Influenza A virus resistance to 4-fluorouridine coincides with viral attenuation in vitro and in vivo

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 LD50 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 SummaryReduced 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.