2.2 BMV virus production
The VLP particles were produced and purified from the native virus as previously reported.[42] The BMV native virus was amplified through plant infection to produce VLPs. First, barley plants (Hordeum vulgare ) were grown under controlled conditions. Then, young barley leaves were inoculated with the BMV virus. Once the leaves showed the infection pattern, they were harvested and stored at -20°C to be later liquefied in a blender (Osterizer) with virus extraction buffer (0.5 M sodium acetate, 80 mM magnesium acetate, pH 4.5). The resulting macerated material was filtered through cheesecloth, then the flowthrough was mixed with chloroform 1:1 v/v. The mixture was centrifuged at 10,000 rpm, 4°C, for 20 min. Since two phases formed in the centrifuge bottle; the upper phase (aqueous phase) was collected and stirred at 4°C overnight. Next, the aqueous extract containing the BMV virions was ultracentrifuged on a 5 mL sucrose cushion in a 32 mL centrifuge tube at 32,000 rpm, 4°C for 2 h. After centrifugation, the supernatant was discarded and a pellet containing the BMV virions was resuspended in a virus suspension buffer (50 mM sodium acetate, 8 mM magnesium acetate, pH 4.5). Then, the BMV virion suspension was poured into a previously prepared centrifuge tube with a sucrose density gradient (10-40%) and centrifuged at 30,000 rpm, 4°C for 2 h. After centrifugation, the tube was examined under white light to confirm the presence of the blue phase containing the viruses. The blue phase was then recovered and diluted 1:4 v/v in a virus suspension buffer. The dilute blue phase was centrifuged at 32,000 rpm, 4°C for 3 h to concentrate the virions in a pellet. After centrifugation, the supernatant was discarded and the pellet containing the virions was resuspended in virus suspension buffer and stored at -70°C.