In response to the rapid global spread of an antigenically novel A/H1N1 influenza virus in 2009, the World Heath Organization (WHO) recommended surveillance and monitoring for antiviral resistance of influenza viruses. We designed and evaluated pyrosequencing (PSQ)-based genotypic assays for high-throughput analysis of the susceptibility of pandemic A/H1N1 influenza viruses to neuraminidase (NA) inhibitors. A total of 1570 samples circulating in Germany between April 2009 and April 2010 were tested for determination of molecular markers of resistance to the NA inhibitors oseltamivir and zanamivir, and 635 of them were evaluated by phenotypic fluorescence-based assay with MUNANA substrate. Eight (0.5%) viruses were resistant to oseltamivir due to the H274Y NA substitution (N2 numbering). Six of these oseltamivir-resistant cases were treatment-related; four of them were selected in immunocompromised patients, two in patients suffered from chronic diseases. The two remaining oseltamivir-resistant viruses seem to have evolved in the absence of drug treatment and were isolated from immunocompetent healthy patients. All tested A/H1N1 pandemic viruses were sensitive to zanamivir. In addition, analysis of 1011 pandemic A/H1N1 virus samples by a PSQ-based assay according to the WHO protocol revealed the presence of mutation S31N in the M2 protein that conferred resistance to M2 ion channel inhibitors. Our data demonstrate a low incidence of oseltamivir-resistant pandemic A/H1N1 influenza variants isolated under drug selection pressure as well as community-acquired or naturally evolving viruses.
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