Abstract
Although σA is an important major core protein of duck reovirus (DRV), the B-cell epitopes of this protein remain unknown to reseacrhers. Six monoclonal antibodies (MAbs) (1A7, 3F4, 5D2, 4E2, 3C7, and 2B7) were developed by using prokaryotic-expressed recombinant His-σA protein. Five of six MAbs (1A7, 3F4, 4E2, 3C7, and 2B7) reacted with His-σA protein in a conformation-independent manner, while 5D2 reacted with σA in a conformation-dependent manner. Immunofluorescence assays showed that the MAbs could specifically bind to DRV infected BHK-21 cells. The MAbs were delineated as three groups by a competitive binding assay. By using 12-mer peptide phage display and mutagenesis, MAb 4E2 was identified to recognize minimal epitope 56EAPYPG61 and MAb 1A7 recognize 341WVV/MAGLI/V347, residues 341V/M and 347I/V are replaceable. Dot blotting and sequence analysis confirmed that EAPYPG and WVV/MAGLI/V are cross-reactive epitopes in both DRV and avian reovirus (ARV). An enzyme-linked immunosorbent assay (ELISA) based on two expressed EAPYPG and WVVAGLI as antigen demonstrated its diagnostic potential by specific reacting with serum samples from DRV- or ARV-infected birds. Based on these observations, an epitope-based ELISA could be potentially used for DRV or ARV surveillance. These findings provide insights into the organization of epitopes on σA protein that might be valuable for the development of epitope-based serological diagnostic tests for DRV and ARV infection.
Highlights
Duck reovirus (DRV) is a member of genus Orthoreovirus in the family of Reoviridae
Muscovy duck reovirus S12 was propagated in duck embryo or embryo fibroblasts (DEF) or BHK-21 cells as described previously [5,7,22]
The epitope reactions to sera against DRV and avian reovirus (ARV) were determined by the dot blotting assay
Summary
Duck reovirus (DRV) is a member of genus Orthoreovirus in the family of Reoviridae. The viral genome contains ten double-stranded RNA segments, including three size classes: large (L1–L3), medium (M1–M3) and small (S1–S4), which encode eight structural proteins (λA, λB, λC, μA, μB, σA, σB, and σC) and several nonstructural proteins (μNS, σNS, p10.8, etc.) [1,2,3,4,5,6,7,8]. The identification of epitopes for viral proteins can provide important information for understanding of immunological responses and pathogenesis in virus infection. Many epitope-based enzyme immunoassays have been successfully developed for the detection of virus-specific antibodies in serum samples [32,33,34,35]. We identified two minimal σA protein epitopes and assessed their cross-reactivity to DRV and ARV. An. ELISA based on expressed epitopes as antigens was evaluated to detect antibodies in serum samples from DRV- and ARV-infected birds. ELISA based on expressed epitopes as antigens was evaluated to detect antibodies in serum samples from DRV- and ARV-infected birds These findings will extend our understanding to σA protein structure–function relationships and provide insights into the improvement of the birds reovirus serodiagnosis and the understanding of the viral pathogenesis
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