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United States Department of Agriculture

Agricultural Research Service

Related Topics

Research Project: GENETIC AND BIOLOGICAL DETERMINANTS OF AVIAN TUMOR VIRUS SUSCEPTIBILITY

Location: Avian Disease and Oncology Laboratory

2008 Annual Report


1a.Objectives (from AD-416)
Identify genetic predictors of Marek's disease virus (MDV) virulence. Identify host-viral genetic determinants that control avian tumor virus pathgenicity and shedding. Elucidate the genetic determinants that modulate MDV interactions within the avian immune system. Elucidate host-viral interactions that drive the evolution of new virulent strains of avian tumor viruses. Discover safe and highly effective vaccine platforms that convey protection against emerging MDV strains.


1b.Approach (from AD-416)
Avian tumor viruses of economic importance include:.
1)Marek’s disease virus (MDV), a herpesvirus that induces a lymphoproliferative disease of chickens that, in the absence of effective control measures, is capable of causing devastating losses in commercial layer and broiler flocks; and 2) avian retroviruses, namely avian leukosis virus (ALV) and reticuloendotheliosis virus (REV), both are associated with neoplastic diseases and other production problems in poultry. Also, both ALV and REV are potential contaminants of live-virus vaccines of poultry. Critical needs are:.
1)better MDV vaccines to protect against the current and next generation of virulent field strains of MDV; and.
2)a long-term strategy designed to reduce the ongoing emergence of new virulent MDV, and creation of recombinant ALVs through multiple barriers or reduction in viral load and shedding. The primary emphasis will be on molecular approaches to better understand which viral genes are important for immunopathogenesis and shedding of MDV. Parallel studies will monitor the virulence of field strains of MDV and ALV. Studies are also aimed at characterization of new virus isolates and on improving assays for their detection; additional efforts will be devoted to better understand MDV immunity. The project also emphasizes studies on:.
1)elucidating factors involved in creation of recombinant ALVs; and.
2)determining whether REV genome insertion into MDV and fowlpox virus influences transmission and epidemiology of REV. The end product will be a better understanding of viral gene function, virus-host interactions and the development of materials and improved methodology for control of avian tumor viruses.


3.Progress Report
Substantial progress was made on all objectives of the project. Brief description of selected accomplishments is listed below. For more detail, see question 4. Field and laboratory trials to evaluate the efficacy of the already patented cosmid clone MEQ deleted Marek’s disease virus (MDV) indicated that the vaccine provided better protection than most commercial vaccines in face of challenge with a highly virulent strain of MDV. Several material transfer agreements with various vaccine manufacturers were established for further evaluation as a commercial vaccine. This year, we used another technique named bacterial artificial chromosome (BAC) to delete both copies of the MEQ gene (gene responsible for oncogenicity) from MDV. The BAC deletion virus was completely attenuated, and when tested as a vaccine, it protected chickens against challenge with a highly virulent MDV. Characterization of two MDV genes, pp38 and vIL8 established that both genes are virulence factors in MDV; also, study of the expression pattern of cytokines genes associated with a Th-2 type immune response indicated that highly virulent MDV strains induce a humoral type of immunity that is ineffective in controlling virus replication and pathogenesis. These studies helped us to better understand pathogenesis of the disease. We have previously reported on the isolation and biological characterization of reticuloendotheliosis virus (REV) from broiler breeder chickens, turkeys and prairie chickens located in various regions of the United States. In order to determine the genetic relationship among these REV isolates, one isolate from each species was chosen for molecular characterization. A high degree (greater than 99.0%) of homology at the amino acid level between the three isolates and strain CSV, a prototype of REV subtype C, suggesting that subtype C REV is the most prevalent subtype of REV among the three avian species in the USA.


4.Accomplishments
1. Generation of a highly effective Marek’s disease virus (MDV) vaccine using bacterial chromosome (BAC) technology. Currently available commercial MDV vaccines are not highly protective against field challenge with very virulent MDV. We deleted both copies of the MEQ gene from a MDV BAC. The deletion virus was completely attenuated. In a protection study, the deleted virus protected chickens from a challenge with a highly virulent MDV. Our deletion virus was a much better MD vaccine than the best commercially available vaccine. In addition, the MEQ deletion was made in a specific manner that will allow any other foreign protein-coding DNA to be cloned into the deletion site. This work resulted in not only a highly protective MDV vaccine that is needed by the poultry industry, but also in the development of a vector that can express foreign proteins from other avian pathogen, such as avian influenza and infectious laryngotracheitis. Thus, we will be able to generate a polyvalent vaccine that can protect against both Marek’s disease and another avian pathogen. This accomplishment addresses NP 103 Component 2 (Genetic and biological determinants of disease susceptibility), and Problem Statement 2B (Avian Tumor Viruses).

2. Characterization of immune response during cytolytic phase of Marek’s disease virus (MDV) infection. The immune response in MDV infection is poorly understood. Using Real-Time PCR analysis of chicken cytokines, chemokines, and other immune-related genes revealed that a Th-1 type of immune activity that is critical for the induction of a successful host antiviral immune response does not predominate in the bursa of Fabricius of chickens infected with a very virulent plus (vv+) strain of MDV. The expression pattern of cytokines genes associated with a Th-2 type immune response indicated that indeed a vv+ MDV strain induces not a cell-mediated immune response but a humoral type of immunity that is ineffective in controlling virus replication and pathogenesis. This observation is critical in understanding the role of cytokines and chemokines in orchestrating the immune responses against MDV infection and will allow scientists to develop better strategies for controlling the disease. This accomplishment addresses NP 103 Component 2 (Genetic and biological determinants of disease susceptibility), and Problem Statement 2B (Avian Tumor Viruses).

3. Efficacy of a MEQ-deleted Marek’s disease virus (MDV) vaccine generated by cosmid clone technology. Currently available commercial MDV vaccines are not highly protective against field challenge with very virulent MDV. We have previously reported on the generation of a MEQ-deleted MDV vaccine. The efficacy of MEQ deletion mutants generated from cosmids was studied in both maternal antibody positive and negative chickens. In four experiments, we found MEQ deleted rMd5 virus protected chickens significantly better than the best commercially available vaccines. This work resulted in information that is necessary for poultry vaccine manufacturers to work on commercialization of this highly protective MDV vaccine. Several Material Transfer Agreements (MTAs) between ARS and several vaccine companies were established to provide the deletion mutant virus for further evaluation by the companies. This accomplishment addresses NP 103 Component 2 (Genetic and biological determinants of disease susceptibility), and Problem Statement 2B (Avian Tumor Viruses).

4. Biological properties of two Marek’s disease virus (MDV) genes (pp38 and vIL8). The molecular basis for MDV pathogenesis is poorly understood. Using gene deletion mutants of recombinant MDV, rMd5, we found that two genes, pp38 and vIL8, are essential for the cytolytic phase of MDV infection. Both deletion viruses established latency and replicated in feather follicle epithelium and were partially attenuated. This study established that pp38 and vIL8 genes are virulence factors in MDV; and shed some light on the molecular basis for MDV pathogenicity and shedding. Understanding the molecular basis for MDV pathogenicity is an essential factor in development of new strategies to control the disease. This accomplishment addresses NP 103 Component 2 (Genetic and biological determinants of disease susceptibility), and Problem Statement 2B (Avian Tumor Viruses).

5. Molecular characterization of reticuloendotheliosis virus (REV) isolates obtained from various avian species located in various regions of USA. Avian tumor viruses are ubiquitous and can cause tumors and other production problems in poultry and other avian species. Biological and molecular characterization of field isolates of avian tumor viruses are essential in development of effective diagnostics and control measures. We have previously reported on the isolation and biological characterization of REV isolated from broiler breeder chickens, turkeys and prairie chickens located in various regions of the United States. In order to determine the genetic relationship among these REV isolates, one isolate from each species was chosen for molecular characterization. A high degree (greater than 99.0%) of homology at the amino acid level between and among the three isolates and strain CSV, a prototype of REV subtype C was noted, suggesting that subtype C REV is the most prevalent subtype of REV among the three avian species in the USA. This information helps poultry breeders and growers, as well as avian disease specialists, to understand molecular epidemiology of the disease. This accomplishment addresses NP 103 Component 2 (Genetic and biological determinants of disease susceptibility), and Problem Statement 2B (Avian Tumor Viruses).

6. Genetic variation among naturally occurring recombinant avian leukosis virus (ALV) isolates. Biological and molecular characterization of recent isolates of naturally recombinant ALVs are essential to better understand the molecular basis for pathogenicity and develop better diagnostics and control measures. Proviral DNA sequence analysis of naturally occurring recombinant avian leukosis virus (ALV) termed ALV-B/J revealed that one isolate exhibited significant amino acid differences in the envelope gp85 SU protein; and another isolate showed nucleotide additions and deletions in 3’ UTR and LTR regions. This information helps scientists to understand the molecular basis for the pathogenicity of such viruses, and development of more specific diagnostics. This accomplishment addresses NP 103 Component 2 (Genetic and biological determinants of disease susceptibility), and Problem Statement 2B (Avian Tumor Viruses).

7. Surveillance of field flocks for Marek’s disease virus (MDV) of unusual pathogenicity. Maintaining surveillance of field flocks for MDV of unusual pathogenicity is a key element in development of effective control measures. In collaboration with poultry breeders and growers, virulent MDVs were isolated from field flocks that had a history of a relatively high incidence of the disease. Isolates were saved for pathotyping using protocol developed by ADOL scientists. This work allowed us to have access to field isolates of MDV, particularly in cases where there is a relatively high incidence of the disease; such access is essential in monitoring any changes in pathogenicity of the virus. This accomplishment addresses NP 103 Component 2 (Genetic and biological determinants of disease susceptibility), and Problem Statement 2B (Avian Tumor Viruses).


5.Significant Activities that Support Special Target Populations
None.


6.Technology Transfer

Number of the New MTAs (providing only)5
Number of New Commercial Licenses Executed4

Review Publications
Fadly, A.M., Witter, R.L., Hunt, H.D. 2008. Oncornaviruses: leukosis/sarcomas and reticuloendotheliosis. In: Dufour-Zavala, L., Swayne, D.E., Glisson, J.R., Pearson, J.E., Reed, W.M., Jackwood, M.W., Woolcock, P.R., editors. A Laboratory Manual for the Isolation, Identification and Characterization of Avian Pathogens. 5th edition. Jacksonville, FL: American Association of Avian Pathologists. p. 164-172.

Gimeno, I.M., Silva, R.F. 2008. Deletion of the Marek's disease virus UL41 gene (vhs) has no measurable effect on latency or pathogenesis. Virus Genes. 36:499-507.

Spatz, S.J., Silva, R.F. 2007. Polymorphisms in the long repeat regions of oncogenic and attenuated pathotypes of Marek's disease virus 1. Virus Genes. 35(1):41-53.

Fadly, A.M. 2008. Neoplastic diseases: Introduction. In: Saif, Y.M., Fadly, A.M., Glisson, J.R., McDougald, L.R., Nolan, L.K., Swayne, D.E., editors. Diseases of Poultry. 12th edition. Ames, IA: Iowa State University Press. p. 449-451.

Fadly, A.M., Nair, V. 2008. Leukosis/sarcoma group. In: Saif, Y.M., Fadly, A.M., Glisson, J.R., McDougald, L.R., Nolan, L.K., Swayne, D.E., editors. Diseases of Poultry. 12th edition. Ames, IA: Iowa State University Press. p. 514-568.

Fadly, A.M., Zavala, G., Witter, R.L. 2008. Reticuloendotheliosis. In: Saif, Y.M., Fadly, A.M., Glisson, J.R., McDougald, L.R., Nolan, L.K., Swayne, D.E., editors. Diseases of Poultry. 12th edition. Ames, IA: Iowa State University Press. p. 568-588.

Lee, L.F., Silva, R.F., Cui, X., Zhang, H., Heidari, M., Reddy, S. 2007. Characterization of LORF11, a unique gene common to the three Marek's disease virus serotypes. Avian Diseases. 51:851-857.

Heidari, M., Fitzgerald, S.D., Zhang, H.M., Silva, R.F., Lee, L.F., Dunn, J.R. 2007. MDV-induced skin leukosis in scaleless chickens: tumor development in the absence of feather follicles. Avian Diseases. 51(3):713-718.

Silva, R.F., Fadly, A.M., Taylor, S.P. 2007. Development of a polymerase chain reaction to differentiate avian leukosis virus (ALV) subgroups: detection of an ALV contaminate in a commercial Marek's disease vaccine. Avian Diseases. 51(3):663-667.

Spatz, S.J., Silva, R.F. 2007. Sequence determination of variable regions within the genomes of Gallid herpesvirus-2 pathotypes. Archives of Virology. Available: http://www.springerlink.com/content/ku4064776312w861/.

Davidson, I., Silva, R.F. 2008. Creation of diversity in the animal virus world by inter-species and intraspecies recombinations: lessons learned from poultry viruses. Virus Genes. 36(1):1-9.

Heidari, M., Huebner, M., Kireev, D., Silva, R.F. 2008. Transcriptional profiling of Marek's disease virus genes during cytolytic and latent infection. Virus Genes. 36:383-392.

Lee, L.F., Lupiani, B., Silva, R.F., Kung, H., Reddy, S.M. 2008. Recombinant Marek's disease virus (MDV) lacking Meq oncogene confers protection against challenge with a very virulent plus strain of MDV. Vaccine. 26:1887-1892.

Ding, J., Zhizhong, C., Lee, L.F. 2007. Marek's disease virus unique genes pp38 and pp24 are essential for transactivating the bi-directional promoters for the 1.8 kb mRNA transcripts. Virus Genes. 35:643-650.

Pandiri, A.R., Gimeno, I.M., Reed, W.M., Lee, L.F., Fadly, A.M. 2008. Distribution of viral antigen gp85 and provirus in various tissues from commercial meat-type and experimental white leghorn line 0 chickens with different subgroup J avian leukosis virus infection profiles. Avian Pathology. 37:7-13.

Last Modified: 9/22/2014
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