RNA viruses accumulate mutations to rapidly adapt to environmental changes. Enterovirus A71 (EV-A71) causes various clinical manifestations with occasional severe neurological complications. However, the mechanism by which EV-A71 evolves within the human body is unclear. Utilizing deep sequencing and haplotype analyses of viruses from various tissues of an autopsy patient, we sought to define the evolutionary pathway by which enterovirus A71 evolves fitness for invading the central nervous system in humans. Broad mutant spectra with divergent mutations were observed at the initial infection sites in the respiratory and digestive systems. After viral invasion, we identified a haplotype switch and dominant haplotype, with glycine at VP1 residue 31 (VP1-31G) in viral particles disseminated into the integumentary and central nervous systems. In vitro viral growth and fitness analyses indicated that VP1-31G conferred growth and a fitness advantage in human neuronal cells, whereas VP1-31D conferred enhanced replication in human colorectal cells. A higher proportion of VP1-31G was also found among fatal cases, suggesting that it may facilitate central nervous system infection in humans. Our data provide the first glimpse of EV-A71 quasispecies from oral tissues to the central nervous system within humans, showing broad implications for the surveillance and pathogenesis of this reemerging viral pathogen.

IMPORTANCE EV-A71 continues to be a worldwide burden to public health. Although EV-A71 is the major etiological agent of hand, foot, and mouth disease, it can also cause neurological pulmonary edema, encephalitis, and even death, especially in children. Understanding selection processes enabling dissemination and accurately estimating EV-A71 diversity during invasion in humans are critical for applications in viral pathogenesis and vaccine studies. Here, we define a selection bottleneck appearing in respiratory and digestive tissues. Glycine substitution at VP1 residue 31 helps viruses break through the bottleneck and invade the central nervous system. This substitution is also advantageous for replication in neuronal cells in vitro. Considering that fatal cases contain enhanced glycine substitution at VP1-31, we suggest that the increased prevalence of VP1-31G may alter viral tropism and aid central nervous system invasion. Our findings provide new insights into a dynamic mutant spectral switch active during acute viral infection with emerging viral pathogens.

RNA病毒会积累突变，以迅速适应环境变化。肠病毒A71（EV-A71）引起各种临床表现，偶有严重的神经系统并发症。但是，EV-A71在人体内进化的机制尚不清楚。通过对来自尸检患者各种组织的病毒进行深度测序和单倍型分析，我们试图确定进化途径，肠病毒A71通过该途径进化为入侵人中枢神经系统的适应性。在呼吸系统和消化系统的初始感染部位观察到了具有不同突变的宽突变谱。病毒入侵后，我们确定了单倍型转换和显性单倍型，甘氨酸在VP1残基31（VP1-31G）传播到外皮和中枢神经系统中。体外病毒生长和适应性分析表明，VP1-31G在人神经元细胞中具有生长和适应性的优势，而VP1-31D则在人结肠直肠细胞中具有增强的复制能力。在致命病例中也发现了更高比例的VP1-31G，这表明它可能促进人类中枢神经系统感染。我们的数据提供了从口腔组织到人体内中枢神经系统的EV-A71准种的第一印象，显示了对该新兴病毒病原体的监视和发病机理的广泛意义。

重要事项EV-A71仍然是全球范围内公共卫生的负担。尽管EV-A71是手足口病的主要病因，但它也会引起神经性肺水肿，脑炎，甚至导致死亡，尤其是在儿童中。了解在人类入侵过程中能够传播并准确估计EV-A71多样性的选择过程，对于在病毒发病机制和疫苗研究中的应用至关重要。在这里，我们定义了呼吸和消化组织中出现的选择瓶颈。VP1残基31处的甘氨酸取代有助于病毒突破瓶颈并侵入中枢神经系统。这种取代也有利于在神经元细胞中进行体外复制。考虑到致命病例在VP1-31处含有增强的甘氨酸替代，我们建议VP1-31G的患病率增加可能会改变病毒的向性并有助于中枢神经系统的侵袭。我们的发现为动态突变谱切换提供了新的见解，该突变在活跃的病毒感染新兴病毒病原体期间活跃。