Ribavirin-Resistant Variants of Foot-and-Mouth Disease Virus: the Effect of Restricted Quasispecies Diversity on Viral Virulence

Abstract

Mutagenic nucleoside analogues can be used to isolate RNA virus high-fidelity RNA-dependent RNA polymerase (RdRp) variants, the majority of which are attenuated in vivo. However, attenuated foot-and-mouth disease virus (FMDV) high-fidelity RdRp variants have not been isolated, and the correlations between RdRp fidelity and virulence remain unclear. Here, the mutagen ribavirin was used to select a ribavirin-resistant population of FMDV, and 4 amino acid substitutions (D5N, A38V, M194I, and M296V) were identified in the RdRp-coding region of the population. Through single or combined mutagenesis using a reverse genetics system, we generated direct experimental evidence that the rescued D5N, A38V, and DAMM mutants but not the M194I and M296V mutants are high-fidelity RdRp variants. Mutagen resistance assays revealed that the higher replication fidelity was associated with higher-level resistance to ribavirin. In addition, significantly attenuated fitness and virulence phenotypes were observed for the D5N, A38V, and DAMM mutants. Based on a systematic quantitative analysis of fidelity and virulence, we concluded that higher replication fidelity is associated with a more attenuated virus. These data suggest that the resulting restricted quasispecies diversity compromises the adaptability and virulence of an RNA virus population. The modulation of replication fidelity to attenuate virulence may represent a general strategy for the rational design of new types of live, attenuated vaccine strains. IMPORTANCEThe ribavirin-isolated poliovirus (PV) RdRp G64S variant, the polymerases of which were of high replication fidelity, was attenuated in vivo. It has been proposed (M. Vignuzzi, E. Wendt, and R. Andino, Nat. Med. 14:154 -161, http://dx.doi.org/10.1038/nm 1726) that modulation of replication fidelity is a promising approach for engineering attenuated virus vaccines. The subsequently mutagen-isolated RdRp variants also expressed the high-fidelity polymerase, but not all of them were attenuated. Few studies have shown the exact correlation between fidelity and virulence. The present study investigates the effect of restricted quasispecies diversity on viral virulence via several attenuated FMDV high-fidelity RdRp variants. Our findings may aid in the rational design of a new type of vaccine strain.

Foot-and-mouth disease virus type O specific mutations determine RNA-dependent RNA polymerase fidelity and virus attenuation

“…To evaluate the effects of increased or decreased fidelity on virulence of FMDV, we chose the well-established 3-days suckling mice as animal model (Rodriguez-Pulido et al, 2011; Zeng et al, 2014). After inoculation with a dose of 1 TCID 50 , all mice died in the WT, O-D5N, O-A38V and T64I mutant inoculated groups, but no mice died in the AMMR mutant inoculated groups (Fig.…”

“…For example, the well-established role of the leader proteinase in the virulence in of swine and cattle (Kleina and Grubman, 1992; Roberts and Belsham, 1995; Skern et al, 1998), the role of RGD sequence in the VP1 G-H loop (Leippert et al, 1997; Mason et al, 1994), and the importance of virus nonstructural proteins (Gladue et al, 2014; Pacheco et al, 2013, 2003; Rai et al, 2017), the 3′ NTR (Rodriguez Pulido et al, 2009) and the codon usage bias (Diaz-San Segundo et al, 2015). We have previously shown that increased 3D pol fidelity of the type Asia1 FMDV strain could reduce its fitness in vitro and attenuate virulence in vivo (Zeng et al, 2014). …”

“…The researchers in this example demonstrated that the mutator strain had decreased fidelity in vivo, which resulted in attenuation in young, aged, and immunocompromised mouse models of human SARS (Graham et al, 2012). With regard to FMDV, it has been shown that either increased or decreased RdRp fidelity attenuates virus growth in animals (Rai et al, 2017; Zeng et al, 2014). Therefore, manipulation of polymerase fidelity is clearly a promising approach to engineer attenuated virus vaccines.…”

“…We have previously demonstrated that increase or decrease in RdRp fidelity can attenuate the type Asia1 FMDV (Xie et al, 2014; Zeng et al, 2014). In this study, we introduced the same RdRp amino acid substitutions that are responsible for increased type Asia1 FMDV polymerase fidelity into the type O FMDV.…”

“… Previous studies have shown that the FMDV Asia1/YS/CHA/05 high-fidelity mutagen-resistant variants are attenuated (Zeng et al, 2014). Here, we introduced the same single or multiple-amino-acid substitutions responsible for increased 3D pol fidelity of type Asia1 FMDV into the type O FMDV O/YS/CHA/05 infectious clone.…”