Sequence and phylogenetic analysis
A Simply P total RNA extraction kit was used to extract RNA according to manufacturer instructions. The reverse transcription was performed in a total volume of 60 μL containing 39 μL of RNA, 3 μL of dNTPs, 3 μL of 12 bp (10 nmol), 1.5 μL of M-MLMLV, 1.5 μL of RRI, and 12 μL of 5*buffer. The liquid was mixed, then placed in a 42°C water bath for 2 to 4 hours. We used published universal primers and primers designed for different subtypes in the literature, as well as primers designed based on the downloaded sequences from the influenza database (www.gisaid.org).
PCR amplification of cDNA was performed in a total volume of 50 μL and contained 25 μL of Premix Taq, 20 μL of dd H2O, 4 μL of upstream and downstream primers, and 1 μL of template cDNA. The PCR program comprised 34 cycles of 95°C for 50 s, 55°C for 50 s, and 72°C for 1 m and 50 s. The PCR products were separated on 1% agarose gels by electrophoresis and visualized using a UV transilluminator. The sequence was measured by generation 1 sequencing technology, and the sequence result was processed with the Lasergene 7.1 software package.
The sequences of partial H5 subtype AIVs with whole genes from 2016 to 2019 were collected from the Global Initiative on Sharing Avian Influenza Data (GISAID) database (www.gisaid.org). We used PhyloSuite data processing software for constructing phylogenetic trees(Dong Zhang & Wang, 2019) . MAFF (Katoh,Misawa,Kuma, & Miyata, 2002), which is included in the software, was used to compare the nucleotide sequences. Modefinder was used to find the best model and using maximum likelihood in IQ-tree (Kalyaanamoorthy,Minh,Wong,von Haeseler, & Jermiin, 2017; Nguyen,Schmidt,von Haeseler, & Minh, 2015), eight phylogenetic trees were constructed. ITOL was used to beautify the phylogenetic tree (https://itol.embl.de).
We calculated the timescale of avian influenza virus clades and evolutionary rates using BEAST 1.1.04 (Suchard et al., 2018). import eight sequence fragments, tag sequences of each sampling time, chose a relaxed molecular clock model, set MCMC chain length at 500000000. The acquisition interval was 50,000 and resulted in 10,000 trees eventually. The generated XML file was run by BEAST, and the log file was opened by Tracer1.7.1(Rambaut,Drummond,Xie,Baele, & Suchard, 2018) to verify that the ESS value of each parameter was > 200. We opened the generated tree file using TreeAnnotator v1.10.4 software, burn in 10% to generate the tree file, and placed it in Figtree 1.4.4 to open(http://tree.bio.ed.ac.uk/software/figtree/).