Figure 4

Expression of N, Gn and Gc proteins produced virus-like particles. A) Sf9 cells were infected with the recombinant baculovirus expressing RVFV N, Gn/Gc proteins and after 4 days both infected cells and the media were harvested. An aliquot from the infected cell lysate was analyzed by SDS-PAGE and stained by Commassie Brilliant blue (lane1). The media was clarified followed by ultracentrifugation on a potassium tartrate-glycerol gradient. An aliquot of purified material was analyzed as before (lane 2). Confirmation of viral proteins in purified samples was performed by Western blotting using monoclonal antibodies against either Gn (lane3) or N (lane 4) or with a polyclonal antibody against RVFV Zinga strain (lane 5). Protein markers were included (lane M) and sizes in kDa are shown on the right. B) Negative staining of purified VLPs. The spiky structures of the particle surface units consisting of glycoproteins Gn and Gc are indicated by arrows (upper panel). The spiky surface units are indicated by arrows (lower panels). The number of the surface unit of each particle is indicated at the upper left corner. Bar represents 100 nm. C) EM of infected cells' section showing VLPs are released into vacuoles. Note the presence of particles (black arrow) within the membrane (white arrow) of the vacuole boundaries. D) Same showing virus inclusion body in the cytoplasm indicated by arrows.