2.2 Genetic, phylogenetic and structural analyses
Viral RNA was extracted with the RNeasy kit (Qiagen, Valencia, CA) and was reverse transcribed. PCR reaction amplification was performed by using segment-specific primers, and the products were purified. The whole virus gene was sequenced by the Illumina Solexa system. The reference sequences were retrieved from GenBank, https://www.ncbi.nlm.nih.gov/genomes/FLU/Database/nph-select.cgi?go=database. The sequence dataset was aligned by Muscle program (PMID: 15318951) followed by the manual adjustment. The best-fit nucleotides substitution model was selected from the 286 model candidates based on Bayesian information criterion (BIC) by using ModelFinder (PMID: 28481363). The Maximum-Likelihoood method was used to construct the phylogenetic trees implanted in the IQ-TREE 2.1.2 (PMID: 25371430). The phylogeny topological structure was supported by 10,000 times Ultrafast Bootstrap (UFBoot) (PMID: 23418397), 10,000 times Shimodaira-Hasegawa approximate likelihood ratio test (SH-aLRT) (PMID: 20525638), and the approximate Bayesian-like test (aBayes) (PMID: 21540409). The receptor-binding site (RBS) structure H6N6 ZZ346 virus was simulated based on H6 HA structure (PDB accession code: 5BR0) with molecular replacement and manual refinement using PyMOL 2.4.2 (Schrödinger, LLC).