Prevalence of amino acid mutations in hepatitis C virus core and NS5B regions among Venezuelan viral isolates and comparison with worldwide isolates

Abstract

Background: Recent reports show that R70Q and L/C91M amino acid substitutions in the core from different hepatitis C virus (HCV) genotypes have been associated with variable responses to interferon (IFN) and ribavirin (RBV) therapy, as well to an increase of hepatocellular carcinoma (HCC) risk, liver steatosis and insulin resistance (IR). Mutations in NS5B have also been associated to IFN, RBV, nucleoside and non-nucleoside inhibitors drug resistance. The prevalence of these mutations was studied in HCV RNA samples from chronically HCV-infected drug-naïve patients.

Methods: After amplification of core and NS5B region by nested-PCR, 12 substitutions were analyzed in 266 Venezuelan HCV isolates subtype 1a, 1b, 2a, 2c, 2b, 2j (a subtype frequently found in Venezuela) and 3a (n = 127 and n = 228 for core and NS5B respectively), and compared to isolates from other countries (n = 355 and n = 646 for core and NS5B respectively).

Results: R70Q and L/C91M core substitutions were present exclusively in HCV G1b. Both substitutions were more frequent in American isolates compared to Asian ones (69% versus 26%, p < 0.001 and 75% versus 45%, p < 0.001 respectively). In Venezuelan isolates NS5B D310N substitution was detected mainly in G3a (100%) and G1a (13%), this later with a significantly higher prevalence than in Brazilian isolates (p = 0.03). The NS5B mutations related to IFN/RBV treatment D244N was mainly found in G3a, and Q309R was present in all genotypes, except G2. Resistance to new NS5B inhibitors (C316N) was only detected in 18% of G1b, with a significantly lower prevalence than in Asian isolates, where this polymorphism was surprisingly frequent (p < 0.001).

Conclusions: Genotypical, geographical and regional differences were found in the prevalence of substitutions in HCV core and NS5B proteins. The substitutions found in the Venezuelan G2j type were similar to that found in G2a and G2c isolates. Our results suggest a high prevalence of the R70Q and L/C91M mutations of core protein for G1b and D310N substitution of NS5B protein for the G3a. C316N polymorphism related with resistance to new NS5B inhibitors was only found in G1b. Some of these mutations could be associated with a worse prognosis of the disease in HCV infected patients.

Figure 1

Phylogenetic analysis of the NS5B partial genomic region (256 nt, 8302–8556) of HCV G1 strains. Genetic distance was estimated by Kimura 2 parameters and phylogenetic tree was constructed with the Neighbor joining method. Bootstrap values over 50% are shown in the tree. Isolates are designated by their GenBank accession number, followed by their country of origin, except for Venezuelan ones, which are numbered and preceded by a C. G1a sequences harboring D310N substitution were included, and a similar number of sequences not harboring these mutations, from Venezuela (n = 23), Brazil (BRA, n = 8) and USA (n = 6). The same criteria of selection was applied to G1b sequences, respect to C316N substitution, from Venezuela (n = 23), Brazil (n = 6) and Asia (n = 7 from HK: Hong Kong, JAP: Japan and CHI: China). Sequences grouped in clades containing D310N or C316N substitutions are shown in brackets. Percent identities over 98% are shown for these clades.