Effect of castanospermine on WNV and DEN secretion and infectivity. BHK-21 cells were infected with WNV or DEN (MOI of 0.1) and treated with castanospermine (0, 100, or 500 μM), and supernatants were harvested and clarified at 48 h after infection. (A) WNV. Supernatants were titrated for infectivity using viral plaque assays and quantitated for viral RNA by fluorogenic RT-PCR. The data are the averages of three separate experiments and are normalized to the medium control. The error bars indicate standard deviations. (B) DEN. Supernatants were titrated for infectivity using viral plaque assays and quantitated for viral RNA by fluorogenic RT-PCR. In addition, DEN particles were measured using a two-MAb (anti-prM and biotinylated anti-E) trap ELISA (see Materials and Methods). The data were analyzed as described above.

(A) Effect of castanospermine on mortality after DEN-2 infection of A/J mice. Four-week-old wild-type A/J mice were inoculated intracranially with 10⁵ PFU of DEN-2. Mice were treated with saline vehicle or increasing concentrations (200 μg, 1 mg, and 5 mg per day) of castanospermine as a single dose for 10 consecutive days. The survival curves were constructed using data from three independent experiments. The number of animals for each castanospermine dose ranged from 30 to 45 for each treatment group. The difference in survival curves was statistically significant for doses of drug shown (P ≤ 0.0001). (B) Effect of castanospermine on WNV-induced mortality in C57BL/6 mice. Seven-week-old C57BL/6 mice were inoculated with 10² PFU of WNV via the footpad. Mice were treated with saline vehicle or 5 mg per day of castanospermine for 10 days. The survival curves were constructed using data from two independent experiments, and there was no significant statistical difference between vehicle and castanospermine treatment (P > 0.6).

(A) Effect of castanospermine on marker gene expression in DEN-2 and WNV subgenomic replicons. The level of luciferase gene expression from either DEN-2 or WNV subgenomic replicons is shown in the absence or presence of various concentrations of castanospermine. As baseline, the WNV replicon replicates at a ∼10-fold-higher rate than does the DEN-2 replicon. The effect of mycophenolic acid (MPA; 10 μg/ml) is shown as an example of an inhibitor that directly blocks replication of DEN-2 and WNV (13). The data are from one representative experiment of three performed in quintuplicate, and the error bars reflect standard deviations. (B) The effect of castanospermine on infectivity of pseudotyped VLP. A stable BHK cell line expressing a WNV subgenomic replicon containing a luciferase gene was transfected transiently with the structural proteins of WNV or DEN-1 and then incubated in the presence or absence of castanospermine (500 μM). Pseudotyped VLP were purified from supernatants by sucrose gradient centrifugation and added to new BHK cells for 40 h. Cells were lysed, and Renilla luciferase activity was measured. Relative infectivity of each VLP preparation was measured using triplicate infections, and the error bars reflect standard deviations. The data are from one representative independent experiment of three performed.

Inhibition of viral spread in flaviviruses after treatment with castanospermine. (A) BHK-21 cells were infected with DEN isolates representing each of the four serotypes at an MOI of 0.01 in the presence or absence of 50 μM castanospermine. The percentage of infected cells was measured by flow cytometry, and the results were normalized to untreated controls (i.e., no drug = 100%). The results are the average of three independent experiments. The percentages of BHK-21 cells infected for the no-drug controls were 58, 14, 8, and 82 for DEN-1, DEN-2, DEN-3, and DEN-4, respectively. (B) Inhibition of virion secretion by low-passage-number isolates by plaque reduction assay. BHK-21 cells were infected with 10² PFU/well in the presence or absence of 50 μM castanospermine, and an agarose overlay was added. Five days later, plaques were fixed, stained with crystal violet, andscored visually. The graph shows one representative experiment of three. (C) Differential inhibition of DEN, WNV, and YFV infection by castanospermine. BHK-21 cells were infected at an MOI of 0.1 in the absence or presence of castanospermine (50 or 500 μM). Two days later, cells were harvested, immunostained with anti-flavivirus MAbs, and processed by flow cytometry. The data represent the average of between three and six independent experiments, and the error bars represent standard deviations. For direct comparison between viruses, the data were normalized to the no-drug control. The percentages of BHK-21 cells infected for the no-drug controls were 46, 98, and 24 for DEN, WNV, and YFV, respectively.

(A and B) Inhibition of DEN-2 production in BHK-21 cells. BHK-21 cells were infected at a range of MOI from 0.01 to 10 with DEN-2 (strain 16681) in the absence or presence of increasing concentrations of castanospermine. The amount of virus released into supernatants after 72 h is shown (A). The data are also expressed as the percent infection compared to no-drug control (B) to more clearly define the IC₅₀. The graphs show one representative experiment of three. (C) Inhibition of DEN-2 production in Huh-7 cells. DEN-2 release was measured from Huh-7 cells at a range of castanospermine concentrations 24 h after infection at an MOI of 10. Virus was quantified in tissue culture supernatants by plaque assay. The results are the average of three independent experiments performed in quadruplicate. The amount of infectious virus in the no-drug control sample was 2.2 × 10⁴ PFU/ml. (D) Western blot of DEN prM protein. BHK-21 cells were infected with DEN (MOI, 0.1) and treated with medium or castanospermine. One day later, cells were harvested, lysed in RIPA buffer, treated with endo H glycosidase, electrophoresed, transferred, and blotted with a rabbit anti-prM polyclonal antibody. The difference in electrophoretic mobility of prM after castanospermine treatment is indicated by the two lines in the center of the blot.