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      Structural investigation of C6/36 and Vero cell cultures infected with a Brazilian Zika virus

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          Abstract

          Zika virus (ZIKV) is a member of the flavivirus genus, and its genome is approximately 10.8 kilobases of positive-strand RNA enclosed in a capsid and surrounded by a membrane. Studies on the replication dynamics of ZIKV are scarce, which limits the development of antiviral agents and vaccines directed against ZIKV. In this study, Aedes albopictus mosquito lineage cells (C6/36 cells) and African green monkey kidney epithelial cells (Vero cells) were inoculated with a ZIKV sample isolated from a Brazilian patient, and the infection was characterized by immunofluorescence staining, phase contrast light microscopy, transmission electron microscopy and real-time RT-PCR. The infection was observed in both cell lineages, and ZIKV particles were observed inside lysosomes, the rough endoplasmic reticulum and viroplasm-like structures. The susceptibility of C6/36 and Vero cells to ZIKV infection was demonstrated. Moreover, this study showed that part of the replicative cycle may occur within viroplasm-like structures, which has not been previously demonstrated in other flaviviruses.

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          Most cited references 34

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          Composition and Three-Dimensional Architecture of the Dengue Virus Replication and Assembly Sites

          Summary Positive-strand RNA viruses are known to rearrange cellular membranes to facilitate viral genome replication. The biogenesis and three-dimensional organization of these membranes and the link between replication and virus assembly sites is not fully clear. Using electron microscopy, we find Dengue virus (DENV)-induced vesicles, convoluted membranes, and virus particles to be endoplasmic reticulum (ER)-derived, and we detect double-stranded RNA, a presumed marker of RNA replication, inside virus-induced vesicles. Electron tomography (ET) shows DENV-induced membrane structures to be part of one ER-derived network. Furthermore, ET reveals vesicle pores that could enable release of newly synthesized viral RNA and reveals budding of DENV particles on ER membranes directly apposed to vesicle pores. Thus, DENV modifies ER membrane structure to promote replication and efficient encapsidation of the genome into progeny virus. This architecture of DENV replication and assembly sites could explain the coordination of distinct steps of the flavivirus replication cycle.
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            Virus factories: associations of cell organelles for viral replication and morphogenesis

            Abstract Genome replication and assembly of viruses often takes place in specific intracellular compartments where viral components concentrate, thereby increasing the efficiency of the processes. For a number of viruses the formation of ‘factories’ has been described, which consist of perinuclear or cytoplasmic foci that mostly exclude host proteins and organelles but recruit specific cell organelles, building a unique structure. The formation of the viral factory involves a number of complex interactions and signalling events between viral and cell factors. Mitochondria, cytoplasmic membranes and cytoskeletal components frequently participate in the formation of viral factories, supplying basic and common needs for key steps in the viral replication cycle.
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              Autophagic machinery activated by dengue virus enhances virus replication

              Autophagy is a cellular response against stresses which include the infection of viruses and bacteria. We unravel that Dengue virus-2 (DV2) can trigger autophagic process in various infected cell lines demonstrated by GFP-LC3 dot formation and increased LC3-II formation. Autophagosome formation was also observed under the transmission electron microscope. DV2-induced autophagy further enhances the titers of extracellular and intracellular viruses indicating that autophagy can promote viral replication in the infected cells. Moreover, our data show that ATG5 protein is required to execute DV2-induced autophagy. All together, we are the first to demonstrate that DV can activate autophagic machinery that is favorable for viral replication.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: Project administrationRole: ResourcesRole: Writing – original draftRole: Writing – review & editing
                Role: Methodology
                Role: Methodology
                Role: Methodology
                Role: Methodology
                Role: Methodology
                Role: Methodology
                Role: Methodology
                Role: Methodology
                Role: Methodology
                Role: Methodology
                Role: Resources
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                12 September 2017
                2017
                : 12
                : 9
                Affiliations
                [1 ] Laboratory of Morphology and Viral Morphogenesis, Instituto Oswaldo Cruz, Fiocruz, Avenida Brasil, Rio de Janeiro, RJ, Brazil
                [2 ] Laboratory of Flavivirus, Instituto Oswaldo Cruz, Fiocruz, Avenida Brasil, Rio de Janeiro, RJ, Brazil
                [3 ] Laboratory of Pathology, Instituto Oswaldo Cruz, Fiocruz, Avenida Brasil, Rio de Janeiro, RJ, Brazil
                [4 ] Laboratory of Virological Technology, Bio-Manguinhos, Avenida Brasil, Rio de Janeiro, RJ, Brazil
                University of Minnesota College of Veterinary Medicine, UNITED STATES
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Article
                PONE-D-17-15841
                10.1371/journal.pone.0184397
                5595330
                28898286
                © 2017 Barreto-Vieira et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                Page count
                Figures: 10, Tables: 1, Pages: 18
                Product
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100004586, Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro;
                Award Recipient :
                This study was funded by Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Faperj, and Ana Maria Bispo de Filippis is supported by the European Union's Horizon 2020 program under grant agreement No. 734857. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and life sciences
                Organisms
                Viruses
                RNA viruses
                Flaviviruses
                Zika Virus
                Biology and Life Sciences
                Microbiology
                Medical Microbiology
                Microbial Pathogens
                Viral Pathogens
                Flaviviruses
                Zika Virus
                Medicine and Health Sciences
                Pathology and Laboratory Medicine
                Pathogens
                Microbial Pathogens
                Viral Pathogens
                Flaviviruses
                Zika Virus
                Biology and Life Sciences
                Organisms
                Viruses
                Viral Pathogens
                Flaviviruses
                Zika Virus
                Research and Analysis Methods
                Biological Cultures
                Cell Lines
                Vero Cells
                Biology and Life Sciences
                Microbiology
                Virology
                Viral Replication
                Biology and Life Sciences
                Cell Biology
                Cellular Structures and Organelles
                Endoplasmic Reticulum
                Biology and Life Sciences
                Cell Biology
                Cell Processes
                Secretory Pathway
                Endoplasmic Reticulum
                Biology and Life Sciences
                Cell Biology
                Cellular Structures and Organelles
                Endoplasmic Reticulum
                Rough Endoplasmic Reticulum
                Biology and Life Sciences
                Cell Biology
                Cell Processes
                Secretory Pathway
                Endoplasmic Reticulum
                Rough Endoplasmic Reticulum
                Biology and Life Sciences
                Microbiology
                Virology
                Viral Structure
                Research and Analysis Methods
                Microscopy
                Electron Microscopy
                Transmission Electron Microscopy
                Biology and Life Sciences
                Cell Biology
                Cellular Structures and Organelles
                Lysosomes
                Custom metadata
                All relevant data are within the paper.

                Uncategorized

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