Genotypic and phenotypic susceptibility of emerging avian influenza A viruses to neuraminidase and cap-dependent endonuclease inhibitors

Abstract

Avian influenza outbreaks, including ones caused by highly pathogenic A(H5N1) clade 2.3.4.4b viruses, have devastated animal populations and remain a threat to humans. Risk elements assessed for emerging influenza viruses include their susceptibility to approved antivirals. Here, we screened >20,000 neuraminidase (NA) or polymerase acidic (PA) protein sequences of potentially pandemic A(H5Nx), A(H7Nx), and A(H9N2) viruses that circulated globally in 2010-2023. The frequencies of NA or PA substitutions associated with reduced inhibition (RI) or highly reduced inhibition (HRI) by NA inhibitors (NAIs) (oseltamivir, zanamivir) or a cap-dependent endonuclease inhibitor (baloxavir) were low: 0.60% (137/22,713) and 0.62% (126/20,347), respectively. All tested subtypes were susceptible to NAIs and baloxavir at sub-nanomolar concentrations. A(H9N2) viruses were the most susceptible to oseltamivir, with IC₅₀s 3- to 4-fold lower than for other subtypes (median IC₅₀: 0.18 nM; n = 22). NA-I222M conferred RI of A(H5N1) viruses by oseltamivir (with a 26-fold IC₅₀ increase), but NA-S246N did not reduce inhibition. PA-E23G, PA-K34R, PA-I38M/T, and the previously unreported PA-A36T caused RI by baloxavir in all subtypes tested. Avian A(H9N2) viruses endemic in Egyptian poultry predominantly acquired PA-I38V, which causes only a <3-fold decrease in the baloxavir EC₅₀ and fails to meet the RI criteria. PA-E199A/D in A(H7Nx) and A(H9N2) viruses caused a 2- to 4-fold decrease in EC₅₀ (close to the borderline for RI) and should be closely monitored. Our data indicate antiviral susceptibility is high among avian influenza A viruses with pandemic potential and present novel markers of resistance to existing antiviral interventions.

Keywords: Antiviral susceptibility; Avian influenza viruses; Baloxavir; Neuraminidase inhibitors; Oseltamivir; Zanamivir.

Figure 1.

Frequencies of avian influenza A viruses carrying PA-A37S and PA-A37 worldwide (A) and within the Western Pacific region (B). The accumulation of PA-I38V among endemic A(H9N2) viruses in Egypt and the Eastern Mediterranean region in 2011–2021 (C), and the total ratio of A(H9N2) viruses with PA-I38V (D).

Figure 2.

NAI IC₅₀ fold-changes for oseltamivir (A) and zanamivir (B), and CENI EC₅₀ fold-changes (C) in avian influenza A viruses with designated NA and PA substitutions. NI, normal inhibition; RI/HRI, reduced/highly reduced inhibition. NAI RI, ≥10-fold; HRI, ≥100-fold; CENI RI, ≥3-fold.