A novel monoclonal antibody differentiates highly pathogenic serotype 4 fowl adenovirus through targeting 188R in the Hexon.

Since 2015, hepatitis-hydropericardium syndrome (HHS) induced by the infection of highly pathogenic serotype 4 fowl adenovirus (HP-FAdV-4) has caused severe economic losses to the poultry industry. Although Hexon protein is closely associated with the pathogenicity of HP-FAdV-4, its antigenic epitopes remain poorly elucidated. In this study, two monoclonal antibodies (mAb) against Hexon were generated, designated as 2C5 and 4H7, respectively. Cross-reactivity with different FAdVs showed that mAb 2C5 only reacted with HP-FAdV-4 but not with low pathogenic FAdV-4 (LP-FAdV-4), whereas mAb 4H7 reacted with both HP-FAdV-4 and LP-FAdV-4. Epitope mapping revealed that the mAb 2C5 and 4H7 recognized 185GPGRNP190 and 161TSTSKDT167 in Hexon, respectively. Moreover, 188R in Hexon was identified as the key site recognized by mAb 2C5, and 162S and 166D in Hexon were identified as the critical sites recognized by mAb 4H7. Using mAb 2C5 as a capture antibody and HRP-conjugated mAb 4H7 as a detection antibody, we developed a novel sandwich ELISA to efficiently differentiate HP-FAdV-4 from LP-FAdV-4 and other serotypes of FAdV. All these data give novel insights into the epitopes and key antigenic sites in Hexon of FAdV-4 and provide efficient differentiating diagnostics approaches for HP-FAdV-4 endemic in China.