Replication of human cytomegalovirus in human retinal glial cells

Eileen M. Burd, Jose S Pulido, Donald G. Puro, William J. O'Brien

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

Purpose. The purpose of these studies was to characterize the replication cycle of human cytomegalovirus (HCMV) in human retinal glial cells in vitro. Methods. Cultured human retinal glial cells were exposed to HCMV strain AD169 or low-passage clinical isolates for a 2-hour adsorption period and then incubated in the appropriate growth medium at 37°C. Cultures were examined by microscopy for cytopathic effect and by immunofluorescence staining using monoclonal antibodies directed against immediate-early, early, and late HCMV proteins. Viral DNA was analyzed by field inversion gel electrophoresis and detected using Southern blot analysis or the polymerase chain reaction. Results. Immunocytochemical staining revealed that the glial cells expressed all three classes of HCMV proteins and that infectious virus could be transferred from the medium of the infected cultures to susceptible MRC-5 cell monolayers. Less than 1% of the glial cells expressed the S-phase enzyme, thymidine kinase, at the time of infection compared to MRC-5 fibroblasts, of which 81% expressed it. Progeny virus was found to be highly cell associated in glial cells (80%) at peak virus titer compared to MRC-5 cells (39% cell associated at peak titer). Four low-passage clinical isolates of HCMV from patients with acquired immune deficiency virus also productively infected cultures of human retinal glial cells. Field inversion gel electrophoresis of HCMV-infected glial cell lysates was performed to identify the replicative forms of DNA. Southern blots probed with HCMV-specific probes showed that HCMV DNA replication proceeds through high molecular weight intermediates before forming the 230-kb unit length genome. Conclusions. The full permissive replication of HCMV in human retinal glial cells indicates that glial cells are a likely site of HCMV replication in the retina and thus may play an important role in the pathogenesis of HCMV retinitis.

Original languageEnglish (US)
Pages (from-to)1957-1966
Number of pages10
JournalInvestigative Ophthalmology and Visual Science
Volume37
Issue number10
StatePublished - Sep 1996
Externally publishedYes

Fingerprint

Cytomegalovirus
Neuroglia
Pulsed Field Gel Electrophoresis
Southern Blotting
Viruses
Cytomegalovirus Retinitis
Staining and Labeling
Thymidine Kinase
Viral DNA
Viral Load
DNA Replication
S Phase
Adsorption
Fluorescent Antibody Technique
Culture Media
Retina
Microscopy
Proteins
Fibroblasts
Molecular Weight

Keywords

  • cytomegalovirus
  • glial cells
  • Muller cells
  • retinitis
  • virus infection

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Replication of human cytomegalovirus in human retinal glial cells. / Burd, Eileen M.; Pulido, Jose S; Puro, Donald G.; O'Brien, William J.

In: Investigative Ophthalmology and Visual Science, Vol. 37, No. 10, 09.1996, p. 1957-1966.

Research output: Contribution to journalArticle

Burd, Eileen M. ; Pulido, Jose S ; Puro, Donald G. ; O'Brien, William J. / Replication of human cytomegalovirus in human retinal glial cells. In: Investigative Ophthalmology and Visual Science. 1996 ; Vol. 37, No. 10. pp. 1957-1966.
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abstract = "Purpose. The purpose of these studies was to characterize the replication cycle of human cytomegalovirus (HCMV) in human retinal glial cells in vitro. Methods. Cultured human retinal glial cells were exposed to HCMV strain AD169 or low-passage clinical isolates for a 2-hour adsorption period and then incubated in the appropriate growth medium at 37°C. Cultures were examined by microscopy for cytopathic effect and by immunofluorescence staining using monoclonal antibodies directed against immediate-early, early, and late HCMV proteins. Viral DNA was analyzed by field inversion gel electrophoresis and detected using Southern blot analysis or the polymerase chain reaction. Results. Immunocytochemical staining revealed that the glial cells expressed all three classes of HCMV proteins and that infectious virus could be transferred from the medium of the infected cultures to susceptible MRC-5 cell monolayers. Less than 1{\%} of the glial cells expressed the S-phase enzyme, thymidine kinase, at the time of infection compared to MRC-5 fibroblasts, of which 81{\%} expressed it. Progeny virus was found to be highly cell associated in glial cells (80{\%}) at peak virus titer compared to MRC-5 cells (39{\%} cell associated at peak titer). Four low-passage clinical isolates of HCMV from patients with acquired immune deficiency virus also productively infected cultures of human retinal glial cells. Field inversion gel electrophoresis of HCMV-infected glial cell lysates was performed to identify the replicative forms of DNA. Southern blots probed with HCMV-specific probes showed that HCMV DNA replication proceeds through high molecular weight intermediates before forming the 230-kb unit length genome. Conclusions. The full permissive replication of HCMV in human retinal glial cells indicates that glial cells are a likely site of HCMV replication in the retina and thus may play an important role in the pathogenesis of HCMV retinitis.",
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AB - Purpose. The purpose of these studies was to characterize the replication cycle of human cytomegalovirus (HCMV) in human retinal glial cells in vitro. Methods. Cultured human retinal glial cells were exposed to HCMV strain AD169 or low-passage clinical isolates for a 2-hour adsorption period and then incubated in the appropriate growth medium at 37°C. Cultures were examined by microscopy for cytopathic effect and by immunofluorescence staining using monoclonal antibodies directed against immediate-early, early, and late HCMV proteins. Viral DNA was analyzed by field inversion gel electrophoresis and detected using Southern blot analysis or the polymerase chain reaction. Results. Immunocytochemical staining revealed that the glial cells expressed all three classes of HCMV proteins and that infectious virus could be transferred from the medium of the infected cultures to susceptible MRC-5 cell monolayers. Less than 1% of the glial cells expressed the S-phase enzyme, thymidine kinase, at the time of infection compared to MRC-5 fibroblasts, of which 81% expressed it. Progeny virus was found to be highly cell associated in glial cells (80%) at peak virus titer compared to MRC-5 cells (39% cell associated at peak titer). Four low-passage clinical isolates of HCMV from patients with acquired immune deficiency virus also productively infected cultures of human retinal glial cells. Field inversion gel electrophoresis of HCMV-infected glial cell lysates was performed to identify the replicative forms of DNA. Southern blots probed with HCMV-specific probes showed that HCMV DNA replication proceeds through high molecular weight intermediates before forming the 230-kb unit length genome. Conclusions. The full permissive replication of HCMV in human retinal glial cells indicates that glial cells are a likely site of HCMV replication in the retina and thus may play an important role in the pathogenesis of HCMV retinitis.

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