Browsing by Author "Leat, Neil"

Now showing 1 - 5 of 5
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    Development of infectious transcripts and genome manipulation of black queen-cell virus of honey bees
    (Microbiology Society, 2002) Benjeddou, Mongi; Leat, Neil; Allsopp, Mike
    The South African isolate of Black queen-cell virus (BQCV), a honey bee virus, was previously found to have an 8550 nucleotide genome excluding the poly(A) tail. Its genome contained two ORFs, a 5«-proximal ORF encoding a putative replicase protein and a 3«-proximal ORF encoding a capsid polyprotein. Long reverse transcription (RT)–PCR was used to produce infectious transcripts for BQCV and to manipulate its genome. Primers were designed for the amplification of the complete genome, the in vitro transcription of infectious RNA and PCR-directed mutagenesis. An 18-mer antisense primer was designed for RT to produce full-length single-stranded cDNA (ss cDNA). Unpurified ss cDNA from the RT reaction mixture was used directly as a template to amplify the full genome by long high-fidelity PCR. The SP6 promoter sequence was introduced into the sense primer to transcribe RNA directly from the amplicon. RNA was transcribed in vitro with and without the presence of a cap analogue and injected directly into bee pupae, which were then incubated for 8 days. In vitro transcripts were infectious but the presence of a cap analogue did not increase the amount of virus recovered.
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    Development of molecular tools for honeybee virus research: the South African contribution
    (Academic Journals, 2003) Davison, Sean; Leat, Neil; Benjeddou, Mongi
    Increasing knowledge of the association of honeybee viruses with other honeybee parasites, primarily the ectoparasitic mite Varroa destructor, and their implication in the mass mortality of honeybee colonies has resulted in increasing awareness and interest in honeybee viruses. In addition the identification, monitoring and prevention of spread of bee viruses is of considerable importance, particularly when considering the lack of information on the natural incidence of virus infections in honeybee populations worldwide. A total of eighteen honeybee viruses have been identified and physically characterized. Most of them have physical features resembling picornaviruses, and are referred to as picorna-like viruses. The complete genome sequences of four picorna-like honeybee viruses, namely Acute Bee Paralysis Virus (ABPV), Black Queen Cell Virus (BQCV), Sacbrood Virus (SBV) and Deformed Wing Virus (DWV) have been determined. The availability of this sequence data has lead to great advances in the studies on honeybee viruses. In particular, the development of a reverse genetics system for BQCV, will open new opportunities for studies directed at understanding the molecular biology, persistence, pathogenesis, and interaction of these bee viruses with other parasites. This review focuses on the contribution of the Honeybee Virus Research Group (HBVRG), from the University of the Western Cape of South Africa, in the development of molecular tools for the study of molecular biology and pathology of these viruses.
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    The evaluation of Y-STR loci for use in forensics
    (University of the Western Cape, 2005) Ehrenreich, Liezle Suzette; Leat, Neil; Davison, Sean; Dept. of Biotechnology; Faculty of Science
    The aim of this study was to investigate the forensic usefulness of various Y-chromosome short tandem repeat loci among South African sub-populations. Three different sets of Y-chromosome short tandem repeat loci were chosen for investigation.
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    Identification, sequence analysis, and phylogeny of the immediate early gene 1 of the trichoplusia ni single nucleocapsid polyhedrosis virus
    (Springer Verlag, 2001) Wang, Weizhou; Leat, Neil; Fielding, Burtram C.; Davison, Sean
    Substantial research has been conducted on the immediate early 1 (ie-1) genes from the prototype baculovirus Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) and the Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV). In both cases ie-1 gene products have been implicated in transcriptional activation and repression. In this study an ie-1 homolog was identified from Trichoplusia ni single nucleocapsid polyhedrosis virus (TniSNPV). Nucleotide sequence analysis indicated that the TniSNPV ie-1 gene consists of a 2217 nucleotide open reading frame (ORF), encoding a protein with a molecular mass of 84.464 kDa. This represents the largest baculovirus ie-1 gene characterised to date. Of the seven ie-1 homologs identified to date, the TniSNPV ie-1 shared most sequence similarity with the ie-1 gene of Spodoptera exigua MNPV (SeMNPV) (41%). At the nucleotide level, expected TATA and CAGT motifs were found to precede each ie-1 ORF. At the protein level, it was confirmed that the N-termini are poorly conserved, but share the characteristic of having a high proportion of acidic amino acids. In addition it was found that N-terminal regions significantly matched the SET domain in the Swiss-Prot prosite database. The C-terminal regions of the deduced IE-1 sequences were found to be substantially more conserved than the N-termini. Several conserved motifs were identified in the C-terminal sequences. A phylogenetic tree of nine baculovirus IE-1 proteins was constructed using maximum parsimony analysis. The phylogenetic estimation of the ie-1 genes shows that TniSNPV is a member of the previously described lepidopteran NPV group II and it is most closely related to SeMNPV.
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    The Internal Validation and Casework Application of MiniSTR Systems
    (University of the Western Cape, 2008) Kleyn, Eugene Lyle; Leat, Neil; Davison, Sean; Dept. of Biotechnology; Faculty of Science
    The objective of the study was to conduct an internal validation on miniSTR systems and apply it to cases received from the South African Missing Persons Task Team (SAMPTT). This was prompted by the fact that miniSTR systems have been shown to out perform some of the commercial kits available in the time of the study and provide an alternative to mtDNA when analysing degraded DNA from skeletal remains and that the DNA extracted from skeletal remains received from the SAMPTT would be degraded due to the remains generally being fragmented or charred and buried for many years. The miniSTR loci chosen for validation comprised the Combined DNA Index System (CODIS) thirteen core loci and were arranged into four triplexes and one uniplex.