Hemagglutination Inhibition (HI) Test

HI tests were carried out as described by Phan et al. (2013). Briefly, a 25-μl aliquot of murine serum was mixed with 25 μl PBS and added to the first well of a V-bottom microtiter plate. Twofold serial dilutions were prepared across the row of 12 wells. Aliquots (25 μl) containing 4HAU of inactivated virus [A/swan/Germany/R65/2006(H5N1)] were added to each well and incubated for 30 min at room temperature. We then pipetted 25 μl of a 1% red blood cells (RBCs) suspension into each well and the plate was again incubated for 30 min at room temperature. The HI titer was defined as the reciprocal of the highest serum dilution that achieved the complete inhibition of hemagglutination.