title: In vitro amplification of ovine prions from scrapie-infected sheep from Great Britain reveals distinct patterns of propagation.
abstract: BACKGROUND
  Protein misfolding cyclic amplification (PMCA) is a method that facilitates the detection of prions from many sources of transmissible spongiform encephalopathy (TSE). Sheep scrapie represents a unique diversity of prion disease agents in a range of susceptible PRNP genotypes. In this study PMCA was assessed on a range of Great Britain (GB) sheep scrapie isolates to determine the applicability to veterinary diagnosis of ovine TSE.
  
  
  RESULTS
  PrPSc amplification by protein misfolding cyclic amplification (PMCA) was assessed as a diagnostic tool for field cases of scrapie. The technique was initially applied to thirty-seven isolates of scrapie from diverse geographical locations around GB, and involved sheep of various breeds and PRNP genotypes. All samples were amplified in either VRQ and/or ARQ PrPC substrate. For PrPSc from sheep with at least one VRQ allele, all samples amplified efficiently in VRQ PrPC but only PrPSc from ARH/VRQ sheep amplified in both substrates. PrPSc from ARQ/ARQ sheep displayed two amplification patterns, one that amplified in both substrates and one that only amplified in ARQ PrPC. These amplification patterns were consistent for a further 14/15 flock/farm mates of these sheep. Furthermore experimental scrapie strains SSBP1, Dawson, CH1641 and MRI were analysed. SSBP1 and Dawson (from VRQ/VRQ sheep) amplified in VRQ but not ARQ substrate. MRI scrapie (from ARQ/ARQ sheep) nor CH1641 did not amplify in ARQ or VRQ substrate; these strains required an enhanced PMCA method incorporating polyadenylic acid (poly(A)) to achieve amplification.
  
  
  CONCLUSIONS
  PrPsc from 52 classical scrapie GB field isolates amplified in VRQ or ARQ or both substrates and supports the use of PMCA as a rapid assay for the detection of a wide range of ovine classical scrapie infections involving multiple PRNP genotypes and scrapie strains.
authors.affiliation: Animal Health Veterinary Laboratories Agency (AHVLA), New Haw, Addlestone, Surrey, UK.
authors.forename: Leigh
authors.initials: L
authors.lastname: Thorne
authors.forename: Thomas
authors.initials: T
authors.lastname: Holder
authors.forename: Andrew
authors.initials: A
authors.lastname: Ramsay
authors.forename: Jane
authors.initials: J
authors.lastname: Edwards
authors.forename: Maged Mohamed
authors.initials: MM
authors.lastname: Taema
authors.forename: Otto
authors.initials: O
authors.lastname: Windl
authors.forename: Ben Charles
authors.initials: BC
authors.lastname: Maddison
authors.forename: Kevin Christopher
authors.initials: KC
authors.lastname: Gough
authors.forename: Linda Ann
authors.initials: LA
authors.lastname: Terry
mesh_terms: D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism*; D020022:Genetic Predisposition to Disease; D005838:Genotype; D007124:Immunoenzyme Techniques; D018620:PrPSc Proteins / Q000235:genetics / Q000378:metabolism*; D011328:Prions / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D012608:Scrapie / Q000453:epidemiology / Q000235:genetics / Q000378:metabolism*; D012756:Sheep; D006113:United Kingdom / Q000453:epidemiology
chemical_list: D018620:PrPSc Proteins; D011328:Prions