Generation of H9N2 AIVs by reverse genetics
The primers, synthesized by Tsingke Biological Technology (Nanjing, China), used to amplify the DNA sequence to add the single mutation in HA protein of F/98 virus were designed using Primer 5.0 software (Primer‑E Ltd., Plymouth, UK) based on the HA gene sequence of the F/98 H9N2 avian influenza virus. The full‑length HA genes containing the single mutation was amplified by PCR, and inserted into a transcriptional/expression vector pHW2000 (Hoffmann, Neumann, Kawaoka, Hobom, & Webster, 2000) by using ClonExpress II One Step Cloning Kit (Vazyme Biotech Co., Ltd., Nanjing, China), resulting in the plasmids pHW204‑HAmutations. Seven dual-promoter plasmids, including pHW201‑PB2, pHW202‑PB1, pHW203‑PA, pHW205‑NP, pHW206‑NA, pHW207‑M, and pHW208‑NS, from the F/98 virus strain, were stocked in our lab at -70 °C (Shi, Ashraf, Gao,Lu, & Liu, 2010). The recombinant viruses were rescued by transfection in the 293T cell as previously described (Peng et al., 2019). Briefly, a total weight of 2.4 ng of the eight plasmids mixture with a rate of 1:1 was mixed with 100 µL Opti‑MEM medium (GIBCO, BRL, Grand Island, USA). Next, 7 µL of PolyFect transfection reagent (QIAGEN, Duesseldorf, Germany) was added. The samples were incubated at room temperature for 10 min and then added to the 70-80% confluent monolayers of 293T cell in 24‑well plates. After incubation at 37°C with 5% CO2 for 6 h, 2 µg/mL of TPCK-trypsin (Sigma, St. Louis, MO, USA) was added to the wells. Thirty hours after transfection, the supernatants were harvested and inoculated into 10‑day‑old SPF embryonated chicken eggs for virus propagation. The rescued virus was analyzed with a hemagglutinin assay, and the HA genes from the rescued virus was sequenced by Tsingke Biological Technology (Nanjing, China) to confirm the accuracy of the designed mutation.