The Betacoronavirus PHEV Replicates and Disrupts the Respiratory Epithelia and Upregulates Key Pattern Recognition Receptor Genes and Downstream Mediators, Including IL-8 and IFN-λ

Figures

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  FIG 1. Morphology of air-liquid interface (ALI)-grown porcine respiratory epithelial cells (PRECs). (A and B) Relief contrast micrograph of nonadherent PRECs on transwell immediately after seeding (A) and on day 27 completely differentiated PRECs on transwell (B). (C) Paraffin-embedded H&E cross section of ALI-PRECs on transwell on day 27. (D) TEM images of day 27 ALI-PRECs, showing cilium axoneme, tight junctions, and desmosomes in a ciliated cell. Electron-dense secretory granules in some nonciliated cells, potential M cells; basal cells; intermediate cells. (E) Note the presence of mitochondria in ciliated cells, the powerhouse for ciliary movement. (F) Goblet cells were rich in mucin granules showing mucus. Representative images from three biological replicates.

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  FIG 2. Cellular and receptor characterization of ALI-PRECs. (A and B) Paraffin-embedded cross sections of day 27 ALI-PRECs stained for periodic acid-Schiff stain (A), with arrowhead showing magenta red for neutral mucins, and alcian blue (B); note arrowhead indicating the blue stain for acidic mucins. (C to E) Immunofluorescence composite images of various cell markers (mouse monoclonal primary antibodies), where nucleus is stained blue (4′,6-diamidino-2-phenylindole [DAPI]) and red shows goat anti-mouse secondary antibody labeled with Alexa Fluor 647. (C) Tight junction marker occludin; (D) cell proliferation marker Ki67; (E) epithelial cell marker pancytokeratin. (F to H) Expression of sialic acids; biotinylated Maackia amurensis II (MAL II) was detected with streptavidin DyLight 650 conjugate, specific for α-2,3-linked sialic acids (F), while the expression of α-2,6-linked sialic acids was detected using fluorescein isothiocyanate (FITC)-labeled Sambucus nigra (SNA) lectin (G); (H) respective composite images showing predominant expression of α-2,6-linked sialic acids on ALI-PRECs. (I to N) Immunocytochemistry images of the surface of day 27 ALI-PRECs in transwell. Cells fixed in 4%…

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  FIG 3. ALI-PREC susceptibility toward PHEV infection. (A to D) Completely differentiated ALI-PRECs (day 30) treated with infection medium only, i.e., without virus (mock inoculated) (A), and (B) with HA (titer of 128) of PHEV 67N for 24 hpi, (C) 36 hpi (arrow, area of CPE), and (D) 48 hpi (arrows, areas of CPE). (F) Magnification (×200) of CPE areas at 48 hpi (note the cytopathic effects on ALI-PRECs: [1] cytoplasmic stranding, [2] rounding of cells, [3] cluster of rounded cells, and [4] vacuolation of cells). Bar, 100 μm. Representative images from two biological and three technical replicates. (E) Detection of PHEV nucleocapsid gene using reverse transcription-qPCR. RNA from the subnatants collected from ALI-PRECs treated with PHEV was analyzed using RT-qPCR developed by ISU and Tetracore. Collection time is shown in hours. A sample volume of 5 μl of extracted sample RNA along with internal control was added to the qPCR master mix. All qPCRs were performed with a negative extraction control (NEC), a positive extraction control (PEC), and a no-template control (NTC) included in each run. Samples from two biological replicates and three technical replicates. Statistical analysis was…

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  FIG 4. Transcriptional regulation of early innate immune genes in ALI-PRECs inoculated with PHEV or mock inoculated with culture medium. Bar graph showing relative quantification (RQ) levels of 14 porcine genes (TLR3, TRL7, MDA5, MyD88, RIPK1, RIG1, IFNλ1, STAT1, OAS1, Mx1, CCL2, CCL5, IL8, and CXCL10) measured in ALI-PRECs treated with HA (titer of 128) of PHEV and mock inoculum at respective hours postinfection (x axes). RQ values were calculated using the 2−ΔΔCT method. The data are normalized against the geometric mean for three endogenous control genes (EIF3K, PPIA, and RPL10). This graph is generated from three technical replicates and two biological replicates (2 pigs). Statistical analysis was performed using Fisher’s LSD multiple-comparison test (GraphPad Prism 9.0.1). *, P value < 0.05; **, P value < 0.001; ***, P value < 0.005; ****, P value < 0.0001.

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  FIG 5. Transcriptional regulation of early innate immune genes in porcine tracheal tissues collected from pigs experimentally inoculated with PHEV or mock inoculated with culture medium. Bar graph showing relative quantification (RQ) levels of 12 porcine genes (CXCL10, IFNα1, IFNβ1, IFNγ, IFNλ1, IL8, MHC1, Mx1, OAS1, STAT1, TRL7, and TRAF6) measured in 5-day-postinfection snap-frozen pig trachea treated with HA (titer of 128) from PHEV and mock inoculum. RQ values were calculated using the 2−ΔΔCT method. The data are normalized against the geometric mean for three endogenous control genes (EIF3K, PPIA, and RPL10). This graph is generated from three technical replicates and two biological replicates (2 pigs). An amount of 20 ng of RNA (cDNA) is used in each SYBR-green PCR. Statistical analysis was performed using Fisher’s LSD multiple-comparison test (GraphPad Prism 9.0.1). *, P value < 0.05; **, P value < 0.005; and ****, P value < 0.0001.

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  FIG 6. IL-8 secretion into the subnatants of ALI-PRECs treated with PHEV. Subnatants from ALI-PRECs inoculated with PHEV (HA titer of 128) or mock inoculated were collected at 6, 24, 36, and 48 hpi, and the secretory levels of IL-8 were estimated using the Milliplex MAP porcine cytokine/chemokine magnetic bead panel (PCYTMG-23K-13PX; Millipore-Sigma). The minimum detectable concentration (MinDC) of IL-8 was 0.005 ng/ml. This graph was generated from three technical replicates and two biological replicates (2 pigs). Statistical analysis was performed using Fisher’s LSD multiple-comparison test (GraphPad Prism 9.0.1). **, P value < 0.005.