FIG 4.
Inhibition of exosome release impairs PRRSV transmission mediated by exosomes. (A) PK-15CD163 cells were treated with the indicated concentrations of GW4869, and the exosomes isolated were subjected to Western blotting for Alix expression. (B) PK-15CD163 cells were infected with PRRSV at an MOI of 1 for 1 h and then maintained in medium containing 10 μM GW4869 for 36 h. TCID50 and real-time PCR assays were performed to determine the viral titers and RNA copy numbers, respectively. (C) Schematic diagram of the Transwell system used to coculture PK-15CD163 and PK-15 cells. The upper-chamber PK-15CD163 cells were infected with PRRSV or treated with purified PRRSV-positive exosomes for 3 h and then treated or not with GW4869 (10 μM). After 36 h, the total RNAs were extracted from cells in both chambers and analyzed for PRRSV RNA with real-time quantitative PCR. (D) Cell samples from both bottom and top chambers of the experiment diagrammed in panel C were collected and subjected to Western blotting with antibody against viral protein nsp2.