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

Location: Endemic Poultry Viral Diseases Research

2021 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. 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.

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
For Objective 1, in collaboration with investigators at the University of California at Davis, the chicken genome was annotated using tissues from birds located at ARS facilities in East Lansing, Michigan. Based on eight tissues, our results indicate 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 smaller size of the chicken genome, which is one third that of a typical mammal, or that chicken enhancers are more flexible. This information is vital in the fundamental understanding of chicken biology and, in particular, genetic improvement of commercial poultry. For Objective 2, in collaboration with an investigator at Simon Fraser University, Vancouver, Canada, recombinant Marek’s disease viruses (MDV) were constructed to express either the wild-type allele of Ikaros or a mutant allele found in Marek’s disease (MD) tumors; Ikaros is a known cancer driver gene in humans. As expected, birds challenged with MDVs expressing the mutant Ikaros allele had significantly higher disease incidence including more tumors. This result proves that Ikaros is a cancer driver gene for chickens as well. This result also supports our “two hit” hypothesis for MDV-induced oncogenesis, which is that both (1) the MDV oncogene (Meq) and (2) somatic mutations in MD cancer driver genes are necessary. MicroRNAs (miRNAs) are small, noncoding RNAs transcribed from the genomes of all multicellular organisms and some viruses that 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 differential expressed between the primary and the 21 and 48 passaged CEF, respectively. There were 60 miRNAs that were uniquely and differentially expressed in the 48-passaged embryo fibroblasts, that is, the immortalized line of cells, in contrast to the primary 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. MicroRNAs were also surveyed in the skin of Marek’s disease virus (MDV)-infected susceptible chickens. Several MDV-encoded miRNAs were identified that were highly up-regulated in the feather follicle epithelial cells (FFE) of the skin. Deletion or insertional mutation of the identified miRNAs 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. For Objective 3, to further examine the role of specific cells in vaccine mediated protection, chicks (donors) were vaccinated at one week of age and their total white blood cells isolated and transferred 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 white blood cells transfer. Tissue samples (spleen, skin, thymus, and bursa) were collected at 5, 10, and 20 days post challenge for DNA and RNA isolation, gene expression analysis, and immunohistochemistry. At termination, the recipient birds did not exhibit any symptoms of Marek’s disease (MD) and were fully protected when compared to the non-vaccinated and challenged birds. Our previous studies have also revealed that B and T cells do not play an essential role in vaccine-mediated protection to MD. This prediction was verified by transferring total leukocytes depleted of B and T cells via magnetic beads coated with B and T cells specific antibodies. These result support the important role of the innate immune system in the initial steps of vaccine-mediated immunity.

Accomplishments

Review Publications
Kern, C., Ying, W., Xu, X., Pan, Z., Halstead, M., Chanthavixay, G., Saelao, P., Waters, S., Xiang, R., Chamberlain, A., Korf, I., Delany, M.E., Cheng, H.H., Medrano, J.F., Van Eenennaam, A.L., Tuggle, C.K., Ernst, C., Flicek, P., Quon, G., Ross, P., Zhou, H. 2021. Functional annotations of three domestic animal genomes provide vital resources for comparative and agricultural research. Nature Communications. 12:1821. https://doi.org/10.1038/s41467-021-22100-8.
Umthong, S., Dunn, J.R., Cheng, H.H. 2020. Depletion of CD8aß+ T cells in chickens demonstrates their involvement in protective immunity towards Marek’s disease with respect to tumor incidence and vaccinal protection. Vaccines. 8(4):557. https://doi.org/10.3390/vaccines8040557.
Bailey, R.I., Cheng, H.H., Chase-Topping, M., Mays, J.K., Anacleto, O., Dunn, J.R., Doeschl-Wilson, A. 2020. Pathogen transmission from vaccinated hosts can cause dose-dependent reduction in virulence. PLoS Biology. 18(3):e3000619. https://doi.org/10.1371/journal.pbio.3000619.
Bai, Y., Yuan, P., Luo, J., Zhang, H., Ramachandran, R., Miao, H., Yang, N., Song, J. 2020. Adiponectin and its receptor genes' expression in response to MDV infection of White Leghorns. Poultry Science. 99(9):4249-4258. https://doi.org/10.1016/j.psj.2020.06.004.
Liao, Z., Zhang, X., Song, C., Lin, W., Chen, Y., Xie, Z., Chen, S., Nie, Y., Li, A., Zhang, H., Li, H., Li, H., Xie, Q. 2020. ALV-J inhibits autophagy through the GADD45ß/MEKK4/P38MAPK signaling pathway and mediates apoptosis following autophagy. Cell Death & Disease. 11:684. https://doi.org/10.1038/s41419-020-02841-y.
Hoencamp, C., Dudchenko, O., Elbatsh, A.M., Brahmachari, S., Raaijmakers, J.A., Van Schaik, T., Sedeño Cacciatore, A., Contessoto, V., Hildebrandt, E.C., Cheng, H.H., Lieberman Aiden, E., Rowland, B.D. 2021. 3D genomics across the tree of life reveals condensin II as a determinant of architecture type. Science. 372(6545):984-989. https://doi.org/10.1126/science.abe2218.
Tixier-Boichard, M., Fabre, S., Dhorne-Pollet, S., Goubil, A., Acloque, H., Vincent-Naulleau, S., Ross, P., Wang, Y., Chanthavixay, G., Cheng, H.H., Catherine, E., Leesburg, V.L., Giuffra, E., Zhou, H. 2021. Tissue resources for the functional annotation of animal genomes. Frontiers in Genetics. 12:666265. https://doi.org/10.3389/fgene.2021.666265.
Yan, Y., Zhang, H., Gao, S., Zhang, H., Zhang, X., Chen, W., Lin, W., Xie, Q. 2021. Differential DNA methylation and gene expression between ALV-J-positive and ALV-J-negative chickens. Frontiers in Veterinary Science. 8:659840. https://doi.org/10.3389/fvets.2021.659840.
Bai, H., He, Y., Ding, Y., Chu, Q., Lian, L., Heifetz, E.M., Yang, N., Cheng, H.H., Zhang, H., Chen, J., Song, J. 2020. Genome-wide characterization of copy number variations in the host genome in genetic resistance to Marek's disease using next generation sequencing. BMC Genetics. 21:77. https://doi.org/10.1186/s12863-020-00884-w.