# Development of recombinant fowl adenovirus serotype 4 harboring the HiBiT-Tag reporter and its utility in antiviral research

## TL;DR

Researchers developed a new tool using a modified fowl adenovirus to detect antibodies and screen antiviral drugs more efficiently.

## Contribution

A recombinant fowl adenovirus 4 with a HiBiT-tag reporter was developed for rapid antiviral screening and antibody detection.

## Key findings

- The HiBiT-tagged virus retains growth kinetics and stability similar to the parental virus.
- The system enables rapid quantification of neutralizing antibodies and antiviral agents in chicken serum.
- The tool improves detection efficiency and reduces testing time for FAdV-4 neutralizing antibodies.

## Abstract

Severe hydropericardium-hepatitis syndrome (HHS) caused by fowl adenovirus 4 (FAdV-4) significantly affects the global poultry industry. However, there is currently no convenient and sensitive platform available for the rapid screening of antiviral drugs and the detection of neutralizing antibodies. Here, we first established an improved reverse genetics system for FAdV-4 Y17215-1 strain and identified the optimal insertion site for expressing foreign genes (between the ORF19A and ORF4 genes). To obtain a quantifiable recombinant reporter FAdV-4, the HiBiT-tag reporter gene was inserted into this site, and recombinant FAdV-4 expressing the HiBiT gene (rFAdV-4-HiBiT) was successfully rescued. Further analysis showed that rFAdV-4-HiBiT had similar growth kinetics to the parental virus and retained luciferase activity and genetic stability after 10 rounds of serial passages. A proof-of-concept test confirmed that rFAdV-4-HiBiT can be used to rapidly quantify anti-FAdV-4 neutralizing antibodies in chicken serum samples and screen for antiviral agents, including antiviral drugs and proteins, based on the intensity of luciferase activity. Collectively, the HiBiT-tagged virus rFAdV-4-HiBiT provides a robust tool for the rapid detection of FAdV-4 and facilitates the development of novel therapeutics and vaccines for FAdV-4.

The epidemic spread of fowl adenovirus 4 (FAdV-4) presents significant challenges for the global poultry industry. However, there are currently few convenient and sensitive platforms available for antiviral drug screening and neutralizing antibody detection. In this study, we first established an improved reverse genetics system for the FAdV-4 and screened for the optimal insertion site for foreign genes between ORF19A and ORF4. Furthermore, the HiBiT gene was further inserted into this site, and rFAdV-4-HiBiT was successfully rescued. A luciferase-based FAdV-4 neutralizing antibody detection method has been successfully established, which can reduce detection time and greatly enhance the efficiency of neutralizing antibody testing. Furthermore, this system can serve as a more convenient screening platform for anti-FAdV-4 drugs. Collectively, rFAdV-4-HibiT represents an important tool with great potential for facilitating the development of novel therapeutics and vaccines for FAdV-4.