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This study reports a novel method for the rapid detection and identification of the four recognized species in the pestivirus genus of the Flaviviridae family, i.e. classical swine fever virus (CSFV), border disease virus (BDV), bovine viral diarrhoea virus type 1 (BVDV1) and type 2 (BVDV2). The analysis of pestivirus PCR products was performed on microarrays by means of magnetic bead detection. The process utilizes an oligonucleotide array, onto which 5' biotinylated PCR products were hybridized, followed by visualization with streptavidin-coated magnetic particles by the naked eye, microscope or biochip reader. The assay was tested on a collection of pestiviruses that included all four species and allowed a specific and sensitive detection. Sensitivity was compared with other post-PCR detection methods, namely gel electrophoresis and suspension microarray. The results indicate that due to its high sensitivity, specificity and simple detection procedure, the magnetic bead assay provides a powerful tool for detection and identification of viral pathogens. Considering the simplicity of the assay, the protocols for hybridization and magnetic bead detection offer an emerging application for molecular diagnoses in virology that is amenable for use in a modestly equipped laboratory.

During the hunting season of 2001-02, blood and spleen samples from 59 red deer (Cervus elaphus), 77 roe deer (Capreolus capreolus), four fallow deer (Dama dama), and five chamois (Rupicapra rupicapra) were collected from nine hunting districts (n = 133) and one deer farm (n = 12) in southern Austria. Sera were tested for antibodies against bovine viral diarrhea virus (BVDV) with an enzyme-linked immunosorbent assay (ELISA) and virus neutralization tests against three BVDVs and one border disease virus strain. Reverse transcriptase polymerase chain reaction was used for detection of pestivirus-specific RNA in spleen samples. Antibodies were detected in one serum sample when using ELISA and virus neutralization tests. Results of the virus neutralization tests of this sample provided strong evidence for the exposure to the BVDV-1 genotype. The spleen samples were negative for pestivirus-specific RNA.

Pestivirus infection was identified in 16 of 17 chamois during an outbreak of a previously unreported disease in Pyrenean chamois (Rupicapra pyrenaica pyrenaica) in northeastern Spain in 2001-02. By analysis of the 5' noncoding regions of the virus, we assigned it to the border disease virus cluster with pairwise similarity values ranging from 82.1% to 88.1%. It will be important to investigate the association of this pestivirus with disease in Pyrenean chamois.

Three pestiviruses in fetal calf serum (FCS), a pestivirus in porcine ST cell line, and two pestiviruses in two of five bovine cell lines were detected by RT-PCR method employing panpestivirus primers selected from the 5'-non-coding region (5'-NCR). The 288 bp products were sequenced in both directions. To identify these pestiviruses, their nucleotide sequences and those of reference pestiviruses were used for construction of a dendrogram. Three viruses present in FCS and a virus present in the bovine RP-15 cell line were identified as bovine viral diarrhea virus I (BVDV-1). Bovine viral diarrhea virus 2 (BVDV-2) was identified in a batch of the bovine MDBK cell line. A pestivirus contaminating the porcine ST cell line was identified as a Border disease virus (BDV).

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.

Antigenic differences between pestivirus isolates of ruminant and porcine origin were characterized by neutralization assays. First, six different clusters of pestiviruses were identified by clustering cross-neutralization results of 13 pestivirus strains tested against 23 sera. Cluster I consisted of four strains of bovine viral diarrhoea virus (BVDV) of bovine origin and two BVDV isolates of porcine origin. Cluster II consisted of one sheep isolate and two porcine BDV isolates. Cluster III consisted of one classical swine fever virus strain and cluster IV, V, and VI each consisted of one strain isolated from a giraffe, a deer, and a pig. After the clusters were identified, one-way neutralization tests were used to test a total of 45 pestivirus isolates. Although the same six groups were found, results of some individual strains differed from previous cross-neutralization results and the results obtained by typing with monoclonal antibodies. The discrepancy between one way neutralization tests and cross-neutralisation tests is demonstrated clearly by recalculation of the cross-neutralization results without the classical swine fever sera. We conclude that neutralization tests are only suitable to characterize antigenic differences when virus strains are tested in a cross-neutralization test.

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.