Susceptibility of Cell Lines from Different Species to Porcine Deltacoronavirus

Figures

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  Figure 1. Effects of trypsin on PDCoV replication in cell lines from distinct host origins. Cells from human, murine, and feline were seeded into 24-well plates and cultured in a humidified atmosphere at 37 °C with 5% CO2. When the cells reached 100% confluency, the cells were infected with PDCoV at an MOI of 0.1. After 1 h of incubation, the cells were washed with PBS for two times to remove unbound viruses. Subsequently, 1 mL of DMEM with serial concentrations of trypsin (0, 0.5, 1, 2.5, 5, and 10 μg/mL) was added to the plates and incubated at 37 °C. At 24 h post-infection, cells were fixed and subjected to IFA to detect PDCoV NP protein (green), with nuclei stained with DAPI (blue). Representative images are shown for each cell line: (A) H460 cells, (B) H1299 cells, (C) Caco-2 cells, (D) HepG2 cells, (E) U251 cells, (F) MCF-7 cells, (G) TE-10 cells, (H) KYSE-30 cells, (I) KYSE-410 cells, (J) CT26.WT cells, (K) MC-38 cells, (L) Neuro-2a cells, (M) CRFK cells, and (N) F81 cells. Scale bar, 100 μm.

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  Figure 2. Susceptibility of human lung and colorectal cells to PDCoV infection. H460 (A–D), H1299 (E–H), and Caco-2 (I–L) cells were seeded in 24-well plates and maintained in a humidified 37 °C incubator with 5% CO2. At 100% confluency, cells were inoculated with PDCoV at an MOI of 0.1. Following 1 h of adsorption at 4 °C, cells were washed twice with PBS to remove unbound virus, then overlaid with 1 mL of DMEM supplemented with 2 μg/mL trypsin and incubated at 37 °C. At the indicated time points post-infection, cells and/or culture supernatants were harvested for IFA, Western blotting, RT-qPCR, and TCID50 assay to characterize the replication kinetics of PDCoV. Scale bar, 100 μm.

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  Figure 3. Susceptibility of human cells originating from different organs to PDCoV infection. HepG2 (A–D), U251 (E–H), MCF-7 (I–L), TE-10 (M–P), KYSE-30 (Q–T), and KYSE-410 (U–X) cells were seeded in 24-well plates and maintained in a humidified 37 °C incubator with 5% CO2. At 100% confluency, cells were inoculated with PDCoV at an MOI of 0.1. Following 1 h of adsorption, cells were washed twice with PBS to remove unbound virus, then overlaid with 1 mL of DMEM supplemented with 2 μg/mL trypsin and incubated at 37 °C. At the indicated time points post-infection, cells and/or culture supernatants were harvested for IFA, Western blotting, RT-qPCR, and TCID50 assay to characterize the replication kinetics of PDCoV. Scale bar, 100 μm.

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  Figure 4. Susceptibility of murine cell lines CT26.WT, MC-38, and Neuro-2a to PDCoV infection. CT26.WT (A–D), MC-38 (E–H), Neuro-2a (I–L) cells were seeded in 24-well plates and maintained in a humidified 37 °C incubator with 5% CO2. At 100% confluency, cells were inoculated with PDCoV at an MOI of 0.1. Following 1 h of adsorption, cells were washed twice with PBS to remove unbound virus, then overlaid with 1 mL of DMEM supplemented with 2 μg/mL trypsin and incubated at 37 °C. At the indicated time points post-infection, samples were harvested for IFA, Western blotting, RT-qPCR, and TCID50 assay to characterize the replication kinetics of PDCoV. Scale bar, 100 μm.

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  Figure 5. Susceptibility of feline cell lines CRFK and F81 to PDCoV infection. CRFK (A–D) and F81 (E–H) cells were seeded in 24-well plates and maintained in a humidified 37 °C incubator with 5% CO2. At 100% confluency, cells were inoculated with PDCoV at an MOI of 0.1. Following 1 h of adsorption, cells were washed twice with PBS to remove unbound virus, then overlaid with 1 mL of DMEM supplemented with 2 μg/mL trypsin and incubated at 37 °C. At the indicated time points post-infection, samples were harvested for IFA, Western blotting, RT-qPCR, and TCID50 assay to characterize the replication kinetics of PDCoV. Scale bar, 100 μm.

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  Figure 6. Phylogenetic and expression profile of APN in cell lines of distinct host origins. (A) Phylogenetic analysis of APN nucleotide sequences from representative vertebrate species. The tree was constructed using the neighbor-joining method with 1000 bootstrap replicates, and bootstrap values are labeled at internal nodes. The scale bar indicates 0.05 nucleotide substitutions per site. (B) Relative APN mRNA expression levels in a panel of mammalian and avian cell lines. Expression was normalized to LLC-PK1 cells (set as 1.0). Data are presented as mean ± SD from at least three independent experiments. Statistical significance relative to LLC-PK1 cells was analyzed by one-way ANOVA test: ** p < 0.01; *** p < 0.001; **** p< 0.0001.