Substrate specificity of avian influenza H5N1 neuraminidase.

AIM To characterise neuraminidase (NA) substrate specificity of avian influenza H5N1 strains from humans and birds comparing to seasonal influenza virus. METHODS Avian influenza H5N1 strains from humans and birds were recruited for characterising their NA substrate specificity by using a modified commercial fluorescence Amplex Red assay. This method can identify the preference of α2,6-linked sialic acid or α2,3-linked sialic acid. Moreover, to avoid the bias of input virus, reverse genetic virus using NA gene from human isolated H5N1 were generated and used to compare with the seasonal influenza virus. Lastly, the substrate specificity profile was further confirmed by high-performance liquid chromatography (HPLC) analysis of the enzymatic product. RESULTS The H5N1 NA showed higher activity on α2,3-linked sialic acid than α2,6-linked (P < 0.0001). To compare the NA activity between the H5N1 and seasonal influenza viruses, reverse genetic viruses carrying the NA of H5N1 viruses and NA from a seasonal H3N2 virus was generated. In these reverse genetic viruses, the NA activity of the H5N1 showed markedly higher activity against α2,3-linked sialic acid than that of the H3N2 virus, whereas the activities on α2,6-linkage were comparable. Interestingly, NA from an H5N1 human isolate that was previously shown to have heamagglutinin (HA) with dual specificity showed reduced activity on α2,3-linkage. To confirm the substrate specificity profile, HPLC analytic of enzymatic product was performed. Similar to Amplex red assay, H5N1 virus showed abundant preference on α2,3-linked sialic acid. CONCLUSION H5N1 virus maintains the avian specific NA and NA changes may be needed to accompany changes in HA receptor preference for the viral adaptation to humans.

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