Evolutionary sites drive avian influenza virus adaptation

Deep mutational scanning (DMS) selection of the A/chicken/Beijing/16/2013 (designated as BJ16) HA library was performed in four immune backgrounds: naive (Cage 1a), A/chicken/Guangdong/SS/1994-vaccinated (SS vax; Cage 1b), BJ16/2013-vaccinated (BJ16 vax; Cage 1c), and BJ16/2013-naturally infected chickens (BJ16 NI; Cage 1d). In each model, three six-week-old donor chickens were intranasally inoculated with 100,000,000 PFU of the DMS virus library.Donor oropharyngeal swabs were collected at 1 and 3 dpi for deep sequencing. Each donor was co-housed with one contact chicken from the same immune background. Contact oropharyngeal swabs were collected at 2 and 4 dpi for sequencing.At 3 dpi, donor palate and lung tissues were also collected for deep sequencing to assess changes in library diversity.