An alternative pathway of enteric PEDV dissemination from nasal cavity to intestinal mucosa in swine

Abstract

Porcine epidemic diarrhea virus (PEDV) has catastrophic impacts on the global pig industry. Although the fecal-oral route is generally accepted, an increased number of reports indicate that airborne transmission may contribute to PEDV outbreak. Here, we show that PEDV could cause typical diarrhea in piglets through a nasal spray. Firstly, PEDV can develop a transient nasal epithelium infection. Subsequently, PEDV-carrying dendritic cells (DCs) allow the virus to be transferred to CD3⁺ T cells via the virological synapse. Finally, virus-loaded CD3⁺ T cells reach the intestine through the blood circulation, leading to intestinal infection via cell-to-cell contact. Our study provides evidence for airborne transmission of a gastrointestinal infected coronavirus and illustrates the mechanism of its transport from the entry site to the pathogenic site.

Fig. 1

PEDV causes typical PED symptoms after intranasal inoculation. a Acute watery diarrhea and gross lesions of the intestine in piglets after PEDV intranasal inoculation at 66 hpi. b Hematoxylin and eosin (H&E) staining and IFA of the intestine of an intranasal inoculated pig at 66 hpi. PEDV antigen was located in the atrophied intestinal villus (white arrowhead). IN: intranasal inoculation. The scale bar represents 50 μm. Blue, DAPI; green, PEDV. c Viral RNA expression in different tissues of the diarrheic piglets after intranasal inoculation, n = 6 from 3 piglets per group. Data are the mean ± SD. Statistical significance was using one-way ANOVA. **P < 0.01. d Protein expression of PEDV in different tissues of diarrheic piglets as determined by Western blotting with a mouse mAb against N protein. At least three independent experiments were performed

Fig. 2

Replication and distribution of PEDV in piglets' nasal cavity. a RNA expression levels of PEDV in different tissues of piglets at different time points after intranasal PEDV challenge, n = 6 from 3 piglets per group. b For FACS analyses, pigs were nasally administered PEDV at indicated times. Then, individual cells isolated from the nasal mucosa (both left and right nasal cavity) were gated based on CK18⁺ (marker of epithelial cells), and viral infection was detected by PEDV N protein staining, n = 6 from 3 piglets per group. c Quantification of the FACS results as shown in (b). d, e IHC results showing the distribution pattern of PEDV in four cross-sections (I, II, III, and IV) of the piglet nasal cavity at 12 h post intranasal infection. The numbers of PEDV-positive cells (black arrowheads) in different part of the cavity were counted in six random fields (40×) from three cross-sections. The scale bar represents 100 μm. All data shown are the mean results ± SD from three independent experiments. Statistical significance was using one-way ANOVA. NS no significance, *P < 0.05, **P < 0.01

Fig. 3

NECM establishment and susceptibility to PEDV evaluation. a Schematic of differentiation and polarization of porcine NECs after seeding onto collagen-coated porous inserts. b Ciliated epithelial cells and the air–liquid interface after 6 days of NECs culture were observed by phase contrast microscopy. Expression of the tight and adherent junction marker protein ZO-1 (green) and a SEM image demonstrating the polarization of NECs. Cell nuclei were stained with DAPI (blue). c The PEDV replication in unpolarized NECs (MOI) = 1, polarized NECs cultures (MOI) = 1, and Vero cells (MOI) = 0.1 were observed by light microscopy. Bars, 20 μm. d The NECM and NECs were infected with PEDV (MOI 0.1) through the apical membrane. Media from both apical and basolateral chambers of the NECM were collected at various time points. e The viral titer was determined by a plaque assay and quantified. The data shown are the mean results ± SD from three independent experiments. The comparisons were performed with t-tests (two groups) or analysis of variance (ANOVA) (multiple groups). *P < 0.05, **P < 0.01

Fig. 4

Capture of luminal PEDV by lamina propria (LP) DCs. a At 12 hpi, virus-loaded DCs in NALTs were analyzed by FACS. DCs cells were gated based on FITC-labeled MHCII and PE-labeled SWC3a, viral uptake by DCs in piglets were further detected by APC-labeled PEDV N mAb. SSC: side scatter. b Moreover, IV sections of the nasal cavity were detected by IFA. DCs located in nasal mucosa were immunolabeled with anti-SWC3a mAb (red) and anti-MHCII mAb (green). Colonization of PEDV was detected by pig anti-PEDV polyclonal antibody (blue); enlarged images of the corresponding region in the white box in panel I (b) was magnified in panel II. Bars, 20 μm. c Schematic of the experimental setting used to study viral uptake in the coculture system. d DCs were collected from the coculture system at 1 hpi and detected by FACS. e The quantified FACS results are shown in (d), data shown are the mean results ± SD, n = 3 per group. Statistical significance was using one-way ANOVA. *P < 0.05, **P < 0.01. f Uptake of PEDV by submucosal DCs was also determined using IFA. DyLight 633-labeled PEDV (green) was observed within DCs (SWC3a⁺, red). Bars, 5 μm. g Schematic of the experimental setting used to study viral capture by DCs in the coculture system. h The filters were processed for CLSM after DyLight 633-labeled PEDV inoculation. In the 3D views, the TEDs of DCs (MHCII, green) were crossing the nasal epithelial cell monolayer (DAPI, blue). Internalization of viruses by TEDs was also observed (red frame) and enlarged in cross-sectional images. In contrast, basolateral DCs showed no response when inoculated with medium. Bars, 20 μm. i After 1 h of PEDV incubation, the filters from the coculture system were processed for TEM. An enlarged image of the region in the black frame in panels I and III was shown in panels II and IV, respectively. PEDV virions were marked with black arrowheads in panels II and IV. Bars, 1 μm (I); 0.5 μm (III); and 200 nm (II and IV). All results are representative of three independent experiments

Fig. 5

Role of CCL25 and NF-κB pathway in DCs recruitment and TED formation. a, b After incubation with medium, PEDV or PEDV plus antiserum on the apical side of the NECs for 20 h, basolateral DCs were collected. The expression of phenotypic marker including CD1a, SWC3a, and MHCII on DCs was analyzed by FACS. n = 3 per group. c After 1 h of PEDV incubation, qRT-PCR was used to determine the relative mRNA expression of a series of chemokines related to DCs in NECs, n = 3 per group. d The protein expression of CCL25, CCL20, and CXCL2 in the medium of the basolateral side were detected using ELISA kits, n = 3 per group. e After PEDV inoculation, the phosphorylation of p65 and nuclear p65 in the NECs of the coculture system was detected at the indicated time by Western blotting. f The coculture system was pretreated with an NF-κB inhibitor for 2 h (DMSO as a negative control) by upper compartment inoculation, followed by inoculation with DyLight 633-labeled PEDV for 1 h. Basolateral DCs were collected and analyzed by FACS. g IFA of filters from the coculture system and a three-dimensional (3D) rendering of representative fields showing that basolateral DCs sent dendrites (MHCII, green) to creep through ECs in response to PEDV and/or inhibitor. h Quantitative analysis of TEDs was performed. The number of TEDs was counted from five random fields of view at a unit area (0.078 mm²) for each of three individual filters and depicted as a dot plot with each dot representing a field. All data are the mean ± SD, comparisons performed with one-way ANOVA. *P < 0.05, **P < 0.01. The results are from at least three different experiments

Fig. 6

CD3⁺ T cells acquire PEDV from DCs and enter the peripheral blood. a After PEDV intranasal inoculation, blood samples were collected at the indicated time during which PBMCs were isolated and analyzed by FACS. The cells were gated based on CD3⁺ T cells (Q1), and gated cells were further selected based on the uptake of PEDV. b Quantification of the FACS results is shown in panel (a), n = 6 from 3 piglets per group. c, d PEDV-pulsed DCs were cocultured with CD3⁺ T cells to detect the transmission of DC-associated PEDV to CD3⁺ T cells. Then, PEDV transmission was quantified by analyzing the CD3⁺ PEDV⁺ population, n = 3 wells per group. e Furthermore, after coculturing with CD3⁺ T cells for 2 h, DCs were separated by magnetic beads separation (MACS). The remaining CD3⁺ T cells remained in culture, and PEDV RNA expression levels were determined at the indicated time; the results of a representative experiment are shown, n = 3 per group. f FACS profiles of conjugate formation between CFSE-labeled T cells (x-axis) and PKH26-labeled PEDV-pulsed DCs (y-axis). Conjugates are apparent in the upper right quadrant, and the percentages of cells are shown. g Conjugation between CFSE-labeled T cells (green) and PKH26-labeled DCs (red) is indicated by CLSM, and the typical conjugation structure between the two cells is enlarged in panel (a). PEDV (blue) was detected in the conjugate structure using an antibody against N protein. Bars, 20 μm. h Electron micrographs of DC-T cell conjugates. PEDV-carrying DCs were cocultured with CD3⁺ T cells for 1 h and collected for TEM analysis. The sites of interaction between DCs and T cells showed firm interactions (black frame). DCs and T cell membranes were closely apposed at the tips of the protrusions (black asterisk). The fine ultrastructure of the virus particles (black arrowheads) was observed in the T cells adjacent to the conjugate structure. Bars, 2 μm. All data are the mean ± SD, comparisons performed with one-way ANOVA. *P < 0.05. The results are representative of three independent experiments

Fig. 7

PEDV-loaded T cells reach the intestine and mediate transfer infection. a Through an autotransfusion competition experiment, the percentage of PEDV-treated (CFSE) and PBS-treated (CM-DiI) cells among PBMCs and in the jejunum were analyzed by FACS. b The numbers of cells shown in (a) were quantified, n = 3 piglets per group. c After the autotransfusion competition experiment, frozen sections of the jejunum from mock-injected and fluorescence-labeled cells injected groups were stained with DAPI (blue) and observed by fluorescence microscopy. PEDV-treated (CFSE) CD3⁺ T cells were marked with white arrowheads. Bars, 50 μm. d Schematic of the model to study PEDV-carrying CD3⁺ T cell transmission of the virus to Vero cells. PEDV-pulsed DCs were cocultured with CD3⁺ T cells for 2 h and donor DCs were removed by MACS. Then, the remaining CD3⁺ T cells were cocultured with Vero cells by two methods (contact and noncontact coculture). e PEDV⁺ CD3⁺ T cells in the contact and noncontact groups were detected by FACS at the indicated times. f In addition, viral titers in the supernatant of the coculture system were measured by a plaque assay, n = 3. g PEDV RNA expression in Vero cells was evaluated by qRT-PCR at 96 h, n = 3. h At 48 hpi, Vero cells in the noncontact and contact groups were observed. LM light microscopy. i Vero cells (contact group) displayed thin cell bodies with long, branched membrane protrusions toward the T cells at 24 and 96 h. Bars, 20 μm. j TEM image showing the morphological features of sorted T cells cocultured with medium or PEDV-treated DCs. Bars, 1 μm. When cocultured for 96 h, Vero cells from the contact group were processed for TEM, which revealed an accumulation of virus particles in the cytoplasm. Some virus particles are indicated by a white asterisk. Bars, 1 μm. All data represent the mean ± SD, comparisons performed with t-tests (two groups) or analysis of variance (ANOVA) (multiple groups). *P < 0.05, **P < 0.01. Data were combined from at least three independent experiments unless otherwise stated

Fig. 8

Schematic of the proposed mechanism for PEDV transportation in swine. When airborne PEDV enters the nasal cavity, the virus accumulates and propagates in NECs and is released from the apical side of NECs. DCs may play an important role in helping the virus enter the nasal mucous and be transferred to CD3⁺ T cells. Furthermore, virus-carrying T cells enter the blood and reach the intestine through lymphocyte recirculation. Finally, the virus-carrying CD3⁺ T cells can transfer the virus to intestinal epithelial cells, causing typical PED symptoms. ELV efferent lymphatic vessels, CLN Cervical lymph node, IECs intestinal epithelial cells, NECs nasal epithelial cells, HEV high endothelial venules