The efficacy of treatments using NS5A inhibitors for patients with chronic hepatitis C virus (HCV) infection can be affected by the presence of NS5A resistance-associated substitutions (RASs). We reviewed results from 35 clinical trials where patients with genotype 1 HCV infection received treatments that included ledipasvir-sofosbuvir to determine how prevalent NS5A RASs are in patients at baseline, and found that ledipasvir-specific RASs were present in 8-16% of patients prior to treatment and had a negative impact on treatment outcome in subset of patient groups, particularly treatment-experienced patients with genotype 1a HCV.

These data demonstrate a uniform susceptibility of subject-derived HCV to sofosbuvir, and also show that selection of sofosbuvir-resistant HCV is exceedingly rare and is associated with a significant reduction in viral fitness.

The development of resistance to direct-acting antivirals (DAAs) targeting the hepatitis C virus (HCV) can compromise therapy. However, mechanisms that determine prevalence and frequency of resistance-conferring mutations remain elusive. Here, we studied the fidelity of the HCV RNA-dependent RNA polymerase NS5B in an attempt to link the efficiency of mismatch formation with genotypic changes observed in vivo. Enzyme kinetic measurements revealed unexpectedly high error rates (approximately 10 −3 per site) for G∶U∕U∶G mismatches. The strong preference for G∶U∕U∶G mismatches over all other mistakes correlates with a mutational bias in favor of transitions over transversions. Deep sequencing of HCV RNA samples isolated from 20 treatment-naïve patients revealed an approximately 75-fold difference in frequencies of the two classes of mutations. A stochastic model based on these results suggests that the bias toward transitions can also affect the selection of resistance-conferring mutations. Collectively, the data provide strong evidence to suggest that the nature of the nucleotide change can contribute to the genetic barrier in the development of resistance to DAAs.A pproximately 170 million people worldwide are infected with hepatitis C virus (HCV) (1). Chronically infected individuals are at risk of developing severe liver disease, including cirrhosis and hepatocellular carcinoma (2). Although the infection can be cured with a combination of interferon-α and ribavirin, severe side effects and complicated treatment schedules can compromise therapy (3). Moreover, the response is particularly poor in patients infected with HCV genotype 1, which is the most prevalent genotype in North America and Europe (4).Several direct-acting antivirals (DAAs) targeting important viral and host factors are currently undergoing clinical evaluation. Drugs acting against the viral NS5B RNA-dependent RNA polymerase (RdRp) or NS3 protease are in advanced clinical trials, with the protease inhibitors (PIs) telaprevir and boceprevir recently gaining FDA approval. Combining interferon-α and ribavirin with either PI can produce increases in sustained virological response when compared with the combination of interferon-α and ribavirin without PI (5, 6). However, the emergence of resistance-conferring mutations can lead to treatment failure (7). Like other RNA viruses, HCV shows quasispecies-like characteristics (8). Accordingly, resistant variants are selected from a genetically highly diverse population. Resistance to PIs is rapidly selected in cell-based replicon systems and in vivo (9). Similar observations have been made with the various classes of nonnucleoside analogue inhibitors (NNIs) of NS5B (9). Moreover, mutations that decrease susceptibility to highly potent inhibitors of the RNA binding protein NS5A also emerge rapidly in the replicon system (10). In contrast, the barrier to the development of resistance to nucleoside analogue inhibitors (NIs) appears to be significantly higher. The 2′-C-methylated NIs were shown to select in v...

c GS-5885 is a novel hepatitis C virus (HCV) NS5A inhibitor. In a 3-day monotherapy study in treatment-naive genotype 1a (GT1a) and GT1b HCV-infected subjects, median viral load reductions ranged from 2.3 to 3.3 log 10 HCV RNA IU/ml across dosing cohorts (1, 3, 10, 30, or 90 mg once daily). Here, we report viral sequencing and phenotypic analysis of clinical isolates from this study. Detection of baseline NS5A amino acid substitutions at positions 28, 30, 31, or 93 in GT1a was associated with a reduced treatment response. In the GT1b cohort, Y93H was detected in 100% of subjects at day 4 or 14. In the Gt1a cohort, population sequencing detected NS5A resistance-associated mutations at day 4 or 14 for 3/10 subjects at the 1-mg dose and for all subjects dosed at >3 mg. A subset of mutants that confer a low level of reduced susceptibility to GS-5885 was not detected by population sequencing at the 30-and 90-mg doses. Subject-derived M28T, Q30R, L31M, and Y93C mutations all conferred >30-fold reductions in GS-5885 and daclatasvir susceptibilities in vitro. Site-directed NS5A mutants also showed reduced susceptibility to GS-5885. However, all NS5A mutants tested remained fully susceptible to other classes of direct-acting antivirals (DAAs), interferon alpha, and ribavirin. Importantly, the nonoverlapping resistance profile and high potency of GS-5885 support its further development with other direct-acting antivirals for the treatment of chronic HCV. (This study has been registered at ClinicalTrials.gov under registration number NCT01193478.) H epatitis C virus (HCV) infection is a global health issue, with approximately 170 million people infected worldwide (1).The standard of care (SOC) has been pegylated alpha interferon and ribavirin until the more recent approval of two NS3 protease inhibitors, telaprevir and boceprevir, for use in conjunction with pegylated alpha interferon and ribavirin (1-3). The last several years have seen a great expansion of new direct-acting antivirals (DAAs) in clinical development to augment or supplant treatment with pegylated alpha interferon and ribavirin. HCV nonstructural protein 5A (NS5A) has emerged as a viable and attractive viral target for small-molecule inhibition. Although there is no known enzymatic activity for NS5A, it is essential for viral replication (4). The first NS5A replication complex inhibitor to show efficacy in the clinic was daclatasvir (BMS-790052) (5). This compound elicited rapid and profound reductions in HCV RNA and validated NS5A as a clinical target. Sequence analysis of clinical isolates following daclatasvir monotherapy identified the main resistanceassociated mutations (RAMs) at NS5A amino acid positions 28, 30, 31, and 93 (6).In addition to daclatasvir, several other NS5A replication complex inhibitors have entered the clinic, including GS-5885, PPI-461, ABT-267, and GSK2336805 (7-10). In addition, preclinical data have recently been described for several other NS5A replication complex inhibitors, including EDP-239, IDX719, MK-4882, , highlighting ...

The incidence of late recurrent viremia was low. Distinguishing reinfection from virologic relapse has implications for determining true treatment efficiency and selecting optimal retreatment strategies.

No abstract

Deep-sequencing analysis confirmed that NS5B variants L159F and V321A emerged in a subset of patients treated with SOF at virologic failure. These variants had no impact on retreatment outcome with SOF, ribavirin, and pegylated interferon. Baseline L159F in genotype 1 did not affect the treatment outcome with LDV/SOF.

Remdesivir is a broad-spectrum antiviral nucleotide prodrug that has been clinically evaluated in Ebola virus patients and recently received emergency use authorization (EUA) for treatment of COVID-19. With approvals from the Federal Select Agent Program and the Centers for Disease Control and Prevention’s Institutional Biosecurity Board, we characterized the resistance profile of remdesivir by serially passaging Ebola virus under remdesivir selection; we generated lineages with low-level reduced susceptibility to remdesivir after 35 passages. We found that a single amino acid substitution, F548S, in the Ebola virus polymerase conferred low-level reduced susceptibility to remdesivir. The F548 residue is highly conserved in filoviruses but should be subject to specific surveillance among novel filoviruses, in newly emerging variants in ongoing outbreaks, and also in Ebola virus patients undergoing remdesivir therapy. Homology modeling suggests that the Ebola virus polymerase F548 residue lies in the F-motif of the polymerase active site, a region that was previously identified as susceptible to resistance mutations in coronaviruses. Our data suggest that molecular surveillance of this region of the polymerase in remdesivir-treated COVID-19 patients is also warranted.