Immune efficacy of two recombinant Turkey herpesviruses expressing the fusion protein of Newcastle disease virus genotype VII and hemagglutinin protein of H9N2 avian influenza virus generated by HDR/NHEJ-CRISPR/Cas9 systems.

Newcastle disease viruses (NDV) and H9N2 avian influenza viruses (AIV) are two major threats to poultry farming. Current vaccination programs against two diseases are complex, requiring considerable labor and resources, and repeated immunizations can induce stress in animals. Therefore, developing a simplified, single-dose strategy capable of providing protection against both infections is highly desirable. Recombinant turkey herpesviruses (rHVT)-based live vaccines provide an attractive and effective platform for controlling avian viral diseases. This study generated two rHVTs, rHVT-OHA-OF(U) and rHVT-OHA-OF(H), each co-expressing F protein of NDV strain aSG10 and HA protein of H9N2 strain G, using a homologous directed repair (HDR) and non-homologous end-joining (NHEJ)-dependent CRISPR/Cas9-based gene editing strategy. In vitro, the two rHVTs correctly expressed the F and HA protein. After 10 passages in primary chicken embryonic fibroblasts (CEF) cells, the exogenous proteins remained stable expressed. In vivo, although rHVT-OHA-OF(H) provided strong protection against H9N2 strain G shedding at 3 and 5 days post-challenge (dpc), it offered no significant protection against mortality following challenge with NDV strain SG10. In contrast, rHVT-OHA-OF(U) provided 100% protection against mortality and significantly suppressed viral shedding following challenge with SG10, as well as achieving a significantly reduction of viral shedding at 3 dpc after H9N2 strain G challenge, indicating a promising vaccine candidate against both viral diseases, which requires further optimization. This study provided a reference for developing rHVT-based live vaccines targeting NDV and/or H9N2, thereby establishing a foundation for the design of dual- or multi-insert rHVTs.