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Journal of Virology, February 2005, p. 2115-2123, Vol. 79, No. 4
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.4.2115-2123.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Human Cytomegalovirus Genes in the 15-Kilobase Region Are Required for Viral Replication in Implanted Human Tissues in SCID Mice

Weijia Wang,¹ Shannon L. Taylor,² Stacey A. Leisenfelder,² Robert Morton,² Jennifer F. Moffat,² Sergey Smirnov,³ and Hua Zhu^1*

Department of Microbiology and Molecular Genetics,¹ Department of Biochemistry and Molecular Genetics, UMDNJ-New Jersey Medical School, Newark, New Jersey,³ Department of Microbiology and Immunology, State University of New York Upstate Medical University, Syracuse, New York²

Received 16 July 2004/ Accepted 1 October 2004

Since animal models for studying human cytomegalovirus (HCMV) replication in vivo and pathogenesis are not available, severe combined immunodeficiency mice into which human tissues were implanted (SCID-hu mice) provide an alternative and valuable model for such studies. The HCMV clinical isolates, including those of the Toledo strain, replicate to high titers in human tissue implanted into SCID mice; however, the attenuated AD169 strain has completely lost this ability. The major difference between Toledo and AD169 is a 15-kb segment, encoding 19 open reading frames, which is present in all virulent strains but deleted from attenuated strains. This fact suggests that crucial genes required for HCMV replication in vivo are localized to this region. In this study, the importance of this 15-kb segment for HCMV replication in vivo was determined. First, Toledo_BAC virus (produced from a Toledo bacterial artificial chromosome) and AD169 virus were tested for growth in SCID-hu mice. Toledo_BAC, like Toledo, grew to high titers in implanted human thymus and liver tissues, while AD169 did not. This outcome showed that the Toledo genome propagated in bacteria (Toledo_BAC) retained its virulence. The 15-kb segment was then deleted from Toledo_BAC, and the resulting virus, Toledo_15kb, was tested for growth in both human foreskin fibroblast (HFF) cells and SCID-hu mice. Toledo_15kb had a minor growth defect in HFF but completely failed to replicate in human thymus and liver implants. This failure to grow was rescued when the 15-kb region was inserted back into the Toledo_15kb genome. These results directly demonstrated that the genes located in the 15-kb segment are crucial for HCMV replication in vivo.

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

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Journal of Virology, February 2005, p. 2115-2123, Vol. 79, No. 4
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.4.2115-2123.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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