The European Virus Archive goes global: A growing resource for research.

The European Virus Archive (EVA) was created in 2008 with funding from the FP7-EU Infrastructure Programme, in response to the need for a coordinated and readily accessible collection of viruses that could be made available to academia, public health organisations and industry. Within three years, it developed from a consortium of nine European laboratories to encompass associated partners in Africa, Russia, China, Turkey, Germany and Italy. In 2014, the H2020 Research and Innovation Framework Programme (INFRAS projects) provided support for the transformation of the EVA from a European to a global organization (EVAg). The EVAg now operates as a non-profit consortium, with 26 partners and 20 associated partners from 21 EU and non-EU countries. In this paper, we outline the structure, management and goals of the EVAg, to bring to the attention of researchers the wealth of products it can provide and to illustrate how end-users can gain access to these resources. Organisations or individuals who would like to be considered as contributors are invited to contact the EVAg coordinator, Jean-Louis Romette, at jean-louis.romette@univmed.fr.

The European Virus Archive: a new resource for virology research.

The European Virus Archive (EVA) was conceived as a direct response to the need for a coordinated and readily accessible collection of viruses that could be made available to academia, public health organisations and industry, initially within Europe, but ultimately throughout the world. Although scientists worldwide have accumulated virus collections since the early twentieth century, the quality of the collections and the viruses collected may vary according to the personal interests and agenda of the scientists. Moreover, when laboratories are re-organised or closed, collections are no longer maintained and gradually cease to exist. The tragedy of 9/11 and other disruptive activities have also meant that some previously available biological reagents are no longer openly exchanged between countries. In 2008, funding under the FP7-EU infrastructure programme enabled the initiation of the EVA. Within three years, it has developed from a consortium of nine European laboratories to encompass associated partners in Africa, Russia, China, Turkey, Germany and Italy. There is every reason to believe that EVA will continue to expand and ultimately exist as a globally networked, quality-controlled non-profit archive for the benefit of science. Organizations or individuals who would like to be considered as contributors are invited to contact the EVA coordinator, Jean-Louis Romette, at jean-louis.romette@univmed.fr.

The VIZIER project: preparedness against pathogenic RNA viruses.

Life-threatening RNA viruses emerge regularly, and often in an unpredictable manner. Yet, the very few drugs available against known RNA viruses have sometimes required decades of research for development. Can we generate preparedness for outbreaks of the, as yet, unknown viruses? The VIZIER (VIral enZymes InvolvEd in Replication) (http://www.vizier-europe.org/) project has been set-up to develop the scientific foundations for countering this challenge to society. VIZIER studies the most conserved viral enzymes (that of the replication machinery, or replicases) that constitute attractive targets for drug-design. The aim of VIZIER is to determine as many replicase crystal structures as possible from a carefully selected list of viruses in order to comprehensively cover the diversity of the RNA virus universe, and generate critical knowledge that could be efficiently utilized to jump-start research on any emerging RNA virus. VIZIER is a multidisciplinary project involving (i) bioinformatics to define functional domains, (ii) viral genomics to increase the number of characterized viral genomes and prepare defined targets, (iii) proteomics to express, purify, and characterize targets, (iv) structural biology to solve their crystal structures, and (v) pre-lead discovery to propose active scaffolds of antiviral molecules.

Purification of GSK-3 by affinity chromatography on immobilized axin.

Glycogen synthase kinase 3 (GSK-3), an element of the Wnt signalling pathway, plays a key role in numerous cellular processes including cell proliferation, embryonic development, and neuronal functions. It is directly involved in diseases such as cancer (by controlling apoptosis and the levels of beta-catenin and cyclin D1), Alzheimer's disease (tau hyperphosphorylation), and diabetes (as a downstream element of insulin action, GSK-3 regulates glycogen and lipid synthesis). We describe here a rapid and efficient method for the purification of GSK-3 by affinity chromatography on an immobilized fragment of axin. Axin is a docking protein which interacts with GSK-3ss, beta-catenin, phosphatase 2A, and APC. A polyhistidine-tagged axin peptide (residues 419-672) was produced in Escherichia coli and either immobilized on Ni-NTA agarose beads or purified and immobilized on CNBr-activated Sepharose 4B. These "Axin-His6" matrices were found to selectively bind recombinant rat GSK-3 beta and native GSK-3 from yeast, sea urchin embryos, and porcine brain. The affinity-purified enzymes displayed high kinase activity. This single step purification method provides a convenient tool to follow the status of GSK-3 (protein level, phosphorylation state, kinase activity) under various physiological settings. It also provides a simple and efficient way to purify large amounts of active recombinant or native GSK-3 for screening purposes.

Production of Sm37-GAPDH, a major therapeutical target in human schistosomiasis.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key enzyme in the glycolytic metabolism and the production of energy. This probably explains why GAPDH was evidenced as a major therapeutical target in several parasitic diseases; either as a vaccine candidate or as a target for chemotherapeutic treatments. Schistosoma mansoni GAPDH (Sm37-GAPDH) is one of the main schistosome vaccine candidates. The production of recombinant Sm37-GAPDH is essential to evaluate the ability of this molecule to induce protective immunity in animals and possibly in humans. The cDNA encoding Sm37-GAPDH has been cloned and sequenced. In addition, five B cell (including the major B-cell epitope Sm35-5) and two T cell epitopes have been localized on the molecule. Different expression systems have been evaluated in respect with the production yield and the GAPDH enzymatic activity. Some of them have led to either a high production of insoluble material (E. coli) or to an inactive enzyme (Pischia pastoris). The present article describes the production setting of rSm37-GAPDH using the baculovirus-insect cell system. Large amounts of soluble rSm37-GAPDH with enzymatic activity were obtained. Most sera from individuals living in an area endemic for S. mansoni recognised the rSm37 molecule and inhibited its catalytic activity.

Purification and interfacial behavior of recombinant human gastric lipase produced from insect cells in a bioreactor.

Recombinant human gastric lipase (rHGL) (EC 3.1.1.3) was produced on a large scale (5-13 mg/liter) from recombinant baculovirus-infected insect cells using a bioreactor apparatus. Here an improved procedure is described for purifying rHGL involving the use of cation exchange chromatography followed by immunoaffinity column methods, which gives a total yield of 62% and a purification factor of 464, using 10% isopropanol in all the purification buffers. The presence of isopropanol was necessary to preserve the stability of the enzyme during the chromatographic separation steps. The specific activity of rHGL on tributyroylglycerol (700 U/mg) was lower than that of native HGL (nHGL) (1080 U/mg). The rHGL interfacial adsorption kinetics were studied by recording the changes in the surface pressure with time in the presence or absence of an egg phosphatidycholine monomolecular film spread at the air/water interface at various initial surface pressures. The surface behavior of rHGL was similar to that of nHGL. It can be concluded that the lipid binding affinity of rHGL is identical to that of the native lipase and, consequently, that the presence of detergents and lipids in the insect cell culture media did not affect the interfacial behavior of the purified rHGL. It will be therefore possible to specifically study the binding step of HGL mutants to a lipid monolayer.

An enzyme electrode for on-line determination of ethanol and methanol.

Since a stable alcohol oxidase with a high specific activity is not commercially available, we propose to produce and purify this enzyme from a strain of the yeast Hansenula polymorpha. This alcohol oxidase was immobilized into a gelatin matrix and its activity was estimated by a pO(2) sensor. The enzyme electrode obtained was then used in a continuous flow system to measure methanol or ethanol concentrations. The sample oxygen content dependence of the signal was minimized by the support properties. Measuring time for each sample were less than two minutes including response data treatment and rinsing step. The enzyme electrode response was set for ethanol from 0.5mM to 15mM and for methanol from 10mM to 300mM. On repeated use, the electrode signal for 10mM of ethanol was stable for at least 500 assays. Analysis have been performed in different beverages such as wine and beer, and the results compared to those obtained with classical methods of analysis.

Immobilized respiratory chain activities from Escherichia coli utilized to measure D- and L-lactate, succinate, L-malate, 3-glycerophosphate, pyruvate, or NAD(P)H.

The respiratory chain (membranous, multienzymatic system) from Escherichia coli, was coimmobilized with gelatin and insolubilized in film form by tanning with glutaraldehyde. The film was fixed onto an oxygen sensor. The enzyme electrode can be used for measuring NAD(P)H, D- and L-lactate, succinate, L-malate, 3-glycerophosphate, or pyruvate. The range of metabolites concentrations was from 1 to 50 mM. It was possible to discriminate between the different metabolites (if mixed): By inducing during bacterial growth the specific flavoproteins necessary for L-lactate, succinate, L-malate, and 3-glycerophosphate respirations. The constitutive activities are unaltered on glucose or glycerol, namely D-lactate, NAD(P)H, and pyruvate respiration. When intact bacteria were immobilized (with or without induction), D- and L-lactate, succinate, 3-glycerophosphate, and L-malate respiration were measured, no activities of pyruvate and NAD(P)H respiration were obtained. For these last activities, French press breakage (see section on Membrane Preparations) of bacteria prior to immobilization was necessary. Products of reactions can be used as enzyme inhibitors: Pyruvate inhibits D- and L-lactate; fumarate inhibits succinate, and oxaloacetate inhibits L-malate respirations. Heat denaturation of the bacteria at 55 degrees C for 1 h maintains full activity of succinate and pyruvate respiration. On the other hand, no activity of D- and L-lactate, L-malate, or NAD(P)H respiration was measurable. These enzyme electrodes have many applications in basic and applied research.

A computerised enzyme immunosensor: application for the determination of antigens.

The present report gives preliminary results of a new sensitive method for the amperometric determination of antigens in serum. This method, developed from the biological model 'hepatitis B surface antigen antibodies' is less time-consuming than most immunochemical techniques, and eliminates many inconveniences arising from use of isotopes. Specific antibodies immobilised onto a gelatin membrane are applied in a solid phase 'sandwich' procedure. The antibodies are labelled with glucose oxidase. The measurement consists of an immunological process and an enzymatic reaction. The second part is carried out by fixing the active membrane onto a pO2 electrode. The sensor is immersed in a standard glucose solution and a signal is obtained by measuring the consumption of oxygen due to the enzyme reaction. This response is correlated to the antigen concentration of the sample. It is a function of both the diffusional and the reactional characteristics of the active layer. Under software conditions, the signal is sampled when the stationary state is obtained. The difference between initial signal and the stationary state signal is measured and compared with the pre-set calibration curve. Use of the computerised enzyme immunosensor could easily be extended to assay of other antigens and haptens that are usually determined by radioimmunoassay.

Enzyme electrode for specific determination of L-lysine.

L-Lysine alpha-oxidase from Trichoderma viride Y244-2 is immobilized in a gelatin support and fixed on a pO(2) sensor. The enzyme electrode obtained is used in a continuous flow system in order to measure the concentration of L-lysine in a fermentor. The sample oxygen-content dependance of the signal is minimized because of the enzyme support properties. The enzyme electrode response is set for lysine concentration from 0.2mM to 4mM. The specificity of lysine is tested with other amino acids. The enzyme membrane for lysine electrode can be used 3000 times or stored six months with good stability.

Determination of substrate concentrations by a computerized enzyme electrode.

A numerical treatment of the signal produced by an electrode onto which an artificial enzyme membrane is mounted can give the concentration of the substrate (glucose, saccharose, lactose, amino acids, etc.) in solution. In the example of a glucose analyzer, in which glucose oxidase catalyzes the oxidation of glucose, the computer receives pO(2) level data from the electrode and calculates the glucose concentration. The transient electrode signal, measured as the enzyme membrane is exposed to a solution of glucose, is least-square approximated by a third-degree polynomial whose slope at inflection point is characteristic of the external glucose concentration. A calibration procedure provides a cubic spline approximation of glucose concentration as a function of slope, thus enabling automatic measurement of samples. The computer performs the calculations, and actuates valves for air rinsing, introduction of the sample, and water rinsing.

An enzyme electrode for specific determination of L-lysine: A real-time control sensor.

Lysine decarboxylase (L-lysine carboxylyase, E.C.4.1.1.18) is immobolized on a carbon dioxide gas-sensing electrode, by copolymerization with gelatin using the bifuncitional agent glutaraldehyde. The enzyme electrodes thus prepared are used in a continuous flow system to measure the concentration of L-lysine in a mixture of amino acids. The measuring time for each sample is about 3 min, including response and rinsing times. The electrode response is linear between 0.01-1 g/L and has a high specificity for L-lysine. The enzyme electrode response to lysine at concentrations below 0.5 g/L is stable on repeated use for at least 500 assays.

Glucose-oxidase electrode. Measurements of glucose in samples exhibiting high variability in oxygen content.

Measurements of glucose in samples exhibiting high variability in oxygen content present an important problem to solve for pO2 detection methods. Different solutions exist, all based on the stabilization of the pO2 sample before or during the measurement. The electrode described in this paper itself contains enough O2 to compensate for the variability of the sample oxygen content. The enzyme is cross-linked with gelatin using the bifunctional agent, glutaraldehyde. Amperometric enzyme electrodes have been constructed using these membranes. Measurements have been done by collecting the derivative in time of the signal given by the electrode for blood or plasma samples.

Experimental evidence for a kinetic and electrochemical memory in enzyme membranes.

The existence of hysteresis phenomena in artificial enzyme membranes due to the coupling of simple kinetic enzyme properties with diffusion transport processes is reported. The intramembrane pH of a urease coating on the surface of a glass pH electrode exhibits a hysteresis loop when the pH of the bulk solution varies cyclically. The steady-state potential of a urease membrane, as a function of the substrate concentration in the bulk solution, also exhibits a memory effect. The influence of the membrane's history on its overall behavior is visualized by electron microscopy. We interpret the results in terms of a coupling between the enzyme reactions and diffusion processes, without taking into account molecular effects.