Vaccine farming in Cape Town

The review details the development of the Subunit Vaccine Group at the University of Cape Town, from its beginnings as a plant virology laboratory in the 1980s. The investigation and development of Human papillomavirus (HPV) and Human immunodeficiency vaccine candidates are covered in detail, with an emphasis on how this work allowed the evolution of a systematic approach to the optimisation of expression of these and other proteins especially in plants, but also in insect cell culture. We discuss various insights gained during our work, such as approaches to codon optimisation, use of different vector systems and plant hosts, intracellular targetting and gene modification. The future prospects for both our work and for the field of plant-made vaccines in general, are discussed. © 2011 Landes Bioscience.

Systematics and biology of the iconic Australian scribbly gum moths Ogmograptis Meyrick (Lepidoptera: Bucculatricidae) and their unique insect-plant interaction

The larvae of particular Ogmograptis spp. produce distinctive scribbles on some smooth-barked Eucalyptus spp. which are a common feature on many ornamental and forest trees in Australia. However, although they are conspicuous in the environment the systematics and biology of the genus has been poorly studied. This has been addressed through detailed field and laboratory studies of their biology of three species (O. racemosa Horak sp. nov., O. fraxinoides Horak sp. nov., O. scribula Meyrick), in conjunction with a comprehensive taxonomic revision support by a molecular phylogeny utilising the mitochondrial Cox1 and nuclear 18S genes. In brief, eggs are laid in bark depressions and the first instar larvae bore into the bark to the level where the future cork cambium forms (the phellegen). Early instar larvae bore wide, arcing tracks in this layer before forming a tighter zig-zag shaped pattern. The second last instar turns and bores either closely parallel to the initial mine or doubles its width, along the zig-zag shaped mine. The final instar possesses legs and a spinneret (unlike the earlier instars) and feeds exclusively on callus tissue which forms within the zig-zag shaped mine formed by the previous instar, before emerging from the bark to pupate at the base of the tree. The scars of mines them become visible scribble following the shedding of bark. Sequence data confirm the placement of Ogmograptis within the Bucculatricidae, suggest that the larvae responsible for the ‘ghost scribbles’ (unpigmented, raised scars found on smooth-barked eucalypts) are members of the genus Tritymba, and support the morphology-based species groups proposed for Ogmograptis. The formerly monotypic genus Ogmograptis Meyrick is revised and divided into three species groups. Eleven new species are described: Ogmograptis fraxinoides Horak sp. nov., Ogmograptis racemosa Horak sp. nov. and Ogmograptis pilularis Horak sp. nov. forming the scribula group with Ogmograptis scribula Meyrick; Ogmograptis maxdayi Horak sp. nov., Ogmograptis barloworum Horak sp. nov., Ogmograptis paucidentatus Horak sp. nov., Ogmograptis rodens Horak sp. nov., Ogmograptis bignathifer Horak sp. nov. and Ogmograptis inornatus Horak sp. nov. as the maxdayi group; Ogmograptis bipunctatus Horak sp. nov., Ogmograptis pulcher Horak sp. nov., Ogmograptis triradiata (Turner) comb. nov. and Ogmograptis centrospila (Turner) comb. nov. as the triradiata group. Ogmograptis notosema (Meyrick) cannot be assigned to a species group as the holotype has not been located. Three unique synapomorphies, all derived from immatures, redefine the family Bucculatricidae, uniting Ogmograptis, Tritymba Meyrick (both Australian) and Leucoedemia Scoble & Scholtz (African) with Bucculatrix Zeller, which is the sister group of the southern hemisphere genera. The systematic history of Ogmograptis and the Bucculatricidae is discussed.

I-vector based speaker recognition using advanced channel compensation techniques

This paper investigates advanced channel compensation techniques for the purpose of improving i-vector speaker verification performance in the presence of high intersession variability using the NIST 2008 and 2010 SRE corpora. The performance of four channel compensation techniques: (a) weighted maximum margin criterion (WMMC), (b) source-normalized WMMC (SN-WMMC), (c) weighted linear discriminant analysis (WLDA), and; (d) source-normalized WLDA (SN-WLDA) have been investigated. We show that, by extracting the discriminatory information between pairs of speakers as well as capturing the source variation information in the development i-vector space, the SN-WLDA based cosine similarity scoring (CSS) i-vector system is shown to provide over 20% improvement in EER for NIST 2008 interview and microphone verification and over 10% improvement in EER for NIST 2008 telephone verification, when compared to SN-LDA based CSS i-vector system. Further, score-level fusion techniques are analyzed to combine the best channel compensation approaches, to provide over 8% improvement in DCF over the best single approach, (SN-WLDA), for NIST 2008 interview/ telephone enrolment-verification condition. Finally, we demonstrate that the improvements found in the context of CSS also generalize to state-of-the-art GPLDA with up to 14% relative improvement in EER for NIST SRE 2010 interview and microphone verification and over 7% relative improvement in EER for NIST SRE 2010 telephone verification.

Impact of Wolbachia on infection with chikungunya and yellow fever viruses in the mosquito vector Aedes aegypti

Incidence of disease due to dengue (DENV), chikungunya (CHIKV) and yellow fever (YFV) viruses is increasing in many parts of the world. The viruses are primarily transmitted by Aedes aegypti, a highly domesticated mosquito species that is notoriously difficult to control. When transinfected into Ae. aegypti, the intracellular bacterium Wolbachia has recently been shown to inhibit replication of DENVs, CHIKV, malaria parasites and filarial nematodes, providing a potentially powerful biocontrol strategy for human pathogens. Because the extent of pathogen reduction can be influenced by the strain of bacterium, we examined whether the wMel strain of Wolbachia influenced CHIKV and YFV infection in Ae. aegypti. Following exposure to viremic blood meals, CHIKV infection and dissemination rates were significantly reduced in mosquitoes with the wMel strain of Wolbachia compared to Wolbachia-uninfected controls. However, similar rates of infection and dissemination were observed in wMel infected and non-infected Ae. aegypti when intrathoracic inoculation was used to deliver virus. YFV infection, dissemination and replication were similar in wMel-infected and control mosquitoes following intrathoracic inoculations. In contrast, mosquitoes with the wMelPop strain of Wolbachia showed at least a 10(4) times reduction in YFV RNA copies compared to controls. The extent of reduction in virus infection depended on Wolbachia strain, titer and strain of the virus, and mode of exposure. Although originally proposed for dengue biocontrol, our results indicate a Wolbachia-based strategy also holds considerable promise for YFV and CHIKV suppression.

Insect odour perception: recognition of odour components by flower foraging moths

Odours emitted by flowers are complex blends of volatile compounds. These odours are learnt by flower-visiting insect species, improving their recognition of rewarding flowers and thus foraging efficiency. We investigated the flexibility of floral odour learning by testing whether adult moths recognize single compounds common to flowers on which they forage. Dual choice preference tests on Helicoverpa armigera moths allowed free flying moths to forage on one of three flower species; Argyranthemum frutescens (federation daisy), Cajanus cajan (pigeonpea) or Nicotiana tabacum (tobacco). Results showed that, (i) a benzenoid (phenylacetaldehyde) and a monoterpene (linalool) were subsequently recognized after visits to flowers that emitted these volatile constituents, (ii) in a preference test, other monoterpenes in the flowers' odour did not affect the moths' ability to recognize the monoterpene linalool and (iii) relative preferences for two volatiles changed after foraging experience on a single flower species that emitted both volatiles. The importance of using free flying insects and real flowers to understand the mechanisms involved in floral odour learning in nature are discussed in the context of our findings.

Wolbachia infection alters olfactory-cued locomotion in Drosophila spp

Wolbachia pipientis is an endosymbiotic bacterium present in diverse insect species. Although it is well studied for its dramatic effects on host reproductive biology, little is known about its effects on other aspects of host biology, despite its presence in a wide array of host tissues. This study examined the effects of three Wolbachia strains on two different Drosophila species, using a laboratory performance assay for insect locomotion in response to olfactory cues. The results demonstrate that Wolbachia infection can have significant effects on host responsiveness that vary with respect to the Wolbachia strain-host species combination. The wRi strain, native to Drosophila simulans, increases the basal activity level of the host insect as well as its responsiveness to food cues. In contrast, the wMel strain and the virulent wMelPop strain, native to Drosophila melanogaster, cause slight decreases in responsiveness to food cues but do not alter basal activity levels in the host. Surprisingly, the virulent wMelPop strain has very little impact on host responsiveness in D. simulans. This novel strain-host relationship was artificially created previously by transinfection. These findings have implications for understanding the evolution and spread of Wolbachia infections in wild populations and for Wolbachia-based vector-borne disease control strategies currently being developed.

Can mechanism help explain insect host choice?

Evolutionary theory predicts that herbivorous insects should lay eggs on plants in a way that reflects the suitability of each plant species for larval development. Empirical studies, however, often fail to find any relationship between an adult insect’s choice of host–plant and offspring fitness, and in such cases, it is generally assumed that other ‘missing’ factors (e.g. predation, host–plant abundance, learning and adult feeding sites) must be contributing to overall host suitability. Here, I consider an alternative theory – that a fitness cost inherent in the olfactory mechanism could constrain the evolution of insect host selection. I begin by reviewing current knowledge of odour processing in the insect antennal lobe with the aid of a simple schematic: the aim being to explain the workings of this mechanism to scientists who do not have prior knowledge in this field. I then use the schematic to explore how an insect’s perception of host and non-host odours is governed by a set of processing rules, or algorithm. Under the assumptions of this mechanistic view, the perception of every plant odour is interrelated, and seemingly bad host choices can still arise as part of an overall adaptive behavioural strategy. I discuss how an understanding of mechanism can improve the interpretation of theoretical and empirical studies in insect behaviour and evolution.

Sampling stored-product insect pests : a comparison of four statistical sampling models for probability of pest detection

Background: Developing sampling strategies to target biological pests such as insects in stored grain is inherently difficult owing to species biology and behavioural characteristics. The design of robust sampling programmes should be based on an underlying statistical distribution that is sufficiently flexible to capture variations in the spatial distribution of the target species. Results: Comparisons are made of the accuracy of four probability-of-detection sampling models - the negative binomial model,1 the Poisson model,1 the double logarithmic model2 and the compound model3 - for detection of insects over a broad range of insect densities. Although the double log and negative binomial models performed well under specific conditions, it is shown that, of the four models examined, the compound model performed the best over a broad range of insect spatial distributions and densities. In particular, this model predicted well the number of samples required when insect density was high and clumped within experimental storages. Conclusions: This paper reinforces the need for effective sampling programs designed to detect insects over a broad range of spatial distributions. The compound model is robust over a broad range of insect densities and leads to substantial improvement in detection probabilities within highly variable systems such as grain storage.

Improving short utterance based I-vector speaker recognition using source and utterance-duration normalization techniques

A significant amount of speech is typically required for speaker verification system development and evaluation, especially in the presence of large intersession variability. This paper introduces a source and utterance duration normalized linear discriminant analysis (SUN-LDA) approaches to compensate session variability in short-utterance i-vector speaker verification systems. Two variations of SUN-LDA are proposed where normalization techniques are used to capture source variation from both short and full-length development i-vectors, one based upon pooling (SUN-LDA-pooled) and the other on concatenation (SUN-LDA-concat) across the duration and source-dependent session variation. Both the SUN-LDA-pooled and SUN-LDA-concat techniques are shown to provide improvement over traditional LDA on NIST 08 truncated 10sec-10sec evaluation conditions, with the highest improvement obtained with the SUN-LDA-concat technique achieving a relative improvement of 8% in EER for mis-matched conditions and over 3% for matched conditions over traditional LDA approaches.

RNAi for insect-proof plants

RNA interference induced in insects after ingestion of plant-expressed hairpin RNA offers promise for managing devastating crop pests

A novel T-DNA vector design for selection of transgenic lines with simple transgene integration and stable transgene expression

Plants transformed with Agrobacterium frequently contain T-DNA concatamers with direct-repeat (d/r) or inverted-repeat (i/r) transgene integrations, and these repetitive T-DNA insertions are often associated with transgene silencing. To facilitate the selection of transgenic lines with simple T-DNA insertions, we constructed a binary vector (pSIV) based on the principle of hairpin RNA (hpRNA)-induced gene silencing. The vector is designed so that any transformed cells that contain more than one insertion per locus should generate hpRNA against the selective marker gene, leading to its silencing. These cells should, therefore, be sensitive to the selective agent and less likely to regenerate. Results from Arabidopsis and tobacco transformation showed that pSIV gave considerably fewer transgenic lines with repetitive insertions than did a conventional T-DNA vector (pCON). Furthermore, the transgene was more stably expressed in the pSIV plants than in the pCON plants. Rescue of plant DNA flanking sequences from pSIV plants was significantly more frequent than from pCON plants, suggesting that pSIV is potentially useful for T-DNA tagging. Our results revealed a perfect correlation between the presence of tail-to-tail inverted repeats and transgene silencing, supporting the view that read-through hpRNA transcript derived from i/r T-DNA insertions is a primary inducer of transgene silencing in plants. © CSIRO 2005.

Marker gene elimination from transgenic barley, using co-transformation with adjacent 'twin T-DNAs' on a standard Agrobacterium transformation vector

We have tested a methodology for the elimination of the selectable marker gene after Agrobacterium-mediated transformation of barley. This involves segregation of the selectable marker gene away from the gene of interest following co-transformation using a plasmid carrying two T-DNAs, which were located adjacent to each other with no intervening region. A standard binary transformation vector was modified by insertion of a small section composed of an additional left and right T-DNA border, so that the selectable marker gene and the site for insertion of the gene of interest (GOI) were each flanked by a left and right border. Using this vector three different GOIs were transformed into barley. Analysis of transgene inheritance was facilitated by a novel and rapid assay utilizing PCR amplification from macerated leaf tissue. Co-insertion was observed in two thirds of transformants, and among these approximately one quarter had transgene inserts which segregated in the next generation to yield selectable marker-free transgenic plants. Insertion of non-T-DNA plasmid sequences was observed in only one of fourteen SMF lines tested. This technique thus provides a workable system for generating transgenic barley free from selectable marker genes, thereby obviating public concerns regarding proliferation of these genes.

Genome segment 5 of rice ragged stunt virus encodes a virion protein

The complete nucleotide sequence of the genome segment 5 (S5) of a Thai isolate of rice ragged stunt virus (RRSV) was determined. The 2682 nucleotide sequence contains a single long open reading frame capable of encoding a polypeptide with a molecular mass of ~91 kDa. Polypeptides encoded by various truncated cDNAs of S5 were expressed using the pGEX fusion protein vector and the highest level of fusion protein was obtained from a construct encoding a hydrophilic region of S5 protein. Antibodies raised against this fusion protein recognized a minor polypeptide, with a molecular mass of ~ 91 kDa, that was present in purified preparations of RRSV particles, infected insect vectors and infected rice plants. This indicates that RRSV S5 encodes a minor structural protein. Comparing the RRSV S5 sequence with sequences of other reo-viruses did not reveal any significant sequence similarities.