New molecular diagnostic and surveillance tools for orthotospoviruses and their thrips vectors

Abstract Collectively, orthotospoviruses and their thrips vectors are some of the most economically important pathogens and pests of crops worldwide. Rapid identification tools are needed, and variants of polymerase chain reaction (PCR) or reverse transcription-PCR (RT-PCR) still have an important role to play, but there are deficiencies in existing assays for both groups of organisms. The number of known orthotospoviruses has rapidly expanded in recent years, and existing universal RT-PCR assays no longer account for all possible sequence variation. The cytochrome c oxidase subunit I gene ( COX1 ) is the international standard for DNA barcoding of insects, but the PCR primers used to amplify this gene were designed many years ago using limited sequence datasets, and these primers have many primer-template mismatches when used with thrips. In this study, a universal RT-PCR assay for orthotospoviruses has been developed targeting the RNA-dependent RNA polymerase gene ( RdRp ) on the L RNA segment by identifying the most conserved protein motifs and taking advantage of the presence of codon usage biases in the virus genus. There is potential for a vector-enabled metabarcoding strategy to monitor the presence of orthotospoviruses using this universal RT-PCR assay. For robust virus testing in thrips, an internal control assay targeting the translation elongation factor 1-alpha RNA transcripts has been developed, which is predicted to be applicable to not only thrips but all hemipteran plant virus vectors. New COX1 DNA barcoding primers for thrips have also been designed that theoretically cover all species in the suborder Terebrantia, which includes all orthotospoviral vectors. All assays were validated using both synthetic nucleic acid templates and biological samples.