Development and validation of an indirect ELISA based on recombinant N-P fusion protein for detecting antibodies against parrot bornavirus in psittacine birds

Parrot bornavirus (PaBV) is a significant pathogen of psittacine birds and the etiological agent of proventricular dilatation disease (PDD). Although asymptomatic infections are common, clinical presentation is variable, posing substantial challenges for diagnosis and disease control. This study developed and validated an indirect enzyme-linked immunosorbent assay (iELISA) based on recombinant nucleoprotein (N), phosphoprotein (P), and an N-P fusion protein derived from PaBV-4 for the serological detection of PaBV-specific antibodies in psittacine birds. Immunodominant, hydrophilic regions were identified using in silico epitope prediction and antigenicity scoring. Recombinant proteins were expressed, purified, and confirmed via Western blot to exhibit specific reactivity with PaBV-positive sera. The N-P fusion protein (48 kDa) demonstrated the highest signal intensity and sensitivity. The optimized iELISA showed 92.9% sensitivity and 96.7% specificity at a sample-to-positive (S/P) ratio cut-off of 0.2064, and no antibody cross-reactivity with other avian pathogens was detected. Receiver operating characteristic (ROC) curve analysis yielded an area under the curve (AUC) of 0.985, indicating excellent diagnostic accuracy. Testing 208 serum samples from captive psittacine birds in Thailand revealed a PaBV seroprevalence of 68.27%, suggesting widespread subclinical infection. The developed iELISA represents a reliable and practical tool for PaBV serological surveillance and may serve as a valuable complement to molecular diagnostic methods for improved disease monitoring and control. • Addresses the lack of commercial PaBV vaccines and ELISAs. • N-P fusion protein yielded superior antigenicity in iELISA. • Optimized iELISA achieved 92.9% sensitivity and 96.7% specificity. • Field serology revealed 68.3% PaBV seroprevalence. • iELISA is a reliable tool for surveillance and carrier identification.