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.
Immunologic Tests/methods , Reoviridae , Viral Proteins , Zea mays/virology , Animals , Camelids, New World/immunology , Enzyme-Linked Immunosorbent Assay/methods , Escherichia coli/genetics , Plant Diseases/virology , Plants , Recombinant Proteins/analysis , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Reoviridae/immunology , Reoviridae/isolation & purification , Reoviridae/metabolism , Viral Proteins/analysis , Viral Proteins/biosynthesis , Viral Proteins/genetics
Las diarreas agudas son una de las afecciones más comunes en personas de todas las edades, especialmente en niños menores de 5 años. Rotavirus (RV) es la principal causa de diarrea en niños a nivel mundial seguido por norovirus (NoV). Sin embargo, a partir de la implementación de la vacunación con RV, NoV pasó a tener un rol preponderante en varios países como EEUU y Nicaragua. En Argentina, luego del inicio de la vacunación obligatoria de RV se logró un alto porcentaje de cobertura vacunal y se evidenció un descenso del 10% en la ocurrencia de diarreas agudas en general y un descenso del 50% de las diarreas causadas específicamente por RV, demostrando el éxito rotundo de la intervención. No existen al momento vacunas contra NoV, lo que lleva al desarrollo de otras estrategias de contención. A través del laboratorio INCUINTA del Instituto Nacional de Tecnología Agropecuaria (INTA), se han desarrollado y patentado nanoanticuerpos VHH contra RV y NoV con propiedades que los transforman en excelentes herramientas para el desarrollo de métodos de diagnóstico y para su utilización en estrategias de inmunidad pasiva oral para el tratamiento y prevención de la infección causada por estos agentes virales. Los nanoanticuerpos anti RV fueron utilizados con éxito para el desarrollo de un kit de ELISA nacional validado junto con el Instituto Malbrán que está siendo registrado para su comercialización. Además, demostraron poseer capacidad neutralizante de la infección contra RV in vitro y protección frente a la diarrea y excreción viralen los modelos de ratones lactantes y cerdos gnotobióticos. Los nano anticueroscontra NoV poseen alta afinidad para la detección del virus y fueron capaces de bloquear la unión del virus a los receptores celulares in vitro, demostrando su potencial como moléculas terapéuticas y de diagnóstico. El objetivo final de este desarrollo es completar la triada diagnostico-prevencióntratamiento para el control de las gastroenteritis virales con una tecnología de vanguardia de industria nacional.
Acute diarrhea is a common affection in people ofall ages, especially in children under five years old. Rotavirus (RV) is the major cause of diarrhea in children worldwide followed by norovirus (NoV). However, afterthe introduction of RV vaccine in the vaccination calendar in several countries like the USA and Nicaragua, NoV is having an important role in gastroenteritis incidence. In Argentina, after the introduction of the RV vaccine, high vaccine coverage was reached and the overall acute diarrhea incidence was reduced in a 10% and RV-associated diarrhea was reduced 50%, leading to complete success of the vaccination strategy. At the moment, there are no vaccines against NoV, thus, it is a need to develop therapies to control this disease. INCUINTA has developed and patented two sets of VHH nanobodies against RV and NoV with unique properties that make them suitable for the development of diagnostic tools, as well as to design oral passive immune strategies to preventand treat these two viral infections associated with diarrhea. VHH against rotavirus were able to neutralize RV infection in a suckling mouse model and in a gnotobiotic pig model of human RV infection and disease. Also, they were used to develop an ELISA kit for RV detection in human and animal samples. The ELISA kit was statistically validated in collaboration with the Malbrán Institute and is being registered for commercialization. VHH to NoVs showed high affinity to the virus and blocked the interaction between the virus and histo-blood antigens in target cells in vitro, showing to be a potential tool to detect and treat NoV infection. The final goal of this project is to complete the diagnostic-prevention-treatment triad to control viral gastroenteritis using a vanguard technology made in Argentina.
Camelids, New World , Rotavirus , Diarrhea , Single-Domain Antibodies
