J Vet Sci.  2018 Jul;19(4):577-581. 10.4142/jvs.2018.19.4.577.

Isolation and characterization of Korean porcine deltacoronavirus strain KNU16-07

• Jang G
• Kim SH
• Lee YJ
• Kim S
• Lee DS
• Lee KK
• Lee C

• KMID: 2417575
• DOI: http://doi.org/10.4142/jvs.2018.19.4.577

Abstract

Porcine deltacoronavirus (PDCoV) has emerged in several pig-raising countries and has been a causative pathogen associated with diarrheal diseases in South Korea since 2014. In the present study, we were able to isolate and cultivate a Korean PDCoV strain (KNU16-07) in cell culture and investigate its pathogenicity. PDCoV-inoculated piglets showed watery diarrhea accompanied by acute enteritis in the natural host. Sequencing analysis demonstrated the genetic stability of KNU16-07 for at least thirty serial passages.

Keyword

cell culture isolation; genome analysis; porcine deltacoronavirus; virulence

MeSH Terms

Cell Culture Techniques
Diarrhea
Enteritis
Korea
Serial Passage
Virulence

Figure

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  Fig. 1 Cytopathology and growth properties of porcine deltacoronavirus (PDCoV) isolate KNU16-07 in infected swine testicle (ST) cells. (A) Cytopathic effect (CPE) formation in ST cells infected with PDCoV KNU16-07-P5 or -P10. PDCoV-specific CPEs were observed daily and were photographed at 24 h post-infection (hpi) using an inverted microscope. The infected cells were subjected to immunofluorescence assay using the indicated N-specific monoclonal antibodies and examined using a fluorescence microscope. (B) Electron microscopy images of PDCoV KNU16-07. Purified virions were negatively stained with 2% phosphotungstic acid and viewed by using transmission electron microscope. (C) Virus titers at selected passages. ST cells were infected with PDCoV KNU16-07 harvested at the indicated passage numbers. At 24 hpi, the virus supernatants were collected and virus titers were determined. (D) One-step growth kinetics for the KNU16-07 strain. At the indicated times post-infection, culture supernatants were harvested and virus titers determined. Results are expressed as mean values from triplicate wells; error bars represent SD. TCID50, 50% tissue culture infective dose; P, passage. 200× (A), 100,000× (B). Scale bar = 100 nm (B).

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  Fig. 2 Macroscopic and microscopic examinations of intestines of piglets inoculated with the Korean porcine deltacoronavirus (PDCoV) strain KNU16-07. (A) Small intestine of an inoculated pig at 3 days post-inoculation (dpi) showing thin and transparent intestinal walls (arrows) and an extended stomach filled with curdled milk. (B and C) H&E stained jejunum of a KNU16-07-inoculated pig at 3 dpi showing acute diffuse and severe atrophic enteritis with moderate vacuolation (arrows) of enterocytes lining the epithelium of atrophied villi. (D) Detection of PDCoV antigen by immunohistochemistry analysis of jejunum tissue sections from a virus-inoculated piglet at 3 dpi. The PDCoV antigen signals (arrows) appear red and were detected in villous epithelial cells. 100× (B and D), 200× (C).

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