A highly efficient fluorescence-based virus neutralizing test for fowl adenoviruses associated with hepatitis-hydropericardium syndrome and inclusion body hepatitis

Fowl adenoviruses (FAdVs), specifically FAdV-4 associated with hepatitis-hydropericardium syndrome (HHS) and FAdV-8a, FAdV-8b, and FAdV-11 associated with inclusion body hepatitis (IBH), pose significant threats to the global poultry industry. The current gold standard for detecting neutralizing antibodies against these viruses is the conventional virus neutralization test (VNT), which relies on the subjective visual assessment of cytopathic effect. This method is time-consuming, low-throughput, and prone to inter-observer variability, highlighting an urgent need for a rapid, objective, and high-throughput alternative. Leveraging reverse genetics systems, we constructed recombinant reporter viruses for FAdV-4, FAdV-8a, FAdV-8b, and FAdV-11, each expressing the green fluorescent protein ZsGreen. These fluorescent-tagged viruses were used to develop a rapid fluorescence-based virus neutralization test (F-VNT). The test quantifies neutralization titers based on the reduction of green fluorescence in infected cell cultures, allowing for objective and automated readout. Notably, this new test delivers results within 24 hours post-infection, significantly faster than the conventional VNT. Additionally, the established method was used to investigate cross-neutralization between anti-FAdV-8a, -8b, -11, and -4 sera. The results showed high cross-neutralization between anti-FAdV-8a and -8b antibodies. Anti-FAdV-11 antibodies exhibited weak neutralization against FAdV-8a and -8b, while anti-FAdV-4 antibodies had no neutralizing effect on FAdV-8a, -8b, or -11. The developed F-VNT provides a rapid, accurate, and high-throughput platform for serological evaluation of FAdVs associated with HHS and IBH. This method represents a substantial improvement over traditional VNT and is well-suited for large-scale seroepidemiological studies and vaccine efficacy evaluations in the poultry industry. This study also provides theoretical guidance for antigen selection in developing multivalent vaccines against HHS and IBH.