Neuraminidase-mediated haemagglutination of recent human influenza A(H3N2) viruses is determined by arginine 150 flanking the neuraminidase catalytic site

Abstract

Over the last decade, an increasing proportion of circulating human influenza A(H3N2) viruses exhibited haemagglutination activity that was sensitive to neuraminidase inhibitors. This change in haemagglutination as compared to older circulating A(H3N2) viruses prompted an investigation of the underlying molecular basis. Recent human influenza A(H3N2) viruses were found to agglutinate turkey erythrocytes in a manner that could be blocked with either oseltamivir or neuraminidase-specific antisera, indicating that agglutination was driven by neuraminidase, with a low or negligible contribution of haemagglutinin. Using representative virus recombinants it was shown that the haemagglutinin of a recent A(H3N2) virus indeed had decreased activity to agglutinate turkey erythrocytes, while its neuraminidase displayed increased haemagglutinating activity. Viruses with chimeric and mutant neuraminidases were used to identify the amino acid substitution histidine to arginine at position 150 flanking the neuraminidase catalytic site as the determinant of this neuraminidase-mediated haemagglutination. An analysis of publicly available neuraminidase gene sequences showed that viruses with histidine at position 150 were rapidly replaced by viruses with arginine at this position between 2005 and 2008, in agreement with the phenotypic data. As a consequence of neuraminidase-mediated haemagglutination of recent A(H3N2) viruses and poor haemagglutination via haemagglutinin, haemagglutination inhibition assays with A(H3N2) antisera are no longer useful to characterize the antigenic properties of the haemagglutinin of these viruses for vaccine strain selection purposes. Continuous monitoring of the evolution of these viruses and potential consequences for vaccine strain selection remains important.

Fig. 1.

Haemagglutination titres of recombinant A(H3N2) viruses with various combinations of 2003 and 2009 HA and NA genes, in the presence (+) or absence (−) of oseltamivir.

Fig. 2.

Schematic representation of the cDNA of chimeric NAs based on the NA of A/NL/109/03 and A/NL/761/09. The seven amino acid differences between the two NA proteins are shown, as well as the EcoRV restriction site that was used to construct the chimeras. NCR, non-coding region.

Fig. 3.

Proportion of human A(H3N2) viruses with substitutions at amino acid position 150 in NA, from 2000 until 2015. A total of 5942 NA sequences available from full genomes, including 900 from MDCK-passaged viruses and 1687 derived from clinical specimens, were analysed. No full-genome sequences were available for human A(H3N2) viruses from 2016. NA genes with histidine (H), arginine (R) or any other amino acid (X) at position 150 are shown in orange, blue and grey respectively.