Novel Thermostabilizing Formulation for Enhanced Stability of Foot-and-Mouth Disease Virus During Storage and Environmental Stress

Figures

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  Figure 1. Evaluation of stabilizer efficacy under different temperatures and storage durations. Four FMDV serotype O strains, (a) O/SKR/Jincheon/2014, (b) O/SKR/Boeun/2017, (c) O1 Manisa (R), and (d) PanAsia-2 (R), were mixed with either stabilizers or cell culture medium (control) and stored at −80 °C, 4 °C, 25 °C, or 37 °C for 2, 4, or 7 d. Following storage under the indicated conditions, viral infectivity was determined by measuring the 50% tissue culture infectious dose (TCID50) in adherent BHK-21 cells. Data are presented as the mean ± standard deviation from three independent experiments (n = 3). Statistical significance was assessed using two-way ANOVA followed by Tukey’s multiple-comparisons test. Statistical significance was defined as * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

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  Figure 2. Evaluation of viral stability following repeated freeze–thaw cycles of FMDV formulated with different stabilizers after storage at −80 °C or in liquid nitrogen (LN2). Viral titers of four FMDV serotype O strains [O/SKR/Jincheon/2014, O/SKR/Boeun/2017, O1 Manisa (R), and O PanAsia-2 (R)] formulated with the indicated stabilizers were compared with those of the serum-free medium control following repeated freeze–thaw cycles between −80 °C or LN2 and 37 °C. Virus samples were stored either (a) at −80 °C or (b) in LN2 prior to freeze–thaw treatment. Viral infectivity was measured after 0, 5, and 10 freeze–thaw cycles. Data are presented as mean viral titers ± standard deviation. Statistical significance was determined by comparison with the serum-free medium control where applicable. Data are presented as the mean ± standard deviation from three independent experiments (n = 3). Statistical significance was assessed using two-way ANOVA followed by Tukey’s multiple-comparisons test. Statistical significance was defined as * p < 0.05, ** p < 0.01, and *** p < 0.001.

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  Figure 3. Effects of stabilizers on BHK-21 suspension cell growth and viability. BHK-21 suspension cells were cultured in CD BHK-21 or ProVero-1 medium and treated with either 3% sucrose or CVS. Cells were collected at specific time points (0, 16, 24, 48, and 72 h) after treatment to determine (a) total cell number and (b) cell viability. For cell number, statistical significance was assessed using two-way ANOVA followed by Tukey’s multiple-comparisons test, with treatment and time as the two factors. In contrast, cell viability (%) was presented for descriptive comparison of viability patterns, and no statistical significance testing was performed. Data are presented as the mean ± standard deviation (SD) of three independent experiments (n = 3), with triplicate measurements performed within each experiment. For cell number, statistical significance was defined as p < 0.05. * indicates p < 0.05.

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  Figure 4. Effects of 3% sucrose and CVS on FMDV growth kinetics in BHK-21 suspension cells. Growth kinetics of four FMDV serotype O strains, (a) O/SKR/Jincheon/2014, (b) O/SKR/Boeun/2017, (c) O1 Manisa (R), and (d) O PanAsia-2 (R), were evaluated in BHK-21 suspension cells. Growth kinetics of viruses derived from 3% sucrose- or CVS-treated virus stocks were compared with those of unstabilized control virus stocks. Data are presented as the mean ± standard deviation of three independent experiments (n = 3), with triplicate measurements performed within each experiment. Statistical significance was assessed using two-way ANOVA followed by Tukey’s multiple-comparisons test. No statistically significant differences were observed among the stabilizer-treated and unstabilized control groups in viral growth kinetics.