Prion strain discrimination in cell culture: The cell panel assay

Abstract

Prions are thought to consist mainly or entirely of misfolded PrP, a constitutively expressed host protein. Prions associated with the same PrP sequence may occur in the form of different strains; the strain phenotype is believed to be encoded by the conformation of the PrP. Some cell lines can be persistently infected by prions and, interestingly, show preference for certain strains. We report that a cloned murine neuroblastoma cell population, N2a-PK1, is highly heterogeneous in regard to its susceptibility to RML and 22L prions. Remarkably, sibling subclones may show very different relative susceptibilities to the two strains, indicating that the responses can vary independently. We have assembled four cell lines, N2a-PK1, N2a-R33, LD9 and CAD5, which show widely different responses to prion strains RML, 22L, 301C, and Me7, into a panel that allows their discrimination in vitro within 2 weeks, using the standard scrapie cell assay (SSCA).standard scrapie cell assay ͉ infectivity ͉ response index ͉ PrP

Spontaneous generation of mammalian prions

“…Given the prion strain selectivity of PK1 cells (14), the spontaneously generated prions must be of a strain type permissive for propagation in these cells. We considered therefore whether in fact iPK1 cells, although originally infected with mouse-passaged RML prions, were in fact propagating a distinct strain that might have arisen by adaptation to this cell line, with strain selection or mutation to a preferred conformer (23,24).…”

“…Although the amount of protein bound was undetectable by conventional methods, the wires were as effective in eliciting disease as injection of approximately 10 6 LD 50 units in the form of brain homogenate. We have isolated an N2a neuroblastoma-derived cell line, N2aPK1 (i.e., "PK1") (13), that is highly susceptible to infection by a wide spectrum of murine strains, including RML and 22L, but not ME7 and 301C (13,14). When PK1 cells were grown on RML prion-coated wires, adherent cells became PrP Sc -positive, in contrast to neighboring cells, which remained negative, implying that adherence to the prion-coated surface is a prerequisite for infectivity transfer (15).…”

Differentiating Prion Strains Using Dendrimers

“…[17,25] Classically, this has been achieved for mouse-adapted prion strains through the determination of incubation times in at least two mouse lines over 6-10 months. [26] As this is a slow and costly process, a number of alternatives have been developed including the cell panel assay (CPA) [27] , the conformational stability assay (CSA) [28] and the differential ELISA system based on Bio-Rad's TSE detection [29] . However, despite their various advantages, all these methods have their limitations: The CPA takes two weeks to generate a result [27] .…”

“…[26] As this is a slow and costly process, a number of alternatives have been developed including the cell panel assay (CPA) [27] , the conformational stability assay (CSA) [28] and the differential ELISA system based on Bio-Rad's TSE detection [29] . However, despite their various advantages, all these methods have their limitations: The CPA takes two weeks to generate a result [27] . The CSA does not distinguish between certain prion strains such as ME7-H and Syrian hamster Sc237 [28] .…”

Prion agent diversity and species barrier

“…Some transgenic lines have been established by a gene replacement method (for review [44]). PrP sequence identity between the transgenic host and donor usually lead to an enhanced susceptibility to TSE as compared to wild-type [58,127] mice. Moreover, in various instances, the TSE agent original properties, in particular PrP Sc molecular profile, appeared to be essentially maintained in the infected transgenic host (see also Sect.…”