Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Summary

Measuring the barrier to the interspecies transmission of prion diseases is challenging and typically involves animal challenges or biochemical assays. Here, we present an in vitro prion protein conversion assay with the ability to predict species barriers.

Abstract

Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they typically rely upon lengthy and costly in vivo animal challenge studies. A number of in vitro assays have been developed to aid in measuring prion species barriers, thereby reducing animal use and providing quicker results than animal bioassays. Here, we present the protocol for a rapid in vitro prion conversion assay called the conversion efficiency ratio (CER) assay. In this assay cellular prion protein (PrP^C) from an uninfected host brain is denatured at both pH 7.4 and 3.5 to produce two substrates. When the pH 7.4 substrate is incubated with TSE agent, the amount of PrP^C that converts to a proteinase K (PK)-resistant state is modulated by the original host’s species barrier to the TSE agent. In contrast, PrP^C in the pH 3.5 substrate is misfolded by any TSE agent. By comparing the amount of PK-resistant prion protein in the two substrates, an assessment of the host’s species barrier can be made. We show that the CER assay correctly predicts known prion species barriers of laboratory mice and, as an example, show some preliminary results suggesting that bobcats (Lynx rufus) may be susceptible to white-tailed deer (Odocoileus virginianus) chronic wasting disease agent.

Introduction

Transmissible spongiform encephalopathies (TSEs, prion diseases) are a group of fatal neurodegenerative diseases with extended incubation periods that affect a variety of animals and humans. The putative etiological agent of TSEs is comprised of a misfolded isomer of the host prion protein (PrP) that is capable of self-propagation by template-driven conversion of the normal cellular form of PrP (PrP^C) into an infectious, disease-associated form (PrP^TSE) that accumulates in central nervous system tissues of the infected host¹. Infectious mammalian prions generally transmit from host-to-host in a species-specific manner, which has given ....

Protocol

Animal work conducted at the USGS National Wildlife Health Center was performed in accordance with the NIH Office of Laboratory Animal Welfare guidelines and under institutional animal care and use committee protocol #EP080716. Tissues from hunter-harvested animals were gifts from the Wisconsin or Michigan Departments of Natural Resources.

1. Solution Preparation

NOTE: The solutions listed below are required for preparation of the CER assay substrate in Section 2 below. Prepare each of these solutions from higher concentration stock solutions; recommended concentrations for stock solutions will be ....

Results

Successful use of the CER assay is largely dependent upon the quality of the substrate pairs used in conversion reactions. For this reason, following procedures to prepare CER substrate pairs, a quality control immunoblot should be performed (Figure 2). For both substrates, assay 10-25 μl by immunoblotting. PrP^C should be easily detectable in each substrate and PrP^C levels must be approximately the same between the two. Immunoreactivity will be mainly visible above 20 kDa, but small.......

Discussion

For successful completion of this protocol, attention should be paid to PrP^C levels in uninfected brain tissue used for substrate preparation (step 2.1.2) and the seed to substrate ratio for conversion reactions (step 4.4.2). In our experience, brains can be extracted for use as CER substrate after a substantial period post-mortem, as long as PrP^C is present by immunoblotting (Figure 4). In fact, some autolysis was observed in the brains of the bobcats used for CER studies. Neverthe.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
12% Bis-Tris SDS-PAGE gels	Life Technologies	NP0342
0.5 mm Zirconium oxide beads	Next Advance	ZROB05	Other varieties of beads are also effective
Antibodies	various suppliers		Select appropriate primary and secondary antibodies for immunoblot detection of PrPres from species of interest
Bead homogenizer	Next Advance	BBY24M
Centrifuge	Beckman Coulter	369434	High speed with temperature control
Conical tubes	any brand
Cotton-tipped applicator	Uline	S-18991
Cuphorn sonicator	Heat Systems-Ultrasonics	W-380	Heat Systems-Ultrasonics, Inc. is now Qsonica, LLC
Densitometry software program	UVP	Vision Works LS Image Acquisition and Analysis software	Other programs, such as NIH ImageJ, will also work
Dounce homogenizer	Kimble Chase	885300
End-over-end mixer	Labnet International	H5600
Ethylenediaminetetra acetic acid	Boston Bioproducts	P-770	Hazardous chemical: eye irritation
Guanidine hydrochloride, 8 M	Thermo Fisher Scientific	24115	Hazardous chemical: acute toxicity, skin irritation, eye irritation
Heating block	Fisher Scientific	11-718
Hydrochloric acid	Sigma-Aldrich	435570	Hazardous chemical: strong acid
Lithium dodecyl sulfate sample buffer, 4x	Life Technologies	NP0008	Hazardous chemical: skin & respiratory irritation, serious eye damage, flammable solid
Methanol	Fisher Scientific	A454-4	Hazardous chemical: acute toxicity, flammable liquid
Microcentrifuge tubes	any brand
Mini-centrifuge	Labnet International	C1301
N-lauroyl-sarcosine (sarkosyl)	Sigma-Aldrich	L-5125	Hazardous chemical: acute toxicity, skin irritation, eye damage
Nonidet P-40	Amresco	M158	Hazardous chemical: skin irritation, eye damage
SDS-PAGE gel system	Life Technologies	NuPAGE electrophoresis system	Other SDS-PAGE systems will also work
PCR tubes (low-binding)	Axygen	PCR-02-L-C
Pestle homogenizer	Fisher Scientific	03-392-106
pH meter	Sentron	SI600
Polyvinyldifluoride membrane	Millipore	IPVH00010
Proteinase K	Promega	V3021	Hazardous chemical: skin & eye irritation, respiratory sensitisation, organ toxicity
Reducing agent for SDS-PAGE samples, 10x	Life Technologies	NP0009
Sodium chloride	Fisher Scientific	7647-14-5
Sodium deoxycholate	Sigma-Aldrich	D6750	Hazardous chemical: acute toxicity
Sodium dodecyl sulfate	Thermo Fisher Scientific	28364	Hazardous chemical: acute toxicity, skin irritation, eye damage, flammable solid
Sodium hydroxide	Sigma-Aldrich	S5881	Hazardous chemical; strong base
Syringe	BD Biosciences	various	Use syringe size appropriate to volumes of substrate to be homogenized
Syringe needles	BD Biosciences	various
Thermoshaker (PCR tube shaker)	Hangzhou All Sheng Instruments	MS-100
Tris base	Bio Basic	77-86-1	Hazardous chemical: skin, eye, respiratory irritation
Triton X-100	Integra Chemical Company	T756.30.30	Hazardous chemical: acute toxicity, eye irritation
Vortexer	Fisher Scientific	12-812