Two H5N6 viruses—namely, A/oriental magpie-robin/Guangdong/SW8/2014 (SW8) and A/Pallas’s sandgrouse/Guangdong/ZH283/2015 (ZH283)—used in this study were grown and purified three times in Madin–Darby canine kidney cells by standard plaque assay. The stocks of H5N6 viruses were propagated in 9-day-old specific pathogen-free (SPF) chicken eggs at 37°C for 72 h per the procedure (Yuan et al., 2014). Allantoic fluid pooled from multiple eggs was taken for centrifugation for 2 min at 8,000 rpm, from which the supernatant was harvested and subsequently frozen in aliquots at -80°C for further characterization. The 50% egg infectious dose (EID50) titer for egg-grown virus was determined by 10-fold serial dilutions and the titration of each virus in 9-day-old SPF eggs using Reed and Muench’s (1938) method. Six-week-old SPF white leghorn chickens (Guangdong Wens Dahuanong Biotechnology Co., Ltd, Yunfu, China) were held in isolator cages with a feeding space of 117 m3 throughout the duration of each experiment.