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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Animal Parasitic Diseases Laboratory » Research » Research Project #441680

Research Project: Immune and Genomic Determinants of Host Responses Associated with Porcine Reproductive and Respiratory Syndrome Virus Infections

Location: Animal Parasitic Diseases Laboratory

2022 Annual Report

Objective 1: Develop immunologic tools to evaluate swine immunity. Sub-objective 1.A: Develop immunological assays and validate their use for assessing host immune responses to infectious diseases and vaccination. Sub-objective 1.B: Use immunological tools to inform the design of novel innate immune intervention strategies to treat respiratory diseases of swine. Objective 2: Elucidate immune and genomic determinants of host responses associated with porcine reproductive and respiratory syndrome virus infection. Sub-objective 2.A: Discover genetic and biological determinants associated with fetal susceptibility, resilience, or resistance to porcine reproductive and respiratory syndrome. Sub-objective 2.B: Discover genetic and biological determinants associated with good responders to porcine reproductive and respiratory syndrome vaccines.

This project will produce new reagents for analyses of swine immunity. This will include characterization of monoclonal antibodies (mAbs) reactive with swine cytokines and chemokines and development of assays that quantify their reactivity. These efforts will be coupled with NIFA grant supported Swine Toolkit efforts. Once characterized commercial access to these mAbs will be coordinated through ARS Technology Transfer Office. Basic studies will be expanded by establishing molecular tools to evaluate gene expression using NanoString Technologies, with codesets developed to explore relative importance of specific tissues and unique cell subsets in disease responses and pathogen resistance. These tools will help to identify alternate biomarkers for risk analyses to assess the impact of respiratory diseases and vaccine efficacy. This project will apply the use of these innovative, next-generation, genomic, transcriptomic, and immunologic techniques to address Porcine Reproductive and Respiratory Syndrome (PRRS), focusing on our unique sample resources collected via the PRRS Host Genetics Consortium and the Canadian Pregnant Gilt Model projects. We will evaluate the influence of host genetics on antiviral response pathways and will aim to identify critical biomarkers of protective vaccine and infection responses. We will use RNAseq data analyses to provide a more complete picture and reveal details of regulatory mechanisms impacting pig responses to vaccination, viral infection, and differential growth effects. Advanced bioinformatics will be combined with new molecular studies, as well as previously collected genotypic and phenotypic data, to identify pathways and biomarkers associated with protective immune responses to respiratory and reproductive diseases that cause significant losses to the pork industry. This project will provide alternate resources for improved respiratory disease control and elimination strategies. Overall, our project will inform vaccine evaluation and virus control strategies, identify disease-resistant pigs, and stimulate advances that may be of broad economic importance to pig breeders and the animal health industry.

Progress Report
This new project’s first objective focuses on the development of new reagents for swine immunity. Scientists at Beltsville, Maryland, obtained, cloned, and expressed swine immune proteins (cytokines, chemokines) from a commercial partner. A second commercial partner developed monoclonal antibodies (mAbs) to selected immune proteins. The USDA team characterized reactivities of 2 panels of resultant mAbs, and started to characterize another 3 panels of mAbs, reactive with different swine interleukins, interferons, and chemokines. We tested for intracellular staining and cross species reactivity, in collaboration with university partners. These mAbs will enable researchers to quantify each immune protein within cells and in body fluids and culture supernatants. We are offering these characterized sets of mAbs to commercial partners for further distribution to researchers and veterinarians. Tools and reagents generated by this project support swine immune, disease, vaccine, and biomedical research efforts worldwide. This new project’s second objective focuses on porcine reproductive and respiratory syndrome (PRRS) which causes an estimated $600 million in annual losses to the U.S. pig industry. We are defining genes that regulate responses of nursery pigs to PRRS virus infection as part of the PRRS Host Genomics Consortium. In addition, ARS scientists in Beltsville, Maryland, are working with scientists at the University of Saskatchewan to probe fetal responses to congenital PRRS infection. These responses can vary greatly; litters can include uninfected fetuses neighboring dead fetuses or piglets harboring high viral levels. We have concentrated on maternal and fetal factors that predict disease severity and fetal resilience, looking for specific biomarkers by targeting gene expression using swine immune focused NanoString arrays.

1. New tools to measure swine immune responses. Losses caused by infectious diseases necessitate effective diagnostics, therapeutics, and vaccine interventions. Unfortunately, researchers lack many commercial reagents to probe pig immunity and host response to pathogen infections and vaccines. Therefore, ARS scientists in Beltsville, Maryland, worked with commercial partners to express important immune proteins, a regulator of disease responses (interleukin-17A) and a determinant of anti-viral immunity (interferon gamma). They generated and characterized monoclonal antibodies to these targets that now enable researchers and clinicians to quantify expression of these cytokines in blood and tissues of healthy and diseased pigs. Therefore, these tools expand opportunities for swine immunology and biomedical research and will help dissect in more detail pig responses to pathogens and vaccines.

2. New biomarker of the swine innate immune system. Researchers and veterinarians lack tools needed to study innate immunity, which contributes greatly to animal health and welfare. Therefore, ARS scientists in Beltsville, Maryland, collaborated with U.S. and Austrian scientists to produce and characterize a species-specific monoclonal antibody (mAb) reactive with a surface marker on an early cell that stimulates innate immune responses (targeting the NKp44 antigen on Natural killer (NK) cells). The team used this antibody to test various blood and tissue-derived NK cells in pigs of various ages, finding NKp44 expression in resting NK cells. This mAb helps to elucidate the mechanisms underlying the differentiation, function, and activation of porcine NK cells and expand the understanding of the role these cells play in pig immune development and disease responses. Such “toolkit” efforts provide fundamental information for studying pig immune, disease and vaccine responses, benefitting veterinarians, immunologists, pork producers, and the public.

3. Thyroid hormones mediate swine immune responses. Thyroid hormones powerfully regulate growth and development; stressed or ill animals experience notable changes in their expression. Therefore, ARS scientists in Beltsville, Maryland, collaborated with U.S. and Canadian scientists to test the role of thyroid hormones, thyroxin (T4) and triiodothyronine (T3), in pig disease responses. They tested serum samples from diseased nursery-aged pigs. Pigs from high health farms grown under typical industry conditions, stably expressed T3 or T4. In pigs experimentally challenged with viral or bacterial infection, expression of these hormones decreased significantly in the ensuing two weeks. Thereafter, thyroid hormones rebounded; those expressing the highest levels tended to gain weight faster but did not harbor more virus through time. These data provide insight into mechanisms by which pigs starve pathogens of resources required for their replication. As such, this work may influence how livestock producers and veterinarians manage swine undergoing disease exposure.

4. New genetic markers for swine viral disease resistance. Pig breeders aim to produce healthy, disease resistant pigs. Today's breeders select the best parents using genetic variants (termed single-nucleotide polymorphisms, or SNPs). Viruses may face difficulty infecting pigs bearing structural changes in the receptors that facilitate viral entry. Therefore, ARS scientists in Beltsville, Maryland, collaborated with Iowa State University scientists to test for natural genetic variants in important disease genes (CD163, CD169, RGS16) that encode receptors for two major viral infections (porcine reproductive and respiratory syndrome virus and porcine circovirus 2b). They identified several new SNPs influencing viral resistance and improved growth traits after infection. Pig breeders are using this information to increase natural resistance to viral infections, which benefits the swine industry and consumers.

Review Publications
Manirarora, J., Walker, K.E., Patil, V., Renukaradhya, G., Labresh, J., Sullivan, Y., Francis, O., Lunney, J.K. 2022. Development and characterization of new monoclonal antibodies against porcine Interleukin-17A and Interferon-gamma. Frontiers in Immunology. 13:786396.
Mair, K., Crossman, A., Wagner, B., Babsyan, S., Noronha, L., Boyd, P., Zarlenga, D.S., Stadler, M., Von Dongen, K., Gerner, W., Sallmueller, A., Lunney, J.K. 2022. The natural cytotoxicity receptor NKp44 (NCR2, CD336) is expressed on the majority of porcine NK cells ex vivo without stimulation. Frontiers in Immunology.
Dong, Q., Dunkelberger, J., Lim, K., Lunney, J.K., Tuggle, C., Rowland, R., Dekkers, J. 2021. Associations of natural variation in the cd163 and other candidate genes on host response of nursery pigs to PRRSV infection. Journal of Animal Science. 1-54.
Pasternak, A., Macphee, D., Lunney, J.K., Rowland, R., Dyck, M., Fortin, F., Dekkers, J., Plastow, G., Harding, J. 2021. Thyroid dysfunction in feeder pigs following polymicrobial or porcine reproductive and respiratory syndrome virus-2 challenge. Journal of Animal Science. 99:(911)1-13.
Rowland, R.R., Doerksen, T., Lu, A., Sheahan, M., Lunney, J.K., Dekkers, J., Palinski, R.M. 2022. Effect of the host genotype at a Porcine Reproductive and Respiratory Syndrome (PRRS) resistance marker on evolution of the modified-live PRRS vaccine virus in pigs. Virus Research.
Herrera-Uribe, J., Wiarda, J., Sivasankaran, S.K., Daharsh, L., Liu, H., Byrne, K.A., Smith, T.P., Lunney, J.K., Loving, C.L., Tuggle, C.K. 2021. Reference transcriptomes of porcine peripheral immune cells created through bulk and single-cell RNA sequencing. Frontiers in Genetics. 12. Article 689406.