Notably, chickens inoculated with SW8 showed high pathogenicity, whereas naïve contact chickens infected showed no deaths. By contrast, chickens inoculated with ZH283 showed high pathogenicity, with a mortality rate of 100% within 2–3 days and efficient horizontal transmission in chickens. The mechanisms of lethality and transmissibility might be associated with mutations at positions K52T, I155T, and A544V of the HA protein, at positions K207R and Y436H of the PB1 protein, and at position T515A of the PA protein (Hulse-Post et al., 2007; Li et al., 2014). However, with the exception of position I155T of the HA protein, no mutations were observed in ZH283, which suggests that differences in the pathogenicity and transmissibility of H5N6 influenza viruses in chickens correlate with the probability of their being at position I155T of the HA protein. In addition, the transmissibility of H5N6 AIV in different birds may also depend on the stability of viral particles and the difference of viral protein structure, relative humidity, and temperature (Webster et al., 1992; Lowen et al., 2007). However, our experiment posed several limitations, meaning that more viral strains isolated from different animals and species need to be tested in order to investigate the correlation between pathogenicity and host immunity. Further investigation is also clearly needed to elucidate the differences of pathogenicity, transmissibility, and host innate immune response to infection with H5N6 AIVs in chickens.