Virus Genes

, Volume 45, Issue 3, pp 526–536 | Cite as

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

  • Stephen J. SpatzEmail author
  • Jeremy D. Volkening
  • Isabel M. Gimeno
  • Mohammad Heidari
  • Richard L. Witter


Attenuation of Gallid herpesvirus-2 (GaHV-2), the causative agent of Marek’s disease, can occur through serial passage of a virulent field isolate in avian embryo fibroblasts. In order to gain a better understanding of the genes involved in attenuation and associate observed changes in phenotype with specific genetic variations, the genomic DNA sequence of a single GaHV-2 virulent strain (648A) was determined at defined passage intervals. Biological characterization of these “interval-isolates” in chickens previously indicated that the ability to induce transient paralysis was lost by passages 40 and the ability to induce persistent neurological disease was lost after passage 80, coincident with the loss of neoplastic lesion formation. Deep sequencing of the interval-isolates allowed for a detailed cataloguing of the mutations that exist within a single passage population and the frequency with which a given mutation occurs across passages. 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 the virulence factors vLipase, vIL8, and RLORF4, as well as a deletion in the promoter of ICP4, appeared between passages 61 and 101. Three mutations in the virus-encoded telomerase which predominated in late passages were also identified. Overall, the frequency of mutations fluctuated greatly during serial passage and few genetic changes were absolute. This indicates that serial passage of GaHV-2 results in the generation of a collection of genomes with limited sequence heterogeneity.


Marek’s disease Marek’s disease virus Sequencing Polymorphisms Serial passage Gallid herpesvirus 2 Pyrosequencing Virulence 



The authors would like to thank Barbara Riegle of the Avian Disease and Oncology Laboratory for her assistance in the propagation of the virus strains.


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Copyright information

© Springer Science+Business Media, LLC (Outside the USA) 2012

Authors and Affiliations

  • Stephen J. Spatz
    • 1
    Email author
  • Jeremy D. Volkening
    • 2
  • Isabel M. Gimeno
    • 3
  • Mohammad Heidari
    • 4
  • Richard L. Witter
    • 4
  1. 1.Southeast Poultry Research Laboratory, United States Department of AgricultureAgricultural Research ServiceAthensUSA
  2. 2.BASE2BIOMadisonUSA
  3. 3.Department of Population Health and PathobiologyNorth Carolina State UniversityRaleighUSA
  4. 4.Avian Disease and Oncology Laboratory, United States Department of AgricultureAgricultural Research ServiceE. LansingUSA

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