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A rapid and efficient method for studies of virus interaction at the host cell surface using enteroviruses and real-time PCR
University of Kalmar, School of Pure and Applied Natural Sciences.
University of Kalmar, School of Pure and Applied Natural Sciences.
University of Kalmar, School of Pure and Applied Natural Sciences.
University of Kalmar, School of Pure and Applied Natural Sciences.ORCID iD: 0000-0003-3841-4826
2009 (English)In: Virology Journal, ISSN 1743-422X, E-ISSN 1743-422X, Vol. 6, no Article ID: 217Article in journal (Refereed) Published
Abstract [en]

Background: Measuring virus attachment to host cells is of great importance when trying to identify novel receptors. The presence of a usable receptor is a major determinant of viral host range and cell tropism. Furthermore, identification of appropriate receptors is central for the understanding of viral pathogenesis and gives possibilities to develop antiviral drugs. Attachment is presently measured using radiolabeled and subsequently gradient purified viruses. Traditional methods are expensive and time-consuming and not all viruses are stable during a purification procedure; hence there is room for improvement. Real-time PCR (RT-PCR) has become the standard method to detect and quantify virus infections, including enteroviruses, in clinical samples. For instance, primers directed to the highly conserved 5' untranslated region (5'UTR) of the enterovirus genome enable detection of a wide spectrum of enteroviruses. Here, we evaluate the capacity of the RT-PCR technology to study enterovirus host cell interactions at the cell surface and compare this novel implementation with an established assay using radiolabeled viruses. Results: Both purified and crude viral extracts of CVB5 generated comparable results in attachment studies when analyzed with RT-PCR. In addition, receptor binding studies regarding viruses with coxsackie- nd adenovirus receptor (CAR) and/or decay accelerating factor (DAF) affinity, further demonstrated the possibility to use RT-PCR to measure virus attachment to host cells. Furthermore, the RT-PCR technology and crude viral extracts was used to study attachment with low multiplicity of infection (0.05 x 10(-4)TCID(50)/cell) and low cell numbers (250), which implies the range of potential implementations of the presented technique. Conclusion: We have implemented the well-established RT-PCR technique to measure viral attachment to host cells with high accuracy and reproducibility, at low cost and with less effort than traditional methods. Furthermore, replacing traditional methods with RT-PCR offers the opportunity to use crude virus containing extracts to investigate attachment, which could be considered as a step towards viral attachment studies in a more natural state.

Place, publisher, year, edition, pages
2009. Vol. 6, no Article ID: 217
National Category
Microbiology
Research subject
Biomedical Sciences, Virology; Natural Science, Microbiology; Natural Science, Biomedical Sciences
Identifiers
URN: urn:nbn:se:lnu:diva-2142DOI: 10.1186/1743-422X-6-217ISI: 000273070600001OAI: oai:DiVA.org:lnu-2142DiVA, id: diva2:309192
Available from: 2010-04-06 Created: 2010-04-06 Last updated: 2017-12-12Bibliographically approved
In thesis
1. Tissue tropism and oncolytic potential of enteroviruses
Open this publication in new window or tab >>Tissue tropism and oncolytic potential of enteroviruses
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[sv]
Vävnadstropism och onkolytisk potential hos enterovirus
Place, publisher, year, edition, pages
Växjö, Kalmar: Linnaeus University Press, 2012
Series
Linnaeus University Dissertations ; 77/2012
Keywords
picornavirus, enterovirus, echovirus, receptor, tropism, viral oncolysis, quantification, receptor interactions, colon cancer
National Category
Other Natural Sciences
Research subject
Natural Science, Biomedical Sciences
Identifiers
urn:nbn:se:lnu:diva-17721 (URN)978-91-86983-32-1 (ISBN)
Public defence
2012-03-09, N2007, Smålandsgatan 26B, Kalmar, 09:00 (Swedish)
Opponent
Supervisors
Available from: 2012-02-28 Created: 2012-02-22 Last updated: 2012-02-28Bibliographically approved

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Jonsson, NinaGullberg, MariaIsraelsson, StinaLindberg, A. Michael

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