Progress 06/01/04 to 05/30/08
Outputs
OUTPUTS: Progress was made on a number of objectives. First, we were able to reproducibly demonstrate that we could isolate material using laser capture microdissection (LCM), and obtain quality results with our DNA microarrays that were confirmed by qRT-PCR. This result alleviates our concerns over limited material to work with. And it shows that are genes that are associated with resistance and susceptibility to Marek's disease. For example, we find that MHC class II genes are surprisingly upregulated in Marek disease virus (MDV) infected cells, which cannot be accounted for by the production of interferons. This is the only virus that we know which does this, and may reflect the unique cell-associated and lymphocyte-specific properties of MDV. Second, we addressed another problem, which are missing values in DNA microarray datasets. Using computational methods that estimate missing values, we can demonstrate meaningful analysis results, which extend our abilities to extract useful results. In short, the experiments are progressing well and materials are being gathered to replicate our initial findings. And third, we had generated fully infectious and virulent MDV BAC clones. The cloning of the entire viral genome combined with the ability to manipulate the sequence at the single base level allows us to make very specific and defined recombinant viruses. As part of this project, we have developed recombinant MDV-BAC clones in which each of several open reading frames (ORFs) are expressed as fusion products with tandem affinity purification (TAP) tags. These TAP tags allow virus-host protein complexes from cultured chicken cells to be recovered from whole cell lysates under near-physiological conditions. Through the use of liquid chromatography-tandem mass spectrometry technology, these studies, when completed, will allow identification of viral and host proteins interacting with each MDV ORF and provide targets for further examination of the role of these proteins in resistance to viral infection and/or pathogenesis. PARTICIPANTS: H. Cheng (Lead) provided overall supervision of the project. J. Burnside and R. Morgan performed the microarray analyses,. H. Hunt, T. Kim, and M. Niikura lead efforts on Marek's disease virus BAC cloning and immunological studies. M. Ouyang did all the bioinformatics. TARGET AUDIENCES: Poultry breeding companies, poultry vaccine companies, and scientists working on poultry or viruses. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Identifying genes that confer resistance to Marek's disease is important for the poultry industry to continue improvement in genetic resistance as well as for scientists to understand the biological pathways. We were able to demonstrate that we can obtain reproducible results from a limited number of infected cells. Furthermore, computation methods were developed that could account for missing data points. These two advances suggest that we have the techniques in place to address our biological questions. Furthermore, we have demonstrated potential pathways that the virus uses to circumvent the chicken's immune system. Finally, by cloning MDV genomes, we could provide direct evidence that host genome acquisition by MDV actually occurs during virus replication, and that one or more such MDV genomes with host sequences may exist within MDV viral stocks which tend to be polyclonal, due to the cell-associated nature of its infection process. The infectious clones of the virus provided the complete sequence for comparative studies, and a platform for understanding virus gene function and developing new recombinant vaccines in the future.
Publications
- Niikura, M., Dodgson, J, and Cheng, H.H. 2006. Stability of Marek's disease virus 132 bp repeats during serial in vitro passages. Archives of Virology 151:1431-1438.
- Cogburn, L.A., Porter, T.E., Duclos, M.J., Simon, S.C., Burgess, S.C., Zhu, J.J., Cheng, H., Dodgson, J.B., and Burnside, J. 2007. Functional genomics of the chicken - a model organism. Poultry Sci. 86: 2059-2094.
- Niikura, M., Kim, T., Hunt, H.D., Burnside, J., Morgan, R.W., Dodgson, J.B., and Cheng, H.H. 2007. Marek's disease virus up-regulates major histocompatibility complex class II cell surface expression in infected cells. Virology 359:212-219.
- Jornsten, R., Ouyang, M., and Wang, H.Y. 2007. A meta-data based method for DNA microarray imputation. BMC Bioinformatics 8:109.
- Mao, W., Niikura, M., Silva, R.F., and Cheng, H.H. 2008. Quantitative evaluation of viral fitness due to a single nucleotide polymorphism in the Marek's disease virus UL41 gene via an in vitro competition assay. J. Virol. Methods 148:125-131.
- Cheng, H., Niikura, M., Kim, T., Mao, W., MacLea, K.S., Hunt, H., Dodgson, J., Burnside, J., Morgan, R., Ouyang, M., Lamont, S., Dekkers, J., Fulton, J., Soller, M., and Muir, W. 2008. Using integrative genomics to elucidate genetic resistance to Marek's disease in chickens. Developments in Biologicals (Karger, Basel), Vol 132.
- Cheng, H.H. Integrating genomics to understand the Marek's disease virrus-chicken host-pathogen interaction. 2008. In Genomics of Disease, 23rd Stadler Symposium. P. Gustafson, G. Stacey, and J. Taylor, eds., in press.
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Progress 10/01/06 to 09/30/07
Outputs
The cDNA microarrays were found to produce reliable, reproducible results only with highly expressed genes. This was due in part to the caveats of cDNA arrays, but also with the repeated amplifications needed to obtain adequate amounts of RNA from laser captured material. Affymetrix GeneChip arrays were made available and we compared cDNA to Affymetrix arrays and found the Affymetrix produced more reliable, reproducible results and extended the list of differentially expressed genes to include moderately and low abundantly expressed genes. However, we were unable to obtain sufficient laser captured RNA to apply to these arrays. The Affymetrix arrays were used to compare gene expression profiles in spleens from resistant and susceptible birds infected with MDV. The Berkeley RMA Express software was implemented for quality control evaluation and analysis. Differential expression of a subset of genes was confirmed using qRT-PCR. Data are now being annotated and analyzed
further. Previously we had shown that MDV has a novel feature in that MHC class II is upregulated on the cell surface of infected cells. The mechanism may be controlled by two MDV proteins, LORF4 and R-LORF10, that directly interaction with MHC class II β chain and Ii, respectively. To test whether these MDV proteins are necessary for MHC class II upregulation, recombinant MDVs were generated that lacked LORF4 or R-LORF10. In vitro, both viruses show delayed and greatly reduced amounts of class II on the cell surface.
Impacts
Identifying genes that confer resistance to Marek's disease is important for the poultry industry to continue improvement in genetic resistance as well as for scientists to understand the biological pathways. We were able to demonstrate that we can obtain reproducible results from a limited number of infected cells. Furthermore, computation methods were developed that could account for missing data points. These two advances suggest that we have the techniques in place to address our biological questions. Furthermore, we have demonstrated one potential pathway that the virus uses to circumvent the chicken's immune system.
Publications
- Cogburn, L.A., Porter, T.E., Duclos, M.J., Simon, S.C., Burgess, S.C., Zhu, J.J., Cheng, H., Dodgson, J.B., and Burnside, J. 2007. Functional genomics of the chicken - a model organism. Poultry Sci. 86: 2059-2094.
- Niikura, M., Kim, T., Hunt, H.D., Burnside, J., Morgan, R.W., Dodgson, J.B., and Cheng, H.H. 2007. Marek's disease virus up-regulates major histocompatibility complex class II cell surface expression in infected cells. Virology 359:212-219.
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