An increased replication fidelity mutant of foot-and-mouth disease virus retains fitness in vitro and virulence in vivo

•Nucleoside analogue mutagen 5-FU-resistant FMDV mutant was isolated.•Single R84H substitution in 3Dpol confers FMDV resistance to three RNA mutagens.•FMDV R84H mutant is a RNA-dependent RNA polymerase high fidelity variant.•The R84H variant retains fitness in vitro and virulence in vivo. In a screen for RNA mutagen-resistant foot-and-mouth disease virus (FMDV) strains, we isolated an FMDV mutant with RNA-dependent RNA polymerase (RdRp) R84H substitution. This mutant, selected under the mutagenic pressure of 5-fluorouracil (5-FU), is resistant not only to 5-FU but also to other two RNA mutagens, 5-azacytidine and ribavirin, suggesting that the RdRp R84H mutant is a high fidelity variant. Subsequently, the increased fidelity of this mutant was verified through analysis of mutation frequency, which revealed a 1.4-fold enhancement in RdRp fidelity compared with the wild-type virus. Further studies indicated that the R84H mutant exhibited slightly increased fitness in vitro, and its virulence was not reduced in suckling mice. These results indicated that an increase in RdRp fidelity does not always correlate with reduced virus fitness and virus attenuation. Thus, this isolated R84H mutant provides a new platform to examine the evolutionary dynamics of fidelity-changing RNA viruses, such as mutagen resistance, fitness and virulence.