A microneutralization assay to detect humoral neutralization responses against influenza was performed as previously described [22, 35] . For mice, two-fold dilutions of heat-inactivated sera were tested for the presence of antibodies that neutralized the infectivity of 100 TCID50 (50% tissue culture infectious dose) of H5N1 viruses on MDCK cell monolayers using two wells per dilution on a 96-well plate as described [35] . After 2 days of incubation, cells were fixed, and ELISA was performed to detect the presence of viral nucleoprotein (NP) and determine the neutralization activity. For ferrets, neutralizing antibody activity was analyzed in an MN assay based on the methods of the WHO Global Influenza Program [36] . Sera were treated with receptor-destroying enzyme by diluting one part serum with three parts enzyme and incubated overnight in a 37uC water bath and heat-inactivated as described for the HAI assay. Virus strains used for the MN assay are lowpathogenic, H5N1-PR8 re-assortants, obtained from Ruben Donis at the CDC Influenza Branch Hemagglutination inhibition (HAI) assay of ferret sera HAI assays were performed using four hemagglutinin units of virus and 1% horse RBC as previously described [37] [38] [39] [40] . Ferret sera were treated with receptor-destroying enzyme by diluting one part serum with three parts enzyme and incubated overnight in a 37uC water bath. The enzyme was inactivated by 30 min. incubation at 56uC followed by addition of six parts PBS for a final dilution of 1/10. Virus strains used for the HAI assay were low-pathogenic, H5N1-PR8 re-assortants obtained from Ruben Donis at the CDC Influenza Branch (Atlanta, GA): Clade 1, A/ Vietnam/1203/2004(H5N1)/PR8-IBCDC-RG and Clade 2.1, A/Indo/5/2005(H5N1)/PR8-IBCDC-RG2. Seed stocks of the re-assortant strains were obtained and expanded at BIOQUAL in 10-day-old embryonated chicken eggs. Virus strains used for the HAI assay were identical to the low pathogenic re-assortants listed for the MN assay.