published_or_final_version / Biological Sciences / Master / Master of Philosophy

PhD / In South Africa, tomato (Solanum /ycopersicum) is an important vegetable crop with considerable nutritional and economic value. Over the last decade, begomovirus (family Geminiviridae) infections associated with an upsurge of the whitefly vector, Bemisia tabaci, on tomato crops has become a serious threat to sustainable tomato production in South Africa. Begomovirus disease control in tomato is challenging and requires an integrated "pest" and "vector" management strategy, primarily based on the use of chemical and cultural practices aimed at reducing the virus vector as well as the use of resistant cultivars. Development of effective disease management practices for South Africa therefore requires detailed information on the complex vector-virus-host cropping system interactions. The aim of the study presented in this thesis was to investigate the South African whitefly vector/begomovirus/tomato-host pathosystem, with emphasis on the virus and vector diversity and distribution, and the identification of possible resistance sources. A survey of tomato-infecting begomoviruses was conducted during a six-year period (2006-2011 ). Techniques used to determine begomoviruses diversity included whole genome amplification using PCR, RCA (rolling circle amplification), conventional as well as next generation sequencing and development of a RCA-RFLP (restriction fragment length polymorphism) for rapid assessment of diversity. Sequence comparisons and phylogenetic analyses revealed the presence of three new monopartite begomovirus species, in addition to ToCSV, all of which belong to the African/South West Indian Ocean (SWIO) begomovirus clade. Recombination analysis indicated that all four tomato-infecting begomovirus species appear to be complex recombinants and suggests that they have evolved within the sub-Saharan Africa region, along with other African begomoviruses and that they are most likely indigenous to the region. Several weed species were also confirmed as symptomless begomovirus reservoirs, supporting their role in the emerging begomovirus epidemics in South Africa.

Primary Epstein-Barr virus (EBV) infection in childhood is typically asymptomatic, but infection later in life results in a mononucleosis with the illness severity ranging from asymptomatic to requiring hospitalisation. The illness is generally short-lived (four to six weeks following onset of symptoms), but persistent disabling symptoms (lasting up to 6 months or longer) are well described in around ten percent of individuals. The aim of this work was to characterise immunological and virological parameters in the peripheral blood, which correlate with persistence of symptoms following EBV-induced mononucleosis. Subjects were recruited prospectively following confirmed primary EBV infection, to allow blood samples and clinical data to be collected at multiple timepoints (baseline, 2 weeks, 4 weeks, 3 months and at least 12 months later). Subjects with 6 months or more of disabling symptoms were defined as ???cases??? with control subjects being those whose illness resolved within 6 weeks of enrolment. Cases were compared with control subjects in terms of: cellular EBV viral load in the peripheral blood by PCR; development of antibodies against EBV VCA (IgG and IgM) and EBNA-1 (IgG) by ELISA; proportions of peripheral blood leucocyte subsets and their activation status by flow cytometry; the magnitude, kinetics of development, and breadth of the CD8+ cytotoxic cell response by interferon-?? Elispot; cytokine levels in serum, and production by peripheral blood mononuclear cells ex vivo; and gene expression patterns in peripheral blood mononuclear cells by microarray. With the exception of gene expression, none of these parameters correlated with early resolution of symptoms or predicted clinical outcome following primary EBV infection. Antibody patterns suggest a tendency to Th2 type immune response may be associated with persistent illness. Preliminary analysis of the gene expression studies indicates that there are many genes involved in this complex disease requiring further investigation. Persistent illness following EBV infection is not associated with uncontrolled viral replication, or chronic immune activation due to an aberrant primary immune response.

Geminiviruses ( family Geminiviridae ) have circular single - stranded genomes encapsidated in twinned quasi - isometric particles and are responsible for major crop losses worldwide. The largest genus, Begomovirus, comprises viruses transmitted by the whitefly Bemisia tabaci. Most begomoviruses have bipartite genomes, termed DNA A and DNA B. The DNA A component encodes proteins required for viral DNA replication and encapsidation whereas the DNA B encodes two proteins that are essential for systemic movement. A small number of begomoviruses have a monopartite DNA genome that resembles the DNA A of bipartite begomoviruses. This DNA carries all gene functions for replication and pathogenesis. Specific small circular single - stranded DNA satellites containing a single open reading frame ( ORF ), termed DNA β, have recently been found in association with certain monopartite begomovirus infections. They comprise about 1350 nucleotides and require a helper begomovirus for replication and encapsidation. DNA β contributes to the production of symptoms and enhanced helper virus accumulation in certain hosts. This study examines the role of DNA β satellite in viral pathogenicity and movement in the host plant. Infectivity analysis of Tomato leaf curl virus and DNA β having mutation in the C1 and V1 ORF indicated that the complementary - sense ORF, βC1, is responsible for inducing disease symptoms in Nicotiana tabacum. An ORF present on the plus strand, βV1, appeared to have no role in pathogenesis. Tobacco plants transformed with the βC1 ORF under the control of the Cauliflower mosaic virus 35S promoter, or with a dimeric DNA β exhibited severe disease - like phenotypes, while plants transformed with a mutated version of βC1 appeared normal. Northern blot analysis of RNA from the transgenic plants using strand - specific probes identified a single complementary - sense transcript. The transcript carried the full βC1 ORF encoding a 118 amino acids product. It mapped to the DNA β nucleotide ( nt ) position 186 - 563 and contained a polyadenylation signal 18 nt upstream of the stop codon. A TATA box was located 43 nt upstream of the start codon. These results indicate that βC1 protein is responsible for DNA β induced disease symptoms. Tomato leaf curl New Delhi virus ( ToLCNDV ) is a bipartite begomovirus in which both DNA A and DNA B are required for systemic infection. Inoculation of tomato plants with ToLCNDV DNA A alone induced local but not systemic infection whereas co - inoculation with DNA A and the DNA β resulted in systemic infection. The presence of both DNA A and the DNA β in systemically infected tissues and the absence of DNA B was confirmed by probe hybridization. DNA β containing a disrupted βC1 ORF did not mobilize the DNA A for systemic infection. Co - inoculation of plants with DNA A and a construct of βC1 ORF, under the control of the Cauliflower mosaic virus 35S promoter, resulted in the systemic movement of the DNA A. βC1 fused to GFP accumulated around and inside the nucleus, at the periphery of tobacco and onion epidermis cells and co - localized with the endoplasmic reticulum. This distribution would be consistent with βC1 mediating intra cellular transport from the nucleus to the plasma membrane. These results showed that the βC1 protein can replace the functions of DNA B in allowing the systemic movement of a bipartite geminivirus DNA A. / Thesis (Ph.D.)--School of Agriculture, Food and Wine, 2006.

Graduation date: 2000

Six 3 to 4 weeks old, cesarian-derived lambs were inoculated with ovine an adenovirus isolate 475N. Inoculated lambs showed moderate clinical signs of respiratory distress, conjunctivitis, and loose feces during the 10-day observation period. Virus was detected from nasal and conjunctival swabs starting on postinoculation day (PID) 2. Virus was detected in the feces in a inconsistent fashion. At necropsy, virus was present in the lung, tonsils, and bronchial and mediastinal lymph nodes of lambs necropsied on PID 5 and 7. Tissue samples from gastrointestinal tract and kidney were negative for the virus. Presence of virus in the feces was believed to be from replication in tonsillar tissue. At necropsy, lambs showed signs of pneumonia and numerous intranuclear inclusion bodies were detected in affected lung tissue. Virus neutralizing antibodies appeared at low levels in serum on PID 6 and reached higher levels by PID 10. Six ovine adenovirus prototype species, three uncharacterized ovine and bovine adenoviruses isolates and two uncharacterized llama adenoviruses isolates were digested with four different restriction enzymes. Digested viral DNA was separated in 0.7% agarose gels. The enzymes Barn HI, Eco RI, Hind III, and Pst I digested viral DNA and produced 2-10 bands. The profile of the band distribution permitted the differentiation of the viruses under study. However, further studies using multiple isolates of each species are required to determine if this procedure will efficiently distinguish different species of ruminant adenoviruses. Ten adenoviruses from sheep (including the six prototype species), one from bovine and one from llama were studied by virus neutralization test to determine their degree of antigenic similarities. Reciprocal virus neutralization tests were performed and the degree of antigenic similarities, i.e., strain differentiation was determined by criteria established by the International Committee for the Nomenclature of Viruses. Isolates 32CN (a bovine adenovirus) and 475N (an ovine adenovirus) were antigenically identical and not neutralized by any of the prototype species antiserum. They are candidates for a new species of ruminant adenoviruses. Ovine adenovirus isolate 47F was shown to be a member of OAV-5 species while the llama adenovirus strain represents a newly recognized species for this animal. / Graduation date: 1996

Monoclonal antibodies were developed against USDA challenge strain of infectious laryngotracheitis virus (ILTV). Indirect immunofluorescence test was used to detect antibodies in supernatants of hybridomas. Hybridoma cells were developed by fusing Sp 2/0 myeloma cells with spleen cells obtained from mice immunized four times with partially purified USDA challenge strain of infectious laryngotracheitis virus. The supernatant of three hybridomas, designated as 2D1D8, 2E11G2, 2C6C7 were found positive for antibody activity against USDA challenge strain of ILTV. Hybridomas producing antibodies were cloned by the limiting dilution method. All three monoclonal antibodies reacted with USDA challenge strain of ILTV, S 88 00224 strain of ILTV, and 86 1169 strain of ILTV in an indirect immunofluorescence test. None of the monoclonal antibodies reacted with avian adenovirus 301 or parrot herpes virus in an indirect immunofluorescence test. The monoclonal antibodies were isotyped, and all three monoclonal antibodies were found to be IgM. / Graduation date: 1993

A safe, potent and broad-spectrum antiviral is urgently needed to combat emerging viral respiratory diseases such as avian influenza H5N1 and H7N9, severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS). Previous studies carried out by PhD students in our lab found that mouse β-defenisn 4 (mBD4) shows highly antiviral activity in vitro. However, the recombinant mBD4 (rmBD4) expressed by E.coli is limited to very small scale of production and is very expensive. Thus, in this study, we firstly screened 16 short peptides derived from mBD4 and other mouse and human β-defensins for identifying their antiviral effects. One short peptide P9 (30 amino acids), derived from mBD4, exhibited potent and broad-spectrum antiviral effects against multiple respiratory viruses, including influenza A viruses H1N1, H3N2, H5N1, H7N7 and H7N9, SARS coronavirus (SARS-CoV)and MERS coronavirus (MERS-CoV). This P9 showed very high selectivity index (970), which was higher than that of the full-length peptide of synthetic mBD4 (smBD4) and rmBD4 in vitro. Secondly, the prophylactic and therapeutic effects of P9 against the infection of H1N1 virus were further detected in animal model. The survival rate of P9-pretreated mice challenged by lethal dose of H1N1 virus was 100%. The therapeutic effects of P9 protecting mice from lethal challenge of H1N1 virus were also statistically significant. The survival rate of mice could reach up to 67% by intranasal inoculation and 56% by intraperitoneal injection, respectively. To investigate the antiviral mechanism, we firstly elucidated that P9 could inhibit viral infection but not viral replication or release. Secondly, we detected whether P9 inhibited viral infection by binding to the surface of target cells or viral particles. The results showed that P9 only bound to viral particles but not to the cell surface. It was further identified that P9 bound to viral surface glycoprotein HA but not NA. Thirdly, we demonstrated that P9 did not inhibit virus binding to its receptor and block the virus entry into cells by endocytosis. Instead, P9 inhibited the acidification in late endosomes and thusP9 blocked virus-membrane fusion and subsequent viral disassembly and viral RNA release. Finally, we elucidated that the antiviral activity of P9 was attributed to its high binding affinity to viral HA and the abundance of basic amino acids in its composition. In this study, we have demonstrated that a short peptide P9, which is derived from mBD4, showed potent antiviral activity against multiple respiratory viruses. This peptide can be developed to a new promising prophylactic and therapeutic agent with broad-spectrum antiviral activity and low possibility to cause drug resistance. Moreover, this study has also revealed a novel antiviral mechanism for P9 and paved a path for the development of new antiviral agents with broad-spectrum antiviral activity against emerging respiratory viruses, such as avian influenza H5N1 and H7N9, as well as SARS-CoV and MERS-CoV. / published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

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published_or_final_version / abstract / toc / Zoology / Master / Master of Philosophy