Sensitive, reproducible and convenient fluorometric assay for the in vitro evaluation of anti-cytomegalovirus agents.
Fluorescein diacetate (FDA), a non-fluorescent diacetyl fluorescein ester that becomes fluorescent upon hydrolysis by cytoplasmic esterases, permitted the easy distinction by fluorometry between non-infected and human cytomegalovirus (CMV)-infected HEL cell cultures. As a result of enhanced cytoplasmic esterase activity after CMV infection, FDA-derived fluorescence intensity was brighter for infected than non-infected HEL cells. A similar increase in fluorescence intensity was observed after loading the cells with Indo-1/AM, a non-fluorescent ester of Indo-1 that becomes fluorescent upon cleavage by cytoplasmic esterases. The 50% effective concentrations of a number of anti-CMV agents as determined by the fluorometric assay were very similar to those obtained by the conventional and more time-consuming microscopic evaluation. The fluorometric assay appears very suitable for an automated evaluation of anti-CMV compounds, and also allows rapid determination of the cytotoxicity of potential antiviral compounds.
Neyts
J
J
Rega Institute for Medical Research, Katholieke Universiteit Leuven, Belgium.
Snoeck
R
R
Schols
D
D
Himpens
B
B
De Clercq
E
E
D000212:Acyclovir / Q000494:pharmacology; D000998:Antiviral Agents / Q000494:pharmacology*; D000077404:Cidofovir; D003587:Cytomegalovirus / Q000187:drug effects*; D003586:Cytomegalovirus Infections / Q000473:pathology*; D003596:Cytosine / Q000031:analogs & derivatives / Q000494:pharmacology; D004950:Esterases / Q000378:metabolism; D005434:Flow Cytometry; D005452:Fluoresceins / Q000378:metabolism; D005470:Fluorometry / Q000379:methods*; D017245:Foscarnet; D015774:Ganciclovir / Q000494:pharmacology; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods*; D063065:Organophosphonates; D009943:Organophosphorus Compounds / Q000494:pharmacology; D010746:Phosphonoacetic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D011092:Polyethylene Glycols / Q000494:pharmacology; D013498:Suramin / Q000494:pharmacology
D000998:Antiviral Agents; D005452:Fluoresceins; D063065:Organophosphonates; D009943:Organophosphorus Compounds; D017245:Foscarnet; D011092:Polyethylene Glycols; D013498:Suramin; D003596:Cytosine; D004950:Esterases; D000077404:Cidofovir; D010746:Phosphonoacetic Acid; D015774:Ganciclovir; D000212:Acyclovir; C018506:diacetylfluorescein