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Deletion of Marek's Disease Virus Large Subunit of Ribonucleotide Reductase Impairs Virus Growth In Vitro and In Vivo

Author:
Sun, Aijun, Lee, Lucy F., Khan, Owais A., Heidari, Mohammad, Zhang, Huanmin, Lupiani, Blanca, Reddy, Sanjay M.
Source:
Avian diseases 2013 v.57 pp. 464
ISSN:
0005-2086
Subject:
Mardivirus, bacterial artificial chromosomes, chickens, ducks, fibroblasts, gene amplification, genes, in vivo studies, microbial growth, monoclonal antibodies, mutants, nonpathogenic strains, open reading frames, polymerase chain reaction, poultry diseases, recombinant DNA, ribonucleotide reductase, vaccines, virus replication, viruses
Abstract:
Marek's disease virus (MDV), a highly cell-associated lymphotropic alphaherpesvirus, is the causative agent of a neoplastic disease in domestic chickens called Marek's disease (MD). In the unique long (UL) region of the MDV genome, open reading frames UL39 and UL40 encode the large and small subunits of the ribonucleotide reductase (RR) enzyme, named RR1 and RR2, respectively. MDV RR is distinguishable from that present in chicken and duck cells by monoclonal antibody T81. Using recombinant DNA technology we have generated a mutant MDV (Md5DRR1) in which RR1 was deleted. PCR amplification of the RR gene in Md5DRR1-infected duck embryo fibroblasts (DEF) confirmed the deletion of the 2.4 kb RR1 gene with a resultant amplicon of a 640-bp fragment. Restriction enzyme digests with SalI confirmed a UL39 deletion and the absence of gross rearrangement. The biologic characteristics of Md5DRR1 virus were studied in vitro and in vivo. The Md5DRR1 replicated in DEF, but significantly slower than parental Md5-BAC, suggesting that RR is important but not essential for replication in fibroblasts. In vivo studies, however, showed that the RR1 deletion virus was impaired for its ability to replicate in chickens. Inoculation of specific-pathogen-free (SPF) chickens with Md5DRR1 showed the mutant virus is nonpathogenic and does not induce MD in birds. A revertant virus, Md5DRR1/R, was generated with the restored phenotype of the parental Md5-BAC in vivo, indicating that RR is essential for replication of the virus in chickens. Protection studies in SPF chickens indicated that the Md5DRR1 virus is not a candidate vaccine against MD.
Agid:
56950
Handle:
10113/56950