Fig. 6: PDCoV infection was significantly reduced in BHK-21 cells overexpressing hAPN mutants (Y316A, K379A E426A, and W429A) compared to overexpression of wild-type hAPN.

a–j BHK-21 cell lines overexpressing wild-type or mutant hAPN proteins in 24-well plates were inoculated with 1 MOI of PDCoV CZ2020 strain. After incubation at 4 °C for 1 h, the plates were washed twice with PBS and then cultured in DMEM containing 0.5 μg/ml trypsin at 37 °C with 5% CO₂. At 8 hpi, the samples were subject to IFA. Porcine polyclonal antibodies were used to detect PDCoV (green), and cell nuclei was stained with DAPI (blue). Scale bar, 100 μm. k Cells from a to j were harvested with lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.1% SDS) supplemented with PMSF (Sigma-Aldrich). The cell lysates were boiled at 100 °C for 10 min then separated by SDS-PAGE. After electroblotting onto polyvinylidene difluoride membranes (Millipore), the membranes were blocked with 5% skimmed milk and then probed with indicated primary and secondary antibodies, and visualized using the chemiluminescent substrate. l The fluorescence intensity in a–j 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 < 0.001; ****p < 0.0001; ns no significance. m At 8 hpi, the samples from a to j 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; ns no significance. Values for fluorescence intensity and GE are provided as a Source Data file.