Detection of Prions

在2013财年：1)我们将RT-QuIC和eQuIC应用于各种小鼠朊病毒，并评估了种子活性如何依赖于糖磷脂酰肌醇（GPI）锚定以及淀粉样斑块和蛋白酶抗性PrPSc （PrPRes）的丰富度。RT-QuIC和eQuIC检测痒病脑稀释度分别为10-8和10-13。野生型小鼠与表达GPI无锚点PrP的转基因小鼠的比较表明，尽管在大脑中积累了相似浓度的种子活性，但淀粉样蛋白含量高的无锚点小鼠组织的种子反应更快。接下来，我们比较了具有相似传染性滴度但PrPRes水平差异很大的小鼠大脑中的播种活动，发现前者相似。因此，在本比较中，RT-QuIC播种活性与传染性的相关性比与PrPRes水平的相关性更密切。我们还发现，结合PrPSc免疫沉淀步骤的eQuIC，可以检测接种了22L、79A和/或RML痒病菌株的野生型和无锚定PrP转基因小鼠血浆中的种子活性。总的来说，我们得出结论，这些新的小鼠适应朊病毒播种试验检测不同类型的PrPSc。2)将RT-QuIC法应用于慢性消耗性疾病鹿唾液中朊病毒种子活性的检测。3)我们还与神经科医生合作，开始评估我们之前开发的RT-QuIC检测和诊断人类散发性克雅氏病的实用性，该检测和诊断使用来自血液或嗅粘膜刷拭的标本。迄今为止的数据表明，RT-QuIC可以灵敏地检测多种人类CJD病例的鼻刷中的朊病毒播种活性。血液测试的结果还没有出来。我们正在继续测试合作者收集的其他样本。关于这些测试的诊断效用的结论必须等待进一步的测试。

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.