Browsing by Author "Davison, Sean"
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Item Characterisation of eight non-codis Ministrs in four South African populations to aid the analysis of degraded DNA(University of the Western Cape, 2009) Ismail, Aneesah; Davison, Sean; d’Amato, Maria Eugenia; Dept. of Biotechnology; Faculty of ScienceIn many forensic cases, such as mass disasters reconstruction cases, the recovered DNA is highly degraded. In such incidences, typing of STR loci has become one of the most powerful tools for retrieving information from the degraded DNA. However, as DNA degradation proceeds, three phenomena occur consecutively: loci imbalance, allele dropout and no amplification. To solve the problem of degraded DNA, redesigned primer sets have been developed in which the primers were positioned as close as possible to the STR repeat region. These reduced primer sets were called Miniplexes. Unfortunately, a few of the CODIS STR loci cannot be made into smaller amplicons. For this reason non-CODIS miniSTRs have been developed. The present study was undertaken for the population genetic analysis of microsatellite variation in four South African populations; Afrikaner, Xhosa, Mixed Ancestry and Asian Indian using eight non-CODIS miniSTR loci. These miniSTRs loci were characterized within the populations by estimating the levels of diversity of the markers, estimating the population genetic parameters, and studying the inter-population relationships. All of the miniSTRs were amplified successfully and the genetic variability parameters across all loci in Afrikaner, Mixed Ancestry, Asian Indian and Xhosa were estimated to be in the range of 3 (D4S2364) to 12 (D9S2157) alleles, the total number of alleles over all loci ranged from 100 to 204, the allelic richness ranged from 3.612 to 10.307 and the heterozygosity ranged from 0.4360 to 0.8073. Genetic distance was least between Afrikaner and Asian Indian and highest between Xhosa and Mixed Ancestry. Deviations from Hardy-Weinberg equilibrium were not observed for most of the loci. The low mean FIS (-0.027) and FIT (-0.010) and FST (0.017) values across the populations indicated low level of inbreeding within (FIS) and among (FST) the populations. The Asian Indian population showed higher levels of the inbreeding coefficient, indicating less gene exchange between it and other populations. These 8 markers can be used for genetic investigations and assessing population structure. The study contributed to the knowledge and genetic characterization of four South African populations. In addition, these MiniSTRs prove to be useful in cases where more genetic information is needed.Item The characterization and phylogenetic relationship of the trichoplusia ni single capsid nuclear polyhedrosis virus polyhedrin gene(Kluwer Academic Publishers, 1999) Fielding, Burtram C.; Davison, SeanThe polyhedrin gene (polh) was identified from the Trichoplusia ni (Tni) single capsid nuclear polyhedrosis virus (SNPV). An EcoRI fragment containing the truncated polyhedrin gene was detected by hybridization with an AcMNPV expression vector probe; the remaining portion of the gene was amplified by reverse PCR. An open reading frame (ORF) of 741 nucleotides (nt), encoding a putative protein of 246 amino acids (a.a) with Mr 28,780 Da was identified. The 50-noncoding region contained the putative late (TAAG) transcription initiation motif. The 30 end, downstream of the translation stop codon, lacked an obvious putative poly (A) signal. Nucleotide and amino acid homology are greater than 80% to that of Mamestra brassicae polyhedrin sequences.Results suggest that T. niSNPV is a member of the group II nuclear polyhedrosis viruses.Item Development of a COMT PCR multiplex to investigate resilience, anxiety and childhood trauma in a South African population(University of the Western Cape, 2020) Jacobs, Sarah; Davison, SeanAnxiety, resilience and childhood trauma can be categorized as functional behavioural categories, with a wealth of research behind each. The research approach adopted for each, in most cases, is either from a genetic or neuropsychological standpoint, with few studies combining both to better understand all three functional behavioural categories as a multidimensional construct A number of candidate genes have been identified as markers for anxiety, resilience and childhood trauma, of which Catechol-methyl-transferase (COMT) and several respective single nucleotide polymorphisms (SNPs) are included. Although COMT SNPs have been linked to at least one of the functional categories, with a handful of haplotypes identified, to our knowledge no study has investigated the combination of SNPs selected for this study (rs6269, rs4818, rs4680, rs4633, rs737865, rs2075507) as a possible haplotype, specifically in a South African population. The use of SNaPshot for the genotyping of genes is an efficient and reliable means of identifying genotype frequencies and haplotypes in large sample groups, yet when selecting more than two SNPs of interest, the development of a multiplex assay is ideal. The first aim of the study was to design and optimize a multiplex assay to genotype several COMT SNPs. The primer design, multiplex optimization and SNaPshot conditions used showed good working parameters that can be utilized and further improved by optimization. Self-report measures are widely used to measure psychiatric disorders, such as anxiety, and has also been used for the measurement of resilience and childhood trauma. With each functional behavioural category well investigated in its respective domain, there is a need to investigate all three as a collective in a South African population due to the high rate of anxiety and childhood trauma exposure in communities. The second aim of the study was to investigate the prevalence of anxiety, resilience and childhood as functional behavioural categories in the full South African sample group; and the role of sex, through established self-report measures and respective normative data. Additionally, this carried over into investigating the correlation between anxiety, resilience and childhood trauma as a multidimensional construct in both the full South African sample and between sexes. There is a clear relationship which exists between all three functional behavioural categories, as they show a correlation in various dimensions independent of one another. Higher anxiety levels amongst females were reported, with no difference between sexes for resiliency. The empirical data collected from both COMT SNP and self-report measures for male and female where explored and reviewed against current literature for better understanding and insight into the association of COMT SNPs with anxiety, resilience and childhood trauma in a South African population. The results of this study to understand the complexity and association of all three functional behavioural categories as a multidimensional construct will be invaluable and may assist in the identification of possible risk factors which are essential for the promotion of better mental health in society.Item Development of molecular tools for honeybee virus research: the South African contribution(Academic Journals, 2003) Davison, Sean; Leat, Neil; Benjeddou, MongiIncreasing 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.Item Differences in visual attention processing: An event-related potential comparative analysis within psychotic disorders(University of the Western Cape, 2019) Williams, Kimberley Clare; Davison, Sean; Howells, FleurINTRODUCTION: Sustained attention is known to be dysfunctional in psychotic disorders. Sustained attention is the ability to remain focused on a specific time-locked stimulus within a task. We aimed to determine whether there are specific group differences between CON and three psychotic disorders: SCZ, MPD and BPD, then to determine differences between these psychotic disorders. This included differences in behavioural performance and prominent electrophysiological event-related potential (ERP) wave components during cueing and target processing of a visual sustained attention task. Further we aimed to characterize ERP waveform component relationships across and within these groups for demographics, substance use, behavioural performance, and clinical variables, the last limited to the psychotic groups. Lastly, we investigated the effects of prescribed medications on ERP wave components within the psychotic groups. METHODOLOGY: 103 participants (29 schizophrenia (SCZ), 28 bipolar disorder with a history of psychosis (BPD), 21 methamphetamine-induced psychotic disorder (MPD), and 30 controls (CON)) underwent electroencephalography (EEG) record while completing a visual continuous performance task. Participants were presented with 60 trials with three consecutive S’s, the presentation of the third S required a behavioural response. Prominent ERP waveform components were extracted from cues and target stimulus. Group differences were determined by ANOVA with Bonferroni post-hoc correction or multivariate Kruskal-Wallis test dependent on data distribution. Relationships between ERP wave components were determined appropriate with Spearman’s Rank order correlation analyses. RESULTS: (1) MPD reported higher use of substances compared to CON, SCZ and BPD. SCZ behavioural performance was poorer compared to CON which was shown by their longer response times, reduced accuracy and increased errors of omission. Clinically, MPD was found to have a shorter duration of illness compared to SCZ. Then SCZ was found to have more positive symptoms compared to BPD whereas BPD had more negative symptoms compared to SCZ. For the first cue, wave component differences were found only over the left hemisphere, for P100 amplitude over the frontal cortex, P300 amplitude over the central cortex, and N170 amplitude over the parietal cortex. For the presentation of the second cue, differences noted for all groups were localised to the frontal and central brain regions, for P100 and N170 ERP waveforms. For the target stimulus wave component differences were found over the prefrontal, frontal and parietal brain regions, within CON, SCZ, BPD and MPD. (2) For the first cue, education positively correlated with the N170 left parietal amplitude in CON and P300 right parietal amplitude in MPD. During the second cue, the left parietal N170 latency in SCZ correlated positively with education and the left central P300 latency correlated negatively with education in MPD. The age on the day of testing correlated positively with the target left frontal P300 latency in MPD. For the first cue, substance use positively correlated with the left and right parietal P300 latency and negatively for the right parietal P100 amplitude in SCZ. In MPD, a negative correlation was noted across left and right prefrontal N170 and P300 amplitudes, and positive correlation for the left prefrontal P300 latency in MPD. For the target stimulus, correlations were evident for the left and right parietal N70, N170 amplitudes, P300 latency, the right parietal P100 amplitude and left central P300 latency in SCZ. For the first cue, in SCZ PANSS total score correlated positively with left and right central P300 amplitudes and the left parietal P300 amplitude. For the second cue; in MPD, the PANSS negative symptom score, positively correlated with the P100 and N170 left parietal amplitude, left and right parietal P150 amplitude, left central and right parietal P300 amplitude. For the target, the Hamilton depression rating scale correlated positively with the left and right frontal P300 amplitude in MPD and then negatively with the right parietal P300 amplitude in SCZ. Behavioural performance in CON, positively correlated with the left parietal N70, P100, P150 and N170 amplitude the number of correct responses, and left central N170 amplitude. While the number of impulsive responses correlated negatively with the left parietal N70, P100, P150 and N170 and the left central N170 amplitude of CON. For the second cue, behavioural performance was related to the fronto-parietal relationship across all groups. For the target stimulus, impulsive responses positively correlated with the left parietal N70 latency in SCZ. Overall response time negatively correlated with the right parietal P300 latency for SCZ. (3) Medication was found to affect ERP wave components during the sustained visual attention task. For the first cue FGA’s increased the left central P100 amplitude in both SCZ and BPD and decreased the left parietal P100 amplitude in SCZ only. The use of antipsychotics increased the right parietal N70 and left central P100 amplitudes in BPD, specifically the right prefrontal N170 amplitude was increased with the use of SGA’s. Then clozapine use increased the left frontal P100 amplitude in SCZ. For the second cue, SGA’s decreased the right parietal P150 amplitude in SCZ but in MPD the right parietal P150 amplitude was increased with haloperidol use, and FGA. SGA’s increased the left parietal P300 latency in BPD and sodium valproate decreased the left prefrontal P300 latency. For the target stimulus, SGA’s decreased the right parietal P100, P150 and left parietal P150 amplitudes and increased the left central P300 latency in BPD. CONCLUSION: (1) sustained attentional performance is poorer in SCZ. Our study adds to previous studies showing attention processing deficits in SCZ, are evident during cueing of a sustained attention tasks; (2) substance use was found to slow cognitive processing, education improved executive function and information processing, and symptom severity was associated with dysfunction of prefrontal and frontal cortices; (3) antipsychotic medication was related to improved processing of salient information. These data support the current literature and provide novel insights to the attentional processing deficits during cueing in the psychotic disorders.Item Differences in visual attention processing: An event-related potential comparative analysis within psychotic disorders(University of the Western Cape, 2018) Williams, Kimberley Clare; Davison, Sean; Howells, FleurIntroduction: Sustained attention is known to be dysfunctional in psychotic disorders. Sustained attention is the ability to remain focused on a specific time-locked stimulus within a task. We aimed to determine whether there are specific group differences between CON and three psychotic disorders: SCZ, MPD and BPD, then to determine differences between these psychotic disorders. This included differences in behavioural performance and prominent electrophysiological event-related potential (ERP) wave components during cueing and target processing of a visual sustained attention task. Further we aimed to characterize ERP waveform component relationships across and within these groups for demographics, substance use, behavioural performance, and clinical variables, the last limited to the psychotic groups. Lastly, we investigated the effects of prescribed medications on ERP wave components within the psychotic groups. Methodology: 103 participants (29 schizophrenia (SCZ), 28 bipolar disorder with a history of psychosis (BPD), 21 methamphetamine-induced psychotic disorder (MPD), and 30 controls (CON)) underwent electroencephalography (EEG) record while completing a visual continuous performance task. Participants were presented with 60 trials with three consecutive S’s, the presentation of the third S required a behavioural response. Prominent ERP waveform components were extracted from cues and target stimulus. Group differences were determined by ANOVA with Bonferroni post-hoc correction or multivariate Kruskal-Wallis test dependent on data distribution. Relationships between ERP wave components were determined appropriate with Spearman’s Rank order correlation analyses.Item A dual analysis of the South African Griqua population using ancestry informative mitochondrial DNA and discriminatory short tandem repeats on the Y chromosome(University of the Western Cape, 2015) Heynes, Kirstie; D’Amato, Maria Eugenia; Davison, SeanThe primary objective of this Masters project was to investigate the maternal ancient substructure of the Griqua population in South Africa. Genetic ancestry was determined by investigating ancestry informative single nucleotide polymorphisms. These are located in the control region of the mitochondrial genome. The auxiliary aim was to test the validity of the UWC 10plex system in relation to a sample group of Griqua males. This short tandem repeat multiplex targets specific mutations confined to paternal lineages. The Khoi Khoi or Hottentots were the first inhabitants in the Cape. Indigenous Khoi Khoi female slaves had offspring with the European settlers in the 1800s which resulted in the Griqua population group. The incorporated European paternal ancestry is what set the Griqua apart from the native population groups at that time. Colonisation events from the mid-17th to 19th Century and the apartheid regime resulted in land dispossession of the native population and an extensively mixed gene pool in South Africa. One hundred and seventy six (N=176) male and female Griqua people were collectively sampled in Kokstad (2012), Vredendal (2012 and 2013) and at the Griqua National Conference in Ratelgat (2013). All 176 samples were analysed using mtDNA control region Sanger sequencing. The sample group (N=176) was separated based on birthplace (Origin sample group and post-colonial sample group). The origin sample group consists of individuals whose ancestors were not part of the Griqua Trek to Northern regions of South Africa and were less likely to be exposed to colonial influences. Mutations within the hypervariable segments of the mtDNA control region were used to infer haplogroups with geographic-specific population data. In this way one can plot the extent of ancient Khoisan (L0d) and Bantu influences (L1-L5) as well as the influence of East (M, A, B, E) and West (N, R, J, H) Eurasian haplogroups in the maternal ancestry of the Griqua population group. The origin sample group showed 91% African ancestry (76.8% L0d) while the post-colonial group had 78% African ancestry (60% L0d). The origin sample group had 2% East Eurasian and 7% West Eurasian ancestry, while the post-colonial group contained 20% Eurasian ancestry. There is greater admixture in the post-colonial group which can be attributed to the integration of surrounding populations during settlement periods in parts of the Northern Cape and KwaZulu-Natal. The UWC 10plex STR kit was tested to see if it could discriminate between male individuals of this admixed sample group (N=91 males). The markers for this multiplex were selected according to their ability to differentiate between individuals of African descent. It proved to be a viable Y chromosome short tandem repeat testing tool, displaying a statistically significant discrimination capacity value of 0.966 and only having 3 shared haplotypes in the sample group of 91 Griqua males.Item Evaluation of insertion-deletion polymorphisms with the kit Qiagen Investigator® DIPplex for forensic application in South Africa(University of the Western Cape, 2015) Jacobs, Gwynneth; D‘Amato, Maria Eugenia; Davison, SeanInsertion-deletion polymorphisms (indels) have been underutilized in forensic identification of individuals in comparison with single nucleotide polymorphisms (SNPs) and short tandem repeat (STRs) systems. The use of indels for the purpose of human identification is more advantageous than previously used methods as it combines desirable characteristics of both the SNPs and STRs i.e. low costs and simplistic typing methods as well as indels having small amplicons size, making them suitable for genotyping highly degraded DNA. Currently there is only one commercial kit available for the forensic community, the Investigator® DIPlex kit (Qiagen), which cover a total of 30 indel loci distributed over 19 autosomal chromosomes. The objective of this study was to evaluate the Qiagen Investigator® DIPplex kit for forensic application in South Africa. The kit‘s performance was evaluated by comparing different extraction methods; sensitivity, robustness and reproducibility were evaluated and forensic parameters (match probability, power of discrimination, polymorphism information content, power of exclusion and typical paternity index) were estimated based on population data generated from five South African populations (Afrikaner, Mixed Ancestry, Indian-Asian, Xhosa and Zulu). Population comparisons were performed using Fst-analysis, factorial component analysis as well as phylogenetic tree construction. DNA was extracted from buccal swabs and whole blood collected from a total of 512 individuals from the five South African population groups and genotyped using the Qiagen Investigator® DIPplex kit. Sanger DNA sequencing and sequence alignments confirmed the presence of a null allele at locus HLD97 which was present in high frequency in the Xhosa and Zulu populations. This observation was made in 14 individuals belonging to the Xhosa and Zulu populations. Null allele frequencies in all five South African populations were also estimated. Null alleles were estimated for all loci using analytical methods i.e. Charkraborty null allele estimator, Brookfield null allele estimators 1 and 2 and ML-NullFreq software program. The kit performed well in the laboratory, not requiring any additional reagents or instrumentation and successfully generating profiles with input DNA amounts as low as 0.2 ng/μL. Although well suited for forensic application, the Qiagen Investigator® DIPplex kit showed some drawbacks with regards to application on South African populations. The presence of a null allele at the HLD97 locus as well as indication of population substructure affects allele frequency estimates for the South African populations. Correction for population substructure as present within the South African populations should be considered using FST analysis and it is recommended that the HLD97 locus should be excluded from any kinship analysis performed on South African populations.Item 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 ScienceThe 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.Item A forensic analysis of genetic variation in the Botswana population(University of the Western cape, 2016) Tau, Tiroyamodimo; D’Amato, Maria Eugenia; Davison, SeanThis thesis has been placed under a long term embargo. Forensic and population genetic parameters were investigated in the Botswana population using autosomal and Y-chromosome short tandem repeat markers. AmpFlSTR Profiler plus markers were used to investigate the genetic diversity and forensic parameters in 773 individuals from Botswana from the reference database of the Botswana Police. The levels of polymorphism found using the AmpFlSTR Profiler Plus markers showed that the nine loci that make up the AmpFlSTR Profiler Plus can differentiate individuals for forensic casework in the Botswana population. AmpFlSTR Identifiler autosomal STR markers were used to investigate the population structure according to ethno-linguistics and geography 990 individuals from Botswana that serve as a reference database for the Botswana Police. Using pairwise genetic distances (Fst), analysis of molecular variance (AMOVA), factorial correspondence analysis (FCA), and the unsupervised Bayesian clustering method found in STRUCTURE and the landscape genetics software TESS, ethno-linguistics were found to have a greater influence on population structure than geography. The patterns of population structure found using these markers highlight the need for regional reference databases that include both ethnolinguistic and geographic location information. These markers have important potential for bio-anthropological studies as well as for forensic applications. The 17 Y-chromosomal short tandem repeats found in AmpFlSTR Y-filer and a highly discriminatory Y-STR genotyping system (the Y-STR 10-plex developed in the Forensics DNA Laboratory at the University of the Western Cape) were analysed in 249 unrelated male individuals from Botswana. Rst, multi-dimensional scaling (MDS) and AMOVA were used to investigate population differentiation in Botswana. The discrimination capacity (DC) was found to be higher using the Y-STR 10-plex as compared to the 17 markers in the Y-filer genotyping system. No geographic regional or ethnic differentiation was observed between the Northern and Southern regions of Botswana using both marker systems. Regional and ethnic variation can be useful in forensic working hypotheses. Cluster analysis using the highly discriminatory Y-STR 10-plex haplotypes may provide information about ancestry and haplogroup information.Item Forensic identification of six of Tanzanian populations using the extended haplotype markers(University of the Western Cape, 2011) Mwema, Hadija Saidi; D’Amato, Maria Eugenia; Davison, Sean; Dept. of Biotechnology; Faculty of ScienceThe aim of the present study was to evaluate the power of discrimination and genetic (diversity) parameters in the Y chromosome extended haploytpe markers in populations of Tanzania for forensic and populations studies. Eleven Y chromosome extended haplotype markers were selected for this study, these includes Minimal haplotypes markers i.e. DYS19, DYS390, DYS391, DYS392, DYS393, DYS385a/b, DYS389I/II and two additional markers DYS438 and DYS439. Six populations of Tanzania were investigated under this study. These populations were selected based on the language family categories; Niger Congo (Kuria and Sukuma), Nilo Saharan (Luo and Maasai) and Afro Asiatic (Iraqw and Alagwa).Item The genetic organisation of a 2,966 basepair DNA fragment of a Single Capsid Nucleopolyhedrovirus isolated from Trichoplusia ni(Springer Verlag, 2002) Fielding, Burtram C.; Khan, Sehaam; Wang, Weizhou; Kruger, Courtney; Abrahams, Rayaana; Davison, SeanIn order to investiagte the genomic organization of the Trichoplusia ni Single Capsid Nucleoplyhedrovirus (TnSNPV) , a 2,966 base pairs (bp) genomic fragment was sequenced. The fragment was found to contain five open reading frames (ORF's) homologous to baculovirus genes, including p26, fibrillin (p10), AcMNPV ORF-29, late expression facor 6 (lef 6) and the C-terminal portion of p74, on either stand of DNA. Predicted amini acid sequences for the ORFs were compared and identity values of between 12% and 54% were observed. Clustering and arrangement of the TnSNPV genes were similar to the clustering reported for SeMNPV, confirming TnSNPV was a Group II NPV.Item Genetic variation and population structure of Botswana populations as identified with AmpFLSTR Identifiler short tandem repeat (STR) loci(Nature Publishing Group, 2017-07-28) Tau, Tiroyamodim; Wally, Anthony; Fanie, Thokozile Patricia; Ngono, Goitseone Lorato; Mpoloka, Sununguko Wata; Davison, Sean; D’Amato, María EugeniaPopulation structure was investigated in 990 Botswana individuals according to ethno-linguistics, Bantu and Khoisan, and geography (the nine administrative districts) using the Identifiler autosomal microsatellite markers. Genetic diversity and forensic parameters were calculated for the overall population, and according to ethno-linguistics and geography. The overall combined power of exclusion (CPE) was 0.9999965412 and the combined match probability 6,28 × 10−19. CPE was highest for the Khoisan Tuu ethnolinguistic group and the Northeast District at 0.9999582029 and 0.9999922652 respectively. CMP ranged from 6.28 × 10−19 (Khoisan Tuu) to 1,02 × 10−18 (Northwest district). Using pairwise genetic distances (FST), analysis of molecular variance (AMOVA), factorial correspondence analysis (FCA), and the unsupervised Bayesian clustering method found in STRUCTURE and TESS, ethno-linguistics were found to have a greater influence on population structure than geography. FCA showed clustering between Bantu and Khoisan, and within the Bantu. This Bantu sub-structuring was not seen with STRUCTURE and TESS, which detected clustering only between Bantu and Khoisan. The patterns of population structure revealed highlight the need for regional reference databases that include ethno-linguistic and geographic location information. These markers have important potential for bio-anthropological studies as well as for forensic applications.Item Identification and characterization of the Trichoplusia ni Single Capsid Nuclear Polyhedrosis Virus p10 gene(Springer Verlag, 2000) Fielding, Burtram C.; Davison, SeanThe p10 gene was identified and characterized from the Trichoplusia ni single capsid nuclear polyhedrosis virus (TniSNPV). The p10 open reading frame (ORF) sequence was identified following sequencing of the ends of the EcoRI-G clone. Subsequent sequencing of an EcoRI-SmaI subclone identified the entire p10 and a portion of a p26 homologue. The p10 ORF of 264 basepairs (bps), encoded a predicted protein of 88 amino acids (aas) with Mr 9527 Da. The putative late transcription initiation motif (TAAG) was found upstream of the translation initiation codon at position ÿ46. Downstream of the translation stop codon, a putative poly(A) signal was identified. The p10 amino acid sequence contained the three conserved domains reported for all other p10 genes. The p10 amino acid sequence was most homologous (85% similarity and 67% identity) to that of Buzura suppresaria NPV p10 sequence.Item 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, SeanSubstantial 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.Item 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 ScienceThe 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.Item Meat trade: need for international standardization?(Elsevier, 2013) D’Amato, María Eugenia; Davison, Sean; Corach, DanielExtensive substitution and undeclared species have been recently detected in meat products in South Africa, Europe and Asia. Here we review the methodologies utilized in the identification of species in red meat products and highlight the advantages and drawbacks of these methods. The problem is of a different nature in countries with easily accessible game meat and poor or nonexistent monitoring systems in place. Recommendations are drawn for meat DNA testing in these two scenarios.Item Molecular detection and characterisation of RNA viruses of honeybees(University of the Western Cape, 2009) Topley, Elize Lindsay; Davison, Sean; Dept. of Biotechnology; Faculty of SciencePropagation methods for honeybee viruses have not changed since these viruses were discovered. There are no suitable cell lines or cell culture techniques available for honeybee viruses. Honeybee viruses have to be manually injected with virus in order for the virus to multiply and be extracted. With the presence of inapparent viruses which could co-infect pupae, a method for pure virus propagations needs to be found. Recombinant baculovirus systems have been used extensively to produce foreign proteins from different viruses using vectors and recombinant technology. In this chapter we inserted the capsid gene from BQCV into a transfer vector under the control of the p10 promoter of Autographa californica. Fractions of the sucrose gradient containing the virus like particles (VLPs) were seen under the electron microscope. A Western blot showed the four capsid proteins at the expected sizes for BQCV capsid. This study therefore has shown that a heterologous system such as baculovirus can be used for virus like particle production. Infectious virus technology has helped gain insight into how viruses work. Using this technology altering honeybee viruses could be used to observe different functionalities of the viruses. An attempt was made to interchange the open reading frames of ABPV and BQCV to observe any changes in virus assembly and infectivity. A fusion PCR strategy was employed to interchange the 5’ and 3’ ORFs of APBV and BQCV. The strategy however was unsuccessful. Alternative strategies could improve the chances of obtaining a chimeric virus.Item Molecular detection and characterisation of RNA viruses of honeybees(University of the Western Cape, 2009) Topley, Elize Lindsay; Davison, SeanHoneybees have evolved through the centuries to inhabit most parts of the world except for the extreme Polar Regions. These insects have also been susceptible to pathogens and disease which has always been part of the honeybees’ ecology and has evolved and adapted accordingly. However disease has spread more rapidly into areas where no disease existed before with the transport and moving of hives. Disease has caused massive losses within the honeybee industry in recent history. Using new technology available to scientists, diseases and parasites can be identified and this information used to prevent damage to hives, the livelihood of many crop farmers and beekeepers around the world. Of these diseases honeybee viruses have become of some concern in recent times. Honeybee viruses’ black queen cell virus (BQCV) and acute bee paralysis virus (ABPV) were found to have genomes consisting of 8550 and 9490 nucleotides respectively. The viruses have two open reading frames (ORFs) which encode a non structural protein at the 5’ ORF and a structural protein at the 3’ ORF. Sacbrood virus (SBV) has a different organisation to BQCV and ABPV where it has a single ORF with the structural genes at the 5’ end and the non structural genes at the 3’ end. In an effort to rapidly identify honeybee viruses a multiplex reverse transcriptase polymerase chain reaction (RT-PCR) was developed Primers were designed within the 3’ open reading frame to amplify fragments of 434bp for SBV, 900bp for ABPV and 316bp for BQCV. RNA was extracted from laboratory infected and naturally infected samples. The PCR products were sequenced and found to be that of the appropriate virus. The primers were tested on naturally infected samples with SBV and BQCV being detected. Another well characterised honeybee virus Kashmir bee virus (KBV) was initially added to the multiplex RT-PCR. However inconsistencies with the multiplex PCR led to the sequencing of a 2 kilobase fragment of the KBV Indian (KBV-in) strain. Three overlapping cDNA fragments of KBV were sequenced and aligned with the full length sequence of KBV and a sequenced capsid region of KBV both from North America. Alignment to ABPV was also completed to observe the homology between KBV-in and ABPV. The KBV-in strain was not highly homologous to the North American strains over the region which was sequenced for KBV-in. ABPV was also not highly homologous over the entire 2 Kb region. However over the region where primers were designed for the RT PCR of KBV, ABPV was highly homologous at 80%. This could have led to the inconsistencies when PCR was done. Primer design and correct strain characterisation is needed before primers are designed to detect more than one virus per reaction. Further characterisation and sequencing of this strain is needed in order to make further comparisons. Propagation methods for honeybee viruses have not changed since these viruses were discovered. There are no suitable cell lines or cell culture techniques available for honeybee viruses. Honeybee viruses have to be manually injected with virus in order for the virus to multiply and be extracted. With the presence of inapparent viruses which could co-infect pupae, a method for pure virus propagations needs to be found. Recombinant baculovirus systems have been used extensively to produce foreign proteins from different viruses using vectors and recombinant technology. In this chapter we inserted the capsid gene from BQCV into a transfer vector under the control of the p10 promoter of Autographa californica. Fractions of the sucrose gradient containing the virus like particles (VLPs) were seen under the electron microscope. A Western blot showed the four capsid proteins at the expected sizes for BQCV capsid. This study therefore has shown that a heterologous system such as baculovirus can be used for virus like particle production. Infectious virus technology has helped gain insight into how viruses work. Using this technology altering honeybee viruses could be used to observe different functionalities of the viruses. An attempt was made to interchange the open reading frames of ABPV and BQCV to observe any changes in virus assembly and infectivity. A fusion PCR strategy was employed to interchange the 5’ and 3’ ORFs of APBV and BQCV. The strategy however was unsuccessful. Alternative strategies could improve the chances of obtaining a chimeric virus.Item Molecular detection and genetic manipulation of the Black Queen Cell Virus(University of the Western Cape, 2002) Benjeddou, Mongi; Davison, Sean; Dept. of Biotechnology; Faculty of ScienceThe South African isolate of the Black Queen-Cell Virus (BQCV), a honeybee 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.A reverse transcriptase PCR (RT -PCR) assay was developed for the detection of BQCV and acute bee-paralysis virus (ABPV). Complete genomes sequences w ere used to design unique PCR primers within a l-kb region from the 3' end of both genomes to amplify a fragment of 70.0 bp from BQCV and 900 bp from ABPV. The combined guanidinium thiocyanate and silica membrane method was used to extract total RNA from samples of healthy and laboratory-infected bee pupae. In a blind test, RT-PCR successfully identified the samples containing BQCV and ABPV. Sensitivities were of the order of 130 genome equivalents of purified BQCV and 1600 genome equivalents of ABPV.