WIN 51711 (5-[7-[4-(4,5-dihydro-2-oxazolyl)phenoxy]heptyl]-3-methylisoxazole), a new antipicornavirus drug, is a potent inhibitor of human entero-and rhinoviruses at concentrations not inhibitory to HeLa cell growth. In plaque reduction assays, WIN 51711 reduced plaque formation by 9 enteroviruses and 33 rhinoviruses, with MICs of 0.004 to 0.17 and 0.004 to 6.2 p,g/ml, respectively. Addition of WIN 51711 to infected cells at concentrations of 0.02 to 5.0 ,ug/ml reduced the yield of picornaviruses by 90%. Other RNA viruses (nonpicornaviruses) and DNA viruses were unaffected by the compound.Currently, no drugs are available for the treatment of human diseases caused by the rhino-and enterovirus members of the picornavirus family (7). Based on the finding that arildone is a potent inhibitor of a limited number of enteroviruses in vitro (1, 2) and in vivo (4), a series of analogs was synthesized in an effort to discover a systemically active compound with broad-spectrum activity against the causative agents of viral meningitis, hepatitis A, acute hemorrhagic conjunctivitis, and the common cold (6, 9). This report describes the potent in vitro activity of WIN 51711 ( Fig. 1)

Arildone (also known as Win 38020), a novel aryl diketone, inhibited replication of herpes simplex virus type 2 in tissue culture by interfering with an event that occurs prior to 6 h postinfection. The inhibition could be partially reversed by washing. Although the exact mechanism of action is unknown, neither viral deoxyribonucleic acid nor viral proteins were synthesized in the presence of arildone. Plaque assay. HSV-2 was quantitated by plaque assay. Serial 10-fold dilutions were made in Leibovitz medium (L-15), and 2-ml portions of the dilutions were pipetted onto 3-or 4-day-old monolayers of BSC1 cells grown in 25-cm2 plastic flasks. After 1 h at 330C, the inoculum was removed and replaced with 5 ml of L-15 supplemented with 5% calf serum (Reheis, Chicago, Ill.) and 0.65% Ionagar (Colab, Chicago Heights, Ill.). Infected cultures were incubated for 3 days at 330C and flooded with a solution of crystal violet stain (0.25%), prepared with 10% Formalin containing 2% sodium acetate. Plaques were counted after the agar was removed from the flasks and expressed as plaqueforming units per flask.Cell viability. showed that DMSO had no effect on HSV-2 replication at the concentrations employed. Three-or 4-dayold monolayers of BSC, cells in test tubes or Falcon flasks were used to determine the effect of arildone when added at various times after infection. HSV-2 Curtis strain, containing from 5 x 105 to 3 x 10' plaque-forming units per ml, was used to inoculate the cells, usually at a multiplicity of infection of about 1. Virus dilutions were prepared in L-15, and inoculum volumes were 0.2 ml and 2 ml for tubes and flasks, respectively. Cultures were incubated at 33°C for 1 h, the inoculum was removed by aspiration, and the monolayers were washed twice with 2-ml or 5-ml volumes of L-15. Fresh L-15 containing 5% calf serum and various concentrations of arildone was added, and incubation was continued at 33°C. In all experiments, 0 h postinfection (PI) refers to the time of addition of the fresh medium. For time of addition experiments, medium without arildone was added at 0 h PI and replaced with medium containing 3 ug of arildone per ml at the indicated time. Reversal of drug action was initiated by removing the culture fluid containing arildone, washing the monolayers twice with 1 or 2 ml of L-15, and finally continuing the incubation with fresh medium devoid of arildone. Virus yield was determined by discarding the culture fluid from monolayers, replacing it with 0.1% EDTA, and then assaying the contents after two cycles of freezing and thawing. Pilot studies had shown that prior to lysis the culture fluid contained less than 2% of the total virus.

Rhinoviruses as a group are notably sensitive to inactivation in solutions with a pH of less than 5.3. Glutaric acid appears to possess virucidal activity in addition to the aciduant effect against rhinoviruses. A model system in which rhinovirus type 14 was incubated in the presence of glutaric acid (GA) (pH 4.0) at 0°C was devised to separate intrinsic virucidal activity from the aciduant effect. Under these conditions, virucidal activity against rhinovirus type 14 was directly related to the concentration of GA present and the proportion of the acid in the diprotonated form. The virucidal activities of GA and several other compounds, including GA analogs and other mono-and dicarboxylic acids, were tested under the conditions described. In general, as the alkane bridge separating two carboxylic acid functions was lengthened, virucidal activity decreased. When 26 additional strains of rhinoviruses were tested in the model system, 19 were inactivated slowly enough to be compared. Of these, 63% were more susceptible to GA than to sodium acetate buffer and 26% were more susceptible to sodium acetate buffer. Eleven percent were resistant to both GA and sodium acetate buffer. The virucidal activity of GA for a majority of strains tested appeared to be due to a combination of low pH and another mechanism of action presumably unrelated to pH.The rhinoviruses are differentiated from other subgroups of the picornaviruses by their sensitivity to inactivation by acidic solutions (pH below 5.3) (2, 5). Korant et al. (2) have demonstrated that VP4 is lost from the protein coat of the virus during exposure to low pH. Many organic acids have been suggested as possible virucidal agents (4). Glutaric acid (GA) is one virucidal organic acid which has demonstrated effectiveness as an agent for the inactivation of rhinovirus on human skin (1). This study was undertaken to determine whether the virucidal activity of GA is solely due to the low pH of solutions in which it is tested or to an intrinsic property of the chemical entity and to determine whether the activity is similar for several strains of rhinoviruses.MATERIALS AND METHODS Tissue culture. HeLa Ohio cells were grown as monolayers in Costar cluster dishes (35-mm-diameter wells) and used after 1 day of growth (75 to 80% confluent) at 37°C in a 5% CO2 atmosphere. The medium was M199 (Flow Laboratories) containing modified Earle salts, glutamine (0.3 g/liter), sodium bicarbonate (1.26 g/liter), penicillin (100,000 U/liter), and streptomycin (0.1 g/liter).Virus strains. Plaque assay. Samples were diluted with M199 to give a final count of >100 PFU/ml. One milliliter of diluted sample was pipetted in duplicate into 35-mm-diameter wells of Costar cluster dishes containing monolayers of HeLa Ohio cells prepared as described above. The virus was allowed to adsorb for 1 h at room temperature (20 to 25°C); inoculum fluid was removed and replaced with 3 ml of overlay medium. Overlay medium consisted of M199 containing 0.5% agarose ME (Seakem), 2.5% bobby calf serum (GIBCO Laborator...

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Tubular structures are released from cells of Cytophaga columnaris after lysis of the cells. To determine the nature of these tubules, they were purified and their composition was determined. Tubules were isolated after treating cell lysates with 1.0% sodium dodecyl sulfate at pH 8.1, which solubilizes all structural components except tubules. Plasma membranes from the same organism were isolated by discontinuous sucrose gradient centrifugation of lysed cells. Both tubules and membranes are composed of lipids and proteins. Lipids extracted from tubules and plasma membranes produced similar patterns when examined by thin-layer chromatography. Proteins solubilized from membranes were separated into 14 bands by polyacrylamide gel electrophoresis, whereas those solubilized from tubules separated into only 5 bands. The presence of lipids in tubules from C. columnaris supports the idea that they are derived from membranes of intact cells. In this respect they are similar to tubules produced by cells of Clostridium botulinum and different from other tubular structures ("rhapidosomes") found in cells of Saprospira grandis.