Separation of Hepatitis C genotype 4a into IgG-depleted and IgG-enriched fractions reveals a unique quasispecies profile

Abstract

Background: Hepatitis C virus (HCV) circulates in an infected individual as a heterogeneous mixture of closely related viruses called quasispecies. The E1/E2 region of the HCV genome is hypervariable (HVR1) and is targeted by the humoral immune system. Hepatitis C virions are found in two forms: antibody associated or antibody free. The objective of this study was to investigate if separation of Hepatitis C virions into antibody enriched and antibody depleted fractions segregates quasispecies populations into distinctive swarms.

Results: A HCV genotype 4a specimen was fractionated into IgG-depleted and IgG-enriched fractions by use of Albumin/IgG depletion spin column. Clonal analysis of these two fractions was performed and then compared to an unfractionated sample. Following sequence analysis it was evident that the antibody depleted fraction was significantly more heterogeneous than the antibody enriched fraction, revealing a unique quasispecies profile. An in-frame 3 nt insertion was observed in 26% of clones in the unfractionated population and in 64% of clones in the IgG-depleted fraction. In addition, an in-frame 3 nt indel event was observed in 10% of clones in the unfractionated population and in 9% of clones in the IgG-depleted fraction. Neither of these latter events, which are rare occurrences in genotype 4a, was identified in the IgG-enriched fraction.

Conclusion: In conclusion, the homogeneity of the IgG-enriched species is postulated to represent a sequence that was strongly recognised by the humoral immune system at the time the sample was obtained. The heterogeneous nature of the IgG-depleted fraction is discussed in the context of humoral escape.

Figure 1

Amino acid alignment of the individual species obtained from each fraction. (A) unfractionated serum, (B) IgG-depleted fraction and (C) IgG-enriched fraction. The black box encloses the 27 amino acid sequence of the HVR1. The asterisk (*) indicate the position of the histidine insertion which is shown in bold (H). The (Δ) indicate the position of the deletion event when present. The closed circles indicate consensus sequence across all species.

Figure 2

Quasispecies complexity within E1/E2 region in the unfractionated serum, the IgG-depleted fraction and the IgG-enriched fraction:(A) at the nucleotide level and (B) at the amino acid level. The vertical bars indicate the proportion of viral variants within each sample. Within the vertical bars, each variant is represented by a different colour. The same colour indicates identity between viral strains present in different fractions. The accession numbers and corresponding viral variant colour code of each strain are shown in the legend box where font colour corresponds to each fraction as follows: red for the unfractionated serum, blue for the IgG-depleted fraction, green for the IgG-enriched fraction [EU482135] was also the subdominant species of the unfractionated fraction), and black for quasispecies present in both unfractionated and IgG-depleted fractions. An accession number followed by an asterisk (*) indicates the presence of an insertion event within the sequence or 2 asterisks (**) indicates the presence of an indel event.

Figure 3

Phylogenetic trees of all viral E1/E2 amino acid sequences encompassing the HVR1 within each fraction. The phylogenetic tree was constructed with the Treecon software and rooted with the unique IgG-enriched species [EU482135]. The genetic distance is shown as a scale bar. A bootstrap analysis using 100 bootstrap replicates was performed to assess the reliability of each branch point. Bootstrap scores are given as percentage value. The values greater than 70% are annotated at appropriate branches. Different font colours are used to represent the different fractions: red for the unfractionated serum species, blue for the IgG-depleted species and green for the IgG-enriched species, [EU482135] was also the subdominant species of the unfractionated fraction. The species with the insertion event are represented in the figure by an asterisk (*) and the species with the indel event are represented by 2 asterisks (**).