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Results for “Pestivirus RT-PCR”

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At least 19 recordsLinked to original sources

PubMed1996

Differentiation of classical swine fever virus from ruminant pestiviruses by reverse transcription and polymerase chain reaction (RT-PCR).

A reverse transcriptase-polymerase chain reaction (RT-PCR) assay was designed to allow the differentiation of pestiviruses by the expected size of the amplified fragments. One oligonucleotide primer, conserved amongst pestiviruses, and two others specific for either classical swine fever virus (CSFV) or bovine viral diarrhea virus (BVDV), were designed from the 5' non-coding region of the genome. CSFV infected cultures (10 strains) amplified a fragment of an expected size of 200 bp; BVDV cultures (23 strains) or border disease virus (BDV) (2 strains) amplified a fragment of an expected size of 260 bp. The specificity of the amplified fragments was confirmed by restriction enzyme analysis. The threshold of sensitivity was 100 TCID50 for CSFV and 1 TCID50 for BVDV. The RT-PCR described here provides a rapid and sensitive diagnostic tool for the detection and differentiation of CSFV from ruminant pestiviruses.

Animals↗

PubMed1994

Evaluation of a 'one tube' reverse transcription-polymerase chain reaction for the detection of ruminant pestiviruses.

A 'one tube' reverse transcription-polymerase chain reaction ('one tube' RT-PCR) using rTth DNA polymerase was compared with an existing RT-PCR using Taq DNA polymerase (Taq RT-PCR) to detect ruminant pestiviruses in infected cell cultures. The technically simpler and more convenient 'one tube' method was relatively insensitive detecting only 11 of the 34 samples tested, all of which were positive by Taq RT-PCR.

Animals↗

PubMed1997

Genetic analysis of classical swine fever virus isolates from a small geographic area.

Twenty-eight field CSFV samples were isolated from outbreaks of CSF which have occurred in three small geographic areas in Slovakia in the period of December 1993-November 1994. All the organ homogenates were positive by virus isolation and RT-PCR assay using general pestivirus 324/326 primers selected from 5'-noncoding (5'-NC) genomic region. Specific discrimination of CSFV was confirmed by the cleavage of amplicons using Bgl1 and Ava 1. Four viral isolates (A, B, C, E) were selected for a comparative sequencing study. Sequencing of amplicons in the 5'-NC, gp55 (E2), p54 (NS2) and NS5B genomic regions revealed that A, B isolates (originated from SR geographic area) were the same but different from identical C (NR geographic area) and E (ER geographic area) isolates. Results of molecular-genetic study were very well supported with epizootological data. All CSFV isolates from Slovakia phylogenetically fell into the group of recent European isolates clearly separated from old European and American strains.

Animals↗

PubMed1993

Detection of hog cholera virus and differentiation from other pestiviruses by polymerase chain reaction.

Reverse transcription coupled with the polymerase chain reaction (RT-PCR) was used for the detection and differentiation of pestiviruses. For this purpose, one primer pair was selected from a highly conserved region of the genome of pestiviruses. Using these primers (PEST 1-PEST 2), DNA fragments of between 72 and 74 bp could be amplified from all pestivirus isolates tested. In order to differentiate hog cholera virus (HCV) from bovine viral diarrhea virus (BVDV) and border disease virus (BDV), we selected a primer pair from a conserved region in the genome of HCV strains that differed from that sequenced in the genome of BVDV strains. By using these primers (HCV 1-HCV 2), a DNA fragment of 478 bp could be specifically amplified from HCV isolates. By these means, viral RNA was detected in extracts of lymph node, spleen, tonsil, and lung. Such extracts were used directly for RT-PCR without prior RNA isolation. We also performed multiplex PCR by using both the PEST 1-PEST 2 and HCV 1-HCV 2 primer pairs in a single reaction. This allowed the differentiation of HCV from BVDV and BDV in one step. To assess the sensitivity of the method, RT-PCR was compared with virus propagation in tissue culture and subsequent detection by immunofluorescence staining. The results show that RT-PCR is useful for the rapid detection and differentiation of pestiviruses.

Animals↗

PubMed2025

Molecular Detection and Characterization of Bovine Diarrhea Virus (BVDV) in Aborted Fetuses and Semen Samples from Paraguay.

Bovine viral diarrhea virus (BVDV) is the most prevalent pathogen in cattle and causes significant economic losses due to its severe clinical manifestations. It belongs to the family Flaviviridae and is distributed in species A, B, and H within the genus Pestivirus. The objective of this study was to detect and characterize BVDV using molecular techniques (RT-PCR) in semen and aborted fetuses samples that were sent to the CEDIVEP (Veterinary Diagnostic Center of Paraguay) laboratory. Seventy-three samples of semen from bulls were analyzed, and 54.7% of the samples were positive for Pestivirus A. The presence of Pestivirus A and H was detected in 2/8 spontaneously aborted fetuses. The genotypes of four individual samples of type A and four samples of type H organs were confirmed by partial sequencing of the 5-UTR region. The presence of BVDV was confirmed by molecular techniques for the first time in our country through its detection in different types of samples, as well as the presence of two genotypes. This suggests that the circulation of this virus can cause significant losses in cattle production in Paraguay.

Diarrhea Viruses, Bovine Viral↗

PubMed1995

Demonstration and genotyping of pestivirus RNA from mammalian cell lines.

We examined 20 cell lines of various animal origins for the presence of pestivirus contamination by the reverse transcription-polymerase chain reaction (RT-PCR), and found 15 (75%) cell lines were positive. The RT-PCR products of the 5' untranslated region (UTR) of pestivirus genome were sequenced and subjected to genotyping. Stem-loop structures at three variable regions in the 5' UTR render genotyping of the contaminated pestiviruses. Bovine cell lines tested were all contaminated with genotypes I, II, or III of bovine diarrhea virus (BVDV). Cell lines of canine, feline, and primate origin were contaminated with genotype II of BVDV. Cell line Ch1Es of caprine origin was contaminated with border disease virus (BDV).

Animals↗

PubMed1993

Distribution of cytopathogenic and noncytopathogenic bovine virus diarrhea virus in tissues from a calf with experimentally induced mucosal disease using antigenic and genetic markers.

A comparative analysis of the distribution of cytopathogenic (cp) and noncytopathogenic (ncp) bovine virus diarrhea disease (BVD) virus in tissues from a calf with experimentally induced mucosal disease was performed using immunohistology and polymerase chain reaction after reverse transcription (RT-PCR) of viral RNA. For immunohistology, an antigenic marker on the superinfecting cp BVD virus defined by a monoclonal antibody (mab) was used, and overall presence of antigen was assessed with a pestivirus specific mab. The primers selected for RT-PCR detected the genomic insertion in the p125 region of the superinfecting cp BVD virus. Both methods gave consistent results.

Animals↗

PubMedDate not supplied

Comparison of RT-PCR assay and virus isolation in cell cultures for the detection of bovine viral diarrhoea virus (BVDV) in field samples.

The virus isolation-immunoperoxidase test (IPX) on cell cultures and the reverse transcription-polymerase chain reaction (RT-PCR) assay were compared for the detection of bovine viral diarrhoea virus (BVDV) directly in serum samples. Material for this study consisted of 403 sera originating from cattle in 41 BVDV-infected Finnish dairy herds and one suckler cow herd. The presence of virus was demonstrated in 48 samples by both assays. In addition, two more samples were found to be positive by the RT-PCR assay. Both methods proved to be extremely sensitive, detecting pestiviruses even in high serum dilutions, and thus to be suitable for demonstrating the occurrence of persistently infected (PI) cattle. In conclusion, the RT-PCR method used had the advantage of ascertaining BVDV nucleic acid sequences in samples in which the virus had been inactivated, eg during transport or due to the presence of neutralising antibodies.

Animals↗

PubMed1996

Evaluation of nucleic acid amplification methods for the detection of hog cholera virus.

A blind panel was tested in a diagnostic evaluation of a reverse transcription (RT) polymerase chain reaction (PCR) method for detecting hog cholera virus (HCV) from pig tissues. The capability of the RT-PCR test to discriminate between HCV and related pestiviruses, bovine viral diarrhea virus (BVDV), and those viruses causing similar diseases in swine, including African swine fever virus (ASFV) and pseudorabies virus (PRV), was also considered. Nucleic acid extraction involved either kit-based or conventional phenol:chloroform:isoamyl alcohol methods. A single-round PCR assay, using primers that hybridize to the conserved p120 nonstructural gene region, was 82.5% sensitive (n = 17) and 100% specific (n = 18) in the detection of the presence of HCV RNA. However, the sensitivity was increased to 100% following a second PCR test. In all, 4 HCV, 7 BVDV, 2 ASFV, and 1 PRV isolates were studied. Novel nucleic acid sequences were generated for 9 HCV strains. Analysis of a portion of the p120 region using these methods was suitable for HCV isolate characterization.

African Swine Fever Virus↗

PubMed1994

Rapid characterization of new pestivirus strains by direct sequencing of PCR-amplified cDNA from the 5' noncoding region.

Reverse transcription coupled with the polymerase chain reaction (RT-PCR) was used for the rapid laboratory diagnosis of pestivirus infections. A direct DNA sequencing method was developed for the analysis of the amplified cDNA from the 5' noncoding region of the viral genome. 70 pestivirus strains were compared in this study. Sequence analysis allowed the characterization of each isolate as either classical swine fever virus (CSFV), bovine viral diarrhea virus, or border disease virus, respectively. The 48 CSFV strains could be further classified into several subgroups, which correlated either with the geographical origin or the date of the first isolation of the respective isolate.

Animals↗

PubMed1995

Further characterization of border disease virus isolates: evidence for the presence of more than three species within the genus pestivirus.

The molecular analysis of three ovine pestivirus strains revealed the existence of two distinct groups of sheep-derived pestiviruses, namely "true" border disease virus strains (BDV) and bovine viral diarrhea virus (BVDV)-like strains. As an extension of these studies RT-PCR and nucleotide sequencing of the autoprotease (Npro) and nucleocapsid protein (C) encoding regions of additional serologically defined ovine pestivirus strains were performed. A comparison of Npro and C revealed that three ovine isolates belong to the group of true BDV while two were clearly different from these as well as from BVDV and CSFV. The amino acid identity between the latter two ovine strains is 85% for Npro and 92% for C and thus similar to values found within the three pestivirus species defined so far. In order to allow comparison with additional pestiviruses the nucleotide sequences of a part of the 5' noncoding region were determined for the ovine pestivirus strains. The comparative analysis showed that recently described BVDV strains associated with acute lethal infection were very similar to two ovine isolates. The latter represent a fourth group within the genus pestivirus different from the so far defined pestivirus species BVDV, true BDV, and CSFV. The data lead to the question whether the current nomenclature of pestivirus species according to the host origin and the induced diseases is still appropriate.

Amino Acid Sequence↗

PubMed1996

Genetic identification of pestivirus strain Frijters as a border disease virus from pigs.

A pig pestivirus isolate, the Frijters strain, was characterized by using reverse transcription-PCR (RT-PCR), followed by RFLP and direct sequencing of the amplicons. Restriction endonuclease enzyme AvaI and Bg/I digestion of 5' NC genomic region PCR products suggested that Frijters strain belongs to the border disease viruses (BDV). This finding was confirmed by nucleotide sequencing of the amplified part of the 5-NC genomic region, which revealed 94% and 95% nucleotide similarity between the Frijters strain and the BDV prototype strains Moredun cp and Moredun ncp, respectively. On the other hand, 55-77% nucleotide identity was observed between the Frijters strain and prototypes of BVDV and CSFV. The amino acid similarity in the Npro (p20) region was 89% between Frijters and the Moredun strains and 70-77% between Frijters and the BVDV of CSFV strains. It was concluded that the Frijters virus is a member of the BDV group of the Pestivirus genus. Considering that this virus circulates in the large swine populations of Europe, the spread of certain pestiviruses to more than one animal species should not be considered sporadic. Since the presence of BDV in swine herds might be a confusing factor during classical swine fever eradication programmes, highly specific methods are required for reliable identification of these isolates. RT-PCR, followed by RFLP and sequencing of the amplicons proved to be a reliable approach for the rapid detection and identification of pestiviruses.

Animals↗

PubMed1995

Comparison of an antigen capture enzyme-linked assay with reverse transcription--polymerase chain reaction and cell culture immunoperoxidase tests for the diagnosis of ruminant pestivirus infections.

A study to compare the merits of three different tests for the diagnosis of ruminant pestivirus infections was carried out. Sensitivity studies using reference strains of bovine viral diarrhoea virus (BVDV) and buffy coat samples from persistently infected (PI) carriers showed the reverse transcription-polymerase chain reaction (RT-PCR) had a greater sensitivity than the other tests. The antigen capture enzyme-linked immunosorbent assay (ELISA) was least sensitive and could only be used on samples containing cells (tissue or blood). When 169 clinical samples were examined, the RT-PCR detected the most positives (42) compared to the ELISA (32) and the immunoperoxidase test (IPT) (20). The RT-PCR was more successful when specific antibody was also present in the sample. The lower sensitivity of the IPT was related to the use of a 1 passage (4-day) test and the testing of toxic or contaminated samples. The ELISA was found to be most suitable for large-scale testing for the diagnosis and control of pestivirus infections.

Animals↗

PubMed1997

Molecular characterization of ovine pestiviruses.

Forty-two ovine pestivirus isolates, collected over a period of 18 years, were compared by phylogenetic analysis. The viruses were mostly field isolates from Britain; two others originated from Sweden and two from New Zealand. RT-PCR products were obtained from two genomic regions, one within the 5'-noncoding (5'-NC) region, and the other encompassing parts of the p20 (Npro) and C coding regions. Direct sequencing of the 5'-NC PCR products, followed by computer-assisted phylogenetic analysis, divided the ovine pestiviruses into three main genotypes. The results demonstrated that sheep may naturally be infected not only with border disease virus (BDV), but also with bovine viral diarrhoea virus (BVDV) types I and II. The BDV isolates segregated into two principal subtypes represented by the Moredun strain from Scotland and the 137/4 strain from England. The BVDV-I group was composed of three clusters, two of them represented by BVDV reference strains NADL and Osloss, respectively, and the third by ovine isolates D1120/1 and D1432/P. The grouping of ovine pestiviruses, based on comparative nucleotide sequence analysis of the 5'-NC region, was confirmed by comparative analysis of the p20 (Npro) and C coding regions, performed both at the nucleotide and at the amino acid level. The presence of three genotypes in sheep, including BVDV-I and BVDV-II, indicates the inadequacy of the current hostspecies-based nomenclature and classification of pestiviruses.

Animals↗

PubMed1997

Detection and identification of ruminant and porcine pestiviruses by nested amplification of 5' untranslated cDNA regions.

Based on published gene sequences of bovine viral diarrhoea virus (BVDV) type I and classical swine fever virus (CSFV), genus- and species-specific primers were designed to detect and identify pestivirus cDNA sequences in a nested polymerase chain reaction (PCR). The PCR primers were validated using cDNA synthesized from 146 pestivirus isolates, comprising representatives of all four so far described genotypes (BVDV type I, BVDV type II, CSFV and border disease virus), as well as others of uncertain classification. PCR products of the predicted size were amplified from all viruses with the genus-specific primers. All 53 cattle isolates, including 5 typed antigenically as BVDV type II were amplified by the internal BVDV-specific primers, but not the CSFV-specific primers. The same result was found for other BVDV type I and II viruses isolated from sheep and pigs. Seventy-seven CSF viruses were amplified by their respective internal primers. Available information strongly indicate that 4 CSF viruses also amplified by the BVDV-specific primers had been contaminated with BVDV in cell cultures. Border disease viruses were mostly not detected by the BVDV-specific primers, but were detected weakly by the CSFV-specific primer pair. Using carrier RNA for extraction of viral RNA, the sensitivity of detection of the single and nested PCR was, respectively, 5 and 50 times higher than obtained with a cell culture assay. The RT-PCR also detected BVDV in all of 15 commercial batches of fetal calf serum examined, and verified three earlier diagnoses of CSFV by detecting specific gene sequences in 30 year old frozen archival organ samples.

Animals↗

PubMed1993

RNA insertions and gene duplications in the nonstructural protein p125 region of pestivirus strains and isolates in vitro and in vivo.

Sixteen cytopathogenic (cp) bovine viral diarrhea virus (BVDV) strains/isolates were screened for the existence of RNA insertions in the p125 gene region and/or for p80 gene duplications using the polymerase chain reaction after reverse transcription. Three strains/isolates were shown to contain insertions, and in three others gene duplications were demonstrated. One strain was shown to contain a gene duplication in addition to an insertion. Insertions or duplications were absent in the genomes of 10 non-cp BVD virus strains/isolates, in 4 hog cholera (HC) virus strains, and in 9 of the 16 cp BVD virus strains/isolates analyzed. These results indicate that RNA insertions and gene duplications, although significant, are not the only mechanism contributing to cytopathogenicity in vitro. The RT-PCR was also applied to RNA isolated from tissues of a calf suffering from mucosal disease. Insertion-containing amplification products could be demonstrated in an organ-specific pattern.

Animals↗

PubMed1997

Variation from cytopathogenic biotype to non-cytopathogenic biotype is correlated with the deletion of cellular sequence from bovine viral diarrhea viruses.

Non-cytopathogenic (NCP) viruses of bovine viral diarrhea (BVD) virus were detected at a low ratio by the reverse plaque formation method from virus samples after several plaque clonings of cytopathogenic (CP) BVD viruses; NADL and Osloss strains. This phenomenon suggests that the NCP BVD viruses are produced at a low ratio during the propagation of CP BVD viruses in vitro. To investigate the differences between the parent CP BVD virus and the NCP BVD virus as a real progeny, the regions flanking the insertion of cellular mRNA in the p125 domain of NADL and Osloss strains were amplified by RT-PCR and cloned into pGEM 3Z plasmid vector, and then sequenced. Consequently, it was confirmed that sequences of cellular mRNA insertion of CP BVD viruses, NADL and Osloss strains, were completely and exactly deleted from the NCP BVD viruses which were real progeny of CP BVD viruses, NADL and Osloss strains. These results suggest that NCP BVD viruses may revert from CP biotype to NCP biotype by the deletion of cellular mRNA insertion in the viral genome of CP BVD viruses (NADL and Osloss strains).

Amino Acid Sequence↗

PubMed1993

Detection of bovine viral diarrhea virus RNA in formalin-fixed, paraffin-embedded brain tissue by nested polymerase chain reaction.

Isolation and amplification of RNA from formalin-fixed, paraffin-embedded tissues is delicate due to its fragility and ubiquitous ribonucleases. For retrospective studies, however, a convenient procedure for the detection of RNA in archived material is of great value. Bovine viral diarrhea (BVD) virus is a member of the pestivirus genus in the family Flaviviridae. Different protocols for the isolation of BVD virus RNA from fresh and autolytic as well as from routinely formalin-fixed and paraffin-embedded brain tissue of BVDV-infected calves were compared. The polymerase chain reaction (PCR) after reverse transcription (RT-PCR) was carried out subsequently for the detection of viral RNA. Using proteinase K digestion of deparaffinized tissue sections without additional ribonuclease inhibitors and subsequent nested PCR, a 803 bp fragment of the gene coding for the nonstructural protein p125 of BVD virus could be consistently detected. In addition, BVD virus RNA was detected by RT-PCR from non-fixed brain tissue after 10 days of autolysis.

Animals↗