Supernatants viral titer was determined by plaque assay as previously described [31], with some modifications. Vero E6 cells (600,000 cells/well) were seeded in a 6-well plate and incubated at 37 °C with 5% CO2 for 24 h. After incubation, the medium was removed and cells were infected with 500 µL of ten-fold serial dilution of supernatant previously obtained, rocking the plates every fifteen minutes. In the meanwhile, the overlay medium (complete medium with Agar 0.1%) was prepared and maintained in a 50 °C water bath. After 1 h of infection, the overlay medium (2 mL) was poured into each well and the plates incubated for 3 days. Finally, the overlay was discarded, cells were fixed for 30 min with 4% formalin and stained with 0.5% crystal violet. Viral titer was determined as plaque-forming units per mL, considering wells with plaques ranging from 2 to 100. For each pool of supernatants, plaque-reduction assay was performed in duplicate.

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