Characterization of grapevine leafroll-associated virus 3 genetic variants and application towards RT-qPCR assay design

Grapevine leafroll-associated virus 3 (GLRaV-3) is the most widely prevalent and economically important of the complex of RNA viruses associated with grapevine leafroll disease (GLD). Phylogenetic studies have grouped GLRaV-3 isolates into nine different monophyletic groups and four supergroups, making GLRaV-3 a genetically highly diverse virus species. In addition, new divergent variants have been discovered recently around the world. Accurate identification of the virus is an essential component in the management and control of GLRaV-3; however, the diversity of GLRaV-3, coupled with the limited sequence information, have complicated the development of a reliable detection assay. In this study, GLRaV-3 sequence data available in GenBank and those generated at Foundation Plant Services, University of California-Davis, was used to develop a new RT-qPCR assay with the capacity to detect all known GLRaV-3 variants. The new assay, referred to as FPST, was challenged against samples that included plants infected with different GLRaV-3 variants and originating from 46 countries. The FPST assay detected all known GLRaV-3 variants, including the highly divergent variants, by amplifying a small highly conserved region in the 3' untranslated terminal region (UTR) of the virus genome. The reliability of the new RT-qPCR assay was confirmed by an enzyme linked immunosorbent assay (ELISA) that can detect all known GLRaV-3 variants characterized to date. Additionally, three new GLRaV-3 divergent variants, represented by four isolates, were identified using a hierarchical testing process involving the FPST assay, GLRaV-3 variant-specific assays and high-throughput sequencing analysis. These variants were distantly related to groups I, II, III, V, VI, VII and IX, but much similar to GLRaV-3 variants with no assigned group; thus, they may represent new clades. Finally, based on the phylogenetic analysis, a new GLRaV-3 subclade is proposed and named as group X.Alfredo Diaz-Lara, Vicki Klaassen, Kristian Stevens, Mysore R. Sudarshana, Adib Rowhani ... Nuredin Habili ... et al

Relative quantitation goes viral: an RT-qPCR assay for a grapevine virus

Accurate detection and quantitation of viruses can be beneficial to plant–virus interaction studies. In this study, three SYBR green real-time RT-PCR assays were developed to quantitate grapevine leafroll-associated virus 3 (GLRaV-3) in infected vines. Three genomic regions (ORF1a, coat protein and 3′UTR) were targeted to quantitate GLRaV-3 relative to three stably expressed reference genes (actin, GAPDH and α-tubulin). These assays were able to detect all known variant groups of GLRaV-3, including the divergent group VI, with equal efficiency. No link could be established between the concentration ratios of the different genomic regions and subgenomic RNA (sgRNA) expression. However, a significant lower virus concentration ratio for plants infected with variant group VI compared to variant group II was observed for the ORF1a, coat protein and the 3′UTR. Significant higher accumulation of the virus in the growth tip was also detected for both variant groups. The quantitation of viral genomic regions under different conditions can contribute to elucidating disease aetiology and enhance knowledge about virus ecology

The experiential modification of a computer software package for graphing algebraic functions

Graphing software and graphics calculators are widely used in most of the world's larger economies to facilitate students' development of conceptual understanding of mathematical function analysis. This has proved to be an extremely effective vehicle in making complex mathematics more accessible to the majority of learners. In contrast, its use in South Africa has been limited. Possible reasons may be the cost of graphics calculators, limited availability of supporting study material, and teachers who lack the necessary skills and confidence. At the School of Teacher Training, University of Pretoria, the Master Grapher for Windows was introduced by way of a pilot study in an effort to adapt the training of mathematics teachers-in-training to meet the specific needs of these students. The experiences of five students were monitored. The aim is to enhance and facilitate trainee-teachers' understanding of mathematics, but also to equip them to develop learner-centred, group-based learning experiences in future teaching situations. Action research was implemented to develop the course

Standing sedation with medetomidine and butorphanol in captive African elephants (Loxodonta africana)

Doses for standing sedation allowing for various procedures in otherwise inaccessible, untrained captive African elephant bulls are presented. Thirty-three standing sedations were performed in 12 males aged 8–30 years (one to four sedations per animal). Each bull received a combination of 0.009 ± 0.002 mg/kg medetomidine and 0.03 ± 0.007 mg/kg butorphanol. Full sedation was reached on average 25.5 min after injection. The addition of hyaluronidase (1000–2000 IU) significantly reduced time to full sedation to 16.5 min (paired t test, P = 0.024). Reversal was induced with intramuscular atipamezole 0.008 (±0.002) and naltrexone 0.035 (±0.015) mg/kg. Recovery took on average 7 min (3–18 min). The medetomidine/ butorphanol combination provided safe standing sedation for smaller procedures.U.S. Fish and Wildlife Service. (African elephant fund AFE 0705), the International Elephant Foundation (Birmingham Zoo, USA).Technology Innovation Agency (Project number TAHC12 - 00042) (Pretoria, South Africa). The German Academic Exchange Service (DAAD, Grant number D/11/44481).http://www.elsevier.com/locate/tvjl2017-03-31hb2016Production Animal Studie