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.     

A natural dominant negative P2X1 receptor due to deletion of a single amino acid residue

The P2X1 receptor belongs to a family of oligomeric ATP-gated ion channels with intracellular N and C termini and two transmembrane segments separating a large extracellular domain. Here, we describe a naturally occurring dominant negative P2X1 mutant. This mutant lacks one leucine within a stretch of four leucine residues in its second transmembrane domain (TM2) (amino acids 351-354). Confocal microscopy revealed proper plasma membrane localization of the mutant in stably transfected HEK293 cells. Nevertheless, voltage-clamped HEK293 cells expressing mutated P2X1 channels failed to develop an ATP or ADP-induced current. Furthermore, when co-expressed with the wild type receptor in Xenopus oocytes, the mutated protein exhibited a dose-dependent dominant negative effect on the normal ATP or ADP-induced P2X1 channel activity. These data indicate that deletion of a single apolar amino acid residue at the inner border of the P2X1 TM2 generates a nonfunctional channel. The inactive and dominant negative form of the P2X1 receptor may constitute a new tool for the study of the physiological role of this channel in native cells.     

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 nanobodies¹. Since poliovirus is sensitive to conformational conversion² 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 test⁵. 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 antibodies⁸ 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 ³⁵S 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.     

Bovine tuberculosis prevalence survey on cattle in the rural livestock system of Torodi (Niger)

Background

Bovine tuberculosis (BTB) is a widespread zoonosis in developing countries but has received little attention in sub-Saharan Africa, especially in Niger. Recent investigations confirmed the high incidence of the disease in cattle slaughtered in an abattoir in Niamey. The fact that most of the animals in which M. bovis has been identified were from the rural area of Torodi implied the existence of a probable source of BTB in this region. This study aimed to determine the prevalence of BTB infection in cattle and to identify risk factors for infection in human and cattle populations in Torodi.

Methods and Principal Findings

A survey was carried out at the level of households keeping livestock (n = 51). The questionnaire was related to the potential risk factors and the presence of clinical signs of TB both in animals and humans. Comparative Intradermal Tuberculin Test was conducted to determine the TB status in cattle (n = 393). The overall apparent individual animal prevalence of tuberculin reactors was 3.6% (CI: 95%, 1.9–5.9), whereas the individual true prevalence was estimated at 0.8% (CI: 95%, 0.0–5.0). Using a multivariate logistic regression analysis and a classification tree analysis, the only household level risk factor that significantly influenced the presence of BTB in cattle was the presence of animals coughing in the herd (OR = 4.7, 95% CI: 1.12–19.71, p-value = 0.034). The lack of the practice of quarantine was borderline significant (OR = 4.2, 95% CI: 0.96–18.40, p-value = 0.056).

Conclusion/Significance

The study confirmed that BTB is endemic in cattle in Torodi and the risk of the transmission of the disease to humans is potentially high. For the control of the disease in livestock, slaughtering of infected animals and the compensation of the owners is needed. Collaboration between the veterinary and the medical sectors, in the diagnosis, monitoring, prevention and control of BTB is strongly encouraged.     

Why Latrines Are Not Used: Communities’ Perceptions and Practices Regarding Latrines in a Taenia solium Endemic Rural Area in Eastern Zambia

Taenia solium cysticercosis is a neglected parasitic zoonosis occurring in many developing countries. Socio-cultural determinants related to its control remain unclear. Studies in Africa have shown that the underuse of sanitary facilities and the widespread occurrence of free-roaming pigs are the major risk factors for porcine cysticercosis. The study objective was to assess the communities’ perceptions, practices and knowledge regarding latrines in a T. solium endemic rural area in Eastern Zambia inhabited by the Nsenga ethno-linguistic group, and to identify possible barriers to their construction and use. A total of 21 focus group discussions on latrine use were organized separately with men, women and children, in seven villages of the Petauke district. The themes covered were related to perceived latrine availability (absence-presence, building obstacles) and perceived latrine use (defecation practices, latrine management, socio-cultural constraints).The findings reveal that latrines were not constructed in every household because of the convenient use of existing latrines in the neighborhood. Latrines were perceived to contribute to good hygiene mainly because they prevent pigs from eating human feces. Men expressed reluctance to abandon the open-air defecation practice mainly because of toilet-associated taboos with in-laws and grown-up children of the opposite gender. When reviewing conceptual frameworks of people’s approach to sanitation, we found that seeking privacy and taboos hindering latrine use and construction were mainly explained in our study area by the fact that the Nsenga observe a traditionally matrilineal descent. These findings indicate that in this local context latrine promotion messages should not only focus on health benefits in general. Since only men were responsible for building latrines and mostly men preferred open defecation, sanitation programs should also be directed to men and address related sanitary taboos in order to be effective.     

An integrated proteomics and genomics analysis to unravel a heterogeneous platelet secretion defect

Eight patients with clinical bleeding problems have evidence for platelet storage pool disease as they present with impaired platelet aggregation and secretion with low concentrations of ADP and collagen and an absence of second phase aggregation with epinephrine. Electron microscopy analysis further showed a reduced but not absent amount of platelet dense granules, and CD63 staining was decreased compared to healthy controls. The presence of alpha granules and CD62P expression after platelet activation was normal. This work aimed at identifying differentially expressed proteins in the platelet releasate and its remaining pellet after activation with A23187 and TRAP in patients and controls using DIGE-based proteomic technology. We identified 44 differentially expressed proteins in patients and the altered expression for some of them was confirmed by immunoblot analysis. Most of these proteins belong to the class of cytoskeleton-related proteins. In addition, 29 cytoskeleton-related genes showed an altered expression in platelet mRNA from patients using a real-time PCR array. In conclusion, our study shows that the dense granule secretion defect in patients with platelet storage pool disease is highly heterogeneous with evidence of an underlying cytoskeleton defect.     

In situ versus laboratory estimations of length–weight regression and growth rate of Daphnia magna (Branchiopoda, Anomopoda) from an aerated waste stabilization pond

The present paper questions the adequacy of using length–weight regressions and growth rates calculated in the laboratory under constant physico-chemical and food conditions for the estimation of biomass and secondary production of animals living in a variable environment from the physico-chemistry and food availability point of view. Length–weight regressions (LWR) and growth rate of Daphnia magna were determined in situat five key periods of the year. In parallel, LWR and growth rate were determined in laboratory incubators at temperature adjusted to the mean temperature measured during the in situexperiments. LWR estimated from pond daphnids collected during the in situ experiments were, on the whole, not significantly different from LWR established during laboratory experiments, indicating that the food availability was globally similar in the laboratory and in situexperiments, even though food items were substantially different between the experiments. In situ algal biomass was indeed low compared to the algal biomass in laboratory experiments, but high biomasses of bacteria, protozoa and detritus were available for daphnid feeding in the tubes incubated in situ. Growth rate of D. magnawas monitored in situusing 50-ml tubes closed with Nylon net (mesh size = 80 m) and in the laboratory using 50-ml glass flasks. The physico-chemical, bacteriological and algological variables were checked to be similar in the tubes and in the pond. Growth rates varied according to the size of the animal and according to the water temperature. The maximum growth rates were observed for juveniles at 20.2 °C. Growth rates were also determined in the laboratory at temperature corresponding to the mean temperature recorded in the pond during the in situ growth experiments. Differences between in situ and laboratory body length–growth rate regressions (LgR) were observed for the experiments conducted at 15.6 °C and 23.6 °C. Due to differences in LWR and LgR between in situ and laboratory experiments, biomass and daily production estimated from laboratory cultures were found to be significantly, but not severely, higher than biomass and daily production estimated on the basis of in situ experiments. It has been, therefore, concluded that, when the constraints linked to the realization of in situ growth experiments are too strong, the laboratory approach is fully justified.