Construction and characterization of recombinant duck enteritis virus expressing duck hepatitis A virus 3 immunogenic genes

Duck enteritis virus (DEV) is considered an ideal vector for waterfowl vaccine development due to its favorable safety profile and multiple genomic sites that accommodate foreign gene insertion. Duck hepatitis A virus (DHAV) causes acute hepatitis, neurological symptoms, and high mortality in young ducklings, and the predominant circulating serotype has shifted from type 1 to type 3, underscoring the urgent need for improved vaccines. In this study, we employed a CRISPR/Cas9 genome editing platform combined with dual single-guide RNAs (sgRNAs) and a homologous directed repair (HDR) strategy to construct two recombinant DEV strains (rDEV-DHAV) expressing the immunogenic VP0 or VP1 proteins of DHAV-3. The recombinants were purified by plaque selection and validated using PCR, Western blotting, indirect immunofluorescence, and animal experiments. Both recombinant viruses replicated efficiently in chicken embryo fibroblasts and exhibited growth kinetics comparable to the parental DEV vaccine strain. The inserted VP0 and VP1 genes remained genetically stable over at least 15 serial passages. Immunization trials in ducklings demonstrated that both recombinants elicited strong humoral responses against DEV and DHAV-3. Safety evaluation showed that neither recombinant virus induced clinical signs, pathological lesions, or abnormal viral shedding, and both displayed safety profiles equivalent to the parental vaccine strain. Overall, the two rDEV-DHAV strains generated in this study are genetically stable, safe, and exhibit good immunogenicity. The HDR-CRISPR/Cas9 strategy employing dual sgRNAs provides an efficient approach for the rapid construction of multivalent DEV vector vaccines, highlighting its substantial potential in poultry vaccine development.