H1N1pdm virus pseudotyped lentiviruses were produced in 293T cells co-transfected with pNL4.3-R 2 E 2 , HA and NA constructs using a polyethylenimine (PEI)-based transfection protocol [50] . Cell culture supernatants were collected 48 hr post-transfection, filtered through a 0.45 mm-pore size filter (Millipore, Billerica, MA ) and used in pseudotype neutralization test. Serum samples, heat inactivated at 56uC for 30 min, were diluted 40-fold in culture medium and mixed with an equal volume of diluted H1N1pdm influenza pseudovirions. After incubation at 37uC for 1 hr, 100 ml of pseudovirions (containing 50 ng/mL of HIV p24 gag protein) and serum mixtures were added into 96-well plates that contained 293T cells grown for 24 hr at initial 1610 4 cells. Infectivity was evaluated at 72 hr post-infection by luciferase assay. The percentage of infectivity of pseudovirions treated by tested serum to that of negative serum (as control) was calculated. 90% reduction in infectivity by serum addition is considered to be neutralizing activity [30] . Tests were run at least as duplicates.