Triplet thermostability mutations HA S84F/G167N/D168N drive A/H9N2 virus transmission
The full-site BJ16 HA saturation mutagenesis (DMS) library was passaged once in MDCK cells to generate a baseline DMS virus library. Thermal screening was performed at the virus TIP90 and at 55 C. Virus aliquots (1000000 PFU/mL) were heat-treated for 30 min, then inoculated into MDCK cells or 10-day-old SPF embryonated chicken eggs for amplification. After three days, viral supernatants were collected and subjected to five rounds of thermal selection, yielding fifth-passage thermally screened virus samples.