The ability of H5-HA to pseudotype lentiviral particles does not correlate with HA protein expression level in producer cells Similar to HA of other subtypes of influenza viruses, H5-HA is highly mutable as a result of antibody-selection pressure, leading to the rise of divergent H5N1 viruses that are categorized into various strains and clades [13, 14, 22] . To ascertain the flexibility and adaptability of H5pp production as an alternative approach for serological studies in the event of novel emerging H5N1 viruses, we sought to develop cladespecific H5pp and compared the ability of three other H5N1 viruses belonging to different clades to pseudotype lentiviral particles. H5-HA from clade 1 (H5Cam), clade 2.1 (H5Ind), clade 2.2 (H5Qin) or clade 2.3 (H5Anh) (see Table 1 ) was expressed in 293T cells together with lentiviral backbone plasmid to allow the production of H5-pseudotyped lentiviral particles (H5pp). Expression levels of H5-HAs in transfected 293T cells was monitored by Western blot using anti-FLAG antibody directed against the C-terminal tag (Fig. 1A , upper panel). Supernatants containing H5pp were harvested 48 hr post-transfection, and used to transduce MDCK cells for luciferase reporter activity assay (Fig. 1A , lower panel), as described in Materials and Methods. Unexpectedly, we observed significant differences in the transduction of MDCK cells by H5pp, depending on the clades of H5-HAs. In particular, H5Anh from A/Anhui/2005/01 resulted in very low luminescence levels after particle transduction in MDCK cells; whereas H5Cam from A/Cambodia/40808/2005 was the most efficient, inducing a consistent 3-4 log increase in luciferase activity compared with H5Anh ( Fig. 1A -B, lower panels). Analysis of cell lysates by Western blots, however, demonstrated that all H5-HAs tested were well expressed in the producer cells and, consequently, that luciferase reporter activity in MDCK target cells did not correlate with the level of HA protein expression in the cells (Fig. 1A) . Two main protein bands were detected, consistent with the expected electrophoretic mobility of the uncleaved protein (HA0) and the C-terminal portion of the cleaved form (HA2 subunit), whereas the N-terminal fragment (HA1 subunit) could not be recognized by the anti-FLAG antibody due to C-terminal tagging (Fig. 1A) . We next decided to compare in detail the behaviour of H5Cam and H5Anh. To determine whether the difference in luciferase reporter activity was due to the level of H5pp production, culture supernatants containing H5Cam-pp and H5Anh-pp were concentrated by ultracentrifugation, and the resulting H5pp pellets were analyzed by Western blotting. Our results showed that the number of particles produced in the culture supernatant was significantly less for H5Anh than for H5Cam in presence of soluble bacterial neuraminidase, as indicated by lower levels of p24 in concentrated supernatants to detect the lentiviral core and lower luciferase reporter activities in MDCK cells (Fig. 1B) . More importantly, incorporation of H5Anh into the pseudotyped lentiviral particles was not observed using anti-FLAG antibody (Fig. 1B, upper panel) . Altogether, these data suggest that H5Anh cannot be efficiently incorporated into pseudotyped particles and released into the cell culture supernatant.