Ex vivo propagation of infectious sheep scrapie agent in heterologous epithelial cells expressing ovine prion protein

Abstract

Transmissible spongiform encephalopathies, or prion diseases, are fatal degenerative disorders of the central nervous system that affect humans and animals. Prions are nonconventional infectious agents whose replication depends on the host prion protein (PrP). Transmission of prions to cultured cells has proved to be a particularly difficult task, and with a few exceptions, their experimental propagation relies on inoculation to laboratory animals. Here, we report on the development of a permanent cell line supporting propagation of natural sheep scrapie. This model was obtained by stable expression of a tetracycline-regulatable ovine PrP gene in a rabbit epithelial cell line. After exposure to scrapie agent, cultures were repeatedly found to accumulate high levels of abnormal PrP (PrPres). Cell extracts induced a scrapie-like disease in transgenic mice overexpressing ovine PrP. These cultures remained healthy and stably infected upon subpassaging. Such data show that (i) cultivated cells from a nonneuronal origin can efficiently replicate prions; and (ii) species barrier can be crossed ex vivo through the expression of a relevant PrP gene. This approach led to the ex vivo propagation of a natural transmissible spongiform encephalopathy agent (i.e., without previous experimental adaptation to rodents) and might be applied to human or bovine prions.

Cell models of prion infection

“…Recent studies confirmed the original finding [146] that non-neuronal cell lines can efficiently propagate prions. Common fibroblast cell lines [147] and a microglial cell line (MG20) established from transgenic mice overexpressing PrP are susceptible to various murine prion strains [61].…”

“…The epithelial rabbit RK13 cell line, expressing no detectable endogenous rabbit PrP C , was genetically engineered to stably express an ovine PrP C variant ( 136 V 154 R 171 Q) associated with a very high susceptibility to scrapie [59]. The resulting cultures, known as Rov cells, can be readily infected with and replicate to high titres some sheep scrapie isolates [146]. Neuroglial cell lines with Schwann-like features [3] and primary cultures of neurons and astrocytes [34], both of which were derived from transgenic mice expressing the VRQ allele of ovine PrP C , were later shown to be permissive to sheep scrapie as well.…”

“…In the absence of nucleic acids, strain biological properties are proposed to be encoded by the abnormal PrP [150]. The PrP res banding patterns of strains generated in cultured cells are often, but not always [34], different from those generated in the brain [3,4,26,90,95,146] but return to the original brain pattern upon animal inoculation [3,4]. This confirmed that the cellular context in which multiplication occurs has a determinant effect on the glycoforms and on the size of the abnormal PrP species and that modifications of the PrP res molecular profile are not necessarily associated with changes in their biological properties [133].…”

“…Monoclonal antibody 6H4 (recognising residues [144][145][146][147][148][149][150][151][152] and Fab fragments of D18 and D13 mAbs (recognising residues 132-156 and 95-103, respectively) potently inhibit prion multiplication in N2a cells [41,102]. SAF34 and SAF61 mAbs, which recognise the octarepeat region and residues 114-152 respectively, impaired prion propagation in infected neuroblastoma cells with an increase of the turn-over of PrP C as a proposed mechanism for prion inhibition [104].…”

“…As a result, very few permissive cell lines were available in the early 2000s and all of them were permissive only to rodent-adapted prion strains [132]. The first exception occurred in 2001 when it was shown that expression of ovine PrP C in rabbit epithelial RK13 cells rendered them permissive to the multiplication of a sheep scrapie agent [146]. This was the first example of successful transmission of a natural transmissible spongiform encephalopathy (TSE) agent (i.e.…”