PMC

(A) Scheme of mouse immunization and virus challenge. (B) Assays performed on lung samples from immunized and mock immunized mice.

Overview of (A) library preparation using unique identifiers (UID) and (B) processing pipeline used for deep sequencing data cleanup.

Identification of variable amino acid positions. (A) Entropy at individual positions. Significant differences of mutation frequencies among groups are highlighted. * p < 0.05, ** p < 0.01, and **** p < 0.0001. p-values were calculated before the Benjamini and Hochberg correction. (B) Frequencies of top 18 mutations in Mock and Immunized groups. * p < 0.05 after the Benjamini and Hochberg correction. Error bars represent SEM.

Lung viruses of vaccinated mice showed a reduction in LAH sequence diversity. (A) Shannon Diversity index of LAH amino acids. †: corresponding to the samples with the same labeling in B and . (B) Nucleotide to amino acid entropy ratios of the Shannon Diversity calculated for each sample. * p < 0.05, ** p < 0.01, and *** p < 0.001. Error bars represent SEM.

Seroconversion elicited by vaccination. Serum reactivity with synthetic long alpha helix (LAH) peptide (A), with group 1 HA proteins H1, H2, H5 and H9 (B) and with group 2 HA proteins H3 and H7 (C). (D) Lung virus titers of mice five (5 dpi) and seven (7 dpi) days post infection. * p < 0.05. Error bars represent standard error of the mean (SEM).

Constrained viral quasispecies evolution under immune pressure. (A) Heatmap and dendrogram of missense mutations detected in at least three out of the 18 samples analyzed by Z score based on frequency. Values from each row (i.e., mutation) have been normalized to have a mean of 0 and SD of 1. Relative expression levels of the same mutation within the individual mice are represented by the color code indicated. (B) Enlarged dendrogram from (A) using hierarchical clustering analysis performed by complete linkage and Euclidean distance measurement. †: corresponding to the samples with the same labeling in A and ; (C) Mean sequence composition of samples per group. The sizes of the bubbles are proportional to the mean percentage sample consensus sequences of each group. * p < 0.05. Error bars represent SEM.