Great Britain and elsewhere have detected atypical scrapie infection in sheep with PrP genotypes thought to be genetically resistant to the classical form of scrapie. DNA sequencing of the PrP gene of British atypical scrapie cases (n=69), classical scrapie cases (n=59) and scrapie-free controls (n=138) was undertaken to identify whether PrP variants, other than the three well-characterized polymorphic codons, influenced susceptibility to atypical scrapie infection. Four non-synonymous changes, M112T, M137T, L141F and P241S, were detected that are most probably associated with the A 136 R 154 Q 171 haplotype. Only the PrP variant containing a phenylalanine residue at amino acid position 141 was found to be associated more commonly with the atypical scrapie cases. In addition to the single nucleotide polymorphisms associated with the ARQ allele, two out of nine atypical scrapie cases with the ARR/ARR genotype were found to contain a 24 bp insertion, leading to an additional octapeptide repeat. In terms of PrP genetics, one classification of the GB scrapie cases examined in this study would place animals carrying any homozygous or heterozygous combination of ARR, AHQ or AF 141 RQ alleles, or any one of these alleles when paired with ARQ, as being susceptible to atypical scrapie infection, and animals heterozygous or homozygous for VRQ or homozygous for ARQ as being susceptible to classical scrapie disease. The AHQ PrP allele was associated with the highest incidence of atypical scrapie (263 per 100 000 alleles), whilst VRQ was associated with the lowest incidence (10 per 100 000 alleles).
Mouse bioassay remains the gold standard for determining proof of infectivity, strain type, and infectious titer estimation in prion disease research. The development of an approach using ex vivo cell-based assays remains an attractive alternative, both in order to reduce the use of mice and to hasten results. The main limitation of a cell-based approach is the scarcity of cell lines permissive to infection with natural transmissible spongiform encephalopathy strains. This study combines two advances in this area, namely, the standard scrapie cell assay (SSCA) and the Rov9 and MovS6 cell lines, which both express the ovine PrP VRQ allele, to assess to what extent natural and experimental ovine scrapie can be detected ex vivo. Despite the Rov9 and MovS6 cell lines being of different biological origin, they were both permissive and resistant to infection with the same isolates of natural sheep scrapie as detected by SSCA. Rov9 subclones that are 20 times more sensitive than Rov9 to SSBP/1-like scrapie infection were isolated, but all the subclones maintained their resistance to isolates that failed to transmit to the parental line. The most sensitive subclone of the Rov9 cell line was used to estimate the infectious titer of a scrapie brain pool (RBP1) and proved to be more sensitive than the mouse bioassay using wild-type mice. Increasing the sensitivity of the Rov9 cell line to SSBP/1 infection did not correlate with broadening susceptibility, as the specificity of permissiveness and resistance to other scrapie isolates was maintained.
The diversity and possible contribution of non-coding regions of the prion protein (PrP) gene (PRNP) to transmissible spongiform encephalopathy susceptibility and PrP regulation are not fully known. This study defined ten ovine PRNP promoters and five untranslated region (UTR) haplotypes found in atypical and classical scrapie cases and healthy control sheep. A greater diversity of promoter and UTR haplotypes was observed in conjunction with the ARQ PrP allele (seven promoter and four UTR haplotypes), while it was observed that the other alleles were linked with a limited number of haplotypes, such as ARR, found to be linked to only two promoter and one UTR haplotypes. In silico analysis identified potential transcription factor binding sites that differed in the promoter haplotype variants. Furthermore, a 59 UTR internal ribosome entry site motif was identified in exon 2 and highlights a possible role for this exon in regulating PrP expression at the translational level.Scrapie is a member of the transmissible spongiform encephalopathy (TSE) family of diseases, whose natural hosts are sheep and goats. TSEs are degenerative disorders of the nervous system and are invariably fatal. Irrespective of whether these diseases are acquired, inherited or of idiopathic origin, the prion protein (PrP) plays a central role (Prusiner, 1998).The PRNP gene codes for the ovine PrP, is over 20 kb long and contains three exons separated by two introns of 2421 and 14031 bp (Lee et al., 1998). The 768 bp coding region or open reading frame (ORF) of PrP is contained entirely within exon 3. The 59 untranslated region (UTR) consists of exon 1, exon 2 and bases 1-10 of exon 3 and the 39 UTR (bases 779-4028) of exon 3 (Fig. 1).Polymorphisms within the PRNP ORF are known to be associated with susceptibility and resistance to classical and atypical scrapie in sheep (Goldmann et al., 1994;Benestad et al., 2003;Moum et al., 2005 (shortened to ARQ when omitting the 141 codon). This allele, plus those generated through the substitution of one of its amino acids, make up the six most common ovine PrP alleles, namely ARQ, VRQ, AHQ, ARR and ARH, which all have leucine at position 141, and AF 141 RQ, which has phenylalanine at this position.Other regions of PRNP could also influence susceptibility, either independently or in synergy with the coding region, and may prove beneficial in future breeding plans, particularly in rare breeds, to maximize genetic diversity in the national flock whilst maintaining scrapie resistance (Dawson et al., 2008). For example, polymorphisms in the PRNP promoter region could destroy or create a transcription factor binding site (TFBS) that in turn leads to the down-or upregulation of PrP and influences disease susceptibility or incubation period. The mechanism by which the 39 UTR region of PRNP could affect scrapie susceptibility and resistance is not known; however, Goldmann et al. (1999) demonstrated that the length of the 39 UTR in mRNA can affect PrP expression levels. In general, the 39 UTR can be involved in ...
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