Yongxiao Chai ^1,2 Qianyue Jin ² Rongfang Zhu ² Zhenhua Guo ² Qingxia Lu ² Shujun Chai ² Yunrui Xing ² Lu Han ³ Guangxu Xing ² Gaiping Zhang ^1,2,4,5*

• ¹ College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China
• ² Key Laboratory of Animal Immunology of the Ministry of Agriculture, Institute for Animal Health, Henan Academy of Agricultural Sciences, Zhengzhou, Henan Province, China
• ³ Henan Husbandry Technology Promotion Station, Zhengzhou, China
• ⁴ Longhu Laboratory, Zhengzhou, China
• ⁵ Jiangsu Co-Innovation Center for the Prevention and Control of Important AnimalInfectious Disease and Zoonoses, Yangzhou, Jiangsu Province, China

Background: Fowl adenovirus serotype 4 (FAdV-4) is the main pathogen of hepatitis-hydropericardium syndrome (HHS), which brings huge economic losses to the poultry industry worldwide. Fiber-1 protein plays an important role in viral infection and pathogenesis by binding directly to cellular receptors of FAdV-4. In particular, the knob domain of fiber-1 protein has been reported to induce the production of neutralizing antibodies and arouse protection against the lethal challenge of chickens with FAdV-4. Methods: The fiber-1 knob (F1K) protein was expressed in a prokaryotic expression system and purified using Ni-NTA affinity chromatography. Monoclonal antibodies (mAbs) against FAdV-4 were generated by immunizing BALB/c mice with the purified F1K protein and screened using a series of immunoassays. Potential B cell epitopes on the knob domain of fiber-1 protein were mapped using enzyme-linked immunosorbent assay (ELISA) and dotblot. Precious location and crucial amino acids of the identified epitopes were determined using peptide array scanning, truncations and alanine-scanning mutagenesis. The epitopes were analyzed and visualized on the knob trimer of FAdV-4 fiber-1 protein using the PyMOL software.Results: Water-soluble recombinant fiber-1 knob (F1K) protein was obtained with the assistance of chaperone.Four monoclonal antibodies (5C10, 6F8, 8D8, and 8E8) against FAdV-4 were generated and characterized using indirect ELISA, Western blot, dot-blot, and immunological fluorescence assay (IFA). The mAbs were demonstrated to be from different hybridoma cell lines based on the sequences of the variable regions. Meanwhile, three distinct novel linear B-cell epitopes ( 319 SDVGYLGLPPH 329 , 328 PHTRDNWYV 336 , and 407 VTTGPIPFSYQ 417 ) on the knob domain of fiber-1 protein were identified and the key amino acid residues in the epitopes were determined.Structural analysis showed that the two adjacent epitopes 319 SDVGYLGLPPH 329 and 328 PHTRDNWYV 336 were exposed on the surface of the fiber-1 knob trimer, whereas the epitope 407 VTTGPIPFSYQ 417 was located inside of the spatial structure.This was the first identification of B-cell epitopes on the knob domain of fiber-1 protein and these findings provided a sound basis for the development of subunit vaccines, therapeutics, and diagnostic methods to control FAdV infections.