An indicator cell assay for detection of human cytomegalovirus based on enhanced green fluorescent protein.

An indicator cell line (ML-UL54-EGFP) for the detection of human cytomegalo virus (HCMV) by a simple and direct method was developed. The stable line w as constructed by introducing into mink lung cells an expression cassette t hat contains the enhanced green fluorescent protein (EGFP) reporter gene un der the control of an HCMV-inducible promoter. The promoter was from the up stream region of the HCMV UL54 (pol) gene, an early gene promoter that is a ctivated in the early phase of HCMV infection. Following infection with HCM V For 48 h, the stable line expressed well detectable level of the EGFP as observed under a fluorescence microscope. The sensitivity of the indicator cell assay is at least comparable with that of a plaque assay as assessed w ith a panel of HCMV strains. There were no detectable fluorescent cells aft er inoculations with several viruses other than HCMV, indicating high speci ficity. Analysis with flow cytometry revealed that the induced fluorescence from the infected cells was proportional to the titer of HCMV inoculated, making it possible to quantify HCMV infectious particles. In summary, the E GFP-based indicator cell line is of potential use for rapid detection and q uantification of HCMV in clinical specimens. (C) 2001 Elsevier Science B.V. All rights reserved.