Location: Produce Safety and Microbiology Research
2025 Annual Report
Objectives
Objective 1: Develop mass spectrometry, immunological, and in vitro prion amplification techniques to detect, structurally define, and distinguish among CWD strains in order to predict their ability to transmit to new animal species- Develop a laboratory test that can be certified as an official method for the USDA CWD Herd Certification Program that is sensitive, CWD-specific, repeatable, reproducible, cost-effective, and can detect CWD in easy to collect samples (e.g., oral fluids, feces, blood, skin) from cervids.
Sub-objective 1.A: Develop mass spectrometry-based methods to improve detection of CWD prions and distinguish among prion strains.
Sub-objective 1.B: Detect covalent modification of prions by Western blot.
Sub-objective 1.C: Improve detection of CWD prions using prion amplification methods and glycosylated recombinant PrP (grPrP).
Objective 2: Develop rapid immunoassays and molecular diagnostic methods for early detection of emerging pathogens-Develop diagnostic tests that can be registered with the USDA-APHIS Center for Veterinary Biologics that is sensitive, specific, reproducible, and cost-effective to detect emerging animal pathogens in easy to collect samples (e.g., oral fluids, feces, blood, skin).
Sub-objective 2.A: Generate monoclonal antibodies (mAbs) against SARS-CoV-2 and SVA antigens to develop immunoassays used for diagnostic detection of viral infection in farm animals.
Sub-objective 2.B: Develop lateral flow and colorimetric assays integrated with highly specific aptamers for rapid detection of SARS-CoV-2, senecavirus A (SVA), and influenza A virus (IAV-S, H1N1) in farm animals.
Approach
The approach will address the development of rapid antemortem tests for the early detection of transmissible spongiform encephalopathies and other animal diseases such as SARS-CoV-2, senecavirus A (SVA), and influenza A virus (IAV-S, H1N1). Objective 1 will develop mass spectroscopy, immunological, and in vitro prion amplification techniques to detect, structurally define, and distinguish CWD strains. Objective 2 will develop pen-side/point-of-care/pre-clinical diagnostic methods involving immunological and non-immunological-based tools targeting emerging and re-emerging viral pathogens, specifically SARS-CoV-2, SVA, and IAV-S (H1N1). Under Objective 1, mass spectrometry-based methods will be developed to improve the detection of CWD prions and distinguish among prion strains by conformation-dependent differences of amino acids. In addition, Western blot will be utilized to detect any covalent modifications present in specific amino groups of lysines present in CWD prions. Prion amplification methods by real-time quaking-induced conversion (RT-QuIC) and glycosylated recombinant prion proteins (grPrP) will also be used to improve detection of CWD prions. Under Objective 2, monoclonal antibodies will be generated against SARS-CoV-2 and SVA antigens, while highly-specific aptamers will be generated via systematic evolution of ligands by exponential enrichment (SELEX) to target SARS-CoV-2, SVA, and IAV-S (H1N1). These recognition elements will be integrated into pen-side diagnostic tools, mainly lateral flow assay (LFA) and gold nanoparticles detection platforms, and ultimately directly applied on animal and environmental samples.
Progress Report
This report documents FY 2025 progress for project 2030-32000-011-000D, “Rapid Antemortem Tests for the Early Detection of Transmissible Spongiform Encephalopathies and Other Animal Diseases”, which began in March 2022.
In support of Sub-objective 1.A, ARS researchers in Albany, California, in collaboration with ARS researchers in Ames, Iowa, used mass spectrometry to quantify the proportion of the polymorphisms at position 132 [leucine (L) or methionine (M)] in different elk chronic wasting disease (CWD) strains. Elk can be infected with two CWD strains, one composed entirely of M/M or M/L at position 132 and another found in elk with only L/L at position 132. This mass spectrometry-based analysis showed that CWD from elk containing M/L at position 132 contained about 40 percent of the L polymorphism. These results indicate that while the 132L can refold into two different strains depending on the prion template, 132M determines the strain.
For Sub-objective 1.B, ARS researchers developed a Western blot-based approach to detecting prion strains. Oxidation with hydrogen peroxide yields prion strain-dependent differences. Cyanogen bromide cleaves unoxidized methionines but not oxidized methionines. Monoclonal antibodies that bind to portions of the prion protein without methionines can be used to analyze the reaction mixtures. In this way, the extent of methionine oxidation can be analyzed using a more accessible Western blot-based analysis as a complement to the mass spectrometry-based approach.
Under Sub-objective 1.C, ARS researchers have an ongoing collaboration with scientists at the State University of New York, Albany, New York. The researchers recently completed the chemical synthesis of bank vole prion protein. ARS scientists in Albany, California, prepared new bank vole prion proteins that are produced by overexpression in cultivated bacteria. These proteins incorporate unnatural amino acids at positions that are suitable for chemical glycosylation. High incorporation rates were verified by mass spectrometry. ARS researchers are working to incorporate sugars into the synthetic and/or overexpressed proteins to produce useful quantities of the desired glycosylated recombinant PrP.
In support of Sub-objective 2.A, novel anti-SARS-CoV-2 monoclonal antibodies were developed into sensitive and selective immunoassays for the detection of the SARS-CoV-2 virus. A sandwich immunoassay format using a complementary pair of novel anti-SARS-CoV-2 antibodies directed against the viral nucleocapsid protein detects all the major SARS-CoV-2 viral variants. These immunoassays were developed to be performed using standard laboratory practices and pen-side methods with visual color reporting of test results.
For Sub-objective 2.B, research has continued on the development and optimization of aptamer-based lateral flow assay (LFA) for the early detection of emerging pathogens [SARS-CoV-2 variants of concern (VOCs): Alpha, Delta, and Omicron variants, Senecavirus A or SVA, and influenza virus A] that cause animal diseases. Three sets of in-house SARS-CoV-2 VOC aptamers were generated and characterized using in silico approaches. The binding affinity analysis showed excellent binding data. Furthermore, a bioluminescent system (NanoBiT) was used to quantitatively evaluate the early-stage viral infection dynamics via the spike protein of a SARS-CoV-2 pseudovirus and human angiotensin-converting enzyme II (hACE2) receptor. Similar to the published aptamers that were initially used in this project, the in-house aptamers (Alpha Apt, Delta Apt, and Omicron Apt) have been used and incorporated onto LFA strips, which were then tested against recombinant proteins and VOCs (inactive). The results showed noticeable LFA signals (two bands – Control and Test Lines); however, additional optimization steps will be conducted to intensify the band signals and enhance stability and sensitivity by concentrating target samples using magnetic beads. After identifying the optimum conditions, LFA strips will be continuously shared with collaborators to test environmental and clinical samples. Future efforts would also include incorporating SVA and influenza virus A in-house aptamers onto LFA and evaluating their specificity and sensitivity.
Accomplishments
1. Sensitive tests detect SARS-CoV-2 viral variants. SARS-CoV-2 virus and its variants, which cause COVID-19 in livestock, remain an evolving threat to animal health and the agricultural supply chain. Testing of animals and other related environmental samples for potential COVID-19 infection is vital to mitigate the spread among animals, slow down the spread, and prevent the emergence of new variants. Rapid and reliable SARS-CoV-2 detection methods are necessary to protect our animal health and ensure food security. ARS researchers at Albany, California, have developed simple and inexpensive portable technologies (1) immunoassay-based and (2) aptamer-based that can be used to facilitate the detection of SARS-CoV-2 viral variants. Both antibodies and aptamers were developed, characterized, and integrated onto lateral flow assay (LFA) strips and microplate format. In addition, a pseudovirus, which is a non-infectious version of SARS-CoV-2, was also constructed to study early-stage viral infection dynamics. These technologies provide improved tools in terms of sensitivity and stability at a lower cost for stakeholders to protect animals against the SARS-CoV-2 virus.
2. A new method for identifying strains of sheep scrapie. Should a new strain of scrapie emerge, it has the potential to adversely impact the 4-billion-dollar American sheep industry that employs nearly 15,000 rural Americans. ARS researchers in Albany, California, used a mass spectrometer to map the surface of the sheep prion protein at six points. The results from the shape of the normal protein were consistent with its known structure. The shape of the scrapie prion differed from that of the normal protein. This method is simpler than existing methods. This approach defines scrapie strains by their shape, thereby allowing stakeholders to determine the origin of a scrapie strain and implement appropriate mitigation strategies.
3. Characterizing the prions involved in chronic wasting disease (CWD) of elk. Chronic wasting disease, a fatal degenerative condition in deer, is caused by a misfolded protein, called a prion. To determine how small differences in the amino acid sequence that comprise this protein impacts animal susceptibility, ARS researchers in Albany, California, developed a mass spectrometry method CWD strain elk by expressing both amino acids leucine and methionine at a certain position of the protein. This analysis showed that the CWD strain from heterozygous elk contained 60% methionine and 40% leucine at that position. It also had the same properties/pathology as CWD from homozygous elk, which only expresses methionine at that position. In contrast, the CWD from homozygous elk expressing only the leucine at that position has different properties, meaning it is a different strain of CWD. These results indicate that methionine determines CWD strain properties and that heterozygous elk do not facilitate the formation of new CWD strains. The information benefits the public by establishing approaches to protecting Americans from potentially problematic CWD strains.
Review Publications
Quintela, I.A., Vasse, T., Jian, D., Harrington, C., Sien, W., Wu, V.C. 2025. Elucidating the molecular docking and binding dynamics of aptamers with spike proteins across SARS-CoV-2 variants of concern. Frontiers in Microbiology. 16. Article 1503890. https://doi.org/10.3389/fmicb.2025.1503890.
Silva, C.J., Erickson-Beltran, M.L., Cassmann, E.D., Greenlee, J.J. 2024. Quantifying the molecular properties of the elk chronic wasting disease agent with mass spectrometry. Pathogens. 13(11). Article 1008. https://doi.org/10.3390/pathogens13111008.
Silva, C.J., Erickson-Beltran, M.L., Requena, J.R. 2025. Comparing the extent of methionine oxidation in the prion and native conformations of PrP. ACS Omega. 10(1):1320-1330. https://doi.org/10.1021/acsomega.4c08892.
Hnasko, R.M., Lin, A.V., McGarvey, J.A., Jackson, E.S. 2025. Immunoassay detection of SARS-CoV-2 using monoclonal antibody binding to viral nucleocapsid protein. Microbial Biotechnology. 18(2). Article e70117. https://doi.org/10.1111/1751-7915.70117.
Lin, M., Lin, C., Chiang, H., Quintela, I.A., Wu, V.C., Lin, C. 2025. Using nano-luciferase binary (NanoBiT) technology to assess the interaction between viral spike protein and angiotensin-converting enzyme II by aptamers. BioTech. 14(1). Article 20. https://doi.org/10.3390/biotech14010020.