Isolation and Identification of Porcine Deltacoronavirus and Alteration of Immunoglobulin Transport Receptors in the Intestinal Mucosa of PDCoV-Infected Piglets

Abstract

Porcine deltacoronavirus (PDCoV) is a porcine enteropathogenic coronavirus that causes watery diarrhea, vomiting, and frequently death in piglets, causing serious economic losses to the pig industry. The strain CHN-JS-2017 was isolated and identified by cytopathology, immunofluorescence assays, transmission electron microscopy, and sequence analysis. A nucleotide sequence alignment showed that the whole genome of CHN-JS-2017 is 97.4%-99.6% identical to other PDCoV strains. The pathogenicity of the CHN-JS-2017 strain was investigated in orally inoculated five-day-old piglets; the piglets developed acute, watery diarrhea, but all recovered and survived. CHN-JS-2017 infection-induced microscopic lesions were observed, and viral antigens were detected mainly by immunohistochemical staining in the small intestine. The neonatal Fc receptor (FcRn) and polymeric immunoglobulin receptor (pIgR) are crucial immunoglobulin (Ig) receptors for the transcytosis ofimmunoglobulin G (IgG), IgA, or IgM. Importantly, CHN-JS-2017 infected five-day-old piglets could significantly down-regulate the expression of FcRn, pIgR, and nuclear factor-kappa B (NF-κB)in the intestinal mucosa. Note that the level of FcRn mRNA in the intestinal mucosa of normal piglets is positively correlated with pIgR and NF-κB. At the same time, the expressions of FcRn, pIgR, and NF-κB mRNA are also positively correlated in infected piglets. These results may help explain the immunological and pathological changes associated with porcine deltacorononirus infection.

Keywords: NF-κB; Neonatal Fc receptor; Porcine deltacoronavirus; polymeric immunoglobulin receptor.

Figure 1

Isolation and characterization of porcine deltacoronavirus (PDCoV) strain CHN-JS-2017. (A) The cytopathic effect (CPE) and immunofluorescence staining (IFA) of LLC-PK cells infected with PDCoV. (a) CPE of LLC-PK cells at 24 h after PDCoV infection. (b) Morphology of the mock-treated LLC-PK cells at 24 h. (c–h) PDCoV-infected or mock-treated LLC-PK cells were examined by IFA at 24 hpi using polyclonal against PDCoV N protein. (c and d) IFA with PDCoV N protein (green), (e and f) IFA with 4′,6-diamidino-2-phenylindole (DAPI)(blue), all views were visualized by using a fluorescence microscope. (B) PDCoV-infected or mock-treated LLC-PK cells were subjected to analysis with polyclonal antibodies against PDCoV N protein by Western blot at 24 hpi. (C) Electron microscopic image of PDCoV particles. The red arrow points to the virus particle.

Figure 2

A phylogenetic tree constructed on the basis of the complete genome (A) of the PDCoV strain from different countries and local areas. CHN-JS-2017 is represented by a triangle. The phylogenetic tree was constructed by the adjacency method in MEGA 7 (http://www.megasoftware.net). Bootstrap analysis was performed using 1000 replicates. (B) Three main deletions or insertions in the complete genome alignment. A multiple sequence alignment was constructed with ClustalW in the DNAStar software. The PDCoV strain CHN-JS-2017 is indicated in bold and highlighted with a box.

Figure 3

Fecal viral shedding and virus distribution in piglets challenged with PDCoV-JS-2017. (A) Diarrhea of piglets in different groups. The severity of diarrhea was scored based on visual examination; 0 = normal and no diarrhea; 1 = mild and fluidic diarrhea; 2 = severe watery diarrhea; scores of 1 or more were considered diarrheic. (B) Fecal virus shedding in PDCoV-challenged piglets. (C) Virus distribution in the small intestine of piglets challenged with PDCoV. DU-P: Duodenum proximal. DU-D: Duodenum distal. JE-P: jejunum proximal. JE-D: jejunum distal. IL-P: ileum proximal. IL-D: ileum distal.

Figure 4

Intestinal changes of infected and uninfected piglets challenged with PDCoV CHN-JS-2017. (A) Macroscopic damage of piglets challenged with PDCoV at 4 dpi. (B) Macroscopic view of 4 dpi negative control piglets. (C,D) Hematoxylin and eosin (H&E)-stained jejunal tissue sections of piglets challenged with PDCoV (C) or negative control piglets (D). (E,F) Immunohistochemically stained jejunal tissue sections of challenged piglets (E) or negative control piglets (F).

Figure 5

Expressions of nuclear factor-kappa B(NF-kB), neonatal Fc receptor (FcRn) and polymeric immunoglobulin receptor (pIgR) mRNA expression in the small intestinal mucosa of piglets induced by PDCoV CHN-JS-2017 at 4 dpi. The mRNA levels of NF-kB (A), FcRn (B), and pIgR (C) in the small intestinal mucosa were determined individually in each animal by RT-qPCR. All data are expressed as the mean ± SEM of three independent experiments. * p < 0.05, ** p < 0.01, relative to the mock-treated piglets. Relative amplification of the target genes expression levels was normalized to GAPDH expression. (D) The protein levels of NF-kB, FcRn, and pIgR at the small intestinal mucosa were determined individually in each animal by Western blot. DU-P: Duodenum proximal. DU-D: Duodenum distal. JE-P: jejunum proximal. JE-D: jejunum distal. IL-P: ileum proximal. IL-D: ileum distal.

Figure 6

Correlations between the mRNA levels of NF-kB, pIgR and FcRn in the intestinal mucosa of infected piglets. Significant positive correlations were found between NF-kB and FcRn (A) and FcRn and pIgR (B), but not between NF-kB and pIgR (C) in uninfected piglets; however, significant positive correlations were found between NF-kB and FcRn (D), FcRn and pIgR (E), and NF-kB and pIgR (F) in infected piglets. FcRn ΔCt = Ct _FcRn − Ct_GAPDH, NF-kB ΔCt = Ct _NF-kB – Ct _GAPDH, pIgR ΔCt = Ct _pIgR – Ct _GAPDH.