A Highly Pathogenic Strain of Porcine Deltacoronavirus Caused Watery Diarrhea in Newborn Piglets

Abstract

Porcine deltacoronavirus (PDCoV) is a newly identified virus that causes watery diarrhea in newborn piglets and results in significant economic losses to the pig industry. Since first reported in Hong Kong in 2012, PDCoV has been subsequently detected in USA, South Korea, Thailand, and mainland China. Here we isolated a strain of PDCoV, named CHN-GD-2016, from the intestinal content of a diseased newborn piglet with severe diarrhea in a pig farm in Guangdong, China. PDCoV CHN-GD-2016 could be identified by immunofluorescence with PDCoV specific rabbit antisera, and typical crown-shaped particles with spiky surface projections of this PDCoV were observed with electron microscopy. Genomic analysis showed that the PDCoV CHN-GD-2016 was closely related to other Chinese PDCoV strains, with the highest sequence similarity with the strain CHN/Tianjin/2016. Importantly, inoculation of newborn piglets with 1 × 10⁵ TCID₅₀ of CHN-GD-2016 by oral feeding successfully reproduced clear clinical symptoms, including vomiting, dehydration, and severe diarrhea in piglets. In addition, the virus RNA in rectal swabs from 1 to 7 days post inoculation was detected, macroscopic and microscopic lesions in small intestine were observed, and viral antigen was also detected in the small intestines with immunohistochemical staining. Collectively, the data show in this study confirms that PDCoV is present in Guangdong, China and is highly pathogenic in newborn piglets.

Keywords: Genomic analysis; Pathogenicity; Porcine deltacoronavirus (PDCoV); Southern China.

Fig. 1

Molecular identification of viral pathogens from intestinal contents. The N gene was amplified by RT-PCR using primers specific for PDCoV (A), PEDV (B). Lanes 1 through 24 represent the PCR products of DNA of 24 samples, PRRSV or PEDV as controls.

Fig. 2

Cytopathic effects (CPE) and IFA staining on PDCoV-inoculated LLC-PK cells. A Mock-inoculated LLC-PK cell culture showing normal cells. B PDCoV inoculated LLC-PK cells at day 6 p.i. showing rounded and clustered cells (indicated by arrows). LLC-PK cells were mock-inoculated (C) or inoculated with PDCoV CHN-GD-2016 (D). At 24 h postinoculation, an immunofluorescence assay (IFA) was performed (PDCoV antigen was indicated by arrows).

Fig. 3

Electron micrographs of PDCoV inoculated LLC-PK cells. Crown-shaped spiked of PDCoV are visible (indicated by arrows). The sample was negatively stain with 3% phosphotungstic acid.

Fig. 4

Sequence alignments partial spike genes of different porcine deltacoronavirus strains and phylogenetic tree constructed on the basis of the whole-genome nucleotide sequences of 27 PDCoV strains from different countries. A A multiple sequence of partial spike (S) genes was constructed with Clustal W in the DNAStar software. Red rectangles indicate the 3-nt deletion sites. B The dendrogram was constructed using the neighbor-joining method in the MEGA software package, version 5 (http://www.megasoftware.net). Bootstrap resampling (1000 replication) was performed, and bootstrap values are indicated for each node. Reference sequence obtained from GenBank are indicated by strain name. The scale bar represents 0.002 nucleotide substitutions per site.

Fig. 5

Reproduction of watery diarrhea and fecal viral shedding in newborn piglets inoculated with PDCoV CHN-GD-2016 strain via oral feeding. A Four newborn piglets uninfected as control. B Watery diarrhea (indicated by arrows) was observed at day 3 p.i. with PDCoV infection. C Ct values of group PDCoV inoculation newborn piglet fecal swabs and viral RNA shedding in fecal swabs after PDCoV inoculation or mock inoculation.

Fig. 6

Intestinal changes in newborn piglets inoculated with PDCoV strain CHN-GD-2016. A Thin-walled small intestines (indicated by arrows) of a PDCoV-challenged newborn piglets at day 7 p.i.. B Macroscopic picture of a control piglet at day 7 p.i.. C Hematoxylin and eosin (H&E)-stained jejunum tissue section of a PDCoV-challenged piglet at day 7 p.i. (Blunt intestinal villus and hyperemia were indicated by arrows). D H&E-stained jejunum tissue section of a control piglet at day 7 p.i.. E Immunohistochemically stained jejunum tissue section of a PDCoV-challenged piglet at day 7 p.i. (PDCoV antigen was indicated by arrows). F Immunohistochemically stained jejunum tissue section of a control piglet at day 7 p.i.