Indubiose

Confluent BHK-21 cell monolayers were infected with 10-fold serial dilutions of virus stock, overlaid with Eagle overlay media supplemented with 5% tryptose phosphate broth solution (Sigma Aldrich), penicillin (100 units/ml) and streptomycin (100 μg/ml) (Sigma Aldrich) and 0.6% Indubiose (MP Biomedicals) and incubated for 48 hours at 37°C. Cells were fixed and stained with 1% (w/v) methylene blue in 10% (v/v) ethanol and 4% formaldehyde in PBS.
Fixed plaques were scanned, and images measured using a GNU Image Manipulation Program IMP (GIMP, available at https://www.gimp.org). For each plaque, horizontal and vertical diameter in pixels was taken and an average of these two values was calculated. All plaques per well were measured.

Confluent monolayers of goat epithelium cells were infected with chimeric and wild type SAT2 viruses at the same MOI (0.01). Unabsorbed virus was removed by acid washing cells with MES-buffered saline (25 mM morpholineethanesulfonic acid (MES]), 145 mM NaCl, pH 5.5) and two subsequent neutralisation washes with culture medium (DMEM/F12, with HEPES, L-Glutamine (Sigma, UK) containing 1% (v:v) fetal calf serum (FCS) and antibiotics). Supernatants were collected at 0, 2, 4, 6, 8 and 24 hours post-infection (hpi) to determine the viral titers by plaque assay. The plaque assay was performed by infecting IBRS-2 cell monolayers with serial 10-fold dilutions of FMDV virus stocks, overlaid with indubiose (MP Biomedicals, UK (Cat # 11AGAI0025) and incubated for 48 h at 37 °C. Cells were fixed and stained with methylene blue staining and plaque forming units (PFU)/ml determined.

Ten-fold virus dilutions were used to infect triplicate wells of confluent ZZ-R 127 cells pre-seeded in 6-well tissue culture plates. Following adsorption at 37 °C for 1 h, the inoculum was removed and 2 mL of indubiose (MP Biomedicals, Santa Ana, CA, USA) overlay was added. Following incubation (48 h, 37 °C, 5%CO₂), cells were fixed by the addition of 10% Tetrachloroauric acid (Merck) for 30 min. indubiose plugs were then removed and cells were stained with methyl blue solution (PBS, 10% formaldehyde, 10% of 1% methyl blue in ethanol) prior to determining plaque forming units/mL (PFU/mL).