Figure 4.

Identification of the inhibitory effect of CS-SeNPs on PRRSV replication in Marc-145 cells. (A) Relative r-PRRSV-EGFP ORF 5 mRNA levels determined by qRT-PCR in Marc-145 inoculated with r-PRRSV-EGFP at 0.1 MOI and treated with CS (1 mg/mL), Na₂SeO₃ (10 μM), and CS-SeNPs (10 μM) for 48 h. (B) Time-of-addition schematic. (C) Relative r-PRRSV-EGFP ORF 5 mRNA levels in Marc-145 inoculated with r-PRRSV-EGFP at 0.1 MOI and treated with CS-SeNPs (10 μM) according to the schematic. **P <0.01 compared with control group cells. (D) Relative ORF 5 mRNA levels determined by qRT-PCR. **P <0.01 compared with r-PRRSV-EGFP infected cells. (E) Viral titers detected by TCID₅₀ of r-PRRSV-EGFP according to group VII treatment pattern in Figure 4B at the indicated times. Results are expressed as the mean ± SD of triplicate experiments, **P <0.01 compared with r-PRRSV-EGFP infected cells. (F) Marc-145 cells infected with r-PRRSV-EGFP at an MOI of 0.1 for 1 h and incubated with or without 10 μM CS-SeNPs for 48 h. Cells were fixed and stained with DAPI (4′,6-diamidino-2-phenylindole) and observed under fluorescence microscopy. Western blot analysis of PRRSV N protein in Marc-145 cells infected with r-PRRSV-EGFP (G) and ZJ-JX/2015 (H) and then incubated with or without 10 μM CS-SeNPs for 24 and 48 h. β-actin was used as the loading control.

Abbreviations: CS, chitosan; CS-SeNPs, chitosan-coated selenium nanoparticles; MOI, multiplicity of infection; ORF5, open reading frame 5; qRT-PCR, Quantitative Real-time Polymerase chain reaction; SD, standard deviation; TCID₅₀, the median tissue culture infectious dose.