Figure 1. Experimental design of parallel studies of mouse adaptation.
The parental strain of A/Hong Kong/1/68 (HK-wt) was clonally derived on MDCK cells and grown in chicken embryos before dilution in PBS to 1×105 pfu/ 0.05 mL to initiate serial mouse passage; followed by 20 serial passages with 6 clones derived by plaque isolation from the passage 12 and 20 populations on MDCK cells. Replicate stocks of HK-wt (HK-#) were generated from individual infectious virions of HK-wt by plaque isolation. Each subclone was amplified in eggs and used without dilution to infect mice (≥1×106 pfu/0.05 mL for each mouse) to initiate 9 parallel MA series as indicated in methods before isolating 3 clones from each of the passage 21 populations.