Serendipitous identification of natural intergenotypic recombinants of hepatitis C in Ireland

Abstract

Background: Recombination between hepatitis C single stranded RNA viruses is a rare event. Natural viable intragenotypic and intergenotypic recombinants between 1b-1a, 1a-1c and 2k-1b, 2i-6p, respectively, have been reported. Diagnostically recombinants represent an intriguing challenge. Hepatitis C genotype is defined by interrogation of the sequence composition of the 5' untranslated region [5'UTR]. Occasionally, ambiguous specimens require further investigation of the genome, usually by interrogation of the NS5B region. The original purpose of this study was to confirm the existence of a suspected mixed genotype infection of genotypes 2 and 4 by clonal analysis at the NS5B region of the genome in two specimens from two separate individuals. This initial identification of genotype was based on analysis of the 5'UTR of the genome by reverse line probe hybridisation [RLPH].

Results: The original diagnosis of a mixed genotype infection was not confirmed by clonal analysis of the NS5B region of the genome. The phylogenetic analysis indicated that both specimens were natural intergenotypic recombinant forms of HCV. The recombination was between genotypes 2k and 1b for both specimens. The recombination break point was identified as occurring within the NS2 region of the genome.

Conclusion: The viral recombinants identified here resemble the recombinant form originally identified in Russia. The RLPH pattern observed in this study may be a signature indicative of this particular type of intergenotype recombinant of hepatitis C meriting clonal analysis of NS2.

Figure 1

The 5'UTR amplicon used in the RLPH assay was generated using the Roche Diagnostics COBAS AMPLICOR HCV MONITOR kit. Hybridization and subsequent chromogenic development were performed as per AutoLipa-Versant protocol. Lane 1, HC9A98987; Lane 2, negative control; M, Interpretation chart; CONJ CTRL, conjugate control; AMP CTRL, PCR amplification control. The HCV specific banding pattern observed was 5, 6 (weak), 9–11, 16. A banding pattern of 5, 9–11 is indicative of a genotype 2a/2c infection, while banding at positions 6 and 16 are usually indicative of a genotype 4a infection and banding at positions 5 and 16 can be indicative of a genotype 4e. The nitrocellulose strip most proximal to the interpretation chart is the negative control.

Figure 2

Phylogenetic tree generated by NEIGHBOR program in the PHYLIP package for NS5B and core amino acid sequences. Genetic distance was calculated with PRODIST program from the PHYLIP package based on Kimura's distance. A bootstrap analysis using 100 bootstrap replicates was performed to assess the reliability of each branch point. Bootstrap values greater than 70% are shown. The corresponding genetic distances are also shown. The arc signifies the sequences generated from HC9A98987. The dashed line reflects a genetic distance of 0.29 between AY587845 (2k/1b) and AB031663 (2k).

Figure 3

Amino acid alignment of sequences from sera (a) HC9A98987 [DQ839386, DQ839390] and (b) HC9A99966 [DQ904446], and reference strain [AB031663] (2k), [AY587845] (2k/1b), [D50485] (1b) and [AF313916] (1b) from the NS2 region. The recombination break point as identified by Kalinina is marked by the arrowhead. The directionality of the genotypes is indicated by the arrow, with subscripted genotype/subtype.