Identification, Characterization, and Natural Selection of Mutations Driving Airborne Transmission of A/H5N1 Virus

•Five substitutions are sufficient for airborne transmission of A/H5N1 between ferrets•Two substitutions in PB1 and PB2 increased RNA transcription and virus replication•Three HA substitutions altered receptor binding preference and lowered pH of fusion•Strong selective advantage of substitutions responsible for airborne transmission

SummaryRecently, A/H5N1 influenza viruses were shown to acquire airborne transmissibility between ferrets upon targeted mutagenesis and virus passage. The critical genetic changes in airborne A/Indonesia/5/05 were not yet identified. Here, five substitutions proved to be sufficient to determine this airborne transmission phenotype. Substitutions in PB1 and PB2 collectively caused enhanced transcription and virus replication. One substitution increased HA thermostability and lowered the pH of membrane fusion. Two substitutions independently changed HA binding preference from α2,3-linked to α2,6-linked sialic acid receptors. The loss of a glycosylation site in HA enhanced overall binding to receptors. The acquired substitutions emerged early during ferret passage as minor variants and became dominant rapidly. Identification of substitutions that are essential for airborne transmission of avian influenza viruses between ferrets and their associated phenotypes advances our fundamental understanding of virus transmission and will increase the value of future surveillance programs and public health risk assessments.

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