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ComparisonofPolymeraseChainReactionand
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Investigation
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Comparison of Polymerase Chain Reaction and Virus Isolation for Detection of Epizootic Hemorrhagic
Disease Virus in Clinical Samples from Naturally Infected Deer
Imadeldin E. Aradaib, Geoffery Y. Akita, James E. Pearson and Bennie I. Osburn
J VET Diagn Invest 1995 7: 196
DOI: 10.1177/104063879500700205
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J Vet Diagn Invest 7:196-200 (1995)
Comparison of polymerase chain reaction and virus
isolation for detection of epizootic hemorrhagic disease
virus in clinical samples from naturally infected deer
Imadeldin E. Aradaib, Geoffery Y. Akita, James E. Pearson,
Bennie I. Osburn
Abstract. We compared our recently reported reverse transcriptase polymerase chain reaction (PCR)-based
assay for detection of epizootic hemorrhagic disease virus (EHDV) in clinical samples with different virus
isolation (VI) procedures. Thirty-six blood samples and 1 spleen sample from deer were assessed by the EHDV
PCR assay and VI in baby hamster kidney (BHK)-21 cells and embryonated chicken eggs (ECE). The EHDV
PCR assay detected EHDV RNA from 6 blood samples obtained from deer during 1988-1989 outbreaks of
epizootic hemorrhagic disease and from the spleen and blood samples of a deer with clinical hemorrhagic disease
in 1992. The 6 blood samples from the 1988-1989 outbreaks and the spleen sample from the 1992 case were
VI positive on BHK-21 cell culture. The blood from the same deer with the PCR- and VI-positive spleen was
VI negative in BHK-21 cells and ECE. All EHDV isolates were identified as EHDV serotype 2 by a plaque
inhibition test. The results of this study indicate that the sensitivity of the previously described EHDV PCR
assay is comparable to or greater than that of the VI method in BHK-21 cell culture or ECE. The EHDV PCR
assays could provide a superior diagnostic alternative to the current cumbersome and time-consuming VI
procedures.
Epizootic hemorrhagic disease virus (EHDV) is an
arthropod-borne, double-stranded RNA virus of the
genus Orbivirus, family Reoviridae, 4,6 that causes fatal
hemorrhagic disease in white-tailed deer (Odocoileus
5,8,9,11
Other domestic and wild ruminants
virginianus).
may also be infected with the virus.13,15 The possibility
of clinical disease in domestic and wild ruminants has
led to restrictions on the international movement of
livestock and their germplasm if the animals are EHDV
positive by serology or virus isolation (VI).22
Diagnosis of EHDV infection by traditional methods includes evaluation of clinical signs, pathologic
changes, serology, and VI. Because clinical signs and
pathologic changes caused by EHDV are indistinguishable from those produced by bluetongue virus (BLU),
an orbivirus related to EHDV, confirmation of EHDV
infection under field conditions by these methods is
unreliable.14,19 Serology may provide a good indication
of EHDV infection if there is a 4-fold increase in the
antibody titer in samples taken 2 weeks apart. However, the difficulty in obtaining paired serum samples
from field-infected animals makes a definitive diagnosis of EHDV infection by serology impractical. De-
From the Department of Veterinary Pathology, Microbiology and
Immunology, School of Veterinary Medicine, University of California, Davis, CA 95616 (Aradaib, Akita, Osburn), and the National
Veterinary Services Laboratories, Ames, IA 50010 (Pearson).
Received for publication December 6, 1993.
finitive diagnosis of EHDV infection presently requires
isolation of the virus. Various laboratories have different methods for isolation of EHDV, including intravenous (i.v.) inoculation of embryonated chicken
eggs (ECE) or isolation on cell lines. The VI technique
is laborious, expensive, and time consuming, and a
final result requires 2-4 weeks.
New techniques in molecular biology have provided
sensitive and specific polymerase chain reaction (PCR)based assays for detection of orbiviruses.1,2,23 Recently
we reported a reverse transcriptase (RT) PCR protocol
that is sensitive and specific for the detection of EHDV
RNA in cell culture and clinical samples.3 The objective of the present study was to determine the ability
of our previously reported EHDV PCR-based assay to
detect EHDV in naturally infected deer and to evaluate
its potential as a rapid, sensitive, and specific diagnostic assay through comparison with standard VI procedures currently used in our laboratory.
Materials and methods
Clinical samples. A total of 36 blood samples were obtained from deer (Table 1). Six blood samples were obtained
from white-tailed deer during outbreaks of epizootic hemorrhagic disease in 1988 and 1989.a The National Veterinary
Services Laboratories isolated EHDV from these samples.
Heparinized blood and a sample of spleen were received from
a black-tailed deer (Odocoileus hemionus) in California with
clinical hemorrhagic disease.b There were 26 heparinized
blood samples from black-tailed deer in California with un-
196
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PCR and virus isolation for EHD virus
197
b
Table 1. Origin and date of collection of samples used for isoknown status. Three heparinized blood samples were obtained from clinically normal captive deer.c Processing of the lation of epizootic hemorrhagic disease virus.
blood and spleen samples for VI was as described previously.2
Blood cells were washed twice with phosphate-buffered saline
(PBS) containing 100 units penicillin/ml and 100 mcg streptomycin/ml. The washed blood cells were restored to the
original volume with 2 mM Tris buffer (pH 8.0) and then
disrupted by sonication. The spleen sample was homogenized 1:10 (w/v) in minimal essential medium (MEM),d and
cell debris were sedimented by centrifugation at 1,500 x g
for 20 min.
Cell culture. Baby hamster kidney (BHK)-21 cells were
prepared in MEM containing 100 units penicillin/ml and 100
mcg streptomycin/ml, 10% tryptose phosphate broth, and
10% fetal bovine serum (FBS) that had been heat inactivated
at 56 C for 30 min. Cell cultures were incubated at 37 C in
a humidified incubator with 5.0% carbon dioxide until confluent monolayers were obtained (usually 2-3 days).
Virus isolation and identification. The BHK-21 cell monolayers were inoculated with lysed blood or spleen homogenate
diluted 1:10 in MEM. After incubation at 37 C for 1 hr, the
inoculated cell cultures were supplemented with MEM, 2%
tryptose phosphate broth, and 2% FBS. The cell cultures were
again incubated at 37 C and observed daily until cytopathic
effect was 80% complete. Cultures with no cytopathic effect
were blind passaged. All cytopathic agents were identified by
a plaque inhibition assay. 20 The remaining lysed blood and
spleen homogenate were stored at 4 C for further analysis by
PCR assay. Six 11-day-old ECE were each inoculated intravenously with 0.1 ml of diluted lysed blood received from a
deer in California with clinical hemorrhagic disease. Inoculated ECE were incubated at 33 C for 7 days. Eggs were
candled daily, and dead embryos were removed. The dead
and the live embryos were harvested, macerated, sonicated,
diluted 1:1,000 in PBS, and used for a second passage.
Nucleic acid extraction from clinical samples. Total nucleic acid was extracted from lysed blood and spleen homogenate as described previously.2 Five microliters of the
total nucleic acid extract was used in the PCR assay.
PCR assay. The RT PCR for EHDV was performed as
previously described3 using primers derived from genome
segment 6 of EHDV-2, which codes for NS1.10 Following
amplification, 20 µl of the PCR product was visualized on a
2% ethidium bromide-stained agarose gel or detected by detected by chemiluminescent hybridization (Fig. 2B)
chemiluminescent hybridization. Southern blot with chemi- from the spleen sample of the deer with clinical hemluminescent hybridization was used as described.2
orrhagic disease.
Results
The specific EHDV PCR product was visualized on
ethidium bromide-stained agarose gels from 4 of the
6 blood samples collected during outbreaks of disease
among susceptible deer during 1988-1989 (Fig. 1A).
Southern blot with chemiluminescent hybridization
detected EHDV in all 6 blood samples from 19881989 and in the blood sample from the deer in California with clinical hemorrhagic disease in 1992 (Fig.
1B). EHDV-specific PCR product was visualized on
ethidium bromide-stained agarose gel (Fig. 2A) and
EHDV was isolated on BHK-21 cell culture from
the 6 blood samples collected during the outbreak of
the disease in 1988-1989. In addition, EHDV was also
isolated on BHK-21 cell culture from the spleen sample
from the deer with clinical hemorrhagic disease. However, VI attempts using the lysed blood of the same
deer on BHK-21 cell culture were negative. All the VI
attempts by the i.v. inoculation of ECE were negative.
All EHDV isolates were identified as EHDV serotype
2 by the plaque inhibition test.20
The 26 blood samples from the field deer and the 3
blood samples from clinically normal deer were EHDV
PCR negative and VI negative (Table 2).
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198
Aradaib et al.
Figure 1. Six blood samples, collected during a 1988-1989 EHDV
outbreak and 1 sample from a deer with clinical hemorrhagic disease.
A. EHDV PCR product on ethidum bromide-stained agarose gel.
Positive signals are seen in 4 of the deer from the 1988-1989 EHDV
outbreak. Lanes: MW, molecular weight marker; 1, 10 pg of EHDV
RNA (positive control); 2-7, 6 blood samples from 1988-1989 outbreak; 8, blood from the deer with clinical disease; 9, blood from
uninfected deer. B. Southern blot with chemiluminescent hybridization of the above gel showing detection of the specific EHDV
PCR product from all 6 blood samples from the 1988-1989 outbreak
and the blood sample from the deer with clinical disease but not
from the blood of the uninfected deer.
Discussion
The EHDV PCR assay with chemiluminescent hybridization detected EHDV RNA in the 6 blood samples collected from deer during outbreaks of disease in
1988-1989 and in blood and spleen samples from a
deer with clinical hemorrhagic disease in 1992. Blood
cells have been reported as the clinical sample of choice
for isolation of EHDV.9 In this study, EHDV was isolated on BHK-21 cell culture from 6 blood samples
collected from deer during the 1988-1989 outbreaks
of the disease. However, the virus isolated on BHK21 cell culture from the spleen of the deer with clinical
hemorrhagic disease virus was not isolated from the
lysed blood of the same deer by ECE or BHK-21 cell
culture. This discrepancy could be due to the -affinity
of the virus for lymphoid tissues in the initial repli-
Figure 2. Spleen sample from a deer with clinical hemorrhagic
disease. A. EHDV PCR product on ethidum bromide-stained agarose gel. Lanes: MW, molecular weight marker; 1, deer with clinical
disease; 2, blood from uninfected deer. B. Southern blot with chemiluminescent hybridization of the above gel.
cation cycle, concentration of lymphoid cells and
erythrocytes in this organ, or the development of antibody response that causes a decrease in the virus titer
of the blood without having a direct influence on the
concentration of the virus in the various tissues. These
findings have been documented in pathogenesis studies
on BLU.12,17 Our study suggests that the spleen from
deer with clinical hemorrhagic disease would be the
appropriate clinical sample for detection of EHDV by
PCR or for isolation on BHK-21 cell culture. There
was no evidence of EHDV infection in the inoculated
ECE. Apparently, the ECE method is relatively insensitive for isolation of EHDV. In a previous study,
EHDV field isolates were recovered on BHK-21 cell
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199
PCR and virus isolation for EHD virus
Table 2. Detection of epizootic hemorrhagic disease virus in
clinical samples by virus isolation (VI) and polymerase chain reaction (PCR).
Sample
no.
VI*
PCR
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
positive
positive
positive
positive
positive
positive
positive†
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
positive
positive
positive
positive
positive
positive
positive
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
* Using embryonated chicken eggs (ECE) or BHK-21 cell culture.
† BHK-21 positive, ECE negative.
culture but not in Vero cell culture or ECE.16 The results of this study agree with those of others that concluded that EHDV is more likely to be isolated directly
on BHK-21 than by i.v. inoculation of ECE, especially
if clinical disease is observed or the animals are ex16
perimentally infected. An epizootiologic study for
isolation of BLU from wild and domestic ruminants
by initial i.v. inoculation of ECE and subsequent adaptation of the isolate to cell culture resulted in recov21
ery of 305 BLU isolates. No EHDV isolate was reported in that study, which suggests that initial i.v.
inoculation of ECE and adaptation of the isolate to cell
culture is a more sensitive procedure for isolation of
BLU than for EHDV. Isolation of EHDV on BHK-21
cell culture is more convenient and inexpensive than
i.v. inoculation of ECE. One disadvantage of the BHK-
21 cell line is that it grows faster and survives for only
5 days without passage; hence, these cultures require
more care than do other cell cultures.
The results of this study indicate that the sensitivity
of the previously described EHDV PCR assay for detection of EHDV3 is comparable to or exceeds that of
the standard VI procedure used for isolation of EHDV.
The VI procedure is laborious and expensive and requires 24 weeks to obtain a definitive diagnosis. The
EHDV PCR assay is a rapid, sensitive, and relatively
inexpensive procedure for the detection of EHDV. A
definitive diagnosis of EHDV infection using Southern
blot with chemiluminescent hybridization can be obtained within 3 working days. This EHDV PCR assay
could provide a superior diagnostic alternative to the
current cumbersome and time-consuming VI procedure.
Acknowledgements
This work was supported by funds from the Livestock Dis-
ease Research Laboratory, School of Veterinary Medicine,
University of California, Davis; USDA/APHIS cooperative
agreement USDA 12-34-93-0234-CA; and the USDA/CSRS
Animal Health and Disease 1433.
Sources and manufacturers
a. National Veterinary Services Laboratory, USDA, APHIS, Ames,
IA.
b. Wildlife Investigations Laboratory, Sacramento, CA.
c. Sacramento Zoo, Sacramento, CA.
d. GIBCO BRL, Gaithersburg, MD.
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