The development of an enzyme linked immunosorbent assay for the detection of the South African strain(s) of grapevine fanleaf nepovirus

[摘要] South Africa is one of the top ten wine producing countries in the world. The South African wineindustry contributes approximately R16.3 billion to South Africa's annual gross domestic productwith 42.8% of wine being exported. To compete with the top wine producing countries and toensure a viable export market, South Africa needs to ensure that healthy, virus free propagationmaterial is produced and sold. One of the viruses that need to be tested for is Grapevine fanleafvirus (GFLV). Grapevine fanleaf virus causes degeneration and malformation of berries, leaves andcanes and is responsible for significant economic losses by reducing crop yields by as much as80%, reducing the longevity of the vines and affecting fruit quality. It is widespread in the BreedeRiver Valley of the Western Cape where the nematode vector, Xiphinema index, is prevalent. TheBreede River Valley contributes approximately 30% of the total production of the local wineindustry, and severe losses in this region could threaten the viticulture. The Plant Improvement Actstates that all propagation material sold must be tested for GFLV by a reputable scientific technique.The technique commonly used in South Africa is the Double Antibody Sandwich - Enzyme-linkedImmunosorbent Assay (DAS-ELISA) and the kits are imported from Europe at a significant cost tothe South African viticulture industry.The objective of this study was to produce a reliable and sensitive diagnostic assay specific for theSouth African strains of GFLV. This project aimed to develop and optimize a DAS-ELISA, byusing recombinant DNA technology to produce antibodies against bacterially expressed viral coatprotein. Total RNA was extracted from GFLV infected grapevine material and the viral coat protein(CP) amplified. The CP was cloned into the pGex-6P-2 expression vector, fusing a Glutathione STransferase(GST) partner to the viral coat protein enhancing solubility and protein purification.Insufficient amounts of the soluble protein were expressed and purified, preventing the productionof antibodies and thus the development of the DAS-ELISA.An alternative diagnostic rapid-direct-one-tube-RT-PCR assay was developed. This rapid-directone-tube-RT-PCR assay was compared to commercially available DAS-ELISA and ImmunoStriptests (Agdia) to assess the reliability, sensitivity and specificity of the rapid-direct-one-tube-RTPCRassay. Twelve GFLV isolates from South Africa were sequenced to investigate the variabilitybetween the isolates as well as the variability between the South African isolates and GFLVsequences available in Genbank. Sequence identities between clones from different GFLV isolatesfrom South Africa were between 86-99% and 94-99% at the nucleotide and amino acid levels,respectively. Phylogenetic analysis based on the coat protein gene sequences showed that the South African isolates form two distinct clades or sub-populations. No significant correlation was foundbetween geographical origin and symptoms, nor between geographical origin and sequencevariability or between grapevine cultivar and symptom expression. Of the 23 samples tested with allthree tests, 21 tested positive with rapid-direct-one-tube-RT-PCR, 19 with the ImmunoStrips and 17with an imported DAS-ELISA kit (Agdia). Rapid-direct-one-tube-RT-PCR was found to be themost reliable technique for GFLV detection.Although the establishment of a DAS-ELISA directed to the South African strain(s) of GFLV wasnot successful, an alternative PCR based diagnostic system was developed, and proved to besensitive and reliable. RT-PCR based diagnostic assays are generally accepted to be more sensitivethan DAS-ELISA, but the latter is still used as the diagnostic assay of choice for routine testing dueto ease of use. This rapid-direct-one-tube-RT-PCR assay is a rapid, sensitive and reliable diagnostictest, reducing the prevalence of false negatives, contributing to a virus free viticulture industry. Therapid-direct-one-tube-RT-PCR assay is as easy to use as DAS-ELISA, faster and can be performedby semi skilled workers, thus providing all the advantages associated with DAS-ELISA.

[发布机构] Stellenbosch University