A natural intergenotypic recombinant of hepatitis C virus identified in St. Petersburg

Abstract

Hepatitis C virus (HCV) evolution is thought to proceed by mutations within the six genotypes. Here, we report on a viable spontaneous HCV recombinant and we show that recombination may play a role in the evolution of this virus. Previously, 149 HCV strains from St. Petersburg had been subtyped by limited sequencing within the NS5B region. In the present study, the core regions of 41 of these strains were sequenced to investigate the concordance of HCV genotyping for these two genomic regions. Two phylogenetically related HCV strains were found to belong to different subtypes, 2k and 1b, according to sequence analysis of the 5' untranslated region (5'UTR)-core and the NS5B regions, respectively. By sequencing of the E2-p7-NS2 region, the crossover point was mapped within the NS2 region, probably between positions 3175 and 3176 (according to the numbering system for strain pj6CF). Sequencing of the 5'UTR-core regions of four other HCV strains, phylogenetically related to the above-mentioned two strains (based on analysis within the NS5B region), revealed that these four strains were also recombinants. Since a nonrecombinant 2k strain was found in St. Petersburg, the recombination may have taken place there around a decade ago. Since the frequency of this recombinant is now high enough to allow the detection of the recombinant in a fraction of the city's population, it seems to be actively spreading there. The reported recombinant is tentatively designated RF1-2k/1b, in agreement with the nomenclature used for HIV recombinants. Recombination between HCV genotypes must now be considered in the classification, laboratory diagnosis, and treatment of HCV infection.

FIG. 1.

Strategy used for amplification and sequencing of overlapping PCR fragments. The nucleotide positions are numbered according to the numbering system for the subtype 2a strain pj6CF. Locations of sequencing primers are shown above the PCR products. Arrows indicate primer polarity.

FIG. 2.

Phylogenetic analysis of 102 HCV strains, performed on 280 nt within the NS5B region (A) and on 548 nt in the 5′UTR-core region (B) by the neighbor-joining method with GBV-B (accession no. NC 001655) as the outgroup. Sequences of 55 HCV isolates from GenBank are defined by the name of the relevant strain or clone (Table 1). Recombinant strains are indicated by the letter R and are shown in bold. Bootstrap values are given on the branches as percentages from 300 replicates. USA, United States of America.

FIG. 2.

Phylogenetic analysis of 102 HCV strains, performed on 280 nt within the NS5B region (A) and on 548 nt in the 5′UTR-core region (B) by the neighbor-joining method with GBV-B (accession no. NC 001655) as the outgroup. Sequences of 55 HCV isolates from GenBank are defined by the name of the relevant strain or clone (Table 1). Recombinant strains are indicated by the letter R and are shown in bold. Bootstrap values are given on the branches as percentages from 300 replicates. USA, United States of America.

FIG. 3.

(A) Similarity plots for two recombinant strains and reference strains based on seven genomic regions: 5′UTR to NS3 (a) and NS5B (b). The query sequence is the 2k/1b recombinant strain 796. An arrow indicates the crossover point at position 3175/3176. (B) Bootscan for 2k/1b recombinant strain 796 based on 5′UTR-to-NS3 region. The parental genotypes are represented by the genotype 1b strain 589, from St. Petersburg, and the genotype 2k strain Vat-96. The genotype 3a strain CB was used as the outgroup. The bootstrap values are based on 1,000 replicates. Nucleotide positions are numbered as described in the legend to Fig. 1.

FIG. 4.

Alignment of the nucleotide sequences within the NS2 region (A) and the amino acid sequences of the E2-p7 and p7-NS2 junctions (B) of reference strains k1-s2, 589, and Vat-96 and recombinant strains 796, 747, 687, 611, and 674. Arrows show the crossover point. Vertical broken lines show putative cleavage sites for the HCV polyprotein according to De Francesco (2). Nucleotide positions are numbered as for Fig. 1.

FIG. 4.

Alignment of the nucleotide sequences within the NS2 region (A) and the amino acid sequences of the E2-p7 and p7-NS2 junctions (B) of reference strains k1-s2, 589, and Vat-96 and recombinant strains 796, 747, 687, 611, and 674. Arrows show the crossover point. Vertical broken lines show putative cleavage sites for the HCV polyprotein according to De Francesco (2). Nucleotide positions are numbered as for Fig. 1.