Cloning Of Marek's Disease Virus Genome As An Infectious Bacterial Artificial Chromosome

Marek's disease virus (MDV) is a member of the Alphaherpesvirinae subfamily of the Herpesviridae, which is a representative oncogenic alphaherpesvirus that induces T-cell lymphomas in poultry, there are three serotypes of MDV. MDV-1 include all virulent and very virulent oncogenic viruses, MDV-2 contain all unoncogenic viruses, and MDV-3 represents the herpesvirus of turkeys (HVT) which has been widely used for vaccination against MD.It is especially true that construction of MDV viral mutants is a difficult and tedious procedure. Fortunately, a completely novel approach for the construction of herpesvirus mutants which is based on cloning of the virus genome as a bacterial artificial chromosome (BAC) in Escherichia coli has been developed. Once the viral genome is cloned as a BAC, mutagenesis can be done by homologous recombination in E. coli which is mainly based on RED/ET mutagenesis system. The novel approach have clear advantages, and the powerful methods of bacterial genetics allow the rapid and efficient introduction of any kind of mutations (deletion, insertion, and point mutation) into the cloned viral genome, even mutagenesis of essential viral genes now becomes feasible.The aim of this study was to clone full-length genome of Marek's disease virus serotype 1 (MDV-1) 814 strain (nature attenuated strain) as an infectious BAC. In this study, Using the 8.8kb fragment containing self-designed selection marker (Eco-gpt, 1.3kb) and the BAC vector pBeloBAC11(7.5kb), a recombination plasmid pUAB-gpt-BAC11 was constructed. For cloning MDV814-BAC,. the recombination plasmid and MDV Total-DNA were cotransfected into secondary CEF by Lipofectamine^TM2000, virus-containing cells were passaged eight rounds in selection medium containing xanthine and mycophenolic acid to obtain purified recombinant viruses. Recombinant viral Genomes was extracted and Electroporated into E.coli and thirty-eight BACs were obtained and identified. Finally, DNA from various MDV-1 BACs was transfected into secondary chicken embryo fibroblasts, recombinant viruses were reconstituted from MDV-BAC2 succesfully. It was found that BAC2-derived viruses have biological characteristics and growth kinetics similar to those of the wildtype parental viruses and recombinant viruses in vitro. Further experiments indicate that BAC2-DNA-based vaccine (rv-MDV814, BAC2-derived MDV814, PBS-BAC2-DNA, Lip-BAC2-DNA, Ca-BAC2-DNA and Live-E.coli-BAC2) containing an infectious Marek's disease virus genome can confer protection against tumorigenic Marek's disease in chicken (challenge infection with J-1 strain). These results suggested that an MDV BAC DNA vaccine has potential to protect chickens against MD. With these cloned genomes in hand, a detailed assessment of genes expressed in the lytic, latent and tumor phases of MDV-1 infection should be possible. In addition, it may be possible to generate a novel generation of MDV-1 vaccines based on BACs.