Abstract
The prion strain 301V, is a mouse passaged form of bovine spongiform encephalopathy (BSE). It has been used as a model of BSE for more than 20 years, in particular in the investigation of tissue distribution of infectivity, the molecular phenotype and transmission properties of BSE, strain typing assays and prion inactivation studies. Most 301V experiments have required murine bioassay as a method for the quantitation of infectivity. To date this model strain has not been studied with the protein misfolding cyclic amplification assay (PMCA) which detects prion-associated PrPSc protein. The detection of BSE PrPSc by PMCA can be more sensitive than mouse bioassay and is carried out in a much shorter time frame of days as opposed to months/years. Here, we describe the development of a new highly sensitive and specific PMCA assay for murine 301V and assess the sensitivity of the assay in direct comparison with murine bioassay of the same material. This in vitro assay detected, in a few days, 301V at a brain dilution of at least 1x10-9, compared to bioassay of the same material in VM mice that could detect down to a 1x10-8 dilution and took >180 days. The 301V PMCA may therefore offer a faster and more sensitive alternative to live animal bioassay when studying the BSE agent in VM mice.
Highlights
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
bovine spongiform encephalopathy (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
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 PrPC during cycles of PrPC to PrPSc seeded conversion followed by sonication with high frequency sound waves that break up aggregates of PrPSc to form new seeds or sites of nucleation
Summary
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. 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 PrPC to the disease isomer PrPSc. 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 PrPC (generally produced from a healthy brain homogenate) during cycles of PrPC to PrPSc seeded conversion followed by sonication with high frequency sound waves that break up aggregates of PrPSc to form new seeds or sites of nucleation. 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
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