Research Project: Enhancing Genetic Resistance and Vaccinal Response to Control Marek's Disease, Infectious Laryngotracheitis, and Infectious Bursal Disease in Chicken

Location: Endemic Poultry Viral Diseases Research

2023 Annual Report

Objectives
1. Enhance chicken genomic resources to support genetic selection, immunological efforts, and other strategies to reduce poultry diseases. 1.1. Enhance the chicken genetic map and its integration with the genome assembly. 1.2. Develop complete T cell receptor (TCR) sequences for various haplotypes. 2. Genetic and epigenetic characterization of Marek’s disease (MD) resistance and vaccine protective efficacy. 2.1. Identify specific alleles in cancer driver genes associated with MD genetic resistance. 2.2. Determine if MD genetic resistance contributes to Marek’s disease virus evolution to higher virulence. 2.3. Determine if DNA methylation of specific genes is associated with MD vaccination response and efficacy. 2.4. Define the role of specific viral miRNAs on Marek’s disease virus (MDV) transmission and vaccinal protection. 3. Identify specific immune response genes that confer resistance to Marek’s disease (MD) or improves vaccinal response. 3.1. Determine the role of interleukin-10 (IL-10) on MD genetic resistance. 3.2. Determine the role of TCR diversity in the MD vaccinal response. 3.3. Validate viral genome polymorphisms associated with the ability of the virus to escape immune surveillance. 4. Determine if there is a host component for resistance to infectious bursal disease (IBD) or infectious laryngotracheitis (ILT). 4.1. Determine if lines that vary for MD genetic resistance or major histocompatibility complex (MHC) haplotype influence IBD or ILT disease incidence. 4.2. Determine if host genetics differentiates IBD vaccinal protection. 4.3. Determine if host genetics contributes to ILT vaccinal protection efficacy.

Approach
Poultry is the fastest growing and most consumed meat both in the U.S. and globally. To achieve economic efficiency, birds are raised at very high densities. Since these conditions promote the spread of infectious diseases, the industry relies heavily on biosecurity and vaccines, if available, for disease prevention and control. Marek’s disease (MD), infectious laryngotracheitis (ILT), and infectious bursal disease (IBD) are three poultry viral diseases of high relevance. A common theme to all these diseases is that while current control measures are able to control disease incidence for the most part, the increasing recurrence of outbreaks in vaccinated flocks and the emergence of new field strains strongly suggest that improved or alternative control strategies are needed for long-term and sustainable disease prevention. This project focuses on host genetics for achieving long-term disease control. Herein we have identified four objectives to help achieve this goal, and to provide basic and applied knowledge to reduce diseases incidence as well as improving overall animal health and welfare. First, we continue to provide critical genomic resources. Second, using various genomic and molecular approaches, we identify specific genes or epigenetic modifications that are associated with MD genetic resistance. Third, we identify specific immune response genes that confer resistance to MD or improve vaccinal protection. And fourth, we will advance our knowledge of vaccine efficacy against infectious diseases that is attributable to genetics and epigenetics, which will greatly empower science-based vaccine design and development. If successful, this project will provide (1) a more complete genetic map that will aid in improving the chicken genome assembly, (2) candidate genes and pathways conferring MD resistance or vaccinal response for evaluation in commercial breeding lines, and (3) knowledge on how to reduce ILT and/or IBD disease incidence. Ultimately, both the poultry industry and US consumers will benefit from the production of safe and economical poultry-based products.

Progress Report
Under Objective 1, in collaboration with investigators at Washington University School of Medicine in St. Louis, a pangenome of the domestic chicken was generated to aid researchers in more confidently calling complex structural variants in the genome compared to the earlier versions that were based on a single bird. Specifically, five breeds of broiler and layer domestic chicken with high levels of coverage using long-read sequencing platforms were used. Based on this pangenome, breed specific variants including those associated with immune function and animal health can be demonstrated. This improved genomic tool should greatly aid both fundamental and applied biologists in understanding and improving chicken traits. Additionally, under Objective 1 and 3, we have been characterizing the immune receptor genes of chicken T cells (TCRs), which are important in response to infections and may contribute to resistance against diseases such as Marek’s disease. We have identified TCR genes in 3 model breeds of layer chickens differing in Marek’s disease susceptibility and resistance using long-read sequencing technologies. We have designed new PCR assays, which have been currently used to characterize and validate genetic diversity of TCRs in inbred and outbred birds. Based on our sequencing findings, we demonstrated substantial diversity existing within the TCR genes of the inbred chickens varied in disease resistance. This enhances the fundamental knowledge of poultry immunity and may enable the industry to explore more rational breeding strategies to improve genetic resistance against infectious diseases caused by severe poultry pathogens in the future. For Objective 2, in collaboration with investigators at the Roslin Institute, Edinburgh, Scotland, and Freie Universitat, Berlin, Germany, several large studies involving natural viral transmissions were conducted in the past several years to determine if Marek’s disease virus (MDV) evolves to higher virulence. Each experiment varies a specific factor namely: 1. Using inbred Marek’s disease (MD) susceptible experiment chickens to compare HVT vaccinated and nonvaccinated birds. 2. Using outbred commercial chickens to compare between MD resistant and MD susceptible lines of birds. 3. Using inbred MD susceptible experiment chickens to compare one-tenth dose of HVT vaccinated and nonvaccinated birds. 4. Compare inbred MD susceptible experiment chickens to outbred, MD resistant commercial chickens. 5. Using inbred MD susceptible experiment chickens and one-tenth dose of HVT, to compare MDV versus a recombinant MDV that has a 2-5 times higher mutation rate. For this past year, Experiments 4 and 5 are underway. For Experiments 4 and 5, six and three biological replicates of groups of 10 birds, respectively, were used. For experiment 4 only, half of the birds were vaccinated at hatch. At one day of age, all birds were infected with virulent MDV. At two weeks of age, these birds were used as donors to transmit to another set of birds. This continued for a total of 10 passages. For each bird, blood and feather samples were collected at multiple times points, which were being analyzed now for the amount of virus present. Also, the viruses recovered in passage 10 are being characterized as to whether they are more virulent or not. This information is important for the sustained control of MD in commercial chickens as well as having implications for other diseases that rely on vaccines to control. Also, for Objective 2, one of the goals is to advance the basic understanding on how genetics and epigenetics factors modulate vaccine protective efficacy against virus-induced lymphomas in chickens. To continue with our previous findings of epigenetic factors that are potentially involved with vaccinal protective efficacy, the specific genetics and epigenetics factors that will be focused on in this study are microRNAs (small non-coding RNAs), target genes (coding genes) of the microRNAs, and DNA methylation modifications that might regulate the microRNA and target gene expression levels upon vaccination of chickens. This is an investigation involving multiple steps as designed, which include identification of the specific microRNAs in cells; then functionally manipulate the specific microRNAs in cells under tissue cultural conditions; then identifying the target genes of those microRNAs and DNA methylation modifications. When completed, the findings would elucidate how the specific genetic and epigenetic factors modulate vaccine protection against tumor formation, which should help develop better control measures in disease prevention in chickens. During the past year, protocols have been developed, by which the targeted microRNAs were detected with expression in chicken embryo-fibroblasts under tissue culture conditions. This paves the road for the next steps of this study. Additionally, under Objective 2, we have successfully deleted one MDV-encoded microRNA. MicroRNAs are commonly found in humans, animals, plants, and viruses, which play critical roles in regulation of gene expression and modulate gene function. Now we are in the process of trying to recover it from infected chicken cells. Also, a second known viral microRNA will be deleted from the recombinant MDV within the next few months. Then, the new recombinant viruses with the microRNAs deleted will be tested in challenge studies as vaccines and the effect of deletion will be evaluated for the MDV replication in the feather follicle epithelium cells and dissemination of the virus in the skin tissue. The goal of the study is to decipher the molecular mechanism of MDV-encoded microRNAs in virus pathogenicity and possible development of new recombinant vaccines. Under Objective 3 in conjunction with Objective 2, we have been characterizing the virulence of MDV strains that have been passaged 9 to 10 times through HVT-vaccinated chickens using pathotyping (standardized bird challenges in different vaccine protection groups) to determine whether vaccine pressure causes detectable changes in virulence. We have also been sequencing the genomes of these viruses to look for changes in the passaged viruses in contrast to the initial challenge viruses. We have performed two replicate pathotyping studies on virus materials from Objective 2, Experiment 1, followed by two additional back-passaging studies in unvaccinated birds (currently underway) to obtain higher virus titers for use in pathotyping. Our key finding so far is that serial passage through vaccinated birds progressively restricts virus shedding, resulting in a very low virus challenge, which was correlated with similarly low rates of disease in birds exposed at the end of a vaccinated “exposure chain.” In contrast, non-vaccinated “exposure chains” maintained significant virus shedding and resulted in relatively higher disease incidence. While this has complicated our attempts to pathotype viruses derived from the late passages by necessitating back-passage and re-isolation of viruses, it is, however, an encouraging finding that supports the continued usefulness of available vaccines (such as HVT) in control of MD and provides relevantly new information in support of the industry’s ongoing practices. Also, for Objective 3, we have tested the binding capacity of an antibody that recognizes chicken interleukin 10 (IL-10). IL-10 has a central role in infection by limiting the immune response to pathogens and preventing damage to the host. The test verified the specificity of the antibody after compared with a commercially available anti-chicken IL-10 antibody. The trial and the actual experiment to block the functional activities of IL-10 in chickens followed by vaccination and/or challenge has been scheduled for FY24. Under Objective 4, the role of host genetics in control of two other virus-induced diseases (Infectious bursal disease virus and infectious laryngotracheitis virus) and in modulating vaccine protection against the disease incidence are to be investigated. The findings from studies under this Objective 4 would provide experimental evidence that host genetics plays important roles in disease prevention against infectious bursal disease and infectious laryngotracheitis virus infection in chickens, which should encourage the poultry industry to pursue genetic improvement through selection to enhance genetic resistance and vaccination protection proficiency in poultry. One pilot study was conducted to select a specific strain of infectious bursal disease virus, by which to sufficiently elucidate the differences of host genetics mediated vaccine prevention efficacy in full-scale experiments in the coming years. This study is still ongoing, which should be completed and would provide the expected data in the coming months. Infectious laryngotracheitis (ILT) and MD are caused by two closely related species of avian viruses. Despite similarity of the two viruses and 70+ years of work on genetic differences in host resistance to MD, there has been very little effort in control of ILT through genetic resistance. The genetic resistance to MD involves MHC, also known as the B complex. Using our unique genetic lines (including B*2, B*5, B*12, B*13, B*19 and B*21 congenic lines), we evaluated the MHC effect against ILT incidence post 63140 and 1874c5 strain challenge. In addition, our Line 6 and 7 birds, which carry the same MHC but differ in non-MHC genes, were also tested. Significant differences in clinical signs and viral loads were observed between the genetic lines of chickens, with the Lines of B*2 and B*5 birds as the most resistance ones. The Line 6 birds also showed higher resistance compared to Line 7. These results provide the basis for genetic improvement by selection in breeding flocks, which will empower a better control, in addition to vaccination, of ILT and should benefit the industry and consumers.

Accomplishments
1. Development of a chicken pangenome assembly. Until recently, like in chicken, most researchers have used a genome reference assembly derived from a single individual to identify polymorphisms (genetic variants) in the DNA sequences obtained from the samples that they are investigating. However, while this approach has been useful for cataloging simple variants, it is incapable of confidently calling more complex variation such as those involving larger structure. To address this need, ARS researchers in East Lansing, Michigan, in collaboration with a researcher at University of Missouri, developed a pangeome assembly using five different breeds of chickens and the most current sequencing platforms. The power of this new tool was demonstrated by identifying a structural variant associated with a feathering locus used to sex chickens, which would not have been possible using the earlier reference of a single bird. As chicken is the primary meat consumed in the U.S. and the world, this improved genomic tool should greatly aid both fundamental and applied biologists in understanding and improving chicken traits, and ultimately benefit consumers and society by increasing productivity, health and well-being of reared birds.

2. Establishment of a needed protocol for depletion of chicken immune cells. The depletion of specific immune cells is essential in understanding the biological function and the potential roles of these immune cells in viral infection and vaccine mediated protection. To advance the understanding of the biological function of the specific cell type, one way is to deplete those cells prior to challenge or vaccination in the lab or in bird experiment. ARS researchers in East Lansing, Michigan, generated antibodies against specific types of T-lymphocytes (T cells), which are an important type of cell involved in adaptive immunity. In a T cell depletion study, ARS researchers successfully depleted several types of T cells from a mixed population of spleen cells using these antibodies. The partially depleted T cell population collected from vaccinated birds were then injected into non-vaccinated birds followed by virus challenge to understand the roles of specific types of T cells in vaccine-mediated protection. The depletion of T cells in the lab and in experimental birds followed by vaccination and/or challenge has shed new light on the potential roles of T cells in Marek’s disease development and in vaccine-induced immunity. This finding advanced the basic understanding on specific T cell’s role in relation to virus infection and vaccination protection. This advancement benefits the poultry industry by paving the road for improvement on better vaccine design and vaccination practice.

Review Publications
Warren, W.C., Rice, E.S., Meyer, A., Hearn, C.J., Steep, A., Hunt, H.D., Monson, M.S., Lamont, S.J., Cheng, H.H. 2023. The immune cell landscape and response of Marek's disease resistant and susceptible chickens infected with Marek's disease virus. Scientific Reports. 13. Article 5355. https://doi.org/10.1038/s41598-023-32308-x.
Guan, D., Halsted, M.M., Islas-Trejo, A.D., Goszczynski, D.E., Cheng, H.H., Ross, P.J., Zhou, H. 2022. Prediction of transcript isoforms in 19 chicken tissues by Oxford Nanopore long-read sequencing. Frontiers in Genetics. 13:997460. https://doi.org/10.3389/fgene.2022.997460.
Heidari, M., Zhang, H., Sunkara, L. 2022. MDV-induced differential microRNA expression in the primary lymphoid organ of thymus. Microbial Pathogenesis. 170. Article 105688. https://doi.org/10.1016/j.micpath.2022.105688.
Heidari, M., Zhang, H., Sunkara, L.T., Ahmad, S. 2023. Role of T cells in vaccine-mediated immunity against Marek’s Disease. Viruses. 15. Article 648. https://doi.org/10.3390/v15030648.
Dong, K., Heidari, M., Mays, J.K., Chang, S., Xie, Q., Zhang, L., Ai, Y., Zhang, H. 2022. A comprehensive analysis of avian lymphoid leukosis-like lymphoma transcriptomes including identification of LncRNAs and the expression profiles. PLOS ONE. 17(8):e0272557. https://doi.org/10.1371/journal.pone.0272557.
Zhu, C., Zhang, L., Heidari, M., Sun, S., Chang, S., Qingmei, X., Ai, Y., Dong, K., Zhang, H. 2023. Small RNA deep sequencing revealed microRNAs’ involvement in modulating cellular senescence and immortalization state. Poultry Science. 102(3):102474. https://doi.org/10.1016/j.psj.2022.102474.
Dar, M.A., Ahmad, S.M., Bhat, B.A., Dar, T.A., Ul Haq, Z., Wani, B.A., Shabir, N., Kashoo, Z.A., Ganai, N.A., Heidari, M., Shah, R.A. 2022. Comparative RNA-Seq analysis reveals insights in Salmonella disease resistance of chicken; and database development as resource for gene expression in poultry. Genomics. 114(5):110475. https://doi.org/10.1016/j.ygeno.2022.110475.
Deng, Q., Li, Q., Li, M., Zhang, S., Wang, P., Fu, F., Zhu, W., Wei, T., Mo, M., Huang, T., Zhang, H., Wei, P. 2022. The emergence, diversification, and transmission of Subgroup J Avian Leukosis Virus reveals that the live chicken trade plays a critical role in the adaption and endemicity of viruses to the yellow-chickens. Journal of Virology. 96(17):e0071722. https://doi.org/10.1128/jvi.00717-22.
Hearn, C.J., Cheng, H.H. 2023. Contribution of the TCR beta repertoire to Marek's disease genetic resistance in chicken. Viruses. 15(3):607. https://doi.org/10.3390/v15030607.
Pan, Z., Wang, Y., Wang, M., Wang, Z., Zhu, X., Gu, S., Zhong, C., An, L., Damas, J., Halsted, M., Cheng, H.H., Zhou, H. 2023. An atlas of regulatory elements in chicken: A resource for chicken genetics and genomics. Science Advances. 9:eade1204. https://www.science.org/doi/10.1126/sciadv.ade1204.
Fu, Y., Hu, J., Erasmus, M.A., Zhang, H., Johnson, T.A., Cheng, H. 2023. Cecal microbiota transplantation: Unique influence of cecal microbiota from divergently selected inbred donor lines on cecal microbial profile, serotonergic activity, and aggressive behavior of recipient chickens. Journal of Animal Science and Biotechnology. 14(1)Article 66. https://doi.org/10.1186/s40104-023-00866-9.