A Heat-Induced Mutation on VP1 of Foot-and-Mouth Disease Virus Serotype O Enhanced Capsid Stability and Immunogenicity

Figures

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  FIG 1. Selection of FMDV thermostable mutants. (A) The flowchart of screening heat-resistant FMDV. (B) The titer of virus incubated at different temperatures was determined by plaque assay, samples were repeated in duplicate, and data are presented as the mean ± SD. (C) The virus was heat treated at 57°C for 60 min or 60°C for 30 min, followed by subpassaging at 37°C until the virus titers were restored; virus pools were named 57P01 and 60P01, respectively. There were 10 repeated cycles of thermal selection and passage. (D) Viruses (57P10 and 60P10) were thermally selected further with the selection pressure increased to 59°C for 60 min or 62°C for 30 min according to the above method. The dashed line represents wt virus grown at 37°C without heat treatment, and the virus titers were all determined by plaque assay. Data are presented as the mean ± SD. Experiments were repeated in duplicate.

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  FIG 2. Genetic stability of thermostable FMDV variants. (A) The dynamics of the virus titer of selected mutants during 15 passage cycles, samples were repeated in duplicate, and data are presented as the mean ± SD. (B) Sequence alignment of the relevant parts of the P1 region of mutants M3, M9, and M10 where amino acid changes occur compared with the wild-type virus. Mutant viruses from passage 1 and passages 11 to 15 were subjected to sequence analysis to show the stability of the mutations.

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  FIG 3. Inactivation kinetics and heat resistance of FMDV serotype O mutants. The percentages of remaining infectious particles of wild-type (wt) virus and mutants M3, M9, and M10 were determined following heat treatment at 4°C for 12 days (A), 25°C for 36 h (B), or 37°C for 10 h (C), and the inactivation rate constants were calculated. (D) The titers of virus incubated at different temperatures were determined by plaque assay. Compared with the wild-type virus, the temperature for complete inactivation of mutants M3, M9, and M10 increased to 64, 63, and 64°C, respectively. Data are presented as the mean ± SD. (E) RNA release from capsid as detected by SYTO 9 fluorescence in PaSTRy assay. The complete RNA release temperature of the wt virus was 49.0°C, M3 was 56.0°C, M9 was 50.0°C, M10 was 56.0°C. (F) Double antibody sandwich ELISA to detect the remaining intact antigens after heat treatment at 37°C. The percentage of intact particle of wt virus and mutants are statistically different (n = 2; ± SD; ns, not significant; ***, P < 0.001; **, P < 0.01).

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  FIG 4. Comparison of antibody titers induced by wild-type virus and mutants M3 and M10. Guinea pigs were immunized with inactivated viruses that were either unheated or heated at 37°C for 12 h or 24 h. Serum samples were collected at 7, 14, 21, and 28 days postvaccination (dpv) to detect the antibody titers by ELISA (A) or VNT (B). The level of unheated and heated samples was statistically different (n = 6; ± SD; ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05).

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  FIG 5. Effect of A1013T mutation on VLP stability. (A) VLPA1013T purification on a 15% to 60% sucrose cushion. (B) VLPA1013Ts were further purified by a 7.5% to 30% sucrose density gradient, and the diluted samples were analyzed by ELISA and Western blotting using rabbit anti-FMDV polyclonal antibodies. (C) TEM image of VLPA1013T. (D) The remaining percentage of VLPwt and VLPA1013T antigens at regular time intervals during incubation at 25°C for up to 84 h were determined by sandwich ELISA. Data are presented as the mean ± SD (n = 2). The significance of differences between VLPwt and VLPA1013T is indicated (ns, not significant; **, P < 0.01).

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  FIG 6. Analysis of amino acid residue interactions. (A) Negative-staining images (left) and cryo-EM images (right) of wild-type virus and mutant M3 particles. (B) Gold-standard Fourier shell correlation (FSC) curves of the final maps. (C) The final maps were analyzed by ResMap, showing a resolution distribution from 2.9 to 5.4 Å of wild-type virus and mutant M3 virus. (D) Distribution of two stabilizing amino acid mutations on the surface of the mutant M3 capsid. The position of the 5-fold (5′), 3-fold (3′), and 2-fold (2′) axis of symmetry is indicated. The VP1, VP2, and VP3 proteins of one protomer are color coded as indicated in the legend, whereas adjacent capsid proteins are indicated in gray. Rectangles 1 and 2 indicate enlargements visible in E. (E) Superimposition of structures of wild-type virus and mutant M3; the wild-type virus is colored gray, and the VP1, VP2, and VP3 proteins of mutant M3 are colored with green, cyan, and magenta, respectively. Hydrogen bonds are shown as yellow sticks. The side chain T1013 on VP1 forms a hydrogen bond with T3156 of an adjacent VP3 protein (E-1). The hydrophilic side chain of Q2057 forms a hydrogen bond with Y2098 of the adjacent VP2…