Development and characterization of a recombinant attenuated duck adenovirus B2 vector expressing the vp3 gene of short beak and dwarfism syndrome virus.

Abstract

Duck adenovirus B2 (DAdV-B2) and short beak and dwarfism syndrome virus (SBDSV) are major pathogens that cause severe economic losses in the global duck industry. To develop a recombinant DAdV-B2 virus capable of expressing SBDSV antigens, we first generated an attenuated DAdV-B2 strain, designated BG18cm20, by serially passaging the virulent BG18 strain in Muscovy duck embryo fibroblast cells. Pathogenicity assessment confirmed that BG18cm20 is non-pathogenic in Muscovy ducklings, validating its safety as a viral vector backbone. Using CRISPR/Cas9-assisted homology-mediated end joining technology, we inserted GFP reporter gene or the SBDSV vp3 gene into the genomic loci (ORF20-ORF53) of BG18cm20, successfully constructing recombinant viruses BG18cm20-GFP and BG18cm20-SBDSV-VP3. In vitro characterization demonstrated that recombinant viruses replicate efficiently and maintain stable expression of the inserted genes over 15 serial passages. Notably, transmission electron microscopy revealed that the VP3 protein expressed by BG18cm20-SBDSV-VP3 self-assembles into SBDSV virus-like particles. Collectively, these findings demonstrate that BG18cm20 serves as a safe and effective viral vector platform for foreign antigen expression. The construction and in vitro characterization of BG18cm20-SBDSV-VP3 provide a foundational basis for future development of bivalent vaccine against DAdV-B2 and SBDSV in ducks.