Study of CWD Deer and Elk Prion Disease in Nonhuman Primates and transgenic mice

为了研究慢性消耗性疾病(CWD)跨物种传播的可能性，用感染CWD的鹿或麋鹿的脑组织材料经口或脑内感染了两种非人类灵长类动物--松猴和食蟹猴。所有的松鼠猴子都出现了临床神经症状，并经脑部生化和病理测试证实患有普恩病毒病。 相比之下，在食蟹猴身上进行的实验，通过这两种接种途径都不容易感染CWD，因为食蟹猴在基因上比松鼠猴子更接近人类。为了测试猕猴的亚临床感染水平，我们用几种方法对脑、脊柱和淋巴组织进行了Prion感染的证据筛查，包括IHC检测Prion蛋白，H&E检测神经病理学，免疫印迹法(检测疾病相关的Prion蛋白)。 尽管我们之前的研究没有令人信服的证据表明CWD感染食蟹猴，但偶尔注射CWD的老年动物在大脑中显示出不寻常的PrP染色区域。因此，在18财年，我们还利用免疫组织化学方法筛选了额外的年龄匹配的未感染食蟹猴，以检测PrP异常。在这些研究中，注射CWD的猕猴和未注射CWD的猕猴体内PrP染色的几种异常形式似乎是相似的，因此表明这些类型的PrP染色与与年龄相关的PrP变化有关，而不是CWD感染的证据。我们还使用了新开发的RT-QuIC检测PrP淀粉样蛋白的种子，该测试已经与人类和许多动物模型中传染性普恩的存在相关。未注射或注射CWD的食蟹猴RT-QuIC检测均为阳性。因此，在我们的研究中，13年后，在临床上或通过使用几种灵敏的普恩病毒疾病筛查试验，没有发现CWD传播给猕猴的证据。 我们的数据显示，两种非人类灵长类动物对CWD的易感性因物种而异。基于人类与食蟹猴之间更紧密的遗传关系，以及之前的工作表明，猕猴与人类具有相似的Pron疾病易感性模式，这些数据表明，人类可能也对CWD感染具有抵抗力。 在FY19-22财年，我们测试了CWD通过脑内接种在过表达人类Pron蛋白的转基因小鼠(tg66和tgrm)中的传播。用传统的Prion检测方法筛选的小鼠为阴性。然而，在22-23财年，在用超灵敏RT-QuIC试验筛选的88只小鼠中，我们发现4只Tg66小鼠产生了不一致的RT-QuIC阳性反应。这些数据可能是假阳性反应、残留输入疫苗或暗示CWD跨物种传播到人类的亚临床感染的指示。在这个模型中，还需要进行额外的实验来了解普里恩播种活动的性质。在这份手稿中，第二次传代实验使用了来自普恩种子活性较弱的小鼠的大脑，结果表明，它们不会感染额外的受体tg66小鼠。清除实验表明，在不能复制PrPKO蛋白的tg66小鼠和PrPKO小鼠中，输入CWD的Prion种子活性在180天后被消除，表明在以后的时间点检测到的种子活性的弱阳性水平不太可能是残余接种。在连续两代后，CWD蛋白在tg66中未能致病，这表明一种强大的物种屏障阻止了CWD感染表达人Prion蛋白的小鼠。这些结果巩固了我们早先的结论，即人类不太可能容易患鹿角型CWD。 ==

To study the possibility of cross-species transmission of Chronic Wasting Disease (CWD), two species of nonhuman primates, squirrel monkeys and cynomolgus macaques, were infected orally or intracerebrally with brain material derived from CWD-affected deer or elk. All of the squirrel monkeys developed clinical neurological signs and were confirmed by biochemical and pathological testing of brain to have a prion disease. In contrast, experiments done in cynomolgus macaques, which are genetically closer to humans than are squirrel monkeys, were not susceptible to CWD by either route of inoculation. To test for a subclinical level of infection in the macaques we screened brain, spinal column and lymphoid tissues for evidence prion infection by several methods including IHC for prion protein, H&E for neuropathology, immunoblot (for disease associated prion protein). Although our previous studies showed no convincing evidence of CWD infection of cynomolgus macaques, occasional older CWD-injected animals showed unusual areas of PrP staining in brain. Therefore, in FY18, we also screened additional age-matched uninfected cynomolgus macaques using immunohistochemistry for detection of abnormal PrP. In these studies, several abnormal forms of PrP staining appeared to be similar in both CWD-injected and uninjected macaques, thus indicating that these types of PrP staining were related to PrP changes associated with aging and were not evidence for CWD infection. We also used the newly developed RT-QuIC test for PrP amyloid seeding which has been correlated with the presence of infectious prions in humans and numerous animal models. None of the uninjected or CWD-injected cynomolgus macaques were positive by RT-QuIC testing. Thus, after 13 years, no evidence for CWD transmission to macaques was detected clinically or by using several sensitive prion disease-screening assays in our studies. Our data showed strong species-specific differences in susceptibility of two species of non-human primates to CWD. Based on the closer genetic relationship between humans and cynomolgus macaques, and previous work demonstrating that macaques have a similar prion disease susceptibility pattern to humans, these data suggest that humans may also be resistant to CWD infection. In FY19-22 we tested CWD transmission via intracerebral inoculation into transgenic mice (tg66 and tgRM) that over-expressed human prion protein. Mice screened by traditional prion detection assays were negative. However, in FY22-23, in a group of 88 mice screened by the ultrasensitive RT-QuIC assay, we identified 4 tg66 mice that produced inconsistent positive RT-QuIC reactions. These data could be false positive reactions, residual input inoculum or indicative of subclinical infections suggestive of cross species transmission of CWD to humans. Additional experiments were required to understand the nature of the prion seeding activity in this model. In this manuscript, second passage experiments using brains from mice with weak prion seeding activity showed they were not infectious to additional recipient tg66 mice. Clearance experiments showed that input CWD prion seeding activity was eliminated by 180 days in tg66 mice and PrPKO mice, which are unable to replicate prion protein, indicating that the weak positive levels of seeding activity detected at later time points was not likely residual inoculum. The failure of CWD prions to cause disease in tg66 after two sequential passages suggested that a strong species barrier prevented CWD infection of mice expressing human prion protein. These results reinforced our earlier conclusions that humans are unlikely to be susceptible to cervid CWD. ===