The wMel Wolbachia strain blocks dengue and invades caged Aedes aegypti populations

Dengue fever is the most important mosquito-borne viral disease of humans with more than 50 million cases estimated annually in more than 100 countries. Disturbingly, the geographic range of dengue is currently expanding and the severity of outbreaks is increasing. Control options for dengue are very limited and currently focus on reducing population abundance of the major mosquito vector, Aedes aegypti. These strategies are failing to reduce dengue incidence in tropical communities and there is an urgent need for effective alternatives. It has been proposed that endosymbiotic bacterial Wolbachia infections of insects might be used in novel strategies for dengue control. For example, the wMelPop-CLA Wolbachia strain reduces the lifespan of adult A. aegypti mosquitoes in stably transinfected lines. This life-shortening phenotype was predicted to reduce the potential for dengue transmission. The recent discovery that several Wolbachia infections, including wMelPop-CLA, can also directly influence the susceptibility of insects to infection with a range of insect and human pathogens has markedly changed the potential for Wolbachia infections to control human diseases. Here we describe the successful transinfection of A. aegypti with the avirulent wMel strain of Wolbachia, which induces the reproductive phenotype cytoplasmic incompatibility with minimal apparent fitness costs and high maternal transmission, providing optimal phenotypic effects for invasion. Under semi-field conditions, the wMel strain increased from an initial starting frequency of 0.65 to near fixation within a few generations, invading A. aegypti populations at an accelerated rate relative to trials with the wMelPop-CLA strain. We also show that wMel and wMelPop-CLA strains block transmission of dengue serotype 2 (DENV-2) in A. aegypti, forming the basis of a practical approach to dengue suppression.

Wolbachia uses host microRNAs to manipulate host gene expression and facilitate colonization of the dengue vector Aedes aegypti

The obligate endosymbiont Wolbachia pipientis is found in a wide range of invertebrates where they are best known for manipulating host reproduction. Recent studies have shown that Wolbachia also can modulate the lifespan of host insects and interfere with the development of human pathogens in mosquito vectors. Despite considerable study, very little is known about the molecular interactions between Wolbachia and its hosts that might mediate these effects. Using microarrays, we show that the microRNA (miRNA) profile of the mosquito, Aedes aegypti, is significantly altered by the wMelPop-CLA strain of W. pipientis. We found that a host miRNA (aae-miR-2940) is induced after Wolbachia infection in both mosquitoes and cell lines. One target of aae-miR-2940 is the Ae. aegypti metalloprotease gene. Interestingly, expression of the target gene was induced after Wolbachia infection, ectopic expression of the miRNA independent of Wolbachia, or transfection of an artificial mimic of the miRNA into mosquito cells. We also confirmed the interaction of aae-miR-2940 with the target sequences using GFP as a reporter gene. Silencing of the metalloprotease gene in both Wolbachia-infected cells and adult mosquitoes led to a significant reduction in Wolbachia density, as did inhibition of the miRNA in cells. These results indicate that manipulation of the mosquito metalloprotease gene via aae-miR-2940 is crucial for efficient maintenance of the endosymbiont. This report shows how Wolbachia alters the host miRNA profile and provides insight into the mechanisms of host manipulation used by this widespread endosymbiont.

Wolbachia-mediated resistance to dengue virus infection and death at the cellular level

Dengue is currently the most important arthropod-borne viral disease of humans. Recent work has shown dengue virus displays limited replication in its primary vector, the mosquito Aedes aegypti, when the insect harbors the endosymbiotic bacterium Wolbachia pipientis. Wolbachia-mediated inhibition of virus replication may lead to novel methods of arboviral control, yet the functional and cellular mechanisms that underpin it are unknown.

Are insect frugivores always plant pests? The impact of fruit fly (Diptera: Tephritidae) larvae on host plant fitness

Herbivory is generally regarded as negatively impacting on host plant fitness. Frugivorous insects, which feed directly on plant reproductive tissues, are predicted to be particularly damaging to hosts. We tested this prediction with the fruit fly, Bactrocera tryoni, by recording the impact of larval feeding on two direct (seed number and germination) and two indirect (fruit decay rate and attraction/deterrence of vertebrate frugivores) measures of host plant fitness. Experiments were done in the laboratory, glasshouse and tropical rainforest. We found no negative impact of larval feeding on seed number or germination for three test plants: tomato, capsicum and eggplant. Further, larval feeding accelerated the initiation of decay and increased the final level of fruit decay in tomatoes, apples, pawpaw and pear, a result considered to be beneficial to the fruit. In rainforest studies, native rodents preferred infested apple and pears compared to uninfested control fruit; however, there were no differences observed between treatments for tomato and pawpaw. For our study fruits, these results demonstrate that fruit fly larval infestation has neutral or beneficial impacts on the host plant, an outcome which may be largely influenced by the physical properties of the host. These results may contribute to explaining why fruit flies have not evolved the same level of host specialization generally observed for other herbivore groups.

Stability studies of HIV-1 Pr55gag virus-like particles made in insect cells after storage in various formulation media

Background: HIV-1 Pr55gag virus-like particles (VLPs) expressed by baculovirus in insect cells are considered to be a very promising HIV-1 vaccine candidate, as they have been shown to elicit broad cellular immune responses when tested in animals, particularly when used as a boost to DNA or BCG vaccines. However, it is important for the VLPs to retain their structure for them to be fully functional and effective. The medium in which the VLPs are formulated and the temperature at which they are stored are two important factors affecting their stability. FINDINGS We describe the screening of 3 different readily available formulation media (sorbitol, sucrose and trehalose) for their ability to stabilise HIV-1 Pr55gag VLPs during prolonged storage. Transmission electron microscopy (TEM) was done on VLPs stored at two different concentrations of the media at three different temperatures (4[degree sign]C, --20[degree sign]C and -70[degree sign]C) over different time periods, and the appearance of the VLPs was compared. VLPs stored in 15% trehalose at -70[degree sign]C retained their original appearance the most effectively over a period of 12 months. VLPs stored in 5% trehalose, sorbitol or sucrose were not all intact even after 1 month storage at the temperatures tested. In addition, we showed that VLPs stored under these conditions were able to be frozen and re-thawed twice before showing changes in their appearance. Conclusions Although the inclusion of other analytical tools are essential to validate these preliminary findings, storage in 15% trehalose at -70[degree sign]C for 12 months is most effective in retaining VLP stability.

Transient expression of Human papillomavirus type 16 L1 protein in Nicotiana benthamiana using an infectious tobamovirus vector

A Tobacco mosaic virus (TMV)-derived vector was used to express a native Human papillomavirus type 16 (HPV-16) L1 gene in Nicotiana benthamiana by means of infectious in vitro RNA transcripts inoculated onto N. benthamiana plants. HPV-16 L1 protein expression was quantitated by enzyme-linked immunosorbent assays (ELISA) after concentration of the plant extract. We estimated that the L1 product yield was 20-37 μg/kg of fresh leaf material. The L1 protein in the concentrated extract was antigenically characterised using the neutralising and conformation-specific Mabs H16:V5 and H16:E70, which bound to the plant-produced protein. Particles observed by transmission electron microscopy were mainly capsomers but virus-like particles (VLPs) similar to those produced in other systems were also present. Immunisation of rabbits with the concentrated plant extract induced a weak immune response. This is the first report of the successful expression of an HPV L1 gene in plants using a plant virus vector. © 2006 Elsevier B.V. All rights reserved.

High level protein expression in plants through the use of a novel autonomously replicating geminivirus shuttle vector

We constructed a novel autonomously replicating gene expression shuttle vector, with the aim of developing a system for transiently expressing proteins at levels useful for commercial production of vaccines and other proteins in plants. The vector, pRIC, is based on the mild strain of the geminivirus Bean yellow dwarf virus (BeYDV-m) and is replicationally released into plant cells from a recombinant Agrobacterium tumefaciens Ti plasmid. pRIC differs from most other geminivirus-based vectors in that the BeYDV replication-associated elements were included in cis rather than from a co-transfected plasmid, while the BeYDV capsid protein (CP) and movement protein (MP) genes were replaced by an antigen encoding transgene expression cassette derived from the non-replicating A. tumefaciens vector, pTRAc. We tested vector efficacy in Nicotiana benthamiana by comparing transient cytoplasmic expression between pRIC and pTRAc constructs encoding either enhanced green fluorescent protein (EGFP) or the subunit vaccine antigens, human papillomavirus subtype 16 (HPV-16) major CP L1 and human immunodeficiency virus subtype C p24 antigen. The pRIC constructs were amplified in planta by up to two orders of magnitude by replication, while 50% more HPV-16 L1 and three- to seven-fold more EGFP and HIV-1 p24 were expressed from pRIC than from pTRAc. Vector replication was shown to be correlated with increased protein expression. We anticipate that this new high-yielding plant expression vector will contribute towards the development of a viable plant production platform for vaccine candidates and other pharmaceuticals. © 2009 Blackwell Publishing Ltd.

Optimization of chimeric HIV-1 virus-like particle production in a baculovirus-insect cell expression system

A baculovirus-insect cell expression system potentially provides the means to produce prophylactic HIV-1 virus-like particle (VLP) vaccines inexpensively and in large quantities. However, the system must be optimized to maximize yields and increase process efficiency. In this study, we optimized the production of two novel, chimeric HIV-1 VLP vaccine candidates (GagRT and GagTN) in insect cells. This was done by monitoring the effects of four specific factors on VLP expression: these were insect cell line, cell density, multiplicity of infection (MOI), and infection time. The use of western blots, Gag p24 ELISA, and four-factorial ANOVA allowed the determination of the most favorable conditions for chimeric VLP production, as well as which factors affected VLP expression most significantly. Both VLP vaccine candidates favored similar optimal conditions, demonstrating higher yields of VLPs when produced in the Trichoplusia ni Pro insect cell line, at a cell density of 1 × 106 cells/mL, and an infection time of 96 h post infection. It was found that cell density and infection time were major influencing factors, but that MOI did not affect VLP expression significantly. This work provides a potentially valuable guideline for HIV-1 protein vaccine optimization, as well as for general optimization of a baculovirus-based expression system to produce complex recombinant proteins. © 2009 American Institute of Chemical Engineers.

Use of the piggyBac transposon to create HIV-1 gag transgenic insect cell lines for continuous VLP production

Background Insect baculovirus-produced Human immunodeficiency virus type 1 (HIV-1) Gag virus-like-particles (VLPs) stimulate good humoral and cell-mediated immune responses in animals and are thought to be suitable as a vaccine candidate. Drawbacks to this production system include contamination of VLP preparations with baculovirus and the necessity for routine maintenance of infectious baculovirus stock. We used piggyBac transposition as a novel method to create transgenic insect cell lines for continuous VLP production as an alternative to the baculovirus system. Results Transgenic cell lines maintained stable gag transgene integration and expression up to 100 cell passages, and although the level of VLPs produced was low compared to baculovirus-produced VLPs, they appeared similar in size and morphology to baculovirus-expressed VLPs. In a murine immunogenicity study, whereas baculovirus-produced VLPs elicited good CD4 immune responses in mice when used to boost a prime with a DNA vaccine, no boost response was elicited by transgenically produced VLPs. Conclusion Transgenic insect cells are stable and can produce HIV Pr55 Gag VLPs for over 100 passages: this novel result may simplify strategies aimed at making protein subunit vaccines for HIV. Immunogenicity of the Gag VLPs in mice was less than that of baculovirus-produced VLPs, which may be due to lack of baculovirus glycoprotein incorporation in the transgenic cell VLPs. Improved yield and immunogenicity of transgenic cell-produced VLPs may be achieved with the addition of further genetic elements into the piggyBac integron.

An investigation into the use of human papillomavirus type 16 virus-like particles as a delivery vector system for foreign proteins: N- and C-terminal fusion of GFP to the L1 and L2 capsid proteins

Development of vaccine strategies against human papillomavirus (HPV), which causes cervical cancer, is a priority. We investigated the use of virus-like particles (VLPs) of the most prevalent type, HPV-16, as carriers of foreign proteins. Green fluorescent protein (GFP) was fused to the N or C terminus of both L1 and L2, with L2 chimeras being co-expressed with native L1. Purified chimaeric VLPs were comparable in size (∼55 nm) to native HPV VLPs. Conformation-specific monoclonal antibodies (Mabs) bound to the VLPs, thereby indicating that they possibly retain their antigenicity. In addition, all of the VLPs encapsidated DNA in the range of 6-8 kb. © 2007 Springer-Verlag.

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.