3.2 Selection of the Best Sampling Time for the Proteomic Analysis Following PDCoV Infection In order to determine the optimal time point for the proteomic analysis following PDCoV infection, IPEC-J2 cells were inoculated with PDCoV at an MOI of 0.1 and microscopically observed for CPE at 4, 8, 12, 24, and 36 hpi. Meanwhile, the cell supernatants at the same time points were collected and used to measure the proliferation dynamics of PDCoV in IPEC-J2 cells. Compared to mock-infected cells, PDCoV-inoculated IPEC-J2 cells began to exhibit slight CPE at 8 hpi, and the CPE gradually became increasingly evident as the infection progressed. Obvious CPEs were observed at 12 hpi and became more evident at 24 and 36 hpi, which were characterized by cell rounding, enlarging, and granular degeneration of the cytoplasm that occurred either singly or in different-sized clusters, usually forming cell masses, followed by cell shrinkage and increased detachment. These CPEs were in agreement with those reported by Jung et al.,8 and resembled those observed in PDCoV-infected swine testicular (ST) and LLC-PK1 cells.34 However, from 36 hpi onward, the majority of the cells became detached and floated in the medium (Figure 1A). The proliferation of PDCoV in IPEC-J2 cells was verified by IFA using a mAb raised against the PDCoV nucleocapsid protein, and the results demonstrated that almost all cells became infected at 24 hpi (Figure 1B). The one-step growth curve further revealed that the virus titer reached a plateau of ∼107.5 TCID50/mL at 24 hpi, followed by a gradual and continuous decline (Figure 1C). In general, the time point at which viral proliferation stays high but no obvious cellular membrane or cytoskeleton rearrangement occurs is the optimal sampling time for a proteomic analysis.22 On the basis of the above experimental results, we therefore chose 24 hpi as the optimal time point for the proteomic analysis of PDCoV-infected IPEC-J2 cells. Figure 1 Proliferation of PDCoV in IPEC-J2 cells. (A) Morphological changes in IPEC-J2 cells infected with the PDCoV CHN-HN-1601 strain at an MOI of 0.1 TCID50/cell or mocked infected for 4, 8, 12, 24, and 36 h, respectively. Scale bars, 10 μm. (B) Confirmation of PDCoV proliferation in IPEC-J2 cells by immunofluorescence assays using the mAb 1A3 specific for PDCoV (α-N) and an Alexa Fluor 488-labeled goat antimouse IgG, and mock-infected cells at 24 h was used as a negative control. Cell nuclei were counterstained with DAPI (blue). Scale bars, 10 μm. (C) One-step growth curve of PDCoV in IPEC-J2 cells at the indicated time points following viral infection. The titer of virus was presented as TCID50/mL, and the data were recorded as means ± SD from three independent experiments.