2.7. Adaptation and Parallel Evolution

We evaluated amino acids under molecular adaptation and parallel evolution in HP H7N9 AIV using Hyphy (Hypothesis Testing using Phylogenies, v2.5.2), Treesub programs (https://github.com/tamuri/treesub, last accessed on 14 December 2021) and ProtParCon [39]. To determine which amino acids in the HA coding region were under positive selection, we used Hyphy to estimate the ratio of non-synonymous to synonymous substitution rates (dN/dS) [40,41]. Since the Mixed Effects Model of Evolution (MEME) [42] considers that selection pressure for each site is pervasive or episodic along the entire phylogenetic tree, we employed MEME to detect diversifying selection in HP H7N9 viruses. Significant levels of p < 0.05 were considered as an indicator of positive selection in MEME. Treesub was used to infer amino acid substitutions along the branches and ProtParCon was used to evaluate the sites that underwent parallel and convergent evolution among the internal node by reconstructing ancestral nodes to the tips based on sequences. PyMol (v2.5) was used to map the amino acid substitutions against the 3D crystal structure of the HA protein (Protein Data Bank code: 6D7U) [43].