Generation of Recombinant Marek’s Disease Viruses In Vivo

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University of Delaware
Marek’s Disease Virus is a highly contagious herpesvirus that causes T-cell lymphomas in chickens. Although there are vaccines which prevent lymphoma formation, MDV has increased in virulence as new strains evolve in the field. Mutations have been identified in the meq gene of MDV strains that appear to directly correspond to the virulence level of the virus. The first part of this project will attempt to create recombinant viruses using MD5Δmeq in which the meq genes have been deleted from the MD5 strain of the virus. MD5Δmeq replicates in chickens, but will not cause disease. The meq gene from three different strains; JM, RB1B and MK will then be combined with MD5 through co-transfection of the MD5Δmeq with the corresponding meq locus from each strain, creating viruses that differ only in the meq gene. For the second part of this project, an MD5-based construct was made that had loxP recombination sites flanking the meq loci. This insert was also co-transfected with the MD5Δmeq virus in order to generate recombinant viruses with meq regions that can be interchanged using Cre recombinase. Since viruses that have recombined the meq gene into their genome cannot be selected in cell culture, chickens were inoculated with co-transfected cells for the selection of recombinants through tumor formation. Using this in vivo selection method, we have previously generated an MD5:RB1B Meq virus. Any recombinants isolated during this experiment will be used to evaluate the effect of meq mutations on changes in MDV virulence.