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PubMed1999

Detection and genotyping of bovine diarrhea virus by reverse transcription-polymerase chain amplification of the 5' untranslated region.

A reverse-transcription polymerase chain reaction (RT-PCR) was developed to differentiate the bovine diarrhea virus (BVDV) from other pestiviruses, and to determine the genotype of the BVDV isolates. For this purpose, primer pairs were selected in the 5' untranslated region (5'UTR). The primers BE and B2 were located in highly conserved regions and were pestivirus-specific. Two primer pairs named B3B4 and B5B6 were specific of BVDV genotypes I and II, respectively. With this technique, an amplification product of the expected size was obtained with either the B3B4 or the B5B6 primer pairs for the 107 BVDV isolates tested but not for BDV or CSFV. For some isolates that were grouped in the genotype II, sequence analysis of the PCR fragments confirmed their classification into this genotype.

5' Untranslated Regions↗

PubMed1999

Serological and molecular diagnosis of bovine viral diarrhoea virus and evidence of other viral infections in dairy calves with respiratory disease in Venezuela.

An investigation based on 2 studies was carried out to assess the involvement of bovine virus diarrhoea virus (BVDV), bovine herpesvirus type 1 (BHV-1), and bovine respiratory syncytial virus (BRSV) in calf respiratory disease in dairy farms in Venezuela. In the first study, 8 farms were selected and paired serum samples from 42 calves with respiratory disease were tested by ELISA for antibodies to the 3 viruses. Seroconversion to BVDV, BHV-1, and BRSV was found to 5, 2, and 6 farms out of the 8, respectively. The proportion of calves that showed seroconversion to BVDV, BHV-1, and BRSV were 19%, 14%, and 26%, respectively. In the second study, another farm having previous serological evidence of BVDV infection was selected. The decline of maternal antibodies against BVDV was monitored in 20 calves and the half-life of maternal antibodies was 34 +/- 12 days presumably indicating an early natural infection with BVDV. Furthermore, sera free of BVDV antibodies that were collected in studies 1 and 2 and were assayed for the presence of BVDV by nested RT-PCR. Two BVDV strains were detected and compared to those of ruminant and porcine pestiviruses. Both strains were assigned to subgroup Ib of type I BVDV. This investigation provides information on BVDV genotypes circulating in Venezuela and may contribute to the establishment of official control programmes against the viruses studied.

Animals↗

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↗

PubMed2000

Evaluation of vaccines, interferons and cell substrates for pestivirus contamination.

Pestiviruses are potential contaminants of biological products produced in bovine or porcine cells or manufactured via processes using animal-derived raw materials such as bovine serum. In order to investigate possible contamination of products including those manufactured and/or licensed in the US, 38 lots of viral vaccines and five lots of interferon alpha (IFNalpha) were tested by reverse transcriptase polymerase chain reaction (RT-PCR) for the presence of bovine viral diarrhoea virus (BVDV). All vaccines and interferons were negative for contaminating BVDV RNA when tested by RT-PCR, with the exception of an experimental live viral vaccine that had been produced in BVDV contaminated rabbit kidney cells. Cell lines commonly used to produce biological products and vaccines were experimentally infected with the NADL strain of BVDV to determine if they were permissive for virus replication. MRC-5 and WI-38 cells were not infected. In contrast, Vero, CHO and CEF cells showed evidence of pestivirus infection. Taken together these data suggested that currently licensed viral vaccines were unlikely to be contaminated with pestiviruses. However, cell banks derived from non-human primate, hamster or rabbit kidney cell lines, or cultures of primary chick embryo fibroblasts, may be infected with BVDV if exposed to pestivirus contaminated raw materials during manufacture.

Animals↗

PubMed1999

Genetic heterogeneity of porcine and ruminant pestiviruses mainly isolated in Japan.

The genetic variability of porcine and ruminant pestiviruses was studied by comparative nucleotide sequence analysis of 73 isolates (42 porcine and 31 ruminant), including 65 Japanese isolates (35 porcine and 30 ruminant). The 5'-untranslated region (UTR) amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) was determined by direct sequencing and phylogenetic analysis was performed from the nucleotide sequence data. Most porcine isolates were divided into two major subgroups, classical swine fever virus (CSFV) subgroup 1 (CSFV-1, represented by Brecia strain) and subgroup 2 (CSFV-2, represented by Alfort strain). However, the Japanese Kanagawa/74, Okinawa/86, Okinawa/86-2 and Thai CBR/93 strains were the most distinct variants and these were assigned to another new disparate subgroup, CSFV-3 (represented by p97 strain). Most ruminant isolates were classified as the bovine viral diarrhoea virus (BVDV) genotype-I (BVDV-I) and subdivided into two subgroups, BVDV-Ia (represented by the NADL strain) and Ib (represented by the Osloss strain). Two bovine isolates (MS-1 and SY-89) and a contaminating strain (V/FLL) from an ovine cell line were classified as BVDV genotype-II (BVDV-II) on genetic characteristics. These data suggested that the detection and phylogenetic analysis of 5'-UTRs are useful for the rapid characterization of field isolates.

Animals↗

PubMed1998

Detection of bovine viral diarrhea virus (BVDV) using reverse transcription polymerase chain reaction assay.

The reverse transcription polymerase chain reaction (RT-PCR) was applied to detect bovine viral diarrhea virus (BVDV) for the rapid diagnosis. The primers were selected from the p80 region of BVDV gene. The RT-PCR assay detected all of the 17 BVDV strains tested including cytopathogenic and non-cytopathogenic strains, while specific amplification was not observed from 17 bovine viruses other than BVDV. Detection limit of the assay was 10(1.5) TCID50/ml. Sera and organ samples were collected from four field bovine viral diarrhea-mucosal disease (BVD-MD) cases; mucosal disease, abortion, diarrhea and persistent infection. The RT-PCR assay detected BVDV from those samples more than conventional virus isolation method.

Abortion, Veterinary↗

PubMed2000

Classical swine fever virus: a second ring test to evaluate RT-PCR detection methods.

Six laboratories participated in a study to compare the sensitivity and specificity of RT-PCR tests for the detection of classical swine fever virus (CSFV). Sets of coded samples were prepared by serial dilution of positive samples and then distributed to each of the laboratories. One set comprised 25 samples of random primed cDNA, synthesised from viral RNA representative of different pestiviruses. The other set comprised samples of blood and serum obtained from virus-free or CSFV-infected pigs. Each laboratory tested the samples using PCR/RT-PCR according to a set of standardised protocols that specified the exact conditions and requirements for inclusion of control samples. Two types of test were evaluated. One amplified a part of the 5'-non coding region of the pestivirus genome by means of a closed, one-tube RT-nested PCR. The other amplified a part of the NS5B gene using non-nested RT-PCR. The results of the laboratories were compared with one another, and with those obtained earlier when similar samples were tested by the same laboratories using non-standardised methods [Paton et al., Classical swine fever virus: a ring test to evaluate RT-PCR detection methods, Vet. Microbiol., in press]. Standardisation of the protocols resulted in a more consistent test sensitivity. Three laboratories avoided significant false positive results. Others that did not, could nevertheless recognise that test specificity was inadequate from the results obtained with the control samples. Minimum requirements for the inclusion of adequate controls and periodic proficiency testing are proposed.

Animals↗

PubMed1997

Genetic heterogeneity of bovine viral diarrhoea viruses isolated in Southern Africa.

Seventy three field isolates of bovine viral diarrhoea virus (BVDV), obtained from cattle in Mozambique and South Africa, were characterised by comparative nucleotide sequence analysis of part of the 5' non-coding region (5'NCR) of the viral genome. The target region was amplified by reverse transcription-polymerase chain reaction (RT-PCR). The amplicons were cloned in pUC 19 plasmid and both strands were sequenced by T7 polymerase dideoxynucleotide chain-termination sequencing or directly by cycle sequencing. The 245 base pair (bp) nucleotide sequences, derived from the 5'NCR, were aligned and compared to the corresponding positions of published sequences of BVDV type I and II strains and of pestiviruses of ovine and porcine origin. The phylogenetic trees, generated from those comparisons, allowed the Southern African isolates to be assigned to two main groups within the BVDV I genotype. Group A could be subdivided into three clusters, two of which grouped with BVDV strains of European and American origin. The third cluster did not group with any known BVDV I strains and it was confirmed in a comparison from the NS3 coding region. Group B contained more divergent isolates which differed by 18-23%, from BVDV I reference strains NADL, Osloss and SD-1 and comprised another distinct subset within the BVDV I genotype. This grouping was consistent in a comparison involving the NS2-NS3 region. It was concluded that BVD viruses occurring in Southern Africa are genetically diverse, comprising different variants within the BVDV I genotype. They include viruses similar to BVDVs found in Europe and America and others apparently rare or absent in those continents, termed here as BVDV Ic and Id. The co-existence of BVDV strains of European and American origin in certain areas both in Mozambique and South Africa, indicates a probable introduction of those viruses through imports of cattle or through potentially infectious bovine products. In addition, the detection of isolates apparently rare or absent from Europe and America may indicate the presence of African variants of BVDV I (Pestivirus 1).

Africa, Southern↗

PubMed2001

Genetic typing of bovine viral diarrhea virus isolates from Argentina.

Genetic typing of 29 Bovine Viral Diarrhea Virus (BVDV) isolates from Argentina was carried out by sequencing 245 nucleotides of the RT-PCR products of the 5'-UTR region. Sequence analysis shows that these Argentinean BVDV include types 1 and 2. The majority (26/29) of the isolates are type 1, which comprises subtypes 1a and 1b, together with an additional subgroup within subtype 1a. This subgroup is close to the South African subgroup Ic of 1a viruses, and to the deer pestivirus strain "Deer". The three type 2 BVDV were isolated from fetal tissues or serum during the 7-8 years before a clinical outbreak in Argentina had been reported. Only inactivated vaccines are used in bovines of the country, thus the analysed viruses are authentic field strains. The long term circulation of type 2 BVDV (situation similar to that of North America before the epidemic of 1993), and the existence of viral populations which differ from the reference strains commonly used in vaccine elaboration should be considered by manufacturers of diagnostic reagents and vaccines.

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↗

PubMedDate not supplied

An RT-PCR assay for the specific detection of classical swine fever virus in clinical samples.

A simple reverse transcriptase-polymerase chain reaction (RT-PCR) assay has been developed for the specific amplification of DNA after reverse transcription of RNA from the classical swine fever virus (CSFV). A pair of oligonucleotides was selected from an area of high homology in the genome of CSFV strains, but which differed from the corresponding sequences in the genome of bovine viral diarrhea virus (BVDV) strains. Using these primers (CSFV1-CSFV2), a CSFV specific DNA band of 174 bp was amplified from the CSFV RNA extracted from four reference strains and 14 field isolates, as well as from 25 organ extracts and eight buffy coats and serum samples of experimentally infected animals. No amplification was observed with the RNA from four BVDV reference and vaccine strains and seven field isolates. This RT-PCR assay made it possible, in a one-step reaction, to detect CSFV rapidly, sensitively and specifically in cell culture supernatants and in clinical specimens.

Animals↗

PubMed1999

An internal duplication in the 5' noncoding region of strain H: a bovine viral diarrhoea virus (BVDV) isolated from pigs.

A pig pestivirus isolate, strain H, was characterized by using reverse transcription-PCR (RT-PCR) and direct sequencing of the amplicons. A duplication of 74 nucleotides was found at the 5' terminus of the 5' noncoding (NC) region, which was also found in RNA isolates from tonsils from two other pigs from the same farm. When the duplication was omitted, the 5' NC region showed 97.8% similarity to bovine viral diarrhoea virus (BVDV) strain Korevaar and 94% to BVDV strain Osloss. Furthermore, the rearrangement of the 5' NC region of strain H was maintained after passaging in different cell lines and is not common for ruminant-like pestivirus isolated from pigs. Phylogenetic analysis based on the deduced amino acid sequence of the E2 gene of strain H confirmed the findings of the 5' NC region and show that this strain belongs to the BVDVIb subgroup. These results show for the first time rearrangements in the 5' NC region of a pestivirus.

5' Untranslated Regions↗

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↗

PubMed1995

A nested polymerase chain reaction assay to differentiate pestiviruses.

Viruses that comprise the Pestivirus genus cause significant losses to the livestock industry. Based on sequence analysis, currently 4 distinct genotypes are identified of which 3 infect cattle and sheep. Distinguishing between bovine and ovine isolates by serological tests has often been difficult because of a high degree of cross reactivity. In this study, a nested polymerase chain reaction (PCR) assay was developed to identify and distinguish between bovine viral diarrhea virus (BVDV) type I, BVDV type II, as well as border disease virus (BDV) genotypes. Consensus oligonucleotide primers were designed to amplify a 826-bp product from any of the 3 pestivirus types in a reverse transcription-PCR (RT-PCR). This product was subjected to a second round of nested PCR with type-specific primers which yielded DNA products of unique size characteristic for each pestivirus genotype. Using this assay, we were able to rapidly characterize several viral isolates and determine that all 3 genotypes can be found among ovine isolates.

Animals↗