title: Ultrasensitive detection of scrapie prion protein derived from ARQ and AHQ homozygote sheep by interspecies in vitro amplification.
abstract: Prions, infectious agents causing TSEs, are composed primarily of the pathogenic form (PrP(Sc)) of the PrP(C). The susceptibility of sheep to scrapie is determined by polymorphisms in the coding region of the PRNP, mainly at codons 136, 154, and 171. The efficiency of in vitro amplification of sheep PrP(Sc) seems to be linked also to the PrP genotype. PrP(Sc) derived from sheep with V(136)R(154)Q(171)-associated genotypes can be amplified efficiently by PMCA in the presence of additional polyanion such as poly A, but there are no reports that cite ultrasensitive detection of PrP(Sc) derived from sheep of other PrP genotypes. We report here that sheep PrP(Sc) derived from ARQ and AHQ homozygotes was amplified efficiently by serial PMCA using mouse brain homogenate as PrP(C) substrate. ARQ/ARQ PrP(Sc) was detected in infected brain homogenates diluted up to 10(-10) after five rounds of amplification, and AHQ/AHQ PrP(Sc) was detected in samples diluted up to 10(-8) after four rounds of amplification. On the other hand, amplification of PrP(Sc) from VRQ/ARQ sheep seemed to be less efficient under the experimental conditions used. The interspecies PMCA developed in this study may be useful in the detailed analysis of PrP(Sc) distribution in classical scrapie-infected ARQ and AHQ homozygote sheep.
authors.affiliation: Prion Disease Research Center, National Institute of Animal Health, Tsukuba, Ibaraki, Japan. ymura@affrc.go.jp
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Murayama
authors.forename: Morikazu
authors.initials: M
authors.lastname: Imamura
authors.forename: Kentaro
authors.initials: K
authors.lastname: Masujin
authors.forename: Noriko
authors.initials: N
authors.lastname: Shimozaki
authors.forename: Miyako
authors.initials: M
authors.lastname: Yoshioka
authors.forename: Shirou
authors.initials: S
authors.lastname: Mohri
authors.forename: Takashi
authors.initials: T
authors.lastname: Yokoyama
mesh_terms: D000818:Animals; D001923:Brain Chemistry; D005838:Genotype; D006720:Homozygote; D051379:Mice; D011328:Prions / Q000032:analysis* / Q000235:genetics; D012608:Scrapie / Q000175:diagnosis*; D012756:Sheep
chemical_list: D011328:Prions