The 2009 pandemic H1N1 D222G hemagglutinin mutation alters receptor specificity and increases virulence in mice but not in ferrets

J Infect Dis. 2011 Oct 1;204(7):1008-16. doi: 10.1093/infdis/jir483.


Background: The D222G (H1 numbering) hemagglutinin (HA) mutation within the receptor-binding site was detected with higher frequencies in severe cases of 2009 pandemic H1N1 (pH1N1) infections. We investigated the impact of this mutation in vitro and in animal models using recombinant pH1N1 viruses.

Methods: The recombinant D222G HA mutant was generated from a wild-type (WT) clinical strain by using reverse genetics and site-directed mutagenesis. Replicative capacities were determined in MDCK and MDCK-α2,6 cells. Antigenicity was characterized by HA inhibition and microneutralization assays. HA titers were determined using human, chicken, and resialylated turkey red blood cells (RBCs). Virulence and contact-transmissibility were analyzed in mice and ferrets.

Results: The recombinant D222G virus grew to significantly higher titers and generated larger viral plaques compared with the WT in MDCK but not in MDCK-α2,6 cells. The mutant also showed a significant reduction in HA titers using α2,6-expressing RBCs. The 2 recombinants were antigenically similar. The D222G mutant virus induced higher lung viral titers and alveolar inflammation in mice whereas the 2 recombinants had similar impacts in ferrets.

Conclusions: The D222G HA mutation alters receptor binding specificity, resulting in higher lung titers in mice. This could contribute to the higher case fatality rates reported in humans.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Binding Sites
  • Cell Line
  • Female
  • Ferrets
  • Hemagglutinin Glycoproteins, Influenza Virus / genetics*
  • Humans
  • Influenza A Virus, H1N1 Subtype / genetics*
  • Influenza A Virus, H1N1 Subtype / pathogenicity*
  • Influenza, Human / virology*
  • Interferon-gamma / metabolism
  • Interleukins / metabolism
  • Lung / metabolism
  • Lung / pathology
  • Lung / virology
  • Male
  • Mice
  • Mice, Inbred BALB C
  • Mutation*
  • RNA, Viral / metabolism*
  • Receptors, Cell Surface / metabolism
  • Viral Load*
  • Virus Attachment


  • H1N1 virus hemagglutinin
  • Hemagglutinin Glycoproteins, Influenza Virus
  • Interleukins
  • RNA, Viral
  • Receptors, Cell Surface
  • sialic acid receptor
  • Interferon-gamma