Amino acids responsible for the absolute sialidase activity of the influenza A virus neuraminidase: Relationship to growth in the duck intestine

The 1957 human pandemic strain of influenza A virus contained an avian viru s hemagglutinin (RA) and neuraminidase (NA), both of which acquired specifi city for the human receptor, N-acetylneuraminic acid linked to galactose of cellular glycoconjugates via an alpha2-6 bond (NeuAc alpha2-6Gal). Althoug h the NA retained considerable specificity for NeuAc alpha2-3Gal, its origi nal substrate in ducks, it lost the ability to support viral growth in the duck intestine, suggesting a growth-restrictive change other than a shift i n substrate specificity. To test this possibility, we generated a panel of reassortant viruses that expressed the NA genes of human H2N2 viruses isola ted from 1957 to 1968 with all other genes from the avian virus A/duck/Hong Kong/278/78 (H9N2). Only the NA of A/Singapore/1/57 supported efficient vi ral growth in the intestines of orally inoculated ducks. The growth-support ing capacity of the NA correlated with a high level of enzymatic activity, comparable to that found to be associated with avian virus NAs. The specifi c activities of the A/Ann Arbor/6/60 and A/England/12/62 NAs, which showed greatly restricted abilities to support viral growth in ducks, were only 8 and 5%, respectively, of the NA specific activity for A/Singapore/1/57. Usi ng chimeric constructs based on A/Singapore/1/57 and A/England/12/62 NAs, w e localized the determinants of high specific NA activity to a region conta ining six amino acid substitutions in A/England/12/62: Ser331-->Arg, Asp339 -->Asn, Asn367-->Ser, Ser370-->Leu, Asn400-->Ser, and Pro431-->Glu. Five of these six residues (excluding Asn400) were required and sufficient for the full specific activity of the A/Singapore/1/57 NA. Thus, in addition to a change in substrate specificity, a reduction in high specific activity may be required for the adaptation of avian virus NAs to growth in humans. This change is likely needed to maintain an optimal balance between NA activity and the lower affinity shown by human virus HAs for their cellular recepto r.