REPLICATION OF HUMAN CYTOMEGALOVIRUS IN HUMAN RETINAL GLIAL-CELLS

Purpose. The purpose of these studies was to characterize the replicat ion 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 P-hour adsor ption period and then incubated in the appropriate growth medium at 37 degrees C. Cultures were examined by microscopy for cytopathic effect and by immunofluorescence staining using monoclonal antibodies direct ed against immediate-early, early, and late HCMV proteins. Viral DNA w as analyzed by field inversion gel electrophoresis and detected using Southern blot analysis or the polymerase chain reaction. Results, Immu nocytochemical staining revealed that the glial cells expressed all th ree classes of HCMV proteins and that infectious virus could be transf erred from the medium of the infected cultures to susceptible MRC-5 ce ll monolayers. Less than 1% of the glial cells expressed the S-phase e nzyme, thymidine kinase, at the time of infection compared to MRC-5 fi broblasts, of which 81% expressed it. Progeny virus was found to be hi ghly 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 deficien cy virus also productively infected cultures of human retinal glial ce lls. Field inversion gel electrophoresis of HCMV-infected glial cell l ysates was performed to identify the replicative forms of DNA. Souther n blots probed with HCMV-specific probes showed that HCMV DNA replicat ion proceeds through high molecular weight intermediates before formin g the 230-kb unit length genome. Conclusions, The full permissive repl ication of HCMV in human retinal glial cells indicates that glial cell s are a likely site of HCMV replication in the retina and thus may pla y an important role in the pathogenesis of HCMV retinitis.