| Summary: | Thesis (MSc (Genetics))--University of Stellenbosch, 2005. === An infectious clone of a viral RNA genome is one that can be used, either as an in vitro transcript or as
cDNA, to produce an infection in a susceptible plant. Infectious clones serve as a tool to study viral
RNA genomes at a molecular level to gain deeper insight into genome organization, viral gene
function, presence of regulatory sequences and gene expression. In the Western Cape (and elsewhere) a
new crippling grapevine disease, known as Shiraz disease, is emerging of which the aetiology and
pathogenic agents involved are not yet fully understood. Grapevine virus A (GVA), genus Vitivirus,
family Flexiviridae, is thought to be the associated with this disease. The aim of this study was to
construct a full-length infectious cDNA clone of GVA, which will aid in the molecular study of the
viral genome. This clone could ultimately be used to investigate GVA’s involvement in Shiraz disease,
which could lead to the unravelling of the aetiology and control of the disease. A full-length clone of
GVA, named GVA-IC2/T7-2972-3, was constructed in several steps using restriction digestion/ligation
and primer overlap extension PCR. Grapevine virus A cDNA fragments were obtained from GVAinfected
Nicotiana benthamiana and Vitis vinifera plants using three different techniques, of which the
Rapid direct-one-tube RT-PCR was most successful. A 5’ T7 promoter and a 3’ poly-A tail were
incorporated and the full-length clone was cloned into pBluescript II SK (+). Full-length sequencing
of the clone, revealed two significant frameshift mutations. The first mutation was a single base pair
insertion (one G) in a slippery site of 6 G’s at position 1380 – 1385 in open reading frame one (ORF 1)
of the viral genome. This mutation was corrected by PCR-based site-directed mutagenesis, which
resulted in pSK-GVA-mutagen-3 and pSK-GVA-mutagen-4. The second mutation was a single base
pair deletion (one G) at position 6959 in ORF4, which coded for the coat protein (CP). Several
techniques were attempted to correct this mutation, but none were successful. Even though the second
mutation could not be corrected, in vitro transcriptions were performed on three clones followed by
subsequent infections of N. benthamiana plants. The three clones included pSK-GVA-mutagen-3, pSKGVA-
mutagen-4 (both hosting the mutation at position 6959) and GVA-IC2/T7-2972-3 (hosting both
mutations). At 21 days post-inoculation no significant visual symptoms were observed in plants
infected with in vitro RNA or in plants infected with wild type GVA. Rapid direct-one-tube RT-PCR
results revealed the presence of viral RNA in infected leaves and apical leaves of infected plants, and
provided preliminary evidence that the mutated clones were still capable of systemic infection and viral
movement. These results are still inconclusive, and several post-infection studies will have to be
performed to confirm these findings. Koch's postulates will also have to be proved in order to confirm the infectious nature of the clones. The effect of the two mutations in the constructed clones will be
investigated further and post-infection analysis performed to deduce whether the viral progeny are
devoid of the mutations. Three full-length GVA cDNA clones (hosting mutations) seemingly capable
of systemic infection in N. benthamiana plants were constructed in this study and have laid the
foundation for molecular and mutational analysis of the GVA genome. This could lead to the study of
pathogen-host interactions in order to unravel the aetiology of Shiraz disease in the future.
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