Fig. 3

Effects of exogenous NO on PCV2 replication in PK-15 cells. When monolayers reached about 50% in each well in 24-well cell culture plates, the cells were incubated with NAP or SNP or VC or SNP plus VC for 6 h, four wells for each treatment. After washing, the cells were infected with PCV2 (1 MOI), and then cultured with the drugs for another 72 h. Nontreated cells served as mock, and the infected-cells without drug treatment were infected control. As for IFA (a), the cells in different groups were washed with PBS, then fixed with cold methanol, and all stained for PCV2. Since FITC-Fluorescence could be observed in PCV2-infected cells, the appearance of PCV2-positive cells was judged by FITC staining intensity, examined under a fluorescence microscope (Olympus, Japan). In addition, supernatant from each well was collected for determination of NO production (b), meanwhile, the cells in each sample were also gathered for assay of relative infected cells (c), virus titers (d), virus DNA copies (e) and virus Cap expression. NO levels were quantitated by Griess reaction. Relative infected cells were determined by flow cytometry, calculated as a percentage of infected control. Virus titers were detected by IFA, evaluated by Reed-Muench method. Virus DNA copies were measured by qPCR. virus Cap expression was determined by western blot assay. Data were presented as means ± SD from three independent experiments (*P < 0.05, **P < 0.01 vs infected control)