RESUMO
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.
Assuntos
Capsídeo/metabolismo , Enterovirus Humano B/fisiologia , Infecções por Enterovirus/metabolismo , Glutationa/metabolismo , RNA Viral/biossíntese , Replicação Viral/fisiologia , Animais , Chlorocebus aethiops , Infecções por Enterovirus/genética , Glutationa/genética , Células HeLa , Humanos , Mutação , RNA Viral/genética , Células VeroRESUMO
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.
Assuntos
Anticorpos Neutralizantes/imunologia , Fragmentos de Imunoglobulinas/imunologia , Mutação/imunologia , Poliovirus/imunologia , Substituição de Aminoácidos/imunologia , Antivirais/farmacologia , Sítios de Ligação/imunologia , Proteínas do Capsídeo/imunologia , Linhagem Celular Tumoral , Células HeLa , Humanos , Poliovirus/efeitos dos fármacos , Proteínas Virais/imunologiaRESUMO
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.
Assuntos
Anticorpos Antivirais/farmacologia , Capsídeo/química , Poliomielite/virologia , Poliovirus/efeitos dos fármacos , Anticorpos de Domínio Único/farmacologia , Antivirais/farmacologia , Capsídeo/efeitos dos fármacos , Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Linhagem Celular , Humanos , Poliovirus/química , Poliovirus/genética , Poliovirus/fisiologia , Replicação Viral/efeitos dos fármacos , Desenvelopamento do Vírus/efeitos dos fármacosRESUMO
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.
Assuntos
Nanoestruturas/química , Poliovirus/química , Eletroforese CapilarRESUMO
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.
Assuntos
Eletroforese Capilar/métodos , Poliovirus/química , Vírion/isolamento & purificação , Anticorpos Monoclonais/química , Anticorpos Antivirais/química , Antígenos Virais/química , Antígenos Virais/isolamento & purificação , Proteínas do Capsídeo/química , Cromatografia por Troca Iônica , Endopeptidase K/metabolismo , Genoma Viral , Temperatura Alta , Ribonucleases/metabolismo , Vírion/químicaRESUMO
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.
Assuntos
Substâncias Macromoleculares/análise , Microfluídica , Nanotecnologia , Preparações Farmacêuticas/análise , HumanosRESUMO
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. A panel convoked by the National Research Council concluded that the use of antiviral drugs may be essential in the polio eradication strategy. We here report on a comparative study of the antipoliovirus activity of a selection of molecules that have previously been reported to be inhibitors of picornavirus replication and discuss their potential use, alone or in combination, for the treatment or prophylaxis of poliovirus infection.
Assuntos
Antivirais/farmacologia , Poliomielite/prevenção & controle , Poliovirus/efeitos dos fármacos , Células HeLa , Humanos , Poliomielite/tratamento farmacológico , Poliovirus/fisiologia , Ligação Proteica , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
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(4.0)-fold, 10(6.2)-fold and 10(6.6)-fold for poliovirus type 1, type 2 and type 3, respectively. R75761 could be considered as a lead compound for development of anti-poliovirus drugs to be used during the late stage of poliovirus eradication and beyond.
Assuntos
Antivirais/farmacologia , Poliomielite/prevenção & controle , Vacina Antipólio Oral , Poliovirus/efeitos dos fármacos , Piridazinas/farmacologia , Efeito Citopatogênico Viral/efeitos dos fármacos , Células HeLa , Humanos , Poliomielite/tratamento farmacológico , Poliomielite/virologia , Poliovirus/classificação , Poliovirus/fisiologia , Piridazinas/química , Replicação Viral/efeitos dos fármacosRESUMO
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. RESULTS: In the present study we explored the potential of diverse compounds to modulate viral persistence in these DRAW cells. Hemin was found to increase viral yields and to induce cell lysis. Enviroxime and neutralizing anti-TMEV monoclonal antibody were shown to decrease viral yields, whereas interferon-alpha and interferon-gamma completely cleared the persistent infection. We also compared the cytokine pattern secreted by uninfected RAW, DRAW and interferon-cured DRAW macrophages using a cytokine protein array. The chemokine RANTES was markedly upregulated in DRAW cells and restored to a normal expression level after abrogation of the persistent infection with interferon-alpha or interferon-gamma. On the other hand, the chemokine MCP-1 was upregulated in the interferon-cured DRAW cells. CONCLUSION: We have identified several compounds that modulate viral replication in an in vitro model system for TMEV persistence. These compounds now await further testing in an in vivo setting to address fundamental questions regarding persistent viral infection and immunopathogenesis.
Assuntos
Antivirais/farmacologia , Infecções por Cardiovirus/tratamento farmacológico , Macrófagos/virologia , Theilovirus/efeitos dos fármacos , Theilovirus/fisiologia , Replicação Viral/efeitos dos fármacos , Animais , Infecções por Cardiovirus/genética , Infecções por Cardiovirus/imunologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Quimiocina CCL5/genética , Quimiocina CCL5/metabolismo , Citocinas/imunologia , Expressão Gênica/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Theilovirus/imunologiaRESUMO
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.e. the Respironics REMstar Auto (RR) and the ResMed Spirit (RS). METHODS: Fifty obstructive sleep apnea patients were recruited for a double-blind randomized crossover trial. Both devices were employed overnight by means of split-night polysomnography. The primary outcome was the AHI. Secondary outcome measures were the snoring index, pressure profiles and subjective appraisal of sleep quality assessed the morning after the sleep study. The Wilcoxon signed rank test for matched pairs was applied to assess differences between treatment conditions. RESULTS: No significant differences were found in sleep parameters, subjective sleep quality and snoring index. The use of the RR was associated with a significantly lower AHI in comparison with the RS [mean (SD) 6.9 (11.6)/h vs. 9.4 (9.2)/h, p = 0.004]. This result was obtained at significantly lower pressure levels [P95 9.2 (2.3) cm H(2)O vs. 10.2 (1.5) cm H(2)O, p = 0.001]. CONCLUSION: While the RR provided a lower AHI than the RS at lower pressure levels, it could not be assessed whether this difference was relevant for clinical outcomes. However, this face-to-face comparison of Auto-adjustable continuous positive airway pressure devices seems useful for the assessment of titration efficacy.
Assuntos
Pressão Positiva Contínua nas Vias Aéreas/instrumentação , Consumo de Oxigênio/fisiologia , Apneia Obstrutiva do Sono/terapia , Ventiladores Mecânicos/normas , Adulto , Resistência das Vias Respiratórias , Análise de Variância , Automação , Gasometria , Pressão Positiva Contínua nas Vias Aéreas/métodos , Estudos Cross-Over , Método Duplo-Cego , Desenho de Equipamento , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Polissonografia , Probabilidade , Troca Gasosa Pulmonar , Medição de Risco , Índice de Gravidade de Doença , Apneia Obstrutiva do Sono/diagnóstico , Estatísticas não Paramétricas , Titulometria , Resultado do TratamentoRESUMO
Xenopus laevis oocytes can be used as an alternative system to study replication of Theiler's murine encephalomyelitis virus (TMEV). We have shown that transcript RNA, containing full-length viral genome, can be directly used to programme the oocytes. In the programmed oocytes, there is correct viral translation, polyprotein processing and assembly of capsid proteins leading to the production of infectious TMEV. The vast majority of de novo synthesised virions were found in the medium in which the programmed oocytes were incubated and not in the oocytes.
Assuntos
Oócitos/virologia , Theilovirus/fisiologia , Animais , Feminino , Genoma Viral , Microinjeções , RNA Viral/administração & dosagem , RNA Viral/genética , RNA Viral/metabolismo , Theilovirus/genética , Proteínas Virais/biossíntese , Proteínas Virais/genética , Replicação Viral , Xenopus laevisRESUMO
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. However, the addition of either 3CDpro mRNA or of purified 3CDpro protein to in vitro reactions, programmed with viral RNA, results in a 100-fold increase in virus yield. Mutational analyses of 3CDpro indicated that RNA binding by the 3Cpro domain and the integrity of interface I in the 3Dpol domain of the protein are both required for function. The aim of these studies was to determine the exact step or steps at which 3CDpro enhances virus yield and to determine the mechanism by which this occurs. Our results suggest that the addition of extra 3CDpro to in vitro translation RNA-replication reactions results in a mild enhancement of both minus and plus strand RNA synthesis. By examining the viral particles formed in the in vitro reactions on sucrose gradients we determined that 3CDpro has only a slight stimulating effect on the synthesis of capsid precursors but it strikingly enhances the maturation of virus particles. Both the stimulation of RNA synthesis and the maturation of the virus particles are dependent on the presence of an intact RNA binding site within the 3Cpro domain of 3CDpro. In addition, the integrity of interface I in the 3Dpol domain of 3CDpro is required for efficient production of mature virus. Surprisingly, plus strand RNA synthesis and virus production in in vitro reactions, programmed with full-length transcript RNA, are not enhanced by the addition of extra 3CDpro. Our results indicate that the stimulation of RNA synthesis and virus maturation by 3CDpro in vitro is dependent on the presence of a VPg-linked RNA template.
Assuntos
Cisteína Endopeptidases/metabolismo , Poliovirus/metabolismo , RNA Viral/biossíntese , Proteínas Virais/metabolismo , Montagem de Vírus , Proteases Virais 3C , Sistema Livre de Células , Regulação Viral da Expressão Gênica , Células HeLa , Humanos , Biossíntese de Proteínas , Transcrição GênicaRESUMO
Quantification of virus-like RNA sequences in biological fluids, like serum and cerebrospinal fluid, requires an RNA extraction method that is both reproducible and fast. Three RNA extraction methods were tested on enteroviruses: (1) the acid guanidine thiocyanate-phenol/chloroform (AGPC) method; (2) a method based on differential precipitation of the RNA and (3) a 'bind-wash-elute' system based on silica-gel membrane binding. The latter two methods yielded a comparable detection limit as measured by RT-PCR ELISA. The detection limit for the AGPC method was 10 times higher. The relative standard deviation for the bind-wash-elute method (3%) was superior to that of the other methods tested (both 20%) and provides a reliable and fast method to extract (viral) RNA from biological fluids for quantification by RT-PCR.
Assuntos
Enterovirus/isolamento & purificação , RNA/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Enterovirus/química , Ensaio de Imunoadsorção Enzimática/métodos , Vírus de RNA/química , Vírus de RNA/isolamento & purificação , Espectrofotometria Ultravioleta/métodosAssuntos
Equinococose Pulmonar/diagnóstico por imagem , Endossonografia/métodos , Cisto Mediastínico/diagnóstico por imagem , Adulto , Albendazol/uso terapêutico , Equinococose Pulmonar/diagnóstico , Equinococose Pulmonar/cirurgia , Endossonografia/instrumentação , Humanos , Masculino , Cisto Mediastínico/diagnóstico , Cisto Mediastínico/cirurgia , Recidiva , Sensibilidade e Especificidade , Som , Toracotomia , Tomografia Computadorizada por Raios X , Resultado do TratamentoRESUMO
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.
Assuntos
Anticorpos/química , Magnetismo , Poliovirus/química , Proteínas/química , Anticorpos/imunologia , Especificidade de Anticorpos , Antígenos Virais/química , Antígenos Virais/imunologia , Cobalto/química , Epitopos , Histidina/química , Oligopeptídeos/química , Poliovirus/imunologia , Poliovirus/isolamento & purificação , Domínios e Motivos de Interação entre Proteínas , Radioisótopos de Enxofre/químicaRESUMO
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.
Assuntos
Eletroforese Capilar/métodos , Vacinas contra Poliovirus/isolamento & purificação , Poliovirus/isolamento & purificação , Tecnologia Farmacêutica/métodos , Carga Viral/métodos , Automação Laboratorial/métodos , Extratos Celulares/isolamento & purificação , Humanos , Fatores de TempoRESUMO
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.
Assuntos
Eletroforese Capilar/métodos , Poliovirus/isolamento & purificação , Vírion/isolamento & purificação , Métodos Analíticos de Preparação de Amostras/métodos , Limite de Detecção , Reprodutibilidade dos Testes , Suspensões/química , Ultrafiltração/métodosRESUMO
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.
Assuntos
Proteínas do Capsídeo/química , Capsídeo/química , Eletroforese Capilar/métodos , Poliovirus/química , Dodecilsulfato de Sódio/química , Tensoativos/química , Proteínas Virais/química , Ânions/química , Eletroforese em Gel de Poliacrilamida/métodosRESUMO
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.
RESUMO
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.