Characterization of changes in the hemagglutinin that accompanied the emergence of H3N2/1968 pandemic influenza viruses

Author summaryPandemic influenza A viruses (IAVs) originate from animal IAVs and always contain the envelope protein hemagglutinin (HA) of the animal parent. Molecular changes essential for the animal-to-human adaptation of the HA are not well defined. To address this problem, we studied phenotypic effects of eight amino acid substitutions that accompanied evolution of the HA of the 1968 pandemic IAV from its inferred avian precursor. We also analysed patterns of evolution of these HA positions of IAVs in birds and mammals. Our results predict that the 1968 pandemic IAV acquired its HA from a wild duck virus. We showed that two substitutions were primarily responsible for increased HA binding to human-type receptors and, in addition, marginally decreased stability of the HA, whereas three other substitutions reduced binding avidity of the HA. We demonstrated that substitutions accompanied by the addition of N-glycan at either position 63 or 81 represent a previously unrecognized common marker of avian-to-mammalian adaptation of IAVs. Our findings highlight the importance of the studies on previous pandemic IAVs for the influenza risk assessment and pandemic preparedness. The hemagglutinin (HA) of A/H3N2 pandemic influenza viruses (IAVs) of 1968 differed from its inferred avian precursor by eight amino acid substitutions. To determine their phenotypic effects, we studied recombinant variants of A/Hong Kong/1/1968 virus containing either human-type or avian-type amino acids in the corresponding positions of HA. The precursor HA displayed receptor binding profile and high conformational stability typical for duck IAVs. Substitutions Q226L and G228S, in addition to their known effects on receptor specificity and replication, marginally decreased HA stability. Substitutions R62I, D63N, D81N and N193S reduced HA binding avidity. Substitutions R62I, D81N and A144G promoted viral replication in human airway epithelial cultures. Analysis of HA sequences revealed that substitutions D63N and D81N accompanied by the addition of N-glycans represent common markers of avian H3 HA adaptation to mammals. Our results advance understanding of genotypic and phenotypic changes in IAV HA required for avian-to-human adaptation and pandemic emergence.

Automated summary

This study revealed the evolutionary pathway of the HA of the 1968 pandemic influenza virus, predicting its origin from wild duck virus and identifying key amino acid substitutions affecting HA binding and stability. The addition of N-glycan at specific positions was also found to be a common marker of avian-to-mammalian adaptation, highlighting the importance of studying past pandemic IAVs for influenza risk assessment and preparedness.