Evolutionary sites drive avian influenza virus adaptation

To assess how receptor-binding preference influences viral adaptation in chickens, we generated two A/chicken/Beijing/16/2013 (BJ16) HA mutants with defined binding properties: T190&Q226 (dual alpha-2,3/alpha-2,6 SA binding) and E190&Q226 (exclusive alpha-2,3 SA binding), in comparison with wild-type BJ16 (T190&L226, exclusive alpha-2,6 SA binding). Three-week-old White Leghorn chickens were divided into three groups (n = 12 per group) and infected with either the alpha-2,6 WT virus (T190&L226), the alpha-2,3/alpha-2,6 variant (T190&Q226), or the alpha-2,3 variant (E190&Q226). Chickens were intranasally inoculated with 10000 PFU of virus. Oropharyngeal swabs, palates, and lungs were collected at 1, 3, 5, and 7 dpi for HA-targeted deep sequencing.