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. Schotte, S. Muyldermans, U. Wernery, G. Hassanzadeh-Ghassabeh, and B. Rombaut, Antiviral Res 87:257-264, 2010, http://dx.doi.org/10.1016/j.antiviral.2010.05.012), and their mechanisms of action have been studied (L. Schotte, M. Strauss, B. Thys, H. Halewyck, D. J. Filman, M. Bostina, J. M. Hogle, and B. Rombaut, J Virol 88:4403-4413, 2014, http://dx.doi.org/10.1128/JVI.03402-13). In this study, neutralization escape mutants were selected for each VHH. Sequencing of the P1 region of the genome showed that amino acid substitutions are found in the four viral proteins of the capsid and that they are located both in proximity to the binding sites of the VHHs and in regions further away from the canyon and hidden beneath the surface. Characterization of the mutants demonstrated that they have single-cycle replication kinetics that are similar to those of their parental strain and that they are all drug (VHH) independent. Their resistant phenotypes are stable, as they do not regain full susceptibility to the VHH after passage over HeLa cells in the absence of VHH. They are all at least as stable as the parental strain against heat inactivation at 44°C, and three of them are even significantly (P < 0.05) more resistant to heat inactivation. The resistant variants all still can be neutralized by at least two other VHHs and retain full susceptibility to pirodavir and 35-1F4.

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. At low nanobody-to-virus ratios, a clear shift in migration time of the viral peak was observed. A broad peak was obtained, indicating the presence of a heterogeneous population of nanobody-virion complexes, caused by the binding of different numbers of nanobodies to the virus particle. At elevated nanobody-to-virus ratios, a cluster of peaks appeared, showing an additional increase in migration times. It was shown that, at these high molar excesses, aggregates were formed. The developed capillary electrophoresis method can be used as a rapid, qualitative screening for the affinity between poliovirus and nanobodies, based on a clearly visible and measurable shift in migration time. The advantages of this technique include that there is no need for antigen immobilization as in enzyme-linked immunosorbent assays or surface plasmon resonance for the use of radiolabeled virus or for the performance of labor- and time-intensive plaque-forming neutralization assays.

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. We show that TP219 binds directly glutathione (GSH), thereby rapidly depleting intracellular GSH levels and that this interferes with virus morphogenesis without affecting viral RNA replication. The inhibitory effect on assembly was shown not to depend on an altered reducing environment. Using TP219, we show that GSH is an essential stabilizing cofactor during the transition of protomeric particles into pentameric particles. Sequential passaging of coxsackievirus B3 in the presence of low GSH-levels selected for GSH-independent mutants that harbored a surface-exposed methionine in VP1 at the interface between two protomers. In line with this observation, enteroviruses that already contained this surface-exposed methionine, such as EV71, did not rely on GSH for virus morphogenesis. Biochemical and microscopical analysis provided strong evidence for a direct interaction between GSH and wildtype VP1 and a role for this interaction in localizing assembly intermediates to replication sites. Consistently, the interaction between GSH and mutant VP1 was abolished resulting in a relocalization of the assembly intermediates to replication sites independent from GSH. This study thus reveals GSH as a novel stabilizing host factor essential for the production of infectious enterovirus progeny and provides new insights into the poorly understood process of morphogenesis.

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. All five VHHs interfere at multiple levels of the viral replication cycle, as they interfere both with attachment of the virus to cells and with viral uncoating. The latter effect is consistent with their ability to stabilize the poliovirus capsid, as observed in a ThermoFluor thermal shift assay, in which the virus is gradually heated and the temperature causing 50% of the RNA to be released from the capsid is determined, either in the presence or in the absence of the VHHs. The VHH-capsid interactions were also seen to induce aggregation of the virus-VHH complexes. However, this observation cannot yet be linked to their mechanism of action. Cryo-electron microscopy (cryo-EM) reconstructions of two VHHs in complex with poliovirus type 1 show no conformational changes of the capsid to explain this aggregation. On the other hand, these reconstructions do show that the binding sites of VHHs PVSP6A and PVSP29F overlap the binding site for the poliovirus receptor (CD155/PVR) and span interfaces that are altered during receptor-induced conformational changes associated with cell entry. This may explain the interference at the level of cell attachment of the virus as well as their effect on uncoating. IMPORTANCE: The study describes the mechanism of neutralization and the capsid-stabilizing activity of five single-domain antibody fragments (VHHs) that have an in vitro neutralizing activity against poliovirus type 1. The results show that the VHHs interfere at multiple levels of the viral replication cycle (cell attachment and viral uncoating). These mechanisms are possibly shared by some conventional antibodies and may therefore provide some insight into the natural immune responses. Since the binding sites of two VHHs studied by cryo-EM are very similar to that of the receptor, the VHHs can be used as probes to study the authentic virus-cell interaction. The structures and conclusions in this study are original and raise interesting findings regarding virus-receptor interactions and the order of key events early in infection.

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. Additionally, simulated samples, obtained from concentrated poliovirus suspensions of high purity and diluted with commonly used lab buffers in order to obtain samples with either high or low conductivities, were also investigated. The goal of the study was to obtain a better understanding of the effect of the sample matrix on the signal enhancement, in order to define a general approach allowing a repeatable capillary electrophoretic (CE) separation of poliovirus from complex samples. This study clearly demonstrates that the sample matrix has an important influence on the sensitivity of the CE poliovirus separations. Translation of these observations into routine practice involves several compromises and a set of rules in order to reduce day-to-day variation and to maximize sensitivity.

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. As the outer surface of the non-enveloped viruses consists of proteins, virus interaction with SDS can be judged from the perspective of SDS-protein interaction. Several studies have indicated that proteins have a different susceptibility to SDS, depending on their secondary structure and number of subunits. Therefore it is not straightforward to estimate what should be expected when intact polioviruses and subviral particles obtained by thermal conversion of the poliovirions, are exposed to SDS during CE separation. In this study it is shown that, during CE separations, SDS has no effect on the integrity of the poliovirion, but the presence of SDS in the separation system influences the poliovirus peak height and shape. The implication of SDS in the CE separation of poliovirus is discussed in detail. On the contrary, the proteinaceous subviral particles, such as the empty capsids, are less stable in the presence of SDS during the CE separation, and aggregates between the individual poliovirus capsid proteins and SDS are formed. Finally, we have proposed an alternative separation approach, involving an SDS gradient, for an improved separation of the subviral particles.

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. A sensitivity improvement is then necessary in many cases for a successful application. However, to date, only highly purified viral samples were examined using capillary electrophoresis. The injection of larger sample volumes, followed by intra-capillary concentration, was used in this study for cell extracts. Poliovirus was successfully detected rapidly, without any laborious staining procedures and incubation times. The method is simple, fast, automatic, requires only minute amounts of samples and reagents, and no expensive dyes or biological reagents. Additionally, the method showed a potential for monitoring the viral load during growth and purification, with obvious prospects for the optimization of the variable and time-consuming virus propagation procedures. The results of this study provide a potential basis for the development of routine methods for viral particles analysis, irrespective of their infective properties. In the future, the capillary electrophoresis test could help study the relationship between the intact poliovirus particles and the D-antigenic properties of a viral suspension, or could represent a supplementary or alternative test for virus concentration and D-antigen assays during vaccine production.

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. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies(1). Since poliovirus is sensitive to conformational conversion(2) when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch(3,4) is the micro protein A-immunoprecipitation test(5). Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies(6,7). However, as another opportunity, these interesting and stable single-domain antibodies(8) can be easily engineered with different tags. The widely used (His)(6)-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with (35)S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

The 2011 PHARMINE report on pharmacy and pharmacy education in the European Union / Informe PHARMINE 2001 sobre la farmacia y la formación en farmacia en la Unión Europea

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. The consortium also looked at how European Union higher education institutions and courses are organised. The PHARMINE survey of pharmacy and pharmacy education in Europe produced country profiles with extensive information for EU member states and several other European countries. These data are available at: http://www.pharmine.org/losse_paginas/Country_Profiles/. This 2011 PHARMINE report presents the project and data, and some preliminary analysis on the basic question of how pharmacy education is adapted to pharmacy practice in the EU (AU)

El consorcio PHARMINE se compone de 50 universidades de estados miembros de la Unión Europea que son miembros de la Asociación Europea de Facultades de Farmacias (EAFP). También hacen parte del consorcio asociaciones representando a las farmacias comunitarias (PGEU), hospitalarias (EAHP), e industrias (EIPG), así como la Asociación Europea de Estudiantes de Farmacia (EPSA). El Consorcio encuestó a farmacias y farmacéuticos en diferentes ámbitos: comunidad, hospital, industria y otros sectores. El consorcio también revisó como se organizan las instituciones de educación superior y los cursos en la Unión Europea. El cuestionario PHARMINE de farmacia y educación farmacéutica en Europa produjo perfiles de países con información exhaustiva de los estados miembros de la UE y otros países europeos. Estos datos están disponibles en http://www.pharmine.org/losse_paginas/Country_Profiles/. Este Informe PHARMINE 2011 presenta el proyecto y los datos, y algunos análisis preliminares sobre la cuestión básica de cómo se adapta la educación farmacéutica a la práctica de la farmacia en la UE (AU)

The PHARMINE study on the impact of the European Union directive on sectoral professions and of the Bologna declaration on pharmacy education in Europe / Estudio PHARMINE sobre el impacto de la directiva EU sobre profesiones sectoriales y de la declaración de Boloña en la educación en farmacia en Europa

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 PHARMINE project (Pharmacy Education in Europe, www.pharmine.org) looked at how the Bologna declaration and the directive influence modern-day pharmacy education and training in Europe (AU)

Unión Europea (UE) 2005/36/EC sobre reconocimiento de cualificaciones profesionales influencian la movilidad de los estudiantes de Farmacia y de los profesionales farmacéuticos, respectivamente. Además, la Declaración de Boloña trata de sintonizar los grados de educación superior, incluyendo el de farmacia, en toda la UE para crear un Espacio Común de Educación Superior Europeo. La Directiva señala los conocimientos, habilidades y cualificaciones requeridos para el ejercicio profesional de la farmacia en la UE. El proyecto PHARMINE (Pharmacy Education in Europe, www.pharmine.org) revise como la Declaración de Boloña y la Directiva influencian la educación y la formación modernas de la farmacia en Europa (AU)

Quality assurance in European pharmacy education and training / Aseguramiento de la calidad en la educación y formación en farmacia en Europa

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. Just above half has a working QA system. QA scores were high concerning matters such as complete curriculum and training, use of European Credit Transfer System, students’ representation and promotion of professional behavior. QA scores were low concerning matters such as evaluation of achievement of mission and goals, and financial resources. The PHARMINE consortium now has a basis upon which to elaborate and promote QA in European pharmacy faculties (AU)

Se realizó bajo los auspicios del consorcio PHARMINE de la Asociación Europea de Facultades de Farmacia una encuesta sobre los sistemas de aseguramiento de la calidad (QA) en las facultades de farmacia europeas. Se envió a las facultades europeas un cuestionario basado en los criterios de calidad de la Federación Internacional de Farmacia y el Consejo de Acreditación para la Educación en Farmacia (USA). Se obtuvieron respuestas de 28 países. Ligeramente más de la mitad tiene un sistema en funcionamiento de QA. Las puntuaciones de QA fueron altas en asuntos como currículo y formación completo, uso del Sistema Europeo de Transferencia de Créditos, representación de los estudiantes y promoción de la actuación profesional. Las puntuaciones de QA fueron bajas en asuntos como la evaluación de la consecución de misión y metas y recursos financieros. El consorcio PHARMINE tiene ahora una base sobre la que elaborar y promover la QA en las facultades de farmacia europeas (AU)

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. The technique is simple, fast, reliable, quantitative and inexpensive and was employed to assess the reactivity of 15 VHHs for native infectious poliovirus (N antigen), heat-denatured virus (H antigen) and 14S subviral particles. Three distinct subsets of VHHs were tentatively distinguished based on their specificity for the antigens: one that binds only to 14S precursors, another that binds to the H antigen and a third that binds to the N antigen. Some VHHs of the latter two subsets bound 14S subviral particles with equal affinity but others had at least 100-fold less affinity for the precursors. All neutralizing VHHs were demonstrated to recognize N antigen and all N-specific VHHs were shown to be neutralizing. This study corroborates the findings that VHHs mainly target conformational epitopes and that they target additional epitopes as compared to classical antibodies. The described technique may be useful for epitope mapping and tracking conformational changes of proteins.

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. For the investigated PV samples, a screening design revealed that injecting larger sample plugs significantly enhanced the analytical signal, but also significantly decreased the separation efficiency. A subsequently executed central composite design determined the largest sample plug that can be injected without compromising the separation. Finally, the sample dilution and the length of the injected plug were used for tuning the intensity of the analytical response. Two combinations of sample dilution and injected plug size, at extreme values, were investigated in detail to define the best procedure for PV analysis using CE. In both situations, PV was effectively separated and quantified in rather complex samples, showing a good repeatability, an acceptable linearity for the PV particles and a decreased limit of detection in comparison with the existing method. In conclusion, intra-column signal enhancement can be successfully applied for viral suspensions, extending the applicability of CE methods to samples with lower virus concentrations, and/or allowing a significant reduction in the minimum required volume of sample. For PV samples, 5µl of sample is necessary instead of the previous 20µl, while the analytical signal was enhanced up to 14 times. The results of this study can provide a basis for the development of routine CE methods for viral particle analysis, especially when rational and reproducible signal enhancement is required.

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. The consortium also looked at how European Union higher education institutions and courses are organised. The PHARMINE survey of pharmacy and pharmacy education in Europe produced country profiles with extensive information for EU member states and several other European countries. These data are available at: http://www.pharmine.org/losse_paginas/Country_Profiles/. This 2011 PHARMINE report presents the project and data, and some preliminary analysis on the basic question of how pharmacy education is adapted to pharmacy practice in the EU.

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 PHARMINE project (Pharmacy Education in Europe, www.pharmine.org) looked at how the Bologna declaration and the directive influence modern-day pharmacy education and training in Europe.

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. Just above half has a working QA system. QA scores were high concerning matters such as complete curriculum and training, use of European Credit Transfer System, students' representation and promotion of professional behavior. QA scores were low concerning matters such as evaluation of achievement of mission and goals, and financial resources. The PHARMINE consortium now has a basis upon which to elaborate and promote QA in European pharmacy faculties.

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. Interestingly, VHHs showed a potent antipolio activity with EC50 values in the low nanomolar range. Moreover, these antibody fragments completely blocked viral multiplication at higher concentrations. Remarkably, no (immune) escape variants against some of these VHHs could be generated. In conclusion, VHHs fulfil several in vitro requirements to be assigned as potential antiviral compounds for further development of an anti-poliovirus drugs.

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. that based on intensive screening of complete sets of gene and protein sequences. A deeper bioanalytical perspective may provide quantitative and qualitative tools, enabling study of various diseases and, eventually, may offer support for the development of accurate and reliable methods for clinical assessment. This would open the way to molecule-based diagnostics, i.e. establish accurate diagnosis and disease prognosis based on identification and/or quantification of biomacromolecules, for example proteins or nucleic acids. Finally, the development of disposable and portable devices for molecule-based diagnosis would provide the perfect translation of the science behind life-science research into practical applications dedicated to patients and health practitioners. This review provides an analytical perspective of the impact of microfluidics on the detection and characterization of bio-macromolecules involved in pathological processes. The main features of molecule-based diagnostics and the specific requirements for the diagnostic devices are discussed. Further, the techniques currently used for testing bio-macromolecules for potential diagnostic purposes are identified, emphasizing the newest developments. Subsequently, the challenges of this type of application and the status of commercially available devices are highlighted, and future trends are noted.

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. As a result of heating, the viral genome is released from the capsid. Here, we show that during this incubation some intermediate particles were found. The latter were identified by enzymatic peak shift analysis. The high salt concentration eluate subviral particles were analyzed with preincubation affinity CE together with their sensitivity for RNase and proteinase K treatment. Electropherograms of the higher salt concentration eluate display a mixture of at least four different subviral particles. One particle proved to have an [N1, H] antigenicity and was resistant to RNase and proteinase K digestion. The remaining particles were all sensitive to proteinase K treatment. This CE method proved to be valuable in the detection, identification and analysis of poliovirions and poliovirus particles offering an alternative powerful, cheap, fast and easy analysis method.

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. The proposed method manages to perform an acceptable separation of poliovirus particles using a 50 mM borate buffer with 25 mM SDS, in an uncoated fused-silica capillary upon application of 10 kV at 30 degrees C. The linearity of the proposed method was investigated for a range of poliovirus dilutions up to 140 microg/mL.