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United States Department of Agriculture

Agricultural Research Service

2009 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. We have inserted reticuloendotheliosis virus (REV) long terminal repeat (LTR) sequences into strain Md5 of Marek’s disease (MD) virus (MDV) using rMd5 bacterial artificial chromosome (BAC). The rMd5 BAC with REV LTR insert was passed in duck-embryo fibroblast for 40 passages. Chickens of ADOL line 15 X 7 with and without MD maternal antibodies were used to study the pathogenicity of rMd5 BAC with REV LTR insert, and to compare it with that of rMd5 BAC without REV LTR insert. The data clearly indicate that partial genomic insertion of REV, namely LTR may influence pathogenicity of MDV. The information is important in understanding immunopathogenesis of MDV, an essential component of any successful program for the control of the disease. Three large-scale vaccine trials with Hy-Line International on a comparative evaluation of rMd5¿Meq with CVI988/Rispens virus in commercial chickens. Result indicated that the rMd5¿Meq vaccine protected 99-100% whereas CVI988/Rispens protected 58-98% when challenged with a very virulent vv+686 strain of MDV. Investigation into the immunological basis for resistance or susceptibility of chickens to MD revealed that a subset of T cell population (CD8+ T cells) are either drastically reduced or a critical cell surface antigen (CD8) is not expressed on the surface of cytotoxic T cell population in MD-susceptible lines. In a related study, our preliminary data indicates that MDV meq oncogene induces an immunosuppressive effect in infected chickens. The expression analysis of selected chicken genes showed that meq oncoprotein was suppressive for most immune related genes in comparison to non-infected control birds. This investigation provides further insight into the molecular mechanism of MDV pathogenesis. In another study, we have identified a housekeeping gene (A gene that has stable expression level under different physiological conditions) that is essential for gene expression analysis in MDV-infected chickens. This gene would be of critical importance for normalization analysis in Real-Time PCR and Microarray investigation of gene expression profiling.

1. Generation of a novel poultry vaccine. We used bacterial artificial chromosome (BAC) technology to generate a highly effective Marek’s disease vaccine. We accomplished this by deleting the tumor-causing MEQ gene from the Marek’s disease virus BAC. The resulting Marek’s disease virus BAC was shown to be a better Marek’s disease vaccine than the best commercially available vaccine. To increase the usefulness of the BAC construct, we inserted genes from another avian pathogen, infectious laryngotracheitis virus (ILTV) into the BAC clone. By expressing genes from ILTV in our MDV BAC, we propose to make an effective and safe vaccine for infectious laryngotracheitis. Infectious laryngotracheitis is a serious disease of poultry that is poorly controlled by existing vaccines. The recombinant constructs should help the poultry industry better control Marek’s disease and infectious laryngotracheitis.

2. Immunological basis for resistance and susceptibility to Marek’s disease. Mechanisms of pathogenecity of MDV, and resistance/susceptibility to MDV infection/oncogenesis, are poorly understood. To better understand the molecular mechanism overriding these events, we have conducted a comparative study of immunological responses to Marek’s disease virus infection between a highly susceptible (72) and a resistant (63) chicken line. The purpose of our study was to determine (1) the extent of MDV infection in tissue samples of the spleen and the bursa of Fabricius based on the detection of MDV protein antigens, (2) the immune cell populations, and (3) the extent and number of immune cell infiltration into the infected tissues of both chicken lines in comparison to age-matched un-infected control birds. Immunohistochemical analysis of tissue samples collected at different time points post inoculation revealed severe infection in line 72, whereas no viral antigen was detected in the spleen or bursa of line 63. Our study also showed that there is indeed a considerable difference in cell population between the susceptible and resistant chicken lines, with the most significant difference being in CD8+ T cells. The resistant line has a large number of CD8+ T cells, while the susceptible line seems to have few to none. In addition, heavy infiltration of macrophages and CD8+ T cells in the tissues of inoculated birds were observed. This observation is critical in understanding the essential role of cytolytic T cells in immune responses against viral infection. It also provides insight into possible modulation of the immune system toward an effective T cell mediated immune response against MDV infection using cytokine and chemokines as genetic adjuvant.

3. In search of a suitable housekeeping gene for normalization of gene expression in MDV-infected chicken cells. Gene expression analysis is becoming an increasingly common research tool in biological fields. Understanding gene expression pattern provides insight into complex biological pathways and will likely lead to the identification of genes implicated in disease or biological processes. Selection of a reliable housekeeping gene for normalization purposes is an essential requirement in gene expression analysis. In this study, we evaluated the reliability of 20 housekeeping genes in Marek’s disease virus (MDV)-infected and uninfected control chicken splenocytes. The 20 genes were ranked for stability of expression based on standard deviation (SD) of the cycle threshold, NormFinder, and geNorm software analsysis. In addition, we investigated the expression levels of a gene of interest, interferon gamma (IFN¿), between MDV-infected and control samples using different housekeeping genes. Both NormFinder and geNorm find GUSB as the most reliable housekeeping genes between the infected and control samples. GAPDH and ¿-Actin , the two most widely used housekeeping genes for normalization, ranked as number 13 and 14 respectively. The fold change in the expression level of IFN¿ was greatly influenced by the housekeeping gene used for normalization. This clearly indicates that the stability of the expression of housekeeping gene used as a reference gene is of critical importance in gene expression profiling. Variation in the expression levels of a reference gene under different experimental conditions will influence the expression pattern of genes of interest and may induce artificial changes in the final analysis. Identification of this most reliable housekeeping gene is critical in gene expression analysis in all poultry disease studies.

4. Immunosuppressive nature of MDV meq oncogene. Meq oncogene is involved in the transformation of CD4+ T cells and tumor development in MDV-infected chickens. Although, mechanism of oncogenecity of meq is well established, its adverse effect on the immunological responses to MDV infection is unknown. Using Real-Time PCR technology, we conducted a comparative analysis of an extensive panel of cytokine, chemokine, and other immune-related gene expression in rMd5- and rMd5¿meq-infected chickens at different time points post inoculation. The preliminary results from this study indicate that the expression levels of most of the tested genes that are critical for a successful antiviral immune response were down regulated in rMd5-infected chickens. The expression pattern of these genes in chickens infected with meq-deleted virus, however, were similar to those in the control birds. This finding provides further insights into meq biological function as an immune suppressive viral antigen. Further studies are needed to elucidate the molecular mechanism of immune suppressive nature of meq oncoprotein.

5. MD vaccine efficacy in commercial chickens. Vaccines have controlled MD for almost four decades. At the present, CVI988/Rispens virus is used worldwide for controlling MD in the field and no better vaccine has appeared on the horizon. The continued evolution of MDV towards greater virulence has prompted concern that the currently available vaccine will ultimately loose efficacy in controlling MD. There is no better vaccine on the horizon. We have developed an oncogene deletion virus, rMd5¿Meq, and showed it to be a superior vaccine candidate in the laboratory challenge model. We just completed three large-scale vaccine trials with Hy-Line International on a comparative evaluation of rMd5¿Meq in commercial chickens. These three trials were carried out under the most stringent conditions that allow the distinguishing of vaccine candidate. These conditions include the immediate exposure of day-old vaccinated chicks in contact exposure with seeder birds challenged with the most virulent plus strain of MDV, vv+686 virus. The rMd¿Meq vaccine was shown in all three trials to be the most efficacious MD vaccine. The data indicated the rMd¿Meq vaccine is a superior vaccine, better than CVI988/Rispens vaccine now widely used in the poultry industry. This information is of significant importance to the poultry industry because there is a new vaccine candidate for the future. Three U.S. companies and 2 other companies from abroad have obtained MTA to initiate their own field trials. In addition, this study contributed to scientific knowledge on the pathogenesis and control of Marek’s disease.

6. Retroviral insertion in Marek’s disease virus. It is known that partial or complete genomic (genetic components) insertion of retroviruses can occur in large DNA viruses. The influence of such insertion on the pathogenicity of such DNA viruses is not known. This year, we successfully inserted part of genomic materials from reticuloendotheliosis virus (REV), an important retrovirus that causes tumors in poultry and other avian species into Marek’s disease virus (MDV), a DNA virus that causes tumors primarily in chickens. The insertion of part of genome of REV, named LTR into MDV was achieved by using state-of the-art molecular technique known as bacterial artificial chromosome (BAC). We compared the pathogenicity (disease inducing potential) of the new MDV containing LTR from REV with that of MDV lacking LTR. The data indicate that insertion of LTR from REV into MDV reduced the pathogenicity of MDV in susceptible chickens. The information should add to the knowledge on pathogenicity of MDV, and consequently should be helpful in developing better strategies for control of Marek’s disease.

7. Genetic variation among naturally occurring recombinant avian leukosis virus (ALV) isolates. Using various assays including DNA-based tests such as polymerase chain reaction (PCR) and DNA sequence analysis, we have completed biological and molecular characterization of seven isolates of naturally recombinant ALVs obtained from field flocks suffering from tumors termed myelocytomatosis. All of seven isolates were confirmed to be recombinant ALVs with different genomic regions representing different subgroups of ALV. For example, some isolates were closely related to the naturally occurring recombinant termed ALV-B/J, containing genetic components of subgroup B, E and J of ALV. The information is essential to better understand the molecular basis for pathogenicity and to develop better diagnostics and control measures.

6.Technology Transfer

Number of the New/Active MTAs (providing only)14

Review Publications
Hunt, Henry D., Fadly, Aly M., Silva, Robert F., Zhang, Huanmin. 2008. Survey of Endogenous Virus and TVB* Receptor Status of Commercial Chicken Stocks Supplying Specific-Pathogen-Free Eggs. Avian Diseases. 52(3):433-440.

Pandiri, Arun K.R., Cortes, Aneg L., Lee, Lucy F., Gimeno, I.M. 2008. Marek's Disease Virus Infection in the Eye: Chronological Study of the Lesions, Virus Replication, and Vaccine-Induced Protection. Avian Diseases. 52(4):572-580.

Abdul-Careem, M.F., Hunter, D.B., Shanmuganathan, S., Haghighi, H.R., Read, L., Heidari, M., Sharif, S. 2008. Cellular and Cytokine Responses in Feathers of Chickens Vaccinated Against Marek's Disease. Veterinary Immunology and Immunopathology. 126(3-4):362-366. Available:

Suchodolski, P., Izumiya, Y., Lupiani, B., Ajithdoss, D.K., Gilad, O., Lee, L.F., Kung, H-J, Reddy, S.M. 2009. Homodimerization of Marek's Disease Virus-Encoded Meq Protein Is Not Sufficient for Transformation of Lymphocytes in Chickens. Journal of Virology. 83(2):859-869.

Abdul-Careem, M.F., Hunter, B.D., Lee, L.F., Fairbrother, J.H., Haghighi, H.R., Read, L., Parvizi, P., Heidari, M., Sharif, S. 2008. Host Responses in the Bursa of Fabricius of Chickens Infected with Virulent Marek's Disease Virus. Virology. 379(2):256-265.

Santos, V.L.S.L., Williams, S.M., Zavala, G., Barbosa, T., Zhang, J., Cheng, S., Shivaprasad, H.L., Hafner, S., Fadly, A.M., Santos, R.L., Brown, C.C. 2008. Detection of Reticuloendotheliosis Virus by Immunohistochemistry and In Situ Hybridization in Experimentally Infected Japanese Quail Embryos and Archived Formalin-fixed and Paraffin-embedded Tumours. Avian Pathology. 37(4):451-456.

Pandiri, A.R., Gimeno, I.M., Reed, W.M., Fitzgerald, S.D., Fadly, A.M. 2009. Subgroup J Avian Leukosis Virus-Induced Histiocytic Sarcomatosis Occurs Only in Persistently Viremic, but Not Immunotolerized Meat-type Chickens. Veterinary Pathology. 46(2):282-287.

Heidari, M., Zhang, H.M., Sharif, S. 2008. Marek's disease virus induces Th-2 activity during cytolytic infection. Viral Immunology. 21(2):203-213.

Abdul-Careem, M.F., Hunter, B.D., Sarson, A.J., Parvizi, P., Haghighi, H.R., Read, L., Heidari, M., Sharif, S. 2008. Host responses are induced in feathers of chickens infected with Marek's disease virus. Virology. 370:323-332.

Ajithdoss, D.K., Reddy, S.M., Suchodolski, P.F., Lee, L.F., Kung, H.J., Lupiani, B. 2009. In Vitro Characterization of the Meq Proteins of Marek's Disease Virus Vaccine Strain CV1988. Virus Research. 142(1-2):57-67.

Gimeno, I.M., Cortes, A.L., Silva, R.F. 2008. Load of Challenge Marek's Disease Virus DNA in Blood as a Criterion for Early Diagnosis of Marek's Disease Tumors. Avian Diseases. 52(2):203-208.

Dudnikova, E., Vlasov, A., Norkina, S., Kireev, D., Witter, R.L. 2009. Factors Influencing the Attenuation of Serotype 1 Marek's Disease Virus by Serial Cell Culture Passage and Evaluation of Attenuated Strains for Protection and Replication. Avian Diseases. 53(1):63-72.

Last Modified: 8/27/2015
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