Purification and characterization of infectious Marek's disease virus genomes using pulsed field electrophoresis

Abstract

Marek's disease virus (MDV) is an acutely oncogenic avian herpesvirus. The tightly cell-associated in vitro growth characteristics of MDV present unique problems when attempting to purify, analyze, and manipulate MDV genomes. To facilitate molecular characterization of MDV, contour-clamped homogeneous electric fields electrophoresis (CHEF) was used to purify infectious MDV genomes. CHEF techniques were optimized for evaluation of total genome size and alterations in structure which occur during in vitro attenuation of oncogenic MDV. Our results indicated that genomes of attenuated serotype 1 MDV strain JM may contain deletions totaling 15 kbp while high-passage serotype 2 nononcogenic MDV strains SB-1 and 281Ml/1 were 5 and 3 kbp larger, respectively, than their low-passage counterparts. Using cell-free CHEF-purified MDV genomes as hybridization probes, we identified a 200-bp deletion in attenuated genomes of the very virulent MDV strain MD11. At present, it is unclear if this 200-bp deletion is related to mutations which lead to loss of oncogenicity or pathogenicity in MD11. This study is the first report which describes procedures for purification of infectious herpesvirus genomes from pulsed-field gels. Our results demonstrate that pulsed-field-purified viral DNA will facilitate molecular characterization of MDV and other cell-associated herpesviruses.