Characterization of Poliovirus Neutralization Escape Mutants of Single-Domain Antibody Fragments (VHHs).

To complete the eradication of poliovirus and to protect unvaccinated people subsequently, the development of one or more antiviral drugs will be necessary. A set of five single-domain antibody fragments (variable parts of the heavy chain of a heavy-chain antibody [VHHs]) with an in vitro neutralizing activity against poliovirus type 1 was developed previously (B. Thys, L.

Affinity capillary electrophoresis to evaluate the complex formation between poliovirus and nanobodies.

It was demonstrated that nanobodies with an in vitro neutralizing activity against poliovirus type 1 interact with native virions. Here, the use of capillary electrophoresis was investigated as an alternative technique for the evaluation of the formation of nanobody-poliovirus complexes, and therefore predicting the in vitro neutralizing activity of the nanobodies. The macromolecules are preincubated offline in a specific nanobody-to-virus ratio and analyzed by capillary electrophoresis with UV detection.

Binding of glutathione to enterovirus capsids is essential for virion morphogenesis.

Enteroviruses (family of the Picornaviridae) cover a large group of medically important human pathogens for which no antiviral treatment is approved. Although these viruses have been extensively studied, some aspects of the viral life cycle, in particular morphogenesis, are yet poorly understood. We report the discovery of TP219 as a novel inhibitor of the replication of several enteroviruses, including coxsackievirus and poliovirus.

Mechanism of action and capsid-stabilizing properties of VHHs with an in vitro antipolioviral activity.

Unlabelled: Previously, we reported on the in vitro antiviral activity of single-domain antibody fragments (VHHs) directed against poliovirus type 1. Five VHHs were found to neutralize poliovirus type 1 in an in vitro setting and showed 50% effective concentrations (EC50s) in the nanomolar range. In the present study, we further investigated the mechanism of action of these VHHs.

Impact of the sample matrix composition on the signal enhancement in the capillary electrophoretic separation of poliovirus samples.

The development of capillary electrophoretic applications aiming to provide reliable stability assessment of viral suspensions, to detect subviral particles from cell extracts or to study the interactions between virus particles and various biomolecules, cannot be done without a thorough understanding of the sample matrix contribution to the observed electrophoretic behaviour. The present study thoroughly investigates the effect of the sample matrix on the electrophoretic behaviour of poliovirus injected as sample plugs of 1%, 5% and 12% effective capillary length. The effect of the sample matrix for three different poliovirus batches was evaluated.

The effect of anionic surfactant on poliovirus particles during capillary electrophoresis.

Because of its essential role in SDS-PAGE, sodium dodecylsulphate (SDS) is generally associated with protein denaturation. However, for SDS-PAGE, proteins are linearized in the presence of SDS, following the exposure to high temperatures and reducing agents. In comparison, the conditions employed during a capillary electrophoretic (CE) separation involve only a limited exposure to SDS, at much lower temperatures.

A liquid phase affinity capture assay using magnetic beads to study protein-protein interaction: the poliovirus-nanobody example.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled.

Poliovirus separation from cell extracts using capillary electrophoresis: potential use in vaccine production and control?

Rapid assessment of the concentration of virus particles in a given sample remains a challenge. Modern separation methods, such as capillary electrophoresis, were proposed recently to study viruses and viral infection or to separate and characterize viral vaccines in a time-efficient manner. Even though capillary electrophoresis is much more rapid than traditional virological methods and has the advantages of automation, increased precision and reliability, it has the drawback of reduced sensitivity for low concentrations.

Quality assurance in European pharmacy education and training.

A survey of quality assurance (QA) systems in European faculties of pharmacy was carried out under the auspices of the European Association of Faculties of Pharmacy PHARMINE consortium. A questionnaire based on the quality criteria of the International Pharmaceutical Federation and the Accreditation Council for Pharmacy Education (USA) was sent out to European faculties. Replies were obtained from 28 countries.

The PHARMINE study on the impact of the European Union directive on sectoral professions and of the Bologna declaration on pharmacy education in Europe.

The Bologna declaration and the European Union (EU) directive 2005/36/EC on the recognition of professional qualifications influence the mobility of pharmacy students and pharmacy professionals, respectively. In addition the Bologna declaration aims at tuning higher education degrees including pharmacy throughout the EU in order to prepare for a harmonised European Higher Education Area. The directive outlines the knowledge, skills and qualifications required for the pursuit of the professional activity of a pharmacy in the EU.

The 2011 PHARMINE report on pharmacy and pharmacy education in the European Union.

The PHARMINE consortium consists of 50 universities from European Union member states or other European countries that are members of the European Association of Faculties of Pharmacy (EAFP). EU partner associations representing community (PGEU), hospital (EAHP) and industrial pharmacy (EIPG), together with the European Pharmacy Students' Association (EPSA) are also part of the consortium. THE CONSORTIUM SURVEYED PHARMACIES AND PHARMACISTS IN DIFFERENT SETTINGS: community, hospital, industry and other sectors.

A simple quantitative affinity capturing assay of poliovirus antigens and subviral particles by single-domain antibodies using magnetic beads.

Recently, single-domain recombinant antibody fragments (VHHs or nanobodies) against poliovirus type 1 were isolated. To examine the antigenicity of poliovirus using these recombinant VHHs, an alternative technique mimicking protein A immunoprecipitation had to be developed that was designed specifically for VHHs. The current study validated an affinity capturing assay that is based on the magnetic separation of unbound antigen and antigen-VHH complexes.

Rational use of stacking principles for signal enhancement in capillary electrophoretic separations of poliovirus samples.

The use of an earlier developed capillary electrophoresis (CE) method, either to investigate poliovirus (PV) samples with a low viral-purity level or to study the less abundant sub-viral particles, revealed the necessity for an intra-column signal enhancement strategy. Although intra-column signal enhancement is a very popular approach to assay small molecules, it is less straightforward for the analysis of biological macromolecules or particles. A reason could be that, for a proper signal enhancement approach, these samples have to be thoroughly studied to understand the factors affecting the separation process.

Identification of poliovirions and subviral particles by capillary electrophoresis.

Poliovirions, purified from infected cell extracts with anion-exchange chromatography, can be analyzed and identified by CE in untreated fused silica capillaries using UV detection. Other subviral particles can be eluted as well from the same infected cell extract using a higher salt concentration buffer on the ion-exchange chromatography. Virions can be identified because of their conversion into empty capsids upon heating at 56°C.

In vitro antiviral activity of single domain antibody fragments against poliovirus.

VHHs or Nanobodies are single-domain antigen-binding fragments derived from heavy chain antibodies found in camelids. It has already been shown that complex protein mixtures and even whole organisms elicit good immune responses in camelids; therefore we hypothesized that VHHs selected from a dromedary immunized with poliovirus type 1 might inhibit the in vitro replication of poliovirus through binding to essential biological sites on the viral capsid. In this study, we aimed to determine whether VHHs inhibit wild-type and vaccine strains of poliovirus type 1.

Microfluidics in macro-biomolecules analysis: macro inside in a nano world.

Use of microfluidic devices in the life sciences and medicine has created the possibility of performing investigations at the molecular level. Moreover, microfluidic devices are also part of the technological framework that has enabled a new type of scientific information to be revealed, i.e.

Improving the capillary electrophoretic analysis of poliovirus using a Plackett-Burman design.

Separation techniques may offer interesting alternatives to classical virological techniques both for fundamental research purposes and for vaccine manufacturing. A capillary electrophoretic method for the analysis of the poliovirus was developed based on conditions for the human rhinovirus taken from literature. The method was optimized using a 12-experiment Plackett-Burman design, applied in order to examine simultaneously the effects of eight factors on responses such as, mobility of the electroosmotic flow, effective mobility of the poliovirus, analysis time and resolution between the virus peak and a system peak.

Modulation of viral replication in macrophages persistently infected with the DA strain of Theiler's murine encephalomyelitis virus.

Background: Demyelinating strains of Theiler's murine encephalomyelitis virus (TMEV) such as the DA strain are the causative agents of a persistent infection that induce a multiple sclerosis-like disease in the central nervous system of susceptible mice. Viral persistence, mainly associated with macrophages, is considered to be an important disease determinant that leads to chronic inflammation, demyelination and autoimmunity. In a previous study, we described the establishment of a persistent DA infection in RAW macrophages, which were therefore named DRAW.

Potential use of antiviral agents in polio eradication.

In 1988, the World Health Assembly launched the Global Polio Eradication Initiative, which aimed to use large-scale vaccination with the oral vaccine to eradicate polio worldwide by the year 2000. Although important progress has been made, polio remains endemic in several countries. Also, the current control measures will likely be inadequate to deal with problems that may arise in the postpolio era.

R75761, a lead compound for the development of antiviral drugs in late stage poliomyelitis eradication strategies and beyond.

In this study the antiviral activity of a panel of 18 out of 240 pyridazinamine analogues was evaluated against the Sabin strains of the three poliovirus types. We found one compound, R75761 which had a comparable 50% effective concentration (EC50) value against all three poliovirus Sabin strains. Virus multiplication was reduced by 10(4.

Titration efficacy of two auto-adjustable continuous positive airway pressure devices using different flow limitation-based algorithms.

Background: Auto-adjustable continuous positive airway pressure devices are widely used in titration procedures to determine therapeutic pressure levels in obstructive sleep apnea patients. However, differences in operational characteristics may influence the effect on the apnea-hypopnea index (AHI).

Objectives: We compared the titration performance of two devices based on detection of inspiratory flow limitation, i.

Theiler's virus strain-dependent induction of innate immune responses in RAW264.7 macrophages and its influence on viral clearance versus viral persistence.

Infection of susceptible mice with the DA strain of Theiler's murine encephalomyelitis virus (TMEV) induces a persistent central nervous system infection accompanied by demyelination that resembles multiple sclerosis. In contrast, Theiler's GDVII strain does not persist, because infected animals either clear the virus or die. Previously, the authors have shown that in vitro infection of RAW264.

Persistent infection of RAW264.7 macrophages with the DA strain of Theiler's murine encephalomyelitis virus: An in vitro model to study viral persistence.

Theiler's murine encephalomyelitis virus (TMEV) is a member of the Picornaviridae family and causes a virus strain dependent pathology in the central nervous system of mice. The GDVII strain induces an acute and mostly fatal encephalomyelitis. In the few mice that survive, the virus is cleared by the immune system.

Stimulation of poliovirus RNA synthesis and virus maturation in a HeLa cell-free in vitro translation-RNA replication system by viral protein 3CDpro.

Poliovirus protein 3CDpro possesses both proteinase and RNA binding activities, which are located in the 3Cpro domain of the protein. The RNA polymerase (3Dpol) domain of 3CDpro modulates these activities of the protein. We have recently shown that the level of 3CDpro in HeLa cell-free in vitro translation-RNA replication reactions is suboptimal for efficient virus production.