GENERATION AND ANALYSIS OF A RECOMBINANT MAREK'S DISEASE VIRUS WITH MUTATION IN THE MEQ AND RIBONUCLEOTIDE REDUCTASE GENES FOR POTENTIAL USE AS VACCINE.

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: ANIMAL HEALTH
• Reporting Frequency: Annual
• Accession No.: 0221494
• Project No.: TEX09423
• Project Start Date: Feb 25, 2010
• Project End Date: Sep 30, 2012

Project Director
Reddy, SA.

Recipient Organization
TEXAS A&M UNIVERSITY
750 AGRONOMY RD STE 2701
COLLEGE STATION,TX 77843-0001

Performing Department
Veterinary Pathobiology

Non Technical Summary
Marek's disease virus (MDV) is a ubiquitous pathogen of chicken and has the potential to cause catastrophic losses to the commercial poultry industry. MDV causes a cancer-like disease in chickens, which has been controlled by the use of vaccines. Although vaccines are able to inhibit the development of tumors, disease causing MDV can still infect vaccinated chickens resulting in evolution of strains capable of overcoming vaccine immunity. TThe overall objective of this application, is to further attenuate a mildly pathogenic MDV strain by introducing mutations in a gene involved in virus replication and to evaluate its use as a potential vaccine.

Animal Health Component

100%

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	4030	1101	100%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
4030 - Viruses;

Field Of Science
1101 - Virology;

Keywords

marek's disease virus

bacterial artificial chromosome

ribonucleotide reductase

pathogenesis

Goals / Objectives
Marek's disease (MD) is a lymphoproliferative and neuropathic disease of chickens, caused by a highly contagious, cell-associated, oncogenic herpesvirus called Marek's disease virus (MDV). Prior to use of MDV vaccines, the losses from carcass condemnation of broilers accounted for about $200 millions per annum, representing condemnation of 1.5% of broilers examined. After the introduction of vaccines, the broiler condemnation rates have steadily declined however; MD still remains an economical important disease because of vaccination breaks and emergence of more virulent strains. As a result, Animal Protection Program of AFRI considers MD research as high priority. Our long-term goal is to understand the molecular mechanisms involved in MDV pathogenesis that will lead to better control measures. The overall objective of this application, which is the next step toward the achievement of our long-term goal, is to clone the MDV genome as a bacterial artificial chromosome (BAC) that will facilitate the generation of improved MDV vaccines. Activities include the training of one post-doctoral fellow.

Project Methods
Objective 1. In order to easily introduce mutations into the genome of the mildly pathogenic Md5-CVI-Meq strain, a BAC clone of this virus will be generated. The BAC vector will be cloned into the US2 region of MDV genome. Transfection of BAC DNA into chicken embryo fibroblasts (CEF) will result in recovery of Md5-CVI-Meq virus. Objective 2. We will test the working hypothesis that deletion MDV ribonucleotide reductase (RR) gene will affect virus replication in lymphocytes resulting in reduced virulence. This hypothesis will be tested by deletion of the large subunit of the RR gene from Md5-CVI-Meq BAC clone to generate Md5-CVI-MeqΔRR BAC DNA. The Md5CVIMeqΔRR virus will be tested for pathogenicity in susceptible chickens. If attenuated, the mutant virus will be evaluated for protection against challenge with highly virulent MDV strain.

Progress 02/25/10 to 09/30/12

Outputs
OUTPUTS: Activities included training of one post-doctoral fellow. The research was presented at the international American Association of Avian Pathologists. Collaborated with scientists at Agriculture Research Service laboratory in East Lansing, Michigan. PARTICIPANTS: Sanjay M. Reddy, Aijun Sun, Lucy Lee, Robert Silva and Mohammad Heidari TARGET AUDIENCES: Target audiences include researchers and poultry vaccine manufacturers. PROJECT MODIFICATIONS: It was expected that deletion of ribonucleotide reductase (RR) mutant Marek's disease virus may have to be further attenuated to develop Marek's disease vaccine. However, results showed that RR deleted virus was unable to grow in chicken. Thus further attenuation by introduction of mutations into Meq protein were not necessary.

Impacts
Marek's disease virus (MDV) bacterial artificial chromosome clone (BAC) was engineered to delete the ribonucleotide reductase (RR) gene. Transfection of the engineered clone resulted in the generation of MDV virus that lacked RR gene. The resulting null mutant was characterized for in vitro growth kinetic, genomic integrity and loss of RR expression. Results showed that the viris was genetically stable and it did not express RR protein as tested by immunofluorescence assay using monoclonal antibodies raised against RR protein. This recombinant replicated with similar kinetics as parental virus in cell culture, however when inoculated into chicken it did not cause disease suggesting that RR was essential for pathogenesis. Protection studies indicated that RR null mutant did not protect chickens when challenged with very virulent field strain suggesting that RR is essential for development of live attenuated vaccines.

Publications

• Lee, L.F., Silva, R.F., Heidari, M., Reddy, S.M. Studies on Marek's Disease Virus Encoded Ribonucleotide Reductase. American Association of Avian Pathologists Symposium and Scientific Program, August 1-4, 2010, Atlanta, Georgia. Paper No. 9410-2.

Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: Activities included the training of one post doctoral fellow. The research was presented at International Marek's disease Workshop. Collaborations were established with Agriculture Research Service laboratory at Michigan, USDA. PARTICIPANTS: Sanjay M. Reddy, Aijun Sun, Blanca Lupiani, Lucy Lee (Collaboratory), Agriculture Research Service, USDA, Michigan TARGET AUDIENCES: Target audiences included researchers and vaccine manufacturers. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Marek's disease virus (MDV) genomic DNA was co-transfected into chicken embryo fibroblasts along with BAC transfer vector modified to insert into MDV US2 region. The resulting virus was propagated in the presence of mycophenolic acid, xanthine and hypoxanthine to enrich for recombinant viruses with insertion of bacterial artificial chromosome (BAC) vector. After four rounds of replication, the resulting viral DNA was electroporated into bacteria and selected on agar plates containing chloramphenicol. We have identified stable BAC clones with expected genomic structure and were used to transfect chicken embryo fibroblast to recover MDV. From the MDV BAC clone we have deleted the large fragment ribonucleotide reductase using two-step homologous recombination system. In the first step, the gene encoding for kanamycin resistance was inserted into the ribonucleotide reductase gene. In the second step, the kanamycin gene was removed from the BAC clone, resulting in a BAC clone unable to express both ribonuclotide reductase and kanamycin genes. Transfection of the BAC clone into chicken embryo fibroblasts resulted in the generation of MDV that is unable to express large fragment of the ribonucleotide reductase gene.

Publications

• L. Lee, A. Sun, R. Silva, M. Heidari, B. Lupiani and S.M. Reddy. Marek's Disease virus encoded ribonucleotide reductase large subunit is not essential for in vitro replication. The 5th international workshop on the molecular pathogenesis of Marek's disease virus and 1st symposium on avian herpesviruses. Oct. 17-20, 2010. Edited by S. Spatz and A, Moody. Page 21.