STAINING CHARACTERISTICS AND ANTIVIRAL ACTIVITY OF SULFORHODAMINE-B AND LISSAMINE-GREEN-B

Purpose. Fluorescein and rose bengal are dyes used routinely in the ex amination of the ocular surface. As part of an ongoing search for a su perior ophthalmic dye with optimal specificity and sensitivity and a l ack of interference with subsequent viral cultures, and as part of stu dies that use chemical dyes to understand better the pathophysiology o f ocular surface disorders, the staining characteristics and antiviral activity of sulforhodamine B and lissamine green B were investigated. Methods. Staining of rabbit corneal epithelial cell cultures by sulfo rhodamine B and lissamine green B was compared to that of fluorescein and rose bengal. Diffusion of each dye through a collagen gel was meas ured. Uptake of lissamine green B by herpes simplex virus type 1 (HSV- 1)-infected Vero cell cultures was compared at several times postinfec tion. The effect of sulforhodamine B and lissamine green B on HSV-1 pl aque formation in Vero cells was determined. The cellular toxicity of sulphorhodamine B and lissamine green B in vitro was examined by a qua ntitative C-14-amino acid uptake assay and by a qualitative cell viabi lity assay. Finally, the effect of sulforhodamine B and lissamine gree n B on viral replication was compared in vivo with that of rose bengal in a rabbit model of herpetic epithelial keratitis. Results. Rose ben gal vividly stained cell monolayers of explant cultures of rabbit corn eal epithelium. By light microscopy, sulforhodamine B and lissamine gr een B, like fluorescein, did not stain the epithelial cells, but did s tain the corneal explant stroma. Pretreatment of epithelial cells with 0.25% trypsin for 5 minutes failed to induce dye uptake; however, pre treatment with 0.5% Triton X-100 for 5 minutes resulted in nuclear sta ining by lissamine green B, but not sulforhodamine B. When added to a collagen gel, the relative diffusion rate was fluorescein > lissamine green B > sulforhodamine B > rose bengal. By spectrophotometric analys is, HSV-1-infected and uninfected Vero cells bound equivalent amounts of lissamine green B until late in infection, when infected cells took up more dye (P < 0.001). A direct neutralization assay showed that 0. 06% lissamine green B or 0.5% sulforhodamine B reduced HSV-1 plaque fo rmation in Vero cells by greater than 50%, when present at the time of viral adsorption. By a quantitative C-14-amino acid uptake assay, lis samine green B was toxic to Vero cells in a dose-dependent manner, whe reas sulforhodamine B was relatively nontoxic at the concentrations te sted. By a cell viability assay, however, neither dye showed significa nt cellular toxicity. In a rabbit model of herpetic epithelial keratit is, rose bengal significantly reduced viral replication and recovery, whereas sulforhodamine B and lissamine green B had no effect. Conclusi ons. Neither sulforhodamine B nor lissamine green B stain healthy, nor mal cells. Lissamine green B stains membrane-damaged epithelial cells, but sulforhodamine B does not. Both sulforhodamine B and lissamine gr een B stain corneal stroma. Lissamine green B inhibits HSV-1 plaque fo rmation at low concentrations of dye in vitro, which correlates with s uppression of cellular metabolism as demonstrated by a C-14-amino acid uptake assay, but does not affect cell viability. Neither sulforhodam ine B nor lissamine green B inhibit viral replication or recovery in v ivo.