scholarly journals Detection of Viral −RNA and +RNA Strands in Enterovirus-Infected Cells and Tissues

2020 ◽  
Vol 8 (12) ◽  
pp. 1928
Author(s):  
Sami Salmikangas ◽  
Jutta E. Laiho ◽  
Kerttu Kalander ◽  
Mira Laajala ◽  
Anni Honkimaa ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
In Situ Hybridization ◽  
Viral Rna ◽  
Quantitative Real Time Pcr ◽  
Virion Assembly ◽  
Rna Molecules ◽  
Branched Dna ◽  
Infected Cells ◽  
Good Signal

The current methods to study the distribution and dynamics of viral RNA molecules inside infected cells are not ideal, as electron microscopy and immunohistochemistry can only detect mature virions, and quantitative real-time PCR does not reveal localized distribution of RNAs. We demonstrated here the branched DNA in situ hybridization (bDNA ISH) technology to study both the amount and location of the emerging −RNA and +RNA during acute and persistent enterovirus infections. According to our results, the replication of the viral RNA started 2–3 h after infection and the translation shortly after at 3–4 h post-infection. The replication hotspots with newly emerging −RNA were located quite centrally in the cell, while the +RNA production and most likely virion assembly took place in the periphery of the cell. We also discovered that the pace of replication of −RNA and +RNA strands was almost identical, and −RNA was absent during antiviral treatments. ViewRNA ISH with our custom probes also showed a good signal during acute and persistent enterovirus infections in cell and mouse models. Considering these results, along with the established bDNA FISH protocol modified by us, the effects of antiviral drugs and the emergence of enterovirus RNAs in general can be studied more effectively.

scholarly journals Replication of Tobacco Mosaic Virus on Endoplasmic Reticulum and Role of the Cytoskeleton and Virus Movement Protein in Intracellular Distribution of Viral RNA

1999 ◽  
Vol 147 (5) ◽  
pp. 945-958 ◽  
Author(s):  
Paloma Más ◽  
Roger N. Beachy
Keyword(s):  
In Situ Hybridization ◽  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Movement Protein ◽  
Viral Rna ◽  
Pharmacological Agents ◽  
Intracellular Targeting ◽  
Cytoplasmic Filaments ◽  
Infected Cells

Little is known about the mechanisms of intracellular targeting of viral nucleic acids within infected cells. We used in situ hybridization to visualize the distribution of tobacco mosaic virus (TMV) viral RNA (vRNA) in infected tobacco protoplasts. Immunostaining of the ER lumenal binding protein (BiP) concurrent with in situ hybridization revealed that vRNA colocalized with the ER, including perinuclear ER. At midstages of infection, vRNA accumulated in large irregular bodies associated with cytoplasmic filaments while at late stages, vRNA was dispersed throughout the cytoplasm and was associated with hair-like protrusions from the plasma membrane containing ER. TMV movement protein (MP) and replicase colocalized with vRNA, suggesting that viral replication and translation occur in the same subcellular sites. Immunostaining with tubulin provided evidence of colocalization of vRNA with microtubules, while disruption of the cytoskeleton with pharmacological agents produced severe changes in vRNA localization. Mutants of TMV lacking functional MP accumulated vRNA, but the distribution of vRNA was different from that observed in wild-type infection. MP was not required for association of vRNA with perinuclear ER, but was required for the formation of the large irregular bodies and association of vRNA with the hair-like protrusions.

scholarly journals In Situ Localization of Barley Yellow Dwarf Virus-PAV 17-kDa Protein and Nucleic Acids in Oats

1998 ◽  
Vol 88 (10) ◽  
pp. 1031-1039 ◽  
Author(s):  
Petra H. Nass ◽  
Leslie L. Domier ◽  
Birute P. Jakstys ◽  
Cleora J. D'Arcy
Keyword(s):  
In Situ Hybridization ◽  
Nucleic Acid ◽  
Barley Yellow Dwarf Virus ◽  
Yellow Dwarf ◽  
Viral Rna ◽  
Barley Yellow Dwarf ◽  
Infected Cells ◽  
Yellow Dwarf Virus ◽  
Filamentous Material

Barley yellow dwarf virus strain PAV (BYDV-PAV) RNA and the 17-kDa protein were localized in BYDV-PAV-infected oat cells using in situ hybridization and in situ immunolocalization assays, respectively. The in situ hybridization assay showed labeling of filamentous material in the nucleus, cytoplasm, and virus-induced vesicles with both sense and antisense nucleic acid probes, suggesting that the filamentous material found in BYDV-PAV-infected cells contains viral RNA. BYDV-PAV negative-strand RNA was detected before virus particles were observed, which indicates that RNA replication is initiated before synthesis of viral coat protein in the cytoplasm. The 17-kDa protein was associated with filamentous material in the cytoplasm, nucleus, and virus-induced vesicles. The labeling densities observed using antibodies against the 17-kDa protein were similar in the nucleus and cytoplasm. No labeling of the 17-kDa protein was observed in plasmodesmata, but filaments in the nuclear pores occasionally were labeled. Since BYDV-PAV RNA and 17-kDa protein colocalized within infected cells, it is possible that single-stranded viral RNA is always associated with the 17-kDa protein in vivo. The 17-kDa protein may be required for viral nucleic acid filaments to traverse the nuclear membrane or other membrane systems.

scholarly journals Multisystemic cellular tropism of SARS-CoV-2 in autopsies of COVID-19 patients

2021 ◽  
Author(s):  
Dickson W.L. Wong ◽  
Barbara M. Klinkhammer ◽  
Sonja Djudjaj ◽  
Sophia Villwock ◽  
M. Cherelle Timm ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
In Situ Hybridization ◽  
Virus Detection ◽  
Viral Rna ◽  
Transmission Electron ◽  
Formalin Fixed Paraffin ◽  
Organ Systems ◽  
Formalin Fixed Paraffin Embedded ◽  
Ffpe Tissues

Background: Multiorgan tropism of SARS-CoV-2 has previously been shown for several major organs. Methods: We have comprehensively analyzed 25 different formalin-fixed paraffin-embedded (FFPE) tissues/organs from autopsies of fatal COVID-19 cases (n=8), using detailed histopathological assessment, detection of SARS-CoV-2 RNA using polymerase chain reaction and RNA in situ hybridization, viral protein using immunohistochemistry, and virus particles using transmission electron microscopy. Finally, we confirmed these findings in an independent external autopsy cohort (n=9). Findings: SARS-CoV-2 RNA was mainly localized in epithelial cells, endothelial and mesenchymal cells across all organs. Next to lung, trachea, kidney, heart, or liver, viral RNA was also found in tonsils, salivary glands, oropharynx, thyroid, adrenal gland, testicles, prostate, ovaries, small bowel, lymph nodes, skin and skeletal muscle. Viral RNA was predominantly found in cells expressing ACE2, TMPRSS2, or both. The SARS-CoV-2 replicating RNA was also detected in these organs. Immunohistochemistry and electron microscopy were not suitable for reliable and specific SARS-CoV-2 detection in autopsies. The findings were validated using in situ hybridization on external COVID-19 autopsy samples. Finally, apart from the lung, correlation of virus detection and histopathological assessment did not reveal any specific alterations that could be attributed to SARS-CoV-2. Conclusion: SARS-CoV-2 could be observed in virtually all organs, colocalizing with ACE2 and TMPRSS2 mainly in epithelial but also in mesenchymal and endothelial cells, and viral replication was found across all organ systems. Apart from the respiratory tract, no specific (histo-)morphologic alterations could be assigned to the SARS-CoV-2 infection.

scholarly journals Multisystemic Cellular Tropism of SARS-CoV-2 in Autopsies of COVID-19 Patients

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1900
Author(s):  
Dickson W. L. Wong ◽  
Barbara M. Klinkhammer ◽  
Sonja Djudjaj ◽  
Sophia Villwock ◽  
M. Cherelle Timm ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
In Situ Hybridization ◽  
Viral Rna ◽  
Transmission Electron ◽  
Formalin Fixed Paraffin ◽  
Organ Systems ◽  
Formalin Fixed Paraffin Embedded ◽  
Ffpe Tissues ◽  
Rna In Situ Hybridization

Multiorgan tropism of SARS-CoV-2 has previously been shown for several major organs. We have comprehensively analyzed 25 different formalin-fixed paraffin-embedded (FFPE) tissues/organs from autopsies of fatal COVID-19 cases (n = 8), using histopathological assessment, detection of SARS-CoV-2 RNA using polymerase chain reaction and RNA in situ hybridization, viral protein using immunohistochemistry, and virus particles using transmission electron microscopy. SARS-CoV-2 RNA was mainly localized in epithelial cells across all organs. Next to lung, trachea, kidney, heart, or liver, viral RNA was also found in tonsils, salivary glands, oropharynx, thyroid, adrenal gland, testicles, prostate, ovaries, small bowel, lymph nodes, skin and skeletal muscle. Viral RNA was predominantly found in cells expressing ACE2, TMPRSS2, or both. The SARS-CoV-2 replicating RNA was also detected in these organs. Immunohistochemistry and electron microscopy were not suitable for reliable and specific SARS-CoV-2 detection in autopsies. These findings were validated using in situ hybridization on external COVID-19 autopsy samples (n = 9). Apart from the lung, correlation of viral detection and histopathological assessment did not reveal any specific alterations that could be attributed to SARS-CoV-2. In summary, SARS-CoV-2 and its replication could be observed across all organ systems, which co-localizes with ACE2 and TMPRSS2 mainly in epithelial but also in mesenchymal and endothelial cells. Apart from the respiratory tract, no specific (histo-)morphologic alterations could be assigned to the SARS-CoV-2 infection.

Ultrastructural analysis of the spatial distribution of MRNA

1992 ◽  
Vol 50 (1) ◽  
pp. 552-553
Author(s):  
Gary Bassell ◽  
Robert H. Singer
Keyword(s):  
Electron Microscopy ◽  
Spatial Distribution ◽  
In Situ Hybridization ◽  
High Resolution ◽  
Nucleic Acids ◽  
Colloidal Gold ◽  
Dna Probe ◽  
Nucleotide Analogs ◽  
Gold Particles

We have been investigating the spatial distribution of nucleic acids intracellularly using in situ hybridization. The use of non-isotopic nucleotide analogs incorporated into the DNA probe allows the detection of the probe at its site of hybridization within the cell. This approach therefore is compatible with the high resolution available by electron microscopy. Biotinated or digoxigenated probe can be detected by antibodies conjugated to colloidal gold. Because mRNA serves as a template for the probe fragments, the colloidal gold particles are detected as arrays which allow it to be unequivocally distinguished from background.

scholarly journals Ultrastructural visualization of cytoskeletal mRNAs and their associated proteins using double-label in situ hybridization.

1989 ◽  
Vol 108 (6) ◽  
pp. 2343-2353 ◽  
Author(s):  
R H Singer ◽  
G L Langevin ◽  
J B Lawrence
Keyword(s):  
In Situ Hybridization ◽  
High Resolution ◽  
Colloidal Gold ◽  
Messenger Rna ◽  
Signal To Noise Ratio ◽  
Simultaneous Detection ◽  
Gold Particles ◽  
Rna Molecules ◽  
Nucleic Acid Molecule

We have been able to visualize cytoskeletal messenger RNA molecules at high resolution using nonisotopic in situ hybridization followed by whole-mount electron microscopy. Biotinated cDNA probes for actin, tubulin, or vimentin mRNAs were hybridized to Triton-extracted chicken embryo fibroblasts and myoblasts. The cells were then exposed to antibodies against biotin followed by colloidal gold-conjugated antibodies and then critical-point dried. Identification of mRNA was possible using a probe fragmented to small sizes such that hybridization of several probe fragments along the mRNA was detected as a string of colloidal gold particles qualitatively and quantitatively distinguishable from nonspecific background. Extensive analysis showed that when eight gold particles were seen in this iterated array, the signal to noise ratio was greater than 30:1. Furthermore, these gold particles were colinear, often spiral, or circular suggesting detection of a single nucleic acid molecule. Antibodies against actin, vimentin, or tubulin proteins were used after in situ hybridization, allowing simultaneous detection of the protein and its cognate message on the same sample. This revealed that cytoskeletal mRNAs are likely to be extremely close to actin protein (5 nm or less) and unlikely to be within 20 nm of vimentin or tubulin filaments. Actin mRNA was found to be more predominant in lamellipodia of motile cells, confirming previous results. These results indicate that this high resolution in situ hybridization approach is a powerful tool by which to investigate the association of mRNA with the cytoskeleton.

Detection of individual virus-infected cells by filter in situ hybridization

1988 ◽  
Vol 2 (3) ◽  
pp. 245-253 ◽  
Author(s):  
Briony Forbes ◽  
Lutz Gissmann ◽  
Michael Pawlita
Keyword(s):  
In Situ Hybridization ◽  
Infected Cells
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