Study of an alternative gene embedded in the West Nile virus genome

RIASSUNTO L'analisi bioinformatica del genoma del virus West Nile (WNV) ha individuato 6 schemi di lettura alternativi, il più lungo dei quali (444bp), denominato WARF4, è presente solamente nei ceppi di WNV appartenenti alla linea virale I. Il gene alternativo è sovrapposto all’estremità 3' del gene NS4B ed in parte alla regione 5’ del gene dell’NS5. L'utilizzo di WARF4 è stato indirettamente dimostrato mediante l'individuazione di anticorpi specifici in sieri equini positivi al WNV. Poiché la sequenza di WARF4 è integrata nel gene della proteina virale NS4B, la nuova proteina è stata rinominata N-NS4B/WARF4. Lo scopo di questo progetto è dimostrare la produzione di N-NS4B/WARF4 in colture cellulari eucariotiche infettate con il virus West Nile. A tal fine abbiamo prodotto una una proteina ricombinante che include l'intera sequenza dello schema di lettura alternativo. Questa proteina è stata utilizzata come immunogeno per la produzione di un anticorpo monoclonale anti-WARF4, chiamato MAb 3A12. E' stata inoltre prodotta una proteina ricombinante che comprende la sequenza COOH terminale del gene NS4B da utilizzare come controllo nei test di validazione dell' anticorpo. MAb 3A12, è stato dapprima utilizzato in saggi immunoenzimatici che hanno confermato la sua specificità: in particolare è stata individuata la regione della proteina riconosciuta dall’anticorpo osservando la sua reattività in western blot con quattro peptidi sintetici che coprono tutta la sequenza aminoacidica codificata da WARF4. L’anticorpo MAb 3A12 ha riconosciuto la nuova proteina in cellule VERO infettate con il WNV appartenente alla linea virale I sia in esperimenti di immunofluorescenza che in western blot , mentre non ha reagito con cellule infettate con la linea virale II del virus. I risultati di analisi in western blot eseguite su lisato di cellule VERO infettate a tempi diversi dimostrano che N-NS4B/WARF4 viene espressa in quantità maggiore nella tarda fase dell’infezione. L’espressione “in vivo” della proteina N-NS4B/WARF4 è stata dimostrata indirettamente, sebbene su un numero limitato di campioni, valutando la sua immunogenicità in individui infettati con il WNV. Al momento non ci sono informazioni sperimentali circa il meccanismo di traduzione della proteina N-NS4B/WARF4, tuttavia si può supporre che la proteina venga prodotta in seguito ad uno scivolamento del ribosoma in posizione -1 durante la traduzione della poliproteina virale. Questo meccanismo di traduzione è già stato descritto per il West Nile ed è promosso da specifiche sequenze dell’RNA (slippery sequences associate a pseudoknot) rilevate dalle analisi bioinformatiche effettuate sui genomi virali analizzati. Come suggeriscono anche i dati ottenuti in western blot, in seguito allo scivolamento del ribosoma in posizione -1 la proteina codificata da WARF4 verrebbe sintetizzata come variante carbossi-terminale dell’antigene virale NS4B. Il possibile ruolo della proteina N-NS4B/WARF4 nel corso dell’infezione da WNV resta tuttora sconosciuto, tuttavia recenti dati di letteratura sembrerebbero suggerire un ruolo nella regolazione traduzionale del genoma virale. ABSTRACT Bioinformatics analysis performed on West Nile virus (WNV) genome revealed the presence of six alternative open reading frames, one of which, entitled WARF4, is the longest (444 bp) and exclusively restricted to the viral lineage I. The alternative gene is overlapping the COOH-terminal region of the NS4B gene and a small N-terminal portion of the NS5 gene. Since WARF4 is embedded in the NS4B gene, we rename the novel protein N-NS4B/WARF4. We previously reported that N-NS4B/WARF4 is able to elicit antibodies in WNV infected horses; however, there was no direct experimental proof of the existence of this novel protein. The purpose of this study was to demonstrate the in vitro production of WARF4 protein following WNV infection of mammalian cultured cells. For this purpose we produced two recombinant proteins : His- WARF4, that was used as immunogen for the production of a monoclonal antibody (MAb 3A12), and a NS4B carboxy - terminal portion protein as negative control for the evaluation of the antibody specificity. MAb 3A12 specificity to alternative protein was confirmed by its reactivity to only one peptide among four analyzed that cover the full WARF4 amino acids sequence. This antibody detected the novel protein in WNV lineage I-infected, cultured VERO cells while it did not react with WNV lineage II infected cells. In addition, WARF4 protein was expressed in the late phase of WNV lineage I infection. We indirectly demonstrated the “in vivo” production of N-NS4B/WARF4 by showing its immunoreactivity with human sera obtained from WNV infected patients. We have no experimental information on N-NS4B/WARF4 protein translation, but it appears reasonable to assume that a -1 ribosomal frameshifting mechanism produces the novel protein. This translation mechanism has already been described for West Nile. Since the proposed model requires the presence of specific RNA structures such as slippery sequences associated with pseudknot was carried out bioinformatics analysis that revealed these structures within the NS4B coding region. The data obtained in western blot assays suggest that following the ribosomal frameshifting in - 1 frame N-NS4B/WARF4 would be synthesized as carboxy-terminal variant of NS4B antigen. The possible role of the protein N-NS4B/WARF4 during WNV infection is still unknown, however recent literature data seem to suggest a possible role in the translational regulation of the viral genome

Embodied carbon mitigation and reduction in the built environment – What does the evidence say?

Of all industrial sectors, the built environment puts the most pressure on the natural environment, and in spite of significant efforts the International Energy Agency suggests that buildings-related emissions are on track to double by 2050. Whilst operational energy efficiency continues to receive significant attention by researchers, a less well-researched area is the assessment of embodied carbon in the built environment in order to understand where the greatest opportunities for its mitigation and reduction lie. This article approaches the body of academic knowledge on strategies to tackle embodied carbon (EC) and uses a systematic review of the available evidence to answer the following research question: how should we mitigate and reduce EC in the built environment? 102 journal articles have been reviewed systematically in the fields of embodied carbon mitigation and reduction, and life cycle assessment. In total, 17 mitigation strategies have been identified from within the existing literature which have been discussed through a meta-analysis on available data. Results reveal that no single mitigation strategy alone seems able to tackle the problem; rather, a pluralistic approach is necessary. The use of materials with lower EC, better design, an increased reuse of EC-intensive materials, and stronger policy drivers all emerged as key elements for a quicker transition to a low carbon built environment. The meta-analysis on 77 LCAs also shows an extremely incomplete and short-sighted approach to life cycle studies. Most studies only assess the manufacturing stages, often completely overlooking impacts occurring during the occupancy stage and at the end of life of the building. The LCA research community have the responsibility to address such shortcomings and work towards more complete and meaningful assessments

Circular economy for the built environment: A research framework

The built environment puts major pressure on the natural environment; its role in transitioning to a circular economy (CE) is therefore fundamental. However, current CE research tends to focus either on the macro-scale, such as eco-parks, or the micro-scale, such as manufactured products, with the risk of ignoring the additional impacts and potentials at the meso-scale of individual buildings. This article sets out to unpack the fundamental defining dimensions of a CE and frame them for CE studies for the built environment. A critical literature review forms the basis for identifying and framing such fundamental dimensions. Our contribution highlights the key roles of interdisciplinary research and of both bottom-up and top-down initiatives in facilitating the transition to ‘circular buildings’. The frame for reference has been used to capture current discourse on the sustainability of the built environment and has proved to be a valuable tool to cluster existing initiatives and highlight missing links for interdisciplinary endeavours. The article represents a contribution to the theoretical foundations of CE research in the built environment and a stepping stone to shape future research initiatives

A Method to Facilitate Uncertainty Analysis in LCAs of Buildings

Life cycle assessment (LCA) is increasingly becoming a common technique to assess the embodied energy and carbon of buildings and their components over their life cycle. However, the vast majority of existing LCAs result in very definite, deterministic values which carry a false sense of certainty and can mislead decisions and judgments. This article tackles the lack of uncertainty analysis in LCAs of buildings by addressing the main causes for not undertaking this important activity. The research uses primary data for embodied energy collected from European manufacturers as a starting point. Such robust datasets are used as inputs for the stochastic modelling of uncertainty through Monte Carlo algorithms. Several groups of random samplings between 10 1 and 10 7 are tested under two scenarios: data are normally distributed (empirically verified) and data are uniformly distributed. Results show that the hypothesis on the data no longer influences the results after a high enough number of random samplings (10 4). This finding holds true both in terms of mean values and standard deviations and is also independent of the size of the life cycle inventory (LCI): it occurs in both large and small datasets. Findings from this research facilitate uncertainty analysis in LCA. By reducing significantly the amount of data necessary to infer information about uncertainty, a more widespread inclusion of uncertainty analysis in LCA can be encouraged in assessments from practitioners and academics alike

Measuring embodied carbon dioxide equivalent of buildings: A review and critique of current industry practice

Lowering the embodied carbon dioxide equivalent (embodied CO2e) of buildings is an essential response to national and global targets for carbon reduction. Globally, construction industry is developing tools, databases and practices for measuring embodied CO2e in buildings and recommending routes to reduction. While the TC350 developed standardized methods for the assessment of sustainability aspects in construction works and Environmental Product Declarations, there is no consensus on how this should be carried out in practice. This paper evaluates the current construction industry practice through a review of both academic and professional literature, and through focus groups and interviews with industry experts in the field. Incentives in the available building codes, standards, and benchmarks are also analysed, as are the existing methodologies, tools and datasets. The multiple data sources are used to identify the barriers to the effective measurement and reduction of embodied CO2e in practice. This paper recommends that Governments mandate for improved data quality and support the development of a transparent and simplified methodology

Comparative life cycle analysis of façade passive systems in the Mediterranean: comfort, energy, and carbon

In the Mediterranean region façade shading systems are used to reduce operational energy, particularly cooling loads. However, operational savings do not necessarily translate into net energy savings unless they outweigh the embodied energy/carbon required to manufacture, install, maintain, and dispose of these systems. This study analyses two shading devices, louvers and meshes, from a whole-life perspective in Malta. We first establish, through dynamic energy modelling, the operational energy and carbon savings achieved, and results show that both louvers and meshes are capable of savings in terms of operational energy—20% to 40% compared to the base case. Secondly, we establish the embodied energy and carbon through a life cycle analysis. Although based on the limited data available for Malta, findings suggest that net energy and carbon savings are only achieved by two of the 22 configurations investigated, both mesh systems. These results highlight the urgent need to investigate shading systems to establish net energy and carbon whole-life balances. The risk is otherwise that we will save less operational energy in the future, from decarbonised energy grids, than we have already spent through a surge of embodied energy from current, carbon intensive grids, therefore exacerbating the climate crisis

Molecular evidence of Plasmodium vivax infection in Duffy negative symptomatic individuals from Dschang, West Cameroon

Background: Plasmodium vivax infection is known to be rare in West/Central Africa, the most accepted explanation being the lack of expression of erythroid Duffy antigen in the local human populations. Duffy negativity prevents the parasite to exploit the entry mechanism on the red blood cell surface. However, there are a growing number of reported vivax infections in Duffy-negative individuals. Data on P. vivax circulation in Cameroon are limited. The aim of the study was to evaluate the P. vivax presence, and its association with the Duffy genotype in West Cameroon. Results: Overall, 484 blood samples were collected consecutively from febrile outpatients attending the Dschang’s Hospital (West Cameroon) during a 3-months period. Plasmodium vivax infection was detected by PCR in 5.6% (n = 27/484) of the cases, representing 38.6% (n = 27/70) of all Plasmodium infections detected. All P. vivax infected individuals showed a Duffy-negative genotype, and the frequency of Duffy-positive individuals in the whole tested population was 1.7%. Conclusions: The results of this study confirm the circulation of P. vivax in Cameroon, as well as that the lack of expression of Duffy-antigen does not confer full protection against vivax malaria acquisition

Embodied carbon of concrete in buildings, Part 2: are the messages accurate?

This paper is the second output of a project that examines the embodied greenhouse gas emissions (‘embodied carbon’) from the use of concrete in buildings. In the current absence of either regulation or widespread industry practice in quantified carbon assessment, it seems likely that messaging will play a powerful role in influencing designers’ perceptions and decisions. Using the UK as a case study, this paper considers the current messages about the carbon implications of concrete in buildings from professional institutions and the cement and concrete trade body. Three mechanisms through which it is claimed carbon emissions are significantly reduced are identified: thermal mass, durability, and carbonation. By assessing each of these in turn against the available scientific literature, it is shown that they are likely to have a far more limited effect on the total impacts than suggested. More accuracy is needed from trade organisations if real carbon reductions are to be achieved. Practice relevance In the current absence of whole life carbon assessment for buildings, messaging about the carbon impacts of different materials is likely to have a strong impact. The paper recommends that the cement and concrete industry should be more accurate with the messages it is sharing. In particular the claims that thermal mass, durability and carbonation are effective mechanisms, which suggest concrete is a low carbon option, should be reconsidered. Meanwhile designers should use the excellent advice being produced by the professional institutions and undertake whole-life carbon calculations to ensure the lowest carbon design. These should include a best estimate of future trends towards warming climates and decarbonisation of electricity, but should also recognise the importance of the immediate effects of the emissions from construction materials. There is also a key role for policymakers in legislating to make the measurement of embodied impacts of buildings mandatory

Buildings for the biosphere (and their indoors for the technosphere) - A circular economy view for the construction industry

One of the founding principles of the Circular Economy is to foster the focus on longer-lasting products. Arguably, extremely few products in the present days last as long as buildings do, yet even so only rarely does the actual end of a building’s life correspond with the end of its potential life. In a context where the built environment contributes the most to materials extraction, final energy consumption, GHG emissions, and waste, this puts additional pressure on the natural environment, through unnecessary consumption of finite natural resources and growing generation of waste flows. This paper discusses in what ways and to what extent current LCA and whole life carbon methodologies can be a measure of circularity for buildings. The aim is to develop a framework for circular buildings through a better combined understanding of their design, technical, and cultural life. The paper sets the scene with a reflection on current trends in buildings LCA and the theoretical foundations laid by Frank Duffy and Stewart Brand (shearing layers of buildings) and William McDonough and Michael Braungart (Cradle2Cradle design), as discussed at events in the UK towards the end of 2015. We conclude that in order to bring about circularity in the built environment the greatest challenge is not technological, but rather the necessary behavioural and cultural changes needed in both the design and use of buildings