Rational Engineering of Recombinant Picornavirus Capsids to Produce Safe,Protective Vaccine Antigen

Foot-and-mouth disease remains a major plague of livestock and outbreaks are often economically catastrophic. Current inactivated virus vaccines require expensive high containment facilities for their production and maintenance of a cold-chain for their activity. We have addressed both of these major drawbacks. Firstly we have developed methods to efficiently express recombinant empty capsids. Expression constructs aimed at lowering the levels and activity of the viral protease required for the cleavage of the capsid protein precursor were used; this enabled the synthesis of empty A-serotype capsids in eukaryotic cells at levels potentially attractive to industry using both vaccinia virus and baculovirus driven expression. Secondly we have enhanced capsid stability by incorporating a rationally designed mutation, and shown by X-ray crystallography that stabilised and wild-type empty capsids have essentially the same structure as intact virus. Cattle vaccinated with recombinant capsids showed sustained virus neutralisation titres and protection from challenge 34 weeks after immunization. This approach to vaccine antigen production has several potential advantages over current technologies by reducing production costs, eliminating the risk of infectivity and enhancing the temperature stability of the product. Similar strategies that will optimize host cell viability during expression of a foreign toxic gene and/or improve capsid stability could allow the production of safe vaccines for other pathogenic picornaviruses of humans and animals.     

Small Hydrophobic Protein of Human Metapneumovirus Does Not Affect Virus Replication and Host Gene Expression In Vitro

Human metapneumovirus (HMPV) encodes a small hydrophobic (SH) protein of unknown function. HMPV from which the SH open reading frame was deleted (HMPVΔSH) was viable and displayed similar replication kinetics, cytopathic effect and plaque size compared with wild type HMPV in several cell-lines. In addition, no differences were observed in infection efficiency or cell-to-cell spreading in human primary bronchial epithelial cells (HPBEC) cultured at an air-liquid interphase. Host gene expression was analyzed in A549 cells infected with HMPV or HMPVΔSH using microarrays and mass spectrometry (MS) based techniques at multiple time points post infection. Only minor differences were observed in mRNA or protein expression levels. A possible function of HMPV SH as apoptosis blocker, as proposed for several members of the family Paramyxoviridae, was rejected based on this analysis. So far, a clear phenotype of HMPV SH deletion mutants in vitro at the virus and host levels is absent.     

Fluctuations in Species-Level Protein Expression Occur during Element and Nutrient Cycling in the Subsurface

While microbial activities in environmental systems play a key role in the utilization and cycling of essential elements and compounds, microbial activity and growth frequently fluctuates in response to environmental stimuli and perturbations. To investigate these fluctuations within a saturated aquifer system, we monitored a carbon-stimulated in situ Geobacter population while iron reduction was occurring, using 16S rRNA abundances and high-resolution tandem mass spectrometry proteome measurements. Following carbon amendment, 16S rRNA analysis of temporally separated samples revealed the rapid enrichment of Geobacter-like environmental strains with strong similarity to G. bemidjiensis. Tandem mass spectrometry proteomics measurements suggest high carbon flux through Geobacter respiratory pathways, and the synthesis of anapleurotic four carbon compounds from acetyl-CoA via pyruvate ferredoxin oxidoreductase activity. Across a 40-day period where Fe(III) reduction was occurring, fluctuations in protein expression reflected changes in anabolic versus catabolic reactions, with increased levels of biosynthesis occurring soon after acetate arrival in the aquifer. In addition, localized shifts in nutrient limitation were inferred based on expression of nitrogenase enzymes and phosphate uptake proteins. These temporal data offer the first example of differing microbial protein expression associated with changing geochemical conditions in a subsurface environment.     

Moving Domain Computational Fluid Dynamics to Interface with an Embryonic Model of Cardiac Morphogenesis

Peristaltic contraction of the embryonic heart tube produces time- and spatial-varying wall shear stress (WSS) and pressure gradients (∇P) across the atrioventricular (AV) canal. Zebrafish (Danio rerio) are a genetically tractable system to investigate cardiac morphogenesis. The use of Tg(fli1a:EGFP)^y1 transgenic embryos allowed for delineation and two-dimensional reconstruction of the endocardium. This time-varying wall motion was then prescribed in a two-dimensional moving domain computational fluid dynamics (CFD) model, providing new insights into spatial and temporal variations in WSS and ∇P during cardiac development. The CFD simulations were validated with particle image velocimetry (PIV) across the atrioventricular (AV) canal, revealing an increase in both velocities and heart rates, but a decrease in the duration of atrial systole from early to later stages. At 20-30 hours post fertilization (hpf), simulation results revealed bidirectional WSS across the AV canal in the heart tube in response to peristaltic motion of the wall. At 40-50 hpf, the tube structure undergoes cardiac looping, accompanied by a nearly 3-fold increase in WSS magnitude. At 110-120 hpf, distinct AV valve, atrium, ventricle, and bulbus arteriosus form, accompanied by incremental increases in both WSS magnitude and ∇P, but a decrease in bi-directional flow. Laminar flow develops across the AV canal at 20-30 hpf, and persists at 110-120 hpf. Reynolds numbers at the AV canal increase from 0.07±0.03 at 20-30 hpf to 0.23±0.07 at 110-120 hpf (p< 0.05, n=6), whereas Womersley numbers remain relatively unchanged from 0.11 to 0.13. Our moving domain simulations highlights hemodynamic changes in relation to cardiac morphogenesis; thereby, providing a 2-D quantitative approach to complement imaging analysis.     

Is Consumer Response to Plain/Standardised Tobacco Packaging Consistent with Framework Convention on Tobacco Control Guidelines? A Systematic Review of Quantitative Studies

Background and Objectives

Standardised or ‘plain’ tobacco packaging was introduced in Australia in December 2012 and is currently being considered in other countries. The primary objective of this systematic review was to locate, assess and synthesise published and grey literature relating to the potential impacts of standardised tobacco packaging as proposed by the guidelines for the international Framework Convention on Tobacco Control: reduced appeal, increased salience and effectiveness of health warnings, and more accurate perceptions of product strength and harm.

Methods

Electronic databases were searched and researchers in the field were contacted to identify studies. Eligible studies were published or unpublished primary research of any design, issued since 1980 and concerning tobacco packaging. Twenty-five quantitative studies reported relevant outcomes and met the inclusion criteria. A narrative synthesis was conducted.

Results

Studies that explored the impact of package design on appeal consistently found that standardised packaging reduced the appeal of cigarettes and smoking, and was associated with perceived lower quality, poorer taste and less desirable smoker identities. Although findings were mixed, standardised packs tended to increase the salience and effectiveness of health warnings in terms of recall, attention, believability and seriousness, with effects being mediated by the warning size, type and position on pack. Pack colour was found to influence perceptions of product harm and strength, with darker coloured standardised packs generally perceived as containing stronger tasting and more harmful cigarettes than fully branded packs; lighter coloured standardised packs suggested weaker and less harmful cigarettes. Findings were largely consistent, irrespective of location and sample.

Conclusions

The evidence strongly suggests that standardised packaging will reduce the appeal of packaging and of smoking in general; that it will go some way to reduce consumer misperceptions regarding product harm based upon package design; and will help make the legally required on-pack health warnings more salient.     

Measurement of the Infection and Dissemination of Bluetongue Virus in Culicoides Biting Midges Using a Semi-Quantitative RT-PCR Assay and Isolation of Infectious Virus

Background

Culicoides biting midges (Diptera: Ceratopogonidae) are the biological vectors of globally significant arboviruses of livestock including bluetongue virus (BTV), African horse sickness virus (AHSV) and the recently emerging Schmallenberg virus (SBV). From 2006–2009 outbreaks of BTV in northern Europe inflicted major disruption and economic losses to farmers and several attempts were made to implicate Palaearctic Culicoides species as vectors. Results from these studies were difficult to interpret as they used semi-quantitative RT-PCR (sqPCR) assays as the major diagnostic tool, a technique that had not been validated for use in this role. In this study we validate the use of these assays by carrying out time-series detection of BTV RNA in two colony species of Culicoides and compare the results with the more traditional isolation of infectious BTV on cell culture.

Methodology/Principal Findings

A BTV serotype 1 strain mixed with horse blood was fed to several hundred individuals of Culicoides sonorensis (Wirth & Jones) and C. nubeculosus (Mg.) using a membrane-based assay and replete individuals were then incubated at 25°C. At daily intervals 25 Culicoides of each species were removed from incubation, homogenised and BTV quantified in each individual using sqPCR (C_q values) and virus isolation on a KC-C. sonorensis embryonic cell line, followed by antigen enzyme-linked immunosorbent assay (ELISA). In addition, comparisons were also drawn between the results obtained with whole C. sonorensis and with individually dissected individuals to determine the level of BTV dissemination.

Conclusions/Significance

C_q values generated from time-series infection experiments in both C. sonorensis and C. nubeculosus confirmed previous studies that relied upon the isolation and detection of infectious BTV. Implications on the testing of field-collected Culicoides as potential virus vectors by PCR assays and the use of such assays as front-line tools for use in diagnostic laboratories in this role are discussed.     

Endothelial Progenitors Exist within the Kidney and Lung Mesenchyme

The renal endothelium has been debated as arising from resident hemangioblast precursors that transdifferentiate from the nephrogenic mesenchyme (vasculogenesis) and/or from invading vessels (angiogenesis). While the Foxd1-positive renal cortical stroma has been shown to differentiate into cells that support the vasculature in the kidney (including vascular smooth muscle and pericytes) it has not been considered as a source of endothelial cell progenitors. In addition, it is unclear if Foxd1-positive mesenchymal cells in other organs such as the lung have the potential to form endothelium. This study examines the potential for Foxd1-positive cells of the kidney and lung to give rise to endothelial progenitors. We utilized immunofluorescence (IF) and fluorescence-activated cell sorting (FACS) to co-label Foxd1-expressing cells (including permanently lineage-tagged cells) with endothelial markers in embryonic and postnatal mice. We also cultured FACsorted Foxd1-positive cells, performed in vitro endothelial cell tubulogenesis assays and examined for endocytosis of acetylated low-density lipoprotein (Ac-LDL), a functional assay for endothelial cells. Immunofluorescence and FACS revealed that a subset of Foxd1-positive cells from kidney and lung co-expressed endothelial cell markers throughout embryogenesis. In vitro, cultured embryonic Foxd1-positive cells were able to differentiate into tubular networks that expressed endothelial cell markers and were able to endocytose Ac-LDL. IF and FACS in both the kidney and lung revealed that lineage-tagged Foxd1-positive cells gave rise to a significant portion of the endothelium in postnatal mice. In the kidney, the stromal-derived cells gave rise to a portion of the peritubular capillary endothelium, but not of the glomerular or large vessel endothelium. These findings reveal the heterogeneity of endothelial cell lineages; moreover, Foxd1-positive mesenchymal cells of the developing kidney and lung are a source of endothelial progenitors that are likely critical to patterning the vasculature.     

Dietary partitioning of three large carnivores in Majete Wildlife Reserve,Malawi

Between 2011 and 2012, the carnivore guild in Majete Wildlife Reserve (MWR), Malawi, was restored following the reintroduction of lion (Panthera leo) and leopard (Panthera pardus). The aim of this study was to describe and compare the diet of lion, leopard and resident spotted hyaena (Crocuta crocuta) using scat analysis. Lions and spotted hyaenas displayed the greatest dietary overlap (O_ab = 0.88) and selected mainly medium- to large-bodied prey species. Lions had a mean preferred prey weight of 120.33 ± 42.14 kg (SE), with warthog (Phacochoerus africanus) and waterbuck (Kobus ellipsiprymnus) making up 60.64% of relative biomass consumed. Spotted hyaenas had a mean preferred prey weight of 102.40 ± 41.69 kg and had a more generalised diet (B_a = 0.46) compared to lions (B_a = 0.36). In contrast, leopards occupied a dietary niche substantially lower than that of lions and spotted hyaenas, selecting relatively smaller prey with a mean preferred prey weight of 27.50 ± 6.74 kg. Our results suggest that coexistence between the resident hyaena and reintroduced lion and leopard in MWR is facilitated by dietary partitioning. We advise long-term monitoring of reintroduced carnivores in small, enclosed reserves to assess their impacts on predator and prey populations.