Isolation and characterization of a highly pathogenic strain of Porcine enteric alphacoronavirus causing watery diarrhoea and high mortality in newborn piglets

Abstract

Porcine enteric alphacoronavirus (PEAV) was first discovered in China in February 2017, and the origin and virulence of this novel porcine coronavirus were not fully characterized. Here, we isolated a strain of PEAV, named GDS04 that is identified by immunofluorescence and typical crown-shaped particles observed with electron microscopy. Genomic analysis reveals that PEAV GDS04 shares a close relationship with SADS-CoV and SeACoV. Furthermore, newborn piglets orally challenged with PEAV GDS04 developed typical clinical symptoms as watery diarrhoea in neonatal piglets. Viral RNA was detected in faeces and various tissues of the infected piglets. Moreover, macroscopic and microscopic lesions in whole intestinal tract were observed, and viral antigen could be detected in the small intestines by immunohistochemical staining and electron microscopy. Importantly, the mortality rate of inoculated-newborn piglets was 100% and half of the cohabiting piglets died. Collectively, we demonstrate that PEAV is highly pathogenic in newborn piglets.

Keywords: Porcine enteric alphacoronavirus (PEAV); newborn piglets; pathogenicity.

Figure 1

Cytopathic effects (CPE) and IFA staining on PEAV‐inoculated Vero cells. (a) Mock‐inoculated Vero cell culture showing normal cells. (b) PEAV‐inoculated Vero cells at 1 d.p.i. showing syncytium and cells detachment (indicated by arrows). Vero cells were mock‐inoculated (c) or inoculated with PEAV GDS04 (d). At 24 hr postinoculation, an immunofluorescence assay (IFA) was performed [Colour figure can be viewed at http://wileyonlinelibrary.com]

Figure 2

Electron micrographs of PEAV‐inoculated Vero cells. Crown‐shaped spikes of PEAV are visible (arrows). The sample was negatively stained with 3% phosphotungstic acid

Figure 3

Phylogenetic trees constructed on the basis of the s genes, n genes, and whole‐genome nucleotide sequences of PEAV GDS04 or other coronaviruses (CoVs). (a) Phylogenetic tree of the s gene. (b) Phylogenetic tree of the n gene. (c) Phylogenetic tree of the whole‐genome. The dendrogram was constructed using the neighbour‐joining method in the MEGA software package, version 5 (http://www.megasoftware.net). Bootstrap resampling (1,000 replication) was performed, and bootstrap values are indicated for each node. Reference sequence obtained from GenBank is indicated by strain name. The scale bar represents 1, 0.2, or 0.5 nucleotide substitutions per site

Figure 4

Reproduction of watery diarrhoea and faecal viral shedding in newborn piglets inoculated with PEAV GDS04 strain P12 via oral feeding. (a & c) Newborn piglets uninfected as control. (b & d) Watery diarrhoea (indicated by arrows) was observed at 3 and 7 d.p.i. with PEAV infection. (e) Average diarrhoea scores after PEAV infection [Colour figure can be viewed at http://wileyonlinelibrary.com]

Figure 5

The survival rate of newborn piglets postinfection with PEAV GDS04 P12. The mortality of newborn piglets in each group was recorded from 1 to 14 d.p.i

Figure 6

Virus shedding in rectal swabs and various tissues of PEAV‐inoculated piglets. (a) Ct values of group PEAV inoculation or as cohabitation contrast newborn piglet faecal swabs and viral RNA shedding in faecal swabs after PEAV inoculation or mock inoculation. (b) Virus distribution at 7 d.p.i. in newborn piglets challenged with PEAV

Figure 7

Electron micrographs of PEAV on jejunum of PEAV‐inoculated newborn piglet. (a) Electron micrographs of jejunum of a control newborn piglet at 7 d.p.i. (b & c) Electron micrographs of PEAV (indicated by arrows) on jejunum of a PEAV‐challenged newborn piglets at 7 d.p.i

Figure 8

Intestinal changes in newborn piglets inoculated with PEAV strain GDS04 P12. (a) Macroscopic picture of a control piglet at 7 d.p.i. (b) Thin‐walled intestinal tract (indicated by arrows) of a PEAV‐challenged newborn piglets at 7 d.p.i. (c‐g) Haematoxylin and eosin (H & E)‐stained intestinal tissue section of a control piglet at 7 d.p.i. (h‐l) H&E‐stained intestinal tissue section of a PEAV‐challenged piglet at 7 d.p.i. (Blunt intestinal villus was indicated by arrows). (m&n) Immunohistochemically stained jejunum or ileum tissue section of a control piglet at 7 d.p.i. (o&p) Immunohistochemically stained jejunum or ileum tissue section of a PEAV‐challenged piglet at 7 d.p.i [Colour figure can be viewed at http://wileyonlinelibrary.com]