Enrichment of viral particles.

RRV (at a multiplicity of infection [MOI] of 5) was adsorbed to MA104 cells for 1 h at 4°C to allow the virus to bind to the cells. The unbound virus was removed, and the cells were incubated at 37°C to allow the infection to proceed. At different times postinfection, cells were washed with EGTA (3 mM) for 5 s to release any viral particles that remained bound to the cell surface, and the cells were lysed by freeze-thawing. Viral lysates were centrifuged at 105,000 × g for 1 h at 4°C in a 45 Ti rotor using a Beckman ultracentrifuge (OptimaL-90). The supernatant was discarded, and the viral particles in the pellet were resuspended in 4 ml TNC buffer (10 mM Tris, pH 7.5, 140 mM NaCl, 10 mM CaCl₂), extracted with trichloromonofluoromethane (Genetron; CYDSA 11, Mexico City, Mexico) and placed on top of a 1-ml 40% sucrose cushion in TNC. The samples were centrifuged at 195,000 × g for 2 h at 4°C in a SW55 Ti rotor. Finally, the resulting pellets containing the semipurified viral particles were dissolved in TNC and analyzed by SDS-PAGE.