Fig. 5: Y316, K379, E426, and W429 are the key residues on hAPN that inhibit PDCoV replication.

a–i PDCoV (MOI = 0.1) was preincubated with soluble wildtype or mutant hAPN proteins at a concentration of 80 μg/ml at 37 °C for 1 h, then the virus-APN protein mixtures were added onto a monolayer of LLC-PK1 cells in 24-well plates. After incubation at 4 °C for 1 h, the plates were washed twice with PBS and then cultured in DMEM containing 7.5 μg/ml trypsin at 37 °C with 5% CO₂. At 8 h post-infection (hpi), the samples were subject to immunofluorescence assay (IFA). Porcine polyclonal antibodies were used to detect PDCoV (green), and cell nuclei was stained with DAPI (blue). Scale bar, 50 μm. j The fluorescence intensity in a–i was determined by software Image J version 1.52i. Data are expressed as mean ± SD, n = 3. Error bars denote standard deviations for triplicate samples. An unpaired two-tailed t-test was used to determine the statistical significance. *p < 0.05; **p < 0.01; ***p < 0.001; ns no significance. k At 2 hpi, the samples from a–i were collected for RT-qPCR to determine the PDCoV genome equivalents (GE). Data are expressed as the mean ± SD, n = 3. Error bars denote standard deviations for triplicate samples. An unpaired two-tailed t-test was used to determine the statistical significance. *p < 0.05; **p < 0.01; ***p < 0.001; ns no significance. Values for fluorescence intensity and GE are provided as a Source Data file.