Swapping of HA2 domain (including the polybasic cleavage site) does not increase production of H5Anh-pp Sequence analysis of the polybasic cleavage site reveals that H5Anh has a deletion of a lysine residue when compared to H5Cam and moreover, there is an additional amino acid difference in the HA2 region at position 533, which is located at the border between the ecto-domain and the transmembrane domain (TMD) ( Fig. 2A) . Thus, H5Cam has an isoleucine at position 533 (I533), while H5Anh has a threonine (T533). Cleavage of HA into HA1 and HA2 subunits by host protease is a critical step for influenza viruses to gain membrane fusion capability [23, 24] ; whereas the TMD of HA is important for its association with lipid rafts at the plasma membrane [25] . To test the potential influence of these differences in the cleavage site and at position 533, we generated several chimerical constructs in which either the entire HA2 region including the cleavage site was replaced with that of H5Cam (AnhCam1), or only the cleavage site (AnhCam2) or a single T533I amino acid change was introduced (AnhCam3). All constructs were FLAG-tagged at the C-terminal end of H5 sequences as described in the Materials and Methods section ( Fig. 2A) . When transfected into 293T cells, all three mutant H5Anh proteins were well expressed in the producer cell lysates (Fig. 2B) ; however, analysis of transduction levels of MDCK target cells by H5pp produced with these H5AnhCam chimerical proteins suggests that none of them was able to increase the production of pseudotyped particles (Fig. 2C) . These data indicate that differences in the HA2 domain cannot account for the reduced ability of H5Anh to form pseudotyped particles.