Blood was collected from mice and allowed to clot at room temperature for 30 minutes before centrifuging for 5 minutes at 3,000 x g. Sera was transferred to a new tube and heat inactivated at 56°C for 30 minutes. A portion of sera was used for serial dilutions in DMEM supplemented with 5% FBS and 1% PSG. Diluted serum was mixed with media containing MAYVCH, MAYVBeAr, CHIKV 181/25, or UnaMac150. Media and virus were incubated for 2 h at 37°C while rocking. Serum and virus containing media was transferred to confluent 12 well plates of Vero cells and rocked for 2 h in a 37°C 5% CO2 atmosphere incubator. One milliliter of CMC DMEM medium containing 5% FBS was added to each well and the plate was incubated for 48 h in a 37°C 5% CO2 incubator. Plaques were fixed by adding 1 ml of 3.7% formaldehyde to each well for 15 minutes, then the supernatant was removed and the wells were washed with cold water and stained with 0.2% methyl blue dye for 15 minutes. Plates were washed with cold water to remove excess dye and dried prior to counting plaques. PRNT50 was calculated by non-linear regression after determining the percent of plaques at each dilution relative to the average plaques in virus only control wells.

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