Location: Virus and Prion Research
2020 Annual Report
Accomplishments
1. Differing genetic backgrounds of prion disease sources do not affect likelihood of disease progression. Prion diseases are fatal neurodegenerative diseases that affect a wide range of livestock and wildlife. The disease process occurs through the misfolding of a normally occurring protein. A recently developed approach for the detection of this misfolded protein uses a technique referred to as Real-time quaking induced conversion (RT-QulC). RT-QulC amplifies the amount of misfolded protein for detection and has been used to differentiate prion diseases through differences in the rate of misfolding. ARS scientists in Ames, Iowa, completed a study comparing the relative rates of misfolding in RT-QulC from different sources of chronic wasting disease using both human and bank vole prion protein as the substrate for amplification. Regardless of the chronic wasting disease source and genotype, the bank vole substrate was equivalently sensitive indicating the utility of this substrate for the detection of CWD regardless of host or genotype. Similarly, with human prion protein substrate no differences were found indicating that at the level of conversion of the human prion protein to the fibril form conformation all tested CWD isolates are equivalent. From a diagnostic perspective this further justifies the use of bank vole prion protein as a universal substrate and indicates that regardless of genotype of the CWD source the risk to humans is likely the same. Both results provide important information for knowledge based regulatory decisions by state and federal agencies.
2. Developed an improved enrichment method for amplification-based prion detection. Prion diseases are fatal neurodegenerative diseases that affect a wide range of livestock and wildlife. The disease process occurs through the misfolding of a normally occurring protein. Detection of this misfolded protein is the only known means by which a prion disease can be diagnosed. A recently developed approach for the detection of this misfolded protein uses a technique referred to as Real-time quaking induced conversion (RT-QulC). RT-QulC amplifies the amount of misfolded protein available for detection but can be inhibited by naturally occurring contaminants in the tissue samples used for the technique. ARS scientists in Ames, Iowa, developed a method to clean the samples prior to analysis and demonstrated the sample cleanup technique enhanced the detection and reduced the time required to assess the results providing additional tools for diagnostic laboratories.
3. Improved understanding of cross-species transmission of the prion disease transmissible mink encephalopathy. This work specifically evaluated susceptibility of sheep to bovine adapted transmissible mink encephalopathy (TME) and evaluated pathobiology of disease. Transmissible spongiform encephalopathies (TSEs), or prion diseases, are fatal brain diseases that affect livestock species. Prion diseases have been shown to jump species as exhibited when classical bovine spongiform encephalitis (BSE) infected cattle products were consumed by humans resulting in variant Creutzfeldt-Jakob disease. Another example of cross-species transmission results in a disease of farmed mink known as TME. The present study was designed to determine the effect of cross-species transmission of TSEs in livestock on the ability to infect mice expressing the cattle prion protein. ARS scientists in Ames, Iowa, found that passing cattle adapted TME prions from cattle to sheep changed the ability of the prions to infect mice. These results were compared to atypical BSE (L-BSE type) and Classical BSE (C-BSE type). Depending on the genotype of sheep used, the disease in mice appeared similar to either L-BSE or C-BSE. These results indicate a shift in the disease outcome based on transmission through sheep with different genotypes. This information gives insight into the genesis of new prion strains. It also supports the hypothesis that TME can originate from feeding mink protein from cattle afflicted with L-BSE providing evidence to support regulatory decisions for non-food animal species.
4. Discovery of a novel strain of chronic wasting disease is present in experimentally inoculated LL132 elk. Chronic wasting disease (CWD) is a fatal disease of deer and elk that causes damaging changes in the brain. The infectious agent is an abnormal protein called a prion that has misfolded from its normal state. Whether or not an elk will get CWD is affected by their genetics. This study evaluated transmission of abnormal prion protein from elk of 3 different genotypes that were infected with CWD to transgenic mice expressing the elk prion protein. Previous work by ARS scientists in Ames, Iowa, demonstrated that there are differences in incubation periods, patterns of abnormal prion accumulation in the brain, and fibril stability features in these different genotypes of elk. This study demonstrates that elk donor genotype-associated differences in relative incubation periods, fibril stability, and lesions in the brain were maintained across first and second passages to mice suggesting that they are different CWD strains that may require different approaches for prevention and eradication. This information is useful to wildlife managers and captive wildlife owners that are selectively breeding animals and could impact future regulations for the control of CWD in the U.S.
5. Development of in vitro modelling system for chronic wasting disease. Prion diseases are invariably fatal neurologic diseases for which there is no known prevention or cure. Because of long incubation times and knowledge gaps in how the disease progresses, there is not a well-defined model for testing potential cures or preventative measures. ARS scientists in Ames, Iowa, developed an ultrasensitive in vitro modeling system for chronic wasting disease (CWD) infectivity of samples from deer or elk. In the first step, prion agents are cultured on a brain slice derived from a prion-susceptible mouse. In the second step, an in vitro prion amplification technique (Real-Time Quaking Induced Conversion or RT-QuIC) is used to test for infectivity of the slices. This work demonstrated that the slice cultures are able to accumulate CWD prions that could be detected by RT-QuIC and more traditional laboratory methods, such as mouse bioassay and immunohistochemistry. In addition, three compounds with potential anti-prion properties were screened using slice culture and RT-QuIC indicating that this model may be useful in developing potential treatment schemes for prion disease. Because mechanisms of neurodegeneration in prion disease are similar to other protein misfolding diseases such as Alzheimer’s disease and Parkinson’s disease, use of this model could have a major impact on improving treatments for other neurodegenerative diseases.
Review Publications
Michael, A.V., Greenlee, J.J., Harm, T.A., Moore, S.J., Zhang, M., Lind, M.S., West Greenlee, M.H., Smith, J.D. 2019. In-situ temporospatial characterization of the glial response to prion infection. Veterinary Pathology. 57(1):90-107. https://doi.org/10.1177/0300985819861708.
Cassmann, E.D., Moore, S.J., Smith, J.D., Greenlee, J.J. 2019. Sheep are susceptible to the agent of TME by intracranial inoculation and have evidence of infectivity in lymphoid tissues. Frontiers in Veterinary Science. 6:430. https://doi.org/10.3389/fvets.2019.00430.
Hwang, S., Greenlee, J.J., Nicholson, E.M. 2020. Role of donor genotype in RT-QuIC seeding activity of chronic wasting disease prions using human and bank vole substrates. Scientific Reports. 15(1):e0227487. https://doi.org/10.1371/journal.pone.0227487.
Mammadova, N., West Greenlee, M.H., Moore, S.J., Sakaguchi, D.S., Greenlee, J.J. 2020. Experimental study using multiple strains of prion disease in cattle reveals an inverse relationship between incubation time and misfolded prion accumulation, neuroinflammation and autophagy. Scientific Reports. 190(7):1461-1473. https://doi.org/10.1016/j.ajpath.2020.03.006.
Hwang, S., Dassanayake, R.P., Nicholson, E.M. 2019. PAD-bead enrichment enhances detection of PrPSc using real-time quaking-induced conversion. Bioscience Reports. 12:806. https://doi.org/10.1186/s13104-019-4842-7.
