Detection of Prions

2013财年：1）我们使RT-QuIC和eQuIC适应各种鼠朊病毒，并评估接种活性如何取决于糖磷脂酰肌醇（GPI）锚定以及淀粉样蛋白斑块和蛋白酶抗性PrPSc（PrPRes）的丰度。通过RT-QuIC和eQuIC分别检测高达10-8和10-13的羊瘙痒病脑稀释液。比较羊瘙痒病感染的野生型小鼠和表达GPI无锚PrP的转基因小鼠表明，尽管在大脑中积累了相似浓度的接种活性，但富含淀粉样蛋白的无锚小鼠组织接种了更快的反应。接下来，我们比较了感染性滴度相似但PrPRes水平差异很大的小鼠大脑中的接种活动，发现前者是相似的。因此，在该比较中，RT-QuIC接种活性与感染性比与PrPRes水平更密切相关。我们还发现，eQuIC，其中包括PrPSc免疫沉淀步骤，检测接种22 L，79 A和/或RML羊瘙痒病菌株的野生型和无锚PrP转基因小鼠的血浆中的接种活性。总的来说，我们得出结论，这些新的小鼠适应性朊病毒接种检测不同类型的PrPSc。2)我们采用RT-QuIC测定法检测感染慢性消耗性疾病的鹿唾液中的朊病毒接种活性。3)我们还与神经科医生合作，开始评估我们先前开发的RT-QuIC检测试剂盒用于检测和诊断人散发性克雅氏病的实用性，该检测试剂盒使用来自血液或嗅粘膜刷拭的标本。 到目前为止的数据表明，RT-QuIC灵敏地检测来自各种人类CJD病例的鼻刷中的朊病毒播种活性。血液检测的结果还在等待中。我们正在继续测试我们的合作者正在收集的其他样本。关于这些试验的诊断效用的结论必须等待进一步的试验。

During FY2013: 1) We adapted RT-QuIC and eQuIC to various murine prions and evaluated how seeding activity depends on glycophosphatidylinositol (GPI) anchoring and the abundance of amyloid plaques and protease-resistant PrPSc (PrPRes). Scrapie brain dilutions up to 10-8 and 10-13 were detected by RT-QuIC and eQuIC, respectively. Comparisons of scrapie-affected wild-type mice and transgenic mice expressing GPI anchorless PrP showed that, although similar concentrations of seeding activity accumulated in brain, the heavily amyloid-laden anchorless mouse tissue seeded more rapid reactions. Next we compared seeding activities in the brains of mice with similar infectivity titers, but widely divergent PrPRes levels, and found that the former were similar. Thus, in this comparison, RT-QuIC seeding activity correlated more closely with infectivity than with PrPRes levels. We also found that eQuIC, which incorporates a PrPSc immunoprecipitation step, detected seeding activity in plasma from wild-type and anchorless PrP transgenic mice inoculated with 22L, 79A and/or RML scrapie strains. Overall, we conclude that these new mouse-adapted prion seeding assays detect diverse types of PrPSc. 2) We adapted the RT-QuIC assay to the detection of prion seeding activity in the saliva of deer infected with chronic wasting disease. 3) We have also collaborated with neurologists to begin evaluating the utility of our previously developed RT-QuIC assay for the detection and diagnosis of human sporadic Creutzfeldt-Jakob disease using specimens derived from blood or olfactory mucosa brushings. The data so far have shown that RT-QuIC sensitively detects prion seeding activity in nasal brushings from a wide variety of human CJD cases. Results from the blood testing are pending. We are continuing to test additional samples that are being collected by our collaborators. Conclusions as to the diagnostic utility of these tests must await further testing.