Isolation and characterization of a highly pathogenic strain ofPorcine enteric alphacoronaviruscausing 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.

Attenuation and characterization of porcine enteric alphacoronavirus strain GDS04 via serial cell passage

“…The maintenance medium for PEAV propagation was DMEM supplemented with 10 μg/mL trypsin (Gibco, USA). The isolation and identification of PEAV GDS04 strain were reported previously by our laboratory (Xu et al, 2019a).…”

“…Serial virus passaging and plaque-purification in Vero cells were performed as previously described with some modifications (Lin et al, 2017;Xu et al, 2019a). Briefly, Vero cells were cultured in 6-well plates, and washed three times with sterile pH 7.4 1 × phosphate buffer saline (PBS) when 90% confluent.…”

“…Viral RNA shedding in piglet feces after infection with PEAV was detected as previously described with some modifications (Xu et al, 2019a). Briefly, total RNA was prepared from the supernatant of rectal swab from each piglet using a RNeasy kit (Magen, China) according to the manufacturer' instruction, and was treated with DNase I.…”

“…Histological and Immunohistochemical staining were performed as previously described with some modifications (Xu et al, 2019a). Briefly, tissue samples of jejunum of the piglets from the challenged and control groups were separated, routinely fixed in 10% formalin for 36 h at room temperature, dehydrated in graded ethanol, embedded in paraffin, cut in 5-μm sectioned, and mounted onto glass slides.…”

“…The clinical symptoms caused by PEAV in newborn piglets are similar to that by other porcine enteric pathogens such as PEDV and transmissible gastroenteritis virus (TGEV), which include vomiting, diarrhea, dehydration, and mortality rate as high as 90% (Gong et al, 2017;Zhou et al, 2018b). Since PEAV was detected (Gong et al, 2017), another two swine enteric HKU2-related CoVs named SADS-CoV and SeACoV were identified in the same region, causing same diarrheal disease as PEAV strain GDS04 by experimental infection (Pan et al, 2017;Xu et al, 2019a;Zhou et al, 2018b).…”