Development and evaluation of a recombinant bivalent HVT-vectored vaccine candidate against H9N2 avian influenza virus and fowl adenovirus serotype 4.

H9N2 subtype avian influenza virus (AIV) is one of the most prevalent AIV subtypes in poultry worldwide. Meanwhile, fowl adenovirus serotype 4 (FAdV-4), associated with hepatitis-hydropericardium syndrome (HHS), has been widely distributed in poultry worldwide and has circulated in China since 2015. Notably, co-infections with H9N2 AIV and FAdV-4 are frequently reported in the field, leading to increased mortality and substantial economic losses in the poultry industry. Herpesvirus of turkeys (HVT) has been extensively used as a viral vector for poultry vaccines. In this study, a recombinant bivalent HVT-vectored vaccine candidate (HVT-H9-FAd) co-expressing the hemagglutinin (HA) gene of H9N2 AIV and the Fiber-2 gene of FAdV-4 was constructed and evaluated. The recombinant virus exhibited plaque morphology and growth kinetics comparable to the parental HVT strain and stably expressed HA and Fiber-2 proteins for 20 serial passages. Immunization of specific-pathogen-free (SPF) chickens with HVT-H9-FAd induced robust antibody responses against H9N2 AIV and Fiber-2, with antibody kinetics comparable to those elicited by the corresponding monovalent HVT-H9 and HVT-FAd. Following challenge with the homologous H9N2 AIV strain, HVT-H9-FAd immunized chickens were completely protected, with no detectable viral shedding. Following challenge with the homologous FAdV-4 strain, complete protection was observed, as evidenced by the absence of mortality, viral replication, and pathological lesions in immunized chickens. Collectively, these findings demonstrate that the recombinant HVT-H9-FAd is a promising bivalent vaccine candidate for controlling H9N2 AIV and FAdV-4 in poultry and provides an effective strategy for preventing co-circulating pathogens in chicken flocks.