Impact of Genomic Variations Found in Enterovirus A71 Virus Population on Viral Properties and Disease Severity

Since 1998, enterovirus A71 (EV-A71) has caused several outbreaks in Taiwan with hundreds of severe cases and deaths. EV-A71 exhibits a high genome mutation rate, resulting in a diverse viral population, called quasispecies. However, the consequence of variations found in quasispecies with EV-A71 viral virulence remains elusive. In this study, we aim to analyze the viral genomes of severe and mild clinical isolates from 2012 EV-A71 outbreak. Clinical isolates underwent isolation, extraction, and amplification, then sent for next-generation sequencing (NGS) using Illumina MiSeq. Using bioinformatics tools, sequence variation analysis identified four distinct, statistical variations, located in the 5'-UTR, 2C, 3A, and 3D of EV-A71 isolates between severe and mild clinical cases. Two variants found in the 3A and 3D polymerase (3Dpol) region, respectively, were found to have higher proportions in severe cases. The 3Dpol variant protein showed reduced polymerase activity compared to wild-type polymerase. In contrast, the 3A mutant virus shows a higher replication rate and greater fitness in both RD and DLD-1 cells. With better growth rates and fitness, the EV-A71 3A-5286C (76S) variation has a greater ability to maintain the fast-replicating populations and may be correlated with the disease severity and virulence of EV-A71.