Evidence for separation of HCV subtype 1a into two distinct clades

Abstract

The nucleotide sequence diversity present among hepatitis C virus (HCV) isolates allows rapid adjustment to exterior forces including host immunity and drug therapy. This viral response reflects a combination of a high rate of replication together with an error-prone RNA-dependent RNA polymerase, providing for the selection and proliferation of the viruses with the highest fitness. We examined HCV subtype 1a whole-genome sequences to identify positions contributing to genotypic and phenotypic diversity. Phylogenetic tree reconstructions showed two distinct clades existing within the 1a subtype with each clade having a star-like tree topology and lacking definite correlation between time or place of isolation and phylogeny. Identification of significant phylogenetically informative sites at the nucleotide level revealed positions not only contributing to clade differentiation, but which are located at or proximal to codons associated with resistance to protease inhibitors (NS3 Q41) or polymerase inhibitors (NS5B S368). Synonymous/nonsynonymous substitution mutation analyses revealed that the majority of nucleotide mutations yielded synonymous amino acids, indicating the presence of purifying selection pressure across the polyprotein with pockets of positive selection also being detected. Despite evidence for divergence at several loci, certain 1a characteristics were preserved including the length of the alternative reading frame/F protein (ARF/F) gene, and a subtype 1a-specific phosphorylation site in NS5A (S349). Our analysis suggests that there may be strain-specific differences in the development of antiviral resistance to viruses infecting patients who are dependent on the genetic variation separating these two clades.

Figure 1. Phylogenetic trees showing evolutionary relationships between HCV isolates

Maximum likelihood phylogenetic trees are presented that demonstrate the separation of the HCV 1a isolates into two distinct clades. An unrooted maximum likelihood phylogenetic tree of 94 representative HCV 1a whole genome sequences together with sequences from (A) all other genotypes and subtypes, or (B) the same representative subtype 1a sequences with outgroup sequences from subtypes 1b and 1c. Sequence names were removed for clarity. Maximum likelihood phylogenetic reconstructions containing 240 HCV 1a sequences and are shown as either (C) unrooted, without sequence names for clarity, or (D) rooted trees.

Figure 2. Genomic Representation Showing Clade-informative Sites with Potential Public Health Impact

The translated polyprotein is depicted with the protease cleavage sites marked by black vertical lines and are shown with nucleotide positions in black referencing the first nucleotide of each mature protein. The mature structural proteins are labeled in red while the nonstructural proteins are labeled in black letters. Cyan symbols indicate the two regions displaying positive selection pressures as measured by SNAP. The amino acid positions corresponding to the positive selection regions are specified in cyan letters above the polyprotein depiction. Magenta symbols represent the fourteen regions containing high concentrations (or “hotspots”) of phylogenetically-informative sites. Stacked symbols indicate overlapping “hotspots”. The individual nucleotide positions (in strain H77) are identified in magenta letters below the polyprotein representation.

Figure 3. Phylogenetic Tree Showing 447 HCV 1a Sequences along with Virahep-C Clinical response data

A maximum likelihood phylogenetic tree reconstruction containing all 447 non-chimeric HCV subtype 1a whole genome sequences that were available in November 2008. Both clades are labeled in black. Sequence names lacking clinical data were removed for clarity. Virahep-C Study Group clinical drug resistance data was also included with isolates being sequenced both before (pre) and after (post) treatment and classified as either responders (R) or nonresponders (N). Sequences were color-coded as follows: cyan = pre-N, blue = post-N, Red = pre-R, and Orange = post-R. Of particular note is the dominant quasispecies isolated from patient 6030 which changed from clade 1 to clade 2 either during or after treatment.