FIG 5.

Vesicle associated astrovirus is infective in Caco-2 and MA104 cells. (A) Large extracellular vesicles (LEV) and small extracellular vesicles (SEV) were purified by differential centrifugation and MagCapture exosome isolation kit PS from supernatants of Caco-2 cells infected with astrovirus (strain Yuc8). Samples were treated with MEM (blue); 0.1% Triton X-100 (cyan); anti-Yuc8 (orange); 0.1% TX-100 followed by anti-Yuc8 (yellow), or they were incubated with a mixture of protease inhibitors (aprotinin, leupeptin, and PMSF) (lila) before addition to Caco-2 or MA104 cells. Treated samples were let to adsorb for 2 h, after which time unbound vesicles were washed and infection was left to proceed for 18 h. Infected cells were detected by immuno-peroxidase staining. Focus forming units (FFU) of each sample were counted in 3 wells of three independent experiments. Bars represent the viral focus forming units (FFU) in each sample ± standard error of the mean. Samples treated with TX-100 and TX-100 plus anti-Yuc8 treatments did not show infected cells. Statistical analysis was done with two-way ANOVA, P value ***, P < 0.001. (B) Dilutions of anti-Yuc8 polyclonal antibody, treated or not with 0.1% TX-100, were incubated with fixed amount of Yuc8 for 1 h, after which time mixture was added to Caco-2 cells grown in 96-well plates. Results of minimum 3 experiments done in duplicate are shown. Infection is compared to infectivity of Yuc8 virus incubated in medium without antibody.