Reconstitution of Marek's disease virus serotype 1 (MDV-1) from DNA clonedas a bacterial artificial chromosome and characterization of a glycoprotein B-negative MDV-1 mutant

The complete genome of Mareks disease virus serotype 1 (MDV-1) strain 584Ap 80C was cloned in Escherichia coli as a bacterial artificial chromosome (BA C). BAC vector sequences were introduced into the U(S)2 locus of the MDV-1 genome by homologous recombination. Viral DNA containing the BAC vector was used to transform Escherichia coli strain DH10B, and several colonies harb oring the complete MDV-1 genome as an F plasmid (MDV-1 BACs) were identifie d. DNA from various MDV-1 BACs was transfected into chicken embryo fibrobla sts, and from 3 days after transfection, infectious MDV-1 was obtained. Gro wth of MDV-1 recovered from BACs was indistinguishable from that of the par ental virus, as assessed by plaque formation and determination of growth cu rves. In one of the MDV-1 BAC clones, sequences encoding glycoprotein B (gB ) were deleted by one-step mutagenesis using a linear DNA fragment amplifie d by PCR. Mutant MDV-1 recovered after transfection of BAC DNA that harbore d a 2.0-kbp deletion of the 2.6-kbp gB gene were able to grow and induce MD V-1-specific plaques only on cells providing MDV-1 gB in trans. The gB-nega tive virus reported here represents the first MDV-1 mutant with a deletion of an essential gene and demonstrates the power and usefulness of BACs to a nalyze genes and gene products in slowly growing and strictly cell-associat ed herpesviruses.