| While the number of human infections with highly pathogenic avian H5N1 influenza viruses continues to rise, these viruses are still unable to transmit efficiently between humans. Recently, we and others introduced mutations into the receptor-binding domain of the influenza viral surface glycoprotein hemagglutinin (HA) that altered the receptor-binding preference of HA from avian- to human-type, but also reduced HA protein stability. Virus passages in ferrets resulted in the emergence of compensatory mutations that reduced the pH of fusion and stabilized HA. The H5 viruses that possessed human-type receptor-binding specificity and additional stabilizing mutations in HA could transmit among ferrets through respiratory droplets. To identify additional stabilizing mutations in H5 HAs, we constructed and screened a mutant virus library possessing two mutations that confer binding to human-type receptors in addition to random mutations in the HA ectodomain. After several rounds of heat treatment and virus amplification, we isolated several mutants with increased thermostability. These mutants also initiated membrane fusion at a lower pH value that was comparable to that of human HA proteins and maintained their human-type receptor-binding property.;In addition, we analyzed publicly available H5 HA sequences for polymorphisms in H5N1 human isolates that were not present in avian isolates. This analysis revealed 59 unique polymorphisms. To date, we have tested a subset of these candidates and identified one mutation that affects HA stability. Our previous ferret transmission study was carried out with an HA protein of clade 1. Most currently circulating H5N1 viruses belong to clade 2 subclades. Therefore, we introduced the transmission-conferring mutations into eight genetically distinct clade 2 viruses. All mutant viruses bound to human-type receptors and underwent membrane fusion at the same pH or a more acidic pH than the corresponding wild-type HA. Overall, these variants match the phenotype of the H5 virus that transmitted via respiratory droplets among ferrets. Collectively, the presented data expand on the understanding of HA stability which has been recently identified to play a key role in the mammalian respiratory droplet transmission of influenza viruses. |
