Viruses | Free Full-Text | Production of OSU G5P[7] Porcine Rotavirus Expressing a Fluorescent Reporter via Reverse Genetics

RV reverse genetics was performed as previously described [3,35,37]. Briefly, BHK-T7 cells in 12-well plates were transfected with the 11 SA11 or OSU T7 plasmids, or combinations thereof, and with the capping enzyme plasmid, pCMV-NP868R, using Mirus TransIT-LT1 transfection reagent (Madison, WI, USA. Transfection mixtures contained 0.8 μg each of the pT7 plasmids, except for pT7/NSP2 and pT7/NSP5, which were used at 3-fold higher concentrations [2]. Two days post transfection, the BHK-T7 cells were overseeded with MA104 cells, and trypsin was added to the medium to a final concentration of 0.5 μg/mL. Three days later, the BHK-T7/MA104 cell mixtures were freeze–thawed thrice, and the lysates were clarified via low-speed centrifugation at 800× g for 5 min at 4 °C. To amplify the recovered viruses, the lysates were adjusted to 10 μg/mL trypsin and incubated for 1 h at 37 °C. MA104 cells in 6-well plates were then infected with 300 μL of the trypsin-treated lysates and incubated at 37 °C in a 5% CO₂ incubator until all cells were lysed (typically 3–5 days). Recombinant viruses were recovered from the lysates via plaque isolation on MA104 cells [34,35]. Plaque-isolated viruses were initially grown on MA104 cells in 6-well plates and then, to generate larger pools, grown on MA104 cells in T175 tissue culture flasks at low multiplicity of infection (₂ incubator for 1 h with rocking to ensure equal coverage of the inoculum over the monolayers. Following adsorption, the inoculum was removed and 25 mL of serum-free DMEM containing 0.5 μg/mL trypsin was added to each flask. The flasks were returned to the incubator until all cells were lysed (typically 3–5 days). The infected cell lysates were collected, clarified via low-speed centrifugation at 800× g for 5 min at 4 °C, and stored at −80 °C. Viral dsRNAs were recovered from the infected cell lysates via extraction with TRIzol (ThermoFisher Scientific, Waltham, MA, USA), resolved via electrophoresis on Novex 8% polyacrylamide gels (ThermFisher Scientific) in Tris-glycine buffer, detected via staining with ethidium bromide, and visualized using a Bio-Rad ChemiDoc MP imaging system (Hercules, CA, USA) [34,35,37]. Peak titers were determined from the infected cell lysates via plaque assay.