Construction and immune efficacy of a recombinant turkey herpesvirus vaccine strain expressing fusion protein of genotype VII Newcastle disease virus

Herpesvirus of turkeys (HVT), a commonly used live vaccine against Marek's disease, has proven to be a highly effective viral vector for the generation of recombinant vaccines that deliver protective antigens of other avian pathogens. In this study, a vaccine designated rHVT-NDV-opti F was constructed by inserting a codon-optimized genotype Ⅶ Newcastle disease virus (NDV) fusion (F) gene into the US2 gene of HVT Fc126 vaccine strain using CRISPR/Cas9 gene-editing technology coupled with two single-guide RNAs (sgRNA). The F protein expression of rHVT-NDV-opti F was detectable by western blotting and an indirect immunofluorescence assay. Compared with wildtype HVT, rHVT-NDV-opti F has similar plaque morphology but lower in vitro replication capacity. The F protein of rHVT-NDV-opti F is genetically stable and predominantly expressed in the cell plasma. Immunization of one-day-old specific pathogen-free chickens with 4000 plaque-forming units of rHVT-NDV-opti F induced NDV-specific antibodies and provided 70% protection against a homologous NDV challenge, effectively reducing virus shedding, clinical signs, tissue viral load, and mortality. These results suggest that rHVT-NDV-opti F could be a potential vaccine candidate against Newcastle disease in chickens and that HDR-CRISPR/Cas9 combined with dual sgRNA can rapidly and efficiently construct HVT-vectored vaccine candidates.