Application of reverse transcription polymerase chain reaction to detect porcine epidemic diarrhea virus in Vero cell culture.. Okjin Kim, Chanhee Chae.[r]
Trang 1Investigation 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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Trang 2J 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 1 5 1-kb DNA ladder;
lane 2 5 PEDV-positive control; lane 3 5 PEDV-negative control;
lane 4 5 Vero adapted field isolate; lane 5 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, 5 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 9,0003 g 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 1 ml of the homogenate per flask After
ad-sorption 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
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 13 RT buffer (50 mM
Tris-HCl, 8 mM MgCl2, 30 mM KCl, 1 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 5 minutes at 99 C to denature the products The mixture was then chilled on ice The sense and antisense primers were 5
9-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, 2 ml of each primer (250 nM), 10 ml
of 103 PCR 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: 1 cycle of 2 minutes at 94 C, 2 minutes at
58 C, and 2 minutes at 72 C; 40 cycles of denaturation at
94 C for 1 minute, annealing at 58 C for 1 minute, and elongation at 72 C for 1 minute; and 1 cycle of 2 minutes
at 94 C, 2 minutes at 58 C, and 2 minutes at 72 C The amplified product was visualized by standard gel electro-phoresis of 10ml of the final reaction mixture on a 2%
Trang 3aga-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
3
6
1
4
3
2
2
1 2 3 4 4 5 6 9 10
2 2 2 2 1 2 2 2 2
2 2 2 2 1 2 2 2 2
Figure 2. Vero-cell adapted PEDV-induced cell fusion, 9 hour postinoculation Hematoxylin and eosin, 200 3.
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 1 Only
1 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 4 passages by
RT-PCR PEDV-specific RNA was not detected by RT-PCR in
culture supernatant in the rest of the samples from 1 to 10
passages One PEDV, SNUVR-971496, was detected by
RT-PCR and isolated from intestinal samples Small syncytial
cells were seen between 6 and 7 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 Zu¨rich, Zu¨rich, 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.
2 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.
5 Wood CN: 1969, Transmissible gastro-enteritis of swine Vet Bull 39:239–248.