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Journal of Virology, September 2007, p. 9024-9033, Vol. 81, No. 17
0022-538X/07/$08.00+0     doi:10.1128/JVI.02666-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Genetic Analysis of Varicella-Zoster Virus ORF0 to ORF4 by Use of a Novel Luciferase Bacterial Artificial Chromosome System{triangledown}

Zhen Zhang,1 Jenny Rowe,2 Weijia Wang,1 Marvin Sommer,3 Ann Arvin,3 Jennifer Moffat,2 and Hua Zhu1*

Department of Microbiology and Molecular Genetics, UMDNJ-New Jersey Medical School, 225 Warren Street, Newark, New Jersey 07101,1 Department of Microbiology and Immunology, State University of New York Upstate Medical University, Syracuse, New York 13210,2 Department of Pediatrics and Microbiology and Immunology, Stanford University, 300 Pasteur Drive, Stanford, California 94305-52083

Received 2 December 2006/ Accepted 11 June 2007

To efficiently generate varicella-zoster virus (VZV) mutants, we inserted a bacterial artificial chromosome (BAC) vector in the pOka genome. We showed that the recombinant VZV (VZVBAC) strain was produced efficiently from the BAC DNA and behaved indistinguishably from wild-type virus. Moreover, VZV's cell-associated nature makes characterizing VZV mutant growth kinetics difficult, especially when attempts are made to monitor viral replication in vivo. To overcome this problem, we then created a VZV strain carrying the luciferase gene (VZVLuc). This virus grew like the wild-type virus, and the resulting luciferase activity could be quantified both in vitro and in vivo. Using PCR-based mutagenesis, open reading frames (ORF) 0 to 4 were individually deleted from VZVLuc genomes. The deletion mutant viruses appeared after transfection into MeWo cells, except for ORF4, which was essential. Growth curve analysis using MeWo cells and SCID-hu mice indicated that ORF1, ORF2, and ORF3 were dispensable for VZV replication both in vitro and in vivo. Interestingly, the ORF0 deletion virus showed severely retarded growth both in vitro and in vivo. The growth defects of the ORF0 and ORF4 mutants could be fully rescued by introducing wild-type copies of these genes back into their native genome loci. This work has validated and justified the use of the novel luciferase VZV BAC system to efficiently generate recombinant VZV variants and ease subsequent viral growth kinetic analysis both in vitro and in vivo.

* Corresponding author. Mailing address: Department of Microbiology and Molecular Genetics, UMDNJ-New Jersey Medical School, 225 Warren Street, Newark, NJ 07101-1709. Phone: (973) 972-4483, ext. 2-6488. Fax: (973) 972-8981. E-mail: zhuhu{at}umdnj.edu

{triangledown} Published ahead of print on 20 June 2007.

Journal of Virology, September 2007, p. 9024-9033, Vol. 81, No. 17
0022-538X/07/$08.00+0     doi:10.1128/JVI.02666-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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