Low impact to fixed cell processing aiming transmission electron microscopy

Barth, Ortrud Monika; Silva, Marcos Alexandre Nunes da

doi:10.1590/0074-02760150433

Cited by 17 publications

(13 citation statements)

References 3 publications

(3 reference statements)

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“…Infected monolayers were trypsinized at 48h p.i. Cell suspensions were fixed in 2.5% glutaraldehyde in sodium cacodilate buffer (0.2 M, pH 7.2), post-fixed in 1% buffered osmium tetroxide, dehydrated in acetone, embedded in epoxy resin and polymerized at 60°C over the course of three days [ 57 , 58 ]. Ultrathin sections (50–70 nm) were obtained from the resin blocks.…”

Section: Methodsmentioning

confidence: 99%

Lipid droplets fuel SARS-CoV-2 replication and production of inflammatory mediators

et al. 2020

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Viruses are obligate intracellular parasites that make use of the host metabolic machineries to meet their biosynthetic needs. Thus, identifying the host pathways essential for the virus replication may lead to potential targets for therapeutic intervention. The mechanisms and pathways explored by SARS-CoV-2 to support its replication within host cells are not fully known. Lipid droplets (LD) are organelles with major functions in lipid metabolism, energy homeostasis and intracellular transport, and have multiple roles in infections and inflammation. Here we described that monocytes from COVID-19 patients have an increased LD accumulation compared to SARS-CoV-2 negative donors. In vitro, SARS-CoV-2 infection were seen to modulate pathways of lipid synthesis and uptake as monitored by testing for CD36, SREBP-1, PPARγ, and DGAT-1 expression in monocytes and triggered LD formation in different human cell lines. LDs were found in close apposition with SARS-CoV-2 proteins and double-stranded (ds)-RNA in infected Vero cells. Electron microscopy (EM) analysis of SARS-CoV-2 infected Vero cells show viral particles colocalizing with LDs, suggestive that LDs might serve as an assembly platform. Pharmacological modulation of LD formation by inhibition of DGAT-1 with A922500 significantly inhibited SARS-CoV-2 replication as well as reduced production of mediators pro-inflammatory response. Taken together, we demonstrate the essential role of lipid metabolic reprograming and LD formation in SARS-CoV-2 replication and pathogenesis, opening new opportunities for therapeutic strategies to COVID-19.

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Section: Methodsmentioning

confidence: 99%

Lipid droplets fuel SARS-CoV-2 replication and production of inflammatory mediators

et al. 2020

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“…Cells were fixed in 1% glutaraldeyde in sodium cacodylate buffer (0.2 M, pH 7.2) (Electron Microscopy Science), post-fixed in 1% buffered osmium tetroxide (Electron Microscopy Science), dehydrated in acetone (Merck), embedded in epoxy resin (Electron Microscopy Science) and polymerized at 60°C over the course of three days [ 25 , 26 , 27 , 28 ]. Ultrathin sections (50–70 nm thick) were obtained from the resin blocks.…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2017

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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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“…Cell suspensions were fixed in 2.5% glutaraldehyde in sodium cacodilate buffer (0.2 M, pH 7.2), post-fixed in 1% buffered osmium tetroxide, dehydrated in acetone, embedded in epoxy resin and polymerised at 60ºC over the course of three days. (23,24) Ultrathin sections (50-70 nm) were obtained from the resin blocks. The sections were picked up using copper grids, stained with uranyl acetate and lead citrate, (25) and observed using Jeol JEM 1011, FEI Titan, FEI Tecnai Spirit, Hitachi HT 7800 transmission electron microscopes.…”

Section: Viral Quantification In Cell Cultures Supernatants -mentioning

confidence: 99%

Morphology and morphogenesis of SARS-CoV-2 in Vero-E6 cells

Silva

Garcia

Miranda

et al. 2021

Mem. Inst. Oswaldo Cruz

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BACKGROUNDThe coronaviruses (CoVs) called the attention of the world for causing outbreaks of severe acute respiratory syndrome (SARS-CoV), in Asia in 2002-03, and respiratory disease in the Middle East (MERS-CoV), in 2012. In December 2019, yet again a new coronavirus (SARS-CoV-2) first identified in Wuhan, China, was associated with a severe respiratory infection, known today as COVID-19. This new virus quickly spread throughout China and 30 additional countries. As result, the World Health Organization (WHO) elevated the status of the COVID-19 outbreak from emergency of international concern to pandemic on March 11, 2020. The impact of COVID-19 on public health and economy fueled a worldwide race to approve therapeutic and prophylactic agents, but so far, there are no specific antiviral drugs or vaccines available. In current scenario, the development of in vitro systems for viral mass production and for testing antiviral and vaccine candidates proves to be an urgent matter.OBJECTIVE The objective of this paper is study the biology of SARS-CoV-2 in Vero-E6 cells at the ultrastructural level.METHODS In this study, we documented, by transmission electron microscopy and real-time reverse transcription polymerase chain reaction (RT-PCR), the infection of Vero-E6 cells with SARS-CoV-2 samples isolated from Brazilian patients.FINDINGS The infected cells presented cytopathic effects and SARS-CoV-2 particles were observed attached to the cell surface and inside cytoplasmic vesicles. The entry of the virus into cells occurred through the endocytic pathway or by fusion of the viral envelope with the cell membrane. Assembled nucleocapsids were verified inside rough endoplasmic reticulum cisterns (RER). Viral maturation seemed to occur by budding of viral particles from the RER into smooth membrane vesicles.MAIN CONCLUSIONS Therefore, the susceptibility of Vero-E6 cells to SARS-CoV-2 infection and the viral pathway inside the cells were demonstrated by ultrastructural analysis.

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Low impact to fixed cell processing aiming transmission electron microscopy

Cited by 17 publications

References 3 publications

Lipid droplets fuel SARS-CoV-2 replication and production of inflammatory mediators

Lipid droplets fuel SARS-CoV-2 replication and production of inflammatory mediators

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

Morphology and morphogenesis of SARS-CoV-2 in Vero-E6 cells

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