A9 Deep sequencing analysis to investigate the importance of within host genetic diversity and evolution of influenza A viruses for the development of resistance against neuraminidase inhibitors

Currently approved neuraminidase inhibitors (NAI) for the treatment of influenza A virus infections are prone to induce viral drug resistance development due to the rapid evolutionary dynamics of the neuraminidase (NA) and hemagglutinin (HA) proteins. Both HA and NA proteins are subject to antigenic drift and the epistatic interactions within and between these proteins can lead to genetic diversity that enables the virus to easily develop resistant mutations under selective pressure in a host. To study NAI resistance and clinical outcome, the global observational Influenza Resistance Information Study (IRIS; NCT00884117) was conducted. Patients that were clinically diagnosed with influenza were enrolled in the study. Nasal and throat swabs taken at baseline and on days 3, 6, and 10 were assessed by semi-quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) to determine the influenza virus type and subtype. NAI resistance was initially determined by mutation specific RT-PCR, Sanger sequencing and phenotypic susceptibility analysis. Genetic resistance mutations to oseltamivir were detected in 61 patients (43 H1N1pdm [H275Y] and 18 H3N2 [R292K]) by mutation specific PCR. Subsequently, samples of 43 patients were subjected to deep sequencing analysis to characterize both the betweenand the within-host diversity and the evolutionary process of the HA and NA proteins of infected patients. The NAI resistance mutations (H275Y and R292K) in the NA protein of the H1N1pdm and H3N2 viruses were either detected in day 3 samples or at later time point. Additionally, viruses in several individuals had mutations that were located across the whole HA and NA proteins. Some low frequency mutations such as D114N, S200P, and D239N, that were located in the antigenic sites or near the receptor binding site of the HA protein, became fixed in the later time point viral samples. Also, some mutations in HA may have occurred in concert with the resistance mutations in NA. Further genetic analyses and phylogeny should provide further insights in the emergence of mutations in individual hosts and the larger population.