Avian influenza viruses represent a growing threat for an influenza pandemic. To develop recombinant vaccine for avian influenza of the H9N2 subtype, we expressed in insect cells virus-like particles (VLPs) consisting of three structural proteins of influenza At Hong Kong/1073/99 (H9N2) virus. Upon infection of SO cells with recombinant baculoviruses, the hemagglutinin (HA), neuraminidase (NA), and matrix (M I) proteins were co-expressed in the infected cells, self-assembled, and released into the culture medium as VLPs of 80-120 nm in diameter. VLPs exhibited functional characteristics of influenza virus including hemagglutination and neuraminidase activities. In BALB/c mice, VLPs elicited serum antibodies specific for influenza A/Hong Kong/1073/99 (H9N2) virus and inhibited replication of the influenza virus after challenge. Thus, VLPs represent a potential strategy for the development of human vaccines against avian influenza H9N2 viruses. (c) 2005 Elsevier Ltd. All rights reserved.