Distance estimation within classes for the novel NS1 classification using nucleotide and protein sequences.

Model selection with the lowest BIC value and distance estimations were conducted in MEGA5 [26]. Gamma distribution parameter value α = 0.9304 and 1.0587 were used for maximum likelihood distance estimations using the GTR+I+Γ4 and the JTT+Γ4 models for nucleotide (db4535) and amino acid (pdb4535) substitutions, respectively. Within class distances for unique NS1 nucleotide and protein sequences, A1 corresponds to Equine 1 sequences, A2 to gull sequences, A3 includes viral sequences grouped under classic swine, A4 includes all former human seasonal viruses with A4.1 comprising H1N1 sequences and A4.2 including H3N2 and H2N2. Within A5 are A5.1 (Eurasian swine virus sequences), A5.2 (sequences associated with H9N2 and H6N1 viral sequences from Pacific Ocean avian species), A5.3 (Equine 2 sequences), A5.4 (Eurasian avian), A5.5 (HPAI H5N1) and A5.6 (American avian). Identification of highly distant sequences within each class (by observation of intraclass pairwise comparison matrices) resulted in sequence relocation and some iteration was conducted to reach robust A5 lineages.