Infection of Cell Lines with Experimental and Natural Ovine Scrapie Agents

Abstract

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.

Cellular Aspects of Prion Replication In Vitro

“…Cell lines that have been successfully infected include microglial cells as well as epithelial cells, fibroblasts and myoblasts, which have all been demonstrated to persistently replicate an array of prion strains in vitro [98,99,100,101,102,103,104,105,106,107,108]. Curiously, a rabbit kidney epithelial cell line genetically engineered to express PrP C of different species was shown to be susceptible to a variety of prion strains isolated from different sources [99,100,101,102,103,104,105,109]. Several primary cell culture models for prion replication have been reported, some of which show cytopathic effects upon infection [110,111,112,113,114,115].…”

Alimentary prion infections

“…Autopsies of Parkinson disease patients revealed that Lewy bodies had formed on healthy are needed. These may include the use of transgenic mice, cell lines which are permissive to infection 12 and experimental animals such as sheep, calves, goats, minks, ferrets and non-human primates (reviewed in ref. 9).…”

Prion Disease and the Innate Immune System

“…An alternate hypothesis is to suggest that in prion infection with low or poor replicative ability ( i.e ., due to the factors mentioned above) the balance is switched in favor of degradation of agent, thereby preventing or dramatically slowing pathogenesis. This may occur both within any given cell (and not exclusively to degradative or phagocytic cell types), in fact it is well known that there are limited in vitro cell infection models in the prion field due to the relevant inability to infect numerous cell types with a variety of prion strains even within the same species [76], and within the complexity of the host innate immune response.…”