Title: Evolutionary Genomics of Marek's Disease Virus Author
Submitted to: Meeting Proceedings
Publication Type: Proceedings
Publication Acceptance Date: October 1, 2009
Publication Date: October 17, 2009
Citation: Spatz, S.J. 2009. Evolutionary Genomics of Marek's Disease Virus. Meeting Proceedings. 1(1):8. Technical Abstract: Marek’s disease virus (MDV) is one of the most oncogenic herpesviruses known and induces a rapid onset T-cell lymphoma and demyelinating disease in chickens. The virus is classified as a member of the genus Mardivirus in the Alphaherpesvirinae subfamily of Herpesviridae. The disease (Marek's Disease, or MD) is largely controlled through mass vaccination of chick embryos or day-old chicks with live-attenuated strains of MDV (serotypes 1 and 2) or in combination with the related virus, Meleagrid herpesvirus type 1. Since their introduction in the 1970s, MD vaccines have reduced economic losses by more than 99%. However, over the last 40 years of intense vaccination virulent strains have reappeared, necessitating the continued introduction of new vaccines at fairly regular intervals. Since the 1990s, the disease has been controlled by vaccination with the attenuated oncogenic strain CVI988 (Rispens). Despite its global usage and exceptional protective efficacy, there are signs of further increases in MDV virulence, as shown by the isolation of very virulent strains in vaccinated birds. Because of this, there is great concern that current vaccination programs may fail in the same way previous generations of MD vaccines have lost their usefulness. Furthermore, little is known about the evolutionary forces responsible for the generation of these “breakout” very virulent plus field strains. In order to prevent future vaccinal failures, a better understanding of the genetics involved in virulence is needed. To this end the USDA has begun a comparative genomics program to identify genes involved in virulence. To date, the nucleotide sequences of five virulent (Md5, GA, Md11, RB-1B, C12/130-10), one mildly virulent (CU-2) and three attenuated (CVI988, 584Ap80, C12/130-15) strains have been determined. Comparative genomics revealed that while MDV is relatively stable genetically, it does possess some degree of variability. Recently, my laboratory has completed a study detailing the accumulation of mutations necessary for attenuation during serial passage of a single very virulent plus strain in cell culture. At defined passage intervals (p10, p30, p40, p60, p80 and p100) the genomic deoxyribonucleic acid (DNA) sequences were determined using high throughput 454 DNA sequencing. 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-isolate DNA generated on average a 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 DNA preparation. This is the first evidence of the quasispecies nature of MDV-1 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, and RLORF4 appeared between passages 60 and 100. Insertions in two novel genes (ORF1 and ORF2.6) started to appear after passage 40 and were most prevalent in p100. Few genetic changes were absolute (present in 100% of the sequences) 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 MDV-1 bacterial artificial constructs containing specifically engineered mutations.