Virus purification by sucrose gradient ultracentrifugation.

For virus purification, confluent monolayers of Vero cells grown in T225 flasks (Nunc, Thermo Fisher, USA) were infected with rDR13^att-ORF3^wt, PEDV DR13^att, and rDR13^att-ΔORF3 at an MOI of 2. After infection for 2 days at 37°C in medium supplemented with 5% FCS, the culture supernatants were harvested and precleared by centrifugation at 1,500 × g for 10 min at 4°C to remove cell debris. Supernatants were then centrifuged at 28,000 rpm for 3 h at 4°C, and the pellets were resuspended in PBS. For additional purification of virus, the resuspended pellets were layered onto 20% to 60% (wt/wt) sucrose gradients (in PBS) and centrifuged for 6 h at 28,000 rpm. From each gradient, the virus particle band visible in the 40% to 50% zone of the sucrose gradient was collected and diluted with PBS. The viruses were finally concentrated by pelleting for 3 h at 28,000 rpm and dissolving in a small volume of PBS. The purified virus was analyzed by SDS-PAGE and Western blotting.