Application of reverse transcription polymerase chain reaction to detect porcine epidemic diarrhea virus in Vero cell culture.

Abstract

Porcine epidemic diarrhea (PED) is a contagious diarrheal disease of swine. The disease is very similar to transmissible gastroenteritis (TGE), with high mortality in swine of all ages and high morbidity in neonates. 3,5 PED virus (PEDV), a coronavirus, was identified in 1978 as the etiologic agent of the disease. 4 PEDV is unable to grow in porcine cell cultures permissive to TGE virus. A method to propagate PEDV in Vero cells was described in 10 years after the first report of PED. 1 However, the high cost and prolonged time required to isolate and propagate PEDV in Vero cells have propelled the search for other diagnostic tests. Virus isolation has important implications in the diagnosis, epidemiology, and control of disease. The development of new techniques for the rapid identification of PEDV adaptation in Vero cells would greatly enhance PEDV detection. The objective of the present study was to describe the application of the reverse transcription polymerase chain reaction (RTPCR) technique to detect PEDV in Vero cell culture. Cell growth medium consisted of minimal essential medium (MEM) buffered with 20 mM HEPES and 0.2% (w/v) bicarbonate, supplemented with 5% (v/v) fetal bovine serum and antibiotics (10,000 IU/ml and penicillin, 10 mg/ml dihydrostreptomycin, 5 mg/ml neomycin, 10,000 IU/ml polymyxin). Virus infection medium consisted of MEM with 30 mM HEPES, a 1% (v/v) 0.3 M NaOH, 0.3% (w/v) tryptose phosphate broth, b 10 mg/ml trypsin (1:250), and antibiotics. Forty-one piglets from 35 farms were used to isolate PEDV. The presence of PEDV in the intestinal tissue of each of the pigs was confirmed by direct immunofluorescence antibody test using an anti-PEDV‐fluorescein isothiocyanate conjugate. c The mucosa and the content of the small intestine were collected and pooled, diluted 1:5 in phosphate-buffered saline (0.01 M, pH 7.2), ground by homogenization, and centrifuged for 20 minutes at 9,000 3 g. Before inoculation, the cell growth medium of monolayered Vero cells grown in 25-cm 2 flasks was removed, and the monolayers were washed twice with cell growth medium. The cells were inoculated with 1 ml of the homogenate per flask. After adsorption in the dark for 2 hours at room temperature, virus infection medium was added (5 ml/flask) without removing the virus inoculum. Because trypsin is thermolabile, 80% of virus infection medium was changed daily. Inoculated cell cultures were checked microscopically for cytopathic effects (CPE) daily. If the cell layer did not show CPE after 4 days of incubation, cells and supernatant fluids were frozen and thawed 3 times to release intracellular virus