Reverse Genetics for H7N7 Influenza Viruses

RNA of H7N7 viruses was extracted using QIAamp Viral RNA Mini Kit (Qiagen). Recombinant viruses were generated using reverse genetics. For this purpose, all gene segments of each virus were amplified and cloned into the plasmid pHWSccdB as previously described45 (link). Introduction of the selected mutations into the HA gene segments of different LP and HP viruses in this study was done using site-directed mutagenesis (primer sequences are available upon request) following the QuikChange protocol (Invitrogen). All recombinant viruses and mutants were rescued in HEK 293 T/MDCKII co-culture45 (link). Supernatants were then inoculated in SPF ECE for 3–5 days. Eggs were candled daily and those with dead embryos were kept in 4 °C and allantoic fluid was collected. Egg fluids with HA titre >16 (4 log₂) were used for further investigations. To exclude any unwanted mutation and confirm the introduced genetic changes in the constructed viruses, viral RNA was extracted and analysed by Sanger sequencing of RT-PCR amplicons as described46 (link).