Engineering viable foot-and-mouth disease viruses with increased acid stability facilitate the development of improved vaccines

Figures

• 

  Fig. 1. Acid sensitivity of the rescued FMDV. a Acid-induced inactivation analysis of the rescued FMDVs. Equal PFU amounts of the rescued viruses were treated with PBS at different pH values for 30 min at room temperature. The samples were neutralized with Tris at 1 M and pH 7.4 and the infectivity was expressed as the percentage of PFU recovered in BHK-21 cells relative to the obtained at pH 7.4. b Acid dissociation kinetic studies of FMDV r N17D, r D86H, r N17D2, and O/HN/93. Same amounts (PFU) of chemically inactivated (BEI) viruses tested were mixed with PBS of different pH values for 30 min at room temperature. Then, the solution was neutralized with 1 M Tris (pH 7.4) and the remaining 146S was determined by the size-exclusion high-performance liquid chromatography (SE-HPLC). The percentage of remaining 146S compared to those acquired at pH 7.4 was determined. Data were presented as means ± standard deviations (SD) of three independent experiments

• 

  Fig. 2. Biological characteristics of modified FMDVs with increased acid stability. a Plaque morphologies of the FMDV mutants and parental virus in BHK-21 monolayer cells. BHK-21 cells were infected with FMDVs and stained with crystal violet at 48 h post-infection (hpi). b One-step growth curves for the mutant viruses and parental virus in BHK-21 cells. BHK-21 cells were infected with FMDV mutants and parental virus at a multiplicity of infection (MOI) of 1 and the virus titers of harvested samples at different times (4 h, 8 h, 12 h, and 20 h) were determined by the plaque assay. c Virulence of the variants and its parental virus in suckling mice. One-day-old mice (5 per group) were inoculated with different 50% tissue culture infective dose (TCID50) of the modified viruses and parental virus and observed for 7 days (c1–c3). The percentage of animals surviving was calculated. Survivors were euthanized at the end of experiment

• 

  Fig. 3. Several biophysical stability analyses of the acid-resistant FMDV mutants (a–c). a Alkali-induced inactivation kinetics of the acid-resistant FMDV mutants. Equal amounts of engineered FMD virions were treated with PBS solution of different pH values. The samples were then neutralized and added to BHK-21 monolayers. The infectivity was calculated as described above. b Thermal-inactivation kinetics of r N17D, r D86H, r N17D2, and O/HN/93 viruses. The different viruses were heated at 42 °C for up to 1 h. Then, the remaining virus in each case was determined in plaque assays and the infectivity was calculated (b1). All data were fitted to single-order exponential processes (b2). c Effect of acid treatment on the inactivation of FMD virions at different ionic strengths. The viruses were incubated with PBS buffer (ranging from pH 6.0 to 7.4) and NaCl solutions (150 mM or 1 M) for 30 min. After neutralization, the recovered virus was determined and the infectivity was calculated

• 

  Fig. 4. The structural model of FMDV. The left sphere is the capsid model of FMDV, and the right cartoon representation is the pentamer containing VP1, VP2, VP3, and VP4. The mutated residues are marked on the pentamer

• 

  Fig. 5. The hydrogen bond analysis. a–j The hydrogen bonds between the former or mutated residues (VP1N17D, VP2H145Y, VP2D86H, VP2D86A, VP3H141G, and VP3H141D) and other residues in FMDV capsid. The red and blue lines represent the hydrogen bonds