Anti-rZH501-M847-G serum demonstrating a PRNT 80 titer of 1:640 to ZH501 was a mixture of the sera, each collected at days 6, 7 and 8 p.i. from eight rZH501-M847-G-infected mice. Similarly, anti-rZH501-M847-A serum demonstrating a PRNT 80 titer of 1:80 to ZH501 was a mixture of the sera collected at 6 days p.i. from four rZH501-M847-A-infected mice. Diluent (HBSS-FBS) and normal mouse serum served as negative controls, while convalescent goat anti-ZH501 serum showing a PRNT 80 titer of 1:5,120 to ZH501 served as a positive control. We incubated at 4uC overnight vials containing 100 ml of approximately 5.0 log 10 PFU/ml of rZH501-M847-G, rZH501-M847-A or ZH501 combined with 100 ml of each serum sample or diluents. After incubation, virus titers were determined by using viral plaque assays.