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Journal of Virology, September 2002, p. 9493-9504, Vol. 76, No. 18
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.18.9493-9504.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

A Recombinant Rhesus Cytomegalovirus Expressing Enhanced Green Fluorescent Protein Retains the Wild-Type Phenotype and Pathogenicity in Fetal Macaques

W. L. William Chang,1 Alice F. Tarantal,2 Shan Shan Zhou,1 Alexander D. Borowsky,1 and Peter A. Barry1*

Center for Comparative Medicine and Department of Medical Pathology,1 California National Primate Research Center and Department of Pediatrics, University of California, Davis, California 956162

Received 25 March 2002/ Accepted 11 June 2002

To facilitate identification of rhesus cytomegalovirus (RhCMV)-infected cells, a recombinant virus expressing enhanced green fluorescent protein (EGFP), designated RhCMV-EGFP, was constructed. An expression cassette for EGFP under the control of the simian virus 40 (SV40) early promoter was inserted into the intergenic region between unique short 1 (US1) and US2 of the RhCMV genome by homologous recombination. RhCMV-EGFP exhibited comparable growth kinetics to that of wild-type virus in rhesus fibroblast cultures and retained its pathogenicity in monkey fetuses. Typical neurologic syndromes caused by CMV infection were observed in all fetuses experimentally inoculated with RhCMV-EGFP, as evidenced by sonographic and gross examinations. Systemic RhCMV infections were established in all fetuses, as viral antigen was detected in multiple organs and virus was isolated from fetal blood samples. The engineered viral genome was stable following rapid serial passages in vitro and multiple rounds of replication in vivo. Infected cells could be readily distinguished by green fluorescence both in tissue cultures and in the fetuses. In addition, EGFP expression was detected in various cell types that were permissive to RhCMV infection, consistent with a broad tissue tropism of the SV40 promoter. These results demonstrate that RhCMV can be successfully engineered without loss of wild-type replication and pathogenic potential. Further, the spectrum of cortical anomalies and the distribution of infected cells in the brain tissues indicated that RhCMV may have preferentially targeted immature neuronal cells. The pattern of RhCMV infection in the central nervous system may offer an explanation for the severe developmental outcomes associated with congenital human CMV infection early in gestation.

* Corresponding author. Mailing address: Center for Comparative Medicine, University of California, Davis, CA 95616. Phone: (530) 752-6912. Fax: (530) 752-7914. E-mail: pabarry{at}ucdavis.edu.

Journal of Virology, September 2002, p. 9493-9504, Vol. 76, No. 18
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.18.9493-9504.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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