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. In the first study (n = 1000), the number of false-positive and false-negative results was 11 samples (1.1%) and 67 samples (6.7%), respectively. In the second study (n = 920), the number of false-positive and false-negative results was 7 samples (0.8%) and 52 samples (5.7%). In a third study, undertaken at l'Agence Française de Sécurité Sanitaire des Aliments (AFSSA), Nancy, France (n = 440), 1 false-positive sample (0.23%) and 91 (20.7%) false-negative samples were identified. Data generated using this prototype ELISA indicate a strong correlation for specificity when compared to the gold standard fluorescent antibody virus neutralisation (FAVN) test. Although the ELISA has a lower sensitivity than the FAVN test, it is a useful tool for rapidly screening serum samples from vaccinated companion animals. Using a cut-off value of 0.6 EU/ml, the sensitivity (R = % from VLA and 79% from AFSSA) and specificity (R = 97.3%) indices between the ELISA compared favourably with data generated using the FAVN test. The major advantages of the ELISA test are that it is a qualitative tool that can be completed in four hours, does not require the use of live virus and can be performed without the need for specialised laboratory containment. This contrasts with 4 days using conventional rabies antibody virus neutralisation assays. Using the current format, the ELISA assay described would be a valuable screening tool for the detection of rabies antibodies from vaccinated domestic animals in combination with other Office International des Epizooties (OIE) accepted serological tests.

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. All genotype 1, 5 and 6 viruses were detected by RT-PCR while none of the genotype 2, 3 and 4 viruses were detected. All the genotype 5 and 6 viruses were detected by the two PCR-ELISA probes when used in combination while none of the genotype 1-4 viruses were detected. When used individually, the PCR-ELISA probes also distinguished between the genotype 5 and 6 viruses. This new discriminatory test should allow the rapid genotyping of all lyssaviruses likely to be encountered in Europe and as such could provide useful epidemiological information in the event of an outbreak.

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. For closely related viruses, the more variable or larger data-sets gave better discrimination, and the most reliable classification was obtained with sequence data from the NS5B region. No evidence was found for intertypic recombination between CSFVs. A larger data-set was also analysed comprising 190 nucleotides of E2 sequence from 100 CSFVs from different parts of the world, in order to assess the extent and global distribution of CSFV diversity. Additional groups of CSFV are evident from Asia and the nomenclature of Lowings et al. (1996) [Lowings, P., Ibata, G., Needham, J., Paton, D., 1996. J. Gen. Virol. 77, 1311-1321] needs to be updated to accommodate these. A tentative assignment, adapting rather than overturning the previous nomenclature divides CSF viruses into three groups with three or four subgroups: 1.1, 1.2, 1.3; 2.1, 2.2, 2.3; 3.1, 3.2, 3.3, 3.4. The expanding data-base of CSFV sequences should improve the prospects of disease tracing in the future, and provide a basis for a standardised approach to ensure that results from different laboratories are comparable.

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. Parallel studies between the duplex and the unlinked lyssavirus assay demonstrated only a minor reduction in the sensitivity of the former test. The ribosomal and viral targets (unlike beta-actin RNA) were shown to have similar degradation kinetics making rRNA amplification a good control for viral target integrity. As a consequence, the use of this system would reduce the likelihood of obtaining false negative RT-PCR results from lyssavirus infected material.

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. Furthermore, the application of automated sequencing of the resultant product enabled a definitive characterisation of classical rabies within 16 h. The utility of this assay was confirmed further by the successful detection of representatives of the six lyssavirus genotypes.

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. To address the difficulties encountered during conventional PCMV detection and characterization a set of nested PCR primers were constructed which generated DNA fragments of 415 and 257 bp from the DNA polymerase gene. The nested PCR system proved specific for PCMV and provided a novel means for the detection of this poorly characterized herpes virus in pig populations, vaccines and in organs used in xenotransplantation.

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. Here, sequence analysis of a recent isolate of virulent TGEV, revealed a variant with an intact spike gene, but a large deletion in ORF 3a. This suggests that ORF 3a is not essential for enteric virulence.

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. The PCR products were subsequently reamplified, in conjunction with a CSFV-specific fluorogenic probe, in a nested-PCR with a second set of panpestivirus PCR primers. During nested PCR, when the target of interest was present, the CSFV probe annealed to the amplicon between the forward and reverse primers and was subsequently cleaved via the 5'-3' nucleolytic activity of the DNA polymerase resulting in the release of the fluorescent reporter dye. Each PCR tube was then placed directly into a luminescence spectrometer to monitor for any increase in fluorescence due to cleavage of the probe. This assay detected representatives of all genetic sub-groups of CSFV, but gave negative results for other pestiviruses. A preliminary assessment showed that the method could be used to detect CSFV RNA extracted from infected pig blood with a sensitivity greater than that of VI.

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. There was a marked tendency of viruses placed in the same group to show similar virulence for chickens. Extension of the panel to 26 mAbs produced 39 distinct patterns, although some of these were seen with only a single virus. Again, viruses inducing similar binding patterns shared similar properties and some binding patterns were specific for viruses causing discrete epizootics. Cluster analysis of the mAb binding patterns did not produce concise, discrete groupings, but did emphasise some relationships between virus properties and antigenicity. Examples of the usefulness of this approach were the ability to link two important outbreaks to the contamination of stored food by infected feral pigeons, and the demonstration of two separate viruses responsible for outbreaks in countries of the European Union during 1991 to 1994 thus preventing erroneous epizootiological tracing.

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. A number of PRRSV isolates, from different outbreaks in the UK between 1991 and 1994, were propagated in pig alveolar macrophages and RNA extracted. The ORF3 and ORF7 regions of the individual viruses were amplified by the polymerase chain reaction (PCR) and their sequences were determined using internal sense and antisense primers. A number of differences among the sequences were noted within specific regions of the ORF3, with a hypervariable area detected at the carboxyterminal end, in the area of overlap with ORF4. With the most divergent isolate, 9.5% of the 84 translated amino acids encoded by the area of overlap were different from Lelystad isolate, translating the sequence in both reading frames. In view of consistent changes elsewhere in the ORF that suggest a common ancestry among the isolates studied, we conclude that this region may be subject to rapid change in comparison to other regions studied, and therefore may be an area subjected to immunoselective pressure.

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. Comparative sequence analysis of the PCR products showed that they were of a genetic type common in Western Europe. Further discrimination of virus isolates was possible, into subgroups that largely coincided with their regions of origin in Poland, Slovakia, Hungary and Estonia. The discriminatory ability of the technique was improved by the analysis of a composite dataset consisting of all of the sequence data from all of the viruses. Using this approach we were able to distinguish between all of the viruses and to group them in a manner that precisely matched their geographical origins, apart from a single Estonian isolate which grouped with viruses from Eastern Poland.

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).

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. Group I (represented by Brescia strain) consisted of old and recent American and Asian viruses, as well as old English isolates from the 1950s. This group was subdivided into three subgroups, termed I.A-I.C. Group II (represented by Alfort strain) consisted of relatively recent isolates from Europe, together with strain Osaka, which was isolated in Japan from a pig of European origin. Based on genetic distances the group was divided into subgroups II.A and II.B. Malaysian isolates were branched into both groups, indicating multiple origins for contemporaneous outbreaks in that country. All ten vaccine strains tested were branched in group I, implying a common ancestor. The Japanese Kanagawa strain, isolated in 1974, and the British Congenital Tremor strain from 1964 were the most distinct variants of CSFV in our collection. The comparison of the nucleotide sequences of the polymerase coding region of 32 European strains distinguished subgroups II.A and II.B which were similar to the corresponding subgroups of the E2 phylogenetic tree. Thus, the results revealed that the E2 region and the polymerase coding regions seem to be appropriate for the grouping of CSFV isolates from all over the world, distinguishing two major groups of the virus. The reliability of these regions for phylogenetic analysis is indicated by the similarity of the results obtained from the two separate parts of the CSFV genome.

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. Epitope conservation between the viruses was studied with a panel of 32 monoclonal antibodies, revealing that all of the isolates were BVDV-like. However, certain epitopes present in isolates from cattle were lost following virus transmission to sheep. In vitro amplification of the 5'-untranslated region of the 13 isolates by the polymerase chain reaction (PCR) and subsequent analyses of amplified products with restriction enzymes also indicated that all 13 isolates belong to the BVDV group of pestiviruses. A fragment of the E2 (gp53) gene of each virus was amplified by PCR and a comparison of the amplified sequence of 188 nucleotides separated the isolates into four groups each of which could be identified with a particular farm of origin. The 13 isolates were thus herd-specific rather than species-specific demonstrating that BVDV is readily transmitted between cattle and 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. One subgroup equated to classical swine fever virus (CSFV) and included most porcine pestiviruses but none from ruminants. A second subgroup contained mainly viruses of bovine origin, including reference bovine viral diarrhoea viruses (BVDV) such as NADL; however viruses from pigs and sheep were also represented. A third subgroup represented by reference strains of border disease virus (BDV) comprised mainly ovine isolates, but also viruses from pigs. The fourth and most recently defined subgroup contained no reference strains of CSFV, BVDV or BDV, but included atypical viruses from cattle, sheep and pigs. The subgrouping scheme was supported by genetic comparisons between representative viruses from the 4 subgroups and by virus neutralisation with polyclonal sera.

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. The data from each set of fragments were compared with one another and with reference strains. This allowed us confidently to assign most of the viruses to one of three subgroups. An analysis of the same viruses with a panel of monoclonal antibodies was much less informative. The subgrouping of the isolates suggested that, in this region of Italy, there had been at least two separate introductions of classical swine fever over a 7 year period and that virus had been transmitted between domestic pigs and wild boar. A consensus nucleotide sequence derived from the glycoprotein fragments of all the viruses examined revealed conservation at the wobble position of some codons.

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. Differentiation between the viruses was achieved by cutting the PCR-amplified products with the restriction endonucleases AvaI and Bg1I. Using this procedure it was possible to distinguish at least 3 genogroups; group 1 (HCV) contained 32 of the pig isolates, group II (BVDV) contained all the cattle isolates tested plus 6 sheep isolates and group III (BDV) contained 11 sheep isolates and 1 pig isolate.