Over 6% of global food production is lost due to pathogens, including plant viruses. Plant viruses in the field are often mismanaged or unmanaged due to difficulties in detection of the appropriate virus. Rapid diagnosis and containment is important to prevent disease spread and crop losses. Unfortunately, for many plant viruses this is not practical as the methods of detection are serology which depends on antibodies, many of which are not available, or nucleic acid extraction, amplification and genome sequencing. Both techniques require shipping samples to distant labs, adding time and expense to diagnosis.
Issues with antibodies in developing diagnostic tests for crops include the requirement for large amounts of target virus to develop antibodies against. In many cases obtaining such quantities of virus can be difficult or in some cases impossible and if sourced present with issues around containment in propagating wild type viruses. For these reasons, a team of researchers at the University of Leeds and their collaborators from the John Innes Centre, employed Affimer reagents in their newly published study in the journal Scientific Reports, ‘Affimer reagents as tools in diagnosing plant virus diseases (link is external)’ where they utilise Affimers in an ELISA to distinguish between infected and uninfected crude leaf extracts, that can be developed into a field-based diagnostic assay.
CPMV has been used across the biotechnology industry as a model for single stranded RNA viruses more generally, though as for lots of virus particles the use of infectious virus is associated with containment issues. Empty virus like particles (eVLP) of cowpea mosaic virus (CPMV) were used to screen the Affimer library identifying seven unique binders, with the best performing reagent being taken forward for further analysis. Identifying binders that recognised both the eVLP-CPMV and the WT CPMV was an important step for the researchers, allowing them to safely research and develop functional assays that could be effective in the field.
Cryo-electron microscopy was used to analyse the structure of the Affimer:CPMV complex to 3.4 Å, demonstrating that the Affimer reagent bound the large (L) viral coat protein subunit at the 2-fold symmetry axis of the capsid structure. To avoid misdiagnosis any reagent used to assay samples must be highly specific to the target virus. For this reason, the study used ELISA to examine whether the Affimer reagent was cross-reactive with other similar icosahedral viral structures, including Murine Norovirus (MNV), Satellite Tobacco Necrosis Virus (STNV), Hepatitis B (Hep B) and Potato Leaf Roll Virus (PLRV). ELISA results showed that the Affimer selectively bound both the eVLP-CPMV and WT-CPMV, but showed no cross-reactivity with the other viruses.
Using Affimers to the CPMV virus an ELISA was established that was able to accurately diagnose infected from uninfected leaf crude extracts in a blind experiment. For the purpose of ensuring the assay was suitable for field-based virus detection the leaf substrate material was subject only to manual grinding and dilution in PBS either at 1:5 or 1:20. Both concentrations of leaf crude extract showed clear signals above background when assayed by the Affimer-based ELISA.
This demonstration that Affimer reagents specifically detect CPMV in crude extracts of infected plant leaves presents the basis of a diagnostic test for development, and offers the potential for a wide variety of similar diagnostic tests for plant virus infections that could permit better management of food resources through improved infected crop handling in the field.
The video below shows a 3.4Å structure of CPMV bound to an Affimer: