Bibliography: leaves 104-130. The aim of this thesis is to characterise subgenomic RNAs of cucumoviruses and the functions of their encoding genes. Strains of cucumber mosaic virus (CMV) are classified into two major subgroups (I and II) on the basis of nucleotide sequence homology. The V strain of tomato aspermy virus (V-TAV) and a subgroup I CMV strain (WAII) are chosen to determine whether the 2b genes encoded by these viruses are expressed 'in vivo'. For further investigation of the 2b gene function, cDNA clones of three genomic RNAs of V-TAV are constructed. Using the infectious cDNA clones of V-TAV, a mutant virus containing only one of the two repeats is constructed.

Typescript. Primarily to identify a virus which was isolated from a single Gladiolus plant early in 1962 which showed some resemblance to Cucumber mosaic virus. Exemplifies the large differences which may occur in the biological properties of viruses, even though their physico-chemical properties may be similar.

Viruses or viruslike particles of eukaryotic algae are ubiquitous in aquatic habitats, however, suprisingly little is known about them. The research presented here focused on one such virus which infects a multicellular filamentous brown alga of the genus Feldmannia. Although preliminary studies had been performed on the genome structure of the Feldmannia sp. Virus (FsV), little was known. The purpose of this study was to analyze the structure of the FsV genome in detail. During the experiments aimed at mapping the FsV genome, cross-hybridization was observed among five BamHI-fragments of the digested FsV DNA. Sequence analysis of one of those fragments revealed the presence of 173 by direct repeats. There are two FsV genomes of different size-classes (158 and 178 kbp). The 173 by repeats in the cross-hybridizing BamHI-fragments were confined to a small region of each virus genome. The number of these repeats in the 178 kbp genome was estimated to be about 109 and in the 158 kbp genome to be about 41. in the 178 kbp genome, the repeats are contained within a 22 kbp region and in the 1.58 kbp genome, the repeats are contained within a 10 kbp region. These viruses are actively replicated in sporophyte plants. A family of related 173 by direct repeats was discovered in an encrypted FsV genome. The family of repeats estimated to be greater than 50 kbp in length were found inserted into a protein kinase gene encoded within the 3.6 kbp viral BamHI-fragment Z. Southern analysis indicates that these repeats in the encrypted FsV genome are distinct from the previously characterized repeats in the amplified FsV genome. The translated protein kinase shares highest homologies to the SNF1 subfamily of serine/threonine protein kinases and contains a potential autophosphorylation site in a region unique to this protein kinase. A DNA polymerase gene was identified in the FsV genome. The predicted peptide sequence of the FsV DNA polymerase gene contains all of the conserved motifs found in B-family (a-like) DNA polymerases. A TTTTTNT sequence motif shown to be a transcription termination signal for Vaccinia virus early genes is found at the 3' end of the DNA polymerase gene. Phylogenetic analysis of the FsV DNA polymerase gene and other viral DNA polymerase genes indicates that FsV belongs to a group of algal viruses recently defined as Phycodnaviridae. / Graduation date: 1998

Bibliography: leaves 104-130. / vi, 130, [25] leaves, [13] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / The aim of this thesis is to characterise subgenomic RNAs of cucumoviruses and the functions of their encoding genes. Strains of cucumber mosaic virus (CMV) are classified into two major subgroups (I and II) on the basis of nucleotide sequence homology. The V strain of tomato aspermy virus (V-TAV) and a subgroup I CMV strain (WAII) are chosen to determine whether the 2b genes encoded by these viruses are expressed 'in vivo'. For further investigation of the 2b gene function, cDNA clones of three genomic RNAs of V-TAV are constructed. Using the infectious cDNA clones of V-TAV, a mutant virus containing only one of the two repeats is constructed. / Thesis (Ph.D.)--University of Adelaide, Dept. of Plant Science, 1997

The sequence of 3553 nucleotides corresponding to the 3'-terminal region of potato virus S (PVS) has been determined from cloned cDNA. The sequence obtained contains six open reading frames with the potential to encode proteins of Mr 10,734, Mr 32,515, Mr 7,222, Mr 11,802, Mr 25,092 and at least Mr 41,052. The amino acid sequence of the 33K ORF has been confirmed to be that of the viral coat protein gene. The nucleotide sequence of this ORF was obtained from expression plasmids which were isolated by binding with a specific monoclonal antibody to PVS, and the expression of coat protein fusion products was verified by Western blots of bacterial cell lysates. The deduced amino acid sequence of a 70 amino acid portion from the central region of the PVS coat protein was 59% identical to the analogous region of potato virus X. In addition, the 7K, 12K and 25K ORF's displayed significant sequence homology with similar sized ORF's from a number of potexviruses. The partial 41K ORF was homologous with the C-terminal portion of the viral replicase proteins of potato virus X and white clover mosaic virus. While the biological functions of the 12K and 25K non-structural proteins coded for by PVS and members of the potexvirus group remain unknown, the 12K protein displays a hydropathicity profile consistent with a membrane associated protein and the 25K protein contains a conserved sequence motif found in a number of nucleoside triphosphate binding proteins. Members of the carlavirus group are distinguished from the potexviruses by the presence of a small [11K (PVS, potato virus M) - 16K (lily symptomless virus)] 3' terminal ORF which appears to contain a sequence motif similar to the 'zinc-finger' domain found in many nucleic acid binding proteins. The coat protein gene from potato virus S (PVS) was introduced into Nicotiana debneyii tobacco as well as a commercial potato cultivar, 'Russet Burbank', by leaf disc transformation using Agrobacterium tumefaciens. Transgenic plants expressing the viral coat protein were highly resistant to subsequent infection following mechanical inoculation with the Andean or ME strains of PVS as indicated by a lack of accumulation of virus in the upper leaves. The coat protein mediated protection afforded by these transgenic plants was sufficient to prevent the accumulation of virus in the tissues of non-transformed 'Russet Burbank' shoots which had been grafted onto transgenic plants inoculated with PVS, and in reciprocal grafts, transgenic shoots accumulated less than 2% (6 weeks after grafting) of the concentration of PVS found in non-transformed shoots similarly grafted onto plants systemically infected with PVS. These transgenic plants also displayed a measure of resistance to inoculation with a related carlavirus from potato, potato virus M. In agreement with previous reports for plants expressing PVX coat protein, plants expressing PVS coat protein were also protected from inoculation with PVS RNA. These results provide further evidence that coat protein mediated protection for these two groups of viruses, which share similar genome organizations, may involve inhibition of some early event in infection, other than, or in addition to, virus uncoating. Specific monoclonal antibodies were prepared against a C-terminal derived 18 kDa portion of the 25K protein of PVS expressed as an in-frame chimeric fusion protein with the glutathione S-transferase gene. The in vivo expression of this non-structural protein in virus infected tissue, as well as tissue from transgenic tobacco (var Xanthi-nc) engineered to contain the entire 25K gene, was verified by Western immunoblot labelling. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Potato virus Y (PVY) is an economically important virus worldwide. In South Africa, PVY has been shown to be a major limiting factor in the production of important solanaceous crops, including potato (Solanum tuberosum L.), pepper (Capsicum annuum L.), tomato (Lycopersicon esculentum Mill.) and tobacco (Nicotiana spp). The variability that PVY displays, wherever the virus occurs, merits the study of the isolates occurring in KwaZulu-Natal (KZN) in the Republic of South Africa (RSA). This characterization will provide a clear understanding of strains/isolates from local vegetables and how they relate to the other PVY strains already identified, as well as information that can be used to manage the diseases they cause. Hence, the aim of this project was to study the biological and genetic properties of PVY isolates infecting potato, tomato and pepper in KZN. Enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies and reverse transcription polymerase chain reaction (RT-PCR) using primers specific to all PVY strains were used to detect the virus in plant material showing PVY-like symptoms collected from various locations in KZN. A total of 39 isolates (18 isolates infecting tomato, 12 infecting potato and 9 infecting pepper) were further differentiated into strains by means of ELISA using strain specific antibodies and RT-PCR using primers specific to the different strains of PVY identified around the world. All PVY isolates infecting tomato and pepper tested positive for the ordinary PVYO strain with both ELISA and RT-PCR. PVY isolates infecting potato were more diverse and comprised the PVYN, PVYNTN and PVYNWilga strains, with mixed infections noted in some cases. The biological properties were studied by mechanically inoculating Chenopodium quinoa, Nicotiana tabacum cv Xanthi, N. tabacum cv Samsun, N. glutinosa, and N. rustica with leaf extracts from plants infected with the different PVY strains detected in this study. All inoculated C. quinoa plants did not show symptoms. All tobacco plants showing symptoms were tested for the presence of PVY by means of ELISA using monoclonal antibodies targeting all strains and electron microscopy using the leaf dip technique. Not all the inoculated tobacco tested positive with ELISA. The symptoms observed were therefore divided into PVY-related and PVY non- related. PVY-related symptoms included vein clearing, mosaic chlorosis, stunting, and vein necrosis. PVY non-related symptoms included wrinkles and leaf distortions. Potyvirus-like particles of about 700 nm were observed under the transmission electron microscope (TEM) from plants showing PVY-related symptoms while rod shaped viral particles of sizes varying between 70 and 400 nm were observed from plants showing non-PVY related symptoms. A portion of the virus genome (1067 bp) covering part of the coat protein gene and the 3’ non-translated region (NTR) of three PVYO isolates infecting tomato, one PVYO isolate infecting pepper and one PVYNWilga isolate infecting potato were amplified, cloned and sequenced. The 5’ NTR, P1, HC-Pro and part of P3 regions (2559 bp) of a PVYN isolate infecting potato were also amplified, cloned and sequenced. Sequence data was compared with selected PVY sequences from different geographical locations around the world. These were available on the NCBI website and subsequently used for phylogenic analyses. The sequenced genomic regions of the PVYN isolate were found to be 99% similar to the New Zealand PVYN isolate (GenBank accession number: AM268435), the Swiss PVYN isolate CH605 (X97895) and the American PVYN isolate Mont (AY884983). Moreover, the deduced amino acid sequence comparison of the genomic regions of the PVYN isolate revealed the presence of five distinct amino acids residues. The three amino acid residues (D205, K400, and E419), which determine the vein necrosis phenotype in tobacco, were also identified. The coat protein and 3’ NTR sequences of all KZN PVYO isolates infecting pepper and tomato were closely similar to each other than to KZN PVYNWilga isolate infecting potato. The phylogenic analysis clustered the KZN PVYN isolate with the European sublineage N, PVYNWilga isolate infecting potato with the American PVYO isolate Oz (EF026074) in the O lineage and all PVYO isolates infecting tomato and pepper in a new sublineage within the O lineage. Taken together, these results point to the presence of PVY in solanaceous vegetables cultivated in KZN and they lay the foundation for the formulation of effective control measure against PVY diseases in KZN. / Thesis (M.Sc.) - University of KwaZulu-Natal, Pietermaritzburg, 2009.