title: Assessment of the genetic susceptibility of sheep to scrapie by protein misfolding cyclic amplification and comparison with experimental scrapie transmission studies.
abstract: The susceptibility of sheep to scrapie is influenced mainly by the prion protein polymorphisms A136V, R154H, and Q171R/H. Here we analyzed the ability of protein misfolding cyclic amplification (PMCA) to model the genetic susceptibility of sheep to scrapie. For this purpose, we studied the efficiency of brain homogenates from sheep with different PrP genotypes to support PrP(Sc) amplification by PMCA using an ARQ/ARQ scrapie inoculum. The results were then compared with those obtained in vivo using the same sheep breed, genotypes, and scrapie inoculum. Genotypes associated with susceptibility (ARQ/ARQ, ARQ/AHQ, and AHQ/ARH) were able to sustain PrP(Sc) amplification in PMCA reactions, while genotypes associated with resistance to scrapie (ARQ/ARR and ARR/ARR) were unable to support the in vitro conversion. The incubation times of the experimental infection were then compared with the in vitro amplification factors. Linear regression analysis showed that the efficiency of in vitro PrP(Sc) amplification of the different genotypes was indeed inversely proportional to their incubation times. Finally, the rare ARQK₁₇₆/ARQK₁₇₆ genotype, for which no in vivo data are available, was studied by PMCA. No amplification was obtained, suggesting ARQK₁₇₆/ARQK₁₇₆ as an additional genotype associated with resistance, at least to the isolate tested. Our results indicate a direct correlation between the ability of different PrP genotypes to undergo PrP(C)-to-PrP(Sc) conversion by PMCA and their in vivo susceptibility and point to PMCA as an alternative to transmission studies and a potential tool to test the susceptibility of numerous sheep PrP genotypes to a variety of prion sources.
authors.affiliation: Department of Veterinary Public Health and Food Safety, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy.
authors.forename: Cecilia
authors.initials: C
authors.lastname: Bucalossi
authors.forename: Gianmario
authors.initials: G
authors.lastname: Cosseddu
authors.forename: Claudia
authors.initials: C
authors.lastname: D'Agostino
authors.forename: Michele Angelo
authors.initials: MA
authors.lastname: Di Bari
authors.forename: Barbara
authors.initials: B
authors.lastname: Chiappini
authors.forename: Michela
authors.initials: M
authors.lastname: Conte
authors.forename: Francesca
authors.initials: F
authors.lastname: Rosone
authors.forename: Luigi
authors.initials: L
authors.lastname: De Grossi
authors.forename: Gaia
authors.initials: G
authors.lastname: Scavia
authors.forename: Umberto
authors.initials: U
authors.lastname: Agrimi
authors.forename: Romolo
authors.initials: R
authors.lastname: Nonno
authors.forename: Gabriele
authors.initials: G
authors.lastname: Vaccari
mesh_terms: D000818:Animals; D001921:Brain; D020022:Genetic Predisposition to Disease; D005838:Genotype; D015151:Immunoblotting; D020641:Polymorphism, Single Nucleotide; D018620:PrPSc Proteins / Q000737:chemistry / Q000235:genetics*; D011328:Prions / Q000737:chemistry / Q000235:genetics*; D017510:Protein Folding; D057165:Proteostasis Deficiencies; D012608:Scrapie / Q000235:genetics* / Q000378:metabolism / Q000635:transmission*; D012756:Sheep / Q000235:genetics
chemical_list: D018620:PrPSc Proteins; D011328:Prions