Reconstruction of H3N2 influenza virus based virosome in vitro

Reconstruction of H3N2 influenza virus based virosome in vitro

Reconstruction of H3N2 influenza virus based virosome in vitro

ABSTRACT
Background and Objectives: 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.
Materials and 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.
Conclusion: 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.

 

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