HI test was carried out as described elsewhere [5] . RDE treated serum samples were inactivated at 56uC for 30 min and two-fold serially diluted at an initial dilution of 1:10. Twenty five ml of the diluted serum were incubated with 25 ml of the four hemagglutination units from reference influenza strains for 30 min at room temperature. The reference H1N1 IAV strains for HI test were A/ Tianjinjinnan/15/2009(H1N1) and A/California/04/2009 (H1N1), respectively. 50 ml of 1% chicken erythrocyte suspension was added to each well and incubated for 30 min at 4uC. Positive reactions were recorded when the HI antibody titer was equal to or greater than 40.