Development of a foot-and-mouth disease virus replicon system for the study of RNA replication

Foot-and-mouth disease (FMD) is a highly infectious disease of wild and domestic cloven–hoofed animals such as cattle, swine and deer. It is caused by one of the most contagious animal diseases known; FMD virus (FMDV). Since the disease is endemic in many countries, transmission by international travel/trade presents an on-going potential threat to the UK. Very little is known at the molecular level about how FMDV replicates within host cells. In this study, FMDV replicons have been used to investigate FMDV RNA replication and to improve our understanding of the viral life cycle: a process which will aid in the production of a new generation of live-attenuated vaccine candidate strains. Sequences encoding the capsid coding region of the genome have been replaced with green fluorescent protein (GFP) such that replication can be monitored in live cells via GFP fluorescence. This provides a rapid, non-invasive screen for replicative fitness that can be used outwith high disease security facilities. Differences between replicating and non-replicating forms could easily be distinguished, highlighting the potential of this system to screen for attenuated genomes. The FMDV replicon system was improved through a series of construct modifications until an optimal system was produced. A range of different methods were used to attenuate the replication of these genomes. Of major significance is the finding that increasing dinucleotide frequencies were shown to decrease growth kinetics of Echovirus 7 – as opposed to altering the codon-pair bias - and the application of this finding to construction of further replicon systems (and RNA viruses in general) is described

Problems in FMD eradication : a way forward?

The authors would like to acknowledge the support of the Wellcome Trust (eGrantsref. No. 8048) and Biotechnology and Biological Sciences Research Council (BBSRC; Grants BB/K003801/1 and BB/L004526/1).Foot and Mouth Disease Virus (FMDV) is one of the most commonly reported OIE-listed pathogens. FMDV is one of the most contagious mammalian viruses known to man: the virus is endemic in many developing countries causing substantial economic loses and the restriction of international trade in animals/animal products. The virus infects domestic animals (cattle, pigs, sheep, goats), but also a wide range of wild-life species, the latter forming reservoirs of disease. The need to diagnose FMDV infections (serotype/strain identification) and vaccine production/testing requires expensive, high disease security/containment facilities. Chemically inactivated (‘killed’) vaccines have been available for decades, but the huge genetic diversity of this virus (7 serotypes with 1000s of subtypes) and the need to periodically re-vaccinate animals to maintain protective levels of antibodies argue for the development of new vaccines. Indeed, in the early 1990s, on the basis of a cost-benefit analysis, the European Union replaced the policy of routine vaccination using the inactivated vaccine with disease control via mass-slaughter of infected and surrounding susceptible animals (plus vaccination in extremis). For various reasons mass-slaughter is unacceptable in many developing countries so in their case vaccination, in one form or another, is the only way forward. In the past few years there have been exciting developments in the production of new types of vaccine and many hold great prospects for improving disease control, but the thesis of this paper is that only the development of a new type of vaccine – live, attenuated, FMDV strains, offers the prospect of eradicating FMDV.Publisher PDFPeer reviewe

How the EAT-Lancet commission report 'Food in the Anthropocene' influenced discourse and research on food systems: a systematic review covering the first two years post-publication

In 2019, the EAT–Lancet Commission's report on food in the Anthropocene presented a planetary heath diet to improve health while reducing the environmental effect of food systems globally. We assessed EAT–Lancet's immediate influence on academic research and debate by conducting a systematic review of articles citing the Commission and others published from January, 2019, to April, 2021. The Commission influenced methods, results, or discourse for 192 (7·5%) of 2560 citing articles, stimulating cross-disciplinary research and debate across life sciences (47%), health and medical sciences (42%), and social sciences (11%). Sentiment analysis of 76 critiquing articles indicated that opinions were, on average, more positive than negative. Positive sentiments centred on benefits for informing policy, public health, and raising public awareness. Negative sentiments included insufficient attention to socioeconomic dimensions, feasibility, and environmental effects other than emissions. Empirical articles predominantly evaluated the effects of changed diets or food production on the environment and wellbeing (29%), compared current diets with EAT–Lancet recommendations (12%), or informed future policy and research agendas (20%). Despite limitations in EAT–Lancet's method, scope, and implementation feasibility, the academic community supported these recommendations. A broad suite of research needs was identified focusing on the effects of food processing, socioeconomic and political drivers of diet and health, and optimising consumption or production for environment and health

FMDV replicons encoding green fluorescent protein are replication competent

The study of replication of viruses that require high bio-secure facilities can be accomplished with less stringent containment using non-infectious 'replicon' systems. The FMDV replicon system (pT7rep) reported by Mclnerney et al. (2000) was modified by the replacement of sequences encoding chloramphenicol acetyl-transferase (CAT) with those encoding a functional L proteinase (Lpro) linked to a bi-functional fluorescent/antibiotic resistance fusion protein (green fluorescent protein/puromycin resistance, [GFP-PAC]). Cells were transfected with replicon-derived transcript RNA and GFP fluorescence quantified. Replication of transcript RNAs was readily detected by fluorescence, whilst the signal from replication-incompetent forms of the genome was >2-fold lower. Surprisingly, a form of the replicon lacking the Lpro showed a significantly stronger fluorescence signal, but appeared with slightly delayed kinetics. Replication can, therefore, be quantified simply by live-cell imaging and image analyses, providing a rapid and facile alternative to RT-qPCR or CAT assays

Greasing the wheels or a spanner in the works?:Regulation of the cardiac sodium pump by palmitoylation

The ubiquitous sodium/potassium ATPase (Na pump) is the most abundant primary active transporter at the cell surface of multiple cell types, including ventricular myocytes in the heart. The activity of the Na pump establishes transmembrane ion gradients that control numerous events at the cell surface, positioning it as a key regulator of the contractile and metabolic state of the myocardium. Defects in Na pump activity and regulation elevate intracellular Na in cardiac muscle, playing a causal role in the development of cardiac hypertrophy, diastolic dysfunction, arrhythmias and heart failure. Palmitoylation is the reversible conjugation of the fatty acid palmitate to specific protein cysteine residues; all subunits of the cardiac Na pump are palmitoylated. Palmitoylation of the pump’s accessory subunit phospholemman (PLM) by the cell surface palmitoyl acyl transferase DHHC5 leads to pump inhibition, possibly by altering the relationship between the pump catalytic α subunit and specifically bound membrane lipids. In this review, we discuss the functional impact of PLM palmitoylation on the cardiac Na pump and the molecular basis of recognition of PLM by its palmitoylating enzyme DHHC5, as well as effects of palmitoylation on Na pump cell surface abundance in the cardiac muscle. We also highlight the numerous unanswered questions regarding the cellular control of this fundamentally important regulatory process

Employing transposon mutagenesis to investigate foot-and-mouth disease virus replication

Probing the molecular interactions within the foot-and-mouth disease virus (FMDV) RNA replication complex has been restricted in part to the lack of suitable reagents. Random insertional mutagenesis has proven an excellent method to reveal domains of proteins essential for viral replication as well as locations that can tolerate small genetic insertions. Such insertion sites can be subsequently adapted by the incorporation of commonly used epitope tags and so facilitate their detection with commercial available reagents. In this study, we use random transposon-mediated mutagenesis to produce a library of 15 nucleotide insertions in the FMDV nonstructural polyprotein. Using a replicon-based assay we isolated multiple replication-competent as well as replication-defective insertions. We have adapted the replication competent insertion sites for the successful incorporation of epitope tags within FMDV non-structural proteins, for the use in a variety of downstream assays. Additionally, we show that replication of some of the replication-defective insertion mutants can be rescued by co-transfection of a 'helper' replicon, demonstrating a novel use of random mutagenesis to identify inter-genomic trans-complementation. Both the epitope tags and replication-defective insertions identified here will be valuable tools for probing interactions within picornaviral replication complexes

Spatial priorities for conserving the most intact biodiverse forests within Central Africa

The forests of Central Africa contain some of Earth's few remaining intact forests. These forests are increasingly threatened by infrastructure development, agriculture, and unsustainable extraction of natural resources (e.g. minerals, bushmeat, and timber), all of which is leading to deforestation and forest degradation, particularly defaunation, and hence causing declines in biodiversity and a significant increase in carbon emissions. Given the pervasive nature of these threats, the global importance of Central African forests for biodiversity conservation, and the limited resources for conservation and sustainable management, there is a need to identify where the most important areas are to orientate conservation efforts. We developed a novel approach for identifying spatial priorities where conservation efforts can maximize biodiversity benefits within Central Africa's most intact forest areas. We found that the Democratic Republic of Congo has the largest amount of priority areas in the region, containing more than half, followed by Gabon, the Republic of Congo and Cameroon. We compared our approach to one that solely prioritizes forest intactness and one that aims to achieve only biodiversity representation objectives. We found that when priorities are only based on forest intactness (without considering biodiversity representation), there are significantly fewer biodiversity benefits and vice versa. We therefore recommend multi-objective planning that includes biodiversity representation and forest intactness to ensure that both objectives are maximized. These results can inform various types of conservation strategies needed within the region, including land-use planning, jurisdictional REDD + initiatives, and performance related carbon payments, protected area expansion, community forest management, and forest concession plans