Mixed Infections Unravel Novel HCV Inter-Genotypic Recombinant Forms within the Conserved IRES Region

Abstract

Hepatitis C virus (HCV) remains a significant global health challenge, affecting millions of people worldwide, with chronic infection a persistent threat. Despite the advent of direct-acting antivirals (DAAs), challenges in diagnosis and treatment remain, compounded by the lack of an effective vaccine. The HCV genome, characterized by high genetic variability, consists of eight distinct genotypes and over ninety subtypes, underscoring the complex dynamics of the virus within infected individuals. This study delves into the intriguing realm of HCV genetic diversity, specifically exploring the phenomenon of mixed infections and the subsequent detection of recombinant forms within the conserved internal ribosome entry site (IRES) region. Previous studies have identified recombination as a rare event in HCV. However, our findings challenge this notion by providing the first evidence of 1a/3a (and vice versa) inter-genotypic recombination within the conserved IRES region. Utilizing advanced sequencing methods, such as deep sequencing and molecular cloning, our study reveals mixed infections involving genotypes 1a and 3a. This comprehensive approach not only confirmed the presence of mixed infections, but also identified the existence of recombinant forms not previously seen in the IRES region. The recombinant sequences, although present as low-frequency variants, open new avenues for understanding HCV evolution and adaptation.

Keywords: HCV; IRES; NGS; inter-genotypic recombination; mixed infections.

Figure 1

Majority and low-frequency variants identified in the viral populations of IRES from samples with suspected mixed infections. IRES 1021 and 05, from Gt1a and 3a samples, respectively, are shown. Nucleotide positions that are different among genotypes 1a and 3a in the non-coding region of IRES are shown. Both low-frequency (1–15% of reads) and majority changes are represented: in blue, those specific to genotype 1a, in red, those specific to genotype 3a, and in black, those accounting for mutations (nucleotides different from those of the reference of one or the other genotype).

Figure 2

Alignment of IRES clones corresponding to sample 05 (genotype 3a) suspected to be recombinant sequences and a putative cross-over region. (A) Full IRES region is shown, including 5′NCR from position 41 onwards, and the core region up to position 18 (nucleotide 359 of the genome). Numbering is according to strain H77 (reference sequence for genotype 1a). All positions indicated with dots correspond to conserved nucleotides. Those highlighted in green indicate polymorphic sites between genotypes 1a and 3a within the 5′NCR or the core regions. Positions 98 to 174 (highlighted in grey) indicate a conserved sequence stretch, where the putative cross-over occurred (clone B12 changes from genotype 1a to 3a, whereas C4 and A33 change from 3a to 1a). The start codon is indicated in yellow. (B) HCV IRES secondary structure indicating the same features as in (A). IRES domains (II, III, IV) and subdomains are indicated with roman numbers (IIIa to IIIe).

Figure 3

Similarity plots and Bootscan analyses of potential HCV recombinants determined on the basis of the IRES complete sequence using Simplot version 3.5.1 [54] and Simplot ++ V1.3 [53] software. Simplot++ analyses (left panels) display the percentage of sequence similarity over the IRES sequences of the HCV genome (nucleotide positions 41 to 359 according to strain 1a H77 numbering). Bootscan analysis (right panels) shows the percentage of permuted trees (y-axis). This approach permits to observe levels of phylogenetic relatedness between a query sequence and a reference sequence in different genomic regions. Query sequences (shown on the upper part of the figure) correspond to (A) clone B12 (putative 1a/3a recombinant), (B) clone C4 (putative 3a/1a recombinant), and (C) clone A33 (putative 3a/1a recombinant). Reference and/or putative parental sequences are indicated in purple for genotype 1a (strain H77, NCBI Accession No. AF009606) and green for genotype 3a (strain NZL1, NCBI Accession No. D17763). All analyses were performed using a 100 bp sliding window and a 10 bp step size. For Simplot++ analyses, a GTR optimized model of nucleotide substitution was employed, whereas for Bootscan, a Kimura two-parameter model was used.