In previous studies, we have reported the generation of H5pp and have characterized it as a safe alternative to the use of replicative H5N1 virus for sero-surveillance [16, 18] . Because H5pp mimics the entry mechanism of the avian virus while carrying only the H5-HA as envelope protein, it offers the advantage to be specifically neutralized only by anti-hemagglutinin antibodies, avoiding the confounding effect of antibodies directed against N1 neuraminidase due to infection of influenza virus subtypes other than H5N1. We report here that the efficiency to generate HA-only H5pp varies with HAs derived from different H5N1 virus clades, regardless of the lentiviral backbone used. Through serial mutagenesis of two H5-HAs, we have uncovered that differences in receptor binding ability, due to mutations in the receptor-binding domain of HA, may be the underlying mechanism.