Abortive PDCoV infection triggers Wnt/β-catenin pathway activation, enhancing intestinal stem cell self-renewal and promoting chicken resistance

Abstract

Porcine deltacoronavirus (PDCoV) is an emerging coronavirus causing economic losses to swine industries worldwide. PDCoV can infect chickens under laboratory conditions, usually with no symptoms or mild symptoms, and may cause outbreaks in backyard poultry and wildfowl, posing a potential risk of significant economic loss to the commercial poultry industry. However, the reasons for such a subdued reaction after infection are not known. Here, using chicken intestinal organoid monolayers, we found that although PDCoV infects them nearly as well as porcine intestinal organoid monolayers, infection did not result in detectable amounts of progeny virus. In ex vivo and in vivo experiments using chickens, PDCoV infection failed to initiate interferon and inflammatory responses. Additionally, infection did not result in a disrupted intestinal barrier nor a reduced number of goblet cells and mucus secretion, as in pigs. In fact, the number of goblet cells increased as did the secreted mucus, thereby providing an enhanced protective barrier. Ex vivo PDCoV infection in chicken triggered activation of the Wnt/β-catenin pathway with the upregulation of Wnt/β-catenin pathway genes (Wnt3a, Lrp5, β-catenin, and TCF4) and Wnt target genes (Lgr5, cyclin D1, and C-myc). This activation stimulates the self-renewal of intestinal stem cells (ISCs), accelerating ISC-mediated epithelial regeneration by significant up-regulation of PCNA (transiently amplifying cells), BMI1 (ISCs), and Lyz (Paneth cells). Our data demonstrate that abortive infection of PDCoV in chicken cells activates the Wnt/β-catenin pathway, which facilitates the self-renewal and proliferation of ISCs, contributing to chickens' resistance to PDCoV infection.IMPORTANCEThe intestinal epithelium is the main target of PDCoV infection and serves as a physical barrier against pathogens. Additionally, ISCs are charged with tissue repair after injury, and promoting rapid self-renewal of intestinal epithelium will help to re-establish the physical barrier and maintain intestinal health. We found that PDCoV infection in chicken intestinal organoid monolayers resulted in abortive infection and failed to produce infectious virions, disrupt the intestinal barrier, reduce the number of goblet cells and mucus secretion, and induce innate immunity, but rather increased goblet cell numbers and mucus secretion. Abortive PDCoV infection activated the Wnt/β-catenin pathway, enhancing ISC renewal and accelerating the renewal and replenishment of shed PDCoV-infected intestinal epithelial cells, thereby enhancing chicken resistance to PDCoV infection. This study provides novel insights into the mechanisms underlying the mild or asymptomatic response to PDCoV infection in chickens, which is critical for understanding the virus's potential risks to the poultry industry.

Keywords: PDCoV; Wnt/β-catenin signaling pathway; abortive infection; chicken; intestinal barrier; intestinal organoids; intestinal stem cells; pig.

Fig 1

Isolation and comparison of chicken and pig intestinal 3D organoids and 2D organoid monolayers. (A) Schematic diagram illustrating intestinal organoid isolation and culture. (B) Time course of development of chicken and pig 3D intestinal organoids as observed by light microscopy. Scale bar = 50 µm. (C) Hematoxylin and eosin staining of chicken (upper) and pig (lower) intestinal organoids. Scale bar = 50 µm. (D) Intestinal organoids were subjected to immunofluorescence assay staining for enterocytes (E-Cad), proliferating cells (Ki67), Paneth cells (Lyz), and intestinal stem cells (Lgr5). Scale bar = 50 µm. (E) Schematic diagram of establishment of 2D intestinal organoid monolayers. (F) The dissociated chicken and pig organoids grow into a confluent intestinal organoid monolayer after 4 days. Scale bar = 100 µm.

Fig 2

Chicken intestinal organoid monolayers are susceptible to PDCoV infection but do not support replication. (A) Chicken and (B) pig intestinal 2D organoid monolayers were infected with PDCoV (multiplicity of infection [MOI] = 5) for 1 and 24 h. PDCoV-N protein appears green or red, and nuclei appear blue in IFA; cytopathic effect (CPE) is seen by light microscopy in infected porcine organoid monolayers (white arrows) but not in infected chicken organoid monolayers. Scale bar = 20 µm. Viral replication in (C) chicken and (D) pig intestinal 2D monolayer organoids was quantified by RT-qPCR at 1 and 24 h post-infection (MOI = 0.1). (E) Culture supernatants from PDCoV-infected chicken and porcine organoid monolayers at 24 hpi were titered by plaque assay in ST cells. (F–O) mRNA levels of interferons and their respective cellular receptors (IFN-β, IFNAR1, IFNAR2, IFN-λ, IFNLR1, and IL10RB), interferon-stimulated genes (MX1 and OASL), and proinflammatory factors (IL6 and TNF-α) from PDCoV-infected chicken and pig enteroids at 24 hpi. ** P < 0.01. ns, not significant.

Fig 3

Effect of PDCoV infection on the intestinal barrier integrity of chicken and pig intestinal organoid monolayers. (A–D) mRNA levels of goblet cell-secreted mucin (MUC2) and tight junction proteins (ZO1, occludin, and claudin-1) from PDCoV-infected chicken and pig intestinal organoid monolayers. (E) Illustration of the measurement of electrical resistance across the organoid monolayers in Transwell plates. TEER of (F) chicken and (G) porcine organoid monolayers over 48 h. Viral replication in (H) chicken and (I) pig intestinal organoid monolayers was quantified by RT-qPCR at 24 hpi. (MOI = 0.1). TEER values were calculated from PDCoV-infected (J) chicken and (K) pig intestinal organoid monolayers. (L) Schematic of the Transwell co-culture system. (M) Cytopathic effect of PDCoV-infected ST cells (in the lower chamber) as observed by light microscope (white arrows). Scale bar = 50 µm. (N) Viral replication in ST cells from panel M was quantified by RT-qPCR. **P < 0.01. ns, not significant.

Fig 4

PDCoV infection activates the Wnt/β-catenin signaling pathway, promoting the self-renewal of chicken intestinal stem cells ex vivo. (A–J) Levels of Wnt3a, Lrp5, β-catenin, TCF4, Lgr5, Cyclin D1, c-myc, PCNA, BMI1, and Lyz from mock- and PDCoV-infected chicken and pig intestinal organoid monolayers at 24 hpi. (K) IFA at 24 hpi of PDCoV-infected chicken and pig intestinal organoid monolayers. Scale bar = 20 µm. *P < 0.05, **P < 0.01. ns, not significant.

Fig 5

No obvious symptoms or pathological changes were observed in PDCoV-infected chickens compared to infected pigs. Necropsy of mock- and PDCoV-infected (A) chicken and (B) pig. The red arrows indicate intestinal bloating and thinning of the intestinal wall with accumulation of yellow fluid. H&E staining of small intestinal segments from uninfected and infected (C) chicken and (D) pig. The black arrows indicate severe pathological changes in the villus. Scale bar = 50 µm. mRNA levels of (E) PDCoV-M and (F) PDCoV-N in the small intestinal segments of mock- and PDCoV-infected chickens and piglets. Distribution of PDCoV in the small intestinal segments of mock- and PDCoV-infected (G) chicken and (H) pig. PDCoV-N protein stained deep yellow-brown, and the black arrows indicate large amounts of PDCoV-N protein staining in PDCoV-infected pig. Scale bar = 50 µm. (I–R) mRNA levels of interferons and their respective cellular receptors (IFN-β, IFNAR1, IFNAR2, IFN-λ, IFNLR1, and IL10RB), interferon-stimulated genes (MX1 and OASL), and proinflammatory factors (IL6 and TNF-α) from the jejunum of mock- and PDCoV-infected chickens and pigs. **P < 0.01. ns, not significant.

Fig 6

PDCoV infection promotes the self-renewal and proliferation of chicken intestinal stem cells via the Wnt/β-catenin pathway in vivo. Jejunum tissues from chickens and piglets were stained for (A) PAS and (B) MUC2. Scale bar = 50 µm. (C–F) mRNA levels of MUC2 (intestinal mucosal barrier) and ZO1, occludin, and claudin-1 (physical barrier) from jejunum tissues of mock- and PDCoV-infected chickens and piglets. (G) Morphology of microvilli as observed by transmission electron microscopy. Scale bar = 1 µm. (H) Morphology of jejunum microvilli as observed by scanning electron microscopy. Scale bar = 200 nm or 1 µm. (I–O) mRNA levels of Wnt3a, Lrp5, β-catenin, TCF4, Lgr5, cyclin D1, and c-myc from the jejunum of mock- and PDCoV-infected chicken and pig. (P) Jejunum tissues from mock- and PDCoV-infected chickens were stained for c-myc by immunochemistry (IHC). Scale bar = 50 µm. (Q) PCNA levels in homogenized jejunum tissues. (R) Jejunum tissues from mock- and PDCoV-infected chickens were stained by IHC for PCNA. Scale bar = 50 µm. (S and T) mRNA levels of BMI1 and Lyz in homogenized jejunum tissues. *P < 0.05, **P < 0.01. ns, not significant.

Fig 7

The Wnt/β-catenin pathway promotes the renewal and regeneration of the intestinal epithelium post-PDCoV infection. Porcine intestinal organoid monolayers were treated with 10 µM BML284 or ICG001 for 24 h. (A) The nuclear translocation of β-catenin was observed in organoid monolayer by confocal microscopy. Scale bar = 20 µm. (B–J) mRNA levels of Lgr5, Cyclin D1, c-Myc, PCNA, BMI1, Ki67, ZO-1, occludin, and claudin-1 in porcine intestinal organoid monolayers treated with dimethyl sulfoxide (DMSO), BML284, or ICG001 for 24 h. (K) TEER of BML284 or ICG001 in porcine intestinal organoid monolayers treated with DMSO, BML284, or ICG001 for 24 h. (L) mRNA levels of PDCoV-M in porcine intestinal organoid monolayers were treated with BML284 or ICG001 for 24 h then infected with PDCoV for 1 h. (M) The nuclear translocation of β-catenin was observed in organoid monolayer by confocal microscopy. Scale bar = 20 µm. (N–V) mRNA levels of Lgr5, Cyclin D1, c-Myc, PCNA, BMI1, Ki67, ZO-1, occludin, and claudin-1 in porcine intestinal organoid monolayers infected with PDCoV for 24 h after 24 h pretreatment with DMSO, BML284, or ICG001. (W) TEER of BML284 or ICG001 pretreated pig intestinal organoid monolayers infected with PDCoV for 24 h. *P < 0.05, **P < 0.01. ns, not significant.

Fig 8

Schematic diagram of abortive infection induced intestinal epithelial regeneration via the Wnt/β-catenin pathway in PDCoV-infected chicken intestine.