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

National Institute Of Allergy And Infectious Diseases

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Abstract

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. ===