- Wild-type PR8 and PR8PB1(S216G) viruses were passaged twice in MDCK cells. The HA cDNA was amplified from viral supernatant, and cloned into pJET1.2/blunt as described in the Methods. The HA mutation frequency was determined by clonal sequencing of 94 to 105 bacterial clones for each experiment were picked and repeated independently three times. *by Student’s two-tailed unpaired t-test; NS Not significant