Progress 10/01/05 to 09/30/06
Outputs
Progress Report 1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? Marek's disease is a cancer of the lymphocytes in chickens caused by a herpesvirus and is the number one disease that negatively impacts chicken production in the world. Economic losses from Marek's disease cost 100's millions dollars per year to American farmers. Since the late 1960's, vaccines have played a very important role in preventing and/or controlling the development of cancer. The original vaccine was a serotype 3 herpesvirus, herpes virus of turkey (HVT). This was effective in the 1970's at preventing Marek's disease, but in the late 1970's, field strains of Marek's disease virus (MDV) arose that could overcome vaccinal protection of HVT. Serotype 2 vaccines were developed and provided good protection until field strains of MDV arose that could overcome vaccinal immunity.
Attenuated serotype 1 MDV vaccines have also been developed and provide good protection but there are currently no alternative next-generation vaccines available. This research will identify through genomics studies, genes involved with MDV virulence and the ability to overcome vaccinal protection. This will facilitate design and development of a new generation of recombinant vaccines to protect against MDV. Marek's disease is primarily transmitted by the shedding of feather epithelial dander containing MDV virions. Birds through contact with the infected feather dander as "dust" in the respiratory tract develop the infection which may progress to formation of tumors. Electrostatic space charge systems (ESCS) have been shown to reduce dust in chick hatching cabinets and animal rooms. This technology may be used to reduce exposure of chicks to MDV by reducing feather dander in the air. Prevention of Marek's disease will improve the farm production of chicken by reducing the cost of
production and prevent animal suffering. The objectives of this project are: 1. Development of environmental control systems that will reduce field exposure of chickens to the Marek's disease virus and result in reduced incidence of Marek's disease. 2. Development of a comparative genomics program that will lead to the identification of predictors of virulence shifts. This project supports National Program 103, Animal Diseases (100%) in the components Microbial Genomics, Mechanisms of Disease, and Strategies to Control Infectious and Non-Infectious Disease. 2. List by year the currently approved milestones (indicators of research progress) Year 1 (FY2003) New project in FY2003 Develop project objectives Year 2 (FY2004) Hire lead scientist Construct a Marek's disease laboratory Equip Marek's disease laboratory Develop a project plan Year 3 (FY2005) Develop and test high voltage electrostatic space charge systems (ESCS) for use in chick rearing areas and poultry houses to reduce feather
dander containing the Marek's disease virus. Conduct comparative sequencing of regions in the MDV genomes suspected in encoding virulence factors from 10 strains of Marek's disease virus Year 4 (FY2006) Determine the function of newly discovered MDV genes encoding virulence factors. Characterize the protein products of virulence factors and determine their role in the infection. 4a List the single most significant research accomplishment during FY 2006. The most significant discovery was the identification of an MDV gene encoding a homologue to the cellular protein Raf. Using a bioinformatics approach, the nucleotide sequences of the published MDV genomes: GA, Md5 and Md11 were compared to those genomes (CVI988 and RB1B) recently sequenced by my group. This approach identified key amino acid residues present in a MDV gene of unknown function, that are collinear with residues in the Raf protein of chickens. This is an important discovery since Raf have been implicated in a wide range
of cellular processes: regulation of cell division, cancer (transformation), tissue differentiation and cell death (or apoptosis). The role this gene plays in the disease cause by Marek's disease virus is unknown, but we are currently trying to unravel its function. One clue in its cellular function comes from the fact that other nononcogenic avian herpesvirus that are non cancer-causing also contain a homologue to this gene. Therefore its role in transformation or the cancer process seems quite limited. Our current focus involves its potential role in regulation of the immune response. Recent studies have indicated that the Raf plays an important role in the regulation of antiviral responses mediated by alpha interferon. Activation of Raf and its protein partners in specific metabolic or signaling pathways negatively regulate this normal antiviral response. It is likely that avian herpesviruses have according Raf homologues over the millenniums and are able to "bypass" the
interferon response in order to survive in host cells. To investigate the role of Marek's virual encoded Raf (termed vRaf) in cellular pathways we have created a cell line expression this gene. Preliminary microscopic data indicates that this protein is expressed in both the cytoplasm and nucleus. These cells appear to have normal behavior with respect to cell division providing further evidence of it limited role in the transformation. Future experiments are planned using these cells to investigate vRaf's role to augmenting virus infection. Additionally, antisera will be generated against this protein for use in applications involving protein characterizations and determining cellular proteins that interact with the virus-encoded Raf. Plans to construct a virus containing a deletion in the Raf gene have been formulated with our collaborators from the Institute of Animal Health, Compton, UK and will be useful in functional characterization assays. 4b List other significant research
accomplishment(s), if any. 1. The complete nucleotide sequence of the common laboratory strain of MDV known as RB1B. The sequence of this genome was determined using a recombinant bacterial artificial chromosome (BAC) as a template. Using bioinformatics, two genes (UL 13 and UL 44) differed slightly in comparison to homologous genes found in related MDV strains. This is of significance since virus reconstituted from the MDV-RB1B/BAC construct is unable to transmit horizontally (from bird to bird). This discovery was further investigated by my collaborators R.F. Silva (ADOL) and N. Venugopal (IAH) to determine which gene is responsible for the horizontal transmission defect. Using targeted mutagenesis of the UL13 gene and the generation of recombinant viruses containing a deletion in this gene, it has been demonstrated that shedding rates are drastically reduced as measured by particle collection using the electrostatic space charge systems (ESCS) and infection in sentinel birds.
Although reduced, some sentinel birds had signs of early MDV disease (nerve enlargement). Perhaps both UL13 and UL44 play a role in transmission and plans are in place to generate recombinants containing a deletion in UL44 and one that contains the double deletion. 2. The identification of UL 36 was a potential virulence factor. Genetic changes or polymorphisms in this gene were initially discovered by a comparative genetic study involving the vaccine strain, CVI988 and the virulent strains GA, Md5, Md11 and RB1B. This result was confirmed by additional sequencing studies involving others strains (13 total) representing the five pathotypes of MDV. To date my research has identified two major loci (the oncogene, meq and UL 36) suspected to encode virulence factors. 3. Development of a quantitative assay based on real time PCR that will enable the differentiation of vaccine and virulent strains in animal challenging experiments. In collaboration with Mark Parcells (University of
Delaware), we have developed a methodology for detecting only the vaccine strain's DNA in animal infected with both vaccine and virulent strains. In order to achieve this, two assays are employed: one that will detect both strains and the other, specific for the virulent strain. Subtraction of the values generated by the two assays will indicate the copy number of the vaccine strains. This assay is currently being used in animal studies involving the Electrostatic Space Charge System (ESCS) developed by Bailey Mitchell. 5. Describe the major accomplishments to date and their predicted or actual impact. 1. Methodology development: We have developed technology to enzymatically amplification both large regions ( > 10,000 nucleotides) using short pieces of DNA that flank the region of interest, as well as whole genome amplification using a battery of MDV specific primers. These technologies were developed to bypass the need of virus propagation in cell culture, a technique know to
introduce mutations. This impacts research not only in the herpesvirus field but in all other fields involving DNA viruses. It is know possible to obtain the nucleotide sequence of the genomes of DNA viruses directly from infected tissues without the need for virus isolation, virus propagation in vitro and in combination with newer sequencing technologies (pioneered by 454 Life Sciences), without cloning. 2. Development of instruments based on the Electrostatic Space Charge System. Two instruments were developed for use in animal studies involving Marek's disease virus. On instrument (termed ESCS) has the potential to dramatically reduce (up to 80%) the transmission rate of infectious dander, the carrier of Marek's virions. This same instrument has been shown to also reduce the transmission rate of other viruses (both DNA and RNA) as well as bacteria (e.g. salmonella). The other instrument (termed ESD) is an air sampling device which has been demonstrated to be extremely useful in
(i) determining the shedding rates of viruses and (ii) measuring the efficiency of vaccines in prevent shedding of virulent challenging virus. 3. Identification of MDV genes suspected to encode virulence factors. We have determined the complete nucleotide sequence of two MDV genomes and numerous regions in the genomes of 13 strains representing the 5 pathotype. Our research nearly doubled the amount of sequencing data available in GenBank for MDV and will form the basis for future studies. These studies will, undoubtedly involve the construction of recombinants containing specific deletions in the identified virulence genes. Furthermore we have developed software for comparative genomic investigations that have impact not only in the MDV field, but in others. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known,
to the adoption and durability of the technology products? The complete nucleotide sequence of CVI988 and RB1B as well as the sequence of the repeat long regions from the genomes of 13 MDV strains and their bioinformatics analyses has been transferred or shared with my collaborators: Bob Silva, USDA ARS ADOL; Venugopal Nair, Institute of Animal Health, Compton, Birkshire, UK; Mark Parcells, Dept. of Animal and Food Science, University of Delaware, Newark, DE and Klaus Osterrieder, Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY. Material transfer agreements have been established between Mark Parcells and Joan Burnside, Delaware Biotechnology Institute, Newark, DE for the transfer of the USDA- generated MDV cosmid clones and clones containing the a-like sequence of MDV, respectively. It is anticipated that the complete nucleotide sequence of four additional strains of MDV will become available to scientists by the end of 2006.
This will aid in their going effort to develop better vaccine strains of Marek's Disease.
Impacts
(N/A)
Publications
- Silva, R.F., Lee, L.F., Heidari, M., Gimeno, I.M., Spatz, S.J., Zelepsky, J. 2006. The role of UL13 in generating cell-free MDV [abstract]. 4th International Workshop on Molecular Pathogenesis of Marek's Disease Virus. p. 28.
- Heidari, M., Huebner, M., Zhang, H.M., Silva, R.F., Spatz, S.J., Kireev, D. 2006. Global gene expression profiling during cytolytic infection of Marek's disease virus: array analysis of host-pathogen interactions [abstract]. 4th International Workshop on Molecular Pathogenesis of Marek's Disease Virus. p. 8.
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Progress 10/01/04 to 09/30/05
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Marek's disease (MD) is a cancer of the lymphocytes in chickens caused by a herpesvirus and is the number one disease that negatively impacts chicken production in the world. Economic losses from MD cost 100s millions dollars per year to American farmers. Since the late 1960's, vaccines have played a very important role in preventing and/or controlling MD. The original MD vaccine was a herpes virus of turkeys (HVT). This was effective at preventing MD, but in the late 1970s variant field strains of MD arose that could overcome protection induce by HVT vaccines. Through genomics analysis this research will identify genes involved with MDV virulence and genetic changes in variant strains involved with vaccination resistance. This will facilitate design and development of a new generation of recombinant
vaccines to protect against Mareks disease virus (MDV). Marek's disease is primarily transmitted by the shedding of feather epithelial dander containing MDV. Birds through contact with the infected feather dander as "dust" in the respiratory tract develop the infection which may progress to formation of tumors. Electrostatic space charge systems (ESCS) have been shown to reduce dust in chick hatching cabinets and animal rooms. This technology may be used to reduce exposure of chicks to MDV by reducing feather dander in the air. Prevention of Marek's disease will improve the farm production of chicken by reducing the cost of production and prevent animal suffering. The objectives of this project are: 1. Development of environmental control systems that will reduce field exposure of chickens to the Marek's disease virus and result in reduced incidence of Marek's disease. 2. Development of a comparative genomics program that will lead to the identification of predictors of virulence
shifts. This project supports National Program 103, Animal Diseases (100%) in the components Microbial Genomics, Mechanisms of Disease, and Strategies to Control Infectious and Non-Infectious Disease. 2. List the milestones (indicators of progress) from your Project Plan. Year 1 (FY2003) New project in FY2003 Develop project objectives Year 2 (FY2004) Hire lead scientist Construct a Marek's disease laboratory Equip Marek's disease laboratory Develop a project plan Year 3 (FY2005) Develop and test high voltage electrostatic space charge systems (ESCS) for use in chick rearing areas and poultry houses to reduce feather dander containing the Mareks disease virus. Conduct comparative sequencing of regions in the MDV genomes suspected in encoding virulence factors from 10 strains of Marek's disease virus. 3a List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. Hire a
lead scientist. (FY 2004) Milestone Fully Met 2. Construct a Marek's disease laboratory. Laboratory modular building designed, constructed and ready for occupancy, March (FY2005) Milestone Substantially Met 3. Equip Marek's disease laboratory. Major research equipment items and supplies have been ordered and 50% received. (FY 2004) Milestone Substantially Met 4. Develop a project plan. (FY-2004) Milestone Fully Met 5. Develop and test high voltage electrostatic space charge systems (ESCS) for use in chick rearing areas and poultry houses to reduce feather dander containing the Mareks disease virus. (FY-2005). Milestone Not Met Progress slowed by resource limitation (human,fiscal,equipment, etc. 6. Conduct comparative sequencing of regions in the MDV genomes suspected in encoding virulance factors from 10 strains of Marek's disease virus. (FY- 2005). Milestone Not Met Progress slowed by resource limitation (human,fiscal,equipment, etc. 3b List the milestones that you expect to
address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? The FY2006 milestones are listed below. The entire project is scheduled to be completed during FY 2006 and a new project will be developed to undergo OSQR review, and subsequent implementation beginning FY 2007. (FY2006) Determine the function of newly discovered MDV genes encoding virulence factors. Characterize the protein products of virulence factors and determine their role in the infection. 4a What was the single most significant accomplishment this past year? Methodologies were developed to facilitate the rapid sequencing of large regions within the MDV genome. This allowed for the enzymatic amplification of viral template without the need for virus propagation in primary cell culture, thus saving time and money. Moreover, since propagation of MDV in cell culture has been shown to introduce mutations that attenuate the virus, the
elimination of this step generated a more accurate record of genetic changes important in virulence. To achieve this accomplishment, various reagents were examined to increase the polymerizing ability of the amplification enzymes; temperature and time, parameter important in amplification schemes were also optimized. This methodology will allow for the characterization of strains of MDV directly from infected birds. 4b List other significant accomplishments, if any. A negative-pressure, flow-controlled 720 ft3 test room was constructed for basic testing of the Electrostatic Space Charge Systems (ESCS), and a new ESCS which could be easily configured for different sized animal rooms and houses in experiments to reduce the incidence and transmission of MDV with economical off-the-shelf components was designed and tested. An ESCS with the new design was installed in an exhaust dust capture enclosure on production house 13S to determine effectiveness for capture of feather dander from
disease-free birds while waiting on facilities to become available for tests with MDV infected birds. Preliminary results show reductions of dust up to 80% -- depending on the particle size. An Electrostatic Sampling Device (ESD) air sampling procedure for the detection of two viral pathogens, Avian Reovirus and Newcastle disease virus (NDV) from chickens in isolation cabinets was successfully developed along with an improved ESD power supply which extended the maximum sampling period from 3 hours to 15 hours. The ability of the ESD to recover sufficient Avian Reovirus or NDV from air in a one hour sampling period suggests that the ESD should be able to recover MDV from the air in isolation rooms with MDV-infected birds. A genetic change in the oncogene meq was identified in MDV vaccine strains. A large region (26,200 nucleotides) of the genome of two MDV strains was sequenced and compared with other sequenced MDV strains. Using this approach it was discovered that a 169-nucleotide
insertion was present within the meq gene of two MDV vaccine strains as well as other strains with mildly virulent phenotypes. These results provide evidence that deleting or insertional-inactivating this gene in the genome of MDV might generate a vaccine capable of protecting poultry against new emerging very virulent field strains. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. This is a new project and some major accomplishments have occurred. One accomplishment is the generation of MDV genomic template for nucleotide sequencing. The ability to amplify large regions of the genome enabled quick generation of sequencing data for comparative analysis. Sequence determination of a region in the MDV genome involved in the circularization of the viral genome during replication was identified. This region, which has never been characterized, is extremely divergent and has been shown to control the gene expression of virulence
factors in herpesviruses. A high degree of variability among MDV strains that differ in virulence was observed. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Nucleic acid sequencing information and their bioinformatics analyses has been transferred or shared with my collaborators: Bob Silva, USDA ARS ADOL; Venugopal Nair, Institute of Animal Health, Compton, Birkshire, UK; Mark Parcells, Dept. of Animal and Food Science, University of Delaware, Newark, DE and Klaus Osterrieder, Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY. It is anticipated that the sequencing data from 10 or more strains of MDV will become available to scientists by the end of 2005. This will aid in their going effort to
develop better vaccine strains of Mareks Disease.
Impacts
(N/A)
Publications
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Progress 10/01/03 to 09/30/04
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Marek's disease is a cancer of the lymphocytes in chickens caused by a herpesvirus and is the number one disease that negatively impacts chicken production in the world. Economic losses from Marek's disease cost 100's millions dollars per year to American farmers. Since the late 1960's, vaccines have played a very important role in prevent and/or control development of the cancer. The original vaccine was a serotype 3 herpesvirus, herpes virus turkey (HVT). This was effective in the 1970s at preventing Marek's disease, but in the late 1970s, field strains of Marek's disease virus (MDV) arose that could overcome vaccinal protection of HVT. Serotype 2 vaccines were developed and provided good protection until field strains of MDV arose that could overcome vaccinal immunity. Attenuated serotype 1 MDV
vaccines have also been developed and provide good protection but there are currently no alternative next generation vaccines available. This research will identify through genomics studies, genes involved with MDV virulence and the ability to overcome vaccinal protection. This will facilitate design and development of a new generation of recombinant vaccines to protect against MDV. Marek's disease is primarily transmitted through MDV replicating in the epithelium of the growing feather and being shed in feather dander. The birds through contact with the infected feather dander as "dust" in the respiratory tract develop the infection which may progress to production of the tumors. Electrostatic space charge systems (ESCS) have been shown to reduce dust in chick hatching cabinets and animal rooms. This technology may be used to reduce exposure of chicks to MDV by reducing feather dander in the air. Prevention of Marek's disease will improve the farm production of chicken by reducing
the cost of production and prevent animal suffering. The objectives of this project are: 1) development of environmental control systems that will reduce field exposure of chickens to the Marek's disease virus and result in reduced incidence of Marek's disease, and 2) development of a comparative genomics program that will lead to the identification of predictors of virulence shifts. This projects supports National Program 103, Animal Diseases (100%) in the components Microbial Genomics, Mechanisms of Disease, and Strategies to Control Infectious and Non-Infectious Disease. 2. List the milestones (indicators of progress) from your Project Plan. Year 1 (FY2003) New project in FY2003 Develop project objectives Year 2 (FY2004) Hire lead scientist Construct a Marek's disease laboratory Equip Marek's disease laboratory Develop a project plan Year 3 (FY2005) Develop and test high voltage electrostatic space charge systems (ESCS) for use in chick rearing areas and poultry houses to reduce
feather dander containing the Mareks' disease virus. Conduct comparative sequencing of four proposed virulence genes for 10 strains of Marek's disease virus and determine the function for two of the genes. viruses. 3. Milestones: A. Year 2 (FY2004) Hire lead scientist. Completed Construct a Marek's disease laboratory. Laboratory modular building designed, constructed and set-up on site. Equip Marek's disease laboratory. Major research equipment items and supplies have been ordered and 50% received. Develop a project plan. In progress. B. The Year 3 milestones are listed below with a description of the anticipated outcomes. The entire project is scheduled to be completed during FY 2005 and a new project will be developed to undergo OSQR review, and subsequent implementation beginning FY 2006. Year 3 (FY2005) Develop and test high voltage electrostatic space charge systems (ESCS) for use in chick rearing areas and poultry houses to reduce feather dander containing the Mareks' disease
virus. Conduct comparative sequencing of four proposed virulence genes for 10 strains of Marek's disease virus and determine the function for two of the genes. 4. What were the most significant accomplishments this past year? Lead scientist has been recruited and reported to duty August 9, 2004. B. None C. None D. None
Impacts
(N/A)
Publications
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