Our findings have also implications for the applicability of H5pp assay in serological surveys. H5pp has several advantages over the microneutralization method, which is the current gold standard serological assay for the detection of antibodies against avian influenza viruses [37, 38] . Pseudotyped particles are produced from synthetic genes without the need to have access to the virus and can be safely used in BSL-2 containment, making them ideal for widespread use, especially in areas where BSL-3 facilities are not available. Moreover, it has been reported that the H5pp assay is more sensitive than micro-neutralization [16, 39] . It appears, however, that its use in sero-epidemiological studies and to monitor the efficacy of candidate vaccines may be limited by the strain under investigation. Although we and others have found that production of mixed HA-NA pseudo-particles is consistently successful using N1 from either H1N1 or H5N1 [17, 20, 21] (also Fig. 6A of this paper) , the production of HA-only pseudotypes would be necessary to eliminate potential cross-reactivity that may be displayed by circulating anti-NA antibodies against N1 from avian H5N1 or seasonal influenza H1N1 [40] . We are cognizant that a single A134V mutation may result in a change of antigenicity but this limitation is not different from that of the microneutralization assay, which is the gold-standard to detect anti-H5 neutralization antibodies and utilizes an available H5N1 virus strain that may not be a perfect match of the viruses associated with the serum samples being tested. In fact, serologic surveys often use a collection of serum samples from human or animals without necessarily knowing the exact virus strain(s) involved. Moreover, if positive, samples shall contain polyclonal antibodies against multiple epitopes to an H5N1 virus, further minimizing the likelihood that a single amino acid substitution would compromise the usefulness of a pseudotype-based serological assay as a safer alternative to the microneutralization test. Careful assessment of the H5pp-based assay should obviously be performed when new strains emerge.