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Background: The last two decades witnessed the spread of the first generation of influenza viruses. Influenza virosomes are promising tools in vaccine and immunotherapy programs because of their applications in various medical fields. The aim of the present study was to construct cationic virosomes derived from influenza virus using dialyzable detergent (DCPC) and cationic lipid (DOTAP) in vitro. Materials and Methods: Influenza A / Puerto Rico / 34.8 (H1N1) strain was propagated in MDCK cell line. The influenza virus envelope was dissolved using DCPC as a dialyzable detergent, and finally it was removed by dialysis and the cationic virosome was synthesized through adding cationic lipid (DOTAP). Cytotoxicity and presence of HA and NA proteins were evaluated by cell viability assay (MTT assay) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), respectively. Also, macroscopic and morphology studies of virosomes were performed by transmission electron microscopy (TEM). Results: The final concentration of virosomes was 1.5 mg/ml. The presence of HA and NA proteins was confirmed by SDS-PAGE. Cell survival was significantly decreased after 48 hours of treatment with different concentrations of cationic virosomes (P<0.05). Conclusion: The use of a detergent (DCPC) and also a cationic lipid (DOTAP) is an effective procedure for reconstruction of influenza virus envelope without any alteration in surface glycoproteins (HA, NA). The methoed used in the present study for producing influenza virosome will be a promising candidate for developing influenza vaccines.