients. Materials and Methods: Fourteen HIV/HCV coinfected patients were treated with peg-INF 2 alfa + ribavirin. All of them were under HAART. Serum HCV-RNA levels before initial dosing (baseline level)and at 24 h, week 4,week 12, week 24, week 48 and week 72 were assessed. Sera and PBMC were separated by Ficoll density gradient centrifugation and HCV RNA was isolated from both compartments. 5´UTR was amplified by RT-nested-PCR and the product was sequenced. The genotype was determined by phylogenetic relatedness as well as by using LiPA and RFLP considering eventual mixed infections. The sequence analysis was performed by Bioedit 5.0 and phylogeny based on distance methods. HCV viral load was carried out by Siemens VERSANT HCV RNA 3.0 Assay (bDNA) Results: Seven out of 14 patients achieved sustained virological response. The genotype (Gt) distribution was for Gt1: 4, Gt2: 1 and Gt3: 2. Those isolates from non respondent patients were Gt1. The genomic characterization of 5´UTR in each sample was not affected by the compartmentalization during the follow up being coincident in serum and PBMC. Regarding the therapy, HCV sequences did no exhibit a particular phylogenetic clustering according to the final response. Patients who experienced relapse of viremia at week 72 showed the HCV 5´UTR genomic sequence previously detected in both serum and PBMC. Conclusions: No effect of peg-IFN + ribavirin therapy was observed in the HCV 5´UTR genomic sequences in both serum and PBMC compartments in HIV-coinfected patients, regardless of the virological response. Despite the minor changes detected, no specific mutations of the 5´UTR appeared to be associated with responsiveness to IFN. Those HCV isolates belonging to Gt 1 from non-respondents were closely related to those found in respondents with regard to the nucleotide sequence of the 5´UTR. However, no selection of HCV 5´UTR variants occurred in non-respondents during the course of therapy.