Differential immunoreactivity of goat derived scrapie following in vitro misfolding versus mouse bioassay

Madsen–Bouterse, Sally A.; Zhuang, Dongyue; O’Rourke, Katherine I.; Schneider, David A.

doi:10.1016/j.bbrc.2012.06.034

Cited by 6 publications

(13 citation statements)

References 45 publications

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“…Previously, PMCA conditions were described for the detection of scrapie prions in sheep (Thorne & Terry, 2008) and goats (Madsen-Bouterse et al, 2012). These studies further confirmed that PMCA is more sensitive than TSE ELISA (O'Rourke et al, 2011) and Western blot (Madsen-Bouterse et al, 2012) assays.…”

Section: Introductionsupporting

confidence: 61%

“…Although the survival times of second-passage Tg338 mice became shorter and more consistent for both PRNP WT and polymorphic genotypes, clinical signs of scrapie were observed at *4 months post-inoculation . Although PMCA is faster compared with bioassay studies in Tg338 mice, it still requires several weeks and multiple rounds of amplification followed by Western blot analysis (Madsen-Bouterse et al, 2012). However, we as well as others have shown that RT-QuIC is much faster than PMCA and is overall less technically challenging (compared with PMCA) due to the lack of maintenance of mouse colonies, addition of Detection of caprine scrapie prions using RT-QuIC substrate during the assay, proteinase K digestion and Western blotting.…”

Section: Discussionmentioning

confidence: 99%

“…Protein misfolding cyclic amplification (PMCA) (Saá et al, 2006) and real-time quaking-induced conversion (RT-QuIC) (Atarashi et al, 2007(Atarashi et al, , 2011Orrú et al, 2009;Wilham et al, 2010) assays demonstrate a sensitivity for the detection of brain-derived prion seeding activity from several different prion strains comparable to animal bioassays. In PMCA, brain homogenates prepared from uninfected animals or transgenic mice expressing prion protein of the species of interest are used as the misfolding substrate (Madsen-Bouterse et al, 2012;Saá et al, 2006;Thorne & Terry, 2008), whereas in RT-QuIC, bacterially expressed (unglycosylated) recombinant prion protein (rPrP Sen ) is used as the substrate (Atarashi et al, 2011;Wilham et al, 2010). Previously, PMCA conditions were described for the detection of scrapie prions in sheep (Thorne & Terry, 2008) and goats (Madsen-Bouterse et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

“…In PMCA, brain homogenates prepared from uninfected animals or transgenic mice expressing prion protein of the species of interest are used as the misfolding substrate (Madsen-Bouterse et al, 2012;Saá et al, 2006;Thorne & Terry, 2008), whereas in RT-QuIC, bacterially expressed (unglycosylated) recombinant prion protein (rPrP Sen ) is used as the substrate (Atarashi et al, 2011;Wilham et al, 2010). Previously, PMCA conditions were described for the detection of scrapie prions in sheep (Thorne & Terry, 2008) and goats (Madsen-Bouterse et al, 2012). These studies further confirmed that PMCA is more sensitive than TSE ELISA (O'Rourke et al, 2011) and Western blot (Madsen-Bouterse et al, 2012) assays.…”

Section: Introductionmentioning

confidence: 99%

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Sensitive and specific detection of classical scrapie prions in the brains of goats by real-time quaking-induced conversion

Dassanayake

Orrú

Hughson

et al. 2016

Journal of General Virology

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Real-time quaking-induced conversion (RT-QuIC) is a rapid, specific and highly sensitive prion seeding activity detection assay that uses recombinant prion protein (rPrP Sen ) to detect subinfectious levels of the abnormal isoforms of the prion protein (PrP Sc ). Although RT-QuIC has been successfully used to detect PrP Sc in various tissues from humans and animals, including sheep, tissues from goats infected with classical scrapie have not yet been tested. Therefore, the aims of the present study were to (1) evaluate whether prion seeding activity could be detected in the brain tissues of goats with scrapie using RT-QuIC, (2) optimize reaction conditions to improve scrapie detection in goats, and (3) compare the performance of RT-QuIC for the detection of PrP Sc with the more commonly used ELISA and Western blot assays. We further optimized RT-QuIC conditions for sensitive and specific detection of goat scrapie seeding activity in brain tissue from clinical animals. When used with 200 mM sodium chloride, both full-length sheep rPrP Sen substrates (PrP genotypes A 136 R 154 Q 171 and V 136 R 154 Q 171 ) provided good discrimination between scrapie-infected and normal goat brain samples at 10 23 dilution within 15 h. Our findings indicate that RT-QuIC was at least 10 000-fold more sensitive than ELISA and Western blot assays for the detection of scrapie seeding activity in goat brain samples. In addition to PRNP WT samples, positive RT-QuIC reactions were also observed with three PRNP polymorphic goat brain samples (G/S127, I/M142 and H/R143) tested. Taken together, these findings demonstrate that RT-QuIC sensitively detects prion seeding activity in classical scrapie-infected goat brain samples.

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Section: Introductionsupporting

confidence: 61%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Sensitive and specific detection of classical scrapie prions in the brains of goats by real-time quaking-induced conversion

Dassanayake

Orrú

Hughson

et al. 2016

Journal of General Virology

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“…11 Prior to the preparation of brain homogenates, PrP Sc in clinically scrapie affected goats and Tg338 mice used in this study was confirmed by IHC, a commercially available TSE ELISA kit (IDEXX), and western blot assays and scrapie infectivity was confirmed in bioassay studies using goats and Tg338 mice. 9,21,22 For the present study, brain homogenates prepared from all 5 scrapieinfected goats and 3 Tg338 mice were again assessed for PrP Sc by TSE ELISA and protein gel blot. As expected, significant amounts of PrP Sc in both the caprine and the murine scrapie and pcRK13 cells.…”

Section: Cprk13 Cells Support Propagation Of Classical Caprine Scrapimentioning

confidence: 99%

A transfectant RK13 cell line permissive to classical caprine scrapie prion propagation

Dassanayake

Zhuang

Truscott

et al. 2016

Prion

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ABSTRACT. To assess scrapie infectivity associated with caprine-origin tissues, bioassay can be performed using kids, lambs or transgenic mice expressing caprine or ovine prion (PRNP) alleles, but the incubation periods are fairly long. Although several classical ovine scrapie prion permissive cell lines with the ability to detect brain-derived scrapie prion have been available, no classical caprine scrapie permissive cell line is currently available. Therefore, the aims of this study were to generate a rabbit kidney epithelial cell line (RK13) stably expressing caprine wild-type PRNP (cpRK13) and then to assess permissiveness of cpRK13 cells to classical caprine scrapie prion propagation. The cpRK13 and plasmid control RK13 (pcRK13) cells were incubated with brain-derived classical caprine scrapie inocula prepared from goats or ovinized transgenic mice (Tg338, express ovine VRQ allele) infected with caprine scrapie. Significant PrP Sc accumulation, which is indicative of scrapie prion propagation, was detected by TSE ELISA and immunohistochemistry in cpRK13 cells inoculated with classical caprine scrapie inocula. Western blot analysis revealed the typical proteinase K-resistant 3 PrP res isoforms in the caprine scrapie prion inoculated cpRK13 cell lysate. Importantly, PrP Sc accumulation was not detected in similarly inoculated pcRK13 cells, whether by TSE ELISA, immunohistochemistry, or western blot. These findings suggest that caprine scrapie prions can be propagated in cpRK13 cells, thus this cell line may be a useful tool for the assessment of classical caprine prions in the brain tissues of goats.

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Antiprion Activity of DB772 and Related Monothiophene- and Furan-Based Analogs in a Persistently Infected Ovine Microglia Culture System

Dinkel

Stanton

Boykin

et al. 2016

Antimicrob Agents Chemother

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eThe transmissible spongiform encephalopathies are fatal neurodegenerative disorders characterized by the misfolding of the native cellular prion protein (PrP C ) into the accumulating, disease-associated isoform (PrP Sc ). Despite extensive research into the inhibition of prion accumulation, no effective treatment exists. Previously, we demonstrated the inhibitory activity of DB772, a monocationic phenyl-furan-benzimidazole, against PrP Sc accumulation in sheep microglial cells. In an effort to determine the effect of structural substitutions on the antiprion activity of DB772, we employed an in vitro strategy to survey a library of structurally related, monothiophene-and furan-based compounds for improved inhibitory activity. Eighty-nine compounds were screened at 1 M for effects on cell viability and prion accumulation in a persistently infected ovine microglia culture system. Eleven compounds with activity equivalent to or higher than that of DB772 were identified as preliminary hit compounds. For the preliminary hits, cytotoxicities and antiprion activities were compared to calculate the tissue culture selectivity index. A structure-activity relationship (SAR) analysis was performed to determine molecular components contributing to antiprion activity. To investigate potential mechanisms of inhibition, effects on PrP C and PrP Sc were examined. While inhibition of total PrP C was not observed, the results suggest that a potential target for inhibition at biologically relevant concentrations is through PrP C misfolding to PrP Sc . Further, SAR analysis suggests that two structural elements were associated with micromolar antiprion activity. Taken together, the described data provide a foundation for deeper investigation into untested DB compounds and in the design of effective therapeutics. P rions are unique proteinaceous, infectious agents that cause fatal neurodegenerative disorders collectively known as the transmissible spongiform encephalopathies (TSEs). TSEs include Creutzfeldt-Jakob disease in humans, chronic wasting disease in cervids, and scrapie in sheep and goats (1, 2). Prions are comprised of protease-resistant, disease-associated isoforms (PrP Sc ) of the prion protein (3). Prion protein in its native cellular form (PrP C ) is a glycoprotein encoded by the host's PRNP gene and is highly expressed in multiple cell types, including neurons, microglia, and certain cells of the immune system (1-3). Misfolding to PrP Sc promotes self-templated replication and accumulation within the central nervous system that lead to slowly progressive neurological dysfunction and eventually death (1-3). Currently, the mechanisms underlying this conversion are incompletely defined, and treatments to prevent or cure disease do not exist (4).Due to the invariable lethality of prion disease, identification of compounds that prevent misfolding, replication, or accumulation is a vital goal (4-6). Previous studies to screen candidate antiprion compounds have primarily employed rodent cell culture systems chronically infe...

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Differential immunoreactivity of goat derived scrapie following in vitro misfolding versus mouse bioassay

Cited by 6 publications

References 45 publications

Sensitive and specific detection of classical scrapie prions in the brains of goats by real-time quaking-induced conversion

Sensitive and specific detection of classical scrapie prions in the brains of goats by real-time quaking-induced conversion

A transfectant RK13 cell line permissive to classical caprine scrapie prion propagation

Antiprion Activity of DB772 and Related Monothiophene- and Furan-Based Analogs in a Persistently Infected Ovine Microglia Culture System

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