Abstract / Summary
Background Oseltamivir is still considered to be the first-line antiviral treatment for severe seasonal influenza. However, resistance to neuraminidase (NA) inhibitors poses significant challenges to influenza management. Since the 2023/24 season, A(H1N1)pdm09 viruses carrying neuraminidase substitutions (NA-I223V and/or NA-S247N) have emerged in Germany, other EU/EEA countries, and some of the World Health Organization (WHO) regions. These viruses have been linked to reduced oseltamivir susceptibility. Methods A rapid RT-PCR/pyrosequencing assay was developed and validated to detect NA-I223X and NA-S247X substitutions. Subsequently, a total of 765 A(H1N1)pdm09 viruses detected in Germany during the 2024/25 and 2025/26 influenza seasons were characterized phenotypically using fluorometric NA inhibition testing and/or genotypically using next-generation sequencing and pyrosequencing. Results The prevalence of A(H1N1)pdm09 viruses bearing the NA-S247N substitution increased from 1.2% in the 2024/25 season to 56.8% in the 2025/26 season, accounting for up to 80% of analyzed viruses by March 2026. Viruses carrying NA-S247N exhibited higher oseltamivir IC 50 values than wild-type viruses. Combinations of NA-S247N with substitutions at position 223 demonstrated an additional increase. In viruses carrying the combined NA-S247N and NA-I223V/T substitutions oseltamivir showed reduced inhibition with increased IC 50 values of up to 17-fold compared to wild type viruses. Conclusions The accumulation of NA substitutions in A(H1N1)pdm09 influenza viruses, particularly NA-S247N in combination with NA-I223V/T, substantially reduces susceptibility to oseltamivir. The marked increase in NA-S247N prevalence suggests that this substitution is becoming persistent rather than transient. Targeted pyrosequencing is a practical tool for the early detection of emerging resistance-associated substitutions.