Location: Endemic Poultry Viral Diseases Research
2024 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, we previously performed whole genome, ~30x coverage, PacBio long-read sequencing at and assembly of the inbred ADOL line 15I5, which provides the non- major histocompatibility complex (MHC) background genome to our MHC congenic lines. The assemblies allowed us to identify germline T-cell receptor (TCR) variants present for downstream TCR repertoire sequence analysis. During this reporting period, we have obtained similar 30x, PacBio whole-genome, long-read sequencing data as well as ~8x coverage Illumina paired end 150bp data for the 7 MHC congenic lines including the MD-susceptible B*19 and MD-resistant B*21 lines. These data have confirmed the overall congenic nature of the MHC congenic lines. Additionally, we have collected flow sorted CD3+ and CD8+ T cells from the B*19 and B*21 MHC congenic lines for TCR repertoire sequencing analysis and obtained DNA for TCR haplotype analysis from the B congenic and a commercial outbred W36 line of layer chickens, which we will use in amplicon sequencing assays to estimate TCR diversity and support the TCR repertoire sequence analysis.
For Objective 2, we have completed all passages of Marek’s disease virus (MDV) in chickens using SPF and commercial layers to examine how host genetic resistance influences MDV transmission and evolutionary dynamics. The transmission design allows for detailed investigation about the direct protective effects of host resistance as well as the indirect protective effects on naïve contact birds. Every bird was sampled three times including at introduction of the recipients, at removal of the recipients and at termination. The results thus far indicate natural virus transmission is greatly diminished upon each subsequent passage and there is a strong dose response in severity of lesions in naïve birds.
Also, under Objective 2, detections of microRNA expressions in the cultured embryo fibroblasts of the inbred line 63 and line 72 chickens were successful. Therefore, the subsequent evaluation of the targeted microRNAs and their expressions will be ready to conduct using the line 63 and 72 chicken embryo fibroblast (CEF) cultures, instead of any other cell lines. Use of the line 63 and 72 CEF culture to evaluate the targeted microRNA function in relation to vaccine efficacy should be more relevant, in contrast to other cell lines, since the drastic differences in vaccine protection and those targeted microRNAs were both identified in the line 63 and line 72 chickens post vaccination and Marek’s disease virus challenge.
Under Objective 3, animal studies for Line 63 and Line 72 TCR repertoire sequencing of naïve, challenged and vaccinated birds were postponed for a second time due to the ADOL lab closure and personnel movement to USNPRC; these animal studies will be performed at USNPRC during the upcoming reporting period. Upcoming TCR repertoire sequencing for Objective 1 studies will support bioinformatics pipeline development for sequence analysis in Objective 3.
In addition, under Objective 3, all Marek’s disease virus (MDV) strains that have been isolated from groups of chickens that were exposed to passaged virus have been pathotyped alongside the original virus that was inoculated into the passage 1 chickens. We have not detected any increase in virulence over the starting virus, which is in contrast to results from previous work at our laboratory where viruses were isolated from each passage, selected for virulence, and then injected into the next passage. This suggests that virus evolution is much slower in our laboratory model that simulates natural transmission by inhalation.
In Objective 4, we performed a replicate of our experiments to compare incidence of infectious laryngotracheitis (ILT) in chickens with different host resistance. Using our unique genetic lines (including the major histocompatibility complex (MHC) B*2, B*5, B*12, B*13, B*19 and B*21 congenic lines), we evaluated the MHC haplotype effect against ILT incidence post 63140 and 1874c5 ILT strain virus challenge. In addition, our Line 63 and 72 birds, which carry the same MHC but differ in non-MHC genome, 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 63 birds, however, showed higher resistance compared to the Line 72. 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.
Also under Objective 4, a pilot study was successfully completed, and a specific strain of infectious bursal disease virus was selected for the subsequent vaccination and challenge trial to elucidate the differences of preventing infectious bursal disease in response to vaccination and the virus challenge among the different genetic lines of chickens.
An animal trial was successfully conducted using sampled experimental chicks from 7 ADOL B-congenic lines, representing the major histocompatibility complex B*2, B*5, B*12, B*13, B*15, B*19, and B*21 haplotypes, respectively, and two highly inbred lines, the line 63 and line 72, which are B*2 haplotype, to evaluation host genetics influence on infectious bursal disease (IBD) vaccine protection against an infectious bursal disease virus challenge. Visual differences in both body weight and lymphoid organ sizes, including bursae and spleens, were observed during necropsy. The weighted data and sample histology data connected this year are still under analyses. The findings, when confirmed, would provide the second piece (we reported the first piece of evidence that host genetics modulate Marek’s disease vaccine protection) of experimental evidence that host genetics does play a role in modulating IBD vaccine protection efficacy in chickens. This basic concept should be important to knowledge-based vaccine development in the near future.
Accomplishments
1. Rare cell line development and epigenetic characterization of the developed cell line. The cell line is known as ZS-1, which was developed from the chicken embryo fibroblasts (CEFs) of a genetic line of birds, known as 0.TVB*S1 developed at ADOL. It is commonly believed that pure CEFs in cultural condition cannot continue to go over 10-15 passages unless it is subjected to some sort of stimulant reagents, such as virus inoculation. Cell lines that are developed from CEFs without use of any stimulant reagent are rare events and are known as Immortalized Cell Lines, which are critical for biomedical research. The mechanisms underlying of the immortalization of such events are poorly understood. ARS researchers in Athens, Georgia, recently deep sequenced the ZS-1 immortalized cell line and three samples of the original CEFs for non-coding small RNAs. Over 400 microRNAs (miRNAs) were identified in the ZS-1 cell line, and 188 of those miRNAs were differentially expressed between the ZS-1 and the CEFs samples. Further bioinformatics analyses indicated that the differentially expressed miRNAs might highly likely modulate the cell immortalization processes and contribute to the maintenance of the immortalization state of the cell line. This is the very first piece of experimental evidence that elucidated the epigenetic factor, miRNAs, involvement in the rare event of cell immortalization. This finding should advance the basic understanding of the rare event and benefit the industry in development of such rare and useful reagents.
Review Publications
He, Y., Taylor, R.L., Bai, H., Ashwell, C.M., Zhao, K., Li, Y., Sun, G., Zhang, H., Song, J. 2023. Transgenerational epigenetic inheritance and immunity in chickens that vary in Marek's disease resistance. Poultry Science. 102:12. https://doi.org/10.1016/j.psj.2023.103036.
Rice, E.S., Alberdi, A., Alfieri, J., Athery, G., Balacco, J.R., Bardou, P., Blackmon, H., Charles, M., Cheng, H.H., Fedrigo, O., Fiddaman, S., Formenti, G., Frantz, L., M. Thomas, G.P., Hearn, C.J., Jarvis, E.D., Klopp, C., Marcos, S., Velez-Irizarry, D., Xu, L., Warren, W.C., Mason, A.S. 2023. A pangenome graph reference of 30 chicken genomes allows genotyping of large and complex structural variants. BMC Biology. https://doi.org/10.1186/s12915-023-01758-0.
Zhang, L., Xie, Q., Chang, S., Ai, Y., Dong, K., Zhang, H. 2024. Epigenetic factor microRNAs likely mediate vaccine protection efficacy against lymphomas in response to tumor virus infection in chickens through target gene involved signaling pathways. Veterinary Sciences. 11(4):139. https://doi.org/10.3390/vetsci11040139.
Fiddaman, S., Klopp, C., Charles, M., Bardou, P., Lebrasseur, O., Derks, M., Schauer, J., Reimer, C., Geibel, J., Gheyas, A., Smith, A., Schnabel, R., Martin Cerezo, M., Nishibori, M., Godinez, C.P., Layos, J.N., Larson, G., Ng'Ang'A, I., Muir, W., Lange, M., Wright, D., Simianer, H., Cheng, H.H., Weigend, S., Warren, W., Crooijmans, R., Hanotte, O., Smith, J., Tixier-Boichard, M., Frantz, L. 2023. Chicken genomic diversity consortium: large-scale genomics to unravel the origins and adaptations of chickens. Cytogenetics and Genome Research. https://doi.org/10.1159/000529376.
Warren, W.C., Fedrigo, O., Tracey, A., Mason, A.S., Formenti, G., Perini, F., Wu, Z., Murphy, T., Schneider, V., Stiers, K., Rice, E.S., Coghill, L., Anthony, N., Okimoto, R., Carroll, R., Mountecastle, J., Balacco, J., Haase, B., Yang, C., Zhang, G., Smith, J., Dreschler, Y., Cheng, H.H., Howe, K., Jarvis, E. 2023. Multiple chicken (Gallus gallus) genome references to advance genetic variation studies. Cytogenetics and Genome Research. https://doi.org/10.1159/000529376.
Kim, T.N., Hearn, C.J., Heidari, M. 2024. Efficacy of recombinant Marek’s disease vaccine 301B/1 expressing membrane-anchored chicken interleukin-15. Avian Diseases. 68(2):117-128. https://doi.org/10.1637/aviandiseases-D-23-00068.
