Intra- and inter-host evolution of H9N2 influenza A virus in Japanese quail

Influenza A viruses are constantly evolving. Crucial steps in the infection cycle, such as sialic acid (SA) receptor binding on the host cell surface, can either promote or hamper the emergence of new variants. We previously assessed the relative fitness in Japanese quail of H9N2 variant viruses differing at a single amino acid position, residue 216 in the hemagglutinin (HA) viral surface protein. This site is known to modulate SA recognition. Our prior study generated a valuable set of longitudinal samples from quail transmission groups where the inoculum comprised different mixed populations of HA 216 variant viruses. Here, we leveraged these samples to examine the evolutionary dynamics of viral populations within directly inoculated quails and between inoculated and naive contact quails. We found that positive selection led to fixation at HA 216 shaping HA gene evolution. Interestingly, we also found that clonal interference interrupted this dynamic. Analysis of the whole genome revealed further evidence of positive selection acting both, within and between hosts. Positive selection drove fixation of variants in non-HA segments within inoculated and contact quails. Importantly, transmission bottlenecks were modulated by the molecular signature at HA 216. Overall, we show that selection strongly shaped the evolutionary dynamics within and between quails. These findings support the notion that poultry allows for selective processes to act effectively and therefore for rapid evolution of influenza A viruses.