The development of an enzyme linked immunosorbent assay for the detection of the South African strain(s) of grapevine fanleaf nepovirus
Date
2008-12
Authors
Liebenberg, Annerie
Journal Title
Journal ISSN
Volume Title
Publisher
Stellenbosch : Stellenbosch University
Abstract
South Africa is one of the top ten wine producing countries in the world. The South African wine
industry contributes approximately R16.3 billion to South Africa’s annual gross domestic product
with 42.8% of wine being exported. To compete with the top wine producing countries and to
ensure a viable export market, South Africa needs to ensure that healthy, virus free propagation
material is produced and sold. One of the viruses that need to be tested for is Grapevine fanleaf
virus (GFLV). Grapevine fanleaf virus causes degeneration and malformation of berries, leaves and
canes and is responsible for significant economic losses by reducing crop yields by as much as
80%, reducing the longevity of the vines and affecting fruit quality. It is widespread in the Breede
River Valley of the Western Cape where the nematode vector, Xiphinema index, is prevalent. The
Breede River Valley contributes approximately 30% of the total production of the local wine
industry, and severe losses in this region could threaten the viticulture. The Plant Improvement Act
states 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-linked
Immunosorbent Assay (DAS-ELISA) and the kits are imported from Europe at a significant cost to
the South African viticulture industry.
The objective of this study was to produce a reliable and sensitive diagnostic assay specific for the
South African strains of GFLV. This project aimed to develop and optimize a DAS-ELISA, by
using recombinant DNA technology to produce antibodies against bacterially expressed viral coat
protein. 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 production
of 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 ImmunoStrip
tests (Agdia) to assess the reliability, sensitivity and specificity of the rapid-direct-one-tube-RTPCR
assay. Twelve GFLV isolates from South Africa were sequenced to investigate the variability
between the isolates as well as the variability between the South African isolates and GFLV
sequences available in Genbank. Sequence identities between clones from different GFLV isolates
from 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 found
between geographical origin and symptoms, nor between geographical origin and sequence
variability or between grapevine cultivar and symptom expression. Of the 23 samples tested with all
three tests, 21 tested positive with rapid-direct-one-tube-RT-PCR, 19 with the ImmunoStrips and 17
with an imported DAS-ELISA kit (Agdia). Rapid-direct-one-tube-RT-PCR was found to be the
most reliable technique for GFLV detection.
Although the establishment of a DAS-ELISA directed to the South African strain(s) of GFLV was
not successful, an alternative PCR based diagnostic system was developed, and proved to be
sensitive and reliable. RT-PCR based diagnostic assays are generally accepted to be more sensitive
than DAS-ELISA, but the latter is still used as the diagnostic assay of choice for routine testing due
to ease of use. This rapid-direct-one-tube-RT-PCR assay is a rapid, sensitive and reliable diagnostic
test, reducing the prevalence of false negatives, contributing to a virus free viticulture industry. The
rapid-direct-one-tube-RT-PCR assay is as easy to use as DAS-ELISA, faster and can be performed
by semi skilled workers, thus providing all the advantages associated with DAS-ELISA.
Description
Thesis (MSc (Genetics))--Stellenbosch University, 2008.
Keywords
GFLV, DAS-ELISA, Grapevine fanleaf virus, Double antibody sandwich-enzyme-linked immunosorbent assay, Virus diseases of grapes, Dissertations -- Genetics, Theses -- Genetics, Grapes -- Diseases and pests -- South Africa, Virus diseases of plants -- South Africa