THF-CAR were transfected with varying MOI’s from 0 to 1000. Cells were harvested 72 hpi after washing with PBS and were pelleted at 391 x g for 15 minutes at 4°C in a refrigerated microcentrifuge. Pelleted cells were lysed in 300 μl cell lysis buffer for 30 minutes on ice. Lysed cells and debris were pelleted at 16,363 x g for 15 minutes in a microcentrifuge and supernatant was transferred to a new tube. 20 μl of lysate was run on a 4–12% Bis/Tris polyacrylamide gel alongside MAYV infected Vero cells and control Vero cell lysate at 200 volts for 37 minutes. Semi-dry transfer was used to transfer proteins onto an activated PVDF membrane at 25 volts for 35 minutes. Membranes were blocked with 3% BSA/TBST, and probed with a 1:250 dilution of serum from 108 prime + boost AdV-MAYV vaccinated mice. Monoclonal antibodies directed against CHIKV E1 and E2 (87.H1 and 133.B4) were kindly provided by Dr. Michael Diamond, Washington University at St. Louis. Anti-alphavirus capsid mAb has been previously described [41]. Membranes were then washed, and a secondary HRP conjugated rabbit ⍺-Mouse IgG was used. Membranes were washed and then revealed with ThermoFisher Pico chemiluminescent developer solution and exposed onto X-ray film. 1.7x107 PFU of MAYVTrVl was also run on a 4–12% Bis/Tris polyacrylamide gel, proteins were transferred, and membranes probed with a 1:250 dilution of serum from naïve or 108 prime + boost AdV-MAYV following the above protocol.

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