Progress 01/23/03 to 09/30/07
Outputs
Progress Report Objectives (from AD-416) Identify and characterize Marek's disease virus-chicken protein-protein interactions that influence the chicken immune response. Approach (from AD-416) Screen MDV proteins using a bacterial two-hybrid system and a chicken cDNA library. Verify putative interactions by in vitro binding assay. For chicken proteins that are involved in the immune response, knockout the MDV gene that encodes the interacting partner. Compare the wild type and MDV mutant for immunological characteristics in cell culture and animals as well as wild type MDV mutant for immunological characteristics in cell culture and animals. Also use immunopurification and mass spectrometry to identify proteins in functional complexes. Significant Activities that Support Special Target Populations This report serves to document research being conducted under a Specific Cooperative Agreement between ARS and Michigan State University. Additional details can be found in the report for the parent project 3635- 31320-007-00D, Genomics and Immunogenetics of Economically Important Traits of Poultry. A bacterial artificial chromosome (BAC) clone was generated that possesses the entire genome of the Marek's disease virus (MDV) Md5 strain. This MDV-BAC clone produces an infectious and fully virulent MDV, and serves as a platform for making defined recombinant MDVs. Previous results suggest that two MDV ORFs (LORF4, R-LORF10) specifically and directly interact with MHC class II molecules (beta and invariant chains), which may account for the novel up-regulation of class II on the cell surface of MDV-infected cells. In order to investigate the role of these two viral proteins, three sets of recombinant MDVs that disrupted the viral ORFs (LORF4 only, both copies of R-LORF10 only, and both LORF4 and R-LORF10) were generated using the MDV-BAC clone. Recombinant viruses lacking R-LORF10 showed reduced virus plaque formation in tissue culture, less class II expression on the surface, and reduced spread to other chickens. Furthermore, RP9 cells expressing R- LORF10 showed increased MHC class II expression at their cell surface. This clone and resulting information greatly aids scientists in their ability to understand how the virus infects and induces tumors in chickens, which should result in better vaccines and more disease- resistant chickens. Weekly meetings are held between the ARS and Michigan State University laboratories, which greatly aids in the monitoring of this project.
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Progress 10/01/05 to 09/30/06
Outputs
Progress Report 4d Progress report. This report serves to document research conducted under a specific cooperative agreement between ARS and Michigan State University. Additional details of research can be found in the report for the parent CRIS 3635-31320-007-00D Genomics and Immunogenetics of Economically Important Traits of Poultry. Infectious bacterial artificial chromosome (BAC) clones were generated from low passage Marek's disease virus (MDV) strains Md5 and Md11. The BAC-derived Md5 virus is fully virulent and can transmit to other birds like the parental strain. Though the parent Md11 strain is virulent, only one BAC clone of the 68 characterized reconstituted oncogenic MDV in animals. These clones greatly aid scientists in their ability to understand how the virus infects and induces tumors in chickens, which should result in better vaccines and more disease-resistant chickens.
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Progress 10/01/04 to 09/30/05
Outputs
4d Progress report. This report serves to document research conducted under a specific cooperative agreement between ARS and Michigan State University. Additional details of research can be found in the report for the parent CRIS 3635-31320-007-00D Genomics and Immunogenetics of Economically Important Traits of Poultry. Infectious bacterial artificial chromosome (BAC) clones were generated from low passage Marek's disease virus (MDV) strain Md11. Though the parent Md11 strain is virulent, only one BAC clone of the 68 characterized reconstituted oncogenic MDV in animals. This clone, designated Md11gDloxP5-12, is the first MDV BAC clone that maintains virulence although the disease incidence was low compared to the original Md11 strain. Interestingly, this clone contains a portion of the duck genome, which must have been incorporated during the short time that the virus was initially isolated in the laboratory using duck cells. This result shows that MDV can acquire
host genes including some that may be advantageous to the virus. This information suggests that the increased pathogenicity of MDV field strains is due in part by the "pirating" of host genes, which may lead to novel methods of MD control.
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Progress 10/01/03 to 09/30/04
Outputs
4. What were the most significant accomplishments this past year? D. Progress Report. This report serves to document research conducted under a specific cooperative agreement between ARS and Michigan State University. Additional details of research can be found in the report for the parent CRIS 3635-31320-007-00D Genomics and Immunogenetics of Economically Important Traits of Poultry. Infectious bacterial artificial chromosome (BAC) clones were generated from low passage Marek's disease virus (MDV) strain Md11. Though the parent Md11 strain is virulent, only one BAC clone of the 68 characterized reconstituted oncogenic MDV in animals. This clone, designated Md11gDloxP5-12, is the first MDV BAC clone that maintains virulence although the disease incidence was low compared to the original Md11 strain. Md11gDloxP5-12-derived MDV replicated to levels comparable to Md11 in infected chickens during the early phase of infection but not in the later stages. The complete
sequence of Md11gDloxP5-12 was determined. While most of the Md11gDloxP5-12 insert was identical or very similar to the reported Md5 MDV sequence, the entire terminal repeat short segment was replaced by a 7.6 kb sequence that exists in the original Md11 virus stock and appears to be derived from a region of the duck genome, apparently during earlier passage of Md11 on duck cells. In addition, UL41, encoding the MDV host shutoff protein, carried a non-synonymous nucleotide sequence difference in Md11gDloxP5-12 compared to the parent Md11 and reported Md5 sequences. Finally, as this clone can be easily manipulated, it allows us a tool to identify viral genes responsible for pathogenesis, and extend our specific virus-host protein interactions.
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