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.

甲型流感病毒始终处于持续进化的过程中。病毒感染周期中的关键环节，如宿主细胞表面的唾液酸（sialic acid, SA）受体结合，既可能促进新型病毒变异株的出现，也可能对其产生阻碍。此前我们针对H9N2亚型流感病毒变异株开展了相对适配性评估，这些变异株仅在病毒表面蛋白血凝素（hemagglutinin, HA）的第216位氨基酸残基位点存在差异，实验宿主为日本鹌鹑。该位点已知可调控唾液酸受体的识别过程。我们此前的研究从鹌鹑传播组中获取了一批极具价值的纵向样本，该组的接种物包含多种HA 216位点变异株的混合种群。本研究借助这批样本，探究了直接接种鹌鹑体内以及接种组与易感接触鹌鹑之间的病毒种群进化动态。研究发现，正选择驱动HA基因第216位点的变异株被固定，进而塑造了HA基因的进化轨迹。值得注意的是，我们同时发现克隆干扰现象会打断这一进化动态。全基因组分析进一步证实，宿主体内及宿主间均存在正选择作用。正选择推动了接种组与接触组鹌鹑体内非HA片段的变异株被固定。尤为关键的是，传播瓶颈受到HA 216位点分子特征的调控。综上，本研究证实选择作用深刻塑造了鹌鹑体内及鹌鹑间的病毒进化动态。上述研究结果佐证了这一观点：家禽可使选择过程高效发挥作用，进而推动甲型流感病毒实现快速进化。