Research Project: Rapid Antemortem Tests for the Early Detection of Transmissible Spongiform Encephalopathies and Other Animal Diseases

Location: Produce Safety and Microbiology Research

2024 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 progress for project 2030-32000-011-000D, titled, “Rapid Antemortem Tests for the Early Detection of Transmissible Spongiform Encephalopathies and Other Animal Diseases”, which started in March 2022. In support of Sub-objective 1A, ARS scientists in Albany, California, developed a mass spectrometry based method of quantifying lysine acylation in the elk prion protein. ARS scientists in Albany, California, have entered into a collaboration with ARS scientists in Ames, Iowa, to use this method to distinguish among elk prion strains. The polymorphism at position 132 (leucine (L) or methionine (M)) influences the propagation of Chronic Wasting Disease (CWD) prions in elk. Transgenic mice expressing the elk prion protein (M at position 132) were inoculated with CWD from experimentally infected elk (M/M, M/L, or L/L at position 132). Two distinct CWD prions emerged from these experiments. The ARS scientists in Albany, California, acquired samples of brain tissue from these transgenic mice and the progenitor elk CWD prions from scientists at Ames, Iowa. These samples will be acylated to determine if this method can be used to distinguish between the two elk CWD strains. In support of Sub-objective 1B, ARS scientists identified monoclonal antibodies (mAbs) that bind to cervid PrP, but not acylated cervid PrP. One antibody is commercially available; the other is a non-commercial antibody provided by a collaborator. Both are suitable for a Western blot-based analysis. Cyanogen bromide reacts with methionine to cleave a protein at that position. The cleavage does not occur when the methionine is oxidized. Using cyanogen bromide to map the locations of unoxidized methionines is a means of providing an alternative. Western blot-based or SDS-PAGE-based analysis are means of quantifying the extent of methionine oxidation. In support of Sub-objective 1C, ARS scientists established a collaboration with scientists at the State University of New York, Albany, New York, to prepare synthetic glycosylated bank vole prion protein. The bank vole recombinant prion protein (PrP) has been shown to propagate the known strains of chronic wasting disease (CWD). The protein will be synthesized in two parts: a non-glycosylated part consisting of amino acids 22-157 and a glycosylated part comprising the remainder of the protein (amino acids 158-210). We are working to complete the synthesis and ensure that the protein is properly folded. The resulting protein will be the first synthetic glycosylated recombinant PrP. In support of Sub-objective 2A, research characterizing the biochemical properties of monoclonal antibodies generated against SARS-CoV-2 nucleocapsid protein and Senecavirus A VP2 protein has been completed. This includes the functionalization of antibodies with molecular reporters, evaluation of antibody binding specificity to biosimilar proteins, binding inactivated or intact viral complex, and the identification of suitable antibody pairs necessary for the construction of sandwich-type enzyme-linked immunosorbent assays and lateral flow immunoassays. In support of Sub-objective 2B, 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 concerns (VOCs): Alpha, Delta, and Omicron variants, Senecavirus A or SVA, and influenza virus A – H1N1 in swine] that cause animal diseases. Target viral recombinant proteins were used as baits to generate highly specific in-house aptamers through SELEX (Systematic Evolution of Ligands by Exponential Enrichment). As an iterative process, approximately eight rounds of SELEX can narrow down a random pool of DNA library to highly specific aptamer sequences that possess excellent binding affinity to the target viral proteins. For SARS-CoV-2 VOC (Delta and Omicron), three sets of in-house aptamers (different from the referenced ones) were generated, screened, and identified by the next-generation sequencing (NGS) approach. These sequences were further analyzed using molecular docking modeling and structural analysis software. The binding affinity analysis (in-silico) showed excellent binding data. To fully understand its binding capabilities, these aptamers will be applied on the Aptamer-Linked Immobilized Sorbent Assay (ALISA) microplate to test recombinant proteins and inactivated viruses. The ALISA results are expected to show an increasing signal pattern when the concentration of target samples is tested. Future efforts would also include incorporating these aptamers onto LFA and evaluating its specificity and sensitivity against active Delta and Omicron VOCs. Published aptamers were also used and incorporated onto LFA strips, which were then tested against recombinant proteins and VOCs (inactive and active) in clean samples (buffer). Preliminary results showed noticeable LFA signals (two bands – Control and Test Lines). Specifically, when Delta recombinant proteins were tested, a preliminary working range of 0.0008 nmol – 0.5 nmol was successfully established with a sensitivity around 0.004-0.02 nmol. More optimization steps will be conducted to intensify the band signals, such as 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.

Accomplishments
1. Successful detection of SARS-CoV-2 recombinant proteins and active viruses in LFA. SARS-CoV-2 variants are still persistent in wild animals. Testing of animals and other related environmental samples for potential COVID-19 infection is vital in mitigating the spread among animals, slowing down, and preventing the emergence of new variants. ARS researchers in Albany, California, have continuously collaborated with stakeholders to develop an aptamer-based lateral flow assay (LFA) for pen-side setting use and portable colorimetric microplate assays. In-house aptamers for all SARS-CoV-2 variants were generated. Molecular simulation modeling analysis showed aptamers have a high affinity towards spike proteins and could act as the main detection component. Universal aptamers from published literature targeting all variants were integrated onto both LFA strips and microplates and could detect recombinant proteins and active SARS-CoV-2 viruses. The aptamer-based LFA and microplate assay can assist USDA APHIS, veterinarians, farmers, and other regulatory agencies in testing various animal and environmental samples suspected of SARS-CoV-2 infections.

2. Methionine sulfoxide can map the surface of prion protein and sheep scrapie. Distinguishing prion protein conformations is essential to detect and distinguish among prion strains. ARS researchers in Albany, California, developed a mass spectrometry method to quantify the extent of methionine oxidation in the six methionines present in the sheep prion protein. A methionine’s surface exposure determines the extent of its reactivity with hydrogen peroxide. This approach is a means of determining the shape of a protein by quantifying the surface exposure of its methionines. This information can be used by stakeholders and other researchers to distinguish between the normal cellular sheep prion protein and a scrapie prion.