Mayaro virus CH was generated from an infectious clone received from Dr. Thomas Morrison (UC-Denver). The following reagents were obtained through BEI Resources, NIAID, NIH as part of the WRCEVA program: Mayaro virus, Guyane, NR-49911; Mayaro virus, TRVL 4675, NR-49913; Mayaro virus, BeAr505411, NR-49910; Mayaro virus, Uruma, NR-49914; Una virus, MAC 150, NR-49912. CHIKV SL15649 and vaccine strain CHIKV 181/25 were generated from their respective infectious clones as previously described [39]. Alphaviruses were grown in C6/36 cells. Viral stocks were prepared from clarified supernatants at 72 h post infection (hpi) by ultracentrifugation over 10% sucrose (SW32Ti, 70 min at 76,755 x g). The virus pellets were resuspended in PBS and stored at -80°C. Viral limiting dilution plaque assays using Vero cells were performed on 10-fold serial dilutions of virus stocks or tissue homogenates. The infected cells were continuously rocked in an incubator at 37°C for 2 h, and then DMEM containing 5% FBS, PSG, 0.3% high viscosity carboxymethyl cellulose (CMC) (Sigma) and 0.3% low viscosity CMC (Sigma) was added to the cells. At 2 dpi, cells were fixed with 3.7% formaldehyde (Fisher) and stained with 0.2% methylene blue (Fisher). Plaques were visualized under a light microscope and counted.

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