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. 2022 May 4;13:879733. doi: 10.3389/fphar.2022.879733

FIGURE 4.

FIGURE 4

ZINC12899676 has antiviral activity against PEDV in vitro. (A,B) Cell viability of Vero and IPEC-J2 cells pretreated with compounds (10 μM) and incubated for 24 h. The results are from one of three independent experiments. (C,D) Vero and IPEC-J2 cells were pretreated with compounds (10 μM) for 1 h and then infected with PEDV for 1 h at 37°C. The cells were washed with PBS and then incubated in a fresh medium containing compounds for 16 h. DMSO served as the treatment control. (E–G) IPEC-J2 cells were pretreated with the indicated concentrations of ZINC12899676 for 1 h and then infected with PEDV for 1 h at 37°C. The cells were washed with PBS and then incubated in fresh medium containing ZINC12899676 for 16 h. DMSO served as the treatment control. (E) Culture supernatants were collected at indicated time points for viral titration. Results are expressed as TCID50. Titers from three independent experiments are shown as means ± SD (error bars). (F) Western blot for the viral N-protein in cells infected with PEDV and treated with indicated concentrations of ZINC12899676 or DMSO, at 16 hpi. (G) Relative PEDV N mRNA levels, determined by qRT-PCR, and expressed relative to that in DMSO-treated cells. The internal loading control was GAPDH. (H) IPEC-J2 cells were incubated with PEDV at 37°C for 1 h and washed three times with PBS to remove free virus. At 4 hpi, the culture medium was replaced with fresh DMEM containing ZINC12899676 or DMSO, and the cultures were incubated at 37°C. PEDV N and GAPDH proteins in the samples collected at 10, 12, and 14 hpi were measured using Western blot. (I) IPEC-J2 cells were incubated with PEDV at 37°C for 1 h and washed three times with PBS to remove free virus. At 6 or 10 hpi, the culture medium was replaced with fresh DMEM containing ZINC12899676 or DMSO, and the cultures were incubated at 37°C. PEDV N and GAPDH proteins in the samples collected at 14 hpi were measured using Western blot. (J) Cell viability of IPEC-J2 cells pretreated with indicated concentrations of ZINC12899676 and incubated for 24 h.