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

Abstract

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. The rescued viruses O-DA and O-DAMM are lower replication fidelity mutants and showed an attenuated phenotype. These results demonstrated that the same amino acid substitution of 3D^pol in different serotypes of FMDV strains had different effects on viral fidelity. In addition, nucleoside analogues were used to select high-fidelity mutagen-resistant type O FMDV variants. The rescued mutagen-resistant type O FMDV high-fidelity variants exhibited significantly attenuated fitness and a reduced virulence phenotype. These results have important implications for understanding the molecular mechanism of FMDV evolution and pathogenicity, especially in developing a safer modified live-attenuated vaccine against FMDV.

Keywords: Fidelity; Fitness; Foot-and-mouth disease virus; Nucleoside analogue; Polymerase; Virulence.

Fig. 1

Generation of the type O FMDV 3D mutants with the Asia1 high-fidelity amino acid substitution. (A) Schematic of the FMDV RNA genome and the mutant sites of 3D^pol. (B)The replication ability of FMDV 3D mutants in BHK-21 cells. The mutants and their parent virus (WT) were passaged ten times in BHK-21 cells, and the titers of progeny viruses were determined by a TCID₅₀ assay. The standard deviations (n = 3) of mean virus titers are shown.

Fig. 2

The type O FMDV 3D mutants with Asia1 3D substitutions are low-fidelity variants. (A) A mean of seventy partial P1 sequences (approximately 35,000 nucleotides per replicate) were obtained. The mean mutation frequencies (number of nucleotide mutations per 10,000 nt sequenced)±SD represent the averages of all the replicates. The same pattern of reduced mutation frequency for other polymerase mutants compared to the WT was observed for each replicate (two-tailed Mann-Whitney test, n = 3, *P<0.05, **P<0.01). (B) Replication ability and of FMDV RdRp low-fidelity mutants. BHK-21 cells were infected with the mutants O-D5N, O-A38V, O-DA, O-DAMM or FMDV WT at an MOI of 0.01. The virus harvested at different times were titrated and expressed as TCID_50. Mean values±SD are shown (repeated-measures ANOVA, n = 3; ns for all mutants compared with WT). (C and D) RNA virus mutagen sensitivity assay of viral strains. BHK-21 cells, treated with different concentrations of RNA mutagen or mock-treated, were infected at MOI of 0.01; 72 h post-infection, the infectivity in the progeny virus was determined by TCID₅₀ assay. Mean virus titers ±SD are shown (Student’s t-test, n = 4; *P<0.05, **P<0.01).

Fig. 3

Generation of the FMDV populations resistant to ribavirin and 5-FU. (A) A mean of seventy partial P1 sequences (approximately 35,000 nucleotides per replicate) were obtained. The mean mutation frequencies (number of nucleotide mutations per 10,000 nt sequenced)±SD represent the averages of all the replicates. The same pattern of reduced mutation frequency for other polymerase mutants compared to the WT was observed for each replicate (two-tailed Mann-Whitney test, n =3, *P<0.05, **P<0.01). (B) Replication ability of the FMDV RdRp high-fidelity mutants T64I and AMMR. BHK-21 cells were infected with the mutants or FMDV WT at an MOI of 0.01. Viruses harvested at different times were titrated and expressed as TCID_50. Mean values ±SD are shown (repeated-measures ANOVA, n = 3; ns for all mutants compared with WT). (C and D) RNA virus mutagen sensitivity assay of viral strains. BHK-21 cells, treated with different concentrations of RNA mutagen or mock-treated, were infected at MOI of 0.01; 72 h post-infection, the infectivity in the progeny virus was determined by TCID₅₀ assay. Mean virus titers±SD are shown (Student’s t-test, n = 4; *P<0.05, **P<0.01).

Fig. 4

The FMDV fidelity variants exhibit reduced fitness in vitro. The mutant O-D5N, O-DA, O-DAMM, T64I and AMMR was mixed with the WT at a ratio of 9:1, 1:1 or 1:9 to inoculate BHK-21 cells at an MOI of 0.1 for three passages, after which the 3D^pol region was sequenced. The abundance of each competitor was measured as the height of the nucleotide peak for the mutant (A or T nt) or the WT (G or C nt) in sequencing chromatograms.

Fig. 5

The O-DAMM and AMMR are attenuated in 3-day old BALB/c suckling mice. A total of 15 groups of 3-day-old BALB/c suckling mice were inoculated cervicodorsally with 200 μL of each mutant or WT FMDV diluted to 0.1 TCID₅₀, 1 TCID₅₀ (B and D) or 10 TCID₅₀ (C and E). Animal deaths were scored for up to 7 days after inoculation, and survivors were euthanized (n = 6–9 for each group).