We prepared seed and substrate homogenates as 10% (wt/vol) homogenates in PMCA conversion buffer (10). Seed homogenates were diluted into substrate homogenates so that all PMCA reactions contained equivalent amounts of PrP^d based on the PrP^res levels in each seed homogenate. PrP^res levels were determined by Western blot titration that used monoclonal antibody (MAb) 6H4 after limited proteinase K digestion. The reaction mixes were split into 2 aliquots; 1 aliquot was stored immediately at –80°C (−PMCA), and the other was subjected to 48 cycles of PMCA (+PMCA) (10). To assess the degree of PrP^d amplification achieved from each seed in each substrate, the samples −/+ PMCA were subjected to limited proteinase K digestion, and PrP^res was detected by Western blotting with MAb 6H4 (which recognizes human, bovine, and ovine PrP) and MAb 3F4 (which selectively recognizes only human PrP and would therefore specifically identify PrP^res formed from human PrP^C).

Using MAb 6H4 to probe Western blots, we noted amplification of vCJD, bovine BSE, and ovine BSE PrP^res in the PRNP-129MM substrate (Figure 1, panel A, top) but not in the PRNP-129VV substrate (Figure 1, panel A, bottom). Semiquantitative assessment of these Western blots by densitometry showed that the degree of amplification of vCJD PrP^res was considerably greater than that of bovine or ovine BSE in the PRNP-129MM substrate (Figure 2, panel A). A more sensitive and discriminatory Western blot conducted by using MAb 3F4 confirmed efficient amplification of vCJD, bovine BSE, and ovine BSE PrP^res in the PRNP-129MM substrate (Figure 1, panel B, top), weaker amplification in the PRNP-129MV substrate (Figure 1, panel B, middle), and little, if any, amplification in the PRNP-129VV substrate (Figure 1, panel B, bottom). In all substrates, the amplified PrP^res retained the electrophoretic mobility and glycoform ratio associated with BSE-related PrP^res. No amplification of ovine scrapie PrP^res was evident after PMCA in any of the PRNP humanized transgenic mouse brain substrates (Figure 1, panels A, B). The difference between ovine scrapie and ovine BSE in ability to seed amplification in PRNP-129MM substrate was a robust phenomenon evident in brain samples from 3 different ARQ/ARQ sheep with each disease (Figure 2, panel B). However, failure of the ovine scrapie seed to amplify was not caused by a general lack of competence to do so or by inappropriate amplification conditions because robust amplification of ovine scrapie PrP^res was evident after PMCA in a substrate prepared from normal ARQ/ARQ sheep brain (Figure 2, panel C).

Our results are best appreciated in terms of the molecular interaction between seed PrP^d and substrate PrP^C, specifically the species-specific amino acid sequence and PRNP polymorphic status of PrP^C and PrP^d and the PrP^d conformers involved (Table). Regardless of the seed PrP amino acid sequence, the PrP^d conformers associated with bovine BSE, ovine BSE, and human vCJD were amplified in the humanized mouse substrate and displayed similar PRNP-129 genotype preferences (PRNP-129MM >PRNP-129MV >PRNP-129VV). In contrast, the PrP^d conformer associated with the ovine scrapie strain, although sharing the same PrP amino acid sequence as the PrP^d in ovine BSE, could not be amplified in any of the PRNP humanized mouse substrates but could be amplified in a sheep brain substrate. These observations are consistent with conformation of a TSE agent’s PrP^d (rather than solely its amino acid sequence) having a role in determining the susceptibility of a host’s PrP^C to conversion. They similarly suggest that these molecular factors could in turn have a powerful influence on disease susceptibility and incubation time.

To date, all clinical cases of vCJD have occurred in persons with the PRNP-129MM genotype, as might be predicted from the efficiency of amplification of BSE-related PrP^d shown here. Extrapolating from these results, one would predict that the next genotypic group most likely to show susceptibility to the BSE agent would be heterozygous (MV) at codon 129 of the PRNP gene, as previously suggested from the corresponding in vivo transmission studies (14).

In the wake of BSE epidemics in the United Kingdom and elsewhere, enhanced surveillance has identified apparently new TSEs (15), raising concerns regarding animal and human health. PMCA with suitable substrate sources could provide a rapid way to estimate the molecular component of transmission barriers for particular TSE agents between species, including humans. These estimates could thus indicate whether, like classical scrapie, the agents represent little risk for human health or whether, like classical BSE, they represent cause for concern.