Submitted to: Meeting Proceedings
Publication Type: Proceedings
Publication Acceptance Date: November 9, 2008
Publication Date: December 7, 2008
Citation: Spatz, S.J., Gimeno, I.M., Heidari, M. 2008. Identification of Marek's disease virus genes mutated during serial passage-induced attenuation.. Meeting Proceedings. 1(1): 188. Technical Abstract: Marek’s disease (MD) is a lymphoproliferative disease of chickens caused by gallid herpesvirus type 2 (GaHV-2). The disease is controlled through mass vaccination with live-attenuated strains. Attenuation of oncogenic GaHV-2 involves the serial passage of a virulent field isolate in avian embryo fibroblast 35 –100 times. Little is known of the genetic changes responsible for attenuation. In order to gain a better understanding of the genes involved in attenuation, the genomic deoxyribonucleic acid (DNA) sequence of a single virulent strain (648A) was determined at defined passage intervals (p10, p30, p40, p60, p80 and p100). Biological characterization of these ‘interval-isolates’ in chickens indicated that the ability to induce transient paralysis was lost between passages 30 and 40 and the ability to induce persistent neurological disease was lost after passage 80, coincident with the loss of neoplastic lesions in peripheral nerves and other visceral organs. Sequencing of the interval-isolates using 454 pyrosequencing generated on average 50 fold base pair coverage. This allowed a detailed analysis of the collections of single nucleotide polymorphisms (SNP)that exist in differing proportions within a single DNA preparation. This is the first evidence of the quasi-species nature of GaHV-2 in vitro. Additionally, gross genetic alterations were identified in both novel and well-characterized genes and cis-acting regions involved in replication and cleavage/packaging. Deletions in genes encoding virulence factors vLipase, vIL8, RLORF4 and an internal repeat sequence unique short (IRS/US) junction gene cluster appeared between passages 60 and 100. Insertions in two novel genes MDV001 and MDV002.6 started to appear after passage 40 and were most prevalent in p100. Few genetic changes were absolute (present in 100% of the sequences aligned) by passage 100. Because of this, correlating a phenotype with changes in a specific gene is at best suggestive and will require additional studies using defined GaHV-2 bacterial artificial constructs containing specifically engineered deletions and SNPs.