A sensitive 301V BSE serial PMCA assay

Introduction

The transmissible spongiform encephalopathies (TSE or prion diseases) form a group of infectious and fatal neurodegenerative diseases affecting several species of mammals for which there is no available treatment or cure. The cause is thought to be a novel infectious agent (the prion), itself a misfolded isomer (PrP^Sc) of a benign cell associated protein known PrP or PrP^C. This group of diseases include scrapie in sheep and goats, Creutzfeldt Jakob disease (CJD) in humans and bovine spongiform encephalopathy (BSE) in cattle. The UK BSE epizootic of the mid-1980s to early 1990s was the result of recycling BSE through the cattle food chain via a high protein feed additive known as meat and bone meal. It is thought that over 460,000 BSE infected UK cattle entered the human food chain before a ban on feeding specified risk materials to cattle came into force¹. As a consequence of this, a new human disease referred to as vCJD, the human form of BSE, began to present in a number of young adults from the mid-1990s. This demonstration of the zoonotic potential of prion diseases generated an accelerated program of research into these diseases and much of this has required animal models. The murine passaged BSE strain known as 301V was first described by Bruce and colleagues² during transmission studies of cattle BSE to wild type mice. BSE 301V is the product of serial passage within the VM mouse line and this combination of 301V/VM has been well characterised and used in numerous studies, including those aimed at understanding the fundamental brain pathology during neuropathogenesis³. In addition, the 301V/VM model has also been important in experiments analysing the effectiveness of various decontamination measures for BSE infectivity. For example, Taylor et al.⁴ demonstrated the effectiveness of formic acid in inactivating both 301V and scrapie in the context of occupational exposure to histological samples. This BSE model has also been used to show the lack of sufficient inactivation of BSE prions during historical rendering processes which resulted in the BSE outbreak in UK cattle⁵. For development of safe procedures in the context of human health, 301V has been used to model vCJD in the fractionation of plasma and the safe manufacture of blood products⁶, and additionally has been used to estimate BSE infectivity that is likely to remain after processes in the derivation of bone gelatine from bovine products⁷. A further study by McLeod et al.⁸ screened a number of different proteases for their ability to reduce the infectious titre of 301V as a novel method for the decontamination of sensitive surgical instruments. More recently, the availability of transgenic mice expressing the bovine PRNP transgene have become available, and with this their high susceptibility to bovine BSE prions has complemented the use of 301V in these types of experiments. A study published by Giles et al.⁹ directly compared the effectiveness of decontamination of both bovine BSE and 301V in transgenic and VM mice, respectively. 301V was more sensitive to both heat and chemical denaturation than cattle BSE, suggesting that the physical properties of the 301V BSE strain have diverged slightly from those of cattle BSE. Despite the more recent availability of these transgenic rodent strains the 301V/VM infection system remains a useful, well characterised model for BSE in TSE research and allows direct comparison with numerous previous studies.

In the last 15 years or so prion research has been revolutionised by the demonstration of in vitro assays that are thought to replicate the molecular events occurring in vivo during prion infection and the conversion of PrP^C to the disease isomer PrP^Sc. First reported by Saborio and colleagues in 2001¹⁰ the protein misfolding cyclic amplification (PMCA) assay is able to replicate prions in vitro within a source of PrP^C (generally produced from a healthy brain homogenate) during cycles of PrP^C to PrP^Sc seeded conversion followed by sonication with high frequency sound waves that break up aggregates of PrP^Sc to form new seeds or sites of nucleation. The products of this sensitive in vitro assay retain the biochemical characteristics of the prion seed and are infectious¹¹. The sensitivity of the PMCA assay was improved by including the dilution of the reaction into fresh PrP^C substrate after an optimal period of amplification. This modification, known as serial PMCA (sPMCA)¹² has been widely adopted by the research community and has been applied to several rodent prion strains¹³, scrapie in sheep^14,15, BSE in cattle¹⁶, and CWD of cervids¹⁷. sPMCA can achieve levels of sensitivity significantly beyond that of animal bioassay¹⁸ and these experiments take days or weeks to perform compared to the months to years of animal bioassay, and at a fraction of the cost. As such, amplification of prions by sPMCA can be used as a surrogate for measuring infectivity in vivo. To date, the 301V strain of BSE has not been used in sPMCA based studies. Here, we describe a high sensitivity 301V sPMCA that can, over a period of 5 days detect higher dilutions of infectivity than are attained by a 170–200 day bioassay within the VM mouse line.

Results and discussion

Figure 1. sPMCA amplification of 301V BSE.

A. Reactions were seeded with 10 µls of 10^-4 to 10^-13 dilution of 301V brain (as indicated). Unspiked samples had 10 µl VM brain substrate only. Sc, scrapie positive brain sample was used as a blotting control. B. The assessment of reproducibility of the 301V sPMCA using three separate batches of VM substrate, each were seeded with 301V brain dilutions 10^-7 to 10^-11 (as indicated). Unspiked PMCA samples were always negative (a total of 15 replicates are shown). Western blots were probed with the anti-PrP antibody SHa31, M, molecular mass markers at 41, 30 and 22 kDa.

A pool of 301V mouse brain homogenate was used to assess the efficacy of a new sPMCA assay alongside conventional 301V/VM bioassay. For the in vitro assay we used a 5 day amplification method and a murine VM substrate. This 301V sPMCA assay demonstrated assay sensitivity to 1×10^-9 dilution of brain homogenate (Figure 1A). The batch-to-batch variability test of a further 3 substrate preparations detected 301V to at least the same level (Figure 1B). The observed variations in sensitivity within these limiting dilution experiments (between 10^-9 and 10^-11) is likely a reflection of differences in individual substrate preparations that will be seen when making small volume preparations from limited numbers of brains.

The same 301V sample was also analysed in a VM mouse bioassay by limiting dilution and this bioassay detected infectivity in 1 out of 12 mice at the 10^-8 dilution of 301V brain material (Table 1), equating to a 301V titre of the original brain pool of 10^8.5 LD₅₀/g (as determined by Karber methodology¹⁹).

The 301V sPMCA assay can therefore detect PrP^Sc at a level at least tenfold more sensitive than the VM mouse bioassay, in a total assay time taking little over a week. Whilst we report sensitivity of the assay at 5 days of amplification, it is very likely that much higher levels of sensitivity could be attained with additional rounds of amplification. The highest dilution of 301V infectivity that could be detected within the VM bioassay was a 1×10^-8 dilution of brain at 184 days post inoculation, or 26 times longer than the sPMCA assay. Maintaining animals within bioassay, including their category 3 containment make these kind of titration experiments very costly and time consuming to carry out. That, coupled with the ethical implications of use of animals means sPMCA could be the method of choice unless there is a good scientific reason for requiring to demonstrate infectivity (the ability to cause disease), or a requirement to monitor strain phenotype, as opposed to the surrogate marker of disease, PrP^Sc protein. A useful way of incorporating these two assays into future studies, could be to assess 301V seeding activity within a wide range of samples to identify those that contain PrP^Sc. Bioassay could then be used on a limited number of sPMCA-positive samples to confirm the presence of BSE infectivity. Another example of the application of sPMCA that has been routinely used for the detection of prions in a rodent prion model is with cervid CWD¹⁷. In this instance, CWD amplification within cervid CNS tissue substrate is notoriously inefficient, and transgenic mice have been used as an animal bioassay model for infectivity studies and also to provide substrate to facilitate efficient in vitro amplification by sPMCA.

In summary, we have developed a reliable in vitro method (sPMCA) for the detection of PrP^Sc resulting from infection with 301V (mouse passaged BSE). The assay is at least as sensitive as mouse bioassay and can derive data on the presence of PrP^Sc in a fraction of the time. This will be useful in studies such as those looking at BSE decontamination where the screening of large numbers of samples is required.

Materials and methods

All use of animals, the collection of animal tissues and the use of such tissue was carried out in accordance with the Animal (Scientific Procedures) Act (ASPA) 1986, under licences from the UK Government Home Office (Project licence 60/2544). All animal experiments were subject to review and approval (01-124) by The Roslin Institute Ethical Review Committee and euthanasia methods were approved by the UK Home Office.

Data availability

F1000Research: Dataset 1. Raw uncropped images of the Western blots shown in Figure 1. 10.5256/f1000research.9735.d138638²³