DETERMINING THE ROLE OF MAREK`S DISEASE VIRUS (MDV) GLYCOPROTEIN C IN HORIZONTAL TRANSMISSION

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 0220678
• Grant No.: 2010-65119-20493
• Proposal No.: 2009-01745
• Project Start Date: Jan 15, 2010
• Project End Date: Jan 14, 2013
• Grant Year: 2010
• Program Code: [92521]- Animal Health and Well-Being: Animal Health

Recipient Organization
CORNELL UNIVERSITY
(N/A)
ITHACA,NY 14853

Performing Department
Microbiology And Immunology

Non Technical Summary
Mareks disease (MD) is a continuing threat to the poultry industry worldwide and is caused by a herpesvirus called Mareks disease virus (MDV). MDV causes a devastating disease in chickens characterized by immune suppression making the chicken more susceptible to other pathogens; neurological symptoms including paralysis; and tumors throughout the body that ultimately lead to death of the chicken. Despite regular immunizations, MDV can still infect vaccinated chickens because current vaccines do not prevent superinfection (infection with multiple viruses) with virulent field viruses. One aspect of the virus life cycle where little information has been obtained is how the virus spreads from chicken-to-chicken, also called horizontal transmission. A major obstacle in designing new vaccines which prevent virus spread from chicken-to chicken is the lack of information on viral genes important for this step. Recently we have identified specific genes essential for chicken-to-chicken spread of MDV, namely the UL13 protein kinase and glycoprotein C (gC). The long term goal of this proposal is to expand our knowledge of horizontal transmission of MDV and utilize this knowledge for the development of vaccines and control measures that reduce superinfection of immunized birds. The specific objectives of this proposal are to elucidate the mechanism(s) by which gC functions during horizontal transmission of MDV. We surmise that gCs role in transmission is through binding chicken complement, an important component of the immune systems response to viral infections, and preventing neutralization of infectious virus by the chickens innate immune response. Our hypothesis will be addressed in three specific aims. The gC (UL44) gene produces three variants, therefore we will first determine which variants are important for horizontal transmission; secondly at which stage of transmission gC is essential; and thirdly examine gC and chicken complement interactions. Information obtained from this proposal will yield valuable information when designing the next generation of MDV vaccine viruses and potentially prevent the spread of virulent virus.

Animal Health Component

50%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3210	1101	50%
311	3220	1101	50%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3210 - Egg-type chicken, live animal; 3220 - Meat-type chicken, live animal;

Field Of Science
1101 - Virology;

Keywords

chickens

mareks disease

vaccination

immune evasion

horizontal transmission tumors

Goals / Objectives
The long term goal of this proposal is to expand our knowledge of horizontal transmission of Mareks disease virus (MDV) and harness this knowledge for the development of more potent MD vaccines and control measures that reduce superinfection (infection with multiple viruses) of immunized birds. The overall objective of this proposal is to elucidate the mechanism(s) by which gC proteins function during horizontal transmission of MDV. The specific objectives of this proposal include: 1) determining which forms of gC (there are three variant forms of gC produced by MDV) are essential for horizontal transmission, 2) the stage(s) at which horizontal transmission-defective viruses are impaired (shedding from chicken versus entry into naive chicken), and 3) the functional role of gC interactions with the innate immune response (complement binding). This proposal addresses an important aspect of MDV pathogenesis and evolution of virulence that has been an enigma for decades. Ultimately, the output of this proposal will be the design and development of new vaccines that will protect chickens from superinfection with virulent viruses, either by blocking the shed of virus from chickens, or blocking virus entry into the host.

Project Methods
We believe at least two, or all three, forms of gC are essential for horizontal transmission. We surmise that gCs role in transmission is through binding complement and evading the hosts innate immune response to infectious virus. We will determine which gC proteins are essential for horizontal transmission by constructing and testing recombinant viruses (rMDV) that express two gC moieties (full-length plus one splice variant or both splice variants), having shown individual forms of gC cannot facilitate horizontal transmission. We will use a model in which ten uninfected (contact) chickens are housed with ten that have been experimentally infected with specific viruses by intra-abdominal inoculation. If the rMDV is capable of horizontal transmission, contact chickens will be infected with the rMDV, thus determining which combination of gC proteins are essential for transmission, and the stage(s) at which transmission-deficient MDV are impaired. We know gC-negative viruses, while unable to spread from chicken-to-chicken, reach the skin and replicate therein. We believe the defect in transmission is caused by an inability of cell-free virus (CFV) to initiate infection. A refined model of virus transmission involving extraction of CFV and different routes of entry into the host will be employed. Briefly, skin tissues will be collected, homogenized in a tissue grinder and distributed into two aliquots of equal volume with one aliquot saved for future use; while the other will be sonicated in an ultrasonic oscillator to destroy the cells, centrifuged to pellet cell debris, and the supernatant filtered through a Millipore filter for pure CFV. To determine if CFV is produced in the skin, unfiltered and filtered materials will be 1) used in virus isolation assays on chicken tissue culture cells to determine if a lack of infectious virus is produced, 2) inoculated into two groups of chickens by intra-abdominal or intra-tracheal inoculation (drop-wise into the trachea) to determine if route of inoculation is important for establishment of infection, and 3) used for DNA extraction to quantify the amount of MDV DNA present in samples using qPCR assays. From these experiments we will be able to determine: 1) if infectious virus is produced in the skin of chickens infected with transmission-deficient viruses, 2) if there is a defect in production of CFV, and 3) whether the route of entry of virus produced in FFE cells is important for infection. Finally, we will address the molecular interactions by which gC proteins facilitate horizontal transmission. We believe gC facilitates horizontal transmission of MDV by binding complement and evading the immune system of the chicken. We will determine which gC proteins are important for inhibition of complement-mediated lysis of sensitized sheep red blood cells (SRBCs), as well as determine if MDV gC proteins directly interact with chicken complement component C3 using in vitro binding assays and enzyme-linked immunosorbent assays (ELISA). Studies defining specific interaction domains will be conducted and their contributions to horizontal transmission will be evaluated in vitro and in vivo.

Progress 01/15/10 to 01/14/13

Outputs
OUTPUTS: Activities: Multiple experiments were performed during this project period including; generation of baculovirus expressed glycoprotein C constructs for functional in vitro assays, generation of recombinant viruses expressing individual gC variant forms and their in vitro and in vivo characterization, and preparation and submission of journal articles for dissemination of the knowledge we've gained to the research communities. Events: Results of this project have been presented to both national and international audiences at scientific conferences. Services: None Products: Generation of recombinant viruses that can be directly visualized in a chicken infected with Marek's disease virus that has been, and will be further, disseminated to the research community as a tool for studying transmission of Marek's disease virus in chickens. PARTICIPANTS: Individuals: Dr. Jarosinski performed all aspects of the project in the first year. No training or professional development was provided by the project during its time at Cornell University, but it is planned that a post-doctoral associate will be trained once the grant has been transferred to the University of Iowa. Partner Organizations: None Collaborators and contacts: Dr. Joel Baines at Cornell University is a collaborator on this project; however his service, which is expertise in electron microscopy studies, has not been needed to date. Dr. Rich Roller, at the University of Iowa, will provide this expertise in the coming year. Training or professional development: No professional development was supplied in the first year of the project, however it is intended that undergraduate, graduate, and post-doctoral associates will be provided mentoring and training in the remaining years. TARGET AUDIENCES: The major target audience for this project is the poultry industry, but also general virologists, herpesvirologists, and persons interested in host-pathogen interactions. PROJECT MODIFICATIONS: There are no major changes to the project design, apart from experiments being performed at the University of Iowa. This project already has approval by the Institutional Biosafety Committee (#110215) and the Institutional Animal Care and Use Committee (#1109208) at the University of Iowa for this work.

Impacts
Change in knowledge: 1)A significant impact generated by this proposal was the development of the fluorescent viruses where we are able to evaluate the potential of co-infection of cells in the skin by two different viruses, in particular vaccine and pathogenic viruses. A long standing question, that could never to be fully addressed in the poultry industry, was whether a vaccine and pathogenic virus could infect the same cells and potential exchange genetic material that may drive increasing virulence in the industry. Increasing virulence of Marek's disease virus has plagued the poultry industry for decades, but the mechanism by which this occurs has never been identified. The viruses generated during the project allow us to directly address this question and we conclusively showed that two viruses can, in fact, infect the same cells in the chicken. This has important implications for understanding how Marek's disease virus has become more virulent throughout the decades by vaccination. It is highly possible exchange of genetic material between different strains would drive this increased virulence. 2)Another important output generated during this project is determining important glycoprotein C variants for transmission of Marek's disease virus in chickens. It has been established that glycoprotein C is essential for transmission of Marek's disease. During our studies of glycoprotein C, we identified alternative splice variants of glycoprotein C and therefore a major goal of this project was to determine if a single form of glycoprotein C was responsible for transmission. Interestingly, we found that all three variants were required for efficient transmission of Marek's disease virus. This information has greatly enhanced our knowledge of the role glycoprotein C variants may play in transmission of Marek's disease virus. Change in actions: At this time, there has been no change in societal conditions based on our results, however the information gained we feel could shed some light onto the vaccination practice of chickens against Marek's disease that could alter the way chickens are vaccinated in the future. That is, generation of new and improved vaccines that will not drive pathogenic Marek's disease virus to increased pathogenicity.

Publications

• Journal Articles: 1)Jarosinski, K.W. and Osterrieder, N. 2012. Marek's disease virus expresses multiple UL44 (gC) variants through mRNA splicing that are all required for efficient horizontal transmission. J. Virol. (Pending).
• 2)Jarosinski, K.W. 2012. Dual infection and superinfection inhibition of epithelial skin cells by two alphaherpesviruses co-occur in the natural host. PloS One (Pending).
• 3)Jarosinski, K.W., S. Arndt, B.B. Kaufer, and N. Osterrieder. 2012. Fluorescently tagged pUL47 of Marek's disease virus reveals differential tissue expression of the tegument protein in vivo. J. Virol. 86(6):2428-2436. PM:22190714
• 4)Jarosinski, K.W. and N. Osterrieder. 2010. Further analysis of Marek's disease virus horizontal transmission confirms UL44 (gC) and UL13 protein kinase activity are essential, while US2 is non-essential. J. Virol. 84(15):7911-7916. PM:20484497
• Abstracts: 1)Jarosinski, K.W. and Osterrieder, N. 2012. Marek's disease virus (MDV) UL44 (gC) splice variants and their importance for efficient horizontal transmission. 37th Annual International Herpesvirus Workshop. Calgary, Alberta, Canada. (Abstract No. Pending).
• 2)Jarosinski, K.W., Kaufer, B.B., and Osterrieder, N. 2012. Pathogenesis of Marek's disease virus. 9th International Symposium on Marek's disease and Avian Herpesviruses. Berlin, Germany (Abstract No. Pending).
• 3)Jarosinski, K.W. 2010. Dual infection of MDV in feather follicle epithelial cells. 5th International Workshop on the Molecular Pathogenesis of Marek's Disease Virus and 1st Symposium on Avian Herpesviruses. Athens, GA, (Abstract No. 1273775888).
• 4)Jarosinski, K.W. 2009. Analysis of Marek's disease virus horizontal transmission. 34th Annual International Herpesvirus Workshop. Ithaca, NY, (Abstract No. 161004).