Cell lines, FV3 Stocks and Animal Infections

Baby hamster kidney-21 cells (BHK-21; ATCC no. CCL-10) were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum (FBS; Invitrogen), penicillin (100 U/mL) and streptomycin (100 μg/mL) with 5% CO₂ at 37 °C. FV3 was grown by a single passage on BHK-21 cells, purified by ultracentrifugation on a 30% sucrose gradient and quantified by plaque assay on BHK-21 monolayers under an overlay of 1% methylcellulose¹³.

Tadpoles were infected by intraperitoneal (i.p.) injection with 1 × 10⁴ PFU of FV3 in 10 μL volume using a glass Pasteur pipette whose small end had been pulled in a flame. Adult frogs were infected by i.p. injection with 1 × 10⁶ PFU of FV3 in 100 μL volume using a 1 ml sterile syringe with a 22 gauge, 1½ inch needle. Controls (0 days post infection, d.p.i.) were mock-infected with the same amount of amphibian phosphate-buffered saline (APBS). At different time points, tadpoles (0 and 6 d.p.i.) or adult frogs (0, 1, 3 and 6 d.p.i.) were euthanized by immersion in 1% tricaine methane sulfonate (TMS-222) buffered with bicarbonate.