Summary auto-generated
This study characterized epitopes on the coat protein of zucchini yellow mosaic potyvirus (ZYMV) using monoclonal antibodies and synthetic peptides. Three monoclonal antibodies (CC11, AB6, and DD2) were shown to recognize linear epitopes in the N-terminal region of the coat protein through epitope mapping with overlapping synthetic dodecapeptides. Point mutations found in natural ZYMV isolates from Martinique were introduced into infectious viral cDNA via site-directed mutagenesis to create two quasi-isogenic mutant strains (A and B). These mutations abolished or reduced recognition by specific antibodies without affecting viral symptomatology or multiplication. The mutants served as neutral genetic markers to study virus strain interactions. Sequential inoculation experiments on melon plants demonstrated that cross-protection between the two mutants was established in less than 48 hours, with the challenging strain unable to establish infection when the protecting strain had a head start of 24 hours or more. This rapid cross-protection was attributed to the close genetic relationship of the quasi-isogenic strains and represents faster protection establishment compared to previous studies with more divergent virus strains.
Key findings
- Three monoclonal antibodies recognized distinct linear epitopes in the N-terminal region of ZYMV coat protein, with specific amino acid sequences critical for antibody binding
- Site-directed mutagenesis successfully created quasi-isogenic ZYMV mutants that could be specifically differentiated by monoclonal antibodies while maintaining wild-type pathogenicity
- Cross-protection between the two mutant strains was rapidly established within 48 hours, with challenging virus unable to overcome infection if protection strain had 24+ hours head start
- The point mutations introduced served as neutral genetic markers for tracking virus strain competition without affecting viral fitness or symptom expression
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Abstract
Monoclonal antibodies (MAbs) raised against the coat protein of zucchini yellow mosaic potyvirus (ZYMV) were characterized by epitope mapping using synthetic oligopeptides. Two mutant viruses with a mutation in the amino acid sequence important for epitope recognition in vitro were obtained by site-directed mutagenesis of a full-length cDNA of ZYMV. Two MAbs, CC11 and DD2, could distinguish specifically between these mutants in mixed infections, or after sequential inoculations of muskmelons. Sequential inoculations of the mutants and analysis with MAbs CC11 and DD2 revealed that cross-protection was established between these quasi-isogenic strains within 48 h.