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Abstract
Mal de Río Cuarto virus (MRCV) is a member of the genus Fijivirus of the family Reoviridae that causes a devastating disease in maize and is persistently and propagatively transmitted by planthopper vectors. Virus replication and assembly occur within viroplasms formed by viral and host proteins. This work describes the isolation and characterization of llama-derived Nanobodies (Nbs) recognizing the major viral viroplasm component, P9-1. Specific Nbs were selected against recombinant P9-1, with affinities in the nanomolar range as measured by surface plasmon resonance. Three selected Nbs were fused to alkaline phosphatase and eGFP to develop a sandwich ELISA test which showed a high diagnostic sensitivity (99.12%, 95% CI 95.21–99.98) and specificity (100%, 95% CI 96.31–100) and a detection limit of 0.236 ng/ml. Interestingly, these Nanobodies recognized different P9-1 conformations and were successfully employed to detect P9-1 in pull-down assays of infected maize extracts. Finally, we demonstrated that fusions of the Nbs to eGFP and RFP allowed the immunodetection of virus present in phloem cells of leaf thin sections. The Nbs developed in this work will aid the study of MRCV epidemiology, assist maize breeding programs, and be valuable tools to boost fundamental research on viroplasm structure and maturation.
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1 Instituto de Agrobiotecnología y Biología Molecular (IABIMO), CICVyA INTA, UEDD INTA/CONICET, Buenos Aires, Argentina
2 INCUINTA, Instituto de Virología e Innovaciones Tecnológicas (IVIT), CICVyA INTA, UEDD INTA/CONICET, Buenos Aires, Argentina
3 Vrije Universiteit Brussel, Lab of Cellular and Molecular Immunology, Brussels, Belgium (GRID:grid.8767.e) (ISNI:0000 0001 2290 8069)
4 Unidad de Fitopatología y Modelización Agrícola (UFYMA), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Córdoba, Argentina (GRID:grid.423606.5) (ISNI:0000 0001 1945 2152); Instituto de Patología Vegetal (IPAVE), Instituto Nacional de Tecnología Agropecuaria (INTA), Centro de Investigaciones Agropecuarias (CIAP), Córdoba, Argentina (GRID:grid.419231.c) (ISNI:0000 0001 2167 7174)
5 INCUINTA, Instituto de Virología e Innovaciones Tecnológicas (IVIT), CICVyA INTA, UEDD INTA/CONICET, Buenos Aires, Argentina (GRID:grid.419231.c)
6 Vrije Universiteit Brussel, Lab of Cellular and Molecular Immunology, Brussels, Belgium (GRID:grid.8767.e) (ISNI:0000 0001 2290 8069); Dalian University of Technology, Liaoning Key Laboratory of Molecular Recognition and Imaging, School of Bioengineering, Dalian, People’s Republic of China (GRID:grid.30055.33) (ISNI:0000 0000 9247 7930)
7 Vrije Universiteit Brussel, Lab of Cellular and Molecular Immunology, Brussels, Belgium (GRID:grid.8767.e) (ISNI:0000 0001 2290 8069); VIB Center for Inflammation Research, Myeloid Cell Immunology Lab, Brussels, Belgium (GRID:grid.510970.a)
8 INCUINTA, Instituto de Virología e Innovaciones Tecnológicas (IVIT), CICVyA INTA, UEDD INTA/CONICET, Buenos Aires, Argentina (GRID:grid.510970.a)
9 Instituto de Agrobiotecnología y Biología Molecular (IABIMO), CICVyA INTA, UEDD INTA/CONICET, Buenos Aires, Argentina (GRID:grid.510970.a)