Novel Approach for Isolation and Identification of Porcine Epidemic Diarrhea Virus (PEDV) Strain NJ Using Porcine Intestinal Epithelial Cells

Abstract

Porcine epidemic diarrhea virus (PEDV), which is the causative agent of porcine epidemic diarrhea in China and other countries, is responsible for serious economic losses in the pork industry. Inactivated PEDV vaccine plays a key role in controlling the prevalence of PEDV. However, consistently low viral titers are obtained during the propagation of PEDV in vitro; this represents a challenge to molecular analyses of the virus and vaccine development. In this study, we successfully isolated a PEDV isolate (strain NJ) from clinical samples collected during a recent outbreak of diarrhea in piglets in China, using porcine intestinal epithelial cells (IEC). We found that the isolate was better adapted to growth in IECs than in Vero cells, and the titer of the IEC cultures was 10^4.5 TCID₅₀/0.1 mL at passage 45. Mutations in the S protein increased with the viral passage and the mutations tended towards attenuation. Viral challenge showed that the survival of IEC-adapted cultures was higher at the 45th passage than at the 5th passage. The use of IECs to isolate and propagate PEDV provides an effective approach for laboratory-based diagnosis of PEDV, as well as studies of the epidemiological characteristics and molecular biology of this virus.

Keywords: isolation and identification; porcine epidemic diarrhea virus (PEDV) NJ strain; porcine intestinal epithelial cells.

Figure 1

Isolation and identification of PEDV strain NJ in Vero cells and intestinal epithelial cell (IEC) cultures. (A) Identification of PEDV cultured in Vero cells at serial passages, by reverse transcription PCR (RT-PCR); (B) Identification of PEDV cultured in IEC at serial passages, by RT-PCR; (C) Control (uninfected) Vero cells; (D) PEDV-infected Vero cells; (E) Control (uninfected) IECs; (F) PEDV-infected IECs.

Figure 2

Viral titers of PEDV strain NJ propagated in IECs post-serial passages. All the results of a representative experiment performed with triplicate samples are shown.

Figure 3

Images of PEDV strain NJ particles and PEDV strain NJ-infected IEC produced by electron microscopy. (A) Virions in culture media of IECs infected with PEDV strain NJ, as shown by the arrow; Bar = 200 nm. Magnification, ×100,000; (B) Thin section of IECs infected with PEDV strain NJ 24 h post-infection; many of the virus particles possessed a dense core and gathered in the cytoplasm as shown by the arrow; Bar = 500 nm. Magnification, ×50,000.

Figure 4

Detection of PEDV strain NJ in IECs by immunofluorescence assay at 24 h post-infection; mouse anti-PEDV S protein monoclonal antibody and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse immunoglobulin G (IgG) were respectively used as primary and secondary antibodies, followed by counterstaining with 4,6-diamidino-2-phenylindole (DAPI). (A) PEDV strain NJ-infected IECs; (B) Non-infected IECs.

Figure 5

Phylogenetic analysis of PEDV strain NJ based on the S gene.

Figure 6

The clinical signs and necropsies of results of pigs infected with IEC-adapted NJ at 45th passage and the survival percentage of piglets after challenge. (A) Piglet with diarrhea and significantly dispirited status; (B) Watery diarrhea; (C) The intestinal tracts of infected piglets were thin and transparent; (D) Mesenteric congestion; (E) Survival percentage of piglets after challenge.