Location: Virus and Prion Research
2021 Annual Report
Objectives
Objective 1: Investigate the mechanisms of protein misfolding in prion disease, including the genetic determinants of misfolding of the prion protein, environmental influences on abnormal prion conversion, and environmental influences on protein misfolding as it relates to prion diseases.
Objective 2: Investigate the pathobiology of prion strains in natural hosts, including the influence of prion source genotype on interspecies transmission and the pathobiology of atypical transmissible spongiform encephalopathies (TSEs) and the presence of CWD prion strains in natural hosts by processing field samples through a strain identification program. Subobjective 2.A: Investigate the pathobiology of atypical TSEs. Subobjective 2.B: Investigate the influence of prion source genotype on interspecies transmission.
Objective 3: Investigate sampling methodologies for antemortem detection of prion disease, including the utility of blood sampling as a means to assess prion disease status of affected animals and the utility of environmental sampling for monitoring herd prion disease status. Subobjective 3.A: Investigate the utility of blood sampling as a means to assess prion disease status of affected animals. Subobjective 3.B: Investigate the utility of environmental sampling for monitoring herd prion disease status.
Objective 4: Determine the association of disease susceptibility or resistance with naturally occurring prion protein genotypes not yet associated with positive cases on infected premises, including genotype associated differences in prion accumulation and excretion, and develop a logic-based decision tree for CWD strain determination.
Objective 5: Develop improved live animal test for the detection of CWD-affected cervids, including a sensitive live animal test to detect CWD prions in individual animals, a sensitive live animal screening test for the purpose of determining a herd’s CWD status, and a sensitive deployable CWD test for use by State diagnostic labs.
Approach
The studies will focus on three animal transmissible spongiform encephalopathy (TSE) agents found in the United States: bovine spongiform encephalopathy (BSE); scrapie of sheep and goats; and chronic wasting disease (CWD) of deer, elk, and moose. The research will address sites of protein folding and misfolding as it relates to prion disease, accumulation of misfolded protein in the host, routes of infection, and ante mortem diagnostics with an emphasis on controlled conditions and natural routes of infection. Techniques used will include spectroscopic monitoring of protein folding/misfolding, clinical exams, histopathology, immunohistochemistry, and biochemical analysis of proteins. The enhanced knowledge gained from this work will help understand the underlying mechanisms of prion disease and mitigate the potential for unrecognized epidemic expansions of these diseases in populations of animals that could either directly or indirectly affect food animals.
Progress Report
This will be the final report for project 5030-32000-114-00D terminating September 30, 2021. Four of the six project plan milestones for FY20 were fully met with two not met.
Research efforts directed toward meeting Objective 1 of our project plan originally centered around the production and use of recombinant prion protein from either bacteria or mammalian tissue culture systems and collection of data on the folding and misfolding of the recombinant prion protein produced. The amount of expressed protein from mammalian expression systems was inherently too low to fully accomplish all aspects of the proposed work. Due to this the approach used to address the research goals were altered to exclusively utilize bacterially produced protein and we were still fully able to accomplish the end goal of understanding the influence of metal ions on prion disease using an alternative approach. Similarly, by utilizing amino acid substitutions in the bacterially expressed bovine prion protein we were able to address themodynamic influences on genetic prion disease with the results published.
For Objective 2, much of the research extends beyond the five-year project plan cycle, however, all project milestones have been met and the research will continue to allow full reporting in the literature. All studies have been initiated and animals are under observation for the development of clinical signs. The animal studies for this objective are long term and will continue until onset of clinical signs. For some studies the end point is 8 years post-inoculation, while others will extend to the full natural, humane lifetime of the cattle in question or to the point of unequivocal clinical signs of a TSE. In vitro studies planned in parallel to the animal's studies have been completed with data analysis ongoing.
Objective 3 of the project plan focuses on the detection of disease associated prion protein in body fluids and feces collected from a time course study of chronic wasting disease inoculated animals. At this time sample collection is complete and preliminary methods have been established. Detection of prion disease using fecal samples has proven exceedingly successful and a manuscript reporting these results has been published. Blood, the primary bodily fluid under investigation has been shown to be unfeasible from a diagnostic standpoint. This is likely due to inhibitory components found in blood rather than a lack of amplifiable material.
Objectives 4 and 5 were recently added to the project and work on both is still in the preliminary stages with much of the research intended to be conducted by not yet hired category 1 scientists. Collectively this progress leaves the current and future staff well prepared to address the objectives of the next five-year project plan cycle.
Accomplishments
1. Real Time Quaking Induced Conversion (RT-QuIC) to detect CWD using fecal samples and bank vole prion protein substrate. Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy (TSE) that is fatal to free-range and captive cervids. CWD has been reported in the United States, Canada, South Korea, Norway, Finland, and Sweden, and the case numbers in both wild and farmed cervids are increasing rapidly. Studies indicate that lateral transmission of cervids likely occurs through the shedding of infectious prions in saliva, feces, urine, and blood into the environment. Therefore, the detection of CWD early in the incubation time is advantageous for disease management. ARS researchers in Ames, Iowa, developed a method that detects CWD using fecal samples as much as 30 months prior to the onset of clinical signs with all samples tested between 6 and 30 months post-inoculation showing RT-QuIC positivity. The combination of a highly sensitive detection tool paired with a sample type such as feces, which can be collected non-invasively allows a useful tool to support CWD surveillance in wild and captive cervids.
2. Rectal biopsy to identify goats naturally infected with scrapie. Scrapie is a prion disease that can infect sheep and goats, leading to irreversible brain damage and death. While natural scrapie in sheep has been widely described, less is known about naturally acquired goat scrapie. Additionally, animals infected with scrapie are generally not diagnosed until the onset of obvious clinical signs, therefore a reliable method to screen asymptomatic animals may aid in diagnosis. Changes in retinal thickness have been reported in asymptomatic cattle infected with prion disease, and antemortem rectal biopsy has been previously described to be effective in identifying sheep and deer naturally infected with prion disease. ARS scientists in Ames, Iowa, evaluated the efficacy of antemortem assessment of retinal thickness, and analysis of recto-anal mucosa-associated lymphoid tissue (RAMALT) biopsies as a pre-clinical test to detect asymptomatic goats positive for the classical scrapie agent. Our results showed that rectal biopsy was 78% effective in identifying goats naturally infected with scrapie. We also found no detectable changes in retinal thickness over the course of the observation period. Early identification of goats infected with scrapie is important to goat herders and regulators with roles in scrapie diagnosis for the purpose of eradicating scrapie.
3. Early accumulation of misfolded prions in the retina of sheep with atypical scrapie. The reason for this experiment was to better understand the pathology of atypical scrapie in sheep. Atypical scrapie is a prion disease that affects sheep. Unlike classical scrapie, atypical scrapie is thought to occur spontaneously, and it is unlikely to transmit between sheep under natural conditions. In addition, atypical scrapie generally occurs in sheep that are genetically resistant to classical scrapie. Atypical scrapie does not have lymphoid involvement like classical scrapie, so antemortem diagnostics that work for classical scrapie like rectal biopsy of lymphoid tissue are not applicable for the antemortem detection of atypical scrapie. Misfolded prions are predominantly found in the cerebellum for atypical scrapie and not in the brainstem as seen with classical scrapie. Atypical scrapie is also a relevant disease because of its potential association with other prion diseases. Atypical scrapie is thought to be a possible cause of classical scrapie or bovine spongiform encephalopathy (BSE). ARS scientists in Ames, Iowa, investigated the transmission of the atypical scrapie agent to sheep with three different prion protein genotypes. A diagnosis of atypical scrapie was made in all three genotypes of sheep. ARS scientists identified that misfolded prion protein was detected earliest in the cerebellum and the retina. The early accumulation prion protein in the retina of sheep with atypical scrapie was previously unknown. Understanding where misfolded prions accumulate in cases of atypical scrapie can lead to earlier disease detection through new antemortem detection methods such as Optical Coherence Tomography. Furthermore, the materials derived from this experiment will aid in investigating origins of other prion diseases.
4. Passing Transmissible Mink Encephalopathy (TME) prions from cattle to sheep changed the ability of the prions to infect mice. 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 encephalopathy (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 transmissible mink encephalopathy (TME), the origins of which were not previously understood; however, one possible source is L- BSE from cattle. 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. We found that passing cattle adapted TME (TME that was previously passaged in cattle) to sheep changed the ability of the prions to infect bovinized mice in a laboratory inoculation. These results were compared to atypical BSE (L-BSE type) and Classical BSE in bovinized mice. Depending on the genotype of sheep used, the disease in mice appeared similar by histologic and western blot analysis to either L-BSE or C-BSE. These results indicate a shift in the disease presentation based on transmission through sheep with different genotypes. This information gives insight into origins and development of new prion strains. Because disease in one of the groups of mice resembled the L-BSE phenotype, it supports the hypothesis that TME can originate from feeding mink protein from cattle afflicted with L-BSE.
Review Publications
Mammadova, N., Baron, T., Verchere, J., Greenlee, J.J., West Greenlee, M.H. 2021. Retina as a model to study in vivo transmission of alpha-synuclein in the A53T mouse model of Parkinson’s disease. In: Singh S.R., Hoffman R.M., Singh A. (eds) Mouse Genetics. Methods in Molecular Biology. New York, NY: Humana. p.75-85. https://doi.org/10.1007/978-1-0716-1008-4_5.
Mammadova, N., West Greenlee, H.M., Moore, J.S., Hwang, S., Lehmkuhl, A.D., Nicholson, E.M., Greenlee, J.J. 2020. Evaluation of antemortem diagnostic techniques in goats naturally infected with scrapie. Frontiers in Veterinary Science. 7. Article 517862. https://doi.org/10.3389/fvets.2020.517862.
Mammadova, N., Cassman, E., Greenlee, J.J. 2020. Successful transmission of the chronic wasting disease (CWD) agent to white-tailed deer by intravenous blood transfusion. Research in Veterinary Science. 133:304-306. https://doi.org/10.1016/j.rvsc.2020.10.009.
Mammadova, N., Cassmann, E., Greenlee, J.J. 2020. Efficient transmission of US scrapie agent by intralingual route to genetically susceptible sheep with a low dose inoculum. Research in Veterinary Science. 132:217-220. https://doi.org/10.1016/j.rvsc.2020.06.010.
Cassmann, E.D., Moore, Sara, J, S.J., Kokemullar, R.D., Balkema-Buschman, A., Groschup, M., Nicholson, E.M., Greenlee, J.J. 2020. Bovine adapted transmissible mink encephalopathy is similar to L-BSE after passage through sheep with the VRQ/VRQ genotype but not VRQ/ARQ. BMC Veterinary Research. 16. Article 383. https://doi.org/10.1186/s12917-020-02611-0.
Cassmann, E.D., Mammadova, N., Greenlee, J.J. 2020. Autoclave treatment of the classical scrapie agent US No. 13-7 and experimental inoculation to susceptible VRQ/ARQ sheep via the oral route results in decreased transmission efficiency. PLoS ONE. 15(12). Article e0243009. https://doi.org/10.1371/journal.pone.0243009.
Hwang, S., Greenlee, J.J., Nicholson, E.M. 2021. RT-QuIC detection of PrPsc in fecal samples from CWD infected White-tailed Deer using Bank Vole Substrate. Frontiers in Veterinary Science. 8. Article 643754. https://doi.org/10.3389/fvets.2021.643754.
Ding, M., Teruya, K., Zhang, W., Lee, H., Yuan, J., Oguma, A., Camacho, M., Mitchell, M., Greenlee, J.J., Zou, W., Kong, Q., Doh-Ura, K., Cui, L. 2021. Decrease in skin prion-seeding activity of prion-infected mice treated with a compound against human and animal prions: a first possible biomarker for prion therapeutics. Molecular Neurobiology. https://doi.org/10.1007/s12035-021-02418-6.
Cassmann, E.D., Frese, R., Greenlee, J.J. 2021. Second passage of chronic wasting disease of mule deer in sheep compared to classical scrapie after intracranial inoculation. Journal of Veterinary Diagnostic Investigation. 33(4):711-720. https://doi.org/10.1177/10406387211017615.
Mammadova, N., Cassmann, E., Moore, S., Nicholson, E.M., Greenlee, J.J. 2020. Experimental inoculation of CD11c+ B1 lymphocytes, CD68+ macrophages, or platelet-rich plasma from scrapie-infected sheep into susceptible sheep results in variable infectivity. Access Microbiology. 2(9). https://doi.org/10.1099/acmi.0.000155.
Cassmann, E.D., Mammadova, J., Benestad, S., Moore, S., Greenlee, J.J. 2021. Transmission of the atypical/nor98 scrapie agent to suffolk sheep with VRQ/ARQ, ARQ/ARQ, and ARQ/ARR genotypes. PLoS ONE. 16(2). Article e0246503. https://doi.org/10.1371/journal.pone.0246503.
