Five of Five VHHs Neutralizing Poliovirus Bind the Receptor-Binding Site.

UNLABELLED: Nanobodies, or VHHs, that recognize poliovirus type 1 have previously been selected and characterized as candidates for antiviral agents or reagents for standardization of vaccine quality control. In this study, we present high-resolution cryo-electron microscopy reconstructions of poliovirus with five neutralizing VHHs. All VHHs bind the capsid in the canyon at sites that extensively overlap the poliovirus receptor-binding site. In contrast, the interaction involves a unique (and surprisingly extensive) surface for each of the five VHHs. Five regions of the capsid were found to participate in binding with all five VHHs. Four of these five regions are known to alter during the expansion of the capsid associated with viral entry. Interestingly, binding of one of the VHHs, PVSS21E, resulted in significant changes of the capsid structure and thus seems to trap the virus in an early stage of expansion. IMPORTANCE: We describe the cryo-electron microscopy structures of complexes of five neutralizing VHHs with the Mahoney strain of type 1 poliovirus at resolutions ranging from 3.8 to 6.3Å. All five VHHs bind deep in the virus canyon at similar sites that overlap extensively with the binding site for the receptor (CD155). The binding surfaces on the VHHs are surprisingly extensive, but despite the use of similar binding surfaces on the virus, the binding surface on the VHHs is unique for each VHH. In four of the five complexes, the virus remains essentially unchanged, but for the fifth there are significant changes reminiscent of but smaller in magnitude than the changes associated with cell entry, suggesting that this VHH traps the virus in a previously undescribed early intermediate state. The neutralizing mechanisms of the VHHs and their potential use as quality control agents for the end game of poliovirus eradication are discussed.

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.

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.

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.

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.

No relationship between chronotype and timing of breeding when variation in daily activity patterns across the breeding season is taken into account.

There is increasing evidence that individuals are consistent in the timing of their daily activities, and that individual variation in temporal behavior is related to the timing of reproduction. However, it remains unclear whether observed patterns relate to the timing of the onset of activity or whether an early onset of activity extends the time that is available for foraging. This may then again facilitate reproduction. Furthermore, the timing of activity onset and offset may vary across the breeding season, which may complicate studying the above-mentioned relationships. Here, we examined in a wild population of great tits (Parus major) whether an early clutch initiation date may be related to an early onset of activity and/or to longer active daylengths. We also investigated how these parameters are affected by the date of measurement. To test these hypotheses, we measured emergence and entry time from/into the nest box as proxies for activity onset and offset in females during the egg laying phase. We then determined active daylength. Both emergence time and active daylength were related to clutch initiation date. However, a more detailed analysis showed that the timing of activities with respect to sunrise and sunset varied throughout the breeding season both within and among individuals. The observed positive relationships are hence potentially statistical artifacts. After methodologically correcting for this date effect, by using data from the pre-egg laying phase, where all individuals were measured on the same days, neither of the relationships remained significant. Taking methodological pitfalls and temporal variation into account may hence be crucial for understanding the significance of chronotypes.

Serotonin transporter (SERT) polymorphisms, personality and problem-solving in urban great tits.

Understanding underlying genetic variation can elucidate how diversity in behavioral phenotypes evolves and is maintained. Genes in the serotonergic signaling pathway, including the serotonin transporter gene (SERT), are candidates for affecting animal personality, cognition and fitness. In a model species, the great tit (Parus major), we reevaluated previous findings suggesting relationships between SERT polymorphisms, neophobia, exploratory behavior and fitness parameters, and performed a first test of the relationship between single nucleotide polymorphisms (SNPs) in SERT and problem-solving in birds. We found some evidence for associations between SERT SNPs and neophobia, exploratory behavior and laying date. Furthermore, several SNPs were associated with behavioral patterns and success rates during obstacle removal problem-solving tests performed at nest boxes. In females, minor allele homozygotes (AA) for nonsynonymous SNP226 in exon 1 made fewer incorrect attempts and were more likely to problem-solve. In both sexes, there was some evidence that minor allele homozygotes (CC) for SNP84 in exon 9 were more likely to problem-solve. Only one SNP-behavior relationship was statistically significant after correcting for multiple comparisons, but several were associated with substantial effect sizes. Our study provides a foundation for future research on the genetic basis of behavioral and cognitive variation in wild animal populations.

The serotonin transporter gene and female personality variation in a free-living passerine.

Quantifying variation in behaviour-related genes provides insight into the evolutionary potential of repeatable among-individual variation in behaviour (i.e. personality). Yet, individuals typically also plastically adjust their behaviour in response to environmental conditions and/or age, thereby complicating the detection of genotype-phenotype associations. Here, using a population of free-living great tits (Parus major), we assessed the association between single nucleotide polymorphisms (SNPs) in the serotonin transporter gene (SERT) and two repeatable behavioural traits, i.e. female-female aggression and female hissing behaviour. For female-female aggression, a trait showing age-related plasticity, we found no evidence for associations with SERT SNPs, even when assessing potential age-dependent effects of SERT genotype on aggression. We also found no strong support for associations between SERT SNPs and hissing behaviour, yet we identified two synonymous polymorphisms (exon 13 SNP66 and exon 12 SNP144) of particular interest, each explaining about 1.3% of the total variation in hissing behaviour. Overall, our results contribute to the general understanding of the biological underpinning of complex behavioural traits and will facilitate further (meta-analytic) research on behaviour-related genes. Moreover, we emphasize that future molecular genetic studies should consider age-dependent genotype-phenotype associations for behavioural trait (co)variation, as this will vastly improve our understanding of the proximate causes and ultimate consequences of personality variation in natural populations.

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.

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.

Nest defence behavioural reaction norms: testing life-history and parental investment theory predictions.

Predation is the primary source of reproductive failure in many avian taxa and nest defence behaviour against predators is hence an important aspect of parental investment. Nest defence is a complex trait that might consistently differ among individuals (personality), while simultaneously vary within individuals (plasticity) according to the reproductive value of the offspring. Both complementary aspects of individual variation can influence fitness, but the causality of links with reproductive success remains poorly understood. We repeatedly tested free-living female great tits (Parus major) for nest defence (hissing) behaviour across the nesting cycle, by presenting them with a model predator. Hissing behaviour was highly repeatable but, despite population-level plasticity, we found no support for individual differences in plasticity. Path analysis revealed that repeatable differences in hissing behaviour had no direct effect on nest success or fledgling number. However, our best supported path-model showed that more fiercely hissing females laid smaller clutches, with clutch size in turn positively influencing fledgling number, suggesting that females are most likely facing a trade-off between investment in nest defence and reproduction. Strong stabilizing selection for optimal plasticity, in combination with life-history trade-offs, might explain the high repeatability of nest defence and its link with reproductive success.

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(4.0)-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.

Personality and artificial light at night in a semi-urban songbird population: No evidence for personality-dependent sampling bias, avoidance or disruptive effects on sleep behaviour.

Light pollution or artificial light at night (ALAN) is an increasing, worldwide challenge that affects many aspects of animal behaviour. Interestingly, the response to ALAN varies widely among individuals within a population and variation in personality (consistent individual differences in behaviour) may be an important factor explaining this variation. Consistent individual differences in exploration behaviour in particular may relate to the response to ALAN, as increasing evidence indicates its relation with how individuals respond to novelty and how they cope with anthropogenic modifications of the environment. Here, we assayed exploration behaviour in a novel environment as a proxy for personality variation in great tits (Parus major). We observed individual sleep behaviour over two consecutive nights, with birds sleeping under natural dark conditions the first night and confronted with ALAN inside the nest box on the second night, representing a modified and novel roosting environment. We examined whether roosting decisions when confronted with a camera (novel object), and subsequently with ALAN, were personality-dependent, as this could potentially create sampling bias. Finally, we assessed whether experimentally challenging individuals with ALAN induced personality-dependent changes in sleep behaviour. Slow and fast explorers were equally likely to roost in a nest box when confronted with either a camera or artificial light inside, indicating the absence of personality-dependent sampling bias or avoidance of exposure to ALAN. Moreover, slow and fast explorers were equally disrupted in their sleep behaviour when challenged with ALAN. Whether other behavioural and physiological effects of ALAN are personality-dependent remains to be determined. Moreover, the sensitivity to disturbance of different behavioural types might depend on the behavioural context and the specific type of challenge in question. In our increasingly urbanized world, determining whether the effects of anthropogenic stressors depend on personality type will be of paramount importance as it may affect population dynamics.

Variation in personality traits across a metal pollution gradient in a free-living songbird.

Anthropogenic contaminants could alter traits central to animal behavioral types, or personalities, including aggressiveness, boldness and activity level. Lead and other toxic metals are persistent inorganic pollutants that affect organisms worldwide. Metal exposure can alter behavior by affecting neurology, endocrinology, and health. However, the direction and magnitude of the behavioral effects of metal exposure remain equivocal. Moreover, the degree to which metal exposure simultaneously affects suites of correlated behavioral traits (behavioral syndromes) that are controlled by common mechanisms remains unclear, with most studies focusing on single behaviors. Using a model species for personality variation, the great tit (Parus major), we explored differences in multiple behavioral traits across a pollution gradient where levels of metals, especially lead and cadmium, are elevated close to a smelter. We employed the novel environment exploration test, a proxy for variation in personality type, and also measured territorial aggressiveness and nest defense behavior. At polluted sites birds of both sexes displayed slower exploration behavior, which could reflect impaired neurological or physiological function. Territorial aggression and nest defense behavior were individually consistent, but did not vary with proximity to the smelter, suggesting that metal exposure does not concurrently affect exploration and aggression. Rather, exploration behavior appears more sensitive to metal pollution. Effects of metal pollution on exploration behavior, a key animal personality trait, could have critical effects on fitness.

The Female Perspective of Personality in a Wild Songbird: Repeatable Aggressiveness Relates to Exploration Behaviour.

Males often express traits that improve competitive ability, such as aggressiveness. Females also express such traits but our understanding about why is limited. Intraspecific aggression between females might be used to gain access to reproductive resources but simultaneously incurs costs in terms of energy and time available for reproductive activities, resulting in a trade-off. Although consistent individual differences in female behaviour (i.e. personality) like aggressiveness are likely to influence these reproductive trade-offs, little is known about the consistency of aggressiveness in females. To quantify aggression we presented a female decoy to free-living female great tits (Parus major) during the egg-laying period, and assessed whether they were consistent in their response towards this decoy. Moreover, we assessed whether female aggression related to consistent individual differences in exploration behaviour in a novel environment. We found that females consistently differed in aggressiveness, although first-year females were on average more aggressive than older females. Moreover, conform life history theory predictions, 'fast' exploring females were more aggressive towards the decoy than 'slow' exploring females. Given that personality traits are often heritable, and correlations between behaviours can constrain short term adaptive evolution, our findings highlight the importance of studying female aggression within a multivariate behavioural framework.

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.

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.

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.

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.

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.