Location: Virus and Prion Research
2019 Annual Report
Objectives
Objective 1: Investigate the mechanisms of protein misfolding in prion disease, including the genetic determinants of misfolding of the prion protein and the environmental influences on protein misfolding as it relates to prion diseases. Subobjective 1.A: Investigate the differences in the unfolded state of wild-type and disease associated prion proteins to better understand the mechanism of misfolding in genetic prion disease. Subobjective 1.B: Investigate the influence of metal ions on the misfolding of the prion protein in vitro to determine if environmental exposure to metal ions may alter disease progression.
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.
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 and a sensitive live animal screening test for the purpose of determining a herd’s CWD status.
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
All seven project plan milestones for FY19 were fully met. Research efforts directed toward meeting Objective 1 of our project plan center around the production 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. Both bacterial and mammalian expression systems have been established. However, the amount of material produced in the mammalian expression system is inherently too low to fully accomplish all aspects of the proposed work. Due to this we have altered our research plan, but are still able to accomplish the end goal of understanding the influence of metal ions on prion disease using animal derived prions rather than mammalian cell culture produced recombinant prion protein. Due to the long-term nature of Objective 2, the progress status of the objective has not changed. 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. In vitro studies planned in parallel to the animals studies have similarly been initiated and are 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. Optimization of those methods is ongoing.
Accomplishments
1. A method for long-term storage of prion protein was developed. Recent advances in prion disease diagnostic methods include the amplification of the amount of the infectious agent (abnormal prion protein) in a sample. One such technique, known as RT-QuIC, requires a steady supply of freshly purified prion protein which necessitates constant production that is not sustainable in a diagnostic laboratory setting. ARS researchers at Ames, Iowa, developed a method to dry and preserve the prion protein for long-term storage (months or years). This allows for production of the protein in larger quantities, and it can be shipped to diagnostic laboratories facilitating widespread use of RT-QuIC as a diagnostic method.
2. The impact of source genotype on scrapie transmission to elk was evaluated. Scrapie is a fatal disease of sheep that causes damaging changes in the brain. The infectious agent is an abnormal form of the prion protein, a protein normally found in animals. The abnormal or misfolded prion protein can cause disease in sheep of a specific genetics. However, some genetic lines of sheep are resistant to scrapie disease, and some are partially resistant. The RT-QuIC test is an assay that can detect and amplify the presence of abnormal prion protein in a tissue sample. This test can detect the difference between resistant, partially resistant, and susceptible sheep. It can be used in mouse models to evaluate if scrapie can be transmitted to other species, and how efficiently it may be transmitted. ARS researchers at Ames, Iowa, evaluated how different prion protein gene sequences in prion disease affected sheep can transmit in mouse models demonstrating genotypic background of both source and recipient can strongly influence susceptibility. Thus, not only the genotype of the recipient animal but also the genotype of the animal used as a source of the abnormal prion inoculum should be considered in the study of prion transmission.
3. Demonstrated that sheep homozygous for lysine-171 in the prion protein are resistant to classical sheep scrapie. Scrapie is a fatal disease of sheep and goats 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 a sheep will get scrapie is determined by its genetics. A change in the deoxyribonucleic acid (DNA) that codes for the amino acid sequence of the host prion protein can cause the animal to be either resistant or susceptible to the disease. A specific location on this gene, codon 171, seems to play the biggest role in determining disease susceptibility. Sheep with the amino acid arginine (R) at 171 are resistant to scrapie while those with glutamine (Q) at that position are not. It has been suggested that lysine (K) at 171 may behave similarly to R because the two amino acids have a similar structure. ARS researchers at Ames, Iowa, tested whether sheep with one K allele at codon 171 (QK171) or two K alleles at codon 171 (KK171) had a different response to the disease than sheep that were QQ171. The study determined that while QK171 were not resistant to the disease after oronasal exposure, they took longer to develop scrapie than QQ171 sheep. While KK171 sheep were susceptible to the scrapie agent after intracranial inoculation, they were resistant to infection by the oronasal route. This work suggests that selective breeding for the K171 allele in sheep breeds where this allele is represented may help prevent the development of scrapie after natural exposure. This information is useful to sheep farmers and breeders that are selectively breeding animals with genotypes resistant to scrapie. Further, this result is important to regulators with roles in designing programs to enhance genetic resistance to scrapie.
4. Demonstrated that raccoons do not accumulate scrapie isoform of the prion protein (PrPSc) following inoculation with atypical scrapie. The prion diseases are fatal diseases of animals and humans that cause damaging changes in the brain. Animal prion diseases include scrapie in sheep, chronic wasting disease (CWD) in cervids, and transmissible mink encephalopathy (TME) in ranch-raised mink. The infectious agent is an abnormal protein called a prion that has misfolded from its normal state. This study tested whether raccoons develop clinical disease and/or accumulate abnormal prion protein after inoculation with prion agents from different species: TME from cattle, raccoons, or hamsters that occurs in two forms with distinct clinical signs and molecular properties called hyper and drowsy; CWD from white-tailed deer or elk; and atypical (Nor-98) scrapie from sheep. All raccoons inoculated with TME from raccoons or cattle developed clinical disease with short survival times. Raccoons inoculated with CWD from white-tailed deer, CWD from elk, or ‘hyper’ TME from hamsters did not develop clinical disease, but abnormal prion protein was detected in the brains of 25 percent of the raccoons in each study. The amount of abnormal prion protein in the brains of these raccoons was much less than in the brains of raccoons inoculated with TME from raccoons or cattle. None of the raccoons inoculated with ‘drowsy’ TME from hamsters or atypical scrapie from sheep developed clinical disease or detectable abnormal prion protein. ARS researchers at Ames, Iowa, work suggests that raccoons are susceptible to prion disease isolates from raccoons, cattle, white-tailed deer, and elk. Raccoons are omnivores that have a widespread geographical distribution and are known to scavenge animal carcasses. Therefore, they could provide a route of transmission of prions disease between farmed and wild animal species. This information is useful to farmers and people involved in control of prion disease in free-ranging animals.
Review Publications
Mammadova, N., Summers, C.M., Kokemuller, R.D., He, Q., Ding, S., Baron, T., Yu, C., Valentine, R.J., Sakaguchi, D.S., Kanthasamy, A.G., Greenlee, J.J., West Greenlee, M.H. 2019. Accelerated accumulation of retinal alpha-synuclein (pSer129) and tau, neuroinflammation, and autophagic dysregulation in a seeded mouse model of Parkinson's disease. Neurobiology of Disease. 121:1-16. https://doi.org/10.1016/j.nbd.2018.09.013.
Hwang, S., Greenlee, J.J., Vance, N.M., Nicholson, E.M. 2018. Source genotype influence on cross species transmission of transmissible spongiform encephalopathies evaluated by RT-QuIC. PLoS One. 13(12):e0209106. https://doi.org/10.1371/journal.pone.0209106.
Hwang, S., Tatum, T., Lebepe-Mazur, S., Nicholson, E.M. 2018. Preparation of lyophilized recombinant prion protein for TSE diagnosis by RT-QuIC. BMC Research Notes. 11:895. https://doi.org/10.1186/s13104-018-3982-5.
