Submitted to: Workshop on Molecular Pathogenesis of Marek's Disease and Avian Immunology
Publication Type: Abstract Only
Publication Acceptance Date: 8/5/2006
Publication Date: 8/5/2006
Citation: Niikura, M., Dodgson, J., Silva, R.F., Cheng, H.H. 2006. Infectious bacterial artificial chromosome clones of Marek's disease virus possessing the complete viral genome derived from Md5 cosmid clones [abstract]. 4th International Workshop on Molecular Pathogenesis of Marek's Disease Virus. p. 37.
Technical Abstract: Infectious bacterial artificial chromosome (BAC) clones are the starting material of choice to investigate individual viral gene function in herpesvirus replication and pathogenesis. Three MDV serotype 1 BAC clones have been reported so far although only one is fully virulent. Due to the need to integrate the BAC vector into the viral genome, all of these MDV-BAC clones lack either the US2 or US6 gene. To overcome potential problems associated with insertional deletions or knockouts, we have generated MDV-BAC clones that contain the complete MDV genome based on the virulent overlapping cosmid clones of strain Md5. A BAC plasmid, pBeloBAC11, was inserted by RecA-assisted restriction endonuclease cleavage and ligation into an AluI site between UL3 and UL4 in cosmid clone SN5. This location is downstream of both UL3 and UL4 and may not be transcribed based on potential polyA signals. To facilitate removal of the BAC vector in the MDV-BAC clones, the inserted BAC sequence was flanked by loxP sequences. After co-transfection with the other 4 overlapping cosmid clones, BAC plasmids with the complete MDV genome were recovered in E. coli. These clones produced infectious virus in both CEF and DEF following transfection. When transfected into a modified DF1 cell line (DF1-Cre1) that constitutively expresses Cre recombinase, the BAC vector was removed from the MDV-BAC genome to a level undetectable on Southern blots. Currently, we are investigating the virulence and ability to spread horizontally of the resulting viruses.