Development of a qualitative indirect ELISA for the measurement of rabies virus-specific antibodies from vaccinated dogs and cats.

A protocol suitable for the detection of rabies virus-specific antibodies in serum samples from companion animals using an enzyme linked immunosorbent assay (ELISA) is described. This method has been used successfully for the qualitative assessment of rabies virus-specific antibodies in serum samples from a cohort of vaccinated dogs and cats. In two initial field studies, a variable population of field samples from the Veterinary Laboratories Agency (VLA), United Kingdom were tested.

A rapid RT-PCR method to differentiate six established genotypes of rabies and rabies-related viruses using TaqMan technology.

A rapid and sensitive reverse transcriptase-polymerase chain reaction (RT-PCR) assay incorporating TaqMan probes has been developed that can distinguish among the six established rabies and rabies-related virus genotypes. TaqMan probes were designed and validated against 106 rabies and rabies-related virus isolates, one isolate of the Australian bat Lyssaviruses (genotype 7), and 18 other non-rabies viruses important in the veterinary field. The N gene was used as the target for the probes as it is well conserved and has been intensively used to genotype rabies isolates.

Molecular methods to distinguish between classical rabies and the rabies-related European bat lyssaviruses.

A rapid and sensitive reverse transcriptase-polymerase chain reaction (RT-PCR) assay for the detection of classical rabies virus (genotype 1) and the rabies related European bat lyssaviruses (EBLs) (genotypes 5 and 6) was developed. When combined with specific oligonucleotide probes and a PCR-enzyme linked immunosorbent assay (PCR-ELISA), genotype 5 and 6 viruses can be distinguished from each other and from genotype 1 viruses. Ninety-two isolates from the six established genotypes of rabies and rabies-related viruses were screened by RT-PCR and PCR-ELISA to determine the specificity of the assays.

Genetic typing of classical swine fever virus.

Three regions of the classical swine fever virus (CSFV) genome that have been widely sequenced were compared with respect to their ability to discriminate between isolates and to segregate viruses into genetic groups. Sequence data-sets were assembled for 55 CSFVs comprising 150 nucleotides of the 5' non-translated region, 190 nucleotides of the E2 envelope glycoprotein gene and 409 nucleotides of the NS5B polymerase gene. Phylogenetic analysis of each data-set revealed similar groups and subgroups.

Assessment of template quality by the incorporation of an internal control into a RT-PCR for the detection of rabies and rabies-related viruses.

A method is described to assess RNA template quality by the incorporation of a ribosomal RNA (rRNA) internal (in tube) control into a standard rabies and rabies-related virus specific RT-PCR. Specific virus and rRNA templates were co-amplified in a duplex reaction from RNA extracts derived from 60 isolates representing all six of the established lyssavirus genotypes. To ensure a wide species applicability of this technique we demonstrated that the rRNA assay was capable of functioning using the cells or tissues of 14 different mammals.

Detection and identification of rabies and rabies-related viruses using rapid-cycle PCR.

The rapid identification of suspect rabies infection is essential in human cases, to ensure appropriate post-exposure treatment of contacts, and in animal cases to allow specific control strategies to be decided and implemented. We describe here the use of high speed air thermo-cycler PCR as a diagnostic tool for the detection of rabies and rabies-related viruses. Using this technique we were able to diagnose rabies in a human within 5 h.

Development of a PCR system for porcine cytomegalovirus detection and determination of the putative partial sequence of its DNA polymerase gene.

After PCR amplification with conservative cytomegalovirus primers, a 520 nucleotide putative partial sequence of the DNA polymerase gene of porcine cytomegalovirus (PCMV) was determined. Sequence comparison revealed homology to DNA polymerase genes from various beta herpes viruses and a dendrogram was constructed depicting the relationship of PCMV to other members of the Herpesviridae family. The dendrogram indicates that PCMV is indeed a beta herpes virus that is more closely related to human herpes virus types 6 and 7 than to type 5.

Characterisation of a recent virulent transmissible gastroenteritis virus from Britain with a deleted ORF 3a.

Analyses of transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV) isolates have suggested that tropism and pathogenicity are influenced by the spike protein and ORF 3. In general, enteric viruses (TGEV) have been shown to contain intact spike and ORF 3 genes, whilst respiratory isolates (PRCV) have major deletions within both regions. Virulence has been correlated to a functional ORF 3.

A novel approach to the detection of classical swine fever virus by RT-PCR with a fluorogenic probe (TaqMan).

Detection of classical swine fever virus (CSFV) and its discrimination from other pestiviruses can be achieved by virus isolation (VI) in cell cultures, antigen detection, or molecular analysis. To simplify the latter, a 5'-nuclease assay (TaqMan) was developed for the rapid and specific detection of CSFV with the minimum of downstream PCR processing. A pair of 5'-non-coding region, panpestivirus-specific PCR primers were assessed in a one-step reverse transcription-PCR with each of 36 diverse pestiviruses.

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.

Genetic heterogeneity of classical swine fever virus in Central Europe.

The aim of this work was to genetically characterize Central European isolates of classical swine fever virus (CSFV) and to evaluate the applicability of molecular analysis in the epizootiology of CSFV infections. Thirty four viruses, derived from Central European pigs or wild boar, were examined. All of these viruses were detected by each of three sets of oligonucleotide primers which had been designed for the specific RT-PCR amplification of different genomic regions.

Antigenic diversity and similarities detected in avian paramyxovirus type 1 (Newcastle disease virus) isolates using monoclonal antibodies.

Newcastle disease (ND) virus (APMV-1) isolates submitted to the International Reference Laboratory for ND were characterised antigenically by their ability to cause binding of mouse monoclonal antibodies (mAbs) to cell cultures infected with the isolate. Since the availability of the mAbs 1526 viruses have been examined using a panel of nine mAbs and 818 with an extended panel of 26 mAbs. Using the nine mAb panel a total of 14 different patterns was seen and viruses grouped by the same pattern showed relationships with each other which were either biological, temporal or geographical or more than one of these.

Variation in open reading frames 3, 4 and 7 among porcine reproductive and respiratory syndrome virus isolates in the UK.

Previous studies using monoclonal antibodies (mAbs) have revealed antigenic variation among UK isolates of porcine reproductive and respiratory syndrome viruses (PRRSV) and the use of in vitro translation products has shown that this variation lies in the protein encoded by open reading frame (ORF) 3. This protein has been shown to be present in purified virion preparations, suggesting that it is a structural protein. The original objective was to investigate the degree of variation of ORF3 among a number of UK isolates of different mAb reactivity and diverse chronology by sequencing and to correlate this with the mAb reactivity, in an attempt to define conserved and variable antigenic sites.

Genetic variability of classical swine fever virus.

The genetic variability of classical swine fever virus was studied by comparative nucleotide sequence analysis of 76 virus isolates, collected during a half century from three continents. Parts of the E2 (gp55) and the polymerase gene coding regions of the viral genome were amplified by RT-PCR and DNA fragments of 254 and 207 bp, respectively, were sequenced. The comparative sequence analysis of the E2 region revealed two main phylogenetic groups of CSFV, indicating that the virus apparently evolved from two ancestor nodes.

Identification of herd-specific bovine viral diarrhoea virus isolates from infected cattle and sheep.

Thirteen pestiviruses isolated from ruminants on four different farms in Sweden were compared antigenically and genetically. On two farms, viruses were isolated from both cattle and sheep, a third farm contained only sheep and a fourth only cattle. Seven viruses were isolated from six different cattle and six viruses were isolated from five different sheep.

A proposed division of the pestivirus genus using monoclonal antibodies, supported by cross-neutralisation assays and genetic sequencing.

Sixty-six pestiviruses from ruminant and porcine hosts were analysed with a panel of 76 monoclonal antibodies raised against 9 different viruses. Reactivity was used to construct epitope similarity maps for all of the viruses. Four principal virus subgroups were demonstrated.

Classical swine fever: genetic detection and analysis of differences between virus isolates.

Two pairs of oligonucleotide primers were designed that specifically amplified regions of the classical swine fever virus genome. These products, corresponding to a 671 bp portion of the genes encoding the E1 and E2 (gp33 and gp55) proteins and a 1090 bp portion of the putative polymerase gene, were amplified from eight virus isolates which had been responsible for a series of classical swine fever outbreaks in Italy involving both domestic pigs and wild boar. For each virus the fragments were partially sequenced to give 475 bp of the E1/E2 glycoprotein and 212 bp of the putative polymerase gene sequences.

Pestiviruses isolated from pigs, cattle and sheep can be allocated into at least three genogroups using polymerase chain reaction and restriction endonuclease analysis.

A polymerase chain reaction-based assay capable of detecting a broad range of pestiviruses from pigs, cattle, or sheep was developed. Of six sets of primers selected from different parts of the pestivirus genome, the best results were provided by a pair from the highly conserved 5' non-coding region which gave amplification with all 129 isolates tested. This panel consisted of 33 isolates from pigs, 79 from cattle, and 17 from sheep.

Epitope mapping of the gp53 envelope protein of bovine viral diarrhea virus.

Epitopes recognized by nine monoclonal antibodies (mAbs) on the envelope protein, gp53, of two strains of bovine viral diarrhoea virus (NADL and Oregon C24V) were mapped by competitive binding assays and by the characterization and sequence analyses of mAb neutralization escape mutants. This defined an antigenic domain on gp53 that was shared by many BVDV strains, while other less conserved epitopes were possibly distinct. Sequencing of escape mutant viruses revealed that a cluster of three amino acids in the N-terminal half of gp53 were involved in the main antigenic domain shared by both NADL and Oregon C24V viruses, while an amino acid 31 residues further toward the N-terminus was involved in a second site present only on the NADL strain.