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Title: Dynamic equilibrium of Marek’s disease genomes during in vitro serial passage

item Spatz, Stephen
item VOLKENING, JEREMY - Former ARS Employee
item GIMENO, ISABEL - North Carolina State University
item Heidari, Mohammad
item Witter, Richard

Submitted to: Virus Genes
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/14/2012
Publication Date: 8/26/2012
Citation: Spatz, S.J., Volkening, J.D., Gimeno, I.M., Heidari, M., Witter, R.L. 2012. Dynamic equilibrium of Marek’s disease genomes during in vitro serial passage. Virus Genes. 45:526-536. DOI: 10.1007/s11262-012-0792-z.

Interpretive Summary: Serial passage of Gallid herpesvirus type 2 results in the attenuation of the virus at some undefined passage level. Attenuation for the generation of vaccine strains in done empirically and requires animal pathogenicity studies. We have serially passaged one very virulent plus strain 100 times is cell culture for over a year. At defined intervals (i.e. 10,20,30, 40 etc) we have characterized the passages in animals to determine the level of virulence. Simultaneously we have sequenced the deoxyribonucleic acid of the passages. A correlation between genotype and phenotype was established. Therefore we could catalog the accumulation of mutations necessary for attuenuation. Using deep seqeuncing technology our data suggest that gallid herpesvirus type 2 exists as a collection of mixed genotypes or quasispecies. This is very unique for deoxyribonucleic acid containing viruses but common for ribonucleic acid viruses. The significance of this find is intriguing.

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 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 that exist in differing proportions within a single deoxyribonucleic acid (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 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 single nucleotide polymorphisms (SNPs).