Evaluation of Potential In Vitro Recombination Events in Codon Deoptimized FMDV Strains

Figures

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  Figure 1. Schematic diagram of the FMDV RNA templates used during transfection. DIVA markers are indicated by open (3B1 deletion, RQKP9-12→PVKV) and closed (3D H27N31→YR) triangles. Deoptimized regions are indicated by stripes. ΔGDD within the active site of RdRp encoded by 3D is indicated by an X.

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  Figure 2. (A). Schematic model of FMDV recombination during negative-strand synthesis with the given templates. ΔGDD within the active site of RdRp encoded by 3D is indicated by an X. Template switching must occur between the two arrows. (B). Number of infectious particles generated 24 and 48 h post-transfection in BHK-21 cells. Error bars represent 1 standard deviation. ND—not detected. (C). Plaque morphologies of WT and recombinants in LFBK-αVβ6 cells stained at 48 hpt.

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  Figure 3. Experimental design schematic of the ΔP1-P2P3Deopt × ΔGDD recombination assay and the proceeding viral quantification, plaque isolation, plaque passing, and sample types that were sequenced by NGS.

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  Figure 4. (A). Schematic model of FMDV recombination during negative-strand synthesis with the given templates. DIVA markers are indicated by open (3B1 deletion, RQKP9-12→PVKV) and closed (3D H27N31→YR) DIVA markers. Deoptimized regions are indicated by stripes. The ΔGDD within the active site of RdRp encoded by 3D is indicated by an X. Template switching must occur between the two arrows. (B). Number of infectious particles generated 24 and 48 h post-transfection in BHK-21 cells. * Statistically significant difference between the number of infectious virions produced from the different donor RNA templates. Error bars represent 1 standard deviation. ND—not detected. (C,D). Plaque morphologies of WT and recombinants in BHK-21 or LFBK-αVβ6 cells respectively, stained at 48 hpt.

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  Figure 5. Sequence analysis of 24-hpt ΔP1-P2P3Deopt × ΔGDD recombinant plaque isolates and ΔP1-P2P3Deopt or ΔGDD transfection total cell supernatant (shaded blue box). Locations (x-axis) and counts (y-axis) of reads that preferentially map to either a A24-WT or A24-P2P3Deopt reference within a 1059 nt section of the 3CD coding region as indicated by the pink shaded box. Triangles indicate the location of the 3B DIVA marker (white), 3D DIVA marker (black), the boundary between deoptimized and non-deoptimized regions (red), and the GDD deletion (blue).

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  Figure 6. Sequence analysis of 48-hpt ΔP1-P2P3Deopt × ΔGDD recombinant plaque isolates. Locations (x-axis) and counts (y-axis) of reads that preferentially map to either a A24-WT or A24-P2P3Deopt reference within a 1059 nt section of the 3CD coding region as indicated by the pink shaded box. Triangles indicate the location of the 3B DIVA marker (white), 3D DIVA marker (black), the boundary between deoptimized and non-deoptimized regions (red), and the GDD deletion (blue).

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  Figure 7. Sequence analysis of total cell culture supernatant of 48-hpt ΔP1-P2P3Deopt × ΔGDD recombinants plaque isolates passed in LFBK-αvβ6 cells. Locations (x-axis) and counts (y-axis) of reads that preferentially map to either a A24-WT or A24-P2P3Deopt reference within a 1059 nt section of the 3CD coding region as indicated by the pink shaded box. Triangles indicate the location of the 3B DIVA marker (white), 3D DIVA marker (black), the boundary between deoptimized and non-deoptimized regions (red), and the GDD deletion (blue).