Specific mutations in H5N1 mainly impact the magnitude and velocity of the host response in mice

Tchitchek, Nicolas; Eisfeld, Amie J; Tisoncik-Go, Jennifer; Josset, Laurence; Gralinski, Lisa E; Bécavin, Christophe; Tilton, Susan C; Webb-Robertson, Bobbie-Jo; Ferris, Martin T; Totura, Allison L; Li, Chengjun; Neumann, Gabriele; Metz, Thomas O; Smith, Richard D; Waters, Katrina M; Baric, Ralph; Kawaoka, Yoshihiro; Katze, Michael G

Having characterized VN1203-WT dynamics, we were now able to determine how each specific VN1203 mutant altered the kinetics of the host responses. In particular, our aim was to better quantify the magnitude differences between WT and mutants that were introduced with Figure 2 . We next developed a new geometrical representation method in order to compare the kinetics of the host response to the various VN1203 mutants relative to the host response to VN1203-WT at the transcriptomic level. Using the MDS representation presented in Figure 3C as a reference, we projected the transcriptomic profiles of samples infected by the four VN1203 mutant viruses individually at 10 4 PFU ( Figure 5A -D) and, in the case of VN1203-NS1trunc and VN1203-PB1F2del, at 10 3 and 10 4 PFU dosages ( Figure 5C and D) . While traditional MDS methods project a set of high dimensional objects into a lower dimensional space for visualization purposes, the MDS method that we developed allows for projection of additional objects over a predefined MDS representation (see Methods section). Thus, the resulting representation allows us to visualize the similarities and differences between the WT and mutant samples, and MC and VC allow us to quantify the kinetic changes in the host response related to magnitude and velocity, as previously described for VN1203-WT. The reference representation is named a MDS Reference Map and the resulting projections are named MDS Projections. Quantification of magnitude and velocity revealed different information about each mutant. For VN1203-PB2627E, both the MDS projection ( Figure 5A ) and the MC and VC profiles (Figure 4 ) for the 10 4 dosage were similar to that observed for VN1203-WT at the 10 2 dosage. Therefore, the main effect of the PB2-627E mutation on the host-response was to decrease it to a level similar to that induced by 100 times less VN1203-WT. This is consistent with the reduced replication we observed for VN1203-PB2627E in the current study ( Figure 1B) and with a previous study showing that the main effect of the PB2-627E mutation in the VN1203 background was to reduce virus replication in the lung [5] .