Pre-existing or rapidly emerging resistance of influenza viruses to approved
antivirals makes the development of novel
therapeutics to mitigate seasonal
influenza and improve preparedness against future
influenza pandemics an urgent priority. We have recently identified the chain-terminating broad-spectrum
nucleoside analog clinical candidate
4'-fluorouridine (4'-FlU) and demonstrated oral efficacy against seasonal, pandemic, and highly pathogenic
avian influenza viruses in the mouse and ferret model. Here, we have resistance-profiled 4'-FlU against a pandemic A/CA/07/2009 (H1N1) (CA09). In vitro viral adaptation yielded six independently generated escape lineages with distinct mutations that mediated moderate resistance to 4'-FlU in the genetically controlled background of recombinant CA09 (recCA09). Mutations adhered to three distinct structural clusters that are all predicted to affect the geometry of the active site of the
viral RNA-dependent
RNA polymerase (RdRP) complex for phosphodiester bond formation. Escape could be achieved through an individual causal mutation, a combination of mutations acting additively, or mutations functioning synergistically. Fitness of all resistant variants was impaired in cell culture, and all were attenuated in the mouse model. Oral 4'-FlU administered at lowest-efficacious (2 mg/kg) or elevated (10 mg/kg) dose overcame moderate resistance when mice were inoculated with 10 LD 50 units of parental or resistant recCA09, demonstrated by significantly reduced virus load and complete survival. In the ferret model, invasion of the lower respiratory tract by variants representing four adaptation lineages was impaired. Resistant variants were either transmission-incompetent, or spread to untreated sentinels was fully blocked by therapeutic treatment of source animals with 4'-FlU.
Author Summary: Reduced sensitivity to FDA-approved
influenza drugs is a major obstacle to effective
antiviral therapy. We have previously demonstrated oral efficacy of a novel clinical candidate
drug, 4'-FlU, against seasonal, pandemic, and highly pathogenic
avian influenza viruses. In this study, we have determined possible routes of influenza virus escape from 4'-FlU and addressed whether resistance imposes a viral fitness penalty, affecting pathogenicity or ability to transmit. We identified three distinct clusters of mutations that lead to moderately reduced viral sensitivity to the
drug. Testing of resistant variants against two chemically unrelated
nucleoside analog inhibitors of influenza virus, conditionally approved
favipiravir and the broad-spectrum SARS-CoV-2
drug molnupiravir, revealed cross-resistance of one cluster with
favipiravir, whereas no viral escape from
molnupiravir was noted. We found that the resistant variants are severely attenuated in mice, impaired in their ability to invade the lower respiratory tract and cause
viral pneumonia in ferrets, and transmission-defective or compromised. We could fully mitigate lethal
infection of mice with the resistant variants with standard or 5-fold elevated oral dose of 4'-FlU. These results demonstrate that partial viral escape from 4'-FlU is feasible in principle, but escape mutation clusters are unlikely to reach clinical significance or persist in circulating influenza virus strains.