Analysis of the PKR-eIF2alpha phosphorylation homology domain (PePHD) of hepatitis C virus genotype 1 in HIV-coinfected patients by ultra-deep pyrosequencing and its relationship to responses to pegylated interferon-ribavirin treatment

Abstract

Chronic coinfection with hepatitis C virus (HCV) and human immunodeficiency virus (HIV) is among the greatest challenges facing public health worldwide. In this population, the response to hepatitis C therapy by treatment with pegylated interferon plus ribavirin (PEG-IFN+RBV) is lower than in HCV-monoinfected patients, particularly in those infected by HCV genotype 1. A PKR/eIF-2α phosphorylation homology domain (PePHD) within the E2 protein has been found to interact with PKR and inhibit PKR in vitro, suggesting a possible mechanism for HCV to evade the antiviral effects of IFN. The aim of this work was to analyze the amino acid conservation in the HCV-E2-PePHD and quasispecies diversity among HCV-HIV-coinfected patients exhibiting sustained virological response, non-response, or partial response with viral relapse to PEG-IFN+RBV by ultra-deep pyrosequencing. For this purpose, HCV-E2-PePHD PCR products were generated and sequenced directly for four patients with a sustained response, seven patients with no virological response, and four patients with viral relapse before and after treatment with PEG-IFN+RBV. HCV-E2-PePHD amino acid sequences were obtained for isolates from serum collected before and during treatment (24 h, 4 weeks, and 12 weeks). Quasispecies analysis of the HCV-E2-PePHD and flanking genomic regions was performed using 454/Roche pyrosequencing, analyzing 39,364 sequence reads in total. The HCV-E2-PePHD sequence at the amino acid and nucleotide level was highly conserved among HCV genotype 1 strains, irrespective of the PEG-IFN+RBV response. This high degree of amino acid conservation and sporadic mutations in the HCV-E2-PePHD domain do not appear to be associated with treatment outcome. The HCV-E2-PePHD sequence before or during treatment cannot be used to predict reliably the outcome of treatment in patients coinfected with HCV genotype 1 and HIV.

Quasispecies variant of pre-S/S gene in HBV-related hepatocellular carcinoma with HBs antigen positive and occult infection

“…The aim of this study was to reflect the quasispecies variants in relation to hepatocarcinogenesis; therefore, we analysed variants detected in > 1% of the total viral population. A similar cut-off was applied in previous studies [ 20 , 21 ]. In addition, > 20% of the total viral population is possible to detect the conventional sequencing method.…”

Kinetics of hepatitis B surface antigen quasispecies during REP 2139‐Ca therapy in HBeAg‐positive chronic HBV infection

“…To reflect the nature of quasispecies variants in the MHR in vivo as accurately as possible in relation to clinical diagnosis by distinguishing the true quasispecies variants from sequencing errors, we analysed the variants detected in ≥1% of the viral population. A similar cut‐off was applied in previous studies …”

Genetic Variability of Hepatitis C Virus (HCV) 5’ Untranslated Region in HIV/HCV Coinfected Patients Treated with Pegylated Interferon and Ribavirin

“…Furthermore, both in HCV/HIV coinfection and HCV monoinfection, treatment outcome was related to HVR1 quasispecies complexity before treatment [ 16 ]. However, some other studies did not find such a correlation [ 28 ].…”