Sindbis virus is a positive single stranded RNA virus of the Togaviridae family. Wild type and a neurovirulent strain, NSV, have provided a valuable model system for studying alphavirus induced encephalomyelitis in mice. [1][2][3] In recent years, there has been a growing interest in search for antiviral substances with high efficacy, low toxicity and minor side effects, one approach is the search for viral inhibitors of plant origin. Flavanones, also called citrus flavonoids, possess many biological activities, 4) previous studies have shown its antiviral activity, hesperetin was found to inhibit the replication of herpes simplex virus type 1, poliovirus type 1, parainfluenza virus type 3 and influenza. 5,6) Hesperidin and Naringin had inhibitory activity on rotavirus infection. 7)Here, we report the effects of hesperetin, naringenin and its glycosides on BHK-21 clone 15 cells, as well as their inhibitory effect on the replication of a neuro-adapted strain of Sindbis (NSV). MATERIALS AND METHODS ChemicalsThe four flavanones studied were hesperidin, naringin and its aglycones hesperetin and naringenin, purchased from Sigma Chemical, St. Louis, Mo, U.S.A. A stock solution of each compound was prepared by dissolved in dimethyl sulfoxide (DMSO) and stored at Ϫ20°C.Cells and Virus BHK-21 clone 15 cells were kindly provided by Dr. Guadalupe Guzmán (Virology Department of the Pedro Kourí Institute, Havana, Cuba). The neurovirulent strain (NSV) of Sindbis virus was donated by Dr. Dianne Griffin (Medical School, John Hopkins University, Baltimore, U.S.A.). BHK-21 cells were grown in Eagle's minimal essential medium (MEM) supplemented with 5% inactivated fetal bovine serum (FBS), 2 mM glutamine and 50 mg/ml gentamicine (growth medium). The cells were maintained in a humidified atmosphere containing 5% CO 2 at 37°C. Virus stock NSV was grown in BHK-21 cells with maintenance medium (MEM, 1% FBS, 2 mM glutamine and 50 mg/ml gentamicine) for 24 h. Viral titer was obtained by plaque assay. 8)Cytotoxic Assay The effect of the compounds over cell viability was determined by a modified MTT assay. 9) Briefly, cells were adjusted to 4ϫ10 4 cells/100 ml in growth medium and seeded in a 96-well plate. Cells were stabilized for 24 h and then incubated with twofold serial dilutions of each compound from 25-0.09 mg/ml in maintenance medium. After 24 h 10 ml (10 mg/ml) of MTT were added to each well; it was metabolized for 3 h, and 100 ml of the lysis buffer (20% SDS, 50% N,N-dimethylformamide) was added. Plates were read at 570 nm after 18 h. Antiviral Activity The effect of these compounds over the mammalian cells was assayed in the same conditions as the cytotoxic assay. BHK cells were infected with NSV at a multiplicity of infection (moi) of 0.1 pfu/cell. The infections were carried out in the presence or absence of different concentrations of compound. After 24 h, the viability of the cells was determined by MTT assay as described before. In addition, plaque reduction assay were carried out. 8) Briefly, BHK-21 clone 15 at a final c...

Type 1 fimbriated (mannose-specific) Escherichia coli cells bind to mannose residues on human polymorphonuclear leukocytes (PMN); this leads to phagocytosis of the bacteria. To identify the mannose-containing receptors on the PMN, the cells were surface labeled with 1251 and lysed in 0.5% Nonidet P-40, and the lysate was fractionated by affinity chromatography on a column of Sepharose-bound fimbriae. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of the material eluted from the column with 500 mM methyl-a-mannoside revealed two radioactive bands of Mr 70,000 to 80,000 (gp7O-80) and 100,000 (gplOO). Another weak band of Mr 150,000 (gpl5O) was observed after prolonged exposure of the gel.Upon blotting of the glycoproteins separated by polyacrylamide gel electrophoresis and overlaying of the blots with concanavalin A, gpl50 appeared as the major band. Membrane preparations of the PMN were enriched in gp7O-80, gplOO, and gpl50, in comparison with the cell homogenates, further suggesting that these glycoproteins are surface components. Fractionation of the membrane preparations on the immobilized fimbriae followed by concanavalin A overlay of blots of the methyl-a-mannoside-eluted material revealed that gplSO was the major component in this fraction. The eluted fraction, obtained from a cell lysate (4.4 ,ug/ml), inhibited by 70% the agglutination of yeasts by the intact bacteria. Our results suggest that the three surface glycoproteins isolated by us serve as receptors for mannose-specific E. coli on PMN and may be involved in the lectin-mediated phagocytosis of the bacteria.

Human peritoneal macrophages isolated from uremic patients undergoing peritoneal dialysis bind type 1 fimbriated Escherichia coli in the absence of opsonins. The number of bacteria bound per macrophage was 6.9, as determined by microscopic examination. Methyl alpha-mannoside (0.1 mM) and p-nitrophenyl alpha-mannoside (0.01 mM) inhibited this binding by about 66%. The ability of peritoneal macrophages to bind E. coli in a mannose-specific manner was confirmed in further experiments using an enzyme-linked immunosorbent assay (ELISA) with an antibacterial antibody, radiolabelled E. coli, and counts of colony-forming units (CFU). The number of bacteria bound per macrophage was 7 to 12 in the ELISA and 5.5-8.5 in the CFU assay. Methyl alpha-mannoside caused 70% inhibition of binding in the ELISA and 84% in the CFU assay, whereas p-nitrophenyl alpha-mannoside showed inhibition of 79% and 90%, respectively. Most bound bacteria (76-80%) were subsequently killed. Nonfimbriated E. coli 827 bound poorly to the macrophages (approximately 22%) as compared to that of the fimbriated bacteria. Although this binding was not inhibited by methyl alpha-D-mannoside or p-nitrophenyl alpha-mannoside, the percentage of bacteria killed was similar to that of the fimbriated phenotype. The peritoneal macrophage is thus able to phagocytose E. coli in the absence of opsonins. This may explain the relative rarity of E. coli as an etiologic agent of peritoneal infections in the dialysed patient.

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