Progress 10/01/23 to 09/30/24
Outputs
PROGRESS REPORT Objectives (from AD-416): 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 (from AD-416): 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. 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. Artificial Intelligence (AI)/Machine Learning (ML) Convolutional Neural Network (CNN), rapid CNN (RCNN), and transfer learning were used to identify the tule elk (Cervus canadensis nannodes) in aerial images of Tomales Point Tule Elk Reserve (Point Reyes National Seashore). The resulting model was used to identify the tule elk in aerial images from the greater Point Reyes National Seashore. The model correctly identified tule elk with high precision (>90%). These results suggest that this software can be used to identify cervids from aerial surveillance images. Machine learning and artificial intelligence were used to analyze publicly available spatial and temporal data from radio-collared elk in a CWD endemic area of the United States and red deer in a CWD free area of Norway. A linear mixed model (LMM), autoencoder, and custom loss function were used to detect anomalous behaviors in the two data sets. Seven of the elk showed significantly anomalous movements relative to their herd mates. No significantly anomalous movement was observed in the Norwegian red deer. These results suggest that spatial and temporal data from radio- collared cervids may be used to detect the presence of CWD-infected animals. ACCOMPLISHMENTS 01 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. 02 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.
Impacts
(N/A)
Publications
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Progress 10/01/22 to 09/30/23
Outputs
PROGRESS REPORT Objectives (from AD-416): 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 (from AD-416): 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. In support of Sub-objective 1A, ARS scientists in Albany, California, developed a method to quantify the methionines present in the known variants of the cervid prion protein. In addition, ARS scientists (Albany, California, and Ames, Iowa) developed a method to quantify the proportion of the lysine polymorphism at position 171 in scrapie-infected heterozygous sheep expressing glutamine (Q) and lysine (K) at position 171. Homozygous sheep expressing the lysine polymorphism at position 171 are resistant to scrapie by oral inoculation. Heterozygous animals (171 Q/ K) are partially resistant to scrapie by oral dosing. ARS scientists established a collaboration with the Animal & Plant Health Inspection Service (APHIS) to acquire samples of obex (brain tissue) and retropharyngeal lymph nodes (RLNs) from wild Wisconsin CWD-infected white- tailed deer. These samples will be used to map the surface of CWD prions from these animals. In support of Sub-objective 1B, ARS scientists have prepared plasmids suitable for an Escherichia coli (E. coli)-based overexpression of white- tailed deer prion protein and both polymorphisms (132L and 132M) of elk prion protein. These plasmids were used to produce recombinant proteins, which were isolated and then reacted with synthetic acylating reagents (which react with the e-amino group of lysine). The reacted proteins were digested with LysC (a protease that selectively cleaves lysine but not lysine with an acylated e-amino group) and analyzed by SDS-PAGE-based analysis. These results suggest that commercially available monoclonal antibodies (mAbs) that bind to epitopes within the PrP protein can be used to identify covalent modifications by detecting changes in the size of protein fragments after reaction with acylating reagents and digestion with LysC. In support of Sub-objective 1C, ARS scientists have synthesized bank vole prion protein ("universal prion acceptor") encoding genes containing the amber stop codon in place of those codons encoding asparagines which are glycosylated in the mature prion protein. These genes are suitable for being repurposed, via synthetic biology, to incorporate 4-Acetyl-L- phenylalanine, which can be glycosylated by "click" chemistry. The codons in the three bank vole genes (one site, the other site, and both sites of glycosylation) were optimized for expression in E. coli. Clones (E. coli, BL21) engineered not to use the amber stop codon were transformed with plasmid encoding the tRNA and tRNA transferase necessary to incorporate 4- Acetyl-L-phenylalanine into the over-expressed synthetic protein. Production of the synthetic proteins, followed by their glycosylation, will yield synthetic glycosylated recombinant PrP. In support of Sub-objective 2A, research continues toward the generation and selection of novel hybridoma cell lines that produce specific monoclonal antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Seneca Valley virus (SVA) antigens. To date, we have identified, selected, and immortalized antibody-producing hybridoma cell lines that recognize and bind epitopes in SARS-CoV-2 nucleocapsid and spike protein variants as well as the structural VP2 protein of SVA. Antibody purification and functionalization efforts are underway as a part of efforts to characterize individual antibody-binding properties and develop applicable immunoassays for the detection of viruses. 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. In the second fiscal year, 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 an excellent binding affinity to the target viral proteins. For SARS-CoV-2 Alpha VOC, three sets of in-house aptamers were generated, which were screened and identified by next generation sequencing (NGS) approach and the Sanger method. These sequences were further analyzed using molecular docking modeling and structural analysis software prior to their synthesis. For binding affinity studies, Alpha VOC aptamers were applied on Aptamer-Linked Immobilized Sorbent Assay (ALISA) microplate to test recombinant proteins and inactivated viruses. Preliminary ALISA results showed an increasing signals pattern when increasing concentration of target samples was tested, suggesting successful binding of aptamers to target samples. Future efforts would include incorporating these aptamers onto lateral flow assay (LFA) and evaluating its specificity and sensitivity against active Alpha VOC. For SARS-CoV-2 Delta VOC, SELEX has already reached Round 6, while aptamer sequences have been identified and are currently being analyzed for SARS-CoV-2 Omicron VOC. In parallel to these newly identified in-house aptamers, published aptamers were also used and incorporated into 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) for positive samples. More optimization steps are being conducted to intensify the band signals. After identifying the optimum conditions, LFA strips will be continuously shared with collaborators to test environmental and clinical samples. Previous optimization activities include changing the running buffer and increasing the drying time of LFA strips. For other viral pathogens, SELEX has reached Round 6 for both SVA and H1N1 samples. All efforts will be directed to construct diagnostic tools for pen-side testing. Artificial Intelligence (AI)/Machine Learning (ML) Deep learning approaches (convolutional Neural Network (CNN), rapid CNN (RCNN), and transfer learning) were used to identify tule elk (Cervus canadensis nannodes) in existing National Agriculture Imagery Program (NAIP) images (viewable on Google Earth). The code runs on a desktop personal computer (PC). This approach permits the rapid identification, segmentation, counting, and location determination (using the Global Positioning System (GPS)) of the tule elk in those images. Our approach can easily distinguish between domestic cattle (Aberdeen Angus and Holstein Friesian), sheep, and tule elk. The embedded GPS coordinates were used to quantify tule elk aggregation. Animals afflicted with chronic wasting disease behave differently from uninfected herd mates. Aggregation analysis may provide a means of determining the herd-level CWD infection rate. It is significantly less expensive and time-consuming to use machine learning to analyze images than to test animals. ACCOMPLISHMENTS 01 Defining CWD prion strains by their characteristic shape. Detecting emerging strains of Chronic Wasting Disease (CWD) prions that infect deer, elk, and moose is important to minimize their impact on the rural economy. ARS researchers in Albany, California, used mass spectrometry to develop a method to detect differences in the shape of CWD prions. This approach may allow researchers to define a prion by its shape instead of relying on phenotypic differences. Such information may allow regulators to appropriately respond to the emergence of new CWD prion strains to minimize their impact on the rural economy. 02 Determining the proportion of protein variants in sheep scrapie. Understanding how different amino acids influence the transmissibility of prions is important to minimize their spread and consequent adverse impact. ARS researchers in Albany, California, and Ames, Iowa, developed a mass spectrometry-based method of determining the proportion of the prion protein containing lysine 171 in sheep that produce both lysine and glutamine at position 171 in their prion protein. When sheep express lysine at position 171 of the prion protein, they are more resistant to scrapie infection. Understanding why lysine has such an influence of sheep scrapie propagation is important to controlling scrapie, an endemic sheep prion disease. 03 New technology enhances pen-side sample collection and detection. Swabs are routinely used for biological sampling. ARS scientists in Albany, California, have designed, engineered, and prototyped a disposable single-use swab control module to simplify pen-side animal testing. This tracible device can be used as a standalone swab management solution or integrated with a biosensor housing a lateral flow test strip to facilitate rapid detection of a target analyte. This technology provides a field portable tool to enhance pen-side sample collection and detection methods for animal disease surveillance. 04 Aptamers bind and detect SARS-CoV-2 variants. The presence and cocirculation of severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2) variants in wild animals, such as white-tailed deer, long after they were last detected in humans have been recently confirmed. However, testing of animals and other related environmental samples for potential coronavirus (COVID) infection requires federally approved laboratories which can take a week or more to provide results; this may be too long to prevent the early spread of infection. ARS researchers in Albany, California, collaborated with stakeholders including USDA Animal Plant Health Inspection Service (APHIS), Southeast Poultry Research Laboratory - U.S. National Poultry Research Center, USDA ARS National Animal Disease Center, and Centers for Disease Control and Prevention One Health to develop an aptamer-based lateral flow assay (LFA) for pen-side setting use as well as portable colorimetric microplate assays. In-house aptamers (Alpha and Omicron VOCs) were generated, and results have shown that aptamers were highly compatible and stable in a microplate setup as its main detection component. Universal aptamers from published literature targeting all variants of concerns (Alpha, Delta, and Omicron) were also integrated onto both LFA strips and microplate and could detect both active and inactivated SARS- CoV-2 viruses in buffer solutions. With future optimizations, it is expected that the aptamer-based LFA and microplate assay can be readily used by USDA APHIS, veterinarians, farmers, and other regulatory agencies to test various animal and environmental samples associated with minks, white-tailed deer, zoo animals, and other species suspected of SARS-CoV-2 infections.
Impacts
(N/A)
Publications
- Silva, C.J., Cassmann, E.D., Greenlee, J.J., Erickson-Beltran, M.L., Requena, J.R. 2023. A mass spectrometry-based method of quantifying the contribution of the lysine polymorphism at position 171 in sheep PrP. Journal of American Society for Mass Spectrometry. 34(2):245-254. https:// doi.org/10.1021/jasms.2c00277.
- Silva, C.J., Erickson-Beltran, M.L. 2023. General method of quantifying the extent of methionine oxidation in the prion protein. Journal of American Society for Mass Spectrometry. 34(2):255-263. https://doi.org/10. 1021/jasms.2c00280.
- Diplock, N., Baudin, M., Harden, L.A., Silva, C.J., Erickson-Beltran, M.L., Hassan, J.A., Lewis, J.D. 2023. Utilising natural diversity of kinases to rationally engineer interactions with the angiosperm immune receptor ZAR1. Plant, Cell & Environment. 46(7):2238-2254. https://doi.org/10.1111/pce. 14603.
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Progress 10/01/21 to 09/30/22
Outputs
PROGRESS REPORT Objectives (from AD-416): 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 (from AD-416): 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. This is a new project that started in March 2022 and continues work from expired project 2030-32000-010-000D. In support of Sub-objective 1A, ARS scientists have optimized conditions to detect methionine oxidation in hamster, bank vole, cervid, and ovine recombinant prion proteins. In addition, the conditions have been optimized to facilitate lysine acylation of prions from sheep and cervids. Brain tissue from homozygous (ARQ) sheep naturally infected with classical scrapie and brain tissue from homozygous (ARR) and heterozygous (AHR/ARR) sheep experimentally infected with atypical scrapie have been acquired. Samples of brain tissue from white-tailed deer experimentally infected with the Wisc-1 and H95+ strains of CWD have been obtained. Well- characterized hamster-adapted scrapie strains have been oxidized with hydrogen peroxide and chloramine T to quantify the extent of methionine oxidation. In support of Sub-objective 1B, ARS scientists have synthesized new acylating agents. The 8G8 and 6D11 monoclonal antibodies (mAbs) have been obtained from commercial vendors. mAb N2 was obtained through an incoming materials transfer agreement. These mAbs have been tested against sheep and deer prion protein (PrPC), derived from brain homogenates and acylated with synthetic reagents. These results indicate that when the lysine (PrPC confirmation) in the epitopes of these mAbs is acylated, the mAbs do not bind, and PrPC is not detected by Western blot. In support of Sub-objective 1C, ARS scientists have synthesized genes to express the native bank vole prion protein (109M) and a polymorphic variant (109I). Both genes have had their codons optimized for expression in Escherichia coli (E. coli). The plasmid has been cloned into the BL21 strain of E. coli. The bank vole (109I) recombinant prion protein (rPrP) has been overexpressed in this strain of E. coli. The protein has been successfully purified and analyzed by mass spectrometry. In support of Sub-objective 2A, immunization of mice with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigenic protein variants for the generation of monoclonal antibody-producing hybridoma cell lines is ongoing. Various adjuvant and immunization regimes have been deployed, and animal immune responses were confirmed by immunoassay. Hybridoma technology, cell cloning, and high-throughput enzyme-linked immunosorbent assay (ELISA) screening are progressing toward the selection of distinct hybridoma cell lines that produce novel monoclonal antibodies against SARS-CoV-2 variants. These anti-SARS-CoV-2 monoclonal antibodies will be used to develop improved animal immunodiagnostic assays. In collaboration with partners, existing anti-SARS-CoV-2 antibody cohorts generated against viral spike proteins have been evaluated. Antibody pairs that selectively bind recombinant spike protein variants from SARS-CoV-2 by lateral flow immunoassay have been identified. Efforts are underway to enhance lateral flow reporter sensitivities as a prerequisite for the detection of native viral particles when targeting spike protein antigens. Recombinant proteins have been generated against the external capsid proteins of the Senecavirus-A. These proteins have been purified, biochemically characterized, and used for immunization of mice and the generation of hybridoma cell lines producing specific monoclonal antibodies. A predominant Senecavirus-A strain has been secured from our partners for propagation in cell culture as a source of native virus for laboratory evaluation of immunoassay performance. To date, a cohort of monoclonal antibodies generated against the capsid VP2 protein has been partially characterized, and a sandwich enzyme-linked immunosorbent assay has been constructed. Current efforts are focused on the applicability of these VP2 antibodies and immunoassay formats for the detection of native viruses. In support of Sub-objective 2B, research has continued on the development of aptamer-based lateral flow assay (LFA) for the early detection of emerging pathogens [influenza virus A � H1N1 in swine, SARS- CoV-2 variants of concerns (VOCs: Alpha, Delta, and Omicron variants), and Senecavirus A or SVA] that cause animal diseases. Target viral proteins were used as baits to generate highly-specific in-house aptamers through the Systematic Evolution of Ligands by Exponential Enrichment (SELEX). As an iterative process, each round of SELEX narrows down the aptamer sequences from a random pool of DNA libraries which can eventually generate aptamer sequences that are highly specific with excellent binding affinity to the target viral proteins. For SVA, aptamers have been developed (SELEX � Round 8) and sequenced using VP1 recombinant protein with further optimizations needed. For H1N1, HA protein (Round 5) was used, while both spike and nucleocapsid proteins (Round 3) were used for SARS-CoV-2 VOCs during SELEX. The generated aptamer sequences would then be incorporated onto the LFA system as capture and detection elements for the rapid and on-site detection of viral agents. In parallel to the in-house aptamer generation, available published aptamer sequences targeting VOCs were also successfully incorporated into the LFA system. These efforts have shown the sensitivity of aptamers and compatibility with LFA platforms. ACCOMPLISHMENTS 01 Detecting prion strains by measuring methionine oxidation. Prions cause chronic wasting disease (CWD), a rapidly spreading disease of wild deer and elk. CWD negatively impacts the $30 billion U.S. hunting industry. Prions impart disease through their distinct shapes. Unfortunately, differences in shape are difficult to characterize by conventional means. ARS researchers in Albany, California, used mass spectrometry to detect shape differences of hamster-adapted prion strains by measuring the surface exposure of the amino acid methionine. This approach allowed the researchers to define a prion strain by its shape instead of relying on more empirical and cumbersome conventional methods. This will provide regulators with the tools to manage the emergence of new prion strains. 02 Normal cellular prion protein enables spread of misfolded proteins. Protein misfolding or prion-like diseases, such as Parkinson�s disease, cost Americans untold misery and at least $50 billion annually. These diseases spread from cell-to-cell by binding to the natively expressed prion protein (PrPC). As part of a large international collaboration, ARS researchers in Albany, California, showed that binding of the misfolded protein that causes Parkinson�s disease to PrPC allows the protein to enter a cell, thereby spreading it from one cell to another. The part of PrPC that the misfolded proteins attaches to was also identified. This information can be used to develop drugs to prevent the binding and consequent intercellular spread of misfolded proteins. In principle, such drugs may be a general means of treating prion and prion-like diseases, such as Parkinson�s disease. 03 Novel VP2 capsid antibodies against Senecavirus-A. No reliable rapid tests are available for detecting Senecavirus A (SVA) infection in swine. ARS researchers in Albany, California, have designed, engineered, and expressed three viral capsid proteins (VP1-3) from Senecavirus-A (SVA) in bacteria. These recombinant viral VP capsid proteins have been affinity purified and biochemically characterized. Mice were immunized with the VP2 protein and hybridoma technology used to generate and select unique hybridoma cell lines expressing anti-VP2 monoclonal antibodies. Novel monoclonal antibodies to viral capsid proteins will be used by researchers to develop pen-side SVA diagnostic immunoassays. 04 Aptamers are an alternative option for lateral flow assay detection of SARS-CoV-2 in animals. In the United States, 17 mink farms have been affected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, all of which have had associated human cases. Currently, there are no federally authorized rapid and handheld detection tools for SARS-CoV-2 testing in animals. Researchers in Albany, California, have collaborated with USDA Animal and Plant Health Inspection Service (APHIS), ARS National Animal Disease Center, and Centers for Disease Control and Prevention One Health to develop aptamer-based lateral flow assays (LFA) for pen-side rapid detection. Aptamers have been shown to be highly compatible and stable in the LFA system as its main detection component to screen for SARS-CoV-2. This inexpensive technology is an excellent option in addition to immune-based detection tools to effectively address current diagnostic needs related to animal diseases by USDA APHIS, veterinarians, farmers, and other regulatory agencies.
Impacts
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Publications
- Silva, C.J. 2022. Chronic wasting disease (CWD) in cervids and the consequences of a mutable protein conformation. ACS Omega. 7(15) :12474�12492. https://doi.org/10.1021/acsomega.2c00155.
- Thom, T., Schmitz, M., Fischer, A., Correia, A., Correia, S., Llorens, F., Pique, A., Mobius, W., Domingues, R., Zafar, S., Stoops, E., Silva, C.J., Fischer, A., Outeiro, T.F., Zerr, I. 2021. Cellular prion protein mediates a-synuclein uptake, localization, and toxicity in vitro and in vivo. Movement Disorders. 37(1):39-51. https://doi.org/10.1002/mds.28774.
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