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King K.,Institute for Animal Health Pirbright Laboratory | King K.,Royal Veterinary College University of London | Chapman D.,Institute for Animal Health Pirbright Laboratory | Argilaguet J.M.,Institute for Animal Health Pirbright Laboratory | And 12 more authors.
Vaccine | Year: 2011

African swine fever (ASF) is an acute haemorrhagic disease of domestic pigs for which there is currently no vaccine. We showed that experimental immunisation of pigs with the non-virulent OURT88/3 genotype I isolate from Portugal followed by the closely related virulent OURT88/1 genotype I isolate could confer protection against challenge with virulent isolates from Africa including the genotype I Benin 97/1 isolate and genotype X Uganda 1965 isolate. This immunisation strategy protected most pigs challenged with either Benin or Uganda from both disease and viraemia. Cross-protection was correlated with the ability of different ASFV isolates to stimulate immune lymphocytes from the OURT88/3 and OURT88/1 immunised pigs. © 2011 Elsevier Ltd.

Gao F.-S.,Dalian University | Bai J.,Dalian University | Zhang Q.,Chinese Academy of Agricultural Sciences | Xu C.-B.,Dalian University | Li Y.,Institute for Animal Health Pirbright Laboratory
Gene | Year: 2012

Six breeds of swine were used to study the structure of swine leukocyte antigen class I (SLA-I). SLA-I complexes were produced by linking SLA-2 genes and β 2m genes via a linker encoding a 15 amino acid glycine-rich sequence, (G4S)3, using splicing overlap extension (SOE)-PCR in vitro. The six recombinant SLA-2-linker-β 2m genes were each inserted into p2X vectors and their expression induced in Escherichia coli TB1. The expressed proteins were detected by SDS-PAGE and western blotting. The maltose binding protein (MBP)-SLA-I fusion proteins were purified by amylose affinity chromatography followed by cleavage with factor Xa and separation of the SLA-I protein monomers from the MBP using a DEAE Ceramic Hyper D F column. The purified SLA-I monomers were detected by circular dichroism (CD) spectroscopy and the 3-dimensional (3D) structure of the constructed single-chain SLA-I molecules were analyzed by homology modeling. Recombinant SLA-2-Linker-β 2m was successfully amplified from all six breeds of swine by SOE-PCR and expressed as fusion proteins of 84.1kDa in pMAL-p2X, followed by confirmation by western blotting. After purification and cleavage of the MBP-SLA-I fusion proteins, SLA-I monomeric proteins of 41.6kDa were separated. CD spectroscopy demonstrated that the SLA-I monomers had an α-helical structure, and the average α-helix, β-sheet, turn and random coil contents were 21.6%, 37.9%, 15.0% and 25.5%, respectively. Homology modeling of recombinant single-chain SLA-I molecules showed that the heavy chain and light chain constituted SLA-I complex with an open antigenic peptide-binding groove. It was concluded that the expressed SLA-I proteins in pMAL-p2X folded correctly and could be used to bind and screen nonameric peptides in vitro. © 2012 Elsevier B.V.

PubMed | Institute for Animal Health Pirbright Laboratory
Type: Journal Article | Journal: Journal of virological methods | Year: 2011

The Global Rinderpest Eradication Program (GREP) aimed to eradicate rinderpest by 2010 and it is widely believed to have been successful. An integral part of the program was the submission of samples from suspect rinderpest positive animals to a local Reference Laboratory for final confirmation. Confirmation of rinderpest in field samples is often hampered because of poor quality of the sample upon receipt. As part of GREP a rapid diagnostic strip test for the detection of rinderpest virus (RPV) in the field was developed allowing a rapid response to suspect outbreaks. The feasibility of extracting viral RNA from the used rapid diagnostic rinderpest devices for final confirmation in the laboratory is described. Viral material contained within used rinderpest devices was stable enough after storage for one week at 21C to extract RNA from five different RPV strains and amplify it by reverse transcriptase polymerase chain reaction (RT-PCR). Temperature did not affect adversely the extraction and amplification of the viral RNA but humidity impaired RNA extraction and amplification. Used rinderpest devices from field diagnosed rinderpest-positive animals could represent an ideal additional sample for submission to the Reference Laboratories for confirmation of preliminary diagnosis in the field.

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