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United States Department of Agriculture

Agricultural Research Service

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Research Project: Functional Genomic Approaches for Controlling Diseases of Swine

Location: Animal Parasitic Diseases

2013 Annual Report


1a.Objectives (from AD-416):
Objective 1: Identify genetic variations associated with disease resistance to PRRS virus and identify biological determinants associated with anti-PRRS virus host responses to discover genetic and biological determinants associated with disease resistance to infectious diseases of swine.

Objective 2: Discover effective immune intervention strategies to prevent and control infectious diseases of swine by developing immunologic tools to enhance our understanding of swine immune system development and novel assays to evaluate pig responses to infectious diseases; and by assessing differential responses to infection versus vaccination to determine what pathways are associated with protective responses of pigs to respiratory pathogens.


1b.Approach (from AD-416):
Assess whether genomic variation in pigs is associated with differential PRRS resistance/susceptibility and biologic responses to PRRSV infection. The PRRS Host Genetics Consortium (PHGC) was developed to assess the genomic basis of PRRS disease resistance. The PHGC is a national effort to assess pig resistance/susceptibility to primary PRRSV infection and associated growth effects. Our research will focus on the PHGC objectives:.
1)use genotyping and phenotyping tools to determine if there are host genes that control resistance/susceptibility to PRRSV infection;.
2)verify genetic variation in response to PRRSV, via improved health, survivability and growth; and.
3)identify relative importance of different phenotypes, and their heritability, that predict response to PRRSV infection.

Identify biological determinants associated with anti-PRRS virus host responses. We hypothesize that there will be functional genomic and proteomic differences that correlate with resistance to PRRSV infections. Using PHGC samples, functional analyses will be performed to identify gene networks and resistance associated biomarkers that differ in high versus low viral load pigs. Our goal would be to identify markers expressed pre-infection, e.g., serum biomarkers in uninfected pigs.

Develop immunologic tools to enhance our understanding of swine immune system development and novel assays to evaluate pig responses to infectious diseases. A portion of the time devoted to this project will involve research to develop and improve immune reagents available to characterize swine immune responses and regulation. Numerous commercial sources provide reagents for swine immunity research; yet, compared to human and mouse, these resources are limited. Working with the U.S. Veterinary Immune Reagent Network (VIRN) we will develop new immunologic reagents and assays to measure swine cytokines, chemokines, and immune cell surface molecules. These will help identify key features of an effective swine immune response to infectious pathogens

Assess differential responses to infection versus vaccination to determine what pathways are associated with protective responses of pigs to respiratory pathogens. Biomarkers are present in pigs that correlate with protective immunity to respiratory pathogens. Determining whether a response to infection or vaccination is protective or pathologic is essential for the design of new vaccines, diagnostics and therapeutics. For pigs this means understanding the genes and proteins, networks and pathways involved in these responses. Based on the newly finished swine genome sequence, we will identify and annotate some of the numerous genes and regulatory sequences encoded in the swine genome. With that information, functional genomic tools will be applied to assess infection or vaccination responses. Research targets will be infection and vaccination of pigs with respiratory pathogens, particularly PCV2b and PRRSV.


3.Progress Report:
A major gene on swine chromosome 4 (SSC4) has been identified as associated with resistance of swine to porcine reproductive and respiratory syndrome (PRRS), the most economically important swine pathogen causing $664 million losses annually in the U.S. Scientists at Beltsville MD, working with scientists at Kansas State University, Iowa State University, the University of Alberta, and with 6 commercial pig breeding companies established the PRRS Host Genetics Consortium. This Consortium has now performed a comprehensive test for viral disease resistance in 200 commercial pigs housed in biosecure facilities. The goal was to determine the role of host genetics in resistance/susceptibility to PRRSV infection and associated growth effects. To this end, viral load and host status and gene expression was monitored for a period of several weeks. A centralized database was constructed to house all these disparate data http://www.animalgenome.org/lunney/index.php. Analyses of the first 5 trials of 3 commercial pig breeds affirmed moderate heritability of traits controlling viral load and weight gain 0.39 and 0.34, respectively. Sophisticated statistical analyses, genome wide association studies have identified regions on the 4th and X chromosomes that influence viral load, and on chromosomes 1, 4, 7, 17 that control weight gain. In infected pigs, variation in one genomic region on chromosome 4 accounts for 15% of variation in viral load and also for 11% of variation in weight gain. Importantly, decreased viral load perfectly correlated with increased weight gain. Onging efforts see, to establish which proteins are differentially expressed in such susceptible and resistant pigs. Overall, our results affirm that pig response to experimental PRRSV challenge has a strong genetic component with a major locus on SSC4 explaining a substantial proportion of the genetic variance. This information has been disseminated to swine breeders, genetics companies, and genotyping services so that sets of these recommended genetic markers can be employed in future breeding programs to enhance PRRS disease resistance.


4.Accomplishments
1. A major resource for evaluating porcine reproductive and respiratory syndrome (PRRS) resistance is now available. PRRS is the most economically important swine pathogen causing $664 million losses to U.S. pig producers annually. Because health traits are difficult to measure and require biosecure facilities a national coalition was developed, the PRRS Host Genetics Consortium. With funding from ARS, NIFA, the National Pork Board (NPB) and Genome Canada, ARS scientists in Beltsville Maryland worked academic collaborators at Kansas State University, Iowa State University and the University of Alberta, to perform infection trials, with each of two viral strains, on pigs from several breeding companies. Blood and tissue samples were collected and stored for detailed screening, termed phenotypic measurements. Detailed molecular genotyping of each pig’s DNA was also performed. The PHGC now has the tools to evaluate the role of host genetics in PRRS resistance.

2. A region of the swine genome confers resistance of to porcine reproductive and respiratory syndrome, the most economically important pig disease worldwide. The team, led by ARS researchers at Beltsville, Maryland, Iowa State and Kansas State universities, and working with 6 major pig breeding companies, used state-of-the-art genome-wide association studies to identify the region on swine chromosome 4 and showed that it accounts for 15% of the variation in viral load and for 11% of variation in weight gain after infection. Decreased viral load and increased weight gain were perfectly correlated. This information has been disseminated to swine breeders, genetics companies, and genotyping services and will enable geneticists to develop plans for marker-assisted selection of pigs with improved response to this viral infection.

3. New tools were developed to assist researchers in probing swine immune responses to infectious diseases and vaccine. Prevention of and recovery from infectious diseases is a goal of animal health workers. The U.S. Veterinary Immunological Reagents Network www.vetimm.org was established to assist in this goal by addressing the dearth of immunological reagents for food animal species. ARS researchers at Beltsville, Maryland, working with Kingfisher Biotech Inc. www.kingfisherbiotech.com/ have targeted serum immune proteins (cytokines and chemokines) known to be critical determinant of disease and vaccine responses. Together they expressed, and affirmed bioactivity, of several immune proteins; these included regulatory proteins, interleukin-17A (IL-17A) and IL-17F, and IL-6. Working with colleagues at Cornell and University of Massachusetts, they used these proteins to develop panels of monoclonal antibodies as tools to identify specific cytokines and develop multiplex bead assays. This work produced a new bead assay for the macrophage chemokine CCL2. Overall, as a result of these efforts swine disease and vaccine researchers have new tools to probe immune responses to infectious diseases and develop better vaccine designs.

4. Researchers in the U.S. partnered with Canadians to control swine viral disease. Porcine reproductive and respiratory syndrome virus (PRRSV) infection is the most economically important swine production disease worldwide. This project helped to build a Canadian component to the U.S. effort to map genes and identify biomarkers associated with healthier, PRRS resistant pigs. ARS researchers at Beltsville, Maryland partnered with scientists from the University of Alberta and with their breeding company organization, PigGen Canada, to characterize host factors that contribute to PRRS disease resistance and growth losses. Biomarkers of disease resistance were sought in serum proteins using an innovative, high- throughput assay. A few serum proteins (the cytokine interferon-alpha, and chemokine CCL2) exhibited unique expression patterns that correlated with lower viral loads indicative of PRRS-resistant pigs. The immunoassay technology was shared with Canadian researchers to aid their study of swine disease and vaccine responses, which will contribute to international efforts to blunt the economic losses stemming from this important viral disease.

5. Reduced background RNA improves accuracy of quantifying viral loads in swine. The major disease of swine worldwide is porcine reproductive and respiratory syndrome (PRRS). ARS researchers at Beltsville, Maryland working with scientists from Iowa State and Kansas State universities partnered with scientists from the University of Alberta and their breeding company organization, PigGen Canada, to characterize host factors that contribute to swine disease resistance and growth losses. In order to accurately characterize and quantify the genes expressed most in pigs that were resistant to the harmful effects of the virus, ARS scientists working with their Canadian colleagues had first to develop new pretreatment protocols to reduce of abundant hemoglobin RNA. That accomplishment now enables the use of next generation sequencing and bioinformatics to probe swine responses to this important viral infection. Differential transcripts and unique pathways in resistant pigs can now be discerned.

6. The swine genome has been sequenced. ARS researchers at Beltsville, Maryland worked closely with Wellcome Trust Sanger Institute on the community annotation of genes in the pig genome. Because of the importance of immune genes in pig health, scientists around the world formed the Immune Response Annotation Group to focus on the detailed manual annotation of genes associated with swine immunity; these constitute 5% of the pig genome and are highly variable within each species. This effort opens new vistas for studying immune responses, and allows the identification of a range of new types of genetic changes, e.g., splice and copy number variants. These results will help animal health researchers identify new pathways involved in disease prevention and control. The availability of the swine genome sequence establishes a strong basis to help identify swine genetic variation which will enable producers to breed healthier pigs and make better quality pork products. Understanding the genome will affirm the importance of the pig as a more accurate model species of human development and disease.

7. Development of a centralized data repository for documenting the responses of various kinds of pigs to exposure to porcine reproductive and respiratory syndrome virus, a major swine pathogen which causes $664 million per year losses to the U.S. pig industry. ARS researchers at Beltsville, Maryland partnered with Michigan State University, Iowa State University, Washington State University and Purdue University scientists to compare gene expression in pigs displaying a range of resistance to this virus. To coordinate the consortium’s effort, a system has been constructed to store experimental data (www.animalgenome.org/lunney/). This enabled identification of biomarkers and gene networks that differ in resistant and susceptible animals. The centralized database will serve as the basis for seeking predictive gene expression pathways and genes that will aid the development of breeds, vaccines, and/or treatments that reduce the health and financial impact of this important viral infection.


Review Publications
Butler, J.E., Wertz, N., Sun, X.Z., Lunney, J.K., Muydermanns, S. 2013. Resolution of an immunodiagnostic dilemma: Heavy chain chimeric antibodies for species in which plasmocytomas are unknown. Molecular Immunology. 53:140-148.

Arceo, M., Ernst, C., Lunney, J.K., Choi, I., Raney, N., Huang, T., Tuggle, C.K., Rowland, R.R., Steibel, J. 2013. Characterizing differential individual response to Porcine Reproductive and Respiratory Virus infection through statistical and functional analysis of gene expression. Frontiers in Livestock Genomics. 3:321.

Rowland, R.R., Lunney, J.K., Dekkers, J. 2012. Control of porcine reproductive and respiratory syndrome (PRRS) through genetic improvements in disease resistance and tolerance. Frontiers in Livestock Genomics. 3:321.

Lunney, J.K., Kai, C., Inumaru, S., Onodera, T. 2012. The Ninth International Veterinary Immunology Symposium. Veterinary Immunology and Immunopathology. 148:1-5.

Zhou, X., Michal, J.J., Zhang, L., Ding, B., Lunney, J.K., Liu, B., Jiang, Z. 2013. Interferon induced IFIT family genes in host antiviral defense. International Journal of Biological Sciences. 9:200-208.

Dawson, H.D., Loveland, J., Pascal, G., Gilbert, J., Uenishi, H., Mann, K., Sang, Y., Zhang, J., Carvalho-Silva, D., Hunt, T., Hardy, M., Hu, Z., Zhao, S., Anselmo, A., Sinkai, H., Chen, C.T., Badaoui, B., Berman, D.J., Amid, C., Kay, M., Lloyd, D., Snow, C., Morozumi, T., Cheng, R., Bystrom, M., Bourneuf, E., Kapetanovic, R., Schwartz, J.C., Kataria, R., Astley, M., Fritz, E., Steward, C., Thomas, M., Wilming, L., Giuffra, E., Archibald, A., Bed'Hom, B., Beraldi, D., Ait-Ali, T., Blecha, F., Botti, S., Freeman, T., Hume, D.A., Lunney, J.K., Murtaugh, M.P., Reecy, J.M., Harrow, J., Rogel-Gaillard, C., Tuggle, C.K. 2013. Structural and Functional Annotation of the Porcine Immunome. Biomed Central (BMC) Genomics. 14:332-370.

Groenen, M.A., Archibald, A.L., Uenishi, H., Tuggle, C., Takeuchi, Y., Rothschild, M.F., Rogel-Gaillard, C., Park, C., Milan, D., Hendrik-Jan, M., Li, S., Larkin, D., Kim, H., Franz, L.A., Caccamo, M., Hyeonju, A., Aken, B.L., Anselmo, A., Anthon, C., Auvil, L., Bouabid, B., Beattie, C.W., Bendixen, C., Berman, D.J., Blecha, F., Blomberg, J., Bolund, L., Bosse, M., Botti, S., Zhan, B., Bystrom, M., Capitanu, B., Silva, D.C., Chardon, P., Chen, C.T., Cheng, R., Choi, S., Chow, W., Clark, R.C., Clee, C., Crooijmans, R.P., Dawson, H.D., Dehais, P., De Sapio, F., Dibbits, B., Drou, N., Du, Z., Eversole, K., Fadista, J., Fairley, S., Faraut, T., Faulkner, G.J., Fowler, K.E., Fredholm, M., Fritz, E., Gilbert, J.G., Giuffra, E., Gorodkin, J., Griffin, D., Harrow, J.L., Hayward, A., Howe, K., Zhi-Liang, H., Humphray, S.J., Hunt, T., Hornshoj, H., Jeon, J., Jern, P., Jones, M., Jurka, J., Kanamori, H., Kapetanovic, R., Jaebum, K., Kim, J., Kim, K., Kim, T., Larson, G., Lee, K., Lee, K., Leggett, R., Lewin, H.A., Li, Y., Liu, W., Loveland, J.E., Lu, Y., Lunney, J.K., Ma, J., Madsen, O., Mann, K., Mathews, L., Mclaren, S., Morozumi, T., Murtaug, M.P., Narayan, J., Nguyen, D., Ni, P., Oh, S., Onteru, S., Rohrer, G.A., et al. 2012. Analysis of pig genomes provide insight into porcine demography and evolution. Nature. 491(7424):393-8.

Last Modified: 9/21/2014
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