Hemagglutination assay PS cells were infected at a moi of 0.1 PFU/cell in 500 ml culture flasks and infectious supernatant medium was collected at day 5pi. The TBEV virions with estimated initial virus titers of 228610 6 PFU/ml were concentrated 100 times using 7% polyethylene glycol (PEG) in the presence of 2.4% NaCl overnight at 4uC and precipitated by centrifugation at 8000 rpm for 3 h. The resulting pellet was resuspended in 500 ml of PBS. For routine hemagglutination test [20, 46] , 50 ml of the concentrated virus sample (10 8 PFU/ml) were placed in the first well of 96-well plates and diluted two-fold in borate buffer (pH 9.0). Then 100 ml of a suspension of 0.5% newborn chick erythrocytes was added to each well. Results are recorded as the reciprocal of the maximum virus dilution that produced agglutination.