In vitro amplification of ovine prions from scrapie-infected sheep from Great Britain reveals distinct patterns of propagation

Abstract

BackgroundProtein misfolding cyclic amplification (PMCA) is a method that facilitates the detection of prions from many sources of transmissible spongiform encephalopathy (TSE). Sheep scrapie represents a unique diversity of prion disease agents in a range of susceptible PRNP genotypes. In this study PMCA was assessed on a range of Great Britain (GB) sheep scrapie isolates to determine the applicability to veterinary diagnosis of ovine TSE.ResultsPrPSc amplification by protein misfolding cyclic amplification (PMCA) was assessed as a diagnostic tool for field cases of scrapie. The technique was initially applied to thirty-seven isolates of scrapie from diverse geographical locations around GB, and involved sheep of various breeds and PRNP genotypes. All samples were amplified in either VRQ and/or ARQ PrPC substrate. For PrPSc from sheep with at least one VRQ allele, all samples amplified efficiently in VRQ PrPC but only PrPSc from ARH/VRQ sheep amplified in both substrates. PrPSc from ARQ/ARQ sheep displayed two amplification patterns, one that amplified in both substrates and one that only amplified in ARQ PrPC. These amplification patterns were consistent for a further 14/15 flock/farm mates of these sheep. Furthermore experimental scrapie strains SSBP1, Dawson, CH1641 and MRI were analysed. SSBP1 and Dawson (from VRQ/VRQ sheep) amplified in VRQ but not ARQ substrate. MRI scrapie (from ARQ/ARQ sheep) nor CH1641 did not amplify in ARQ or VRQ substrate; these strains required an enhanced PMCA method incorporating polyadenylic acid (poly(A)) to achieve amplification.ConclusionsPrPsc from 52 classical scrapie GB field isolates amplified in VRQ or ARQ or both substrates and supports the use of PMCA as a rapid assay for the detection of a wide range of ovine classical scrapie infections involving multiple PRNP genotypes and scrapie strains.

Seeded Amplification of Chronic Wasting Disease Prions in Nasal Brushings and Recto-anal Mucosa-Associated Lymphoid Tissues from Elk by Real-Time Quaking-Induced Conversion

“…Increased surveillance efforts during the past several years have led to the detection of new cases in U.S. states previously thought to be outside the area where CWD is endemic (e.g., Texas, Iowa, and Pennsylvania in 2012 and Ohio in 2014) (9)(10)(11). As this devastatingly fatal disease spreads across the United States and beyond, the importance of highly sensitive antemortem detection becomes increasingly evident.…”

“…The disease has since been documented in 23 U.S. states, 2 Canadian provinces, and, via exportation of farmed cervids, the Republic of Korea (5)(6)(7)(8). Four of the 23 states (Texas, Iowa, Pennsylvania, and Ohio) were considered CWD free prior to 2012, with primary cases in three of these states reportedly arising in farmed cervids (9)(10)(11). With the movement of cervids across state and national borders, these new epidemic foci illustrate the increased need for highly sensitive surveillance methods and appropriate antemortem tissue collection in order to potentially mitigate both natural and anthropogenic spread and more accurately estimate prevalence.…”

Enhanced Virulence of Sheep-Passaged Bovine Spongiform Encephalopathy Agent Is Revealed by Decreased Polymorphism Barriers in Prion Protein Conversion Studies

“…It is generally accepted that prion protein conversion studies, similar to transgenic-mouse studies, resemble the trend of incubation periods in intracerebral inoculations, and that for sheep (and some other species) they also seem to be biologically relevant for oral and natural transmission routes (61,62). Even though PrP Sc accumulation in the brain is considered the prime indicator for successful TSE agent spread, other peripheral organs like the liver, spleen, and gut can also significantly contribute to agent spread (2,59,63).…”

Scientific Opinion on a request for a review of a scientific publication concerning the zoonotic potential of ovine scrapie prions

“…The newly generated PrP Sc produced in homologous conversion assays shares the same biochemical, biological and structural properties as the parental isolate (Castilla et al, 2008a;Green et al, 2008;Thorne et al, 2012;Vidal et al, 2013a;Levavasseur et al, 2014;Priem et al, 2014), although exceptions have been reported . This reproduction of strain adaptation and molecular patterns indicate again that the strain determinants are largely encoded in the structure and folding of PrP Sc (Castilla et al, 2008a).…”

“…As seen in vivo the strain and source of PrP Sc , in particular the PRNP genotype of seed and substrate, has a clear influence on the amplification pattern obtained by PMCA (Bucalossi et al, 2011;Thorne et al, 2012;Krejciova et al, 2014;Priem et al, 2014). In this regard it is of interest that a switch of the conversion profile has been shown by the amplification of an ARR scrapie isolate in different sheep genotypes, which resulted in a conversion profile that resembled mostly that of ARQ scrapie (Bucalossi et al, 2011;Priem et al, 2014).…”