We have found that the A134V mutation not only exerts a critical influence in the determination of pseudotyping efficiency, but has also an impact on H5N1 viruses. Both A/Cambodia/ 408008/2005 and A/Cambodia/V0401301/2011, two different H5N1 isolates carrying the same A134V mutation could agglutinate human and guinea pig RBCs but failed to agglutinate horse RBCs [30] (also Figure 7 of this paper); whereas two other strains of H5N1 viruses without the A134V mutation could also agglutinate horse RBCs (Figure 7) . These observations indicate that A134V mutation in H5-HA reduces virus binding to alpha-2,3-sialic acid. Although co-transfection with viral NA enables efficient lentiviral pseudotyping by H5Anh (Fig. 6A) , the differential RBC binding properties observed at the whole virus level, when both HA and NA are present, support the idea that A134V mutation in H5-HA can be biologically relevant. Interestingly, alanine at position 134 (A134) is highly conserved in avian H5N1 viruses and so far A134V mutation has only been found in human isolates of H5N1 viruses, both clade 1 and clade 2 viruses isolated from 2004 to 2011. Almost all avian H5N1 isolates possess A134 in the HA. So far only one avian H5N1 virus in the NCBI database has serine instead at position 134 of the HA protein. Notably, more diversity is observed at this position for human isolates of H5N1 viruses: three H5N1 viruses isolated from human patients have a threonine and eleven a valine at position 134 [36] . At least in two cases (A/Cam/408008/2005 and A/ Thailand/676/2005), viruses found in the original patient specimens were mixtures of both wild type, containing A134 in the HA, and mutant virus, containing V134 [30, 35] . It is possible that other human isolates of H5N1 viruses may actually contain the A134V mutation but failed to be detected in the process of either virus isolation or traditional capillary sequencing of viral genomes. Thousands of H5-HA sequences are available in the NCBI Influenza Database (http://www.ncbi.nlm.nih.gov/ genomes/FLU/FLU.html) from non-human isolates of H5N1 viruses, none of which contains this particular mutation. Altogether, these observations suggest that a valine at residue 134 of the receptor-binding domain is unlikely to be a random sequence variation but may be selected as the avian H5N1 viruses adapt for replication in human hosts. We speculate in general terms that changes in cell surface receptor binding of H5-HA, as a result of A134V mutation, may lead to changes in virus entry and virus release and, therefore, be considered an important factor for determination of host range. It is not clear whether intracellular sialic acid content and distribution may also influence this feature. As our data focus on the pseudotyping system, further studies are required to understand more precisely the biological consequences of A134V mutation and its potential influence on the adaptation of H5N1 viruses in humans.