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Biomedical subjects

D J Paton

Publications and source records attributed to D J Paton.

At least 73 records · Page 4Linked to original sources

Border disease virus: delineation by monoclonal antibodies.

Many ovine pestiviruses from Britain and a number of atypical porcine isolates are largely unrecognised by monoclonal antibodies (mAbs) specific for reference strains of classical swine fever virus and bovine viral diarrhoea virus (BVDV). Additional mAbs have therefore been produced using some of these "unreactive" pestiviruses. Two of the viruses used were atypical porcine isolates (strains 87/6 and Vosges), whilst another had been isolated from a sheep (59386). Thirty-three mAbs were selected, none of which recognised two reference strains of BVDV, but three of which recognised the Alfort strain of classical swine fever. On the basis of radioimmunoprecipitation they were considered to be directed at one of three different pestivirus proteins (gp 53, gp 48 or p 125). Three virus subgroups were evident when the mAbs were used to type 16 ovine and two atypical porcine pestiviruses. One subgroup contained the Vosges and 59386 viruses and four ovine field isolates. The second subgroup comprised the 87/6 virus, the Moredun and Aveyron reference strains of border disease virus and four further ovine field isolates. Three of four ovine viruses making up the third subgroup had been previously categorised as BVDV-like and were largely unrecognised by the new mAbs. The findings were in agreement with previous attempts to segregate some of the same viruses using partial genomic comparisons or cross-neutralization tests.

Animals↗

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.

Animals↗

Congenital infection of pigs with ruminant-type pestiviruses.

Congenital infections of pigs were induced with two ruminant-type pestiviruses isolated from pigs. One of the viruses was bovine viral diarrhoea virus-like and the other border disease virus-like. Both produced symptoms similar to those observed with low virulence strains of classical swine fever virus. A striking effect of persistent virus infection in post-natal life was stunting in viraemic animals. It was also shown that a congenitally infected pig shed virus for 2.5 years and in sufficient quantity to infect other pigs, even by indirect contact. Unlike ruminants, congenitally infected pigs sometimes had persistent viraemia but eventually eliminated the virus. Clearance of virus from the blood was related to the appearance of neutralizing antibodies. However, clearance from the tissues sometimes took as much as 5 months longer than from the blood.

Animals↗

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.

Animals↗

Infection of pigs and cattle with bovine viral diarrhoea virus on a farm in England.

Deaths within a litter of sucking pigs led to a suspicion of hog cholera, but pestiviruses isolated from both dead and live piglets appeared to be bovine viral diarrhoea virus. Persistent viraemia with bovine viral diarrhoea virus was demonstrated in living littermates and also in a bought-in calf, housed in a nearby pen on the same farm. Only two of the littermates survived, both of which had been virus negative and seropositive from the outset of testing. Porcine and bovine virus isolates grew well in calf testicular cells and were neutralised equally by sera collected at the farm from cattle and pigs. However, a comparison by means of their reactivity to monoclonal antibodies showed that they were similar but not identical, and only the porcine isolates grew well in a porcine kidney cell line.

Animals↗

Pathogenicity of concurrent infection of pigs with porcine respiratory coronavirus and swine influenza virus.

Combinations of porcine respiratory coronavirus (PRCV) and either of two swine influenza viruses (H1N1 or H3N2) were administered intranasally and by aerosol to six- to eight-week-old specific pathogen-free pigs. The clinical responses, gross respiratory lesions and growth performances of these pigs were studied and compared with those of single (PRCV, H1N1 or H3N2) and mock-infected animals. PRCV infection caused fever, growth retardation and lung lesions, but no respiratory symptoms. Infection with swine influenza viruses caused rather similar, mild symptoms of disease, with H1N1 infection being the least severe. Combined infections with influenza viruses and PRCV did not appear to enhance the pathogenicity of these viruses. Furthermore, viruses were isolated more frequently from tissues and nasal swabs taken from 'single' than 'dual' infected animals, suggesting a possible in vivo interference between replication of PRCV and swine influenza virus.

Animals↗

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. Since other amino acids defining these epitopes were located at distant positions within the gp53 protein, it is likely that a major domain [corrected] on gp53 consist of composite, conformation-dependent epitopes.

Amino Acid Sequence↗

Isolation of a Lelystad virus-like agent from British pigs and scanning electron microscopy of infected macrophages.

Six, one-week-old gnotobiotic piglets were inoculated with tissues or sera collected from field cases of porcine reproductive and respiratory syndrome. The piglets showed little or no illness, and two that were necropsied at 8 and 9 days post infection appeared grossly normal. However, a Lelystad virus-like agent was isolated from most of the inoculated pigs using porcine alveolar macrophage cultures. Seroconversion to the Lelystad virus was observed and some animals developed microscopically detectable interstitial pneumonias. Scanning electron microscopy was used to study the in vitro cytopathic effect of the Lelystad virus on porcine alveolar macrophages.

Abortion, Veterinary↗

Serological studies of transmissible gastroenteritis in Great Britain, using a competitive ELISA.

A competitive ELISA which differentiates between transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV) was used to detect non-neutralising antibodies to the peplomer protein of TGEV in porcine sera. The test was shown to be TGEV specific, having a relative specificity of 100 per cent, and to have a relative sensitivity of 94.9 per cent when compared with the virus neutralisation test. The prevalence of TGEV in Great Britain is low; only 0.6 per cent of sows sampled in 1990 were seropositive to TGEV. Seroconversion to the virus neutralisation test occurred in a closed herd in 1984, with no apparent spread, but later testing by the ELISA did not detect any blocking antibodies. The possibility of the existence of a less contagious strain of PRCV is discussed. All British isolates of TGEV tested by the indirect fluorescent antibody test were recognised by the monoclonal antibody 1D.B12, the indicator antibody in the ELISA.

Animals↗

BVD monoclonal antibodies: relationship between viral protein specificity and viral strain specificity.

Seventeen monoclonal antibodies raised against bovine viral diarrhoea virus were divided into three groups on the basis of radioimmunoprecipitation results. Seven monoclonal antibodies precipitated a polypeptide of 80kD and defined four domains, all of which showed considerable conservation amongst the 180 pestivirus strains and isolates examined. Nine monoclonal antibodies, including six with virus neutralizing activity, precipitated a 53kD polypeptide and all appeared to be directed towards a single domain of clustered epitopes. Several of these epitopes were present in many ruminant virus strains and isolates, but not in hog cholera viruses. A single monoclonal antibody precipitated a 48kD polypeptide, defining an epitope that was also present on many ruminant viruses, but not hog cholera viruses. Most pestiviruses from cattle and some from sheep shared a number of epitopes located on three different proteins.

Animals↗

Sows infected in pregnancy with porcine respiratory coronavirus show no evidence of protecting their sucking piglets against transmissible gastroenteritis.

Eighteen litters of sucking piglets were challenged with one of two strains of transmissible gastroenteritis virus (TGEV). During pregnancy, their seronegative dams had been either inoculated intranasally with porcine respiratory coronavirus (PRCV), inoculated orally with TGEV or left untreated. On the basis of weight gain, clinical signs and survival, no differences in response to challenge was detected when piglets suckled by PRCV inoculated sows were compared with those suckled by uninoculated sows. Such a difference was evident when the litters of sows successfully pre-immunized with TGEV were compared with those of uninoculated or PRCV-inoculated sows. The possibility of transplacental transmission of PRCV was investigated in two litters born to sows that had been inoculated with this virus in late pregnancy. All sixteen live-born piglets were seronegative for the virus at birth and PRCV was not isolated from tissues taken from two stillborn piglets.

Animals↗

An ELISA detecting antibody to conserved pestivirus epitopes.

A monoclonal antibody based competition-ELISA is described for the detection of pestivirus antibodies directed against conserved epitopes on the p80 viral protein. The ELISA detected increases in serum antibody following experimentally induced infections of pigs, cattle and sheep with a wide range of pestiviruses, although the sensitivity of the test was not uniform for the different viruses studied. The ELISA was compared with virus neutralization tests for the assessment of porcine, bovine and ovine field sera. At a cut-off value of 50% inhibition, the ELISA showed a high specificity relative to virus neutralization tests, but appeared less sensitive for the detection of some weakly positive samples from pigs. Sera from both ruminants and pigs could be assessed without any modification of the test.

Animals↗