In vivo pathogenesis studies of wild bird H5N6 influenza viruses were designed as previously described (Zhang et al., 2008, 2009; Pu et al., 2015). In brief, groups of 12 6-week-old SPF chickens were intranasally inoculated with 0.2 mL of 105 EID50 of SW8 or ZH283, while a control group of 12 chickens was inoculated with 0.2 mL of phosphate buffered saline (PBS) using the same route. Three days later, six inoculated chickens from each group were humanely euthanized to test for viral replication in lung, kidney, spleen, cecal tonsils, bursa of Fabricius, trachea, pancreas, liver, heart, brain, duodenum, ileum, descending colon, and jejunum tissue. The remaining chickens were observed twice daily, at 8:00 and 20:00, for clinical symptoms, morbidity, and mortality for 14 days according to the protocol provided by the World Organisation for Animal Health2.