Development of nanobodies against Mal de Río Cuarto virus major viroplasm protein P9‑1 for diagnostic sandwich ELISA and immunodetection

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.

Instituto de Biotecnología

Fil: Llauger, Gabriela. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina

Fil: Llauger, Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Fil: Monti, Demian Esteban. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina

Fil: Monti, Demian Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Fil: Aduriz Guerrero, Matí­as. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología e Innovaciones Tecnológicas; Argentina

Fil: Aduriz Guerrero, Matí­as. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Fil: Romão, Ema. Vrije Universiteit Brussel. Lab of Cellular and Molecular Immunology; Bélgica

Fil: Dumon, Analia Delina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; Argentina

Fil: Dumon, Analia Delina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Fil: Mattio, Maria Fernanda. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; Argentina

Fil: Mattio, Maria Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Fil: Wigdorovitz, Andres. Instituto Nacional de Tecnología Agropecuaria (INTA). INCUINTA. Instituto de Virología e Innovaciones Tecnológicas; Argentina

Fil: Wigdorovitz, Andres. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Fil: Muyldermans, Serge. Vrije Universiteit Brussel. Lab of Cellular and Molecular Immunology; Bélgica

Fil: Muyldermans, Serge. Dalian University of Technology. School of Bioengineering. Liaoning Key Laboratory of Molecular Recognition and Imaging; China

Fil: Vincke, Cécile. Vrije Universiteit Brussel. Lab of Cellular and Molecular Immunology; Bélgica

Fil: Vincke, Cécile. VIB Center for Inflammation Research. Myeloid Cell Immunology Lab; Bélgica

Fil: Parreño, Viviana. Instituto Nacional de Tecnología Agropecuaria (INTA). INCUINTA. Instituto de Virología e Innovaciones Tecnológicas; Argentina

Fil: Parreño, Viviana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Fil: Del Vas, Mariana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina

Fil: Del Vas, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina