Detection of BVD viruses using synthetic oligonucleotides

Lewis, Terry L.; Ridpath, Julia F.; Bolin, Steven R.; Berry, Eugene S.

doi:10.1007/bf01310770

Cited by 19 publications

(17 citation statements)

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“…17 The BT cells were maintained at 37 Table 2. Age distribution for BVD-infected cattle C in MEM supplemented with 10% horse serum.…”

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“…Nine oligonucleotide probes were selected from across the genome of BVD virus by comparison with the genomic sequences of NADL and Osloss strains of BVD virus. 17 The oligonucleotides, all 20-mers, were synthesized on an Applied Biosystems Model 381A DNA synthesizer, which utilizes phosphoramidite chemistry. The oligonucleotides were end-labeled and prepared for use in hybridizations as previously described.…”

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confidence: 99%

“…The oligonucleotides were end-labeled and prepared for use in hybridizations as previously described. 17 The dot blots were prehybridized at 65 C for 4-5 hours in 6 x NET, 0.1% SDS, 5 x Denhardt's, and 100 µg/ml of denatured salmon sperm DNA. The hybridization reaction was carried out at 50 C for 2 hours in 6 x NET, 0.1% SDS, 5 x Denhardt's, and 1.5-2.0 x 10 7 cpm of end-labeled oligonucleotide (5-10 ng/ml of probe).…”

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Diagnostic Approaches for the Detection of Bovine Viral Diarrhea (BVD) Virus and Related Pestiviruses

et al. 1993

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Diagnostic approaches for the detection of bovine viral diarrhea (BVD) virus and related pestivirusesThe detection of bovine viral diarrhea (BVD) virus infections and diseases has evolved with other areas of viral diagnosis in veterinary medicine. 2,4,7,11,14,20 Our initial concerns were to confirm that a disease process was caused by BVD virus. We have satisfactorily attained a level of diagnostic expertise whereby we can diagnose clinical forms of BVD virus infection with relative ease, but it takes a prolonged period of time to detect BVD by conventional virus isolation in cell culture and subsequent identification by immunofluorescence or immunocytochemistry. 7,8,14,23,25 The questions that have arisen over the past decade are directed at achieving a diagnosis of BVD virus infection more rapidly when faced with a clinical situation and detecting animals with subclinical forms of BVD infection.Perhaps the most subtle form of BVD infection is the case of an immunotolerant animal that appears clinically normal, but harbors and sheds BVD virus throughout its life.18 This animal is regarded as being persistently infected, in contrast to a transiently infected animal, which is undergoing a primary BVD virus infection, but is immunocompetent and capable of developing a protective immune response against the virus.2 The persistently infected animal needs to be identified for at least 2 reasons. First, the animal is at risk to develop the fatal form of BVD, referred to as mucosal disease, if it should come in contact with a heterologous strain of BVD virus either by way of vaccination with modified live From the Washington Animal Disease Diagnostic Laboratory (Evvirus vaccine or by field infection.7,23 More recent evidence even suggests that mucosal disease may result following mutation of BVD, so that contact with other animals shedding heterologous BVD may not be required. 24 Second, the animal poses a risk of infecting other susceptible cattle and small ruminants because it is shedding virus in high concentration from body secretions and excretions. 3,5,15,26 Other subtle forms of disease in which diagnostic tools may be useful in determining whether BVD virus is an important etiologic entity include diseases of the endocrine system; prolonged or intermittent forms of immunosuppression; and concurrent viral and/or bacterial infections, which may have an underlying BVD virus pathogenesis. 2,6,19,23 The purposes of this paper are to present an update on the occurrence of BVD virus infection in the northwestern United States and to present some diagnostic strategies to consider when attempting to detect BVD virus infection and related pestiviruses.Bovine turbinate (BT) cells were obtained from the American Type Culture Collection a and maintained in minimal essential media (MEM) consisting of 5% fetal bovine serum (FBS) b and 5% fetal equine serum. c The FBS was negative for BVD virus and BVD virus antibody. The FBS was heat inactivated at 56 C for 90 minutes as an added precaution against low levels of BVD virus c...

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“…17 The BT cells were maintained at 37 Table 2. Age distribution for BVD-infected cattle C in MEM supplemented with 10% horse serum.…”

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confidence: 99%

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confidence: 99%

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confidence: 99%

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Diagnostic Approaches for the Detection of Bovine Viral Diarrhea (BVD) Virus and Related Pestiviruses

et al. 1993

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“…Other reports have described similar procedures but with primers (Schroeder & Balassu, 1990;Lopez et al, 1991;Ward & Misra, 1991) or probes (Kwang et al, 1991b;Lewis et al, 1991) located in the coding part of the genome. The choice of the 5' non-coding region has, however, been described for detecting hepatitis C viruses by PCR (Bukh et al, 1992) and for pestiviruses (Boye et al, 1991).…”

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Nucleotide sequence of the bovine viral diarrhoea virus Osloss strain: comparison with related viruses and identification of specific DNA probes in the 5' untranslated region

Moerlooze

Lecomte

Brown-Shimmer

et al. 1993

Journal of General Virology

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“…The genomic diversity of BVDV is well documented and presents a challenge to researchers looking for a nucleic acid-based test that will detect all isolates. 13,14,22 Further work in the development of a diagnostic PCR procedure for detection of BVDV should focus on the selection of a single universal set or mixture of primers that can detect all strains of BVDV. Also, reet al…”

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Evaluation of PCR for Diagnosis of Bovine Viral Diarrhea Virus in Tissue Homogenates

et al. 1994

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Abstract. Tissue homogenates from 60 specimens submitted to the Veterinary Diagnostic Center were evaluated by polymerase chain reaction (PCR) for detection of bovine viral diarrhea virus (BVDV). Conventional virus isolation procedures showed the specimens contained BVDV. The BVDV RNA was extracted from the homogenates and subjected to a reverse transcription reaction followed by PCR amplification. The PCR product was blotted onto a nylon membrane and hybridized with a 30-base pair oligonucleotide probe labeled with 32 P. One set of PCR primers detected BVDV in 46/60 (77%) of the tissue homogenates. An additional set of primers was used to detect 10/11 samples that had escaped detection with the first set of primers. The results indicate that BVDV can be detected by PCR directly out of tissue homogenates generated in a diagnostic setting.Bovine viral diarrhea virus (BVDV) is an important using the polymerase chain reaction (PCR) techpathogen of cattle causing a variety of diseases that nique. 1,2,5,6,10,11,15,23,24,26 range in severity from mild inapparent infections to Application of PCR in a diagnostic setting requires fatal mucosal disease. 20,21 BVDV is classified within adaptation for use on clinical samples. Previous reports the family Flaviviridae and is a member of the genus on the use of PCR for detection of BVDV have not Pestivirus. The viral genome consists of a single-strand-addressed the problems associated with clinical speced nonpolyadenylated RNA about 12.5 kb in length. imens. The purpose of this study was to evaluate PCR Isolates of BVDV are divided into 2 biotypes, cyto-detection of BVDV in field specimens using primers 15,22 that amplify sequences near opposite ends of the BVDV genome.pathic and noncytopathic, based on effects in cell culture. 25 Infections of cattle with noncytopathic BVDV during early gestation may cause abortion or birth of calves immunologically tolerant to the infecting virus.12,17 These calves are persistent carriers of BVDV and can be a source of infection to the herd. 4 Superinfection of persistently infected cattle with a cytopathic BVDV may induce mucosal disease.4,7 Detection of persistently infected cattle is important for control of BVD. In addition, contamination of fetal bovine serum and other bovine products used in cell culture work with BVDV presents a problem in preparing veterinary biologics. 3,19 Traditionally, virus isolation with confirmation by immunofluorescence has been the method used to detect BVDV in clinical samples and in contaminated cell cultures and biological products. These techniques are time consuming and require the use of cell culture. Several reports have described rapid diagnostic tests for BVDV Materials and MethodsPreparation of clinical samples. Tissue samples from cattle with clinical signs of BVD were submitted to the University of Nebraska Veterinary Diagnostic Center virology laboratory. All samples came from the state of Nebraska between 1988 and 1990. The samples were prepared as follows. Minimum essential medium (MEM) with...

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Detection of BVD viruses using synthetic oligonucleotides

Cited by 19 publications

References 19 publications

Diagnostic Approaches for the Detection of Bovine Viral Diarrhea (BVD) Virus and Related Pestiviruses

Diagnostic Approaches for the Detection of Bovine Viral Diarrhea (BVD) Virus and Related Pestiviruses

Nucleotide sequence of the bovine viral diarrhoea virus Osloss strain: comparison with related viruses and identification of specific DNA probes in the 5' untranslated region

Evaluation of PCR for Diagnosis of Bovine Viral Diarrhea Virus in Tissue Homogenates

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