However, single-mutation H41Y or E42Q could dramatically increase the binding of marmoset (25.9% or 52.9%) or tufted capuchin (56.9% or 61.8%) ACE2 orthologs with S1-Fc; double-mutations YQ (H41Y and E42Q) further strengthen their binding with S1-Fc (87.3% or 87.6%, respectively) (Fig. 4C); in parallel to the authentic virus infection, at 48 h postinfection the complemented A549 cells were subjected to immunofluorescent staining for ACE2 ortholog protein and viral nucleocapsid for quantification of the infection efficiency by the Operetta High Content Imaging System. Here, ACE2 is linked to infection.