A Sequence‐Based Update on Amino Acid Substitutions in Influenza Polymerase Acidic Protein in Europe That Alter Baloxavir Susceptibility From 2009 to 2025

BACKGROUND: Influenza causes 650,000 deaths, 3-5 million hospitalizations, and 1 billion cases worldwide each year. There is a limited repertoire of antivirals available to tackle this burden, highlighting the risk of developing resistance to current drugs. An effort to develop new influenza antivirals has been made, leading to the approval of baloxavir. Some mutations leading to reduced susceptibility to baloxavir have been reported, but information about their epidemiology is scarce. Thus, we aim to evaluate the prevalence of substitutions in the polymerase acidic (PA) subunit associated with baloxavir susceptibility over 16 epidemiological seasons in Europe. METHODS: We evaluated 87,266 sequences collected from 2009 to 2025 in Europe, annotated the mutations, and identified all known amino acid substitutions related to changes in baloxavir susceptibility to assess their prevalence. RESULTS: A total of 149 (0.2%) sequences with at least one substitution were identified, 81 (0.09%) exhibiting reduced susceptibility. Overall, 17 different substitutions were detected, located both inside and outside baloxavir binding site. The number of substitutions detected ranged from 0 to 15 per season, with E23K, E23R, K34R, A36V, I38F, I38L, and E120D emerging after baloxavir approval in Europe. Double and triple concurrent substitutions were also identified. CONCLUSION: While the prevalence of sequences with substitutions that alter baloxavir susceptibility remains stable, the number of circulating substitutions increases over time. This implies the emergence of amino acid substitutions that did not circulate before and the concurrence of double and triple substitutions that might synergize their individual effects. These results highlight the need for virological surveillance and novel antiviral treatments.