UL45–46 as the preferred insertion site for herpesvirus-of-turkeys-vectored H9N2 avian influenza vaccines: comparative analysis of immunogenicity and protection

H9N2 avian influenza virus (AIV) has evolved into a threat to the global poultry industry, causing huge economic losses and persistent public health risks. Due to inadequate cellular and mucosal immunity, current inactivated vaccines fail to prevent infection and block virus transmission. The recombinant herpesvirus-of-turkeys (HVT)-vectored vaccine is a promising control strategy, but the insertion site with the higher immunogenicity of the hemagglutinin (HA) gene is still unclear. This study compared the immunogenicity and protective efficacy of recombinant HVT vaccines expressing the H9N2 HA gene at the unique long (UL)45–46 and unique short (US)2 sites. The HVT-HA UL45–46 virus maintained replication kinetics consistent with the parental HVT and demonstrated genetic stability through 20 consecutive passages. Compared with HVT-HA US2 , HVT-HA UL45–46 induced significantly higher hemagglutination inhibition (HI) antibody titers and stronger HA-specific cellular immunity. Following the H9N2 challenge, the UL45–46 vaccine completely prevented intestinal viral shedding and significantly reduced lung viral loads and histopathological lesions. These findings establish UL45–46 as a better insertion site for HVT-based H9N2 vaccines.