914 resultados para virus-host interaction


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Le virus du syndrome reproducteur et respiratoire porcin (VSRRP) est un pathogène d’importance dans l’industrie porcine et est responsable d’importantes pertes économiques. Il n’existe pas d’antiviral efficace contre celui-ci. Il a récemment été mis en évidence que le surnageant de culture d’Actinobacillus pleuropneumoniae, l’agent étiologique de la pleuropneumonie porcine, possédait une activité antivirale in vitro contre le VSRRP dans la lignée cellulaire SJPL. Les objectifs de mon projet sont (i) d’étudier les mécanismes cellulaires menant à l’activité antivirale causée par le surnageant de culture d’A. pleuropneumoniae, et (ii) de caractériser les molécules actives présentes dans le surnageant de culture d’A. pleuropneumoniae. Dans un premier temps, des analyses de protéome ont été effectuées et ont permis d’observer que le surnageant de culture modulait la régulation du cycle cellulaire. Dans le but d’analyser le cycle cellulaire des cellules SJPL, la cytométrie en flux a été utilisée et a permis de démontrer que le surnageant de culture induisait un arrêt du cycle cellulaire en phase G2/M. Deux inhibiteurs de la phase G2/M ont alors été utilisé. Il s'est avéré que ces inhibiteurs avaient la capacité d’inhiber le VSRRP dans les cellules SJPL. Enfin, la spectrométrie de masse a été utilisée dans le but de caractériser les molécules actives présentes dans le surnageant de culture d’A. pleuropneumoniae et d’identifier deux molécules. Ce projet a permis de démontrer pour la première fois qu’A. pleuropneumoniae est capable de perturber le cycle cellulaire et que ce dernier était un élément important dans l’effet antiviral contre le VSRRP.

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Coronavirus nucleoproteins (N proteins) localize to the cytoplasm and the nucleolus, a subnuclear structure, in both virus-infected primary cells and in cells transfected with plasmids that express N protein. The nucleolus is the site of ribosome biogenesis and sequesters cell cycle regulatory complexes. Two of the major components of the nucleolus are fibrillarin and nucleolin. These proteins are involved in nucleolar assembly and ribosome biogenesis and act as chaperones for the import of proteins into the nucleolus. We have found that fibrillarin is reorganized in primary cells infected with the avian coronavirus infectious bronchitis virus (IBV) and in continuous cell lines that express either IBV or mouse hepatitis virus N protein. Both N protein and a fibrillarin-green fluorescent protein fusion protein colocalized to the perinuclear region and the nucleolus. Pull-down assays demonstrated that IBV N protein interacted with nucleolin and therefore provided a possible explanation as to how coronavirus N proteins localize to the nucleolus. Nucleoli, and proteins that localize to the nucleolus, have been implicated in cell growth-cell cycle regulation. Comparison of cells expressing IBV N protein with controls indicated that cells expressing N protein had delayed cellular growth. This result could not to be attributed to apoptosis. Morphological analysis of these cells indicated that cytokinesis was disrupted, an observation subsequently found in primary cells infected with IBV. Coronaviruses might therefore delay the cell cycle in interphase, where maximum translation of viral mRNAs can occur.

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Infection of young poults with turkey coronavirus (TCoV) produces a syndrome characterized by acute enteritis, diarrhea, anorexia, ruffled feathers, decreased body weight gain and uneven flock growth. The objective of this study was to standardize an intestinal organ culture (IOC) in order to assess host-virus interaction related to apoptosis. For this purpose the Brazilian strain (TCoV/Brazil/2006 with GenBank accession number FJ188401), was used for infection. Infected IOC cells had mitochondrial dysfunction and initial nuclear activation with MTT value of 90.7 (± 2.4) and apoptotic factor 2.21 (± 2.1), considered statistically different from uninfected IOC cells (p > 0.05). The kinetics of TCoV antigens and viral RNA was directly correlated to annexin-V, caspases- 2 and -3, p53, BCl-2 antigens at 24, 72 and 96 h post-infection (p.i.). Morphological and biochemical features of apoptosis, such as in situ nuclear fragmentation (TUNEL and annexin-V) and DNA ladder formation were also detected in infected cells at all assayed p.i. intervals. Moreover, different from other coronaviruses, the expression of both effective caspase-2 and - 3 and p53 antigens were considered lower. However, at all p.i., the BCl-2 antigens were expressed quantitatively and qualitatively as viral antigen measured by immunofluorescence microscopy analysis. Because the diagnosis of TCoV infection is only performed by infecting embryonated poult eggs, the pathological characteri tics related to host-virus interaction remain unclear. This is the first report on apoptosis of TCoV infected IOC, and reveals that it may be useful immunological method to assess virus pathogenesis.

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Beet necrotic yellow vein virus (BNYVV), the leading infectious agent that affects sugar beet, is included within viruses transmitted through the soil from plasmodiophorid as Polymyxa betae. BNYVV is the causal agent of Rhizomania, which induces abnormal rootlet proliferation and is widespread in the sugar beet growing areas in Europe, Asia and America; for review see (Peltier et al., 2008). In this latter continent, Beet soil-borne mosaic virus (BSBMV) has been identified (Lee et al., 2001) and belongs to the benyvirus genus together with BNYVV, both vectored by P. betae. BSBMV is widely distributed only in the United States and it has not been reported yet in others countries. It was first identified in Texas as a sugar beet virus morphologically similar but serologically distinct to BNYVV. Subsequent sequence analysis of BSBMV RNAs evidenced similar genomic organization to that of BNYVV but sufficient molecular differences to distinct BSBMV and BNYVV in two different species (Rush et al., 2003). Benyviruses field isolates usually consist of four RNA species but some BNYVV isolates contain a fifth RNA. RNAs -1 contains a single long ORF encoding polypeptide that shares amino acid homology with known viral RNA-dependent RNA polymerases (RdRp) and helicases. RNAs -2 contains six ORFs: capsid protein (CP), one readthrough protein, triple gene block proteins (TGB) that are required for cell-to-cell virus movement and the sixth 14 kDa ORF is a post-translation gene silencing suppressor. RNAs -3 is involved on disease symptoms and is essential for virus systemic movement. BSBMV RNA-3 can be trans-replicated, trans-encapsidated by the BNYVV helper strain (RNA-1 and -2) (Ratti et al., 2009). BNYVV RNA-4 encoded one 31 kDa protein and is essential for vector interactions and virus transmission by P. betae (Rahim et al., 2007). BNYVV RNA-5 encoded 26 kDa protein that improve virus infections and accumulation in the hosts. We are interest on BSBMV effect on Rhizomania studies using powerful tools as full-length infectious cDNA clones. B-type full-length infectious cDNA clones are available (Quillet et al., 1989) as well as A/P-type RNA-3, -4 and -5 from BNYVV (unpublished). A-type BNYVV full-length clones are also available, but RNA-1 cDNA clone still need to be modified. During the PhD program, we start production of BSBMV full-length cDNA clones and we investigate molecular interactions between plant and Benyviruses exploiting biological, epidemiological and molecular similarities/divergences between BSBMV and BNYVV. During my PhD researchrs we obtained full length infectious cDNA clones of BSBMV RNA-1 and -2 and we demonstrate that they transcripts are replicated and packaged in planta and able to substitute BNYVV RNA-1 or RNA-2 in a chimeric viral progeny (BSBMV RNA-1 + BNYVV RNA-2 or BNYVV RNA-1 + BSBMV RNA-2). During BSBMV full-length cDNA clones production, unexpected 1,730 nts long form of BSBMV RNA-4 has been detected from sugar beet roots grown on BSBMV infected soil. Sequence analysis of the new BSBMV RNA-4 form revealed high identity (~100%) with published version of BSBMV RNA-4 sequence (NC_003508) between nucleotides 1-608 and 1,138-1,730, however the new form shows 528 additionally nucleotides between positions 608-1,138 (FJ424610). Two putative ORFs has been identified, the first one (nucleotides 383 to 1,234), encode a protein with predicted mass of 32 kDa (p32) and the second one (nucleotides 885 to 1,244) express an expected product of 13 kDa (p13). As for BSBMV RNA-3 (Ratti et al., 2009), full-length BSBMV RNA-4 cDNA clone permitted to obtain infectious transcripts that BNYVV viral machinery (Stras12) is able to replicate and to encapsidate in planta. Moreover, we demonstrated that BSBMV RNA-4 can substitute BNYVV RNA-4 for an efficient transmission through the vector P. betae in Beta vulgaris plants, demonstrating a very high correlation between BNYVV and BSBMV. At the same time, using BNYVV helper strain, we studied BSBMV RNA-4’s protein expression in planta. We associated a local necrotic lesions phenotype to the p32 protein expression onto mechanically inoculated C. quinoa. Flag or GFP-tagged sequences of p32 and p13 have been expressed in viral context, using Rep3 replicons, based on BNYVV RNA-3. Western blot analyses of local lesions contents, using FLAG-specific antibody, revealed a high molecular weight protein, which suggest either a strong interaction of BSBMV RNA4’s protein with host protein(s) or post translational modifications. GFP-fusion sequences permitted the subcellular localization of BSBMV RNA4’s proteins. Moreover we demonstrated the absence of self-activation domains on p32 by yeast two hybrid system approaches. We also confirmed that p32 protein is essential for virus transmission by P. betae using BNYVV helper strain and BNYVV RNA-3 and we investigated its role by the use of different deleted forms of p32 protein. Serial mechanical inoculation of wild-type BSBMV on C. quinoa plants were performed every 7 days. Deleted form of BSBMV RNA-4 (1298 bp) appeared after 14 passages and its sequence analysis shows deletion of 433 nucleotides between positions 611 and 1044 of RNA-4 new form. We demonstrated that this deleted form can’t support transmission by P. betae using BNYVV helper strain and BNYVV RNA-3, moreover we confirmed our hypothesis that BSBMV RNA-4 described by Lee et al. (2001) is a deleted form. Interesting after 21 passages we identifed one chimeric form of BSBMV RNA-4 and BSBMV RNA-3 (1146 bp). Two putative ORFs has been identified on its sequence, the first one (nucleotides 383 to 562), encode a protein with predicted mass of 7 kDa (p7), corresponding to the N-terminal of p32 protein encoded by BSBMV RNA-4; the second one (nucleotides 562 to 789) express an expected product of 9 kDa (p9) corresponding to the C-terminal of p29 encoded by BSBMV RNA-3. Results obtained by our research in this topic opened new research lines that our laboratories will develop in a closely future. In particular BSBMV p32 and its mutated forms will be used to identify factors, as host or vector protein(s), involved in the virus transmission through P. betae. The new results could allow selection or production of sugar beet plants able to prevent virus transmission then able to reduce viral inoculum in the soil.

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Dengue fever is one of the most important mosquito-borne diseases worldwide and is caused by infection with dengue virus (DENV). The disease is endemic in tropical and sub-tropical regions and has increased remarkably in the last few decades. At present, there is no antiviral or approved vaccine against the virus. Treatment of dengue patients is usually supportive, through oral or intravenous rehydration, or by blood transfusion for more severe dengue cases. Infection of DENV in humans and mosquitoes involves a complex interplay between the virus and host factors. This results in regulation of numerous intracellular processes, such as signal transduction and gene transcription which leads to progression of disease. To understand the mechanisms underlying the disease, the study of virus and host factors is therefore essential and could lead to the identification of human proteins modulating an essential step in the virus life cycle. Knowledge of these human proteins could lead to the discovery of potential new drug targets and disease control strategies in the future. Recent advances of high throughput screening technologies have provided researchers with molecular tools to carry out investigations on a large scale. Several studies have focused on determination of the host factors during DENV infection in human and mosquito cells. For instance, a genome-wide RNA interference (RNAi) screen has identified host factors that potentially play an important role in both DENV and West Nile virus replication (Krishnan et al. 2008). In the present study, a high-throughput yeast two-hybrid screen has been utilised in order to identify human factors interacting with DENV non-structural proteins. From the screen, 94 potential human interactors were identified. These include proteins involved in immune signalling regulation, potassium voltage-gated channels, transcriptional regulators, protein transporters and endoplasmic reticulum-associated proteins. Validation of fifteen of these human interactions revealed twelve of them strongly interacted with DENV proteins. Two proteins of particular interest were selected for further investigations of functional biological systems at the molecular level. These proteins, including a nuclear-associated protein BANP and a voltage-gated potassium channel Kv1.3, both have been identified through interaction with the DENV NS2A. BANP is known to be involved in NF-kB immune signalling pathway, whereas, Kv1.3 is known to play an important role in regulating passive flow of potassium ions upon changes in the cell transmembrane potential. This study also initiated a construction of an Aedes aegypti cDNA library for use with DENV proteins in Y2H screen. However, several issues were encountered during the study which made the library unsuitable for protein interaction analysis. In parallel, innate immune signalling was also optimised for downstream analysis. Overall, the work presented in this thesis, in particular the Y2H screen provides a number of human factors potentially targeted by DENV during infection. Nonetheless, more work is required to be done in order to validate these proteins and determine their functional properties, as well as testing them with infectious DENV to establish a biological significance. In the long term, data from this study will be useful for investigating potential human factors for development of antiviral strategies against dengue.

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Since the discovery of Trypanosoma cruzi and the brilliant description of the then-referred to "new tripanosomiasis" by Carlos Chagas 100 years ago, a great deal of scientific effort and curiosity has been devoted to understanding how this parasite invades and colonises mammalian host cells. This is a key step in the survival of the parasite within the vertebrate host, and although much has been learned over this century, differences in strains or isolates used by different laboratories may have led to conclusions that are not as universal as originally interpreted. Molecular genotyping of the CL-Brener clone confirmed a genetic heterogeneity in the parasite that had been detected previously by other techniques, including zymodeme or schizodeme (kDNA) analysis. T. cruzi can be grouped into at least two major phylogenetic lineages: T. cruzi I, mostly associated with the sylvatic cycle and T. cruzi II, linked to human disease; however, a third lineage, T. cruziIII, has also been proposed. Hybrid isolates, such as the CL-Brener clone, which was chosen for sequencing the genome of the parasite (Elias et al. 2005, El Sayed et al. 2005a), have also been identified. The parasite must be able to invade cells in the mammalian host, and many studies have implicated the flagellated trypomastigotes as the main actor in this process. Several surface components of parasites and some of the host cell receptors with which they interact have been described. Herein, we have attempted to identify milestones in the history of understanding T. cruzi- host cell interactions. Different infective forms of T. cruzi have displayed unexpected requirements for the parasite to attach to the host cell, enter it, and translocate between the parasitophorous vacuole to its final cytoplasmic destination. It is noteworthy that some of the mechanisms originally proposed to be broad in function turned out not to be universal, and multiple interactions involving different repertoires of molecules seem to act in concert to give rise to a rather complex interplay of signalling cascades involving both parasite and cellular components.

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2. We documented the within-host distribution of two vector species that differ in transmission efficiency, the leafhoppers Draeculacephala minerva and Graphocephala atropunctata, and which are free to move throughout entirely caged alfalfa plants. The more efficient vector D. minerva fed preferentially at the base of the plant near the soil surface, whereas the less efficient G. atropunctata preferred overwhelming the top of the plant. 3. Next we documented X. fastidiosa heterogeneity in mechanically inoculated plants. Infection rates were up to 50% higher and mean bacterial population densities were 100-fold higher near the plant base than at the top or in the taproot. 4. Finally, we estimated transmission efficiency of the two leafhoppers when they were confined at either the base or top of inoculated alfalfa plants. Both vectors were inefficient when confined at the top of infected plants and were 20-60% more efficient when confined at the plant base. 5. These results show that vector transmission efficiency is determined by the interaction between leafhopper within-plant feeding behaviour and pathogen within-plant distribution. Fine-scale vector and pathogen overlap is likely to be a requirement generally for efficient transmission of vector-borne pathogens.

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We report the complete molecular characterization of the DNA-A and DNA-B of a Brazilian tomato isolate of Tomato severe rugose virus (ToSRV) and the experimental host range of the virus determined using white-fly transmission tests. Genome analysis showed that ToSRV has a close evolutionary relationship with Tomato rugose mosaic virus. Of 33 plants species inoculated with viruliferous Bemisia tabaci biotype B, 13 species were susceptible to ToSRV, nine asymptomatically. Therefore, ToSRV disease management strategy should include the control of infected weeds close to tomato fields.

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Feline immunodeficiency virus (FIV) causes a slow progressive degeneration of the immune system which eventually leads to a disease comparable to acquired immune deficiency syndrome (AIDS) in humans. FIV has extensive sequence variation, a typical feature of lentiviruses. Sequence analysis showed that diversity was not evenly distributed throughout the genome, but was greatest in the envelope gene, env. The virus enters host cells via a sequential interaction, initiated by the envelope glycoprotein (env) binding the primary receptor molecule CD134 and followed by a subsequent interaction with chemokine co-receptor CXCR4. The purpose of this study was to isolate and characterize isolates of FIV from an open shelter in Sao Paulo, Brazil. The separated PBMC from 11 positive cats were co-cultured with MYA-1 cells. Full-length viral env glycoprotein genes were amplified and determined. Chimeric feline x human CD134 receptors were used to investigate the receptor utilization of 17 clones from Brazilian isolates of Fly. Analyses of the sequence present of molecular clones showed that all clones grouped within subtype B. In contrast to the virulent primary isolate FIV-GL8, expression of the first cysteine-rich domain (CRD1) of feline CD134 in the context of human CD134 was sufficient for optimal receptor function for all Brazilian FIV isolates tested. (C) 2011 Elsevier B.V. All rights reserved.

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Infectious bronchitis (IB) is a highly aggressive disease for poultry in terms of symptoms and economic losses, and the control of this disease is difficult if flocks are not protected against type-specific challenges by the Avian infectious bronchitis virus (IBV). This article summarizes data presented by the author at the Workshop on Infectious Bronchitis 2009 on IB and IBV, including future developments on the field.

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During investigation of an outbreak of Japanese encephalitis (JE) in the Torres Strait, Australia, in 2000, mosquitoes were collected in Badu Island community and at a newly established communal piggery about 3 km from the community. A total of 94285 mosquitoes, comprising 91240 (96.8%) unengorged females, 1630 (1.7%) blood-engorged females and 1415 (1.5%) males, were processed for virus isolation. One isolate of JE virus was obtained from Culex gelidus, with a minimum infection rate of 12.4:1000. This is the first isolate of JE virus from Cx. gelidus in the Australasian region. No isolates were obtained from Cx. annulirostris, the primary implicated Australian JE vector. Analysis of mosquito host-feeding patterns, using gel diffusion, demonstrated that Cx. annulirostris and 5 other species fed predominately on mammals, Analysis of blood-fed mosquitoes collected within the community demonstrated that the proportion of Cx. annulirostris feeding on pigs in 2000 (2.3%) was significantly lower than that for the 1995-97 period (31.3%). The removal of the pigs from Badu Island community has limited the contact between potential amplifying hosts and mosquitoes, thus potentially reducing the risk of transmission of JE virus to the human population.

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The initial step in viral infection is the attachment of the virus to the host cell via an interaction with its receptor. We have previously shown that a receptor for human papillomavirus is the alpha6 integrin. The alpha6 integrin is involved in the attachment of epithelial cells with the basement membrane, but recent evidence suggests that ligation of many integrins results in intracellular signaling events that influence cell proliferation. sere we present evidence that exposure of A431 human epithelial cells to human papillomavirus type 6b L1 virus-like particles (VLPs) results in a dose-dependent increase in cell proliferation, as measured by bromodeoxyuridine incorporation. This proliferation is Lost if VLPs are first denatured or incubated with a monoclonal antibody against L1 protein. The MEK1 inhibitor PB98059 inhibits the VLP-mediated increase in fell proliferation, suggesting involvement of the Ras-MAP kinase pathway, Indeed, VLP binding results in rapid phosphorylation of the beta4 integrin upon tyrosine residues and subsequent recruitment of the adapter protein She to beta4, Within 30 min, the activation of Ras, Raf, and Erk2 was observed. Finally, the upregulation of c-myc mRNA was observed at 60 min, These data indicate that human papillomavirus type 6b is able to signal cells via the Ras-MAP kinase pathway to induce cell proliferation. We hypothesize that such a mechanism would allow papillomaviruses to infect hosts more successfully by increasing the potential pool of cells they are able to infect via the initiation of proliferation in resting keratinocyte stem and suprabasal cells.

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Japanese encephalitis (JE) virus spread to northern Australia during the 1990s, transmitted by Culex annulirostris Skuse and other mosquitoes (Diptera: Culicidae). To determine the relative importance of various hosts for potential vectors of JE virus, we investigated the host-feeding patterns of mosquitoes in northern Australia and Western Province of Papua New Guinea, with particular attention to pigs, Sus scrofa L. - the main amplifying host of JE virus in South-east Asia. Mosquitoes were collected by CDC light traps baited with dry ice and 1-octen-3-ol, run 16.00-08.00 hours, mostly set away from human habitations, if possible in places frequented by feral pigs. Bloodmeals of 2569 mosquitoes, representing 15 species, were identified by gel diffusion assay. All species had fed mostly on mammals: only 30%) were trapped where domestic pigs were kept close to human habitation. From seven of eight locations on the Australian mainland, the majority of Cx. annulirostris had obtained their bloodmeals from marsupials, probably the Agile wallaby Macropus agilis (Gould). Overall proportions of mosquito bloodmeals identified as marsupial were 60% from the Gulf Plains region of Australia, 78% from the Cape York Peninsula and 64% from the Daru area of Papua New Guinea. Thus, despite the abundance of feral pigs in northern Australia, our findings suggest that marsupials divert host-seeking Cx. annulirostris away from pigs. As marsupials are poor JE virus hosts, the prevalence of marsupials may impede the establishment of JE virus in Australia.

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Guimarães, in the northwest of Portugal, is a city of strong symbolic and cultural significance and its nomination by UNESCO as world heritage, in 2001, enlarged its tourism potential. In this paper we present a few results of a survey that envisaged capturing the Guimarães residents’ perceptions of tourism impacts and their attitudes towards tourists. Specifically, one analyzes the type of relationship that exists between some socio-demographic groups and the perceived tourism impacts, as well as their socio-characteristics and the existing level of interaction between residents and tourists. The survey was implemented between January and March 2010 to a convenience sample of 540 inhabitants of the municipality of Guimarães resulting in 400 questionnaires with complete data. For this, we made use of various statistical techniques. Using a factorial analysis, we can conclude that the three factors used explain 52.3% of the variance contained in the original variables obtained from the survey. By another side, using a logit model in the analysis and taking as the dependent variable the frequent or very frequent contact with tourists, we found that only the variables referred to perceived positive impacts of tourism, education and the place of residence in urban areas have shown to be statistically significant. We are aware of the multiple ways the issue of residents’ perceptions and attitudes towards tourism can be approached and of the difficulties to get useful policy-oriented insights. This paper is a step in that trail.

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In the face of a growing human population and increased urbanization, the demand for pesticides will simply rise. Farmers must escalate yields on increasingly fewer farm acres. However, the risks of pesticides, whether real or perceived, may force changes in the way these chemicals are used. Scientists are working toward pest control plans that are environmentally sound, effective, and profitable. In this context the development of new pesticide formulations which may improve application effectiveness, safety, handling, and storage can be pointed out as a solution. As a contribution to the area, the microencapsulation of the herbicide oxadiargyl (OXA) in (2-hydroxypropyl)-β-cyclodextrin (HP-β-CD) was performed. The study was conducted in different aqueous media (ultrapure water and in different pH buffer solutions). In all cases an increment of the oxadiargyl solubility as a function of the HP-β-CD concentration that has been related to the formation of an inclusion complex was verified. UV-Vis and NMR experiments allowed concluding that the stoichiometry of the OXA/HP-β-CD complex formed is 1 : 1. The gathered results can be regarded as an important step for its removal from industrial effluents and/or to increase the stabilizing action, encapsulation, and adsorption in water treatment plants.