Figure 2. Cryptoporus volvatus extract inhibits PRRSV entry into Marc-145 cell but not attachment.

(A) Effects of the Cryptoporus volvatus extract on virus attachment. Marc-145 cells were inoculated with Ch1a (MOI = 1) at 4°C for 2 h with different concentrations of the extract, and then cell lysates were prepared by freeze-thaw three times after cells were washed 3 times with cold PBS. Virus titer (TCID₅₀) was determined. (B) Inhibition of PRRSV entry by the Cryptoporus volvatus extract. Marc-145 cells were incubated with Ch1a (MOI = 1) at 4°C for 2 h. Unbound virus was removed by washing three times with cold PBS, and the temperature was switched to 37°C (this time point was set up as 0 h). Cell medium was replaced with fresh medium containing different concentrations of the extract at 0, 1, 2, or 3 h following temperature switch. Five hours after temperature switch, medium was replaced with fresh medium, and cells were further incubated at 37°C. Twenty-four hours later, supernatants were harvested for virus titration. (C) Confocal analysis showing that Cryptoporus volvatus extract inhibits PRRSV entry into Marc145 cells. Marc-145 cells were incubated with Ch-1a (MOI = 50) at 4°C for 2 h. And then, cells were fixed with cold methanol-acetone after 3 washes with cold PBS (negative control) or continued to be cultured in fresh medium with or without (positive control) the Cryptoporus volvatus extract at 37°C for another 3 hours before being fixed with cold methanol-acetone after 3 washes with cold PBS. Fixed cells were stained for PRRSV N protein and labeled with Phalloidin -TRITC (Sigma). Immunofluorescence was observed using Leica Microsystems CMS GmbH. Data are representative of three independent experiments (mean ± SD). Statistical significance was analyzed using Student’s t test.