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Journal of Virology, December 2000, p. 11088-11098, Vol. 74, No. 23
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Reconstitution of Marek's Disease Virus Serotype 1 (MDV-1) from DNA Cloned as a Bacterial Artificial Chromosome and Characterization of a Glycoprotein B-Negative MDV-1 Mutant

Daniel Schumacher, B. Karsten Tischer, Walter Fuchs, and Nikolaus Osterrieder*

Institute of Molecular Biology, Friedrich-Loeffler-Institutes, Federal Research Centre for Virus Diseases of Animals, D-17498 Insel Riems, Germany

Received 14 July 2000/Accepted 15 September 2000

The complete genome of Marek's disease virus serotype 1 (MDV-1) strain 584Ap80C was cloned in Escherichia coli as a bacterial artificial chromosome (BAC). BAC vector sequences were introduced into the US2 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 harboring the complete MDV-1 genome as an F plasmid (MDV-1 BACs) were identified. DNA from various MDV-1 BACs was transfected into chicken embryo fibroblasts, and from 3 days after transfection, infectious MDV-1 was obtained. Growth of MDV-1 recovered from BACs was indistinguishable from that of the parental virus, as assessed by plaque formation and determination of growth curves. In one of the MDV-1 BAC clones, sequences encoding glycoprotein B (gB) were deleted by one-step mutagenesis using a linear DNA fragment amplified by PCR. Mutant MDV-1 recovered after transfection of BAC DNA that harbored a 2.0-kbp deletion of the 2.6-kbp gB gene were able to grow and induce MDV-1-specific plaques only on cells providing MDV-1 gB in trans. The gB-negative 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 analyze genes and gene products in slowly growing and strictly cell-associated herpesviruses.

* Corresponding author. Mailing address: Institute of Molecular Biology, Friedrich-Loeffler-Institutes, Federal Research Centre for Virus Diseases of Animals, Boddenblick 5a, D-17498 Insel Riems, Germany. Phone: 49-38351-7266. Fax: 49-38351-7151. E-mail: klaus.osterrieder{at}rie.bfav.de.

Journal of Virology, December 2000, p. 11088-11098, Vol. 74, No. 23
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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