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).