Abstract
Recently, the outbreaks of hydropericardium-hepatitis syndrome (HHS) caused by the highly pathogenic fowl adenovirus serotype 4 (FAdV-4) have resulted in huge economic losses to the poultry industry globally. Although several inactivated or subunit vaccines have been developed against FAdV-4, live-attenuated vaccines for FAdV-4 are rarely reported. In this study, a recombinant virus FA4-EGFP expressing EGFP-Fiber-2 fusion protein was generated by the CRISPR/Cas9 technique. Although FA4-EGFP shows slightly lower replication ability than the wild type (WT) FAdV-4, FA4-EGFP was significantly attenuated in vivo compared with the WT FAdV-4. Chickens infected with FA4-EGFP did not show any clinical signs, and all survived to 14 day post-infection (dpi), whereas those infected with FAdV-4 showed severe clinical signs with HHS and all died at 4 dpi. Besides, the inoculation of FA4-EGFP in chickens provided efficient protection against lethal challenge with FAdV-4. Compared with an inactivated vaccine, FA4-EGFP induced neutralizing antibodies with higher titers earlier. All these data not only provide a live-attenuated vaccine candidate against the highly pathogenic FAdV-4 but also give a potential insertion site for developing FAdV-4-based vaccine vectors for delivering foreign antigens.