Optimizing formaldehyde and binary ethylenimine combinations for inactivation of foot-and-mouth disease virus GR12: insights from Indonesia's third outbreak for local vaccine development.

Background and Aim: Foot-and-mouth disease (FMD) is a highly contagious transboundary livestock disease that poses serious economic and food security threats. In Indonesia, recurrent outbreaks since 2022 have highlighted the urgent need for localized vaccines to ensure sustainable control. Inactivation is a critical step in the development of inactivated FMD vaccines. While formaldehyde (FA) and binary ethylenimine (BEI) have been used individually or in combination for virus inactivation, their efficacy against new outbreak strains requires reevaluation. This study aimed to determine the optimal FA-BEI concentration and incubation time for inactivating the FMD virus (FMDV) Gresik sample no.12 (GR12) strain, isolated during the third outbreak in Gresik, East Java. Materials and Methods: FMDV serotype O GR12 was propagated in baby hamster kidney (BHK-21) cells, with titers determined by tissue culture infectious dose (TCID50). Four FA-BEI formulations were evaluated: F1 (0.04% FA + 2 mM BEI), F2 (0.1% FA + 1 mM BEI), F3 (0.1% FA + 2 mM BEI), and F4 (0.2% FA + 1 mM BEI). Inactivation was conducted at 37°C with sampling at 24, 48, and 72 h. Validation was performed through three sequential blind passages on BHK-21 monolayers, and cytopathic effects (CPEs) were scored and statistically analyzed. Results: FMDV GR12 propagated successfully in BHK-21 cells with titers of approximately 1.9 × 108 TCID50/mL. All FA-BEI combinations reduced CPE formation at 48 and 72 h; however, only F4 (0.2% FA + 1 mM BEI) achieved complete inactivation, showing no CPE across all passages after 72 h. Increasing BEI concentration alone did not significantly enhance inactivation. Statistical analysis confirmed that F4 was significantly more effective (p < 0.05) than other formulations. Conclusion: The combination of 0.2% FA and 1 mM BEI at 37°C for 72 h effectively inactivated FMDV GR12, establishing a baseline protocol for strain-specific inactivation in Indonesia. This study underscores the necessity of tailoring inactivation strategies to emerging FMDV strains and provides a practical foundation for localized vaccine production. Limitations include reliance solely on CPE validation; future studies should assess antigenic integrity and immunogenicity of inactivated viral proteins to ensure vaccine efficacy.