Serological detection of tomato spotted wilt virus (TSWV) proteins and identification of components of TSWV acquisition by thrips

Tomato spotted wilt virus (TSWV) has enormous economic impact on agriculture due to its geographical distribution and wide host range. It replicates in and is transmitted by the western flower thrips (WFT) (Frankliniella occidentalis). High affinity monoclonal antibodies (Mabs) were made to the non-structural protein (NSs) encoded by the small RNA of TSWV using antigen coated magnetic beads. These Mabs were used to develop an antigen coated plate enzyme-linked immunosorbent assay (ACP-ELISA) for rapid detection of TSWV. Identification of viruliferous thrips (thrips that acquired the virus as larvae and thus as adults can transmit the TSWV) from a population of thrips that includes viruliferous and non-viruliferous thrips (thrips that acquire TSWV as adults as a result of feeding on infected plants and thus cannot transmit the TSWV to a healthy host) would allow vector populations to be used as a predictor of TSWV incidence in a preplanting forecast. Since the presence of NSs is indicative of replication of TSWV in its vector, an ACP-ELISA was developed employing the anti-NSs Mabs to differentiate transmitters from non-transmitters. Addition of Zwitterionic detergent Empigen-BB at 0.1% in antibody dilution buffer reduced the nonspecific background. Electron microscope observations and circumstantial evidence indicate insect acquisition of TSWV is receptor mediated and the TSWV membrane glycoproteins (GP1 and GP2) serve as virus attachment proteins (VAP). To investigate the putative role of GPs as VAP and to identify the corresponding cellular receptor site (CRS), Mabs to GP1 and GP2 were produced against GP1 and GP2 antigens isolated using ChromaPhor{dollar}spcircler{dollar} protein recovery method. By employing gel overlay assays a 50 kDa protein in the excised insect guts was identified as a CRS. This 50 kDa protein specifically hybridized with anti-idiotype antibodies to anti-GP1 and anti-GP2 Mabs on a Western blot supporting the hypothesis that the 50 kDa protein is a cellular receptor for TSWV.