Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Analysis of IAV Replication and Co-infection Dynamics by a Versatile RNA Viral Genome Labeling Method
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).ORCID iD: 0000-0001-7727-1712
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Show others and affiliations
2017 (English)In: Cell reports, ISSN 2211-1247, E-ISSN 2211-1247, Vol. 20, no 1, p. 251-263Article in journal (Refereed) Published
Abstract [en]

Genome delivery to the proper cellular compartment for transcription and replication is a primary goal of viruses. However, methods for analyzing viral genome localization and differentiating genomes with high identity are lacking, making it difficult to investigate entry-related processes and co-examine heterogeneous RNA viral populations. Here, we present an RNA labeling approach for single-cell analysis of RNA viral replication and co-infection dynamics in situ, which uses the versatility of padlock probes. We applied this method to identify influenza A virus (IAV) infections in cells and lung tissue with single-nucleotide specificity and to classify entry and replication stages by gene segment localization. Extending the classification strategy to co-infections of IAVs with single-nucleotide variations, we found that the dependence on intracellular trafficking places a time restriction on secondary co-infections necessary for genome reassortment. Altogether, these data demonstrate how RNA viral genome labeling can help dissect entry and co-infections.

Place, publisher, year, edition, pages
2017. Vol. 20, no 1, p. 251-263
Keywords [en]
RNA viruses, viral genome labeling and localization, influenza A virus, RNA labeling, vRNAs, IAV entry, IAV cell co-infections, IAV replication cycle, single-cell IAV genome trafficking, single-nucleotide specificity
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-146349DOI: 10.1016/j.celrep.2017.06.021ISI: 000404899700022OAI: oai:DiVA.org:su-146349DiVA, id: diva2:1136793
Available from: 2017-08-29 Created: 2017-08-29 Last updated: 2020-03-05Bibliographically approved
In thesis
1. Nucleic acid analysis tools: Novel technologies and biomedical applications
Open this publication in new window or tab >>Nucleic acid analysis tools: Novel technologies and biomedical applications
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Nucleic acids are fundamental molecules of living organisms functioning essentially as the molecular information carriers of life. From how an organism is built to how it responds to external conditions, all of it, can be found in the form of nucleic acid sequences inside every single cell of every life form on earth.

Therefore, accessing these sequences provides key information regarding the molecular identity and functional state of any living organism, this is very useful for areas like biomedicine, where accessing and understanding these molecular signatures is the key to develop strategies to understand, treat and diagnose diseases.

Decades of research and technological advancements have led to the development of a number of molecular tools and engineering technologies that allow accessing the information contained in the nucleic acids. This thesis provides a general overview of the tools and technologies available for nucleic acid analysis, and proposes an illustrative concept on how molecular tools and emergent technologies can be combined in a modular fashion to design methods for addressing different biomedical questions. The studies included in this thesis, are focused on the particular use of the molecular tools named: padlock and selector probes, rolling circle amplification, and fluorescence detection of single molecules in combination with microfluidics and portable microscopy. By using this combination, it became possible to design and demonstrate novel approaches for integrated nucleic acid analysis, inexpensive digital quantification, mobile-phone based diagnostics and the description of viral infections.

These studies represent a step forward towards the adoption of the selected group of tools and technologies, for the design and building of methods that can be used as powerful alternatives to conventional tools used in molecular diagnostics and virology. 

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2017
Keywords
Nucleic acid analysis, padlock probes, selector probes, rolling circle amplification, microfluidics, single-molecule detection, digital quantification, fluorescence microscopy, mobile phone microscopy, molecular diagnostics, virology
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-146334 (URN)978-91-7649-905-4 (ISBN)978-91-7649-906-1 (ISBN)
Public defence
2017-10-12, Nordenskiöldsalen, Geovetenskapens hus, Svante Arrhenius väg 12, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 1: Manuscript.

Available from: 2017-09-20 Created: 2017-08-29 Last updated: 2020-05-06Bibliographically approved
2. Influenza A Virus: Spatial analysis of influenza genome trafficking and the evolution of the neuraminidase protein
Open this publication in new window or tab >>Influenza A Virus: Spatial analysis of influenza genome trafficking and the evolution of the neuraminidase protein
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Influenza A viruses (IAVs) are a common infectious agent that seasonally circulates within the human population that causes mild to severe acute respiratory infections. The severity of the infection is often related to how the virus has evolved with respect to the pre-existing immunity in the population. For IAVs, the most common mechanisms to avoid the immune response are to vary the surface antigens, hemagglutinin (HA) and neuraminidase (NA), by processes known as antigenic drift and shift.

Antigenic drift refers to point mutations that accumulate in HA and NA as a result of the antibody-mediated selection pressure that exists in the population. The majority of the changes attributed to antigenic drift localize to HA and NA surface exposed regions, however this does not exclude that drift can also result in the selection of residues that are not exposed. One region where non-exposed residues have potentially been selected for is the NA transmembrane domain (TMD) of human H1N1 IAVs, where a temporal bias exists for the accumulation of polar residues. By examining these sequence changes in the NA TMD, we found that the polar residues contribute to the amphipathic characteristic of the NA TMD, which mediates the oligomerization of the N-terminus. As more polar residues became incorporated, the strength of the TMD-TMD interaction increased, presumably to benefit the NA head domain assembly into a functional tetramer. We determined that the amphiphilic drift in the NA TMD is able to bypass the strict hydrophobicity required for membrane insertion at the endoplasmic reticulum because it can utilize the co-translational translocation process to facilitate the insertion and inversion of its non-ideal TMD. The contribution of the TMD to proper NA assembly was traced to the formation of the Ca2+ binding pocket that is located at the center of the tetrameric assembly, as this pocket lies above the stalk linker regions and must be occupied for NA to function.

In addition to antigenic drift, NA and HA can also undergo antigenic shift. Antigenic shift occurs when either of the gene segments encoding NA or HA are exchanged with ones from another IAV encoding another subtype of NA or HA. Different from antigenic drift, antigenic shift can only occur when a cell is co-infected and most investigations on the process of reassortment have been made at the protein level due to the methodological issues for labeling the RNA genome in situ. To overcome these technical limitations, we developed an in situ RNA labeling approach that provides highly specific spatial resolution of the IAV genome throughout the infection process. By applying this approach to temporally analyze the co-infection process, we found that the entry of a second IAV is stalled in the cytoplasm if another IAV has begun to replicate. Together, these results provide insight into the low frequency of antigenic shift in nature and provide evidence that non-exposed residues may make an underappreciated contribution to NA antigenic drift in human H1N1 viruses.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2019. p. 40
Keywords
Influenza A virus, IAV, neuraminidase, NA, IAV genome trafficking, viral entry, viral replication, co-infection, antigenic drift, antigenic shift, NA assembly, transmembrane domain, evolution
National Category
Microbiology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-175202 (URN)978-91-7797-885-5 (ISBN)978-91-7797-886-2 (ISBN)
Public defence
2019-12-02, Vivi Täckholmsalen (Q-salen), NPQ-huset, Svante Arrhenius väg 20, Stockholm, 10:00 (English)
Opponent
Supervisors
Available from: 2019-11-07 Created: 2019-10-15 Last updated: 2020-05-22Bibliographically approved

Open Access in DiVA

fulltext(8921 kB)258 downloads
File information
File name FULLTEXT01.pdfFile size 8921 kBChecksum SHA-512
6881ec4d8215bc52e0534069c8a1927173567a30238776a66ad251c4587e70c95fa1f0709cf79092d177af4c99efe86768987f8bcbca0e51ecf2f40ba6a82da3
Type fulltextMimetype application/pdf

Other links

Publisher's full text

Search in DiVA

By author/editor
Dou, DanHernández-Neuta, IvánWang, HaoÖstbye, HenrikQian, XiaoyanNilsson, MatsDaniels, Robert
By organisation
Department of Biochemistry and BiophysicsScience for Life Laboratory (SciLifeLab)
In the same journal
Cell reports
Biochemistry and Molecular Biology

Search outside of DiVA

GoogleGoogle Scholar
Total: 258 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

doi
urn-nbn

Altmetric score

doi
urn-nbn
Total: 149 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf