Generation of VLPs

Titers of baculovirus stocks were determined using a rapid titration kit (BacPakBaculovirus Rapid Titer Kit; Clontech, USA). Sf9 insect cells were maintained as suspension cultures in serum-free SF900II medium (Life technologies, USA) at 27°C, with agitation at a speed of 120 rpm. MERS-CoV VLPs were produced by infecting Sf9 cells with recombinant baculovirus at a multiplicity of infection (MOI) of 0.5, and harvested at 96 h post infection (hpi). Suspensions were first centrifuged to remove cell debris, and the supernatants were then ultracentrifuged at 100,000 g for 1 h to obtain the VLPs pellet, before purification by a 30–40–50% discontinuous sucrose gradient. Bands between 30-40% sucrose, which represent MERS-CoV VLPs, were collected.