Targeted mutagenesis of the ICP4 transactivation domain generates a protective DIVA vaccine against duck plague

Duck plague, caused by duck plague virus (DPV), causes significant economic losses to the global waterfowl industry. The control of this disease is hampered by the limitations of conventional vaccines, which include the risk of virulence reversion, short-lived immunity, and the inability to differentiate infected from vaccinated animals (DIVA). To address these issues, we explored the rational design of a novel live-attenuated DIVA vaccine by targeting the essential immediate-early gene ICP4. A quadruple mutation was introduced into the N-terminal transactivation domain of ICP4. The resulting mutant virus, ICP4-mutNEEEC, exhibited significantly attenuated virulence in ducklings, with reduced viral loads in key organs and diminished shedding compared to the wild-type virus. Importantly, a single dose of ICP4-mutNEEEC elicited a strong neutralizing antibody response and provided complete protection against a lethal DPV challenge, with efficacy comparable to a commercial vaccine. The introduced mutation serves as a stable genetic marker, enabling the development of a companion DIVA assay. This study presents ICP4-mutNEEEC as a promising, safe, and effective vaccine candidate that fulfills a critical need for a DIVA-compliant strategy in duck plague control programs.