Viruses, cells, eggs and plasmids
Human embryonic kidney 293T (HEK-293T) cells were obtained from the research center for swine diseases of Sichuan Agricultural University, and Madin-Darby canine kidney (MDCK) cells were obtained from Zigong Center for Disease Control and Prevention of Sichuan province (Zigong CDC). Both cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % fetal calf serum at 37oC with 5% CO2. Specific pathogen free (SPF) chicken embryos were obtained from the Beijng Merial Vital Laboratory Animal Technology Co., Ltd (Beijing, China). Avian origin H9N2 virus A/Chicken/Sichuan/CQY/2014 (abbreviated as CQY-2014) was isolated by the author’s group in 2014. Viruses were propagated in allantoic cavities of 10-day-old SPF embryonated chicken eggs for 48 hours and stored at -80 oC. The dual-promoter plasmid, pHW2000 was provided by Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences.