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Transmission electron microscopy (TEM) has been essential to study virus-cell interactions. The architecture of viral replication factories, the principles of virus assembly and the components of virus egress pathways are known thanks to the contribution of TEM methods. Specially, when studying viruses in cells, methodologies for labeling proteins and other macromolecules are important tools to correlate morphology with function. In this review, we present the most widely used labeling method for TEM, immunogold, together with a lesser known technique, metal-tagging transmission electron microscopy (METTEM) and how they can contribute to study viral infections. Immunogold uses the power of antibodies and electron dense, colloidal gold particles while METTEM uses metallothionein (MT), a metal-binding protein as a clonable tag. MT molecules build gold nano-clusters inside cells when these are incubated with gold salts. We describe the necessary controls to confirm that signals are specific, the advantages and limitations of both methods, and show some examples of immunogold and METTEM of cells infected with viruses.
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Vírus , Microscopia Eletrônica de Transmissão , Proteínas , Replicação Viral , Montagem de VírusRESUMO
Replication and assembly of many viruses occur in specific intracellular compartments known as 'virus factories'. Our knowledge of the biogenesis and architecture of these unique structures has increased considerably in the last 10 years, due to technical advances in cellular, molecular and structural biology. We now know that viruses build replication organelles, which recruit cell and viral components in a macrostructure in which viruses assemble and mature. Cell membranes and cytoskeleton participate in the biogenesis of these scaffolds and mitochondria are present in many factories, where they might supply energy and other essential factors. New inter-organelle contacts have been visualized within virus factories, whose structure is very dynamic, as it changes over time. There is increasing interest in identifying the factors involved in their biogenesis and functional architecture, and new microscopy techniques are helping us to understand how these complex entities are built and work. In this review, we summarize recent findings on the cell biology, biogenesis and structure of virus factories.
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Eucariotos/virologia , Interações Hospedeiro-Parasita , Montagem de Vírus , Fenômenos Fisiológicos Virais , Replicação Viral , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Mitocôndrias/metabolismoRESUMO
Transmission electron microscopy significantly contributed to unveil the course of virus entry, replication, morphogenesis, and egress. For these studies, the most widely used approach is imaging ultrathin sections of virus-infected cells embedded in a plastic resin that is transparent to electrons. Before infiltration in a resin, cells must be processed to stabilize their components under the observation conditions in an electron microscope, such as high vacuum and irradiation with electrons. For conventional sample preparation, chemical fixation and dehydration are followed by infiltration in the resin and polymerization to produce a hard block that can be sectioned with an ultramicrotome. Another method that provides a superior preservation of cell components is high-pressure freezing (HPF) followed by freeze substitution (FS) before resin infiltration and polymerization. This chapter describes both procedures with cells infected with Bunyamwera virus (BUNV), a well characterized member of the Bunyavirales, and compares the morphological details of different viral structures imaged in the two types of samples. Advantages, disadvantages, and applications of conventional processing and HPF/FS are also presented and discussed.
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Substituição ao Congelamento , Microscopia Eletrônica de Transmissão , Substituição ao Congelamento/métodos , Microscopia Eletrônica de Transmissão/métodos , Orthobunyavirus , Animais , Congelamento , Humanos , Manejo de Espécimes/métodos , Linhagem CelularRESUMO
OBJECTIVES: Hepatitis C virus (HCV) antiviral therapy might lead to decreased chronic immune activation and endothelial dysfunction associated with cardiovascular risk. The aim was to evaluate the effect of HCV eradication on serum markers of inflammation and endothelial dysfunction markers in HIV/HCV co-infected patients. METHODS: We carried out a retrospective study of 69 HIV/HCV co-infected patients on interferon (IFN)-α plus ribavirin. In addition, 47 HIV-infected subjects were selected as a control group. A sustained virological response (SVR) was defined as an undetectable HCV viral load up to 24 weeks after the end of treatment. Tumour necrosis factor (TNF) receptor-1 (TNF-R1), soluble E-selectin (sE-selectin), soluble P-selectin (sP-selectin), soluble intercellular adhesion molecule 1 (sICAM-1) and soluble vascular cell adhesion molecule 1 (sVCAM-1) were measured using a multiplex immunoassay kit. RESULTS: HIV/HCV co-infected patients had higher values of soluble TNF-R1 (sTNF-R1), sE-selectin and sICAM-1 than HIV mono-infected patients (Pâ<â0.05). SVR patients had a decrease in sTNF-R1, sP-selectin, sE-selectin and sICAM-1 during anti-HCV treatment (Pâ<â0.05) and, at the end of treatment, SVR patients had lower values of sTNF-R1, sE-selectin and sVCAM-1 than non-responder patients (Pâ<â0.05), although the values of sTNF-R1, sP-selectin, sE-selectin and sICAM-1 remained higher than in HIV mono-infected patients (Pâ<â0.05). Moreover, we found a significant positive relationship between an increase in sTNF-R1 and increases in sP-selectin, sE-selectin and sICAM-1 during anti-HCV therapy. CONCLUSIONS: Chronic hepatitis C infection induces alterations of markers of inflammation and endothelial dysfunction. Eradication of HCV, following IFN-α and ribavirin therapy, reduces immune activation as well as markers of inflammation and endothelial dysfunction.
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Antivirais/administração & dosagem , Infecções por HIV/complicações , Infecções por HIV/patologia , Hepatite C/complicações , Hepatite C/patologia , Interferon-alfa/administração & dosagem , Ribavirina/administração & dosagem , Adulto , Biomarcadores/sangue , Selectina E/sangue , Células Endoteliais/fisiologia , Feminino , Infecções por HIV/tratamento farmacológico , Hepatite C/tratamento farmacológico , Humanos , Molécula 1 de Adesão Intercelular/sangue , Masculino , Pessoa de Meia-Idade , Selectina-P/sangue , Receptores Tipo I de Fatores de Necrose Tumoral/sangue , Estudos Retrospectivos , Resultado do Tratamento , Carga ViralRESUMO
Mammalian orthoreoviruses (reoviruses) are nonenveloped, double-stranded RNA viruses that replicate and assemble in cytoplasmic membranous organelles called viral inclusions (VIs). To define the cellular compartments involved in nonlytic reovirus egress, we imaged viral egress in infected, nonpolarized human brain microvascular endothelial cells (HBMECs). Electron and confocal microscopy showed that reovirus mature virions are recruited from VIs to modified lysosomes termed sorting organelles (SOs). Later in infection, membranous carriers (MCs) emerge from SOs and transport new virions to the plasma membrane for nonlytic egress. Transmission electron microscopy (TEM) combined with electron tomography (ET) and three-dimensional (3D) reconstruction revealed that these compartments are connected and form the exit pathway. Connections are established by channels through which mature virions are transported from VIs to MCs. In the last step, MCs travel across the cytoplasm and fuse with the plasma membrane, which facilitates reovirus egress. This bio-protocol describes the combination of imaging approaches (TEM, ET, and 3D reconstruction) to analyze reovirus egress zones. The spatial information present in the 3D reconstructions, along with the higher resolution relative to 2D projections, allowed us to identify components of a new nonlytic viral egress pathway.
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The great majority of infections caused by the pandemic variant of the influenza virus (nvH1N1) are self-limited, but a small percentage of patients develop severe symptoms requiring hospitalization. Bermejo-Martin and colleagues have presented a pilot study describing the differences in the early immune response for patients both mildly and severely infected with nvH1N1. Patients who develop severe symptoms after nvH1N1 infection showed Th1 and Th17 'hypercytokinemia', compared to mildly infected patients and healthy controls. The mediators involved with the Th1 and Th17 profiles are known to be involved in antiviral, pro-inflammatory and autoimmune responses. This is the first work reporting the association of a pro-inflamatory immune response with a severe pandemic infection, although it is likely that more studies are needed to understand the detrimental or beneficial roles these cytokines play in the evolution of mild and severe nvH1N1 infection.
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Inflamação/patologia , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Influenza Humana/patologia , Estudos de Casos e Controles , Citocinas/sangue , Humanos , Inflamação/sangue , Inflamação/virologia , Influenza Humana/sangue , Influenza Humana/virologiaRESUMO
Positive-stranded (+) RNA viruses are intracellular pathogens in humans, animals and plants. To build viral replicase complexes (VRCs) viruses manipulate lipid flows and reorganize subcellular membranes. Redesigned membranes concentrate viral and host factors and create an environment that facilitates the formation of VRCs within replication organelles. Therefore, efficient virus replication depends on the assembly of specialized membranes where viral macromolecular complexes are turned on and hold a variety of functions. Detailed characterization of viral replication platforms in cells requires sophisticated imaging approaches. Here we present a protocol to visualize the three-dimensional organization of the tombusvirus replicase complex in yeast with MEtal-Tagging Transmission Electron Microscopy (METTEM). This protocol allowed us to image the intracellular distribution of the viral replicase molecules in three-dimensions with METTEM and electron tomography. Our study showed how viral replicase molecules build replication complexes within specialized cell membranes.
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Transmission electron microscopy (TEM) has been instrumental for studying viral infections. In particular, methods for labeling macromolecules at the ultrastructural level, by integrating biochemistry, molecular biology, and morphology, have allowed to study the functions of viral macromolecular complexes within the cellular context. Here, we describe a strategy for imaging influenza virus ribonucleoproteins in infected cells with two complementary labeling methods, metal-tagging transmission electron microscopy or METTEM, a highly sensitive technique based on the use of a metal-binding protein as a clonable tag, and immunogold labeling on thawed cryosections, a very specific labeling method that allows to study the distribution of different proteins simultaneously. The combination of both labeling methods offers new possibilities for TEM analysis of viral components in cells.
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Imuno-Histoquímica , Vírus da Influenza A/fisiologia , Vírus da Influenza A/ultraestrutura , Influenza Humana/virologia , Metais , Microscopia Eletrônica de Transmissão , Ribonucleoproteínas/metabolismo , Proteínas Virais/metabolismo , Células Cultivadas , Crioultramicrotomia , Genoma Viral , Humanos , Microscopia Eletrônica de Transmissão/métodos , Transporte de RNA , RNA Viral , Coloração e Rotulagem , Montagem de VírusRESUMO
Three-dimensional (3D) imaging technologies are beginning to have significant impact in the field of virology, as they are helping us understand how viruses take control of cells. In this article we review several methodologies for 3D imaging of cells and show how these technologies are contributing to the study of viral infections and the characterization of specialized structures formed in virus-infected cells. We include 3D reconstruction by transmission electron microscopy (TEM) using serial sections, electron tomography, and focused ion beam scanning electron microscopy (FIB-SEM). We summarize from these methods selected contributions to our understanding of viral entry, replication, morphogenesis, egress and propagation, and changes in the spatial architecture of virus-infected cells. In combination with live-cell imaging, correlative microscopy, and new techniques for molecular mapping in situ, the availability of these methods for 3D imaging is expected to provide deeper insights into understanding the structural and dynamic aspects of viral infection.
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OBJECTIVE: To quantify serum levels of intercellular adhesion molecule-1 (sICAM-1) and vascular cell adhesion molecule-1 (sVCAM-1) in HIV/HCV coinfected patients to examine their association with several clinical and epidemiological characteristics and the therapeutic responsiveness to interferon (IFN)-alpha and ribavirin therapy (IFN-alpha + RBV). DESIGN: Retrospective study. METHODS: We carried out a cross-sectional study with 183 IFN-alpha-naive patients on HAART, and 24 healthy controls. We also analyzed 30 out of 183 patients on IFN-alpha + RBV for the duration of 48 weeks. RESULTS: HIV/HCV coinfected patients had higher levels of sICAM-1 and sVCAM-1 than the healthy control group (P < 0.05). Patients with HCV-genotype 1, advanced fibrosis (F>or=3) or moderate to severe activity grade (A>or=2) had the highest values of sICAM-1 and sVCAM-1. When we carried out a multivariate analysis, we found a significant positive relationship between both HCV-genotype 1 and advanced fibrosis (F>or=3) with sICAM-1 (R = 0.549; P < 0.001); and a significant positive relationship between HCV-genotype 1 and advanced fibrosis (F>or=3) with sVCAM-1 (R = 0.624; P < 0.001). We also found a positive relationship of sICAM-1 or sVCAM-1 levels with transaminases and alkaline phosphatase circulation levels (P < 0.05). Nonresponder patients had higher sICAM-1 and sVCAM-1 serum levels, and patients with sustained virologic response had significantly lower levels of sICAM-1 (P = 0.001) and sVCAM-1 (P = 0.019). CONCLUSION: HIV and HCV coinfection induces alterations in sICAM-1 and sVCAM-1 serum levels, which were higher in patients with HCV-genotype 1 and advanced stage of HCV infection. However, response to IFN-alpha + RBV may reduce these cardiovascular risk markers.