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Application of reverse transcription polymerase chain reaction to detect porcine epidemic diarrhea virus in Vero cell culture.. Okjin Kim, Chanhee Chae.[r]

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Journal of Veterinary Diagnostic

http://vdi.sagepub.com/content/11/6/537 The online version of this article can be found at:

DOI: 10.1177/104063879901100610 1999 11: 537

J VET Diagn Invest

Okjin Kim and Chanhee Chae Virus in Vero Cell Culture

Application of Reverse Transcription Polymerase Chain Reaction to Detect Porcine Epidemic Diarrhea

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537 Brief communications

J Vet Diagn Invest 11:537–538 (1999)

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

Okjin Kim, Chanhee Chae

Figure 1. Agarose gel electrophoresis of RT-PCR-amplified PEDV DNA products From left to right: lane 151-kb DNA ladder; lane 25PEDV-positive control; lane 35PEDV-negative control; lane Vero cell-adapted field isolate; lane 5 no Vero cell-adapted field isolate

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.3,5PED virus (PEDV),

a coronavirus, was identified in 1978 as the etiologic agent of the disease.4PEDV is unable to grow in porcine cell

cul-tures permissive to TGE virus A method to propagate PEDV in Vero cells was described in 10 years after the first report of PED.1However, the high cost and prolonged time

required to isolate and propagate PEDV in Vero cells have propelled the search for other diagnostic tests Virus isola-tion has important implicaisola-tions in the diagnosis, epidemiol-ogy, and control of disease The development of new tech-niques for the rapid identification of PEDV adaptation in Vero cells would greatly enhance PEDV detection The ob-jective of the present study was to describe the application of the reverse transcription polymerase chain reaction (RT-PCR) technique to detect PEDV in Vero cell culture

Cell growth medium consisted of minimal essential me-dium (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 di-hydrostreptomycin, mg/ml neomycin, 10,000 IU/ml poly-myxin) Virus infection medium consisted of MEM with 30 mM HEPES,a1% (v/v) 0.3 M NaOH, 0.3% (w/v) tryptose

phosphate broth,b 10mg/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 an-tibody test using an anti-PEDV–fluorescein isothiocyanate conjugate.cThe 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 9,0003g Before inoculation,

the cell growth medium of monolayered Vero cells grown in 25-cm2 flasks was removed, and the monolayers were

washed twice with cell growth medium The cells were in-oculated with ml of the homogenate per flask After ad-sorption in the dark for 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 days of incubation, cells and supernatant fluids were frozen and thawed times to release intracellular virus

From the Department of Veterinary Pathology, College of Veter-inary Medicine, Seoul National University, Suwon 441–744, Kyounggi Do, Republic of Korea

Received for publication December 7, 1998

into the medium The fluid was clarified by low-speed cen-trifugation (1,0003 g for 10 minutes) RNA was extracted

from the fluid with a commercial reagentd according to the

manufacturer’s instructions

RT-PCR was performed as previously described with slight modifications.2 For the first-strand cDNA synthesis, 1 ml of the RNA extracted from fluid was supplemented in a total reaction volume of 20ml with 13RT buffer (50 mM Tris-HCl, mM MgCl2, 30 mM KCl, mM dithiothreitol

[pH 8.3]), 0.5 mM (each) deoxynucleotide triphosphates (dNTPs), 2.5 mM random hexanucleotide mixture, 20 U of RNase inhibitor, and 50 U of Moloney murine leukemia vi-rus reverse transcriptase.eAfter incubation for 15 minutes at

42 C, the mixture was incubated for minutes at 99 C to denature the products The mixture was then chilled on ice The sense and antisense primers were 59 -GGACACATTC-TTGGTGGTCT-39 (nucleotides 1318–1338) and 59 -GTTTA-GACTAAATGAAGCACTTTC-39 (nucleotides 1665–1688), respectively.2 The primer set resulted in an amplified

frag-ment of 370 base pairs The composition of the PCR mixture was 20 ml of cDNA, ml of each primer (250 nM), 10ml of 103PCR buffer (10 mM Tris-HCl, 40 mM KCl, 1.5 mM MgCl2 [pH 8.3]), 0.8 ml of dNTP (0.2 mM), 2.5 U of Taq

polymerase, and 60ml of distilled water The PCR reaction for PEDV proceeded under the following conditions in a thermal cycler: cycle of minutes at 94 C, minutes at 58 C, and minutes at 72 C; 40 cycles of denaturation at 94 C for minute, annealing at 58 C for minute, and elongation at 72 C for minute; and cycle of minutes at 94 C, minutes at 58 C, and minutes at 72 C The amplified product was visualized by standard gel electro-phoresis of 10ml of the final reaction mixture on a 2%

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538 Brief communications

Table 1. Adaptation of PEDV to Vero cells during subsequent passages

No samples

RT-PCR-positive passages

Appearance of syncytia

Vero cell adaptation

10 10 2

1 4 10

2 2 2 2

Figure 2. Vero-cell adapted PEDV-induced cell fusion, hour postinoculation Hematoxylin and eosin, 2003

rose gel Amplified DNA fragments of specific sizes were located by UV fluorescence after staining with ethidium bro-mide Fragment lengths were verified by comparison with a digested lamda DNA standard on the same gel If the specific band exhibiting amplified PEDV nucleic acids was detected by RT-PCR (Fig 1), fluids were reinoculated onto new mon-olayered Vero cells Passage was done if the specific PCR-amplified band was not detected by RT-PCR The cell cul-ture-adapted PEDV strain V215/78fwas used as the standard

strain and grown in Vero cells as described above

The results of the study are summarized in Table Only PEDV (strain 971496) out of 41 samples was isolated from Vero cells The rest of the samples failed to grow in Vero cells PEDV-specific RNA was detected in culture superna-tant in sample 971496 from each of the passages by RT-PCR PEDV-specific RNA was not detected by RT-PCR in culture supernatant in the rest of the samples from to 10 passages One PEDV, SNUVR-971496, was detected by RT-PCR and isolated from intestinal samples Small syncytial cells were seen between and hours By 10 to 24 hours after inoculation, most cells had fused to form syncytia con-taining 10 to 20 nuclei in the fourth passage of Vero cells (Fig 2) These giant syncytia detached from the culture plate and died

The successful and rapid cultivation of the PEDV will greatly enhance our ability to study this important disease, which continues to adversely affect the productivity of the Korean swine industry The results represent the first report confirmation of the propagation of PEDV from a Korean field strain in a stable cell line and the formation of syncytia PEDV could be allowed to propagate in cell culture and provide larger quantities of virus for the development of vi-rus assays and serologic tests PEDV replicates in Vero cells in the presence of trypsin, as revealed by RT-PCR However, PEDV cannot be cultured in Vero cells without adaptation

after several passages Detection of PEDV RNA by RT-PCR is useful for determining whether blind passages were done Isolation and propagation of PEDV in cell culture is essential to provide large quantities of virus for detailed characteriza-tion of virus and control of disease RT-PCR is a useful technique to determine whether PEDV is still undergoing adaptation in Vero cells

Acknowledgment This work was supported by contract

research funds of the Research Institute for Veterinary Sci-ence (RIVS) from the College of Veterinary Medicine, Seoul National University, Republic of Korea

Sources and manufacturers a Sigma Chemical Co., St Louis, MO

b Difco Laboratories, Detroit, MI

c M B Pensaert, University of Ghent, Ghent, Belgium d Trizol LS Reagent, Gibco BRL, Grand Island, NY e Perkin-Elmer Cetus, Norwalk, CT

f Dr M Ackermann, University of Zuărich, Zuărich, Switzerland References

1 Hofmann M, Wyler R: 1988, Propagation of the virus of porcine epidemic diarrhea in cell culture J Clin Microbiol 26:2235–2239 Kweon CH, Lee JG, Han MG, Kang YB: 1997, Rapid diagnosis of porcine epidemic diarrhea virus infection by polymerase chain reaction J Vet Med Sci 59:231–232

3 Pensaert MB: Porcine epidemic diarrhea virus In: Virus Infec-tions of Porcines, ed Pensaert MB, pp 167–176 Elsevier, Am-sterdam, The Netherlands, 1989

4 Pensaert MB, Debouck P: 1978, A new coronavirus-like particle associated with diarrhea in swine Arch Virol 58:243–247 Wood CN: 1969, Transmissible gastro-enteritis of swine Vet Bull

39:239–248

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