A novel recombinant serotype 4 fowl adenovirus expressing HA of H3N8 avian influenza virus.

The recombinant H3N8 avian influenza virus (AIV) continues to circulate among poultry in China, with sporadic human infections reported, posing a significant threat to both the poultry industry and public health. However, no commercial vaccine against H3N8 AIV is currently available. Our previous studies have showed that serotype 4 fowl adenovirus (FAdV-4) with the deletion of fiber-2 gene is highly attenuated and serves as an ideal vaccine vector for expressing foreign genes. In this study, a novel recombinant FAdV-4 carrying HA of H3N8 was generated by targeting fiber-2 through the CRISPR/Cas9 and Cre-LoxP systems, designed as FA4-CMV-H3. In vitro, studies demonstrated that FA4-CMV-H3 not only efficiently expressed the HA protein of H3N8 but also replicated efficiently in LMH cells stably expressing Fiber-2 protein of FAdV-4. Chicken studies showed that FA4-CMV-H3 is highly attenuated in vivo and induces high neutralizing antibodies against FAdV-4 and high HI titers against H3N8. Moreover, FA4-CMV-H3 could provide full protection against both H3N8 and FAdV-4 challenges. All these demonstrate that the generated recombinant virus FA4-CMV-H3 is a promising bivalent efficient vaccine candidate against both FAdV-4 and H3N8.