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Vol. 32, No.

JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1994, p. 2225-2231


0095-1 137/94/$04.00+0

Development of Nested PCR Assays for Detection of Bovine


Respiratory Syncytial Virus in Clinical Samples
S. VIL(cEK,'t M. ELVANDER,I 2* A. BALLAGI-PORDANY,' AND S. BELAK'
Department of Virology, The National Veterinary Institute, S-751 23 Uppsala,' and Department of
Cattle and Sheep, The National Veterinary Institute, S-750 07 Uppsala,2 Sweden
Received 23 February 1994/Returned for modification 4 May 1994/Accepted 9 June 1994

Two nested PCR assays were developed for the detection of bovine respiratory syncytial virus (BRSV).
Primers were selected from the gene encoding the F fusion protein (PCR-F) and the gene encoding the G
attachment protein (PCR-G). Biotinylated oligonucleotide probes, termed F and G, were selected for the
hybridization of the respective PCR products. The sensitivities of the PCR-F and PCR-G assays were similar,
both detecting 0.1 tissue culture infective dose of the virus. The PCR-F assay amplified all bovine strains and
one human strain (RS32) tested. No cross-reactions were observed with nine heterologous respiratory viruses.
PCR-F products of bovine and human RSV strains were discriminated by using endonuclease restriction
enzyme Scal, which specifically cleaved products of BRSV. Oligonucleotide probe F was also specific for
products of BRSV. The PCR-G assay detected all bovine strains and none of the human strains tested. A faint
electrophoretic band was also observed with products of Sendai virus. However, probe G did not hybridize with
this product, only with products of BRSV. Nasal swabs collected from cattle with no symptoms and cattle in
the acute stage of respiratory disease were analyzed for BRSV by the immunofluorescence (IF) method and by
the PCR-F and PCR-G assays. The virus was detected by the PCR assays in 31 of 35 (890%) samples tested. Only
23 samples (66%) were positive by the IF method, and these samples were also positive by both the PCR-F and
PCR-G assays. The 31 samples detected as positive by PCR originated from cattle presenting clinical signs of
acute respiratory disease; the four PCR-negative samples originated from clinically asymptomatic neighboring
cattle. All sampled animals subsequently seroconverted and became reactive to BRSV. Thus, the detection of
BRSV by PCR correlated with clinical observations and was considerably more sensitive (66 versus 89%) than
IF. These results indicate that both nested PCR assays provide rapid and sensitive means for the detection of
BRSV infection in cattle. Considering its higher specificity, the PCR-F assay can be recommended as the
method of choice in the analysis of clinical specimens of BRSV.

antigen enzyme-linked immunosorbent assay (ELISA) (11,


25). These methods are faster than cultivation of the virus, but
their sensitivity and specificity are often low or variable (9, 30,
32).
Human RSV (HRSV), the main cause of severe lower
respiratory tract disease in infants, is antigenically closely
related to BRSV (17). Despite antigenic similarity, there are
major differences in their reactivity patterns with monoclonal
antibodies. Surface glycoprotein G shows no cross-reactivity
between the two viruses (22).
The PCR assay provides a new approach to BRSV detection
(5). Combined reverse transcription-PCR methods have been
published for the detection of HRSV and BRSV (8, 19-21, 24).
The HRSV PCR assay has been evaluated both on viruses
grown in cell culture and on clinical specimens, but the BRSV
PCR assay has not yet been applied to clinical material.
In the present study, we describe the development of two
nested PCR assays which amplify different regions of the
BRSV genome. These assays were applied for the detection of
BRSV in clinical samples obtained during natural outbreaks of
respiratory disease in cattle. The detection range of the PCR
assays and of a conventional IF technique was compared and
correlated with the clinical signs and seroconversions observed
in the outbreaks studied.

Bovine respiratory syncytial virus (BRSV) is a nonhemagglutinating pneumovirus of the Paramyxoviridae family. This
virus plays an important role in outbreaks of acute respiratory
disease in cattle and is also known to be involved in the atypical
pneumonia complex (2). BRSV was first isolated from cattle
with respiratory disease in Switzerland in 1967 (23) and has
since been associated with outbreaks of respiratory disease
worldwide (26). Severe outbreaks of respiratory tract disease
caused by BRSV have occurred every year since 1989 in
Sweden (10). In contrast to most reports, lactating cows, not
calves, are more severely affected. The main symptoms are
fever, coughing, respiratory distress, and a marked drop in milk
production. The morbidity rate is high in the majority of
diseased herds, whereas the mortality rate in some herds with
confirmed acute infection varies between 5 and 20%.
Although BRSV is a major pathogen of the respiratory
disease syndrome in cattle, virus detection in clinical specimens
is still poor because of inadequate laboratory techniques. Since
the virus replicates slowly, classical virus isolation is laborious
and several blind passages are often required before any
cytopathic effect can be seen. Isolation attempts often fail
because of the lability of the virus (27). Direct virus detection
can also be attempted by immunofluorescence (IF) (30) or by
*
Corresponding author. Mailing address: Department of Cattle &
Sheep, The National Veterinary Institute, SVA, P.O. Box 7073, S-750
07 Uppsala, Sweden. Phone: (46) 18 674000. Fax: (46) 18 309162.
t Permanent address: Department of Infectology and Tropical
Veterinary Medicine, University of Veterinary Medicine, 04181 Ko,ice, Slovakia.

MATERIALS AND METHODS


RSV strains and culture. Six BRSV strains and three HRSV
strains were used.
The BRSV reference strain, RB94 (33), was propagated on
2225

2226

VILtEK ET AL.

primary bovine turbinate cells and maintained in FDMEM


(Eagle's minimum essential medium containing HEPES [N-2hydroxyethylpiperazine-N'-2-ethanesulfonic acid] and NaH
C03) supplemented with 2% fetal calf serum, 0.1% kanamycin, 0.1% L-glutamine, and 20 mg of trypsin per ml. The virus
was harvested when showing 90 to 100% cytopathic effect. The
titer was determined as 50% tissue culture infective dose
(TCID90). Two bovine field strains, RS504/93 and RS576/93,
were isolated from acute outbreaks in Sweden. Bovine virus
strain Lelystad was kindly provided by M. Merza (The National
Veterinary Institute; originally received from J. T. van Oirschot, Central Veterinary Institute, Lelystad, The Netherlands). Bovine strains WBH 30/86 and Lelystad 37 were kindly
provided by J. A. Kramps (Central Veterinary Institute).
Human RSV strains RS32 and RS5 were generously donated by M. Grandin (Swedish Institute for Infectious Disease
Control, Stockholm, Sweden) and the RS Long strain (18) was
kindly provided by C. Orvell (Central Microbiological Laboratory, Stockholm, Sweden).
Bovine clinical samples. The clinical samples included nasopharyngeal swabs, obtained from animals in a beef herd
(BH), a dairy herd (DH), and a bull station (BS) in Sweden. A
majority of the animals in these herds showed acute symptoms
of respiratory disease, including fever, nasal discharge, and
respiratory distress associated with BRSV (12). In herds DH
and BS, only animals with acute respiratory distress and a
rectal temperature above 39C were sampled. In herd BH, the
respiratory outbreak was not as severe and not all animals were
affected. Samples were obtained from animals with high fever,
nasal discharge, and signs of respiratory distress, but also from
afebrile asymptomatic neighboring animals. Samples were also
collected from clinically healthy animals at the Veterinary
Medical Faculty, Swedish University of Agricultural Sciences,
Uppsala, Sweden. The nasopharyngeal samples were collected
with Culturette cotton swabs (Marion Scientific, Kansas City,
Kans.) saturated in transport medium in accordance with the
manufacturer's instructions. The swabs were either transferred
to the laboratory at room temperature within 1 day or transported in liquid nitrogen or on dry ice. (The mode of transportation did not influence the results of the compared methods). At the laboratory, the samples were stored in liquid
nitrogen or in -70C until analyzed.
All samples were examined in parallel by the IF technique
and by PCR detection assays. Virus isolation was not attempted, since it is not considered to be a practical diagnostic
method of choice (9).
Two serum samples were collected 3 weeks apart from each
animal. The sera were analyzed by an indirect ELISA for
antibodies to BRSV.
Isolation of RNA. Sample (500 ml of virus suspension or
nasal swab material diluted 1:1 in phosphate-buffered saline
[PBS]), 2 ng of yeast tRNA, 5 ml of proteinase K (14 mg/ml;
Boehringer, Mannheim, Germany), and 50 ml of 10% sodium
dodecyl sulfate (SDS) were mixed and incubated at 56C for 25
min. RNA was extracted with an equal volume of saturated
phenol (pH 4.3)-chloroform mixture (1:1 [vol/vol]; Sigma, St.
Louis, Mo.) and precipitated overnight with 2 volumes of cold
95% ethanol in the presence of 0.3 M sodium acetate (pH 5.2)
at -20C. The precipitated RNA was centrifuged at 14,000 x
g for 30 min, and the pellet was dissolved in 10 ml of
double-distilled H20 (ddH2O) and stored at -20C.
Synthesis of cDNA. Synthesis of cDNA was carried out in
25-ml reaction volumes as follows: 2 ml of RNA was diluted
with 5 ml of ddH2O, to which 0.02 U of random hexamers
(Pharmacia, Uppsala, Sweden) had been added. RNA was
denaturated at 65C for 5 min and then cooled on ice. One

J. CLIN. MICROBIOL.

milliliter (24 U) of RNAguard (Pharmacia), 2.5 ml of deoxynucleoside triphosphates (dNTPs) (2 mM [each]; Pharmacia), 5 ml of S x reaction buffer (0.25 M Tris-HCl [pH 8.3],
0.375 M KCl, 15 mM MgCl2), and 1 ml (200 U) of Moloney
murine leukemia virus reverse transcriptase (Gibco BRL,
Bethesda, Md.) were added. The reaction mixture was incubated at 37C for 90 min, and then enzyme was inactivated by
incubation at 98C for 5 min.
Design of primers. The gene encoding the F fusion glycoprotein of BRSV was chosen for the selection of primers for
the PCR-F assay. Primers were selected by nucleotide sequence analysis using the GCG program package (Genetics
Computer Group, Inc., Madison, Wis.). Selection was made
from highly conserved regions of two bovine strains (14, 31)
and three human strains (6, 13, 15). The outer primers, termed
Bi and B2A, flanked a 711-bp DNA region, while the inner
primers, termed B3 and B4A, flanked a 481-bp region (Table 1).
In the PCR-G assay, primers were selected from the gene
encoding for the G attachment glycoprotein of BRSV. By
comparison of eight sequences deposited in the GenEMBL
data bank (accession numbers L08410 to L08417) and sequences published by Mallipeddi and Samal (16), a strongly
conserved region was identified. The outer primers, termed
B5A and B6A, flanked a 603-bp DNA region, while the inner
primers, termed B7A and B8, flanked a 371-bp region (Table
1). All primers were synthesized in our laboratory with a PCR
Mate DNA synthesizer (Applied Biosystems, Warrington,
United Kingdom).
PCR. The PCR-F and PCR-G assays were performed by the
same protocol.
The first stage of PCR was carried out in 50 ml of a reaction
mixture that contained 5 ml of lOX reaction buffer (100 mM
Tris-HCl [pH 9.0], 500 mM KCl, and 1 mg of bovine serum
albumin [BSA] per ml), 5 ml of 25 mM MgCl2, 5 ml of dNTPs
(2 mM [each]; Pharmacia), 1.5 pmol of the outer primers (Bi
and B2A or BSA and B6A), 1 U of Taq DNA polymerase
(Perkin-Elmer Cetus, Norwalk, Conn.), and 5 ml of cDNA;
ddH2O was added to adjust the final volume to 50 ml. This
aqueous phase was overlaid with 2 or 3 drops of mineral oil
(Sigma). Amplification was performed with a Perkin-Elmer
Cetus DNA Thermal Cycler in a three-step cycling program
consisting of denaturation at 94C for 45 s, annealing at 50C
for 45 s, and elongation of DNA at 72C for 1.5 min. This
cycling method was repeated 25 times.
The second stage of PCR was performed by amplifying 5 ml
of the first-stage PCR product under the same reaction conditions, with the exception that the concentration of inner
primers (B3 and B4A or B7A and B8) was 15 pmol per
reaction and 35 cycles were performed.
In the last cycle, to ensure a complete synthesis of PCR
products, the elongation step at 72C was prolonged to 7 min
in both stages of the PCR.
To avoid carryover or cross-contamination, the most common problems of diagnostic PCR, our routine precautions and
safety methods were applied as reported elsewhere (4).
Sensitivity studies. Dilutions of BRSV reference strain
RB94 (initial titer, 104 TCID50s) were prepared in PBS or in
BRSV-negative nasal swab material obtained from clinically
asymptomatic animals and previously tested by PCR. The virus
was serially diluted 10-fold six times in PBS. Each dilution
(10-1 to 10-6) was tested simultaneously in both PCR systems
(PCR-F and PCR-G).
Specificity studies. The specificity of the PCR was evaluated
with the following viruses: parainfluenza 3 virus (Umea strain),
Sendai virus, bovine coronavirus, bovine viral diarrhea virus
(New York strain), mammalian reovirus type 1 (Jones strain),

DETECTION OF BRSV BY NESTED PCR ASSAYS

VOL. 32, 1994

TABLE 1. PCR
Primer or
probe

2227

primer and probe sequences

Sequence 5'3'

Position'

Size (bp) of
product

PCR-F
Primers

T7FT GGT CAT TCG TFA TAG GCAT

114-135
824-803

711

B2A
B3
B4A

GTG CAG TTA GTA GAG GTT ATC GIA GT


TAG lTFC TT1F AGA TCA AGT ACT 1FTG CT

126-151

481

606-581

B"-CAG TAG AGC AAA AAG AGG GAT ACC AGA GT-B

325-354

CCA CCC TAG CAA TGA TAA CCT TGA C


AAG AGA GGA TGC (T/C)TT GCT GTG G

110-134
712-691

603

CAT CAA TCC AAA GCA CCA CAC TGT C


GCT AGT TCT GTG GTG GAT TGT TGT C

281-305
651-627

371

B-GAG CAC CAA GCA GAG CCC CTA CAA TCA CCC T-B

554-584

BI

Probe F

AAT CAA CAT GCA GTG CAG TTA G

PCR-G
Primers

B5A
B6A

B7A

B8
Probe G

"Position in the F gene for Bi, B2A, B3, and B4A or in the G gene of BRSV for B5A. B6A. B7A, and B8.
"B, biotinylation.

bovine adenovirus serotype 2 (strain 19), bovine herpesvirus


type 1 (Los Angeles strain), and bovine herpesvirus type 4
(Movar 33/63 strain). All viruses used were grown in cell
cultures.
Cleavage and electrophoresis of the PCR products. The
PCR-F products originating from bovine (BRSV) strains were
cleaved by restriction enzyme Scal (Boehringer). Unpurified
PCR product corresponding to 0.5 to 1 mg of DNA (usually 2
to 3 ml) was digested with 8 to 10 U of Scal in 25-ml volume
for 1.5 h. The cleavage buffer supplemented with the enzyme
was used according to the manufacturer's recommendations.
PCR products and cleaved fragments were separated by
electrophoresis in 2.5% agarose gels using 0.5x TBE (20X
TBE is 1 M Tris base, 1 M boric acid, and 20 mM EDTA) as
running buffer. Ten-milliliter portions of the samples were
loaded and run at 300 V for 35 min. The ethidium bromidestained bands were visualized by UV light and recorded with a
video camera (CYBERTECH, Berlin, Germany). The molecular sizes of fragments were compared with those of a 100-bp
ladder (Gibco BRL).
Southern blot hybridization of PCR products. Two oligonucleotide probes were selected from the genomic regions,
flanked by the inner primers. The sequences are given in Table
1. The probes were biotinylated at the 5' and 3' positions
during synthesis (Scandinavian Gene Synthesis AB, K6ping,
Sweden).
After agarose gel electrophoresis, the DNA was transferred
onto nylon membranes (Hybond N+; Amersham) by electroblotting. The DNA was denatured in a solution containing 0.5
M NaOH and 1.5 M NaCl for 15 min at room temperature and
neutralized by immersion in a solution of 1 M Tris HCl (pH
7.5) containing 0.5 M NaCl. Denatured DNA was immobilized
by irradiation of the membranes with a transilluminator using
302-nm-wavelength UV light for 7 min. Samples were hybridized (without prehybridization) in plastic bags containing 3 ml
of hybridization solution (0.2% BSA, 0.2% Ficoll 400, 0.2%
polyvinylpyrrolidone, 1.7 mg of yeast tRNA per ml in 2x SSC
[1 x SSC is 0.15 M NaCl plus 0.015 M sodium citrate]) and 33
nM biotinylated F probe or G probe for 30 min at 45C. The
filters were briefly washed in 2x SSC and incubated in

blocking buffer (1% BSA and 1% SDS in 2x SSC) for 5 min at


45C. The biotinylated probes were visualized by using streptavidin-alkaline phosphatase conjugate (Boehringer) and BCIP
(5-bromo-4-chloro-3-indolylphosphate toluidinium) plus Nitro
Blue Tetrazolium (Western Blue) as substrates according to
the manufacturer's instructions (Promega, Madison, Wis.).
IF. Bovine nasal specimens were soaked for 30 min in 1 .5-ml
Eppendorf centrifuge tubes containing 200-ml portions of
PBS. Each tube was punctured, inserted into a larger tube, and
centrifuged at 14,000 rpm for 10 s in an Eppendorf centrifuge.
Each pellet was dissolved in 50 ml of PBS, placed on a glass
slide, air dried, fixed for 10 min in cold acetone, and then
washed in PBS for 10 min. Bovine antibody to BRSV that had
been conjugated to fluorescein isothiocyanate (CVL, Weybridge, United Kingdom), and diluted according to the instructions of the manufacturer was applied, and the slides were
incubated for 30 min in a humid chamber at 37C. The slides
were washed three times in PBS for 5 min each time, mounted
in PBS-glycerol, and examined under a fluorescence microscope (Nikon UFX, Yokohama, Japan).
Detection of BRSV antibodies. An indirect ELISA was used
in accordance with the manufacturer's recommendations
(Svanova Biotech, Uppsala, Sweden).
RESULTS
Determination of optimal PCR conditions. The optimal
annealing temperature was 50C for all primer pairs. To
decrease competition of amplification in the nested PCR, the
amount of outer primers in the first stage of PCR was reduced
10-fold, i.e., 1.5 pmol of each primer (BI and B2A or B5A and
B6A) was used in the first stage and by 15 pmol of each primer
(B3 and B4A or B7A and B8) in the second stage of the nested
PCR. Under these conditions, the number of nonspecifically
amplified bands was minimal.
All bovine strains were detected by the nested PCR-F and
PCR-G assays, yielding 481-bp (Fig. IA, lanes 1 to 6) and
371-bp (Fig. ID, lanes I to 6) products, respectively. One of the
three human strains, RS32, was also amplified by the PCR-F
assay (Fig. IA, lanes 7 to 9).

VIL(EK ET AL.

2228

J. CLIN. MICROBIOL.
Ml

;;

-----81 bp

AN

- I-81 byp

13

13
i

3-- 7 1

481 bp
-

2 5 bp
10)1 an ct I l 5 b p

L)
1 an;7
b1y)

1.ij 7

FIG. 2. Sensitivity of detection of the nested PCR-F and PCR-G


methods. BRSV strain RB94 (initial titer, 104 TCID50) was diluted
10-fold in PBS. Isolated RNA was transcribed into cDNA and
amplified by the nested PCR-F (A) or PCR-G (B) assay (25 cycles in
the first stage and 35 cycles in the second stage of PCR). Tenmicroliter amounts of the PCR products were loaded on agarose gels.
Lanes 1 to 5, 100, 10, 1, 0.1, and 0.01 TCID5(, respectively; Lane M;
100-bp ladder (Gibco BRL).

bp

FIG. 1. Detection of six bovine and three human RSV strains by


nested PCR assays. Amplicons obtained by nested PCR-F (A, B, and
C) or PCR-G (D and E) were analyzed by electrophoresis (A, C, and
D) and by Southern blot hybridization with biotinylated probe F (B) or
probe G (E). The PCR-F products were cleaved by restriction endonuclease Scal (C). The following virus strains were tested: BRSV
strains RB94 (lanes 1), RS504/93 (lanes 2), RS576/93 (lanes 3),
Lelystad (lanes 4), WBH 30/86 (lanes 5), Lelystad 37 (lanes 6), 100-bp
ladder (Gibco BRL) (lanes M), HRSV strains RS5 (lanes 7), Long
(lanes 8), and RS32 (lanes 9).

The PCR-G assay did not amplify any of the three human
strains (Fig. ID, lanes 7 to 9).
Specificity studies. In the Southern blot hybridization, positive signals were observed with PCR products originating from
all bovine RSV strains tested (Fig. 1B and E, lanes 1 to 6), but
not with the products originating from human RSV strains
(Fig. 1B and E, lanes 7 to 9).
The nucleotide sequences analyzed by the GCG program
predicted that the F-gene PCR products of bovine strains
should be cleaved by the Scal enzyme into four fragments with
expected sizes of 257, 105, 101, and 18 bp. The experiments
verified this prediction, but because of the limited resolution of
the DNA bands in agarose gels, only two electrophoretic bands
were observed (a 257-bp band and a 101- to 105-bp band [Fig.
IC, lanes 1 to 6]). The amplified product of the human RS32
strain, in agreement with computer analysis, was not cut by
Scal (Fig. 1C, lane 9).
The PCR assays showed no cross-reactivity with the heterologous viruses examined, with one exception, a faint fragment
of approximately 370 bp amplified by the PCR-G assay from
the genome of the Sendai virus. To exclude the possibility of
misinterpretation of the results of the PCR-G assay, hybridization probe G was used to confirm the specificities of the
amplified products. We found that this probe recognized only
BRSV strains but not the PCR product of the Sendai virus.
Sensitivity studies. The single PCR-F and PCR-G assays,
performed in 35 cycles, detected 1.0 TCID50 of the virus. The
nested PCR-F and PCR-G assays were 10 times more sensitive
than the single PCR or virus isolation, detecting 0.1 TCID50 of
the virus (Fig. 2).
Testing of clinical samples. BRSV was detected by the IF

technique in 23 of 35 (66%) nasal swabs from diseased


animals. By contrast, the PCR-F and PCR-G assays detected
the virus in 31 (89%) cases. All 23 samples positive by the IF
method were also positive in both PCR assays. The 31 samples
positive in the nested PCR-F assays were also positive by single
PCR-F, using inner primers B3 and B4A (Table 2).
In positive cases, the PCR-F yielded products with the
predicted size (Fig. 3A). These amplicons gave positive hybridization signals with probe F (Fig. 3B). Furthermore, they were
cleaved with restriction endonuclease Scal (Fig. 3C). The
PCR-G assay yielded amplicons of 371 bp (Fig. 3D), which
hybridized with probe G (Fig. 3E).
Serological results. All but two animals were negative for
BRSV antibodies at the first sampling. All animals showed
high antibody titers at the second sampling 3 weeks later
(Table 2).
DISCUSSION

Molecular biological methods offer new means for laboratory diagnosis of RSV infections. Synthetic oligonucleotide
probes have been used for the detection of HRSV in nasopharyngeal cells by in situ hybridization (8), and hybridization
techniques have been developed for the differentiation of
HRSV subgroups (28, 29). Paton et al. (24) reported a reverse
transcription-PCR assay for the detection of HRSV with
primers from the gene encoding the Fl subunit of the fusion F
glycoprotein. The same gene was amplified by Okamoto et al.
(21), to detect HRSV in otitis media by nested PCR. Cubie et
al. (7) developed a nested PCR for HRSV subgroup A with the
N gene.
For BRSV, however, data on the application of diagnostic
PCR is still limited. Recently, a single RT-PCR assay was
reported in which the primers were chosen from the gene
encoding the viral fusion protein F (19, 20). This assay was
demonstrated with viruses grown in cell cultures but not on
bovine clinical specimens.
Our aim was to develop PCR assays of wide detection range
and of high sensitivity and to test their diagnostic applicability
on clinical specimens. In order to avoid false-positive interpretation of the results, a common problem in PCR diagnostics,
two assays were developed in parallel. Primers were selected
from two essential genes (G and F) of the BRSV genome.
Nucleotide sequence analysis predicted that the PCR-F assay

DETECTION OF BRSV BY NESTED PCR ASSAYS

VOL. 32, 1994

% I `
l
S

TABLE 2. Detection of BRSV in nasal swabs both from cattle in


the acute stage of BRSV infection and from asymptomatic
neighbors by IF and by the PCR assays
BRSV detection" by:

Herd and
animal no.

IF

Single
PCR-F'

Herd BH
1
2

3d
4
5

+
+
+

21
22
23
24
25

Hlerd BS
60
61
62
63
64
65
66
67
68

+
+

+
+
+
+

0/0.92
0/0.45
0.25/0.75
0/0.56
0/0.75

+
-

0/0.40
0.19/1.00
0/0.86
)0/1.16
0/0.76
0/0.76
0/0.86

+
+

+4

(+)

+
+

+
+

+
+

16
17
18
19
20

in serum (acute!
convalescence)

+
+
+

10
11
12

Herd DH
13
14
15

Nested
PCR-G

+
+
+
+

9gc

+
+
+

Nested
PCR-F

+
+
+
+

+
-

7dl

BRSV antibodies

(+)

+
+
+
+

+
+
+

+
+

+
+

(+)

+
+
+
+
+

+
+
+

+
+
+

+
+
+
+

+
+

ND
(+)

+
-

()
-

(+)

+
+
+

+
+

0/1.01
0/0.97
0/0.74

0/0.66
0/1.23
0/1.21
0/0.97
0/1.22

0/0.54
0/1.19
0/0.97
0/died
0/1.13

0/1.03
0/1.29
0/1.18
0/died
0/0.94
0/0.87
0/1.39
0/0.93
0/1.39

+, positive; (+), weakly positive; -, negative; ND, not done.


Acute sera obtained in the febrile phase; convalescence sera obtained 3
weeks later. The presence of antibodies expressed as optical density at 450 nm in
ELISA. Values above 0.1 are regarded as positive.
With primers B3 and B4A.
"Not febrile. All others had temperatures of >39C.
a

could theoretically detect most bovine and some human RSV


strains (6, 13-15, 31). Primers selected from the G gene should
theoretically identify only bovine strains. Since both PCR
assays detected all six bovine laboratory strains but only the
PCR-F assay amplified the human strain RS32, the predictions
were confirmed.
In order to obtain a high sensitivity, we developed nested
PCR assays. It is generally accepted that nested PCR is more
sensitive than single PCR (1, 3). By using identical samples of
diluted virus, comparative analysis revealed that nested PCR
was 10 times more sensitive than single PCR. In the case of
clinical specimens, single and nested PCRs yielded identical

1112I
I-

--- 48

.B

I
I

H.

2229

bp

--481 bp

257 p
-1 O1 anld lU' hp5t![

-;-- 71 bp

--

37 bhp

FIG. 3. Detection of BRSV in bovine nasal swabs by nested PCR-F


(A, B, and C) and PCR-G (D and E). The amplicons were analyzed by
electrophoresis (A, C, and D) or by Southern blot hybridization with
biotinylated probe F (B) or G (E). Amplicons of the PCR-F assay were
cleaved by restriction endonuclease ScaI (C). Lanes 1 to 12 in each
panel correspond to the numbers of animals given in Table 2. Lane M,
100-bp ladder (Gibco BRL).

results. However, even here the superiority of nested PCR was


evident, since the results of single PCR were sometimes
difficult to interpret because of the low intensities of the
electrophoretic bands. The nested PCR yielded greater
amounts of products, providing the opportunity to apply other
detection and identification techniques such as restriction
endonuclease cleavage analysis.
The specificity of the amplicons is usually confirmed by
nucleic acid hybridization. For example, Oberst et al. (19) used
a radioactive probe to discriminate amplicons of BRSV from
HRSV and ovine RSV. Our strategy was to develop nonradioactive probes. The biotinylated probes F and G proved to be
specific for BRSV. Because of the production of large amounts
of amplified products, it was possible to shorten the hybridization and visualization procedure to 2 h. The diagnostic procedure could be further shortened by replacing Southern blot
hybridization with the more rapid technique of dot blot
hybridization.
Alternatively, confirmation can be carried out by cleavage of
the PCR products. In our PCR-F system, ScaI restriction
endonuclease cleavage provided the means to discriminate
between amplicons of BRSV and HRSV. The Scal recognition
sites in the viral genome, flanked by primers B3 and B4A, are
strongly conserved not only among the published sequences
but also in all laboratory strains, isolates, and field samples of
BRSV tested in this study. It is likely that this cleavage site
pattern is a characteristic feature of BRSV.
The main goal of this study was to evaluate the diagnostic
applicability of the nested PCR assays in detecting BRSV in
clinical specimens from cattle. To our knowledge, there is no
prior report on the evaluation of diagnostic PCR for BRSV in
clinical samples.

2230

VILDEK ET AL.

However, more data are available from studies on HRSV.


Comparative analysis of clinical samples by PCR and by
classical methods yielded contradictory results. A good correlation between single reverse transcription-PCR, virus isolation, and enzyme immunoassay was observed by Paton et al.
(24) when analyzing a wide range of nasopharyngeal aspirates
collected from children. On the other hand, Cubie et al. (7)
concluded that their nested RT-PCR system was not as
sensitive as expected.
In the present study, three herds were suffering from an
acute outbreak of respiratory disease caused by BRSV. All
tested animals showed high antibody titers to BRSV approximately 3 weeks later. The nested PCR assays detected a higher
percentage of BRSV-positive samples (89%) than the IF
method did (66%). The PCR results, in contrast to IF results,
were in full agreement with clinical symptoms and with positive
serology, which indicates that the PCR assays are without
doubt more sensitive than the IF method. One explanation for
the lower sensitivity of the IF method is that it requires nasal
samples of good quality with enough epithelial cells to permit
the reliable detection of viral antigens. Furthermore, the IF
method is more subjective than the PCR assays.
The acute febrile phase of respiratory disease and detection
of virus by the PCR assays showed good agreement in herds
DH and BS (Table 2). All sampled animals were positive by the
PCR assays in both herds, and each of them seroconverted
approximately 3 weeks later. In herd BH, only diseased
animals were PCR positive, whereas the four clinically asymptomatic neighboring animals (animals 3, 6, 7, and 9) were all
PCR negative (Table 2). However, animals 3 and 7 showed
positive serological reactions at the first sampling and were
therefore likely to have passed the virus excretion phase of the
infection. Animals 6 and 9 were both PCR negative and
serologically negative and therefore presumably not yet infected at the time of sampling. Further evidence of the
reliability of the PCR assays was indicated by the nine animals
in herd BS, all PCR positive in the acute phase of infection.
One week after the outbreak started, only two of the nine
animals were BRSV positive by PCR in nasal swabs, whereas 3
weeks later, all nine animals were negative. This is as would
have been predicted, since the virus is present in the nasal
mucosa during the acute phase of infection (12).
Although two nested PCR assays were developed for the
detection of BRSV, there is no need to analyze samples by
both assays. The PCR-F assay is more specific than the PCR-G
assay, and therefore we prefer to use it on clinical specimens.
The PCR-G assay can be useful in analysis of genomic diversity
of the BRSV genome, since the G gene is very variable among
the BRSV isolates (16).
We conclude that BRSV infection can be accurately diagnosed by using PCR on nasal swab samples taken in the acute
phase of a suspected outbreak. The nested PCR assays provide
a sensitive, specific, and rapid means of diagnosing BRSV
infection. Furthermore, these assays permit a new approach to
study the pathogenesis of BRSV infection.
ACKNOWLEDGMENTS
We thank Maria Persson and Annie Persson for excellent technical
assistance.
This research was supported by grants from AGRIA Insurance
Company Ltd., Stockholm, Sweden, and the Farmer's Research Council for Information and Development, Stockholm, Sweden.
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