Duck Tembusu virus (DTMUV), an emerging avian pathogenic flavivirus, causes severe neurological disorders and acute egg drop syndrome in ducks. Currently, several clusters of DTMUV exhibiting significant antigenic variation are circulating in Asia with distinct geographical distributions. Therefore, developing vaccines based on virus clusters specific to regions is essential. To generate an effective inactivated vaccine, the virus inactivation procedure must be optimized for each virus strain. However, no information is available on the optimal inactivation protocol for cluster 2.1 DTMUV, which predominantly circulates in several Asian countries. This study aimed to determine the effect of beta-propiolactone (BPL) inactivation on the infectivity, antigenic integrity, and immunogenicity of cluster 2.1 DTMUV. Our results demonstrated that all conditions of BPL treatment (1:2000, 1:3000, and 1:4000 (vol/vol) concentration
24, 48, and 72 h of incubation) could completely inactivate cluster 2.1 DTMUV, as evidenced by the absence of cytopathic effect (CPE) and DTMUV antigens after 3 passages in baby hamster kidney (BHK-21) cells. However, BPL at 1:4000 (vol/vol) concentration with 24 h of incubation preserved both the total protein content and the antigenic integrity of cluster 2.1 DTMUV more effectively than other conditions. Furthermore, we found that cluster 2.1 DTMUV inactivated with BPL under this condition was safe and highly immunogenic in ducks. This was evidenced by the absence of clinical signs and the robust induction of DTMUV-specific neutralizing antibodies and T helper lymphocyte responses in immunized ducks. Overall, these findings suggest that a 1:4000 dilution of BPL with 24 h of incubation is the optimal condition for complete inactivation of cluster 2.1 DTMUV without significant loss of antigenicity and immunogenicity. This protocol can serve as a guideline for efficient cluster 2.1 DTMUV inactivation, which is valuable for vaccine and immunoassay development.