Foot-and-Mouth Disease Virus Low-Fidelity Polymerase Variants Are Attenuated

Foot-and-mouth disease (FMD), caused by foot-and-mouth disease virus (FMDV), is an acute, highly contagious disease. The disease affects cloven-hoofed animal species, notably cattle, pigs, sheep, and goats. The severe economic losses resulting from FMD are due to the high morbidity of infected animals and stringent trade restrictions imposed on affected countries and regions. Therefore, FMD is the first disease listed on the World Organisation for Animal Health (OIE) notifiable diseases. FMDV is an Apthovirus within the Picornaviridae family. The virus genome is a single-strand positive-sense RNA. Replication of the picomavirus genome is catalysed by the RNA-dependent RNA polymerase, also termed3D polymerase (3Dpol). SDpol is the key catalytic component of the replication machinery, along with other viral genes encoding proteins and host cell factors, playing an important role in viral RNA replication cycle.RNA viruses have high mutation rates, and the genetic variability is due to the low fidelity and the absence of error-repair mechanisms of the RdRp. Thus, RNA virus populations exist as collections of similar but genetically distinct genomes named quasispecies. Studies have shown that RdRp fidelity is a determinant of viral pathogenesis and virulence. A virus population encoding RdRp with either high or low fidelity may be attenuated due to the change of genetic diversity and virus fitness. A strategy to rationally engineer live attenuated virus strain has been proposed based on modulating the fidelity of RdRps.In this study, a total of39FMDV RdRp mutant recombinant plasmids were constructed based on the structure and biochemical properties of FMDV polymerase using site-directed mutagenesis via a reverse genetic system. Among the39recombinant FMDV mutant plasmids, only five mutants with RdRp single point mutation were rescued and resulted in viable, replication-stable viruses, including D165E, K172R, K177R, Y241F and G361S. Mutagen resistance assays demonstrated that all five mutants were more sensitive to mutagenic nucleoside analogues, suggesting they have polymerase with low-fidelity. Subsequent mutation frequency analysis confirmed that these mutator strains had decreased fidelity polymerase in comparison with WT. In addition, decreased fitness was observed for the RdRp lower fidelity mutants. Importantly, these mutants are less pathogenic than the FMDV WT in sucking mice, especially for D165E and K172R mutants presented10-fold reduction in virulence. Our study lay an theoretical contribution to basic virology for molecular determinants of FMDV virulence, and break an new approach of FMDV attenuation.