title: Vaccinia virus-free rescue of fluorescent replication-defective vesicular stomatitis virus and pseudotyping with Puumala virus glycoproteins for use in neutralization tests.
abstract: 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, rVSVΔG*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 105-108, 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 (rs = 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.
authors.affiliation: Department of Virology, Medicum, Helsinki, Finland.
authors.forename: Rommel
authors.initials: R
authors.lastname: Paneth Iheozor-Ejiofor
authors.affiliation: Department of Virology, Medicum, Helsinki, Finland.
authors.forename: Lev
authors.initials: L
authors.lastname: Levanov
authors.affiliation: Department of Virology, Medicum, Helsinki, Finland.
authors.forename: Jussi
authors.initials: J
authors.lastname: Hepojoki
authors.affiliation: Department of Virology, Medicum, Helsinki, Finland.
authors.forename: Tomas
authors.initials: T
authors.lastname: Strandin
authors.affiliation: Department of Medical Biochemistry and Microbiology, Microbiology-Immunology, Uppsala University, Sweden.
authors.forename: Åke
authors.initials: Å
authors.lastname: Lundkvist
authors.affiliation: Department of Virology, Medicum, Helsinki, Finland.|Department of Medical Biochemistry and Microbiology, Microbiology-Immunology, Uppsala University, Sweden.
authors.forename: Alexander
authors.initials: A
authors.lastname: Plyusnin
authors.affiliation: Department of Virology, Medicum, Helsinki, Finland.|Department of Virology and Immunology, HUSLAB, Helsinki University Hospital, Helsinki, Finland.|Department of Veterinary Biosciences, University of Helsinki, Helsinki, Finland.
authors.forename: Olli
authors.initials: O
authors.lastname: Vapalahti
mesh_terms: D006023:Glycoproteins / Q000737:chemistry / Q000145:classification / Q000378:metabolism*; D008958:Models, Molecular; D009500:Neutralization Tests / Q000379:methods*; D011487:Protein Conformation; D029262:Puumala virus / Q000378:metabolism*; D014616:Vaccinia virus / Q000502:physiology*; D014721:Vesicular stomatitis Indiana virus / Q000145:classification / Q000235:genetics / Q000502:physiology*; D014779:Virus Replication / Q000502:physiology*
chemical_list: D006023:Glycoproteins