Duch, M.

Mateiu, Ramona Valentina

Pedersen, F.S.

Thomsen, J.

Wagner, Jakob Birkedal

Online Research Database In Technology

                       General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.  • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal   If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.     Downloaded from orbit.dtu.dk on: Nov 08, 2017Transmission Electron Microscopy for Morphology Characterization of Virus likeParticlesMateiu, Ramona Valentina; Thomsen, J.; Pedersen, F.S.; Duch, M.; Wagner, Jakob BirkedalPublication date:2015Document VersionPublisher's PDF, also known as Version of recordLink back to DTU OrbitCitation (APA):Mateiu, R. V., Thomsen, J., Pedersen, F. S., Duch, M., & Wagner, J. B. (2015). Transmission ElectronMicroscopy for Morphology Characterization of Virus like Particles. Poster session presented at 13thProtein.DTU Workshop, Lyngby, Denmark.Transmission Electron Microscopy for Morphology Characterization of  Virus like Particles   R.V. Mateiu1, J. Thomsen2, F.S. Pedersen2, M. Duch2, and J. B. Wagner1 1DTU Cen, Center for Electron Nanoscopy, Technical University of Denmark 2Department of Molecular Biology and Genetics, Aarhus University  Acknowledgments   PIGVAC  (2014-2018) is supported by Det Strategiske Forskningsråd.  Further information  ramona.mateiu@cen.dtu.dk Research Area  Denmark produces about 4500 tons pig meat per day and about 90 % of this is exported. This results in an annual export of 4 billion EUR and represents about 5% of the total Danish export.  Porcine reproductive respiratory syndrome virus (PRRSV) causes a loss of about 43.6 million EUR per year in Denmark, and therefore controlling the virus is a top priority. The commercially available PRRSV vaccines are not effective in the prevention and extermination of the virus and therefore new development routes, such as virus like particles (VLPs) are approached for obtaining efficient and safe vaccines, capable of addressing prevalent strains.   The project is a consortium between 8 partners from academia and industry (for details see www.pigvac.dk). DTU Cen’s role in the project is to apply electron microscopy for morphological characterization of VLPs.  PRRSV infected pig lung and piglets born from a PRRSV infected sow. Images from Charlotte Sonne Kristensen, Danish Pig Research Center. Virus like Particles (VLPs)  Virus like Particles are composed of a subset of structural proteins and are attractive because: 1. They present high immunogenicity- they induce a humoral and/or cell mediated immune response similar to that produced for a virus. 2. They are safe; they do not contain genomic material. 3. They are high adaptable for inclusion of new viral proteins.  In the current project we use pseudotyped VLPs: Murine Leukemia Virus pseudotyped with PRRSV GP5 . Schematic drawing showing the pseodotype synthesis of VLPs and a Murine Leukemia Virus like particle pseudotyped with PRRSV envelope particles. Negative Staining  Cu grid TEM image Dried Stain Before Staining After Staining Sample Water x 3 Stain Dry Water (106 particles/ml)  Tweezers Cu Grid We are looking for   I would like to meet other people currently using electron microscopy for visualization and characterization of viruses and large proteins.  Electron Microscopy of Virus like Particles  Control  stained with 2% uranyl acetate on Collodion film. VLPs  stained with 2% uranyl acetate on Collodion film. Size Distribution   Diameter (nm) Particles Measured (%) Conclusions   Negative staining is a quick and easy sample preparation method for morphological electron microscopy analsyis of VLPs. However, the VLPs are not stabilized enough by the uranyl acetate and the particles collapse. Moreover, the control sample contains structures with size comparable to that of VLPs, and therfore specific labelling is needed in order to identify the VLPs by TEM.  Future sample preparation methods aim at using immulabelling, which will allow for the identification of the VLPs, and cryo electron microscopy, which will ensure the preservation of the particle morphology.  051015202530354045"0-50" "50-100" "100-150" "150-200" "200-250" "250-300" "300-350"ControlVLPs

Transmission Electron Microscopy for Morphology Characterization of Virus like Particles

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Transmission Electron Microscopy for Morphology Characterization of Virus like Particles

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