Detection of Viral −RNA and +RNA Strands in Enterovirus-Infected Cells and Tissues
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 t...
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doaj-dddf4308a1f34cc49b2794cf5f861f472020-12-05T00:04:47ZengMDPI AGMicroorganisms2076-26072020-12-0181928192810.3390/microorganisms8121928Detection of Viral −RNA and +RNA Strands in Enterovirus-Infected Cells and TissuesSami Salmikangas0Jutta E. Laiho1Kerttu Kalander2Mira Laajala3Anni Honkimaa4Iryna Shanina5Sami Oikarinen6Marc S. Horwitz7Heikki Hyöty8Varpu Marjomäki9Department of Biological and Environmental Science/Nanoscience Center, University of Jyväskylä, Survontie 9C, FI-40500 Jyväskylä, FinlandFaculty of Medicine and Health Technology, Tampere University, FI-33520 Tampere, FinlandDepartment of Biological and Environmental Science/Nanoscience Center, University of Jyväskylä, Survontie 9C, FI-40500 Jyväskylä, FinlandDepartment of Biological and Environmental Science/Nanoscience Center, University of Jyväskylä, Survontie 9C, FI-40500 Jyväskylä, FinlandFaculty of Medicine and Health Technology, Tampere University, FI-33520 Tampere, FinlandDepartment of Microbiology and Immunology, Life Sciences Institute, University of British Columbia, Vancouver, BC V6T1Z3, CanadaFaculty of Medicine and Health Technology, Tampere University, FI-33520 Tampere, FinlandDepartment of Microbiology and Immunology, Life Sciences Institute, University of British Columbia, Vancouver, BC V6T1Z3, CanadaFaculty of Medicine and Health Technology, Tampere University, FI-33520 Tampere, FinlandDepartment of Biological and Environmental Science/Nanoscience Center, University of Jyväskylä, Survontie 9C, FI-40500 Jyväskylä, FinlandThe 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.https://www.mdpi.com/2076-2607/8/12/1928antiviral drugsbranched DNAenterovirusin situ hybridizationnegative RNApositive RNA |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Sami Salmikangas Jutta E. Laiho Kerttu Kalander Mira Laajala Anni Honkimaa Iryna Shanina Sami Oikarinen Marc S. Horwitz Heikki Hyöty Varpu Marjomäki |
spellingShingle |
Sami Salmikangas Jutta E. Laiho Kerttu Kalander Mira Laajala Anni Honkimaa Iryna Shanina Sami Oikarinen Marc S. Horwitz Heikki Hyöty Varpu Marjomäki Detection of Viral −RNA and +RNA Strands in Enterovirus-Infected Cells and Tissues Microorganisms antiviral drugs branched DNA enterovirus in situ hybridization negative RNA positive RNA |
author_facet |
Sami Salmikangas Jutta E. Laiho Kerttu Kalander Mira Laajala Anni Honkimaa Iryna Shanina Sami Oikarinen Marc S. Horwitz Heikki Hyöty Varpu Marjomäki |
author_sort |
Sami Salmikangas |
title |
Detection of Viral −RNA and +RNA Strands in Enterovirus-Infected Cells and Tissues |
title_short |
Detection of Viral −RNA and +RNA Strands in Enterovirus-Infected Cells and Tissues |
title_full |
Detection of Viral −RNA and +RNA Strands in Enterovirus-Infected Cells and Tissues |
title_fullStr |
Detection of Viral −RNA and +RNA Strands in Enterovirus-Infected Cells and Tissues |
title_full_unstemmed |
Detection of Viral −RNA and +RNA Strands in Enterovirus-Infected Cells and Tissues |
title_sort |
detection of viral −rna and +rna strands in enterovirus-infected cells and tissues |
publisher |
MDPI AG |
series |
Microorganisms |
issn |
2076-2607 |
publishDate |
2020-12-01 |
description |
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. |
topic |
antiviral drugs branched DNA enterovirus in situ hybridization negative RNA positive RNA |
url |
https://www.mdpi.com/2076-2607/8/12/1928 |
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