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

ABSTRACT 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.