Cloning Of Marek's Disease Virus CVI988/Rispens Genome As Bacterial Artificial Chromosome

Marek's disease (MD) is a highly contagious lymphoproliferative disease of poultry caused by Marek's disease virus (MDV).Although it had caused devastating losses in commercial poultry flocks recent years, The disease has been successfully controlled by vaccination with attenuated or non-pathogenic MDV strains since 1970s. However, Marek's disease continues to provide a significant challenge to the current vaccination strategies as field strains continue to evolve towards pathotypes of greater virulence. Marek's disease virus (MDV) is a dsDNA virus,α-herpesvirus of chickens. MDV has three serotypes and only Serotype 1 MDV is pathogenic .MDV CVI988/Rispens was the most successful and widely used live herpersvirus vaccines in the world ,which isolated from a nature low virulent strain .An understanding of MDV gene structure,functions and mechanism is the first step for effectively control of MD , but construction of MDV viral mutants using classic technology of gene is difficult as very long genome sequence and the feature of cell-associated. The construction of the full genome infective clones using bacterial artificial chromosome (BAC) technology can deal with this problem, which has already been a powerful tools for gene manipulation of MDV genome.The aim of this study was to clone full-length genome of Marek's disease virus serotype 1 (MDV-1) CVI988 strain as an infectious BAC. First, a recombination plasmid pUA1B1-gpt-BAC self-designed containing selection marker (Eco-gpt,1.3kb) and MDV infected CEF total-DNA were co-transfected into CEF by LipofectamineTM 2000. After six rounds in selection medium, the purified recombinant viruses was obtained and named rv-BAC-MDV CVI988/Rispens, It was found that rv-BAC-MDV CVI988 viruses have similar biological characteristics and growth kinetics to the wildtype parental viruses in vitro. Recombinant viral Genomes were extracted and electroporated into DH10B and thirty-seven BACs were obtained. Finally, ten BACs were identified as MDV CVI988-BAC by PCR.In this study, we successfully constructed MDV serotypeⅠCVI988/Rispens strain as an bacterial artificial chromosome and laid a good foundation for MDV BAC reverse genetic system.