Evolution and antigenic phenotypes of N2 neuraminidase genes from human seasonal influenza A viruses following two separate introductions into swine in the United States
Figures and analysis files associated with phylogenetic analysis and downsampling the tree for visualziaiton in the manuscript:
Kaplan, B.S., Anderson, T.K., Chang, J., Santos, J., Perez, D., Lewis, N.S., and Vincent, A.L. (in review). Evolution and antigenic phenotypes of N2 neuraminidase genes from human seasonal influenza A viruses following two separate introductions into swine in the United States.
Two separate introductions of human seasonal N2 neuraminidase genes were sustained in United States swine since 1998 (N2-98) and 2002 (N2-02). Herein, we characterized the antigenic evolution of the N2 of swine influenza A virus (IAV) across two decades following each introduction. The N2-98 and N2-02 expanded in genetic diversity, with two statistically supported monophyletic clades within each lineage. To assess antigenic drift in swine N2 following the human-to-swine spillover events, we generated a panel of swine N2 antisera against representative N2 and quantified the antigenic distance between wild-type viruses using enzyme-linked lectin assay and antigenic cartography. The antigenic distance between swine and human N2 was smallest between human N2 circulating at the time of each introduction and the archetypal swine N2. However, sustained circulation and evolution in swine of the two N2 lineages resulted in significant antigenic drift, and the N2-98 and N2-02 swine N2 lineages were antigenically distinct. Although there was within-lineage antigenic diversity, the differences between viruses within each identified genetic clade was not always indicative of significant antigenic drift. These data represent the first quantification of the antigenic diversity of neuraminidase of IAV in swine and demonstrated significant antigenic drift from contemporary human seasonal strains as well as antigenic variation among N2 detected in swine. These data suggest that antigenic mismatch may occur between circulating swine IAV and vaccine strains. Consequently, consideration of the diversity of N2 in swine IAV vaccines will likely result in more effective control, and aid public health initiatives where candidate vaccine viruses are selected for pandemic preparedness.
All available swine IAV NA-N2 sequences from H1N2 and H3N2 subtype were downloaded from the Influenza Research Database on October 24, 2018. To restrict to wild-type field viruses, we excluded data with “lab” or “laboratory” in the host record. As our goal was to describe genetic diversity in circulating swine NA N2 genes, strains that reflected single outbreak events collected as part of active surveillance at agricultural fairs were removed from analyses. These data were compiled with a temporally matched set of N2 genes from the World Health Organization (WHO) recommended human seasonal vaccine sequences. The sequences were aligned with default settings in MAFFT v7.294, and sequences with 100% identity, those that were missing more than 50% of the gene, or those with more than 5 nucleotide base ambiguities were removed. A maximum-likelihood phylogeny for the alignment was inferred, following automatic model selection, using IQ-TREE v2. Monophyletic clades were identified using statistical support (SH aLRT > 90; ultrafast bootstrap > 70), and detection frequency (a minimum of 10 N2 genes from multiple locations across years). From each monophyletic clade, we deduced amino acids for the N2 gene, aligned the data using MAFFT v7.294, and generated a clade consensus sequence using custom python code in the flutile package. We subsequently identified the best-matched field strain to the clade consensus sequence, and this strain was used in subsequent wild-type assays, or in the constriction of reverse engineered IAV viruses. For visualization purposes, we downsampled the inferred maximum likelihood tree using smot: we implemented paraphyletic sampling, where monphyletic trees were identified within each named genetic clade, and a random selection within each clade were identified with the scale factor set to 0.5 (excluding human seasonal N2 and swine antigen genes) and maintained for presentation in Figure 1.
smot clean N2-allData-v1.tre |
smot factor prepend --impute --factor-by-capture="\\|(98B[^|]*|98A[^|]*|02A[^|]*|02B[^|]*|Human_seasonal_N2|98|02)\\|" |
smot factor prepend --impute --patristic --factor-by-capture="^(98B[^|]*|98A[^|]*|02A[^|]*|02B[^|]*|Human_seasonal_N2|98|02)\\|" |
smot sample para -s 0.5 --seed=42 --min-tips=3 --keep-regex="REF\\|" --factor-by-capture="^([^|]+)" > n2-all-sampled.tre