Resistance analysis of genotype 3 hepatitis C virus indicates subtypes inherently resistant to nonstructural protein 5A inhibitors

Abstract

Hepatitis C virus (HCV) genotype (gt) 3 is highly prevalent globally, with non-gt3a subtypes common in Southeast Asia. Resistance-associated substitutions (RASs) have been shown to play a role in treatment failure. However, the role of RASs in gt3 is not well understood. We report the prevalence of RASs in a cohort of direct-acting antiviral treatment-naive, gt3-infected patients, including those with rarer subtypes, and evaluate the effect of these RASs on direct-acting antivirals in vitro. Baseline samples from 496 gt3 patients enrolled in the BOSON clinical trial were analyzed by next-generation sequencing after probe-based enrichment for HCV. Whole viral genomes were analyzed for the presence of RASs to approved direct-acting antivirals. The resistance phenotype of RASs in combination with daclatasvir, velpatasvir, pibrentasvir, elbasvir, and sofosbuvir was measured using the S52 ΔN gt3a replicon model. The nonstructural protein 5A A30K and Y93H substitutions were the most common at 8.9% (n = 44) and 12.3% (n = 61), respectively, and showed a 10-fold and 11-fold increase in 50% effect concentration for daclatasvir compared to the unmodified replicon. Paired RASs (A30K + L31M and A30K + Y93H) were identified in 18 patients (9 of each pair); these combinations were shown to be highly resistant to daclatasvir, velpatasvir, elbasvir, and pibrentasvir. The A30K + L31M combination was found in all gt3b and gt3g samples. Conclusion: Our study reveals high frequencies of RASs to nonstructural protein 5A inhibitors in gt3 HCV; the paired A30K + L31M substitutions occur in all patients with gt3b and gt3g virus, and in vitro analysis suggests that these subtypes may be inherently resistant to all approved nonstructural protein 5A inhibitors for gt3 HCV. (Hepatology 2018).

Figure 1

Summary of daclatasvir and sofosbuvir RASs identified in the literature. Key RAS positions for daclatasvir and sofosbuvir are shown (not to scale) along the NS5A domain 1 and NS5B (polymerase) proteins, respectively, labeled according to the H77 gt1 HCV reference protein sequence. The WT amino acid for gt3 is shown above. and the relevant RASs are indicated below. Data taken from Nelson et al.,7 Leroy V et al.,21 Lontok E et al.,25 and Itakura J et al.39 Abbreviation: aa, amino acid.

Figure 2

Prevalence of RASs to daclatasvir and sofosbuvir. (A) The prevalence of individual daclatasvir and sofosbuvir RASs, categorized by frequency within viral quasi‐species. (B) Histograms with a bin size of 10% showing the distribution of substitution frequency within individual patient viral quasi‐species for the daclatasvir RASs A30K, P58S, and Y93H.

Figure 3

(A) Replication capacity of S52 ΔN replicon carrying candidate RASs. The top dashed and bottom dotted lines indicate the replication capacity of the unmodified WT S52 ΔN replicon and the pSGR‐JFH1/GND replicon, respectively. (B) Fold changes in resistance (based on EC50 measurements) for daclatasvir or sofosbuvir against S52 ΔN replicons carrying candidate RASs. The dotted line indicates WT susceptibility; the dashed line indicates a 5‐fold increase in EC50 when compared to the WT S52 ΔN replicon. Error bars show standard error of the mean calculated from at least two experiments.

Figure 4

Prevalence of RAS combinations to daclatasvir. Each column represents a RAS site, and each row is a patient. The frequency of the substitution within the viral quasi‐species is shown in each block.

Figure 5

(A) Replication capacity of S52 ΔN replicon carrying candidate NS5A RAS combinations. The top dashed and bottom dotted lines indicate the replication capacity of the unmodified WT S52 ΔN replicon and the pSGR‐JFH1/GND replicon, respectively. (B) EC50 fold change for daclatasvir, velpatasvir, elbasvir, or pibrentasvir against S52 ΔN replicon carrying candidate RAS combinations. The dotted line indicates no change in EC50 compared to WT; the dashed line indicates a 5‐fold increase in EC50 when compared to the WT S52 ΔN replicon.

Figure 6

The distribution of RASs within gt3 subtypes. Each column represents an individual RAS, and each row is a patient; each block is colored according to the gt3 subtype of the virus.

Figure 7

Whole‐genome phylogeny of gt3 subtypes labeled with NS5A position 30 and 31 amino acids. A neighbor‐joining, midpoint rooted phylogenetic tree was estimated using the whole‐genome sequence of the gt3b, gt3g, and gt3i subtypes from our cohort and the gt3a, gt3d, gt3e, gt3k, gt3h, gt3i, gt3g, and gt3b sequences downloaded from the National Center for Biotechnology Information database and annotated with the NS5A position 30 and 31 amino acids. The A30K + L31M RASs are in bold and italics.