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

Location: Endemic Poultry Viral Diseases Research

2020 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
Under Objective 1, in collaboration with investigators at Washington University School of Medicine in St. Louis, the Rockefeller University, and the University of Arkansas, new reference assemblies were generated based on a commercial layer and a commercial broiler line. These new assemblies promise to advance chicken genetic research by resolving much of the sequence structure presently fragmented and misappropriated in the current GRCg6a reference, which is based on a female Red Jungle Fowl. In collaboration with investigators at the University of California at Davis, the chicken genome is being annotated using tissues from birds located at ARS facilities in East Lansing, Michigan. Based on eight tissues, our results suggest that the chicken genome has about half of the number of active regulatory elements, including enhancers and promoters, compared to mammals such as cattle and pig. This difference, especially on the number of enhancers, may be due to the small size of the chicken genome, which is one third that of a typical mammal. For Objective 2, we previously identified Ikaros, the master regulator of the development of the immune B and T cells, as the first Marek’s disease (MD) driver gene. To confirm this hypothesis, recombinant Marek’s disease viruses (MDV) were made that expressed either normal Ikaros or a mutant allele with somatic mutations found in MD tumors. Our results demonstrate that birds infected with the MDV expressing the mutant Ikaros allele exhibited significantly higher and more MD tumors compared to other groups infected with the normal Ikaros allele or the control MDV. This validates that Ikaros is an MD driver gene and that MD tumors require “two hits”: (1) infection with MDV and the expression of Meq to prevent cells from undergoing apoptosis (also known as programmed cell death) and (2) perturbations in Ikaros (or other MD driver genes) that lead to unregulated growth. Although MDV induces a T cell lymphoma, it first infects B cells. Thus, the bursa, which is the organ that produces B cells, was examined in chicken lines that are MD resistant or susceptible to determine if there might be differences in this organ to account for the genetic differences. We found that a number of microRNAs, a molecule that regulates gene expression, at 26-days after MD vaccine inoculation. Specifically, 693 microRNAs were identified with over 70 percent not previously reported in chicken. We also identified 631 microRNAs between the same MD resistant and susceptible inbred lines of chickens in bursae 26- days post MD vaccination and MDV challenge. Again, the majority of those were novel microRNAs, and 169 of the 631 microRNAs were only identified in the lines of chickens vaccinated then challenged with MDV. These analyses suggest that both MD vaccine and vaccination followed by MDV challenge treatments were coupled with a significant number of microRNAs with highly detectable expression but each of the treatments (vaccination only and vaccination plus MDV challenge) also induced expression of an unique subset of microRNAs, which might be dominantly involved in vaccine-induced immunity and vaccine protection against tumor formation, respectively. For Objective 3, to further examine the role of specific cells in vaccine mediated protection, we vaccinated donor chicks at one week of age, isolated their total white blood cells, and transferred them into naïve birds (recipients) at one-week post vaccination. The recipient birds, along with two control groups (vaccinated and non-vaccinated), were challenged two days post adoptive lymphocyte transfer. Tissue samples (spleen, skin, thymus, and bursa), were collected at 5, 10, and 20 days post challenge for DNA/RNA isolation, gene expression analysis, and immunohistochemistry. At termination, the adoptive lymphocyte recipient birds did not exhibit any symptom of MD and were fully protected when compared to the non-vaccinated/challenged birds. Studies are ongoing to fully assess the outcome of this study.

Accomplishments
1. Differences in the usage of the two T cell receptor (TCR) gene families is associated with Marek’s disease (MD) genetic resistance. 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 researchers in 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 more specific TCR members compared to the susceptible birds. This information will aid future efforts to select birds for superior disease resistance to MD and improved MD vaccines. As chicken is the primary meat consumed in the United States, this will benefit consumers and society by reducing the amount of feed and waste produced while increasing health and well-being of reared birds.

Review Publications
He, Y., Han, B., Ding, Y., Zhang, H., Chang, S., Zhang, L., Zhao, C., Yang, N., Song, J. 2019. Linc-GALMD1 regulates viral gene expression in the chicken. Frontiers in Genetics. 10:1122. https://doi.org/10.3389/fgene.2019.01122.
Dong, K., Chang, S., Xie, Q., Zhao, P., Zhang, H. 2019. RNA Sequencing revealed differentially expressed genes functionally associated with immunity and tumor suppression during latent phase infection of a vv+MDV in chickens. Scientific Reports. 9:14182. https://doi.org/10.1038/s41598-019-50561-x.
Mays, J.K., Black-Pyrkosz, A., Mansour, T., Schutte, B.C., Chang, S., Dong, K., Hunt, H.D., Fadly, A.M., Zhang, L., Zhang, H. 2019. Endogenous avian leukosis virus in combination with serotype 2 Marek’s disease virus boosted the incidence of LL-like bursal lymphomas in susceptible chickens. Journal of Virology. 93:e00861-19. https://doi.org/10.1128/JVI.00861-19.
Zhang, L., Zhu, C., Heidari, M., Dong, K., Chang, S., Xie, Q., Zhang, H. 2020. Marek’s disease vaccines-induced differential expression of known and novel microRNAs in primary lymphoid organ bursae of White Leghorn. BioMed Central (BMC) Veterinary Research. 51:19. https://doi.org/10.1186/s13567-020-00746-4.
Deng, C., Tan, H., Zhou, H., Wang, M., Lu, Y., Xu, J., Zhang, H., Han, L., Ai, Y. 2019. Four cysteine residues contribute to homodimerization of chicken interleukin-2. International Journal of Molecular Sciences. 20(22):5744. https://www.doi.org/10.3390/ijms20225744.
Lin, J., Ai, Y., Zhou, H., Lv, Y., Wang, M., Xu, J., Yu, C., Zhang, H., Wang, M. 2020. UL36 encoded by Marek’s Disease Virus exhibits linkage-specific deubiquitinase activity. International Journal of Molecular Sciences. 21(5). https://doi.org/10.3390/ijms21051783.
Bai, H., He, Y., Ding, Y., Carrillo, J.A., Selvaraj, R.K., Zhang, H., Chen, J., Song, J. 2019. Allele-specific expression of CD4+ T cells in response to Marek’s disease virus infection. Genes. 10(9):718. https://doi.org/10.3390/genes10090718.
Bai, H., He, Y., Ding, Y., Chang, S., Zhang, H., Chen, J., Song, J. 2019. Grandparental lineage in a reciprocal cross showed no detectable effect on survival days of F2 White Leghorns in response to a very virulent plus Marek’s disease virus challenge. Poultry Science. 98.10: 4498-4503. https://doi.org/10.3382/ps/pez209.
Heidari, M., Zhang, L., Zhang, H. 2020. MicroRNA profiling in the bursae of MDV-infected resistant and susceptible chicken lines. Genomics. 112:2564-2571. https://doi.org/10.1016/j.ygeno.2020.02.009.
Umthong, S., Dunn, J.R., Cheng, H.H. 2019. Towards a mechanistic understanding of the synergistic response induced by bivalent Marek’s disease vaccines to prevent lymphomas. Vaccine. 37(43):6397-6404. https://doi.org/10.1016/j.vaccine.2019.09.003.