VeroE6 cells in 6-well plate were infected with a series of diluted virus samples in 400 ml. After 1 h adsorption at 37uC, we removed the inocula and added 2 ml of MEM containing 0.6% noble agar (Difco Laboratories), 5% FBS and 5% tryptose phosphate broth. The cells were incubated at 37uC for 3 days. Then, 2 ml of MEM containing 0.6% agar, 100 mg of neutral red (N2889, Sigma), 5% FBS and 5% tryptose phosphate broth were added into the wells, and incubated for 16 h. The virus titers were determined in triplicate.