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United States Department of Agriculture

Agricultural Research Service

Research Project: GENOMIC STRATEGIES FOR CONTROL OF MAREK'S DISEASE VIRUS Title: Genotypic characterization of two bacterial artificial chromosome clones derived from a single DNA source of the very virulent gallid herpesvirus-2 strain C12/130

Authors
item Spatz, Stephen
item Smith, Lorraine -
item Baigent, Susan -
item Petherbridge, Lawrence -
item Nair, Venugopal -

Submitted to: Journal of General Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 30, 2011
Publication Date: July 5, 2011
Citation: Spatz, S.J., Smith, L.P., Baigent, S.J., Petherbridge, L., Nair, V. 2011. Genotypic characterization of two bacterial artificial chromosome clones derived from a single DNA source of the very virulent gallid herpesvirus-2 strain C12/130. Journal of General Virology. 92:1500-1507

Interpretive Summary: The complete deoxyribonucleotide sequence of two virulent strains of Gallid herpesvirus type 2 was determined. These strains were from the European continent and their genetic material was cloned as a bacterial artificial chromosome. Although the two strains shared a common background, biologically they are different. We have identified the genes that are responsible for the biological differences.

Technical Abstract: Gallid herpesvirus type 2 (GaHV-2) is the causative agent of Marek’s disease, a lymphoproliferative disease of chickens. GaHV-2 strains vary greatly in pathogenicity. Identification of specific genetic changes associated with differences in pathogenicity has been a formidable task due to the large number of mutations and single nucleotide polymorphisms occurring within in vitro serially passaged attenuated strains. High throughput sequencing of viral deoxyribonucleic acid (DNA) from these strains has suggested that GaHV-2 genomes exist as a collection of quasispecies. Nucleotide sequencing of GaHV-2 genomes contained within bacterial artificial chromosomes (BAC) will allow for mapping of genetic changes responsible for a given phenotype within a single genome. To this end we have generated BAC clones from viral DNA isolated from a GaHV-2 hypervirulent strain known as C12/130. Reconstituted viruses from individual BACs failed to exhibit differences in replication kinetics in vitro and in vivo and did not significantly differ in the level of virus shed from infected chickens. Two reconstituted viruses (vC12/130-10 and vC12/130-15) did, however, show differences in virulence relative to each other and to the parental strain. Although both viruses were able to induce tumor formation, survival rates differed significantly. These studies indicated that vC12/130-15 was attenuated. Sequence analysis of both clones has identified mutations within mononucleotide reiterations which clustered mainly within the novel genes (MDV084.5/99.5, MDV085/99, MDV085.3/98.9, MDV086/98, MDV086.2/97.6 and MDV086.2) in the repeat short/unique short junctions. One diploid gene in the repeat long (MDV03.6/78.2) and one haploid gene (MDV086.6) in the unique short also contained mutations. A 494 base pair insertion in repeat elements between MDV071 (LORF10) and MDV072 (LORF11) was identified within the genome of the virulent C12/130-10 clone. Only seven nonsynonymous substitutions distinguished virulent C12/130-10 from attenuated C12/130-15. These occur in the tegument protein, UL37 (S719P), ribonucleotide reductase UL39, (A756V), ICP4 (A578V, S1634K), MDV083 (R58G) and MDV090 or SORF3 (M182I, A222V). The identification of these mutations will hopefully provide insight into the genes involved in virulence and facilitate the generation of improved GaHV-2 vaccines.

Last Modified: 12/21/2014
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