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Vaccinia virus-free rescue of fluorescent replication-defective vesicular stomatitis virus and pseudotyping with Puumala virus glycoproteins for use in neutralization tests
Univ Helsinki, Dept Virol, Medicum, Helsinki, Finland..
Univ Helsinki, Dept Virol, Medicum, Helsinki, Finland..
Univ Helsinki, Dept Virol, Medicum, Helsinki, Finland..
Univ Helsinki, Dept Virol, Medicum, Helsinki, Finland..
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2016 (English)In: Journal of General Virology, ISSN 0022-1317, E-ISSN 1465-2099, Vol. 97, 1052-1059 p.Article in journal (Refereed) PublishedText
Abstract [en]

Puumala virus (PUUV) grows slowly in cell culture. To study antigenic properties of PUUV, an amenable method for their expression would be beneficial. To achieve this, a replication-defective recombinant vesicular stomatitis virus, rVSV Delta G*EGFP, was rescued using BSRT7/5 and encephalomyocarditis virus (EMCV) internal ribosomal entry site (IRES)-enabled rescue plasmids. Using these particles, pseudotypes bearing PUUV Sotkamo strain glycoproteins were produced, with titres in the range 10(5)-10(8), and were used in pseudotype focus reduction neutralization tests (pFRNTs) with neutralizing monoclonal antibodies and patient sera. The results were compared with those from orthodox focus reduction neutralization tests (oFRNTs) using native PUUV with the same samples and showed a strong positive correlation (r(s)=0.82) between the methods. While developing the system we identified three amino acids which were mutated in the Vero E6 cell culture adapted PUUV prototype Sotkamo strain sequence, and changing these residues was critical for expression and neutralizing antibody binding of PUUV glycoproteins.

Place, publisher, year, edition, pages
2016. Vol. 97, 1052-1059 p.
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
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URN: urn:nbn:se:uu:diva-298240DOI: 10.1099/jgv.0.000437ISI: 000375837800003OAI: oai:DiVA.org:uu-298240DiVA: diva2:945560
Available from: 2016-07-01 Created: 2016-07-01 Last updated: 2016-07-01Bibliographically approved

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Lundkvist, Åke
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Department of Medical Biochemistry and Microbiology
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Journal of General Virology
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)

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