Impact of Variations in Quasispecies on Enterovirus A71 Viral Properties

Enterovirus A71 (EV-A71) is an RNA virus of the Picornaviridae family, Enterovirus genus. Since 1998, it has caused several outbreaks in Taiwan with hundreds of severe cases and deaths. EV-A71 exhibits a higher genome mutation rate and results in a pool of viral populations, called quasispecies. However, the consequence of quasispecies with EV-A71 viral virulence remains elusive. In this study, we analyzed viral genomes of 8 severe and 8 mild clinical isolates from 2012 EV-A71 subtype B5. To obtain the genome information on the viral quasispecies, we conducted next-generation sequencing (NGS) using Illumina Miseq platform. LoFreq and QuasiRecomb programs were used for sequence variation analysis. The results indicate 4 distinct, statistical variations, located in the 5'-UTR, 2C, 3A, and 3D of EV-A71 isolates between severe and mild clinical cases. Viruses with unique variations were generated via a reverse genetics system for further characterizations, including replication rate, fitness, and thermal stability. The virus with variations in 3A region, which is prominent in severe cases, shows a higher replication rate and greater fitness in both RD and DLD-1 cells. Hence, the 3A substitution may inherent a better ability to maintain the fast-replicating populations in RD and DLD-1 cells and to invade into the CNS. In conclusion, EV-A71 viral quasispecies show definitive variations associated with severe and mild clinical cases. The gain-of-function variations in EV-A71 quasispecies display different growth rates and fitness and may be correlated with the disease severity and virulence of EV-A71.