Identification and Characterization of Linear Epitopes of Monoclonal Antibodies Against the Capsid Protein of Goose Astrovirus Genotype 2.

Abstract

Since 2015, outbreaks of a disease causing severe visceral gout in goslings had resulted in substantial economic losses to the goose farming industry in China. Subsequently, the disease, characterized by extensive visceral urate deposition and renal swelling, was determined to be caused by a novel astrovirus, designated as goose astrovirus (GAstV). The capsid protein (Cap) of GAstV, encoded by ORF2, is the sole structural protein of the virus and holds potential for developing therapeutic antibodies and diagnostic tools. Based on genetic divergence in the ORF2 gene, GAstV is classified into two serotypes: GAstV-1 and GAstV-2. Despite the critical role of the GAstV Cap in viral pathogenesis, research on generating and characterizing monoclonal antibodies (mAb) against this antigen remains scarce. In this study, six mAbs (1B3, 1B4, 1B6, 1D4, 1E2, 1F1) specifically recognizing GAstV Cap were screened using Western blotting (WB), indirect immunofluorescence assay (IFA), and indirect enzyme-linked immunosorbent assay (ELISA). For epitope mapping, sequential truncations of the GAstV Cap protein fused to glutathione S-transferase (GST) were generated using bacterial expression systems. Ultimately, antigenicity analysis of the prokaryotically expressed, GST-tagged Cap truncations via indirect ELISA and WB delineated two minimal linear epitopes: epitopeTDPEED, recognized by mAbs 1B3, 1B4, 1B6, 1D4, and 1E2, and epitopeDRAVAPREK, recognized by mAb 1F1. Amino acid sequence alignment revealed that the sequences of epitopesTDPEEDandDRAVAPREKare highly conserved in GAstV-2 but exhibit significant divergence in the GAstV-1 serotype. This study provides essential tools for both fundamental research and diagnostics of GAstV-2.