Viral titers from the cell culture medium were determined by plaque-forming assays, as previously described (15). Briefly, virus-containing medium was serially diluted and then added to confluent Vero cells. After 1-hour incubation, supernatants were removed, cells were washed with PBS, and culture medium containing 2% (w/v) methylcellulose was overlaid for 24 hours. Then, cells were fixed for 30 min with 0.5% (v/v) glutaraldehyde and then stained with 1% (w/v) crystal violet dissolved in 70% ethanol for 30 min. After washing twice with double-distilled water, plaques were counted, and average counts were multiplied by the dilution factor to determine the viral titer as PFU per milliliter.

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