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Human amoebiasis still represents a major health problem worldwide. Metronidazole has been used as the most common drug to treat the disease; however, it is also known that the drug causes undesirable side effects. This has led to the search for new pharmacological alternatives which include phytochemical compounds with antiamoebic effects. We analyzed the amoebicidal activity of stevioside (STV), a diterpene glycoside present in Stevia rebaudiana, on trophozoites of E. histolytica. Different concentrations of STV were tested, and an inhibitory concentration of 50% of cell viability (IC50) was determined with an exposition of 9.53 mM for 24 h. Trophozoites exposed to STV showed morphological changes evidenced by the decrease in the basic structures related to the movement and adherence to the substrate, as well as ultrastructural features characterized by a loss of regularity on the cell membrane, an increase in cytoplasmic granularity, and an increase in apparent autophagic vacuoles. Also, the decrease in cysteine protease expression and the proteolytic activity of trophozoites to degrade the cell monolayer were analyzed. A histological analysis of hamster livers inoculated with trophozoites and treated with STV showed changes related to the granulomatous reaction of the liver parenchymal tissue. Our results constitute the first report related to the possible use of STV as a therapeutic alternative in amoebiasis.

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BACKGROUND Giant viruses have brought new insights into different aspects of virus-cell interactions. The resulting cytopathic effects from these interactions are one of the main aspects of infection assessment in a laboratory routine, mainly reflecting on the morphological features of an infected cell. OBJECTIVES In this work, we follow the entire kinetics of the cytopathic effect in cells infected by viruses of the Mimiviridae family, spatiotemporally quantifying typical features such as cell roundness, loss of motility, decrease in cell area and cell lysis. METHODS Infections by Acanthamoeba polyphaga mimivirus (APMV), Tupanvirus (TPV) and M4 were carried out at multiplicity of infection (MOI) 1 and MOI 10 in Acanthamoeba castellanii. Monitoring of infections was carried out using time lapse microscopy for up to 72 hours. The images were analyzed using ImageJ software. FINDINGS The data obtained indicate that APMV is the slowest virus in inducing the cytopathic effects of rounding, decrease in cell area, mobility and cell lysis. However, it is the only virus whose MOI increase accelerates the lysis process of infected cells. In turn, TPV and M4 rapidly induce morphological and behavioral changes. MAIN CONCLUSIONS Our results indicate that mimiviruses induce different temporal responses within the host cell and that it is possible to use these kinetic data to facilitate the understanding of infection by these viruses.

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The cytopathic effect comprises the set of cellular alterations produced by a viral infection. It is of great relevance since it constitutes a direct marker of infection. Likewise, these alterations are often virus-specific which makes them a phenotypic marker for many viral species. All these characteristics have been used to complement the study of the dynamics of virus-cell interactions through the kinetic study of the progression of damage produced by the infection. Various approaches have been used to monitor the cytopathic effect, ranging from light microscopy, immunofluorescence assays, and direct labeling with fluorescent dyes, to plaque assay for the characterization of the infection over time. Here we address the relevance of the study of cytopathic effect and describe different experimental alternatives for its application.

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BACKGROUND: Dengue virus (DENV) is a Flaviviridae member classified into four antigenically distinct serotypes (DENV 1, 2, 3, and 4) and further subdivided genotypes. DENV3 is subdivided into four or five genotypes, depending on the classification adopted. Despite their high genetic proximity, as revealed by phylogenetic complete polyprotein analysis, DENV3 MG-20 and DENV3 PV_BR showed different neurovirulence in mice models. Our group identified six amino acid mutations in protein E, including the E62K and E123Q, which may affect interactions of hydrophobic clusters on domain II, thus leading to the observed differences in the studied viruses. METHODS: Human glioblastoma cells (U251) derived from a malignant glioblastoma tumor by explant technique were infected by the DENV3 GIL1 isolates DENV3 MG-20 and DENV3 PV_BR and analyzed by plaque assays and titration, optical, immunofluorescence, and transmission electronic microscopy. RESULTS: The two isolates showed different cytopathic effects (CPE) and fusogenic patterns, further confirmed by indirect immunofluorescence. Transmission electron microscopy revealed intense cytopathic effects in DENV3 MG-20 infected U251 cells, displaying endoplasmic reticulum hypertrophy and turgid vesicles with proteins and multiple viruses, distinct from DENV3 PV_BR infected cells. It is hypothesized that the different amino acids in the DENV3 MG-20 isolate are related to an increased membrane fusion ability in viral infection, thus facilitating immune system evasion and increased chances of central nervous system cell infection. CONCLUSION: These results emphasize the biological differences between the isolates, which could be a critical factor in host-virus interaction and severe dengue development. Our study presents comparative results of highly similar isolates with the potential to generate more subsidies for a deeper understanding of the DENV pathogenesis. The neurotropism of the isolate DENV3 MG-20 (belonging to the DENV3 GI L1 genotype) showing infection of nervous system cells (U251) could contribute to understanding neurological dengue disease.

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Acanthamoeba castellanii genotype T4 is a clinically significant free-living amoeba that causes granulomatous amoebic encephalitis and amoebic keratitis in human beings. During the initial stages of infection, trophozoites interact with various host immune responses, such as lactoferrin (Lf), in the corneal epithelium, nasal mucosa, and blood. Lf plays an important role in the elimination of pathogenic microorganisms, and evasion of the innate immune response is crucial in the colonization process. In this study, we describe the resistance of A. castellanii to the microbicidal effect of bovine apo-lactoferrin (apo-bLf) at different concentrations (25, 50, 100, and 500 µM). Acanthamoeba castellanii trophozoites incubated with apo-bLf at 500 µM for 12 h maintained 98% viability. Interestingly, despite this lack of effect on viability, our results showed that the apo-bLf inhibited the cytopathic effect of A. castellanii in MDCK cells culture, and analysis of amoebic proteases by zymography showed significant inhibition of cysteine and serine proteases by interaction with the apo-bLf. From these results, we conclude that bovine apo-Lf influences the activity of A. castellanii secretion proteases, which in turn decreases amoebic cytopathic activity.

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This article describes a practical method for prospecting and isolating giant viruses based on direct inoculation of environmental samples into amoeba cultures of Acanthamoeba castellanii. The giant viruses that infect amoebas have already been isolated from various environmental samples in several countries worldwide, including in extreme environments. Here we describe the methodologic procedures regarding the prospecting of giant viruses in A. castellanii, including the preparation of environmental samples, the culture of amoebas, and the observation of cytopathic effects that can indicate the presence and potential isolation of giant viruses. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Sample collection Support Protocol: Propagation of Acanthamoeba castellanii Basic Protocol 2: Prospecting of giant viruses in environmental samples by cytopathic effect analysis.

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Human Cytomegalovirus (HCMV) is a frequent opportunistic pathogen in immunosuppressed patients, which can be involved in kidney allograft dysfunction and rejection. In order to study the pathophysiology of HCMV renal diseases, we concentrated on the impact of HCMV infection on human renal tubular epithelial HK-2 cells. Our aim was to develop a model of infection of HK-2 cells by using the viral strain TB40/E, that contains the extended cell tropism of clinical isolates and the efficient viral multiplication in cell culture of laboratory-adapted strains. We observed that HK-2 cells can be infected by HCMV and expressed viral antigens, but they do not produce extracellular viral particles. We then studied the interplay of HCMV with ciliogenesis and autophagy. Primary cilium (PC) is a stress sensor important to maintain renal tissue homeostasis that projects from the apical side into the lumen of tubule cells. PC formation and length were not modified by HCMV infection. Autophagy, another stress response process critically required for normal kidney functions, was inhibited by HCMV in HK-2 cells with a reduction in the autophagic flux. HCMV classically induces an enlargement of infected cells in vivo and in vitro, and we observed that HCMV infection led to an enlargement of the HK-2 cell volume. Our results constitute therefore an excellent starting point to further explore the role of these mechanisms in renal cells dysfunction.

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Acanthamoeba is a genus of free-living amoebae widely distributed in nature, associated with the development of encephalitis and keratitis. Despite the fact that it is common to find genotype T5 in environmental samples, only a few cases have been associated with clinical cases in humans. The wide distribution of Acanthamoeba, the characteristic of being amphizoic and the severity of the disease motivate researchers to focus on the isolation of these organisms, but also in demonstrating direct and indirect factors that could indicate a possible pathogenic potential. Here, we performed the characterization of the pathogenic potential of an Acanthamoeba T5 isolate collected from a water source in a hospital. Osmo- and thermotolerance, the secretion of proteases and the effect of trophozoites over cell monolayers were analyzed by different methodologies. Additionally, we confirm the secretion of extracellular vesicles (EVs) of this isolate incubated at two different temperatures, and the presence of serine and cysteine proteases in these vesicles. Finally, using atomic force microscopy, we determined some nanomechanical properties of the secreted vesicles and found a higher value of adhesion in the EVs obtained at 37 °C, which could have implications in the parasite´s survival and damaging potential in two different biological environments.

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Acanthamoeba spp. are free-living amoebae with a worldwide distribution. These amoebae can cause granulomatous amoebic encephalitis and amoebic keratitis in humans. Proteases are considered virulence factors in pathogenic Acanthamoeba. The objective of this study was to evaluate the behavior of Acanthamoeba mauritaniensis, a nonpathogenic amoeba. We analyzed the cytopathic effect of A. mauritaniensis on RCE1(5 T5) and MDCK cells and compared it to that of Acanthamoeba castellanii. A partial biochemical characterization of proteases was performed in total crude extracts (TCE) and conditioned medium (CM). Finally, we evaluated the effect of proteases on tight junction (TJ) proteins and the transepithelial electrical resistance of MDCK cells. The results showed that this amoeba can induce substantial damage to RCE1(5T5) and MDCK cells. Moreover, the zymograms and Azocoll assays of amoebic TCE and CM revealed different protease activities, with serine proteases being the most active. Furthermore, A. mauritaniensis induced the alteration and degradation of MDCK cell TJ proteins with serine proteases. After genotyping this amoeba, we determined that it is an isolate of Acanthamoeba genotype T4D. From these data, we suggest that A. mauritaniensis genotype T4D behaves similarly to the A. castellanii strain.

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Plaque assay has been used for a long time to determine infectious titers and characterize prokaryotic and eukaryotic viruses forming plaques. Indeed, plaque morphology and dimensions can provide information regarding the replication kinetics and the virulence of a particular virus. In this work, we present ViralPlaque, a fast, open-source and versatile ImageJ macro for the automated determination of viral plaque dimensions from digital images. Also, a machine learning plugin is integrated in the analysis algorithm for adaptation of ViralPlaque to the user's needs and experimental conditions. A high correlation between manual and automated measurements of plaque dimensions was demonstrated. This macro will facilitate reliable and reproducible characterization of cytolytic viruses with an increased processing speed.