Sean Davison

Detection of Acute Bee Paralysis Virus and Black Queen Cell Virus from Honeybees by Reverse Transcriptase PCR

ABSTRACT A reverse transcriptase PCR (RT-PCR) assay was developed for the detection of acute bee paralysis virus (ABPV) and black queen cell virus (BQCV), two honeybee viruses. Complete genome sequences were used to design unique PCR primers within a 1-kb region from the 3′ end of both genomes to amplify a fragment of 900 bp from ABPV and 700 bp from BQCV. 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 ABPV and BQCV. Sensitivities were approximately 1,600 genome equivalents of purified ABPV and 130 genome equivalents of BQCV.

Characterization of the highly discriminatory loci DYS449, DYS481, DYS518, DYS612, DYS626, DYS644 and DYS710

During the study of genetic diversity at non-core Y-STRs in South African population groups, we identified loci with high discrimination capacity. In this study we present a detailed account of the allele diversity, allele sequence data, gene diversity, allele frequency spectrum and informativeness for assignment in the European English, Asian Indian and Xhosa population groups at loci DYS449, DYS481, DYS518, DYS612, DYS626, DYS644 and DYS710. The suitability of these loci for forensic, genealogical and evolutionary studies is discussed, and nomenclature for loci DYS518, DYS612, DYS626 and DYS644 is suggested.

Where is the game? Wild meat products authentication in South Africa: a case study

BackgroundWild animals’ meat is extensively consumed in South Africa, being obtained either from ranching, farming or hunting. To test the authenticity of the commercial labels of meat products in the local market, we obtained DNA sequence information from 146 samples (14 beef and 132 game labels) for barcoding cytochrome c oxidase subunit I and partial cytochrome b and mitochondrial fragments. The reliability of species assignments were evaluated using BLAST searches in GenBank, maximum likelihood phylogenetic analysis and the character-based method implemented in BLOG. The Kimura-2-parameter intra- and interspecific variation was evaluated for all matched species.ResultsThe combined application of similarity, phylogenetic and character-based methods proved successful in species identification. Game meat samples showed 76.5% substitution, no beef samples were substituted. The substitutions showed a variety of domestic species (cattle, horse, pig, lamb), common game species in the market (kudu, gemsbok, ostrich, impala, springbok), uncommon species in the market (giraffe, waterbuck, bushbuck, duiker, mountain zebra) and extra-continental species (kangaroo). The mountain zebra Equus zebra is an International Union for Conservation of Nature (IUCN) red listed species. We also detected Damaliscus pygargus, which is composed of two subspecies with one listed by IUCN as ‘near threatened’; however, these mitochondrial fragments were insufficient to distinguish between the subspecies. The genetic distance between African ungulate species often overlaps with within-species distance in cases of recent speciation events, and strong phylogeographic structure determines within-species distances that are similar to the commonly accepted distances between species.ConclusionsThe reliability of commercial labeling of game meat in South Africa is very poor. The extensive substitution of wild game has important implications for conservation and commerce, and for the consumers making decisions on the basis of health, religious beliefs or personal choices.Distance would be a poor indicator for identification of African ungulates species. The efficiency of the character-based method is reliant upon availability of large reference data. The current higher availability of cytochrome b data would make this the marker of choice for African ungulates. The encountered problems of incomplete or erroneous information in databases are discussed.

Design and validation of a highly discriminatory 10-locus Y-chromosome STR multiplex system

The Y-chromosome STRs (short tandem repeat) markers are routinely utilized in the resolution of forensic casework related to sexual assault. For this, the forensic community has adopted a set of eleven (core) Y-STR that is incorporated in all commercial diagnostic systems. Our previous studies of Y-STR polymorphisms in the South African population identified low levels of diversity and discrimination capacity for many commercial marker sets, determining a limited applicability of these systems to the local population groups. To overcome this shortcoming, we designed a Y-STR 10-plex system that shows higher discriminatory capacity (DC) than available commercial systems. The markers were selected from a population group of 283 individuals with African, European and Asian ancestry genotyped at 45 Y-STRs, applying an optimization based selection procedure to achieve the highest possible DC with the minimal number of markers. The 10-plex was satisfactorily subjected to developmental validation tests following the SWGDAM guidelines and shows potential for its application to genealogical and evolutionary studies.

A comparison of the structural polypeptides of three iridescent viruses (types 6, 9, and 16) and the mapping of the DNA region coding for their major capsid polypeptides

SummaryThe iridoviruses fromWiseana cervinata (WIV, type 9),Costelytra zealandica (CzIV, type 16) andChilo suppressalis (CIV, type 6) were compared by SDS-PAGE and Western protein blotting for antigenic determinants. The major capsid proteins were isolated and oligonucleotide probes were synthesized from the partial amino acid sequences. The DNA regions coding for the major capsid proteins of WIV (VP52), CzIV (VP53) and CIV (VP50) were located by hybridization of the oligonucleotide probes to blots of the viral DNA. The major capsid protein was used as the zero point for the proposed linearized maps of these viruses.Using antibody and125I-labelling, several proteins were identified as being on the surface of the virion. It was also shown that CIV was not as antigenically distinct from these two viruses as previously reported.

Allele frequencies of six non-CODIS miniSTR loci (D1S1627, D3S4529, D5S2500, D6S1017, D8S1115 and D9S2157) in three South African populations

Abstract The allele frequency of six non-CODIS miniSTR loci: D1S1627, D3S4529, D5S2500, D6S1017, D8S1115 and D9S2157 were investigated in three South African populations (Afrikaner, Asian Indian and Mixed Ancestry). Deviation from Hardy–Weinberg equilibrium was observed in the Mixed Ancestry population for the locus D9S2157. All loci showed a moderate degree of polymorphism with heterozygosity values >0.6 for all populations. The combined power of discrimination was: 0.999997723, 0.999997163 and 0.99999961 for Afrikaner, Asian Indian and Mixed Ancestry populations, respectively. The respective values for the combined power of exclusion in these populations were: 0.99, 0.99 and 0.98.

Bee health report: Varroa in South Africa

The relative isolation of subsaharan Africa that has largely spared it the spread of most of the important honey bee pathogens and parasites seems to be a thing of the past. We report on the latest international honey bee parasite to be found in subequatorial Africa.

Forensic performance of Investigator DIPplex indels genotyping kit in native, immigrant, and admixed populations in South Africa

The utilization of binary markers in human individual identification is gaining ground in forensic genetics. We analyzed the polymorphisms from the first commercial indel kit Investigator DIPplex (Qiagen) in 512 individuals from Afrikaner, Indian, admixed Cape Colored, and the native Bantu Xhosa and Zulu origin in South Africa and evaluated forensic and population genetics parameters for their forensic application in South Africa.

Functional characterization of the ecdysteroid UDP-glucosyl transferase gene of Helicoverpa armigera single-enveloped nucleopolyhedrovirus isolated in South Africa

The ecdysteroid UDP-glucosyltransferase (egt) gene of a single enveloped nucleopolyhedrovirus was located using an Hz-SNPV gene-specific probe. This SNPV was found infecting a colony of Helicoverpa armigera (HaSNPV) in the Western Cape region of South Africa. The open reading frame of the HaSNPV-SA egt is 1.548 nucleotides long and encodes a predicted protein of 516 amino acids with a Mr of 58,897-kDa. The 5′-noncoding region contained an early transcription initiation motif (CAGT) and a baculovirus late transcription motif (ATAAG). A transcription enhancer sequence (GATA) was also identified. Two possible TATA boxes together with am AT rich region were also recognized. A putative signal peptide of 20 residues was present at the N-terminus of the predicted EGT sequence. A polyadenylation signal (AATAAA) was found downstream of the translation stop codon. Five Helicoverpa NPV EGTs that have an extremely high degree of nucleotide and amino acid sequence homology were used in this study. Single nucleotide polymorphisms (SNPs) within the gene were tabulated. The Helicoverpa NPV egts seem to be closely related to the egt genes of Mamestra configurata NPV (MacoNPV), Buzura suppressaria NPV (BusuSNPV) and Spodoptera exigua NPV (SeMNPV) with amino acid identities of approximately 50%. The Helicoverpa NPV EGTs show ten conserved motifs with other EGTs. A phylogenetic tree of 27 baculovirus EGTs and a human UDP-glucoronosyltransferase was constructed using Neighbour-joining within CLUSTAL X. That a secreted and active EGT is encoded by HaSNPV-SA was confirmed by assay of infected cell culture medium.

The characterization and phylogenetic relationship of the trichoplusia ni single capsid nuclear polyhedrosis virus polyhedrin gene

The 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 5′-noncoding region contained the putative late (TAAG) transcription initiation motif. The 3′ 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.