Blunt3 vector

DNA was isolated using a cetyltrimethylammonium bromide method. DNA methylation assay was performed using DNA Bisulfite Conversion Kit (Tiangen). PCR products amplified with Methylation specific PCR kit (Tiangen) were cloned into Blunt3 vector (TransGen Biotech) and sequenced. Bisulfite sequencing data were analyzed by the CyMATE software. The results returned by CyMATE were put into GraphPad Prism 9.0 to illustrate DNA methylation frequency at CG, CHG and CHH (where H = A, C or T), respectively. Primers were list in Supplementary Table 1.

pHW291-PB2, pHW292-PB1, pHW293-PA, pHW294-HA, pHW295-NP, pHW296-NA, pHW297-M, pHW298-NS (GenBank Taxonomy ID: 1082519), and pHW298-NS1-128 were previously constructed by Hoffmann’s method (Hoffmann et al., 2001 (link); Chen et al., 2017 (link)). The ORF of the HA gene with synonymous mutations was amplified from the pHW294-HA plasmid and then transferred into the Blunt3 vector (TransGen Biotech, China). The pHW298-NS1-128 plasmid was subjected to site-directed mutagenesis to mutate four ATGs (A27T, A76T, A727T, and A795T) and one splice site (G57C). The NS1 packaging signal was amplified from the site-directed mutation pHW298-NS1-128. The ORF-mut of the TX HA [1,683 base pairs (bp)], pHW2000 (2,961 bp), 3′ NS packaging signal (104 bp), and 5′ NS packing signal (130 bp) were amplified and ligated by one step cloning kit (Beijing Biodragon, China), and the recombinant plasmid was named NS-HAmut-NS. Recombinant plasmid HA-NS1-128mut-HA was generated by the method that is similar to NS-HAmut-NS. Briefly, the ORF mutation of TX NS1-128 with synonymous mutation amplified from pHW298-NS1-128 and the HA packaging signal with site-directed mutagenesis (five ATGs: A33T, A82T, A1647T, A1677T, and A1697T) were ligated to pHW2000 together (Figure 1A and Table 1).