Progress 09/01/04 to 08/31/09
Outputs
OUTPUTS: Activities included the training of two graduate students. The research was presented at International Marek's disease Workshop and annual meeting of American Association of Poultry Pathologists (3 presentations). The data was presented in ten high impact refereed publications including, two publications in Journal of Virology, two publications in Vaccine, three publications in Virus Gene, one publication each in Avian Disease, Virus Research and an International Journal. Service included consultation with vaccine manufactures from Merial to potentially commercialize the vaccine product. Collaborations were established with University of California at Davis and Agriculture Research Service laboratory at Michigan, USDA. PARTICIPANTS: The principal investigators who worked on this project are Sanjay Reddy and Blanca Lupiani. Paulette Suchodolski finished her PhD training under Sanjay Reddy and is currently a post-doctoral fellow.Dharani Ajitdoss completed his PhD training under Blanca Lupiani and is currently under residency training in Pathology. Hannah Atkins, an undergraduate student from Penn State University was also trained on this project The work was done in collaboration with partnering institutes at University of California at Davis and Avian Disease and Oncology Laboratory, Agriculture Research Service, USDA. TARGET AUDIENCES: The target audience included poultry virologists and pathologists interested in poultry health. The gained here has relevance not only to animal health but also to human health, as it elucidated the molecular mechanism of herpesvirus pathogenesis. The vaccine products developed in this proposal is of potential commercial use by vaccine manufactures. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Gallid herpesvirus 2 (GaHV-2), commonly known as Marek's disease virus serotype-1 (MDV-1), causes T cell lymphomas in chickens. Vaccines prepared from the attenuated CVI988/Rispens MDV-1 strain currently offer the best protection. Although attenuated CVI988/Rispens is non-oncogenic, it codes for at least two forms of the MDV oncoprotein Meq, and these proteins (CVI-Meq and CVI-LMeq) have not been fully characterized. We showed that both CVI-Meq proteins, like the Meq protein of Md5 (a very virulent oncogenic strain), were capable of transforming Rat-2 and NIH3T3 cells. Both CVI-Meq and CVI-LMeq proteins activated the meq promoter only in the presence of chicken c-Jun (CK-Jun) whereas Md5-Meq activated the same promoter irrespective of CK-Jun co-expression. However, Meq proteins of both Md5 and CVI988 bound the meq promoter in a ChIP assay regardless of whether CK-Jun was co-expressed. To understand the role of Meq DNA binding and transactivation/repression domains in transcription, we constructed three chimeric Meq proteins, namely, Md5-CVI-Meq, CVI-Md5-Meq, and Md5-CVI-L by exchanging domains between Md5 meq and CVI meq genes. Although these chimeric Meq proteins, unlike CVI-Meq proteins, transactivated the meq promoter, the activation was significantly less than Md5-Meq. To determine the role of individual amino acids, point mutations were introduced corresponding to the amino acid changes of CVI-Meq into Md5-Meq. Amino acid residues at positions 71 and 320 of the Md5-Meq protein were found to be important for transactivation of the meq promoter. All three Meq proteins activated the MDV gB, MMP-3 and Bcl-2 promoters and suppressed transcription from the MDV pp38/pp14 bidirectional promoter. Although no significant differences were observed, decreased transactivation activity was observed with CVI-Meq proteins when compared to Md5-Meq. Collectively, the data presented here indicate that CVI-Meq proteins are generally weak transactivators, which might contribute to the non-oncogenic phenotype of CVI988 virus in chickens. We have earlier developed a Meq-null virus, in which the oncogene meq was deleted from a pathogenic strain of MDV. Vaccine efficacy experiments under laboratory conditions indicated that Meq-null virus provided superior protection than CVI988/Rispens when challenged with the very virulent MDV strain. We extended our finding to field conditions by performing three large-scale field experiments, in which seeder chickens were inoculated with a very virulent plus strain of 686, vv+ MDV. Experimental results showed that Meq-null virus was a better or equal vaccine compared to any of the CVI988/Rispens vaccines tested under stringent conditions used including early exposure of day-old vaccinated chicks by contact with maternal antibody positive seeder chickens challenged with a very virulent plus strain of MDV, vv+ 686 virus. In conclusion, results from field trials show that Meq-null virus is a potential candidate vaccine capable of providing protection against challenge with a vv+ MDV strain in commercial chickens under field conditions.
Publications
- Lee L.F, Kreager K.S., Arango J., Paraguassu A., Beckman B., Zhang H., Fadly A., Lupiani B. and Reddy S.M. 2009 Comparative evaluation of vaccine efficacy of recombinant Marek's disease virus vaccine lacking meq oncogene in commercial chickens. Vaccine. Nov 23. (Epub ahead of print]). doi:10.1016/j.vaccine.2009.11.022
- Ajithdoss D., Reddy S.M., Suchodolski P.F., Lee L.F., Kung H.J. and Lupiani B. 2009. In vitro characterization of the Meq proteins of Mareks disease virus vaccine strain CVI988. Virus Research 142 (1-2): 57-67.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: Mareks disease virus (MDV), the etiologic agent of Marek's disease, is a potent oncogenic herpesvirus. MDV is highly contagious and elicits a rapid onset of malignant T-cell lymphomas in chickens within several weeks after infection. MDV genome codes an oncoprotein, Meq, which shares resemblance with the Jun/Fos family of bZIP transcription factors. Similar to Jun, the leucine zipper region of Meq allows the formation of homo- and heterodimers. Meq homo- and heterodimers have different DNA binding affinities and transcriptional activity; therefore, they may differentially regulate transcription of viral and cellular genes. PARTICIPANTS: Suchodolski, P, Texas A&M University Kung, H.J., University of California, Davis Lupiani, B, Texas A&M University Lee, L, Agriculture Research Service TARGET AUDIENCES: Researchers and poultry industry PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
In this study we investigated the role of Meq homodimers in the pathogenicity of MDV by generating a chimeric meq gene, which contains the leucine zipper region of the yeast transcription factor GCN4 (meqGCN). A recombinant virus (rMd5-MeqGCN) containing the chimeric meqGCN gene in place of parental meq was generated with overlapping cosmid clones of Md5, a very virulent MDV strain. The rMd5-MeqGCN virus replicated in vitro and in vivo but was unable to transform T cells in infected chickens. These data provide the first in vivo evidence that Meq homodimers are not sufficient for MDV-induced transformation.
Publications
- Suchodolski, P., Izumiya, Y., Lupiani, B., Ajithdoss, D., Gilad, O., Lee, L.F., Kung, H-J., and Reddy, S.M. (2009). Homodimerization of Mareks disease virus encoded Meq protein is not sufficient for transformation of lymphocytes in chicken. Journal of Virology 83 (2): 859-69.
- Lee L.F., Lupiani B., Silva R.F., Kung H.J., and Reddy S.M. (2008). Recombinant Mareks disease virus (MDV) lacking the Meq oncogene confers protection against challenge with a very virulent plus strain of MDV Vaccine 26(15): 1887-1892.
- Ding J., Cui, Z., Lee, L.F., Cui, X., Reddy, S.M. (2006). The role of pp38 in regulation of Mareks disease virus bi-directional promoter between pp38 and 1.8-kb mRNA. Virus Genes, 32 (2): 193-201.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: Marek's disease is a major problem facing the poultry industry resulting in annual economic losses of $160 million for the US poultry industry. Marek's disease is a lymphoproliferative disease resulting in T-cell lymphomas in infected chicken. Marek's disease is controlled by vaccination but the causative agent continues to mutate to greater virulence, resulting in reduced efficacy of the available vaccines. In this project we evaluated how the vaccines are able to confer protective immunity against challenge with highly virulent strains of Marek's disease virus. The recombinant vaccines were generated in which genes from the vaccine strains were introduced into the pathogenic strains. The enhancement effect of pp38 gene product on the activity of its upstream bi-directional promoter in Marek's disease virus was studied. Homologues of Marek's disease virus genes are present in other herpesviruses, however, it also encodes a proteins unique to Marek's disease virus serotypes.
We analyzed the role of this open reading frame 11 in pathogenesis.
TARGET AUDIENCES: Poultry academia and industry
PROJECT MODIFICATIONS: We have also looked into other genes that may have a role in protection of Marek's virus.
Impacts
The unique open reading frame 11 (LORF11) of Marek's disease virus (MDV) is present in all three serotypes of MDV and is located in the unique long region of the MDV genome. In the serotype 1 Md5 genome, LORF11 comprises 2711 nucleotides and encodes a predicted protein of 903 amino acids. Three rMd5DeltaLORF11 mutants were generated and their biological functions were studied in vitro and in vivo. In vitro growth characteristics of rMd5DeltaLORF11 viruses were similar to those of parental rMd5, indicating that LORF11 is not essential for replication in vitro. In vivo studies of rMd5DeltaLORF11 mutants showed that they were impaired in viral replication in the lymphoid organs and had 100x lower viremia than chickens infected with the parental rMd5 virus. Furthermore, rMd5-infected chickens horizontally transmitted the virus to contact controls whereas no horizontal transmission occurred in rMd5DeltaLORF11-infected chickens. Therefore the LORF11 gene of MDV is essential
for normal virus replication in chickens and deletion of LORF11 renders an attenuated virus
Publications
- Lee LF, Silva RF, Cui X, Zhang H, Heidari M, Reddy SM. (2007). Characterization of LORF11, a unique gene common to the three Marek's disease virus serotypes. Avian Dis.51(4):851-7.
- Ajithdoss D., Lupiani B., Lee L.F. and Reddy S.M. (2007). In vitro transformation properties of the Meq protein of MDV. AVMA/AAAP Washington DC. July 2007.
- Suchodolski P. Lupiani B. and Reddy S.M. (2007). Mutations in the leucine zipper region of Mareks disease virus encoded Meq protein results in loss of T-cell transformation. ASV, Corvallis OR, July 2007.
- Lee, L.F., Reddy, S.M. and Kreager, K. (2007). Recombinant Mareks disease virus lacking the oncogene Meq as a candidate for future control of Mareks disease in chickens. AVMA/AAAP Washington DC. July 2007.
- Ding J., Cui Z., Jiang S., Reddy, S. (2006). The enhancement effect of pp38 gene product on the activity of its upstream bi-directional promoter in Mareks disease virus. Science in China: Series C Life Sciences 49 (1): 53-62.
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Progress 01/01/06 to 12/31/06
Outputs
Marek's disease is a lymphoproliferative disease resulting in a cancer like disease in chicken. Marek's disease is effectively controlled by vaccination but the causative agent continues to mutate to greater virulence, resulting in reduced efficacy of the available vaccines. In this project we evaluated how the vaccines are able to confer protective immunity against challenge with highly virulent strains of Marek's disease virus. The recombinant vaccines were generated in which genes from the vaccine strains were introduced into the pathogenic strains. We have currently generating several revertant viruses in which the original genes are swapped back, so conclusively prove that the observed phenotype is due to the specific gene and not due to unintended consequence of recombination. To date we have purified over 450 plaques to generate the revertant viruses. We have also started sequencing the DNA fragments to see if there is the inserted mutation and no other
consequential mutations. The role of pp38 (using chimeric viruses) was examined on bi-directional promoter between pp38 and 1.8-kb mRNA.
Impacts
Introduction of genes from vaccine strain of Marek's disease virus into a pathogenic strain is a powerful tool to study how vaccines are able to confer protection against challenge with pathogenic strains. Identification of individual genes will help us design better vaccines that are able to confer sterilizing immunity. The results have also shown the role of pp38 on Marek's disease bidirectional promoter, this will help scientists to design vaccines that can control the replication of the viruses, which is critical for improving the safety of vaccines.
Publications
- Ding, J., Cui, Z., Lee, L.F., Cui, X., Reddy, S.M. (2006). The role of pp38 in regulation of Marek's disease virus bi-directional promoter between pp38 and 1.8-kb mRNA. Virus Genes. 32(2): 193-201.
- Gimeno, I, Witter, R.L., Hunt. H.D., Reddy, S.M., Lee, L.F. Silva, R.F. (2005). The pp38 gene of Mareks disease virus (MDV) is necessary for cytolytic infection of B cells and the maintenance of the transformed state but not for cytolytic infection of feather follicle epithelium and the horizontal spread of MDV. Journal of Virology 79(7): 4545-9.
- Lee, L.F., Cui, X., Cui, Z., Gimeno, I, Lupiani, B., Reddy, S.M. (2005). Characterization of a very virulent Marek's disease virus mutant expressing the pp38 Protein from the serotype 1 vaccine strain CVI988/Rispens. Virus Genes. 31(1): 73-80.
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Progress 01/01/05 to 12/31/05
Outputs
Marek's Disease virus (MDV) encodes a gene responsible for a transformation of lymphocytes leading to cancer like disease in chickens. This gene was identified in pathogenic strain of MDV and is called Meq. We have shown that vaccine strains belonging to serotype 1 also encode a homologue of Meq gene. Sequence analysis of the Meq gene from vaccine strain, CVI988, showed that there are differences in the transactivation and DNA binding domains. To identify the role of Meq gene from vaccine strain we have substituted the Meq gene from a vaccine strain into the pathogenic strain of MDV. This mutant virus appears to grow efficiently like the parental strain of MDV.
Impacts
These studies are expected to idenify why Meq gene found in the vaccine strain is not capable of efficiently transforming lymphocytes in chickens. Identifying the role of this critical gene in transformation will led us to design vaccines capable of protecting chickens against highly virulent strains of Marek's disease.
Publications
- No publications reported this period
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Progress 01/01/04 to 12/31/04
Outputs
Chimeric meq genes between MDV serotype 1 viruses, Md5 and CVI 988 were generated. These chimeric genes were cloned into an avian leukosis virus expression vector. The CVI988 Meq gene was also cloned into the SN5 and A6 cosmid clones of Md5 virus. The avian leukosis virus vectors expressing wild type and chimeric meq genes are being evaluated for their ability to transform CEF and lymphocytes in vitro. During the next year these mutants will be tested for their ability to transform in vivo.
Impacts
Since MD is an economically important agent of poultry, these studies will aid in long term improvement and sustainability of US poultry industry.
Publications
- No publications reported this period
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