4.3. Virus Titration and Focus Forming Unit (FFU) Staining

To examine the anti-DENV activity of cordycepin or C. militaris extract on virus production, the newly synthesized virus in the culture supernatant was measured by FFU staining. Vero cells were plated in 96-well format (2 × 10⁴ cells per well) overnight before the experiment. The cells were infected with DENV1, DENV2, DENV3, or DENV4 (1.0 × 10⁴ FFU/mL of DENV2, 100 μL). The plate was incubated for 2 h to allow the virus to enter the cells. The unbound virus was removed and a fresh medium containing 25, 50, or 100 μM of cordycepin was added to the cells. The new viral progenies were harvested at 24 or 48 h, and determined for their virus titer by FFU staining.

To determine DENV titers, the Vero cells were plated 2 × 10⁴ cells per well in 96-well format. A 10-fold serial dilution of DENV was added to the cells. After 2 h, the infected cells were overlaid with 2% carboxymethylcellulose medium, and the plate was further incubated for 72 h. The infected cells were fixed and permeabilized using 3.8% formaldehyde and 0.2% Triton X-100, respectively. The 4G2 monoclonal antibody, which is specific to DENV E proteins, was added to the cells and the plate was incubated overnight. The cells were washed three times with phosphate-buffered saline (PBS) containing 0.1% Tween-20 (PBST) before addition of the horseradish peroxidase (HRP)-conjugated secondary antibody (at 1:2000 dilution), which was incubated at room temperature (RT) for 30 min. The DENV-infected cells shown as DENV foci were detected by adding 3,3′-diaminobenzidine (DAB) substrate (Sigma-Aldrich Corporation, St. Louis, MO, USA). The DENV foci were counted manually under a light microscope (20 × magnification) (Nikon Instruments, Inc., Melville, NY, USA).