Documente Academic
Documente Profesional
Documente Cultură
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.
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.
2226
VILtEK ET AL.
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),
TABLE 1. PCR
Primer or
probe
2227
Sequence 5'3'
Position'
Size (bp) of
product
PCR-F
Primers
114-135
824-803
711
B2A
B3
B4A
126-151
481
606-581
B"-CAG TAG AGC AAA AAG AGG GAT ACC AGA GT-B
325-354
110-134
712-691
603
281-305
651-627
371
B-GAG CAC CAA GCA GAG CCC CTA CAA TCA CCC T-B
554-584
BI
Probe F
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.
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
bp
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
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
% I `
l
S
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
1112I
I-
--- 48
.B
I
I
H.
2229
bp
--481 bp
257 p
-1 O1 anld lU' hp5t![
-;-- 71 bp
--
37 bhp
2230
VILDEK ET AL.
J. CLIN. MICROBIOL.
2.
3.
4.
5.
6.
81:7683-7687.
7. Cubie, H. A., J. M. Inglis, E. E. Leslie, A. T. Edmunds, and B.
Totapally. 1992. Detection of respiratory syncytial virus in acute
bronchiolitis in infants. J. Med. Virol. 38:283-287.
8. Cubie, H. A., J. M. Inglis, and A. M. McGowan. 1991. Detection of
respiratory syncytial virus antigen and nucleic acid in clinical
specimen using synthetic oligonucleotides. J. Virol. Methods 34:
27-35.
9. Edwards, S., R. H. Newman, and M. Stanly. 1984. Respiratory
syncytial virus diagnosis. Vet. Rec. 114:101.
10. Elvander, M., S. Alenius, and S. 0. Jacobsson. 1990. Severe
outbreaks of respiratory disease in dairy herds caused by respiratory syncytial virus, p. 461-465. Proceedings of the XVI World
Buiatrics Congress, Bahia, Brazil, vol. 1. Interlink Consultoria &
Eventos Ltd., Salvador, Bahia, Brazil.
11. Fernando, A. O., G. A. Anderson, J. Sanders, and D. Grotelueschen. 1989. Detection of bovine respiratory syncytial virus using a
heterologous antigen-capture enzyme immunoassay. J. Vet. Diagn. Invest. 1:210-214.
12. Inaba, Y., Y. Tanaka, K. Sato, T. Omori, and M. Matumoto. 1972.
Bovine respiratory syncytial virus. Studies of an outbreak in Japan,
1968-69. Jpn. J. Microbiol. 16:373-383.
13. Johnson, P. R., and P. L. Collins. 1988. The fusion glycoproteins
of human respiratory syncytial virus of subgroups A and B:
sequence conservation provides a structural basis for antigenic
relatedness. J. Gen. Virol. 69:2623-2628.
14. Lerch, R. A., K. Anderson, V. L. Amann, and G. W. Wertz. 1991.
Nucleotide sequence analysis of the bovine respiratory syncytial
virus fusion protein mRNA and expression from a recombinant
vaccinia virus. Virology 181:118-131.
15. Lopez, J. A., N. Villanueva, J. A. Melero, and A. Portela. 1988.
Nucleotide sequence of the fusion and phosphoprotein genes of
human respiratory syncytial (RS) virus Long strain: evidence of
subtype genetic heterogenicity. Virus Res. 10:249-262.
16. Mallipeddi, S. K., and S. K. Samal. 1993. Sequence variability of
the glycoprotein gene of bovine respiratory syncytial virus. J. Gen.
Virol. 74:2001-2004.
17. Mallipeddi, S. K., S. K. Samal, and S. B. Mohtanty. 1990. Analysis
of polypeptides synthesized in bovine respiratory syncytial virusinfected cells. Arch. Virol. 115:23-36.
18. Mufson, M. A., C. Orvell, B. Rafnar, and E. Norrby. 1985. Two
distinct subtypes of human respiratory syncytial virus. J. Gen.
Virol. 66:2111-2124.
19. Oberst, R. D., M. P. Hays, J. F. Evermann, and C. L. Kelling. 1993.
Characteristic differences in reverse transcription-polymerase
chain reaction products of ovine, bovine and human respiratory
syncytial viruses. J. Vet. Diagn. Invest. 5:322-328.
20. Oberst, R. D., M. P. Hays, K. J. Hennessy, L. C. Stine, J. F.
Evermann, and C. L. Kelling. 1993. Identifying bovine respiratory
syncytial virus by reverse transcription-polymerase chain reaction
and oligonucleotide hybridizations. J. Clin. Microbiol. 31:12371240.
21. Okamoto, Y., K. Shirotori, K. Kudo, E. Ito, K. Togawa, I. Saito, I.
Moro, and P. L. Ogra. 1991. Genomic sequences of respiratory
syncytial virus in otitis media with effusion. Lancet 338:1025-1026.
22. Orvell, C., E. Norrby, and M. A. Mufson. 1987. Preparation and
characterization of monoclonal antibodies directed against five
23.
24.
25.
26.
27.
28.
structural components of human respiratory syncytial virus subgroup B. J. Gen. Virol. 68:3125-3135.
Paccaud, M. F., and C. Jacquier. 1970. A respiratory syncytial virus
of bovine origin. Arch. Gesamte Virusforsch. 30:327-332.
Paton, A. W., J. C. Paton, A. J. Lawrence, P. N. Goldwater, and
R. Y. Harris. 1992. Rapid detection of respiratory syncytial virus in
nasopharyngeal aspirates by reverse transcription and polymerase
chain reaction amplification. J. Clin. Microbiol. 30:901-904.
Rodgers, S. J., and C. A. Baldwin. 1990. The rapid detection of
bovine respiratory syncytial virus antigens by use of a commercial
enzyme immunoassay. Bovine Pract. 25:76-81.
Sharma, R., and Z. Woldehiwet. 1991. Bovine syncytial virus: a
review. Vet. Bull. 61:1117-1131.
Smith, M. H., M. L. Frey, and R. E. Dierks. 1975. Isolation,
characterization and pathogenicity studies of a bovine respiratory
syncytial virus. Arch. Virol. 47:237-247.
Sullender, W. M., L. J. Anderson, K. Anderson, and G. W. Wertz.
1990. Differentiation of respiratory syncytial virus subgroups with
29.
30.
31.
32.
33.
2231