EV-A71 is a common viral pathogen that causes hand, foot and mouth disease. It is a single-stranded RNA virus that has a low fidelity RNA polymerase and, as a result, spontaneous mutations frequently occur in the EV-A71 genome. The mutations within the genome give rise to quasispecies within the viral population that could be further defined by haplotypes. In vitro virulence of EV-A71 was shown by plaque size in Rhabdomyosarcoma (RD) cells, which was substantiated by in vitro characterizations of growth, RNA replication, binding, attachment and host cell internalization. Viruses could exhibit different host cell adaptations in different cell lines during viral passaging. The EV-A71/WT (derived from EV-A71 subgenotype B4) was shown to comprise six haplotypes through next-generation sequencing, where only EV-A71/Hap2 was found to be cultivable in RD cells, while EV-A71/Hap4 was the only cultivable haplotype in Vero cells. The EV-A71/WT produced plaques of four different sizes (small, medium, big, huge) in RD cells, while only two plaque variants (small, medium) were present in Vero cells. The small plaque variant isolated from RD cells displayed lower RNA replication rates, slower in vitro growth kinetics, higher TCID50 and lower attachment, binding and entry ability when compared against EV-A71/WT due to the mutation at 3D-S228P that disrupted the active site of the RNA polymerase, resulting in low replication and growth of the variant.
Keywords: enterovirus 71; in vitro virulence; next-generation sequencing; quasispecies; spontaneous mutations; structural analysis.