2013 Annual Report
1a.Objectives (from AD-416):
Objective 1: Enhance the chicken genetic map and integration with the genome sequence specifically chromosomes 16, 25, 29-38, and W to enhance genetic/genomic opportunity for control of MD.
Objective 2: Identify and characterize chicken genes and pathways that confer resistance to Marek’s disease or vaccinal response including determination of the relationship between gut microbes and MHC haplotype of the chicken or immune response to MDV infection.
Subobjective 2.1: Genomic selection for MD resistance in chickens.
Subobjective 2.2: Genetic resistance and Meq regulated pathways.
Subobjective 2.3: Determine if host MHC haplotype, MD genetic resistance, or MDV infection alters gut microbial composition.
Subobjective 2.4: Identify genes conferring MD vaccinal protective efficacy.
Subobjective 2.5: Identify differentially expressed host genes and DNA methylation patterns induced by MD vaccination.
Objective 3: Development of a laboratory model for MDV evolution to higher virulence to develop effective industry tools for the control of Marek’s disease.
Subobjective 3.1: Identify the genomic changes in MDV selected through resistant and susceptible host genotypes.
Subobjective 3.2: Compare in vivo virus replication of the Md5B40BAC with the Md5B40BAC viruses passed in through the MHC-B21 and MHC-B13 hosts.
Subobjective 3.3: Determine if in vivo back passage will increase the pathotype of the Md4B40BAC.
1b.Approach (from AD-416):
Control of Marek’s disease (MD), a T-cell lymphoma induced by the Marek’s disease virus (MDV), is of particular concern to the poultry industry. Since the 1960s, MDV has evolved to higher virulence probably due to the selective pressure of MD vaccines that do not prevent viral replication or spread. Consequently, there is a need to (1) understand how MDV evolves and evades the immune system, and (2) develop alternative strategies to augment current MD control methods. In this project, we define three interrelated objectives to help achieve these goals. 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. Second, we use genomic approaches to identify quantitative trait loci (QTL) and candidate genes that confer genetic resistance or vaccinal immunity to MD. In addition, we explore the potential role of the gut microbial community on the chicken immune response. And third, we evaluate an in vivo model for MDV virulence evolution and if successful, ask the question whether increased virulence is restricted to specific major histocompatibility (MHC) haplotypes. Our efforts are greatly enhanced by the availability of characterized inbred chicken lines and genomic tools especially next generation sequencing (NGS). If successful, this project will provide a number of products including (1) more genetic markers and an enhanced genetic map, (2) candidate genes conferring MD resistance or vaccinal response for evaluation in commercial breeding lines, (3) a laboratory model for MDV evolution, and (4) specific knowledge on how MDV evolves and evades the host. Ultimately, the poultry industry and US consumers will benefit by the production of safe and economical products.
This report shows that substantial progress was made on all three objectives and their sub-objectives. Under Objective 1, in collaboration with investigators at Washington University School of Medicine in St. Louis, Michigan State University, and Cobb-Vantress, DNA libraries of different sizes were prepared and sequenced using an alternative next generation sequencing technology. Along with the genotype data (15K SNP chip) from 1,000+ birds, we should be able to improve both the chicken genetic map and the sequence assembly, both of which provide the basis and tools for understanding chicken biology including genetics and immunology of disease and disease resistance. For Objective 2, using a combination of molecular genetics techniques and computational analyses, we identified the motifs and genes directly regulated by Marek’s disease virus (MDV) protein Meq, a transcription factor and the viral oncogene as well as revealing host pathways that are altered by the expression of Meq and by coupling with genetic polymorphisms, we identified a number of genes that contribute to genetic resistance to Marek’s disease (MD). In a related approach that also takes advantage of the growing importance of variation in transcriptional regulation in complex traits, using a genome-wide scan with genetic markers (SNPs) exhibiting allele-specific expression (ASE) in response to MDV infection, we demonstrate that these SNPs account for all the genetic variation between two inbred experimental lines that differ greatly in MD genetic resistance. Tests are currently underway to validate this conclusion based on predictions derived from these ASE SNPs. We also compared the turkey herpesvirus (HVT) vaccine, a low efficacy MD vaccine, to CVI988/Rispens, a high efficacy one, in the ability to protect against highly virulent MDV strains in commercial layer-type chickens. Our data confirmed what has been observed in our experimental lines of chickens, which is HVT conveys equal or greater protection against highly virulent MDV field strains in MD-resistant lines of chickens. This finding suggests that the HVT vaccine might provide similar protection as the CV1988/Rispens vaccine in chicken lines known to be relatively resistant to MD as well as providing a cost savings over the Rispens vaccinations. Projects to explore mechanisms underlying vaccine protective efficacy are underway. For Objective 3, advances were made to enhance our understanding of the chicken immune response when it encounters MDV. We have completed the back passage of a cloned virus in two chicken lines that differ in their MHC haplotype (B13 and B21); this is the genetic locus that has the largest genetic influence on MD resistance. Interestingly, both in vivo passed viruses were more virulent in chickens with the B13 MHC haplotype but neither caused increased disease incidence in chicken with the B21 MHC haplotype. This result suggests that selection for resistance to MD is effective in controlling disease but virus evolution continues in the MD resistant chicken lines and this may lead to future shifts in virulence.
Genomic screen identifies the chicken genes and pathways targeted by the Marek’s disease virus Meq oncoprotein. Marek’s disease (MD) is an economically significant disease in chickens that is caused by the highly oncogenic Marek’s disease virus (MDV). A major unanswered question is the mechanism of MDV-induced tumor formation. Using modern genomic approaches, an ARS scientist in East Lansing, Michigan identified the binding sites, genes, and biological pathways that are targeted by the MD virus oncogene termed Meq. This basic knowledge will aid future efforts to improve MD genetic resistance in elite poultry flocks or design improved vaccines to reduce the loss of commercial chickens to MDV, thereby economically benefitting the poultry industry and consumers.
Variation in transcription regulation is the underlying mechanism for variation in genetic resistance to Marek’s disease (MD). Enhancing genetic resistance to Marek’s disease in chicken, a complex trait controlled by many genes, is highly desired to augment current controls through vaccines and biosecurity. Using genetic markers spaced throughout the genome, an ARS scientist in East Lansing, Michigan, in collaboration with another scientist at Purdue University demonstrated that those markers associated with variation in gene expression account for all the genetic variation in an experimental population. If confirmed in commercial poultry lines, this would prove that genomic selection with these types of genetic markers would be very efficient at improving elite poultry against MD, and is likely to be the case for genetic resistance to other infectious pathogens. Ultimately, poultry companies and US consumers would benefit with healthier birds yielding more economical poultry products.
Yuan, P., Yu, Y., Luo, J., Tian, F., Zhang, H., Chang, S., Ramachandran, R., Song, J. 2012. Lipoprotein metabolism differs between Marek's disease susceptible and resistant chickens. Poultry Science. 91:2598-2605. Available: http://ps.fass.org/content/91/10/2598.
Hunt, H.D., Dunn, J.R. 2013. The influence of host genetics on Marek's disease virus evolution. Avian Diseases. 57(2):474-482.
Mitra, A., Luo, J., Zhang, H., Cui, K., Zhao, K., Song, J. 2012. Marek’s disease virus infection induces widespread differential chromatin marks in inbred chicken lines. Biomed Central (BMC) Genomics. 2012(13):557. Available: http://www.biomedcentral.com/1471-2164/13/557.
Luo, J., Yu, Y., Mitra, A., Chang, S., Zhang, H., Liu, G., Yang, N., Song, J. 2013. Genome-wide copy number variant analysis in inbred chicken lines with different susceptibility to Marek’s disease. Genes, Genomes, and Genomics. 3:217-223. Available: http://www.g3journal.org/lookup/suppl/doi:10.1534/g3.112.005132/-/DC1.
Ji, J., Li, H., Zhang, H., Xie, Q., Shang, H., Ma, J., Bi, Y. 2012. Complete genome sequence of an avian leukosis virus isolate associated with hemangioma and myeloid leukosis in egg-type and meat-type chickens. Journal of Virology. 86(19):10907-10908.
Kumar, S., Kunec, D., Buza, J.J., Chiang, H.I., Zhou, H., Subramaniam, S., Pendarvis, K., Cheng, H.H., Burgess, S.C. 2012. Nuclear Factor kappa B is central to Marek’s Disease herpesvirus induced neoplastic transformation of CD30 expressing lymphocytes in-vivo. BMC Systems Biology. 6(123). Available: http://www.biomedcentral.com/1752-0509/6/123.
Luo, J., Chang, S., Zhang, H., Li, B., Song, J. 2013. DNA methylation down-regulates EGFR expression in chicken. Avian Diseases. 57(2):366-371.
Tian, F., Zhan, F., Vanderkraats, N.D., Hiken, J.F., Edwards, J.R., Zhang, H., Zhao, K., Song, J. 2013. DNMT gene expression and methylome in Marek’s disease resistant and susceptible chickens prior to and following infection by MDV. Epigenetics. 8(4):431-444.
Perumbakkam, S., Muir, W.M., Black Pyrkosz, A.A., Okimoto, R., Cheng, H.H. 2013. Comparison and contrast of genes and biological pathways responding to Marek’s disease virus infection using allele-specific expression and differential expression in broiler and layer chickens. Biomed Central (BMC) Genomics. 14:64. Available: http://www.biomedcentral.com/content/pdf/1471-2164-14-64.pdf.
Subramaniam, S., Johnston, J., Preeyanon, L., Brown, C., Kung, H., Cheng, H.H. 2013. Integrated analyses of genome-wide DNA occupancy and expression profiling identify key genes and pathways involved in cellular transformation by the Marek's disease virus oncoprotein Meq. Journal of Virology. 87(16):9016-9029. Available at: http://jvi.asm.org/content/87/16/9016.full.
Mao, W., Kim, T., Cheng, H.H. 2013. Visualization of Marek’s disease virus in vitro using enhanced green fluorescent protein fused with US10. Virus Genes. 47(1):181-183. Available: http://link.springer.com/journal/11262/47/1/page/2.