Effect of D 222 G Mutation in the Hemagglutinin Protein on Receptor Binding , Pathogenesis and Transmissibility of the 2009 Pandemic H 1 N 1 Influenza Virus

Influenza viruses isolated during the 2009 H1N1 pandemic generally lack known molecular determinants of virulence associated with previous pandemic and highly pathogenic avian influenza viruses. The frequency of the amino acid substitution D222G in the hemagglutinin (HA) of 2009 H1N1 viruses isolated from severe but not mild human cases represents the first molecular marker associated with enhanced disease. To assess the relative contribution of this substitution in virus pathogenesis, transmission, and tropism, we introduced D222G by reverse genetics in the wild-type HA of the 2009 H1N1 virus, A/California/04/09 (CA/04). A dose-dependent glycan array analysis with the D222G virus showed a modest reduction in the binding avidity to human-like (a2-6 sialylated glycan) receptors and an increase in the binding to avian-like (a2-3 sialylated glycan) receptors in comparison with wild-type virus. In the ferret pathogenesis model, the D222G mutant virus was found to be similar to wild-type CA/04 virus with respect to lethargy, weight loss and replication efficiency in the upper and lower respiratory tract. Moreover, based on viral detection, the respiratory droplet transmission properties of these two viruses were found to be similar. The D222G virus failed to productively infect mice inoculated by the ocular route, but exhibited greater viral replication and weight loss than wild-type CA/04 virus in mice inoculated by the intranasal route. In a more relevant human cell model, D222G virus replicated with delayed kinetics compared with wild-type virus but to higher titer in human bronchial epithelial cells. These findings suggest that although the D222G mutation does not influence virus transmission, it may be considered a molecular marker for enhanced replication in certain cell types. Citation: Belser JA, Jayaraman A, Raman R, Pappas C, Zeng H, et al. (2011) Effect of D222G Mutation in the Hemagglutinin Protein on Receptor Binding, Pathogenesis and Transmissibility of the 2009 Pandemic H1N1 Influenza Virus. PLoS ONE 6(9): e25091. doi:10.1371/journal.pone. 0025091 Editor: Man-Seong Park, College of Medicine, Hallym University, Korea, Republic of Received July 13, 2011; Accepted August 26, 2011; Published September 22, 2011 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding: The source of funding for this work was the Centers for Disease Control and Prevention. This work was funded in part by a merit award to RS (R37 GM057073-13) from the National Institutes of Health and the Singapore-MIT Alliance for Research and Technology. The authors acknowledge the Consortium for Functional Glycomics for providing the glycan standards. No additional external funding was received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: tft9@cdc.gov