Research Project: Enhancing Genetic Resistance to Marek’s Disease in Poultry

Location: Endemic Poultry Viral Diseases Research

2019 Annual Report

Objectives
1. Enhance the chicken genomic resources to support genetic selection and other strategies to reduce Marek’s disease. 1.1. Enhance the chicken genetic map and its integration with the genome assembly. 1.2. Improve the annotation of the chicken genome. 2. Identify and characterize chicken genes and pathways that confer resistance to Marek’s disease or improve vaccinal efficacy. 2.1. Identify driver mutations associated with genetic resistance to Marek’s disease. 2.2. Characterize long-range enhancer-promoter interactions, especially for those involved in genetic resistance to Marek’s disease. 2.3. Validate genes and polymorphisms that confer Marek’s disease vaccine protective efficacy. 2.4. Identify non-coding RNA genes that confer genetic resistance to Marek’s disease and vaccinal protective efficacy.

Approach
Poultry is the primary meat consumed in the U.S. To achieve economic efficiency, birds are raised at very high density. Since these conditions promote the spread of infectious diseases, the industries rely heavily on biosecurity and vaccines for disease prevention and control. Control of Marek’s disease (MD), a T-cell lymphoma induced by the Marek’s disease virus (MDV), routinely ranks as a major disease concern to the industries. Since the 1960s, field strains of MDV have evolved to higher virulence. Consequently, there is a need to develop alternative and sustainable strategies to augment current MD control methods. We define two objectives to help achieve this goal. First, we continue to enhance and curate the East Lansing (EL) chicken genetic map, which provides the foundation for the chicken genome assembly and many of our molecular genetic studies. In addition, we will aid in the annotation of the chicken genome to allow more efficient understanding and the subsequent use of genomic variation. Second, we use and integrate various genomic approaches to (1) identify genetic and epigenetic variation associated with genetic resistance to MD or MD vaccinal efficiency, and (2) mutations associated with MD tumors. If successful, this project will provide a number of products including (1) a more complete genetic map that will aid in improving the chicken genome assembly, and (2) candidate genes and pathways conferring MD resistance or vaccinal response for evaluation in commercial breeding lines. Ultimately, the poultry industries and U.S. consumers will benefit by the production of safe and economical products.

Progress Report
ARS researchers continued work on the importance of host mutations for aiding the induction of tumors associated with Marek’s disease virus (MDV) infection. In Marek’s disease (MD) tumors that we examined, they had either mutations in the gene known as Ikaros or had low Ikaros expression; Ikaros is the master regulator of the development of immune cells including both B and T cells. Based on this result, we speculate a “two-hit” model for tumorigenesis where (1) perturbations in Ikaros lead to unregulated growth and (2) the MDV gene known as Meq prevents cells from undergoing apoptosis (also known as programmed cell death). Finally, as cancer is considered a “pathway disease” and to aid in our identification of genes that promote tumors, sequencing of RNA was performed. 1,394 and 361 genes were significantly up-and down-regulated, respectively, in MD tumors compared to control cells. Analysis of the differentially-expressed genes identified a number of biologically-relevant pathways including those involved in organismal signaling; white blood cell proliferation, differentiation, and leukocyte activation; the production of blood cells; and regulation of the immune response. The T cell receptor (TCR) recognize specific proteins presented by the immune system to up- or down-regulate the response. Because of this important role, the potential contribution of TCR on genetic resistance to MD was characterized. Using genetically defined chicken lines, we find that chickens resistant to MD show a higher usage of specific TCR families known as Vbeta-1. Our results suggest that selection for resistance to MD has optimized the TCR repertoire. Although MD is a T cell lymphoma, MDV first infects B cells. Thus, the bursa from chicken lines that are MD resistant or susceptible were examined for microRNAs, a molecule that both directly and indirectly regulates gene expression, at 26-days after MD vaccine inoculation. We identified 693 microRNAs expressed in the two chicken lines post MD vaccine inoculation and over 70 percent have never been reported in chicken. Analysis of the number of microRNAs suggests that these molecules are involved in modulating the chicken response to MD vaccines. For Objective 3, to examine alterations in the expression pattern of viral genes, we sequence RNA of skin samples from birds that are susceptible to MD at 10, 20, and 30 days post infection; the skin is the only anatomical site where infectious MDV virions that are cell free are produced. These expression patterns were compared to spleen samples from MD resistant and susceptible birds. The majority of the genes of interest are involved in viral replication and particle formation. This study lays the groundwork for development of mutant recombinant viruses as vaccine against the highly pathogenic strains of MDV. A recombinant vaccine known as rMd5delta-Meq (a recombinant MDV with Meq gene deleted) protects chickens against highly pathogenic strains of MDV better than all the commercially existing vaccines. However, this vaccine also causes the atrophy of immune tissues, which limits commercial adaptation. To identify viral genes that might reduce the atrophy while maintaining vaccinal protection, a number of recombinant constructs were produced and evaluated. The virus that deleted that gene known as pp38 provided 100% protection against very virulent strains of MD and induced no atrophy in the lymphoid organs. This result suggest that it is possible to selectively remove the negative aspects of MD vaccines while maintain the high levels of vaccinal protection. To determine the role of B cells in vaccine-mediated protection, birds had the bursa removed by surgery. Our repeated results provide evidence that B cells do not play a critical role in vaccine-induced protection.

Accomplishments
1. Differences in the usage of the two T cell receptor (TCR) gene families is associated with Marek’s disease (MD) genetic resistance. Understanding the biological mechanism(s) for Marek’s disease virus (MDV) to induce T cell lymphomas is critical for future control using vaccines or genetic resistance in chickens. To address this question, ARS scientists at East Lansing, Michigan, measured the expression of specific families of the T cell receptor in chickens that were either genetically resistant or susceptible to MD. It was determined that MD resistant birds expressed specific TCR members more compared to the susceptible birds. This information will aid future efforts to select birds for superior disease resistance to Marek’s disease (MD) and improved MD vaccines. As chicken is the primary meat consumed in the US, this will benefit consumers and society by reducing the amount of feed and waste produced, and increasing health and well-being of reared birds.

Review Publications
Zhang, X., Yan, Y., Lin, W., Li, A., Zhang, H., Lei, X., Dai, Z., Li, X., Li, H., Chen, W., Chen, F., Ma, J., Xie, Q. 2019. Circular RNA Vav3 sponges gga-miR-375 to promote epithelial-mesenchymal transition. RNA Biology. 16(1):118-132. https://doi.org/10.1080/15476286.2018.1564462.
Li, H., Wang, P., Lin, L., Shi, M., Gu, Z., Huang, T., Mo, M., Wei, T., Zhang, H., Wei, P. 2018. The emergence of the infection of subgroup J avian leukosis virus escalated the tumor incidence in commercial Yellow chickens in Southern China in recent years. Transboundary and Emerging Diseases. 66(1):312-316. https://doi.org/10.1111/tbed.13023.
Rexroad III, C.E., Vallet, J.L., Matukumalli, L.K., Ernst, C., Van Tassell, C.P., Cheng, H.H., Reecy, J., Fulton, J., Taylor, J., Lunney, J.K., Liu, J., Cockett, N., Smith, T.P., Van Eenennaam, A., Clutter, A., Telugu, B., Purcell, C., Bickhart, D.M., Blackburn, H.D., Neibergs, H., Wells, K., Boggess, M.V., Sonstegard, T. 2019. Genome to phenome: improving animal health, production, and well-being: a new USDA blueprint for animal genome research 2018–2027. Frontiers in Genetics. 10:327. https://doi.org/10.3389/fgene.2019.00327.
Xu, L., He, Y., Ding, Y., Liu, G., Zhang, H., Cheng, H.H., Taylor, R.L., Song, J. 2018. Genetic assessment of inbred chicken lines indicates genomic signatures of resistance to Marek’s disease. Journal of Animal Science and Biotechnology. 9:65. https://doi.org/10.1186/s40104-018-0281-x.
Dar, M.A., Urwat, U., Ahmad, S.M., Ahemd, R., Kushoo, Z.A., Dar, T.A., Shah, R.A., Heidari, M. 2018. Gene expression and antibody response in chicken against Salmonella Typhimurium challenge. Poultry Science. 98(5):2008-2013. https://doi.org/10.3382/ps/pey560.
Sunkara, L., Ahmad, M., Heidari, M. 2019. RNA-seq analysis of viral gene expression in the skin of Marek’s disease virus infected chickens. Veterinary Immunology and Immunopathology. 213:1-9. https://doi.org/10.1016/j.vetimm.2019.109882.
Kern, C., Wang, Y., Chitwood, J., Korf, I., DeLany, M., Cheng, H.H., Medrano, J.F., Van Eenennaam, A.L., Ernst, C., Ross, P., Zhou, H. 2018. Genome-wide identification of tissue-specific long non-coding RNA in three farm animal species. BMC Genomics. 19:684. https://doi.org/10.1186/s12864-018-5037-7.
McPherson, M.C., Cheng, H.H., Smith, J.M., Delany, M.E. 2018. Vaccination and host Marek's disease-resistance genotype significantly reduce oncogenic gallid alphaherpesvirus 2 telomere integration in host birds. Cytogenetics and Genome Research. 156(4):204-214. https://doi.org/10.1159/000495174.
Liao, Z., Dai, Z., Cai, C., Zhang, X., Li, A., Zhang, H., Yan, Y., Lin, W., Wu, Y., Li, H., Li, H., Xie, Q. 2019. Knockout of Atg5 inhibits proliferation and promotes apoptosis of DF-1 cells. In Vitro Cellular and Developmental Biology - Animals. 55(5):341-348. https://doi.org/10.1007/s11626-019-00342-7.
Dunn, J.R., Black Pyrkosz, A., Steep, A., Cheng, H.H. 2019. Identification of Marek’s disease virus genes associated with virulence of US strains. Journal of General Virology. 100(7):1132-1139. https://doi.org/10.1099/jgv.0.001288.
Chu, Q., Ding, Y., Cai, W., Liu, L., Zhang, H., Song, J. 2019. Marek’s disease virus infection induced mitochondria changes in chickens. International Journal of Molecular Sciences. 20(13): 3150. https://doi.org/10.3390/ijms20133150.