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ARS Home » Southeast Area » Athens, Georgia » U.S. National Poultry Research Center » Endemic Poultry Viral Diseases Research » Research » Research Project #433072

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

Location: Endemic Poultry Viral Diseases Research

2022 Annual Report

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. 3. Characterizing and defining innate defense mechanisims that contribute to Marek's Disease resistence. 3.1 Role of the innate defense mechanisms that drive Marek’s disease resistance, including defining and characterizing innate defense mechanisms that contribute to Marek’s disease resistance.

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
This report shows that substantial progress was made on all objectives for the duration of this project. Under Objective 1, the chicken genome assembly was improved. First, in collaboration with investigators at Washington University School of Medicine in St. Louis, the initial chicken reference assembly, based on Red Jungle Fowl, was updated with genes associated with the immune system, especially the T cell receptor (TCR) locus. Second, in collaboration with investigators at Washington University 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. These new assemblies promise to advance chicken genetic research by resolving much of the sequence structure presently fragmented and misappropriated in the current reference. Since the release of the chicken genome assembly in 2004, a major goal has been to associate sequence variation in the genome with phenotypic (trait) variation, e.g., differences in growth rate or disease resistance. In collaboration with investigators at the University of California at Davis, 23 different tissues from experimental birds were subjected to a number of assays (e.g., traditional and long-read RNA sequencing, sequencing to detect various epigenetic marks) and the results analyzed. Key finding were (1) over 74,000 transcripts were identified with ~40% not previously identified, (2) 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, which may be due in part to the small size of the chicken genome, (3) on average, each chicken gene is linked to 18 enhancers, which are regulatory elements, while each enhancer targets about 3 genes, and (4) tissue-specific enhancers are significantly involved in phenotype changes during domestication and artificial selection (e.g., increased muscle growth). This effort greatly increases the power of the chicken genome, which will ultimately result in more accurate methods to breed and rear poultry. Under Objective 2, we validated the importance of somatic mutations beyond Marek’s disease virus (MDV) infection in the formation of T cell tumors associated with Marek’s disease (MD). Specifically, ~80% of the tumors had mutations in key regions of Ikaros, which is the master regulator of lymphocyte development. To confirm the role of Ikaros, recombinant MDVs (rMDVs) 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 rMDV 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 (other MD driver genes) that lead to unregulated growth. One unexpected result was that the rMDV engineered to express the wild-type allele of Ikaros did not produce tumors in susceptible birds. To test whether this recombinant virus could act as a MD vaccine, in collaboration with an investigator at Simon Fraser University, Vancouver, Canada, this virus was compared to two commercial MD vaccines: HVT, the first generation MD vaccine and CVI988 (aka Rispens), the most current and most protective MD vaccine. We find that rMDV expressing Ikaros provides more and equal protection compared to HVT and CVI988, respectively. This suggests that rMDV expressing Ikaros should be evaluated further as this might be an efficient method to produce highly protective MD vaccines in a single step. Also under Objective 2, gene expression profiles of birds that are MD resistant or susceptible in response to challenge with MDV were systematically examined. Over 100 and over 200 differentially expressed (DE) genes were identified in response to MDV challenge at the early and the post early infection stage of infection, respectively. This information provides experimental evidence on the underlying basis for genetic differences in MD resistance. MicroRNAs (miRNAs) are small, noncoding RNAs transcribed from the genomes of all multicellular organisms and many viruses including MDV, and play key roles in regulating gene expression. To probe if miRNAs are involved in cellular senescence and immortalization status, miRNA profiles of chicken embryo fibroblasts (CEF) were surveyed after isolation and following 21 and 48 passages in culture. A total of 531 known and novel miRNAs were identified among all the samples and of these, 175 and 188 were significantly DE between the primary and the 21 and 48 passaged CEF, respectively. There were 60 miRNAs that were uniquely and DE in the 48-passaged embryo fibroblasts, i.e., the immortalized line of cells, in contrast to CEF. These findings suggest that miRNAs, an epigenetic factor, may be involved with the cellular differentiation and immortalization, which paves the road for further investigation of genetic and epigenetic mechanism underlying the important biological processes of cellular senescence and immortalization. Long non-coding RNAs (lncRNAs) are a class of RNA, which are generally defined as transcripts more than 200 nucleotides in length and are not translated into proteins, however, regulate protein-coding genes and are emerging as an important new player in various diseases, including cancers. Reported studies include those designed to understand the function of lncRNAs and miRNAs in tumorigenesis induced by avian leukosis virus (ALV) or MDV, both tumor-inducing viruses in chicken. However, a systematical understanding of the molecular events that occur during tumorigenesis in the lymphoid leukosis-like lymphomas resulted from co-infections of ALV and MDV was not explored. A comprehensive genomic and bioinformatics analysis was conducted to identify lncRNAs from whole genome RNA sequencing datasets of both lymphoid leukosis-like lymphoma samples and normal control samples. A total of 1,692 lncRNAs were identified of which 39 lncRNAs were detected with significant DE between the tumor and normal samples. This finding is the very first evidence that lncRNAs may be involved in the avian lymphoid leukosis-like lymphoma incidence and development. This finding also provides additional paths for genetic improvement via breeding to better control and prevent such tumorous diseases in poultry and benefit the general public in the future. Also under Objective 2, we investigated the role of miRNAs in MDV-induced bursal/thymic atrophy; the bursa and thymus are primary lymphoid organs that are required to produce B and T cells, respectively. In this study, we performed sequencing of the thymus and bursa from control and MDV-infected chickens that were from either MD-resistant or susceptible lines at 21 days post infection. Sequence analysis in the thymus identified 658 chicken miRNAs in the thymuses of control and MDV-infected birds of both lines. Of these, 453 were novel and 205 were known miRNAs. Comparative analysis between the thymuses of control and infected birds of resistant and susceptible lines identified 78 DE miRNAs that might provide insights into mechanism of thymic atrophy. In addition to some known miRNAs, over 300 novel miRNAs were also identified in bursas of each group that mapped to the chicken genome with 54 showing differential expression between the two chicken lines. Among the DE miRNAs, gga-mir-6631 was highly up-regulated in the thymuses and bursas from the MD resistant line. This result is of interest as this miRNA was previously reported to be involved in programmed cell death, which is one way for the immune response to control intracellular pathogens like viruses. Thus, this miRNA may play an important role in conferring MD genetic resistance. Finally, miRNAs were also surveyed in the skin of MDV-infected susceptible chickens, the only site where infectious virions are produced. Several MDV-encoded miRNAs were identified that were highly up-regulated in the feather follicle epithelial cells (FFE). Future experiments with recombinant MDVs that alter the identified miRNAs will provide insights into the role of virus-encoded miRNAs in virus replication in the FFE, dissemination of the viral particles into the environment, and pathogenicity of MDV. Under Objective 3, we investigated the role of B cells and CD4 and CD8 T cells, which are involved in specific immune responses, in vaccine-mediated protection. In this study, the bursa of day-old chicks was surgically removed to prevent the development of antibodies. These bursectomized birds were vaccinated on day 5 post hatch and then challenged with a virulent strain of MDV 1 week post vaccination. Data provided evidence that vaccination induced 100% protection in the absence of B cells, strongly suggesting that B cells and, thus, antibodies, do not play a role in vaccine-mediated protection. To shed light on the possible role of T cells in vaccine-mediated immunity, we depleted CD4 and/or CD8 T cells using specific antibodies. Once again, the birds with depleted T cells were vaccinated and challenged as the bursectomized birds above. Data showed that vaccination induced 100% protection in the birds with depleted T cells, indicating that T cells also play no necessary role in vaccine-induced protection. In contrast, birds that received total white blood cells, which include B and T cells, from naïve or MD-vaccinated birds were protected for MDV challenge. Thus, there are specific immune cell populations that do respond and are necessary for vaccinal protection. Ongoing studies should provide further insights as to what cells and immune mechanisms are important.

1. Expression of Ikaros. Expression of Ikaros, a cancer driver gene, in a recombinant Marek’s disease virus (MDV) produces a highly protective Marek’s disease (MD) vaccine. Due to the repeated emergence of more virulent MDV strains in MD-vaccinated chicken flocks, there is urgent need to generate more protective MD vaccines. To address this need, ARS researchers in East Lansing, Michigan, and Athens, Georgia, in collaboration with a researcher at Simon Fraser University, Vancouver, Canada, developed a highly protective MD vaccine by generating a recombinant MDV that expresses the wild-type allele of Ikaros. This information provides a very promising method to develop improved MD vaccines which would reduce the amount of feed and waste produced and increase the health and well-being of reared birds.

Review Publications
Yan, Y., Chen, S., Liao, S., Gao, S., Pang, Y., Zhang, X., Zhang, H., Xie, Q. 2022. ALV-miRNA-p19-01 promotes viral replication via targeting dual specificity Phosphatase 6. Viruses.
Cheng, H.H., Warren, W.C., Zhou, H. 2021. Avian genomics. Book Chapter. In: Scanes, Collin G., Dridi, Sami, editors. Sturkie's Avian Physiology. 7th edition. San Diego, CA: Academic Press. p. 7-16.
Heidari, M., Zhang, H., Hearn, C.J., Sunkara, L. 2021. B cells do not play a role in vaccine-mediated immunity against Marek's disease. Vaccine. 10:100128.
Zhou, Z., Xu, J., Li, Z., Lv, Y., Wu, S., Zhang, H., Song, Y., Ai, Y. 2021. Viral deubiquitinases and innate antiviral immune response in livestock and poultry. Journal of Veterinary Medical Science.
Stephan, T., Burgess, S.M., Cheng, H.H., Danko, C., Gill, C.A., Jarvis, E.D., Koepfli, K., Koltes, J.E., Lyons, E., Ronald, P., Ryder, O.A., Schriml, L.M., Soltis, P., Vandewoude, S., Zhou, H., Ostrander, E.A., Karlsson, E.K. 2022. Darwinian genomics and diversity in the tree of life. Proceedings of the National Academy of Sciences(PNAS). 119:4.
Glass, M.C., Smith, J.M., Cheng, H.H., Delany, M.E. 2021. Marek’s disease virus telomeric integration profiles of neoplastic host tissues reveal unbiased chromosomal selection and loss of cellular diversity during tumorigenesis. Genes. 12(10):1630.
Liao, L., Wu, Z., Chen, W., Zhang, H., Li, A., Yan, Y., Xie, Z., Li, H., Lin, W., Ma, J., Zhang, X., Xie, Q. 2021. Anti-CD81 antibodies block vertical transmission of avian leukosis virus subgroup J. Veterinary Microbiology. 264:109293.
Steep, A., Hildebrandt, E.C., Xu, H., Hearn, C.J., Frishman, D., Niikura, M., Dunn, J.R., Kim, T.N., Conrad, S.J., Muir, W.M., Cheng, H.H. 2022. Identification and validation of Ikaros (IKZF1) as a cancer driver gene for Marek’s disease virus-induced lymphomas. Microorganisms. 10(2):401.