A recombinant rhesus cytomegalovirus expressing enhanced green fluorescent protein retains the wild-type phenotype and pathogenicity in fetal macaques

W. L William Chang, Alice F Tarantal, Shan Shan Zhou, Alexander D Borowsky, Peter A Barry

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)9493-9504
Number of pages12
JournalJournal of Virology
Volume76
Issue number18
DOIs
StatePublished - Sep 2002

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Cytomegalovirus
Macaca
green fluorescent protein
Virulence
pathogenicity
Phenotype
phenotype
fetus
Cytomegalovirus Infections
Fetus
Simian virus 40
Viruses
viruses
promoter regions
cells
tissue tropism
Serial Passage
Intergenic DNA
Tropism
genome

ASJC Scopus subject areas

  • Immunology

Cite this

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title = "A recombinant rhesus cytomegalovirus expressing enhanced green fluorescent protein retains the wild-type phenotype and pathogenicity in fetal macaques",
abstract = "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.",
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T1 - A recombinant rhesus cytomegalovirus expressing enhanced green fluorescent protein retains the wild-type phenotype and pathogenicity in fetal macaques

AU - Chang, W. L William

AU - Tarantal, Alice F

AU - Zhou, Shan Shan

AU - Borowsky, Alexander D

AU - Barry, Peter A

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