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Journal of Clinical Microbiology, June 1999, p. 1858-1862, Vol. 37, No. 6
UMR Institut National de la Recherche
Agronomique-Ecole Nationale Vétérinaire de Toulouse de
Physiopathologie Infectieuse et Parasitaire des
Ruminants1 and UMR Institut National de
la Recherche Agronomique-Ecole Nationale Vétérinaire de
Toulouse de Microbiologie Moléculaire,
Received 17 November 1998/Returned for modification 8 February
1999/Accepted 1 March 1999
The first nested reverse transcription (RT)-PCR based on the
nucleoprotein gene (n RT-PCR-N) of the bovine respiratory syncytial virus (BRSV) has been developed and optimized for the detection of BRSV
in bronchoalveolar lavage fluid cells of calves. This test is
characterized by a low threshold of detection (0.17 PFU/ml), which is
506 times lower than that obtained by an enzyme immunosorbent assay
(EIA) test (RSV TESTPACK ABBOTT). During an experimental infection of
17 immunocompetent calves less than 3 months old, BRSV RNA could be
detected up to 13 days after the onset of symptoms whereas isolation in
cell culture was possible only up to 5 days. Compiling results obtained
by conventional techniques (serology, antigen detection, and culture
isolation) for 132 field samples collected from calves with acute
respiratory signs revealed that n RT-PCR-N showed the highest
diagnostic sensitivity and very good specificity. This n RT-PCR-N with
its long period of detection during BRSV infection thus provides a
valuable tool for diagnostic and epidemiological purposes.
Bovine respiratory syncytial virus
(BRSV), like human respiratory syncytial virus (HRSV), is a pneumovirus
in the Paramyxoviridae family (7). The
epidemiology of BRSV is very similar to that of HRSV (34).
Spontaneous infection in young cattle is frequently associated with
severe respiratory signs (10, 31, 37), whereas experimental
infection generally results in milder disease with slight pathologic
changes (4). Diagnosis of BRSV infection is based solely on
antigen detection and serology (3). The method of choice in
dead cattle consists of the detection of BRSV antigens in pulmonary
samples by immunofluorescence (20) or immunoperoxidase
(23) technique. BRSV antigen detection is less frequently
undertaken in live cattle. Nasal secretion sampling (NS), which is
slightly invasive and currently used for human infants, has sometimes
been described in calves (3). Bronchoalveolar lavage (BAL)
(21, 33) can be more sensitive, since the lung is usually
extensively infected in naturally occurring cases (39). BRSV
lability makes routine isolation in cell culture laborious and
time-consuming (21). To our knowledge, comparison of
sampling of the upper respiratory tract and lower respiratory tract has only rarely been conducted in infants (9) and no comparison is available in cattle. As previously demonstrated, in young calves under 3 months, maternally transmitted immunoglobulin (Ig) G1 does not
prevent BRSV infection but hinders serological diagnosis (21,
33). However, the combined use of serology, antigen detection, and culture, which could potentially increase the proportion of positive diagnoses, is laborious and expensive.
Reverse transcription (RT)-PCR is potentially useful for improving the
sensitivity of RSV detection. Several RT-PCRs for HRSV detection based
on the fusion (F) gene (17, 28), the 1B gene (36), the polymerase (L) gene (13), and the
nucleoprotein (N) gene (8, 15) have been developed and have
been evaluated in studies involving numerous field specimens. The
sensitivity results are controversial. Some authors consider RT-PCR
less sensitive than expected when compared with conventional techniques
of HRSV detection (8, 13, 28), whereas for others, the
sensitivity was slightly (36) or more-strongly improved
(15, 17). The RT-PCR tests reported for BRSV are directed
against F (25, 26, 38) and glycoprotein (G) (38)
genes. None based on the N gene from BRSV has been described even
though this is one of the most conserved of the pneumovirus genes
(19). Furthermore, these BRSV studies involved small numbers
of strains, diseased calves, and infected herds (26, 38).
The aims of this work were therefore (i) to evaluate the threshold of
detection of a nested RT-PCR developed based on the N gene (n
RT-PCR-N), (ii) to determine the duration of presence of the BRSV in
BAL fluid during an experimental infection, and (iii) to evaluate the
sensitivity and specificity of the RT-PCR obtained during a large field
study and compare them with those of currently used laboratory
techniques (serology, antigen assays, and isolation in cell culture).
Animals. (i) Spontaneous infections.
Between 1991 and 1998, 111 live calves and 21 dead calves (n = 132; average
age, 58 days; minimum age, 6 days; and maximum age, 480 days) from 49 different herds were studied to compare sites of sampling and
laboratory techniques used to diagnose BRSV infection. Calves were in
cow-calf herds located in Belgium (9 herds) and in central and
southwestern France (40 herds). Animals exhibited acute respiratory
signs compatible with BRSV infection. Dead calves were necropsied
within 36 h of death.
(ii) Experimental infection.
Seventeen male calves of
Prim'Holstein breed, with a BRSV-seronegative status, were reared in
isolation. When they were 2 to 3 months old, they were used for
challenge experiments after their BRSV serological status had been
confirmed to be negative. Eleven calves were inoculated with BRSV (BRSV
A2 gelfi), and six calves were inoculated with bovine turbinate (BT)
cells free of virus (controls), on 3 consecutive days (D0, D1, and D2).
Each day, half of a 20-ml portion of inoculum containing no BRSV or 2.5 × 107 PFU of BRSV was instilled intranasally and
the other half was injected endobronchially. BAL was performed at D2 on
all the animals and then every 2 days for five calves from D4 to D18
and every 2 days for six calves from D5 to D19 and for some of the
animals at D30 and D36 postinoculation (p.i.). A standardized clinical examination was carried out every day from D2 to D36, and the mean
clinical score was calculated (12).
Viral strains.
A field BRSV strain (A2 gelfi) isolated in
1994 during a BRSV outbreak in France involving adults and calves was
propagated in BT cells in minimum essential medium with Earle's salt
and glutamine (MEM) (Gibco BRL, Cergy Pontoise, France) supplemented with 3% fetal calf serum (FCS). The sixth passage was aliquoted and
stored at
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Evaluation of a Nested Reverse Transcription-PCR
Assay Based on the Nucleoprotein Gene for Diagnosis of Spontaneous and
Experimental Bovine Respiratory Syncytial Virus Infections
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
80°C. The titer was 1.25 × 106 PFU/ml.
This stock provided the challenge inoculum and positive controls for
PCR, isolation in cell culture, and antigen detection. The absence of
common viral respiratory pathogens, including bovine viral diarrhea
virus (BVDV), in the inoculum was checked.
Clinical samples.
Clinical samples included serum, NS, BAL
fluid cells, and lung samples. Two blood samples were collected at a 2- to 3-week interval, and the serum was stored at 20°C until
analysis. Nasal cells and NS were collected with sterile cotton swabs,
placed in storage at 4°C less than 2 h after collection, and
stored until virological examination. Lung samples from necropsied
calves were kept at 4°C for less than 1 h and then stored at
80°C. Virological examinations were simultaneously performed less
than 1 month after sampling.
80°C until culture isolation or RT-PCR, both of which
were performed within the first month following sampling. BAL fluid
cells from experimentally infected animals were kept at 4°C and then
seeded on BT cells less than 2 h after sampling for culture
isolation. They were stored in TRIzol Reagent (Gibco BRL) at 80°C
until analysis by n RT-PCR-N. In all cases, enzyme immunosorbent assays
(EIA) and cell cytocentrifugation for indirect immunofluorescence (IIF)
were performed just after sampling. Slides for IIF were stored at
20°C until immunolabelling.
Serology.
Paired serum samples were analyzed with a
commercially available enzyme-linked immunosorbent assay (ELISA) kit
(LSI/VRS; LSI, L'arbresle, France) according to the manufacturer's
recommendations. The increase in antibodies was considered significant
when the corrected optical density at 450 nm (cOD) of the second serum sample was 
0.2 and when the ratio of the cOD of the second serum sample to the cOD of the first serum sample (collected 2 to 3 weeks
before) was 2.
BRSV isolation on BT cells. For BRSV culture isolation, BT cells were cultivated at 37°C in 5% CO2 and in culture medium (CM) (MEM [Gibco BRL] containing NEAA [Gibco BRL], penicillin [Gibco BRL] [100 IU/ml], dihydrostreptomycin [Gibco BRL] [100 µg/ml], Fungizon [Gibco BRL] [2.5 µg/ml], and 3% FCS). The cells contained in 10 ml of BAL fluid or stored in FCS and dimethyl sulfoxide were centrifuged (1,000 × g for 10 min at 4°C), frozen in 1 ml of CM, thawed, and then seeded on BT cells. For virus isolation from lung, 1 cm3 from the densified part (approximately 4 g) was crushed in a mortar and suspended in 10 ml of the medium used for BT cell culture. After centrifugation (700 × g for 10 min at 4°C), 1 ml of the supernatant was seeded on BT cells. After 2 h of adsorption, the inoculum was removed and the BT cells were washed once with 5 ml of CM and incubated with 5 ml of fresh CM. To eliminate bacterial contamination, the BT cells were washed once again 24 h after inoculation. If bacterial contamination was observed the BT cells were washed twice within the next 12 h. Three subculture passages were made in the absence of any characteristic cytopathic effect. BRSV was identified by an IIF test by using the monoclonal antibody 18 B2 directed against the F protein of HRSV (Argen Biosoft, Varilhes, France), which cross-reacts with BRSV (23). The secondary antibody was an anti-mouse rabbit polyclonal antibody-labelled fluorescein isothiocyanate (Sanofi Pasteur, Aulnay sous bois, France).
Antigen detection. The detection of BRSV antigen was carried out by IIF (with the same antibodies as described for identification of BRSV on BT cells) or by a commercial EIA, EIA RSV TESTPACK ABBOTT (EIA-TP) (Abbott, Rungis, France). IIF was performed on cytospin preparations of BAL fluid cells or on cryosections of lung. For cytospin, BAL fluid cells were filtrated through gauze, adjusted to a total number of 5 × 104, and cytocentrifuged (450 rpm for 8 min) by using a Cytospin 3 (Shandon, Pittsburgh, Pa.). EIA-TP was performed according to the manufacturer's recommendations on BAL fluid cells contained in 10 ml of BAL fluid, on NS, or on clarified homogenate of lung used for culture isolation.
RNA extraction.
The procedure for RNA extraction was adapted
from a method previously described (6). RNA extractions were
performed on BAL fluid cells contained in 10 ml of BAL fluid and on
approximately 1 g of crushed lung sample. Each sample was frozen
at 80°C in 1 ml of a solution of phenol and guanidine
isothiocyanate (TRIzol Reagent; Gibco BRL). It was then thawed and
incubated at room temperature for 5 min, and 250 µl of chloroform was
added to each sample. The tubes were centrifuged (12,000 × g for 15 min at 4°C) after vigorous shaking for 2 min and then
allowed to stand at room temperature for 10 min. The supernatant was
collected, and 1 ml of a more-concentrated solution of phenol and
guanidine isothiocyanate (Trizol LS Reagent; Gibco BRL) was
added to each tube. After purification by chloroform (vol/vol) and DNA
decontamination by isopropanol alcohol (1/10 volume), the RNA was
precipitated in isopropanol (vol/vol) with 0.3 M sodium acetate (pH
5.2) (Sigma) and 2 µl of Pellet Paint Co-Precipitant (Novagen,
Abingdon, England) overnight at 4°C. The sample was then centrifuged
(12,000 × g for 10 min at 4°C), the supernatant was
removed, and the pellet was washed in 1 ml of 70% ethanol. The sample
was again centrifuged (7,500 × g for 5 min at 4°C),
the supernatant was removed, the pellet was dried for 10 min, and the
RNA was resuspended in 36 µl of diethylpyrocarbonate-treated (DEPC) water.
RT-PCR. Primers used for RT-PCR-N were based on a multiple alignment, performed by using the ClustalW 1.60 program (18), of available sequences of N genes of RSVs. These sequences were from the following strains: three BRSV strains, RB-94 isolated in Belgium in 1970, A51908 isolated in Maryland in 1975, and 391.2 isolated in North Carolina in 1985 (GenBank accession no. L27840, M35076, and S40504, respectively); one ovine RSV (GenBank accession no. U07233); and two HRSV strains representing the HRSV subtypes A and B, strain A2 and strain 18537 (GenBank accession no. M11486 and D00390, respectively). PRIMER version 0.5 (22) was used for primer selection to ensure an optimal primer length of 20 nucleotides, to be compatible with the annealing temperature, and to have no more than eight bases of self-complementarity or no more than four bases of self-complementarity in the 3' terminal matching region. Primers were selected according to the consensus sequence of BRSV strain in conserved regions with HRSV strains and ovine RSV.
For RT, 1 µl (2 pmol) of primer N2.1 (5'-ATGGCTCTCAGCAAGGTCA-3'; positions 1 to 19 on the N coding region of the BRSV genome [30]) mixed with 9 µl of DEPC-treated water containing RNA was incubated at 68°C for 10 min and then at 58°C for 10 min and finally chilled on ice. Each tube received 4 µl of a solution containing each nucleotide triphosphate (10 mmol), 1 µl of RNasin (40 U) (Promega, Charbonnières, France), 2 µl of dithiothreitol (0.1 M) (Gibco BRL), 4 µl of SuperScript TM II buffer (250 mM Tris HCl, 375 mM KCl, 15 mM MgCl2) (Gibco BRL), and 1 µl of SuperScript TM II (200 U) (Gibco BRL) and was incubated at 42°C for 50 min. The reverse transcriptase was inactivated by heating at 70°C for 15 min. The RNA-cDNA hybrids were diluted 10 times in DEPC-treated water. Ten microliters of diluted cDNA was mixed in a final volume of 100 µl with 5 µl of primer N2.1 (50 pmol) and 5 µl of primer N2.2 (50 pmol) (5'-TCTTGGTTTCTTGGTGTACCTC-3'; positions 1034 to 1013 on the N coding region of the BRSV genome [30]) and used for the first round of PCR, which was carried out in a solution containing 200 µM of each nucleotide triphosphate, 10 mM Tris HCl (pH 8.3), 50 mM KCl, 1 mM MgCl2, and 2.5 IU of AmpliTaq Gold polymerase (Perkin Elmer Cetus, Roissy-Charles de Gaulle, France). The first round of PCR, which amplified 1,034 nucleotides (nt), was performed in a GeneAmp PCR System 480 (Perkin Elmer Cetus) by using the following program: 94°C for 12 min followed by 35 cycles of denaturation at 94°C for 60 s, annealing at 58°C for 60 s, and elongation at 72°C for 90 s and ending with a final elongation for 10 min. Ten microliters of the PCR products diluted 10 times was used to perform the second round of PCR with the same mix but containing the internal primers N2.3 (5'-CATCTCAATAAGTTGTGTGG-3'; positions 127 to 146 of the N coding region of the BRSV genome [30]) and N2.4 (5'-TCTACAACCTGTTCCATTTC-3'; positions 857 to 838 on the N coding region of the BRSV genome [30]). The second round of PCR amplified 731 nt and used the following program: 94°C for 12 min followed by 35 cycles of denaturation at 94°C for 45 s, annealing at 49°C for 60 s, and elongation at 72°C for 60 s and ending with a final elongation for 10 min. The PCR products were detected by electrophoresis on a 2% agarose gel containing ethidium bromide (0.1 µg/ml).| |
RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Threshold of detection of n RT-PCR-N. In order to improve the detection in BAL samples, an n RT-PCR was developed to amplify a region of the N gene. Different preliminary tests concerning the number of PCR cycles (it was found that 35 cycles produced better results than 25) and hybridization temperatures (it was found that for the first step 58°C produced better results than 56 and 60°C and for the second step 49°C produced better results than 50 and 52°C) were performed to optimize the protocol. When optimized, the threshold of detection for the n RT-PCR-N was determined by four independent serial 10-fold dilutions of the suspension of virus A2 gelfi. It was 506 times lower (mean = 0.17 PFU, standard deviation [SD] = 0.16 PFU) than that for EIA-TP (mean = 88.12 PFU, SD = 16.88 PFU).
Duration of detection of BRSV in BAL fluid during an experimental
infection.
Eleven calves were inoculated with BRSV, and six calves
were mock infected. All the animals in the inoculated group developed clinical signs. The mean clinical score from D5 to D13 p.i. was significantly higher (P < 0.001) in the
BRSV-inoculated group. The first clinical signs were observed on D4 and
D5 p.i., and individual scores increased to a maximum at D7 or D8 p.i.
and decreased slowly thereafter. One hundred and seven BAL fluid
samples were sequentially obtained from BRSV-infected calves from D2 to D36 p.i. The results obtained by n RT-PCR-N, isolation on cell culture,
and IIF were compared (Table 1). None of
the six control calves was positive during the trial. All the
inoculated calves were identified as positive by each of the three
techniques during the acute phase of the disease (D4 to D7 p.i.). At D8
and D9 p.i., 4 (36.4%) of 11 samples and 3 (50.0%) of 6 samples were
found to be positive by isolation on cell culture and IIF,
respectively, whereas all 11 samples (100%) tested by n RT-PCR-N were
positive. After the 9th day p.i., all the results for IIF and isolation on cell culture were negative. BRSV was detected until D17 p.i. in 2 of
11 animals (17%) by RT-PCR. BRSV was not detected at D30 and D36 p.i.
The average duration of detection of BRSV in BAL fluid by n RT-PCR was
12.7 days (n = 11). This was significantly longer than
the duration of detection by IIF (8.0 days, n = 6) or
by isolation (7.3 days, n = 11) (P < 0.01, 
2 test).
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Comparison of n RT-PCR-N, serology, EIA-TP, IIF, and isolation on
cell culture for field specimens.
During the acute phase of a
respiratory syndrome characterized by signs or lesions compatible with
BRSV infection, n RT-PCR-N (with BAL fluid cells and lung), EIA-TP
(with NS, BAL fluid cells, and lung), IIF (with BAL fluid cells), and
serology were performed by using field specimens obtained from a total
of 132 calves. To ensure clarity of the results, EIA-TP, IIF, isolation
on cell culture, and n RT-PCR-N were successively chosen as the
reference technique to which each of the other techniques was compared. The numbers of samples used for each of these comparisons were not the
same. For each of these comparisons the prevalence calculated on the
basis of the reference technique and the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of
each of the confirmatory techniques were determined (Table 2).
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2 test) than that of
serology (Table 2). Moreover, three calves were positive by serology
assay and negative by antigen detection (Table 2). IIF and EIA-TP with
BAL fluid cells gave similar results except for three calves (Table 2).
In contrast to the calf positive only by IIF, the only two calves that
were positive by EIA-TP could be considered true positives according to
their serological results or by the detection of BRSV in other calves
from the same herd.
For 28 field BAL fluid samples, the comparison of cell culture and
EIA-TP gave similar results (Table 2). It might seem that the highest
sensitivity was obtained by isolation on cell culture followed by
EIA-TP but these results were not significantly different (2 test). The results obtained with necropsy specimens
of lungs were slightly different from those obtained during the life of the calves. BRSV detection, with 16 lungs, by IIF and by EIA-TP gave
identical results (Table 2). However, isolation on cell culture had a
significantly lower sensitivity than IIF and EIA-TP (P < 0.01, 2 test) and a low NPV (Table 2).
The nested RT-PCR was then compared to EIA-TP (28 BAL fluid cell
specimens and 21 lungs) and to isolation on cell culture (28 BAL fluid
cell specimens and 13 lungs) for 49 naturally occurring cases of
respiratory disease in young cattle (Table 2). For BAL fluid cells,
results were in concordance except for four samples which were positive
by n RT-PCR-N and negative by EIA-TP. Three of them were also negative
by isolation on cell culture. Nineteen of 21 lungs were found to be
positive by n RT-PCR-N; among them 17 were simultaneously identified as
positive by EIA-TP.
Isolation on cell culture was also attempted on 13 of the 21 lungs.
Eleven of these were simultaneously positive by IIF, EIA-TP, and n
RT-PCR-N. However, BRSV was isolated from only one of these lungs.
Compared to n RT-PCR-N, the sensitivity of isolation on cell culture
was 0.87 for BAL fluid cells and 0.09 for lung.
The spectrum of detection of the n RT-PCR-N was also verified by using
nine other field isolates (W6, FV 160, 220/69, MRV533, 5761, RB-94,
Lelystadt, V347, and 127) originating from three European countries.
All of them were correctly amplified. The specificity of this n RT-PCR
assay was also tested by using different pathogens (IBRV, PI3, and
BVDV) not related to BRSV and associated with common respiratory
disorders. The results were negative.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Differences in prevalence (ranging between 25.3 and 90.5%) were observed in our field study depending on the series of calves studied. These differences can be explained by numerous factors, such as choice of herds and animal inclusion or diagnostic tools.
Nasal swabs and, even more often, nasopharyngeal aspirates rather than BAL fluid are commonly used to detect HRSV in infants (1). However, in BRSV infections (this study) as in HRSV infections (9), the sensitivity of EIA-TP on BAL fluid seems higher. Several hypotheses can be put forward. RSV is currently recovered from alveolar macrophages (24). Nasal swabs may sometimes collect an insufficient quantity of infected mucosal cells. However, nasal sampling is less invasive and easier to perform than BAL.
Results obtained by EIA-TP, IIF, and culture isolation on BAL fluid cells were similar. Furthermore, the sensitivities of EIA-TP and IIF were similar and significantly higher than that obtained for culture isolation by using lung specimens. These data could be explained by the lability of the virus several hours after death. Antigen capture ELISAs, such as EIA-TP, are frequently used in the diagnosis of HRSV infection in infants. Many of the antibodies developed against HRSV cross-react with BRSV (27). Results are easier and faster to obtain by EIA-TP than by IIF (assay times, 30 min and 4 h 30 min, respectively).
The sensitivity of antigen detection (IIF or EIA-TP) appeared to be significantly higher than that obtained by serology, thus confirming previous results (5, 21, 33). This low sensitivity of serology can be explained by the interference between maternally acquired antibodies and postinfection immune response (21). Specific IgM ELISA is potentially useful for overcoming this problem (40). However, in some calves no antigens were detected although a seroconversion was observed. Furthermore, respiratory signs could in some cases persist without BRSV detection. This suggests that antigen detection gives positive results only during a short time, which coincides with the acute phase of respiratory disease (4). More-sensitive diagnostic tools are thus needed to monitor BRSV infection.
The high conservation of the N gene in HRSV (19) and BRSV (2, 30) enabled us to define efficient and conserved primers. We therefore developed and optimized a nested RT-PCR whose target was the N gene to ensure high sensitivity and specificity. The ability of this n RT-PCR-N to detect a very small amount of virus was demonstrated by the very low threshold of detection (0.17 PFU/ml). The duration of the detection of BRSV in BAL fluid cells was investigated during an experimental infection of calves, which developed moderate to severe clinical signs. The duration of shedding obtained by n RT-PCR-N (13 days) was approximately more than twice that by isolation on cell culture or by IIF (5 days). Other authors reported that the detection of BRSV by isolation in cell culture (4, 32, 35) or by n RT-PCR targeting the F gene (11, 38) for more than 6 days was not possible. According to our data, the period of shedding of BRSV in calves is thus similar to that of HRSV in infants (8). Whether BRSV can be detected for as long as HRSV in immunocompromised infants (14, 16) is still open to debate.
The sensitivities and specificities of different laboratory techniques were also analyzed by using a series of clinically and epidemiologically identified field specimens. The sensitivity of n RT-PCR-N was higher than those of all the other techniques tested. We did not find any specimen identified as positive by conventional assay and negative by RT-PCR-N, in contrast to other reports for HRSV (13, 15, 17). These results could be linked to the elimination of potential inhibitory factors or to the fitness of the primers to the RNA template. On the other hand and despite potential RNA degradation, identification of BRSV RNA on necropsied lung samples was possible until 36 h after death. The specificity of n RT-PCR-N was checked. No amplification could be observed when n RT-PCR-N was performed by using control BAL fluid cells in the experimental model or the different common respiratory pathogens. Moreover, the sequences of portions of amplified N, G, and F genes were determined for each of 13 instances of discordance between results of n RT-PCR-N and isolation from BAL fluid cells or lungs. Pairwise comparisons indicated that these sequences differed by at least 1 nucleotide (not shown). This strongly supports the idea that a different BRSV isolate is present in each of the specimens.
In conclusion, the n RT-PCR presented in this study seems to be more sensitive than and at least as specific as IIF, EIA, or isolation in cell culture. It will allow (i) a broadening of the detection period for clinical specimens and (ii) study of the molecular epidemiology of field BRSV isolates.
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ACKNOWLEDGMENTS |
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We are grateful to S. Bonhoure, F. Lasserre, and M. Moulignié for their technical assistance. We thank S. Bertagnoli for helpful discussion and D. Desmecht for lung specimens.
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FOOTNOTES |
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* Corresponding author. Mailing address: UMR INRA-ENVT de Physiopathologie Infectieuse et Parasitaire des Ruminants, ENVT, 23 Chemin des Capelles, 31076 Toulouse cedex 3, France. Phone: 33 05-61-19-38-33. Fax: 33 05-61-19-38-34. E-mail: f.schelcher{at}envt.fr.
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REFERENCES |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Journal of Clinical Microbiology, June 1999, p. 1858-1862, Vol. 37, No. 6
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