A New Model to Produce Infectious Hepatitis C Virus without the Replication Requirement

Numerous constraints significantly hamper the experimental study of hepatitis C virus (HCV). Robust replication in cell culture occurs with only a few strains, and is invariably accompanied by adaptive mutations that impair in vivo infectivity/replication. This problem complicates the production and study of authentic HCV, including the most prevalent and clinically important genotype 1 (subtypes 1a and 1b). Here we describe a novel cell culture approach to generate infectious HCV virions without the HCV replication requirement and the associated cell-adaptive mutations. The system is based on our finding that the intracellular environment generated by a West-Nile virus (WNV) subgenomic replicon rendered a mammalian cell line permissive for assembly and release of infectious HCV particles, wherein the HCV RNA with correct 5′ and 3′ termini was produced in the cytoplasm by a plasmid-driven dual bacteriophage RNA polymerase-based transcription/amplification system. The released particles preferentially contained the HCV-based RNA compared to the WNV subgenomic RNA. Several variations of this system are described with different HCV-based RNAs: (i) HCV bicistronic particles (HCVbp) containing RNA encoding the HCV structural genes upstream of a cell-adapted subgenomic replicon, (ii) HCV reporter particles (HCVrp) containing RNA encoding the bacteriophage SP6 RNA polymerase in place of HCV nonstructural genes, and (iii) HCV wild-type particles (HCVwt) containing unmodified RNA genomes of diverse genotypes (1a, strain H77; 1b, strain Con1; 2a, strain JFH-1). Infectivity was assessed based on the signals generated by the HCV RNA molecules introduced into the cytoplasm of target cells upon virus entry, i.e. HCV RNA replication and protein production for HCVbp in Huh-7.5 cells as well as for HCVwt in HepG2-CD81 cells and human liver slices, and SP6 RNA polymerase-driven firefly luciferase for HCVrp in target cells displaying candidate HCV surface receptors. HCV infectivity was inhibited by pre-incubation of the particles with anti-HCV antibodies and by a treatment of the target cells with leukocyte interferon plus ribavirin. The production of authentic infectious HCV particles of virtually any genotype without the adaptive mutations associated with in vitro HCV replication represents a new paradigm to decipher the requirements for HCV assembly, release, and entry, amenable to analyses of wild type and genetically modified viruses of the most clinically significant HCV genotypes

Semiochemical-based alternatives to synthetic toxicant insecticides for pollen beetle management

There is an urgent need to develop sustainable pest management systems to protect arable crops in order to replace the current over-reliance on synthetic insecticides. Semiochemicals are insect- or plant-derived chemicals that are used by organisms as information signals. Integrated pest management tools are currently in development that utilise semiochemicals to manipulate the behaviour of pest insects and their natural enemies to provide effective control of pests within the crop. These innovative tools usually require fewer inputs and can involve multiple elements therefore reducing the likelihood of resistance developing compared with use of synthetic toxicants. We review here the life cycle of the pollen beetle Brassicogethes aeneus (previously known as Meligethes aeneus) which is a pest insect of oilseed rape (Brassica napus) and describe the current knowledge of any behaviour mediated by semiochemicals in this species. We discuss the behavioural processes where semiochemical-based control approaches may be appropriate and consider how these approaches could be integrated into an integrated pest management strategy for this important arable crop

Floral and insect-induced volatile formation in Arabidopsis lyrata ssp. petraea, a perennial, outcrossing relative of A. thaliana

Volatile organic compounds have been reported to serve some important roles in plant communication with other organisms, but little is known about the biological functions of most of these substances. To gain insight into this problem, we have compared differences in floral and vegetative volatiles between two closely related plant species with different life histories. The self-pollinating annual, Arabidopsis thaliana, and its relative, the outcrossing perennial, Arabidopsis lyrata, have markedly divergent life cycles and breeding systems. We show that these differences are in part reflected in the formation of distinct volatile mixtures in flowers and foliage. Volatiles emitted from flowers of a German A. lyrata ssp. petraea population are dominated by benzenoid compounds in contrast to the previously described sesquiterpene-dominated emissions of A. thaliana flowers. Flowers of A. lyrata ssp. petraea release benzenoid volatiles in a diurnal rhythm with highest emission rates at midday coinciding with observed visitations of pollinating insects. Insect feeding on leaves of A. lyrata ssp. petraea causes a variable release of the volatiles methyl salicylate, C11- and C16-homoterpenes, nerolidol, plus the sesquiterpene (E)-β-caryophyllene, which in A. thaliana is emitted exclusively from flowers. An insect-induced gene (AlCarS) with high sequence similarity to the florally expressed (E)-β-caryophyllene synthase (AtTPS21) from A. thaliana was identified from individuals of a German A. lyrata ssp. petraea population. Recombinant AlCarS converts the sesquiterpene precursor, farnesyl diphosphate, into (E)-β-caryophyllene with α-humulene and α-copaene as minor products indicating its close functional relationship to the A. thaliana AtTPS21. Differential regulation of these genes in flowers and foliage is consistent with the different functions of volatiles in the two Arabidopsis species

Use of an innovative T-tube maze assay and the proboscis extension response assay to assess sublethal effects of GM products and pesticides on learning capacity of the honey bee Apis mellifera L.

Transgenic Cry1Ac+CpTI cotton (CCRI41) is a promising cotton cultivar throughout China but side effects and especially sublethal effects of this transgenic cultivar on beneficial insects remain poorly studied. More specifically potential sublethal effects on behavioural traits of the honey bee Apis mellifera L. have not been formally assessed despite the importance of honey bees for pollination. The goal of our study was to assess potential effects of CCRI41 cotton pollen on visual and olfactory learning by honey bees. After a 7-day oral chronic exposure to honey mixed with either CCRI41 pollen, imidacloprid-treated conventional pollen (used as positive sublethal control) or conventional pollen (control), learning performance was evaluated by the classical proboscis extension reflex (PER) procedure as well as a T-tube maze test. The latter assay was designed as a new device to assess potential side effects of pesticides on visual associative learning of honey bees. These two procedures were complementary because the former focused on olfactory learning while the latter was involved in visual learning based on visual orientation ability. Oral exposure to CCRI41 pollen did not affect learning capacities of honey bees in both the T-tube maze and PER tests. However, exposure to imidacloprid resulted in reduced visual learning capacities in T-tube maze evaluation and decreased olfactory learning performances measured with PER. The implications of these results are discussed in terms of risks of transgenic CCRI41 cotton crops for honey bees

Herbivory by a Phloem-Feeding Insect Inhibits Floral Volatile Production

There is extensive knowledge on the effects of insect herbivory on volatile emission from vegetative tissue, but little is known about its impact on floral volatiles. We show that herbivory by phloem-feeding aphids inhibits floral volatile emission in white mustard Sinapis alba measured by gas chromatographic analysis of headspace volatiles. The effect of the Brassica specialist aphid Lipaphis erysimi was stronger than the generalist aphid Myzus persicae and feeding by chewing larvae of the moth Plutella xylostella caused no reduction in floral volatile emission. Field observations showed no effect of L. erysimi-mediated floral volatile emission on the total number of flower visits by pollinators. Olfactory bioassays suggested that although two aphid natural enemies could detect aphid inhibition of floral volatiles, their olfactory orientation to infested plants was not disrupted. This is the first demonstration that phloem-feeding herbivory can affect floral volatile emission, and that the outcome of interaction between herbivory and floral chemistry may differ depending on the herbivore's feeding mode and degree of specialisation. The findings provide new insights into interactions between insect herbivores and plant chemistry

The Kinase Inhibitor SFV785 Dislocates Dengue Virus Envelope Protein from the Replication Complex and Blocks Virus Assembly

Dengue virus (DENV) is the etiologic agent for dengue fever, for which there is no approved vaccine or specific anti-viral drug. As a remedy for this, we explored the use of compounds that interfere with the action of required host factors and describe here the characterization of a kinase inhibitor (SFV785), which has selective effects on NTRK1 and MAPKAPK5 kinase activity, and anti-viral activity on Hepatitis C, DENV and yellow fever viruses. SFV785 inhibited DENV propagation without inhibiting DENV RNA synthesis or translation. The compound did not cause any changes in the cellular distribution of non-structural 3, a protein critical for DENV RNA synthesis, but altered the distribution of the structural envelope protein from a reticulate network to enlarged discrete vesicles, which altered the co-localization with the DENV replication complex. Ultrastructural electron microscopy analyses of DENV-infected SFV785-treated cells showed the presence of viral particles that were distinctly different from viable enveloped virions within enlarged ER cisternae. These viral particles were devoid of the dense nucleocapsid. The secretion of the viral particles was not inhibited by SFV785, however a reduction in the amount of secreted infectious virions, DENV RNA and capsid were observed. Collectively, these observations suggest that SFV785 inhibited the recruitment and assembly of the nucleocapsid in specific ER compartments during the DENV assembly process and hence the production of infectious DENV. SFV785 and derivative compounds could be useful biochemical probes to explore the DENV lifecycle and could also represent a new class of anti-virals

Field Attractants for Pachnoda interrupta Selected by Means of GC-EAD and Single Sensillum Screening

The sorghum chafer, Pachnoda interrupta Olivier (Coleoptera: Scarabaeidae: Cetoniinae), is a key pest on sorghum, Sorghum bicolor (L.) Moench (Poaceae), in Ethiopia. At present there is a lack of efficient control methods. Trapping shows promise for reduction of the pest population, but would benefit from the development of attractive lures. To find attractants that could be used for control of P. interrupta, either by mass trapping or by monitoring as part of integrated pest management, we screened headspace collections of sorghum and the highly attractive weed Abutilon figarianum Webb (Malvaceae) for antennal activity using gas chromatograph-coupled electroantennographic detection (GC-EAD). Compounds active in GC-EAD were identified by combined gas chromatography and mass spectrometry (GC-MS). Field trapping suggested that attraction is governed by a few influential compounds, rather than specific odor blends. Synthetic sorghum and abutilon odor blends were attractive, but neither blend outperformed the previously tested attractants eugenol and methyl salicylate, of which the latter also was part of the abutilon blend. The strong influence of single compounds led us to search for novel attractive compounds, and to investigate the role of individual olfactory receptor neurons (ORNs) in the perception of kairomones. We screened the response characteristics of ORNs to 82 putative kairomones in single sensillum recordings (SSR), and found a number of key ligand candidates for specific classes of ORNs. Out of these key ligand candidates, six previously untested compounds were selected for field trapping trials: anethole, benzaldehyde, racemic 2,3-butanediol, isoamyl alcohol, methyl benzoate and methyl octanoate. The compounds were selected on the basis that they activated different classes of ORNs, thus allowing us to test potential kairomones that activate large non-overlapping populations of the peripheral olfactory system, while avoiding redundant multiple activations of the same ORN type. Field trapping results revealed that racemic 2,3-butanediol is a powerful novel attractant for P. interrupta

Syndromics: A Bioinformatics Approach for Neurotrauma Research

Substantial scientific progress has been made in the past 50 years in delineating many of the biological mechanisms involved in the primary and secondary injuries following trauma to the spinal cord and brain. These advances have highlighted numerous potential therapeutic approaches that may help restore function after injury. Despite these advances, bench-to-bedside translation has remained elusive. Translational testing of novel therapies requires standardized measures of function for comparison across different laboratories, paradigms, and species. Although numerous functional assessments have been developed in animal models, it remains unclear how to best integrate this information to describe the complete translational “syndrome” produced by neurotrauma. The present paper describes a multivariate statistical framework for integrating diverse neurotrauma data and reviews the few papers to date that have taken an information-intensive approach for basic neurotrauma research. We argue that these papers can be described as the seminal works of a new field that we call “syndromics”, which aim to apply informatics tools to disease models to characterize the full set of mechanistic inter-relationships from multi-scale data. In the future, centralized databases of raw neurotrauma data will enable better syndromic approaches and aid future translational research, leading to more efficient testing regimens and more clinically relevant findings

Cellular therapies for treating pain associated with spinal cord injury

Spinal cord injury leads to immense disability and loss of quality of life in human with no satisfactory clinical cure. Cell-based or cell-related therapies have emerged as promising therapeutic potentials both in regeneration of spinal cord and mitigation of neuropathic pain due to spinal cord injury. This article reviews the various options and their latest developments with an update on their therapeutic potentials and clinical trialing