Location: Produce Safety and Microbiology Research
2023 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
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.
Accomplishments
1. 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.
2. 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.
3. 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.
4. 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.
Review 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.
