Two-fold serial dilutions of PBS or heat inactivated serum from PBS or AdV-MAYV vaccinated, as well as AdV-MAYV MAYV challenged mice were prepared in DMEM (1:80 to 1:81920). For pre-attachment assays, diluted serum was mixed with 11 PFU of MAYVBeAr and rocked for 1 h at 4°C. Serum-virus complexes were then added to confluent 12-well plates of Vero cells and rocked for 1 h at 4°C. Non-adsorbed complexes were removed by 3 washes of DMEM and plates were moved to a 37°C incubator for 15 minutes to allow for internalization of bound virus. Plates were then overlaid with 1 ml of CMC DMEM medium containing 5% FBS. Post-attachment assays were conducted in a similar manner, but initially 11 PFU of MAYVBeAr was added to 12-well plates and plates were rocked at 4°C for 1 h. After washing to remove unbound virus, serum dilutions were added to the wells. Plates were again rocked for 1 h at 4°C, followed by washing, and plates were moved to a 37°C incubator for 15 minutes and overlaid with 1 ml of CMC DMEM medium containing 5% FBS. At 48 hpi, cells were fixed with 3.7% formaldehyde, stained with 0.5% methylene blue, and virus plaques were counted.

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