Regarding the potential influence of mutations at position 134 of H5-HA on receptor binding properties, there have been reports with contradictory results. First, Yamada et al. [34] found that A134T mutation did not change alpha-2,3 sialic acid binding preference of H5-HA. Then, Auewarakul et al. [35] reported that L129V/A134V allowed for dual binding to both alpha-2,3 and alpha-2,6-sialic acid receptors, although in their study, the effect of A134V mutation alone was not assessed. More recently, using virus elution assay, Imai and colleagues [36] found that H5N1 viruses containing alanine at position 134 (A134) show stronger binding than those harbouring threonine (T134) to both chicken erythrocytes (expressing both alpha-2,3 and alpha-2,6-sialic acid) and horse erythrocytes (expressing only alpha 2,3-sialic acid). Similar to the observation by Imai et al., we found in the current study that H5Anh which contains A134 displayed a strong binding to both MDCK and MDCK-SIAT-1 cells (expressing an increased level of alpha-2,6 and a decreased level of alpha-2,3-sialic acid than parental MDCK) [26] . As predicted by these observations, the A134V mutation reduced H5Anh binding to a dramatically lower level in both cell lines. It is likely that strong binding of H5Anh to cell surface sialic acid receptors makes it difficult to release H5pp from the producer cells even in the presence of exogenous bacterial NA; and the A134V mutation reduces binding, thus allowing for the release of H5pp. In keeping with this hypothesis, co-transfection with the viral NA gene from H5N1 led to the production of similar amounts of mixed HA-NA pseudoparticles for both H5Anh and H5Cam. Moreover, we did not observe an increase in binding to MDCK-SIAT-1 cells, which contain more alpha-2,6-sialic acids on the cell surface. In fact both sH5Anh and sH5CamM2 bind with slightly lower efficiency to MDCK-SIAT-1, compared with parental MDCK cells, suggesting that A134V mutation probably leads to a decreased binding of H5-HA to alpha-2,3-sialic acid rather than a switch to alpha-2,6sialic acid binding. Consistent with this notion, we observed an increased level of H5Anh-pp production in Lec2 sialylationdeficient cells, when compared with parental CHO cells (Fig. 6B) .