title: Reconstruction of H3N2 influenza virus based virosome in-vitro.
abstract: OBJECTIVE
  Virosomes are Virus Like Particles (VLP) assembled in-vitro. Influenza virosomes maintain the cell binding and membrane fusion activity of the wild type virus but are devoid of viral genetic material or internal proteins. Influenza virosomes mimic the natural antigen presentation route of the influenza virus.
  
  
  METHODS
  Virosomes were prepared by membrane solubilization and reconstitution. Briefly, the Madine-Darby Canine kidney (MDCK) cell line was cultivated on microcarrier beads inoculated with influenza virus strain A/X-47 (H3N2). The culture medium was harvested and clarified. Subsequently, virus was concentrated and purified by ultrafiltration and ultracentrifugation. The purified viral membrane was dissolved by adding 375 µl of 200 mM 1, 2-dicaproyl-sn-glycero-3-phosphocholine (DCPC) in HEPES-buffered saline (HBS). Nucleocapsid was removed by ultracentrifugation. The supernatant consisting of phospholipids and glycoproteins of the influenza virus was reconstituted by removal of DCPC using overnight dialysis against Hank's Buffered Saline (HBS) solution at 4°C. After dialysis, crude virosome preparation was layered over a discontinuous sucrose gradient in order to separate non-incorporated material from the reconstituted virus membranes.
  
  
  RESULTS
  The virosome harvested from the boundary of the two sucrose layers successfully was identified by the Hemagglutination assay and western blotting.
  
  
  CONCLUSIONS
  Use of a dialyzable short-chain phospholipid (DCPC) is an efficient procedure for solubilization and reconstitution of influenza virus virosomes and has not caused structural changes in a major envelope glycoprotein (hemagglutinin protein) on the surface of virosome.
authors.affiliation: Department of Virology, Faculty of Medical Science, Tarbiat Modares University, Tehran, Iran.
authors.forename: Asghar
authors.initials: A
authors.lastname: Abdoli
authors.forename: Hoorieh
authors.initials: H
authors.lastname: Soleimanjahi
authors.forename: Masoumeh Tavassoti
authors.initials: MT
authors.lastname: Kheiri
authors.forename: Abbas
authors.initials: A
authors.lastname: Jamali
authors.forename: Hesam
authors.initials: H
authors.lastname: Sohani
authors.forename: Mohsen
authors.initials: M
authors.lastname: Abdoli
authors.forename: Hamid Reza
authors.initials: HR
authors.lastname: Rahmatollahi
keywords: Influenza virus; Reconstruction; Virosomes