Respiratory syncytial virus diagnosis

Edwards, S.; Newman, R.H.; Stanley, Margaret

doi:10.1136/vr.114.4.101

Cited by 8 publications

(4 citation statements)

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“…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).…”

Section: Methodsmentioning

confidence: 99%

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Development of nested PCR assays for detection of bovine respiratory syncytial virus in clinical samples

et al. 1994

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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.

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Section: Methodsmentioning

confidence: 99%

“…Direct virus detection can also be attempted by immunofluorescence (IF) (30) or by 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).…”

mentioning

confidence: 99%

Development of nested PCR assays for detection of bovine respiratory syncytial virus in clinical samples

et al. 1994

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Development of Nested PCR Assays for Detection of Bovine Respiratory Syncytial Virus in Clinical Samples

1994

J Clin Microbiol

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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.

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“…Similarly, successful isolations of virus from typical clinical cases of BRSV infection are often unsuccessful and can take from 11 to 21 days (with reports of >45 to 50 days [18,24]) because of the late appearance of any noticeable cytopathic effect. Because of these difficulties, isolation of BRSV is not recommended as a routine approach to diagnosis (8).…”

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Identifying bovine respiratory syncytial virus by reverse transcription-polymerase chain reaction and oligonucleotide hybridizations

et al. 1993

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An assay to identify tissue culture cells infected with bovine respiratory syncytial virus (BRSV) that utilizes reverse transcription (RT), the polymerase chain reaction (PCR), and a synthetic oligonucleotide hybridization probe has been developed. The RT-PCR assay uses a BRSV-specific negative-sense oligonucleotide primer to synthesize cDNA from a BRSV fusion protein mRNA template and another BRSV-specific oligonucleotide primer (positive sense) upstream from the negative-sense primer for PCR amplification. In the presence of mRNA templates of BRSV isolates originating from locations throughout the United States, the BRSV RT-PCR assay resulted in amplified products (381 bp) that were specific to BRSV, as demonstrated in hybridizations with a positive-sense oligonucleotide probe complementary to internal sequences and in sequence comparisons with the F protein of BRSV 391-2. In analysis of the BRSV RT-PCR assay with prototype strains of human RSV subgroups A and B, amplification of a similar 381-bp RT-PCR product was not evident, and no RT-PCR product hybridized with the internal probe. We conclude that the specific ability to amplify DNA sequences of BRSV F protein mRNA by RT-PCR and then to demonstrate the presence of the amplified product with a BRSV-specific oligonucleotide probe will greatly add to the speed, sensitivity, and specificity of BRSV diagnostics. Bovine respiratory syncytial virus (BRSV), a pneumovirus in the family Paramyxoviridae, is an important cause of acute respiratory disease in postweaning calves and feedlot cattle in the United States (1-3, 6). In Europe, BRSV infection is considered one of the most significant causes of bovine respiratory disease (11, 23). Although most infections are inapparent (2, 6, 12), the high prevalence of seropositive * Corresponding author. t Contribution no. 93-146-J from the Kansas Agricultural Experiment Station.

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Respiratory syncytial virus diagnosis

Cited by 8 publications

References 0 publications

Development of nested PCR assays for detection of bovine respiratory syncytial virus in clinical samples

Development of nested PCR assays for detection of bovine respiratory syncytial virus in clinical samples

Development of Nested PCR Assays for Detection of Bovine Respiratory Syncytial Virus in Clinical Samples

Identifying bovine respiratory syncytial virus by reverse transcription-polymerase chain reaction and oligonucleotide hybridizations

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